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pimpinella anisum l. , a plant belonging to the umbelliferae family , is one of the oldest medicinal plants .
it is an annual grassy herb with 3050 cm high , white flowers , and small green to yellow seeds , which grows in the eastern mediterranean region , west asia , the middle east , mexico , egypt , and spain .
p. anisum is primarily grown for its fruits ( aniseeds ) that harvested in august and september .
aniseeds contain 1.55% essential oil and used as flavouring , digestive , carminative , and relief of gastrointestinal spasms .
consumption of aniseed in lactating women increases milk and also reliefs their infants from gastrointestinal problems . in the food industry , anise is used as flavoring and aromatic agent for fish products , ice cream , sweets , and gums [ 1 , 3 ] . despite the various studies which were conducted on pimpinella anisum , there is no comprehensive review study on constituents and effectiveness of this plant .
so the objective of this study was collecting all published articles about the chemical constituents and pharmacological properties of aniseeds with literature search of googlescholar , pubmed , sciencedirect , scopus , and sid database from 1970 up to 2011.the agricultural studies and investigations on aniseeds tissue culture were excluded .
anise seeds are used as analgesic in migraine and also as carminative , aromatic , disinfectant , and diuretic in traditional medicine .
aniseed has warm and dry nature and can increase milk production , menstruation , urine , and sweat secretion and also making good complexion .
anise is mentioned for melancholy , nightmare , and also in treatment of epilepsy and seizure [ 5 , 6 ] .
aniseed contains 1.56.0 mass % of a volatile oil consisting primarily of trans - anethole and also as much as 811 mass % of lipids rich in fatty acids , such as palmitic and oleic acids , as well as approximately 4 mass % of carbohydrates , and 18 mass % of protein .
other studies have demonstrated the presence of eugenol trans - anethole , methylchavicol , anisaldehyde , estragole , coumarins , scopoletin , umbelliferone , estrols , terpene hydrocarbons , polyenes , and polyacetylenes as the major compounds of the essential oil of anise seed .study of the essential oil of pimpinella anisum l. fruits by gc and gc - ms showed the presence of trans - anethole ( 93.9% ) and estragole ( 2.4% ) .
other compounds that were found with concentration higher than 0.06% were ( e)-methyleugenol , -cuparene , -himachalene , -bisabolene , p - anisaldehyde , and cis - anethole . in another study for determination of the composition of essential oil of pimpinella
anisum l. fruits obtained from different geographical areas of europe , in addition to the major components ( trans - anethole ( 76.993.7% ) and -himachalene ( 0.48.2% ) , some other compounds such as trans - pseudoisoeugenyl 2-methylbutyrate , p - anisaldehyde , and methylchavicol were also identified in essential oil .
study of components of the whole plants and the seeds of pimpinella anisum from alberta showed that the major oil constituent ( trans - anethole ) was 57.4% of whole plant and 75.2% of seed oil .
the other constituents of plant oil , present in amounts of 15% were cis - anethole , carvone , -caryophyllene , dihydrocarvyl acetate , estragole and limonene .
the chemical constituents of aniseed extract obtained by supercritical extraction using co2 were determined by gc - ms .
the major compounds were anethole ( ~90% ) , -himachalene ( 24% ) , p - anisaldehyde ( < 1% ) , methylchavicol ( 0.91.5% ) , cis - pseudoisoeugenyl 2-methylbutyrate ( ~3% ) , and trans - pseudoisoeugenyl 2-methylbutyrate ( ~1.3% ) .
4-(-d - glucopyranosyloxy ) benzoic acid which is one of the phenolic glycosides of the umbelliferae family was also isolated from aniseed . in a study by fujimato et al .
, four aromatic compound glucosides , an alkyl glucoside , and a glucide were isolated as new compounds from the polar portion of methanolic extract of anise fruits .
the structures of the new compounds were clarified as ( e)-3-hydroxy - anethole -d - glucopyranoside , ( e)-10-(2-hydroxy-5-methoxyphenyl ) propane -d - glucopyranoside , 3-hydroxyestragole -d - glucopyranoside , methyl syringate
4-o--d - glucopyranoside , hexane-1,5-diol 1-o--d - glucopyranoside , and 1-deoxy - l - erythritol 3-o--d - glucopyranoside .
isolation and structure elucidation of flavonoid constituents from anise , caraway , coriander , and fennel by means of chromatography on cellulose columns lead to isolation of quercetin 3-glucuronide , rutin , luteolin 7-glucoside , isoorientin , and isovitexin as crystalline compounds and apigenin 7-glucoside and a luteolin glycoside as noncrystalline compounds from anise . in another study ,
a silver ion hplc procedure was used to determine the fatty acids composition of aniseed oil .
the results showed the positionally isomeric 18:1 fatty acids oleic acid ( cis 918:1 ) , petroselinic acid ( cis 618:1 ) , and cis - vaccenic acid ( cis 1118:1 ) , in aniseed oil by a single gradient run on a single cation exchange column laboratory converted to the silver ion form .
also three lignin - carbohydrate - protein complexes were isolated from a hot water extract of the seeds of pimpinella anisum by combination of anion - exchange , gel filtration , and hydrophobic interaction column chromatographies .
the antibacterial activities of the aqueous , 50% ( v / v ) methanol , acetone and petroleum ether extracts of pimpinella anisum l. fruits were tested against 4 pathogenic bacteria ( staphylococcus aureus , streptococcus pyogenes , escherchia coli , and klebsiella pneumoniae ) by disc diffusion method .
the results showed that only aqueous and methanol extracts exhibited fair antibacterial activity against all of the test bacteria and the aqueous extract was found to be more effective than methanolic extract , whereas acetone and petroleum ether extracts can not inhibit the growth of the pathogenic test bacteria .
antimicrobial effects of water and ethanolic extracts of aniseed were studied by gulcin et al .
against 10 bacterial species and also candida albicans with disc diffusion method . in this study ,
ethanolic extract showed significant inhibitory activity against all tested bacteria but not effective on candida albicans .
however , the antimicrobial effect of water extract was not detected against gram - negative bacteria , pseudomonas aeruginosa , and escherichia coli , but it was effective against candida albicans .
the alcoholic extracts of pimpinella anisum seeds also showed antibacterial activity against micrococcus luteus and mycobacterium smegmatis . in another study ,
synergic antibacterial activity between thymus vulgaris and pimpinella anisum essential oil and methanol extract was evaluated against 9 pathogenic bacteria .
essential oil and methanol extract of these plants exhibited antibacterial activity against most tested pathogens , and the maximum effect was observed against staphylococcus aureus , bacillus cereus , and proteus vulgaris .
however , combination of essential oil and methanol extracts of these plants showed an additive effect against most tested bacteria especially pseudomonas aeruginosa .
the antibacterial potential of aqueous decoctions of black pepper , bay leaf , aniseed , and coriander against 176 bacterial isolates belonging to 12 different genera were detected by the mean of disc diffusion technique .
the findings showed that the aqueous decoction of black pepper was the most bacterial - toxic exhibited 75% antibacterial activity and decoction of aniseed exhibited 18.1% antibacterial activity ( maximum antibacterial activities exhibited against micrococcus roseus ) .
in addition to antibacterial activity , the essential oil of aniseed showed significant inhibitory activity against fungi , and the most active component of it was anethol . in a study by kosalec et al . , antifungal activities of fluid extract and essential oil of anise fruits studied on seven species of yeasts and four species of dermatophytes by the means of diffusion method with cylinders and the broth dilution method .
the findings revealed that the fluid extract of aniseed showed antimycotic activity against candida albicans , c. parapsilosis , c. tropicalis , c. pseudotropicalis , and c. krusei , and the largest inhibition zone was observed in c. albicans .
it is also showed inhibitory effect against dermatophyte species ( trichophyton rubrum , t. mentagrophytes , microsporum canis , and m. gypseum ) , and the largest inhibitory zone was detected for t. mentagrophytes .
the essential oil of anise exhibited strong antifungal activity against yeasts and dermatophytes , and the largest inhibition zone was found in c. parapsilosis ( 30 mm ) , followed by c. albicans , c. glabrata , and geotrichum spp . in this study , anise essential oil exhibited stronger antifungal activities rather than its extract against yeasts and dermatophytes .
antifungal activity of anise essential oil also was reported against aternaria alternata , aspergillus niger , and aspergillus parasiticus , and the most effected fungus was a. parasiticus .
investigation of antifungal activity of methanolic extracts of aniseed against four dermatophyte species , and one saprophyte fungus showed that the extract inhibited only dermatophyte species included candida albicans , trichophyton mentagrophyte , and microsporum canis but demonstrated no inhibitory effect on saprophyte a. niger .
the relaxant effect of pimpinella anisum on isolated guinea pig tracheal chains and its possible mechanism was studied by boskabady and ramazani - assari . in this research
, the bronchodilatory effects of aqueous and ethanol extracts and essential oil of anise were examined on precontracted isolated tracheal chains of the guinea pig by 10 mm methacholine in two different conditions including nonincubated tissues ( group 1 ) and incubated tissues with 1 mm propranolol and 1 mm chlorpheniramine ( group 2).the results showed that aqueous and ethanol extracts , essential oil , and theophylline ( 1 mm ) showed significant relaxant effects compared to those of controls .
the relaxant effects of aqueous and ethanol extracts were not significantly different from that of theophylline , but the effect of essential oil was significantly lower than theophylline .
there was also no significant difference between the relaxant effects obtained in group 1 and 2 experiments .
the results also showed that the relaxant effect of this plant is due to inhibitory effects on muscarinic receptors . in another study ,
antispasmodic and relaxant effects of three hydroalcoholic extracts of the aerial parts of pimpinella anisum ( ethanol : water ; 40 : 60 , 60 : 40 , and 80 : 20 ) were investigated on rat anococcygeus smooth muscle . the entire three hydroalcoholic extracts attenuated acetylcholine - induced contraction .
the finding of this study described that only the extract contains 60% ethanol ( ha60 ) showed concentration dependently relaxed acetylcholine - precontracted tissues , but two other hydro alcoholic extracts can not produce relaxation . studying the possible mechanisms underlying the relaxant effect showed that this effect is mainly dependent on the activation of the no - cgmp pathway .
anticonvulsant effects of an essential oil of the fruits of pimpinella anisum were studied against seizures induced by pentylenetetrazole ( ptz ) or maximal electroshock ( mes ) in male mice .
this study revealed that p. anisum increases the threshold of clonic seizures induced by i.v .
moreover , p. anisum possesses anticonvulsant activity against tonic seizures induced by mes . in another study by heidari and ayeli ,
the effect of methyl alcoholic extract of anise on picrotoxin - induced seizure in mice was studied .
the results showed that anise extract caused an increased delay in the onset of seizure in the mice which had been pretreated with different doses of the extract , and the most effective dose was 200 mg / kg ( p < 0.05 ) .
in addition , this dose delayed the time of death in mice ( p < 0.01 ) more satisfactory than phenobarbital ( 40 mg / kg ) on delaying death time
. results of the other study for investigating the effect of the aqueous extracts of leaves and stems of some arab medicinal plants including pimpinella anisum on the picrotoxin - induced seizures in mice revealed that the extracts of rosmarinus officinalis , pimpinella anisum , matricaria chamomilla , and artemisia vulgaris were found to delay the onset of picrotoxin - induced seizures and to decrease the mortality rate . in year 2008 , janahmadi et al . studied the cellular mechanisms of antiseizure effect of anise fruits . in this study , they determined whether the fruit essential oil of anise affect the bioelectrical activity of snail neurons in control condition or after pentylenetetrazol-(ptz- ) induced epileptic activity .
the results indicate that the candidate cellular mechanisms probably underlying the hyperexcitability produced by anise oil include enhancement of ca channels activity or inhibition of voltage and/or ca dependent k channels activity underlying post - hyperpolarization potential . for studying the effect of aqueous suspension of anise against gastric ulcers in rat ,
the results showed that anise significantly inhibited gastric mucosal damage induced by necrotizing agents and indomethacin .
the antiulcer effect was further confirmed histologically . in a case study , an aromatherapy treatment containing pimpinella anisum , foeniculum vulgare , var .
dulce , anthemis nobilis , and mentha piperita was examined in twenty - five patients suffering from the symptoms of nausea in a hospice and palliative care program .
however , all patients in this study were also using a variety of other treatments for their symptoms .
the laxative efficacy of a phytotherapic compound containing pimpinella anisum l. , foeniculum vulgare miller , sambucus nigra l. , and cassia augustifolia was studied in a randomized clinical trial included 20 patients presenting with chronic constipation according to the criteria of the american association of gastroenterology .
secondary endpoints included number of evacuations per day , perception of bowel function , adverse effects , and quality of life .
the results of the study revealed significant laxative effects of phytotherapic compound when compared with placebo .
this effect was demonstrated by a decrease in colonic transit time as well as an increase in the number of daily evacuations .
although quality of life did not show significant differences among the study periods and no significant differences were observed in terms of adverse effects throughout the study period , so this compound can be a safe alternative option for the treatment of constipation .
the effects of essential oil of pimpinella anisum on the expression and acquisition of conditioned place preference ( cpp ) induced by morphine in mice were studied .
the findings showed that subcutaneous injections of morphine ( 25 mg / kg ) produced place preference in a dose - dependent manner and injection of essential oil of p. anisum may induce conditioned place aversion in mice , that is , the essential oil has some aversive effects as investigated by place conditioning paradigm .
screening of some iraqi medicinal plants for analgesic activity showed that the extracts of tribulus terrestris and pimpinella anisum exhibited significant analgesic activity versus benzoquinone - induced writing and in thermal tests . in a study by tas , essential oil of pimpinella anisum showed significant analgesic effect similar to morphine and aspirin .
also fixed oil of anise was investigated for anti - inflammatory and analgesic activity in mice .
the finding showed that the fixed oil of anise has anti - inflammatory effect as strong as indomethacin and it showed analgesic effect comparable to that of 100 mg / kg aspirin and 10 mg / kg morphine at 30th min .
in a double blind clinical trial , the effect of anise extract on menopausal hot flashes in 72 postmenopausal women was examined . in this study ,
consumption of 3 capsules of anise extract ( each capsule contains 100 mg of extract ) for 4 weeks leads to significant reduction in hot flash frequency and intensity and in postmenopausal women . in a study by khoda karami et al .
, the effectiveness of a herbal capsule containing dried extracts of celery , saffron , and anise was compared with mefenamic acid capsule in 180 female students ( with age 1728 ) with primary dysmenorrhea .
the results showed significant reduction in pain intensity in both herbal and mephnamic acid group compare to placebo group . also the results revealed that the effectiveness of herbal capsule was better than mephnamic acid in pain relief and can be a suitable alternative in primary dysmenorrhea . in a study by gulcin et al .
, the antioxidant properties of water and ethanolic extracts of aniseeds were evaluated using different antioxidant tests , and antioxidant activities were compared with synthetic antioxidants such as butylated hydroxyanisole ( bha ) , butylated hydroxytoluene ( bht ) , and -tocopherol .
both extracts of aniseeds showed strong antioxidant activity , reducing power , dpph radical and superoxide anion scavenging , hydrogen peroxide scavenging , and metal chelating activities compared to bha , bht , and -tocopherol , and water extract exhibited greater antioxidant capacity than ethanolic extract . also investigation of in vitro and in vivo antioxidant potential of aniseeds showed that ethanolic extract of aniseed displayed scavenging activity against nitric oxide , superoxide and 1,1-diphenyl , 2-picryl hydrazyl ( dpph ) radicals and reducing power in a concentration - dependent manner .
the antioxidant activities were assessed by inhibition of linoleic acid peroxidation , 1,1-diphenyl-2-picrylhydrazyl ( dpph ) radical scavenging , fe reducing power , and various lipid peroxidation assays .
the findings showed that the anise oil and its methanol oleoresin showed highest antioxidant activity , even higher than bha and bht .
screening of antioxidant properties of some umbelliferae fruits from iran ( including pimpinella anisum ) by the dpph ( 2,2-diphenyl-1-picrylhydrazyl ) radical scavenging test showed that all the extracts exhibited antioxidant capability , and p. anisum extract ( ic50 = 109.80 ) exhibited the strongest activity and the ethyl acetate fraction of the extract exhibited the highest activity and flavonoid content .
a positive correlation was found between the antioxidant potency and flavonoid content of the fractions .in another study the antioxidant activity of water and alcohol extracts of chamomile flowers , anise seeds , and dill seeds was investigated .
the extracts showed marked antioxidant activity in both linoleic acid and liposome model systems , although the water extracts showed higher antioxidant activity than the corresponding alcohol extracts .
the finding indicate that anise tea showed weak antioxidant activity in trolox - equivalent antioxidant capacity ( teac assay ) and moderate effect in hypochlorite quenching assay and peroxynitrite quenching assay rather than other herbal teas .
plant essential oils from 40 plant species including pimpinella anisum were tested for their insecticidal activities against larvae of lycoriella ingenua using a fumigation bioassay . some of the essential oil including anise and garlic essential oil showed good insecticidal activity against the larvae . among the identified compound in effective essential oils ,
allyl isothiocyanate was the most toxic against larvae of l. ingenua followed by trans - anethole , diallyl disulfide , and p - anisaldehyde .
showed that the essential oils of juniperus macropoda and pimpinella anisum were highly effective as larvicidal and ovicidal against three mosquito species .
also the anise essential oil showed repellency against mosquito culex pipiens . in another study ,
the exposure to vapours of essential oils from anise and cumin resulted in 100% mortality of the eggs of two stored - product insects ( the confused flour beetle , tribolium confusum , and the mediterranean flour moth , ephestia kuehniella ) .
the acaricidal activity of p - anisaldehyde derived from anise seed oil and commercially available components of anise seed oil were studied against the house dust mites , dermatophagoides farina , and d. pteronyssinus .
the results showed that the compound most toxic to these dermatophagoides were p - anisaldehyde followed by benzyl benzoate and , therefore , p - anisaldehyde may be useful as a lead compound for the selective control of house dust mites .
the effects of the essential oil of foeniculum vulgar and pimpinella anisum were examined against pvx ( potato virus ) , tmv ( tobacco mosaic virus ) and trsv ( tobacco ring spot virus ) , on the hypersensitive host chenopodium amaranticolor .
the essential oil is at 3000 ppm completely inhibited pvx , tmv , and trsv .
three lignin - carbohydrate - protein complexes ( lc1 , lc2 , and lc3 ) with antiviral and immunostimulating activity were isolated from a hot water extract of seeds of pimpinella anisum by combination of anion exchange , gel filtration , and hydrophobic interaction column chromatographies .
these complexes showed antiviral activities against herpes simplex virus types 1 and 2 , human cytomegalo virus , and measles virus .
after raw 264.7 , murine macrophage cells had been incubated with these compounds for 20 h , and the nitric oxide ( no ) production was enhanced in dose - dependent manner .
the effects of these compounds on mrna and protein expression of inducible nitric oxide synthase ( inos ) in raw 264.7 cells showed that they induced mrna inos expression in a time - dependent manner .
these results suggest that the lignin - carbohydrate - protein complexes from p. anisum possessed potency as functional food ingredients against infectious diseases .
the antidiabetic , hypolipidemic , and antioxidant activities of aniseeds and coriander seeds were compared in type 2 diabetic patients .
the seed powders ( 5 g / day ) were administered to two groups of type 2 diabetes patients for 60 days .
the results indicated 11% rise of fasting blood glucose in control and 36% decrease in aniseed - treated , and 13% decrease in coriander - treated type 2 diabetics . also significant decrease in serum cholesterol and triglycerides in aniseed treated and coriander seed - treated patient
protein oxidation in serum and lipid peroxidation in erythrocytes and plasma was decreased in both treated groups as compared with the initial values .
both the groups showed rise in serum -carotene and vitamin a levels which could have resulted in a significant decrease in lipid peroxidation in rbc and plasma , and also rise in vitamin c was detected in both anise and coriander group .
so both the seeds have antidiabetic , hypolipidemic and antioxidant effects in diabetic patient [ 45 , 46 ] .
the effect of aniseed oil on the absorption of glucose from the jejunum and water from the colon and kidney tubules and also its mechanism of action were studied by kreydiyyeh et al .
the findings showed that aniseed oil increased significantly glucose absorption in the rat jejunum , because the oil enhanced the activity of the na - k atpase which increases the sodium gradient that gears the mucosal glucose transport .
however , adding of anise oil at a 0.05% concentration did not have any significant effect on colonic water absorption , and it seems thus that the atpase in the colon is resistant to the oil effect . furthermore , adding aniseed oil to drinking water , reduced the volume of urine produced in the rat and increased the activity of the renal na - k atpase even at extremely low concentrations . in the study by ciftci et al .
, adding 400 mg / kg of anise oil to the diet of day - old broilers was improved feed conversion ratio by approximately 6% compared to antibiotic group .
the effects of diet supplementation with aniseed and fenugreek seeds on the performance does and kits were studied.finding revealed that the daily milk intake of kits in anisum - fenugreek group was equivalent to that of control rabbits .
also , the 17 days body weight did not differ significantly between two groups . at 35 days of lactation , the differences between anisum - fenugreek group and control groups were not significant in litter size , litter weight , kit weight and 135 day weight , gain . in conclusion ,
further studies are needed to investigate the palatability and optimal level of these spices in the feed of lactating rabbits .
pharmacological evaluations of plants may be due to new natural agents for treatment of disease . furthermore ,
identification of the active principles of these medicinal plants has an important role in introducing new drugs .
pimpinella anisum is one of the medicinal plants which have been used for different purposes in traditional medicine of iran .
so far , different studies were performed on the extracts and essential oil of pimpinella anisum to identify the chemical compounds and pharmacological properties of this plant , and various properties such as antimicrobial , antifungal , antiviral , antioxidant , and insecticidal effects have been reported of aniseeds .
the findings also revealed that aniseeds can cause gastric protection , muscle relaxant , and affect digestive system .
. furthermore , aniseeds showed anticonvulsant effect , reduced morphine dependence , and induced conditioned place aversion in mice .
the most important compounds of aniseeds essential oil were trans - anethole , estragole , -hymachalen , p - anisaldehyde , and methyl chavicol .
due to broad spectrum of pharmacological effects of this plant , and very few clinical studies performed on this plant , more clinical trials are recommended to evaluate the beneficial effects of pimpinella anisum in human models and identification of active compounds of this plant which can lead to synthesis of new drugs from the active ingredients in future . | pimpinella anisum ( anise ) , belonging to umbelliferae family , is an aromatic plant which has been used in iranian traditional medicine ( especially its fruits ) as carminative , aromatic , disinfectant , and galactagogue . because the wide traditional usage of pimpinella anisum for treatment of diseases , in this review published scientific reports about the composition and pharmacological properties of this plant
were collected with electronic literature search of googlescholar , pubmed , sciencedirect , scopus , and sid from 1970 to 2011 .
so far , different studies were performed on aniseeds and various properties such as antimicrobial , antifungal , antiviral , antioxidant , muscle relaxant , analgesic and anticonvulsant activity as well as different effects on gastrointestinal system have been reported of aniseeds .
it can also reduce morphine dependence and has beneficial effects on dysmenorrhea and menopausal hot flashes in women . in diabetic patients
, aniseeds showed hypoglycemic and hypolipidemic effect and reduce lipid peroxidation .
the most important compounds of aniseeds essential oil were trans - anetole , estragole , -hymachalen , para - anisaldehyde and methyl cavicol . due to broad spectrum of pharmacological effects , and very few clinical studies of pimpinella anisum , more clinical trials
are recommended to evaluate the beneficial effects of this plant in human models and synthesis of new drugs from the active ingredients of this plant in future . |
the ability of temporal resolution is fundamental for comprehension of human speech and is a prerequisite for linguistic abilities.1
2 this ability acts in the auditory perception of verbal and nonverbal sounds ; in the perception of music , rhythms , and periodicity ; and in the discrimination of steps , duration , and phonemes.3
alteration of the auditory perception may bring about problems in speech and language development as well as in learning and socialization of children , adults , and the elderly .
the decoding of the spoken message involves the analysis of many signal components including the acoustical , phonetic , phonological , lexical , suprasegmental , syntactical , and semantic components.4
5 for this decoding to occur , the acoustical cues of frequency , intensity , and timing must be processed in a precise manner by the auditory system.6
7
phonemes , the isolated sounds that make up the syllables , represent these rapid sound stimuli in speech .
hence , the individual with altered perception would have difficulty perceiving that words can be decomposed in those smaller isolated sounds .
this ability , called phonological conscience , is essential for one to master the alphabetical system in which the phonemes are mapped as letters ( graphemes ) .
therefore , the learning of this ability can be considered essential when learning how to read8 and at the same time is the main manifestation of the reading and writing disorders . according to the world health organization,9
dyslexia is defined as a disorder that manifests itself by specific difficulties in learning how to read and write that are not attributable to any other kind of deficit related to intelligence , motivation , learning opportunity , or sensorial acuity .
reading and writing disorder is described as a manifestation that features the difficulty in acquiring and/or developing written language by children and presents both deficits in phonological decoding and oral and/or written language comprehension.10
keeping this relation in mind , subjects with phonological deviation and/or reading and writing difficulties may present alterations in temporal auditory processing and may need more time to detect gaps between auditory stimuli . the goal of this study was to verify and compare the temporal resolution ability in individuals diagnosed with a reading and writing disorder to those with dyslexia , which may help in speech and language rehabilitation .
this study was approved by the universidade federal de so paulo ethics in research committee ( unifesp / epm ) , process no .
1726/09 . all the selected subjects were volunteers and signed a free and willing consent form .
participating subjects were divided in two groups : 11 with the diagnosis of dyslexia ( gd ) and 15 with the diagnosis of reading and writing disorder ( gsd ) .
subjects in the gsd group were receiving speech and language treatment at the nucleus for teaching , assistance and research of reading and writing disorders neapel unifesp . for patients with a diagnosis of dyslexia ,
the brazilian association of dyslexia and infant interdisciplinary nucleus of neuropsychological treatment were contacted . to form this study sample ,
volunteers had to be between the ages of 10 and 15 , of either gender , without any history of evidence of psychiatric or neurologic impairment or of hearing loss . to constitute the control group ,
the work of perez and pereira11 was used to establish normalcy reference criteria for youngsters of 11 and 12 years of age of both genders . the threshold measure considered as the normal value followed the criteria of a positive response for four of six presentations : a 5.0-millisecond threshold for the right ear and 5.11 milliseconds for the left ear , and a total 71.99% correct response for the right ear and 71.41% for the left . a stratified lottery based on the studied age groups
all subjects completed a basic hearing assessment , including collection of a case history , tonal threshold audiometry , speech audiometry , and tympanometry .
the procedure used to measure the behavior of temporal resolution was the gaps - in - noise ( gin ) test proposed by musiek et al,12 with a total of 60 silence intervals or gaps inserted in a noise segment , with a time variation of silence between 2 and 20 milliseconds .
two kinds of responses were analyzed : the temporal acuity threshold , which is the least value in milliseconds in which the subject perceived the silence interval , and the total number of correctly identified gaps , in percentage values .
the gin test , presented from a cd , was applied using an interacoustic ma-41 audiometer attached to a sony cd player ( interacoustics as drejervaenget , assens0 denmark ) in an acoustic cabin at a 50-db sl ( sensation level ) intensity ( according to the mean auditory thresholds for 500 , 1,000 , and 2,000 hz ) .
each test track is composed of diverse stimuli of 6 seconds of white noise with 5-second intervals between the stimuli .
the training track was applied before the beginning of the test to ensure that the subject clearly understood what needed to be done .
the test was applied to one ear at a time . in the right ear , test track 2 was used , and in the left ear , test track 3 .
the instructions to the subjects were : you will hear a noise and within this noise there will be spaces or gaps in which the noise will be absent .
the gaps will vary in size and you will have to listen carefully , because some of them will be extremely small . occasionally there will be no gaps .
the threshold considered was the perception of four of six presentations of the same gap , that is , 66.66% of identification of a specific silence interval in milliseconds according to the criteria of musiek et al.12
to assess the answers in this study , in the three groups ( control , diagnosis of reading and writing disorder , diagnosis of dyslexia ) , the nonparametric test of kruskal - wallis was used , followed by a procedure of multiple nonparametric comparisons .
a nonparametric test was applied because it was not possible to find a transformation that could stabilize the variance in the responses of the three groups .
the criteria adopted to determine the significance was the level of 0.05 with the construction of confidence intervals of 95% .
table 1 shows the descriptive statistics for the responses obtained by the three groups : the group with dyslexia , the group with reading and writing disorders , and the control group .
the gin threshold was not the same for all groups for both the right ( p < 0.001 ) and the left ears ( p = 0.0015 ) ; it was significantly lower for the control group than for the other groups ( p < 0.05 ) .
there was no difference between the group with dyslexia and the group with reading and writing disorders ( p > 0.05 ) .
abbreviations : gd , group with dyslexia ; gin , gaps - in - noise test ; gsd , group with specific reading and writing disorder ; le , left ear re , right ear ; sd , standard deviation ; t , threshold .
the percentage of correct responses for the gin was not the same for the three groups , for both the right ( p < 0.001 ) and left ears ( p < 0.001 ) .
the gin percentage was significantly higher for the control group than for the other groups ( p < 0.05 ) .
there was no difference between the group with dyslexia and the group with reading and writing disorders ( p > 0.05 ) .
analysis of the gin test in our subjects with dyslexia and subjects with a reading and writing disorder reinforce studies that indicate that phonological alterations need a longer temporal processing , that is , need a longer interval to perceive the difference between sounds .
some researchers indicated that school - aged children with dyslexia may present temporal processing problems.13 the perceptual abilities related to speech , language , and reading are extremely dependent on the ability of temporal resolution , which may be assessed by the gin test . in the present study ,
school - aged children in the group with dyslexia and the group with reading and writing disorders presented a deficit in auditory temporal resolution compared with the control group .
there was no statistically significant difference between the responses for the study group with dyslexia and the group with reading and writing disorders .
some studies verified that the phonological deficit must be caused by an auditory processing deficit due to the fact that the altered auditory temporal processing may hinder the perception of subtle signals in speech resulting in the observed difficulties in phonological processing.13
14
15
16 boscariol et al studied the random gap detection test ( rgdt ) and found results of 13 milliseconds for the control group and 32.39 milliseconds for the gd group.13 the rgdt and gin tests assess the temporal resolution ability ; however , they have diverse investigation natures , hence there were differences in threshold limits for the same sample between test protocols.17
simes and schochat assessed 40 children aged 7 to 12 years of age , 20 with dyslexia and 20 in a group with auditory processing disorders.14 the tests involved the abilities of auditory closure , figure - ground for linguistic sounds , and temporal ordering .
the subjects in the dyslexia group presented altered responses only for the test that assessed temporal processing .
these researches agree with other studies that associate reading and writing abilities to auditory temporal processing abilities.15
18
regarding the descriptive measures of the auditory responses for the gin test , we observed a gap threshold for the right ear of 7.8 milliseconds for the group with reading and writing disorders and a gap of 7.1 milliseconds for the group with dyslexia .
the left ear obtained the threshold of 7.4 milliseconds for the group with reading and writing disorders and 7.2 milliseconds for the group with dyslexia .
the found percentage was 55.60% for both ears for the dyslexia group , and for the reading and writing disorders was 57.44% for the right ear and 60.77% for the left ear .
the literature points to gap thresholds less than 6 milliseconds in subjects from 7 to 46 years of age .
this confirms that the subjects with dyslexia and with reading and writing disorders present worse results when compared with the control group .
samelli and schochat19 observed similar gap detection thresholds for both ears when studying 100 young adults age 18 to 31 .
the general mean of the gap thresholds was 3.98 milliseconds and the mean percentage of correct gap detection was 78.89% .
however , the author considered a gap threshold to be 50% of presentations , that is , three correct responses of the six presented .
a study in school - aged children found values of 4.7 milliseconds and 73.6% in children of 8 , 9 , and 10 years of age20 and 5.05 milliseconds and 71.70% in children age 11 and 12 years.11
the group with dyslexia and the group with the diagnosis of reading and writing disorders in our study had lower mean response values than those obtained for the control group and lower than that in the reviewed literature , indicating the presence of the inability of temporal resolution in individuals with reading / writing difficulties .
after analyzing the data obtained in this study , subjects in the group with dyslexia and in the group with reading and writing disorders presented worse responses in the gin test than the control group .
this result can indicate a correlation between the temporal processing abilities and the reading and writing abilities . | introduction the gaps - in - noise ( gin ) test assesses the hearing ability of temporal resolution .
the development of this ability can be considered essential for learning how to read .
objective identify temporal resolution in individuals diagnosed with reading and writing disorders compared with subjects with dyslexia .
methods a sample of 26 subjects of both genders , age 10 to 15 years , included 11 diagnosed with dyslexia and 15 diagnosed with reading and writing disorders .
subjects did not display otologic , neurologic , and/or cognitive diseases .
a control group of 30 normal - hearing subjects was formed to compare thresholds and percentages obtained from the gin test .
the responses were obtained considering two measures of analysis : the threshold gap and the percentage of correct gap .
results the threshold was lower in the gin for the typical group than for the other groups .
there was no difference between groups with dyslexia and with reading and writing disorders .
the gin results of the typical group revealed a higher percentage of correct answer than in the other groups .
no difference was obtained between the groups with dyslexia and with reading and writing disorders .
conclusion the gin test identified a difficulty in auditory ability of temporal resolution in individuals with reading and writing disorders and in individuals with dyslexia in a similar way . |
the study of the impact of ionizing radiation ( ir ) on biological objects began almost with the discovery of radiation . immediately , besides its beneficial use , its destructive effects on living organisms , including human , were discovered and documented .
application of ir in various fields of human activity is progressively increasing in parallel with growing risk of biological effects .
exposure of humans to radiations may occur not only from atomic bomb , nuclear accidents , radiation contamination of environment , or human errors but also through routine diagnostic and therapeutic procedures .
many reports focus on low - dose effects as they do not cause clinical changes and appear only after a long time and more often , by way of cancer or associated diseases .
being a strong mutagen , ir may cause changes in genome of the living organisms .
estimation of the radiation dose is one of the most important steps for the treatment of victims of radiation accidents . among different types of radiation - induced lesions ,
dna double - strand breaks are considered the most relevant of the deleterious effects of ir .
comet assay , also known as single - cell gel electrophoresis assay , is a rapid and sensitive method which is employed in the detection of dna double - strand breaks in individual cells .
this assay can also be applied to determine the dna repair efficiency , which involves the incision and subsequent rejoining of damaged strands .
therefore , this assay may provide important information regarding the molecular mechanisms that are responsible to counteract radiation impact
. there has been significant increase in the use of ir for medical procedures both in diagnostics and treatment in recent times .
although the general radiobiological principles underlying external beam and radionuclide therapy are similar , there exist significant differences in the biophysical and radiobiological effects caused by these two types of radiations . due to this ,
it is important to accurately assess the absorbed dose for the physician to have a real picture of changes in patients caused by irradiation using either external beam or radionuclide .
such information will help better management of the patient as well as its follow - up .
the main contingent for investigation of radiation impact is professionals , being influenced due to their occupation , inhabitants of regions with increased radiation background , and patients undergoing radiotherapy or multiple diagnostic procedures .
here , we compared the response of human cells in the form of chromosomal and dna damage dynamics to comparable radiation doses from whole - body or local irradiation .
radiation contamination was detected in georgia due to safety lapses in the regulation of the territorial military bases in the late 1990s , following the withdrawal of soviet troops from georgia . in 20022004 in two regions of georgia , daba vaziani and dedoplistskaro in the frame of istc project g-564 , it was established by cytogenetic investigations that 19 individuals had received radiation doses exceeding 0.2 gy . increased dicentric frequency exceeding the background was also observed in another group of 30 persons .
hence , these individuals were included in the risk group but were not under any follow - up monitoring .
in our project , we have examined 37 inhabitants from the risk group of two above - mentioned regions and 12 head and neck cancer patients undergoing radiotherapy after their informed consent .
blood and buccal cells were collected and subjected to detection of chromosomal aberrations , dna strand breaks in lymphocytes , and yield of micronuclei in buccal cells . in each case , about 150300 metaphases , 150200 lymphocytes for dna - comet assay , and 1000 buccal cells for micronuclei were analyzed .
biodosimetry was carried out with conventional dicentric chromosomal assay in the first mitosis of peripheral blood lymphocyte in 4852-h cultures . according to standard method and by our own calibration curve
, we estimate the absorbed dose of ir at 0.2 gy or above . in addition
this method enables the detection of dna strand breaks in cells . for the detection of dna damage through comet assay
, blood leukocyte cells were suspended in low melting point agarose and embedded within a thin agarose gel on a microscope slide .
later , slides were electrophoresed and stained with luminescent dyes ( ethidium bromide or 4',6-diamidino-2-phenylindole ) for light microscopic study . for the cancer patients , we investigated the exfoliative cells of oral cavity , henceforth referred to as buccal cells , for micronuclei in buccal cells ( mnb ) and other nuclear anomalies .
these cells were evenly distributed on the slides , dried , and fixed ( 3:1 ; ethyl alcohol : acetic acid ) .
the preparations underwent weak acidic hydrolysis 1 n hcl at room temperature . after this , preparations were washed with distilled water and stained with schiff 's reagent , followed by fast green .
light microscopy was used for the analysis . to determine the possible physiological criteria of assessment of cancer risk and predictors of individual radiosensitivity , the functional state of the red blood system ( rbs ) was studied in cancer patients .
the functional state of rbs was evaluated by specially developed method based on analysis of the dynamics of population spectrum of erythrocytes of peripheral blood ( epb ) epb distribution according to their living resources . as minimally sufficient set of parameters , describing living resources of epb , the following two parameters were applied : spherulation degree ( q ) and volume ( v ) .
the first parameter is a good approximation and could be viewed as a degree of the erythrocytes spherulation ( q ) , determined as the relation of cell volume ( v ) to the volume of sphere with the same surface area ( s ) .
v. spherulation degree characterizes cell deformability and along with the volume determines the probability of overcoming by them of a barrier of reticuloendothelial system . in 12 cancer patients undergoing radiotherapy , biodosimetry was performed to determine the levels of chromosome aberrations and micronuclei and extent of dna damage before and after first and last irradiations .
radiotherapy was performed on linear accelerator ( 6 mv photon energy ) with three - dimensional conformal planning system 2 gy / fraction ( 2033 fractions ) with a total 4070 gy .
we have not been able to reexamine all individuals , living in areas where the radioactive sources were discovered 1012 years ago and who were included in the risk group in 2004 .
this examined group included individuals who had received an estimated dose 0.20.7 gy or had increased number of chromosomal aberrations , though insufficient to determine a dose .
out of 19 people subjected to dose estimation in 2004 , eight ( estimated doses 0.3 , 0.5 gy ) died from various cancers .
we could reexamine nine persons who were subjected to dose estimation and also 28 inhabitants from the risk group . in total ,
biodosimetry was carried out in all above - mentioned cases . in a 31-year - old man ,
whose previous established dose was 0.3 gy , we found 3 dicentrics ( 0.01 per cell ) in a 300 analyzed metaphases and stable chromosome aberrations ( marker chromosomes ) . according to our calibration curve
, the possible dose of exposure should be < 0.2 gy but more than our control data ( 0.0015 0.0006 ) . two dicentrics were found ( 0.008 per cell ) in 250 metaphases of a 38-year - old man , a native resident of daba vaziani .
clinical examination of this patient has revealed slight enlargement of lymph nodes , gastritis , colitis , and lymphocytosis .
we did not observe any significant difference in the dna damage produced in the exposed residents compared to the unexposed individuals ( 8%12% ) . since the comet assay detects the dna break breaks , it is possible that the breaks formed due to initial irradiation might have been eliminated from the system .
all investigated individuals were examined by physician - oncologist , and peripheral blood tests were conducted . in some of them ,
cancer as a cause of death among the eight individuals deserves particular attention . comparing the chronic irradiation of 1012 years ago ( 0.20.7
gy ) and the current clinical outcome , differential response was seen in patients who received similar amounts of radiation .
the effect of local irradiation was studied in cancer patients with tumors of the same anatomical localization .
six patients were in the first group ( five males and one female , 5065 years ) with cancers of planocellulare of larynx and nasopharynx , first - stage local disease . in the second group , there were six patients with the same sex and age distribution , with the same localization of cancer , but with ii
investigation before irradiation did not show any difference compared with our control data on all parameters .
even though the patients displayed cancer clinically , chromosomal abnormalities and dna damage remained well within the baseline data determined for healthy unexposed individuals .
however , following the first course of irradiation ( as part of radiotherapy ) , all end - points studied showed significant increase with differential sensitivity among patients ( mnb level 4.33 0.99 ; amount of comets 26%30% , chromosomal aberrations [ acentric single and paired fragments ] 0.020.05 per cell , number of dicentrics - 0.020.03per cell ) .
after the last irradiation , most of the patients did not show increase in the tested parameters . only in patient
number 4 and 6 , higher dicentrics ( 0.05 and 0.07per cell ) were detected . according to our data ,
this corresponds to the absorbed dose of 0.6 and 0.8 gy , respectively , during the whole - body irradiation .
together with unstable aberrations in patient number 6 , marker chromosomes in 4 metaphases were detected and amount of micronuclei increased from 0.5 up to 2 .
after 4 months of radiotherapy , clinical data were obtained on the patients ' conditions .
as can be expected from the chromosomal data obtained in patient number 4 and 6 , postradiation complications were seen from the clinical data .
for patient number 4 ( cancer of larynx 44 gy in 22 fractions ) , swelling and pain in the throat were observed . for patient number 6 ( cancer of larynx 66 gy in 33 fractions ) , tumors decreased marginally . after a gap of 1 month
dynamics of estimated parameters of first group cancer patients undergoing radiotherapy in the second group , changes in all estimated parameters were observed even before the irradiation [ table 2 ] .
the dynamics of radiation - induced cytogenetic changes is reflected in figure 1 , and the testing of the statistical significance in table 3 .
dynamics of estimated parameters of second group cancer patients undergoing radiotherapy the dynamics of radiation - induced cytogenetic changes in groups i and ii x - axis : stage of irradiation , y - axis : mean values : dna - comet in % , dicentrics per cell , mnb per 1000 cells ( standard error , 0.95 confidence interval ) statistical significance of radiation - induced changes dna - comet , micronuclei , and dicentric values in the first and second group of patients it is apparent that there was a significant difference in the cytogenetic parameters studies in patients from groups 1 and 2 .
a difference in response was visible among the two groups during the process of radiotherapy . in the second group of patients , a sharp statistically significant increase of all cytogenetic characteristics
further , the attention should be paid to the fact that in the first group of patients the level of background dna - comet was significantly lower compared to group 2 [ figure 1 , before , table 3 ] , while in the initial levels of the micronuclei and dicentric chromosomes , inter - group difference was not observed .
taking into account the above stated , correlations in peripheral blood erythrocytes ( pbe ) morphometric characteristics ( volume , shape ) with background values of cytogenetic properties were studied .
it was identified that standard deviation of volume of pbe young fractions ( pbe with low sperulation degree ) has a tendency of correlation with dna - comets background values ( r = 0.7 , p = 0.064 ) . correlations with other cytogenetic characteristics are not observed .
detection of pattern of cytogenetic changes among individuals exposed to low doses of radiation in contaminated environment and patients undergoing radiotherapy enabled us to compare effect of ir on different human beings after whole - body and local irradiations .
whole - body irradiated persons were residents of the regions where the radioactive sources were detected 1012 years ago . absorbed dose in range of 0.20.7 gy was determined by biodosimetry in 19 persons and selected persons with high level of chromosomal aberrations . in this work , we have analyzed the most recent results of these individuals and find that whole - body irradiation with identical doses is causing very heterogeneous response in different individuals .
this might depend on different factors , such as immunological stage , age , and sex .
there are several data that organism response on ir can depend also on genetic polymorphism . to estimate the effect of local irradiation
, we have monitored the dynamics of chromosomal and dna damage in patients with head and neck cancer undergoing fractionated radiotherapy with total dose of 4070 gy .
we observed more evident individual differences among estimated specific radiation biomarkers : dicentrics and other chromosomal damage , micronuclei in exfoliate buccal cells , and amount of dna - comets . despite one and
the same tumor localization and identical received dose of radiation , changes in the studied parameters were not homogeneous .
considering the heterogeneity in the response of patients as well as individuals to ir , caution should be exercised and appropriate treatment regimen should be planned for effective therapeutic outcome .
individual radiosensitivity might play a significant role in the management of radiotherapy patients even for similar cancer types .
we believe that the biomarkers we have chosen are more appropriate for the determination of geno- and cyto - toxic effects of ir .
the methods used in the present study were successfully used in the past to determine the individual radiosensitivity . moreover , our approach of using buccal exfoliated cells for micronucleus analysis may provide a complementary method for measuring dna damage and cytotoxic effects .
there is a strong association between dna damage and radiosensitivity as observed by other authors .
the correlation between initial cytogenetic parameters and functional stage of rbs substantiates as observed in our study highlights the importance of further research to estimate the individual radiation risk .
as can be seen from the current study , even after 1012 years of chronic general exposure to small doses of radiation and during the process of radiotherapy , radiation response may depend on individual sensitivity .
it is believed that the application of multiple / appropriate biomarkers will help in optimizing and individualizing subsequent medical management of radiotherapy in the future .
| dynamic changes of the chromosomal aberrations and the dna damage were analyzed in individuals exposed to low and therapeutic doses of radiation .
the investigation included 37 persons living in areas where the radioactive sources were discovered 1012 years ago .
it was established by biodosimetry methods that the examined persons had absorbed dose of 0.20.7 gy or had increased number of chromosomal aberrations , though insufficient to determine a dose .
clinical examination , chromosomal analysis , and assay of dna damage by the comet ( single - cell gel electrophoresis ) assay were carried out .
there was no correlation between the doses received 10 years ago and the cytogenetic changes with clinical outcome .
the effect of the local fractionated gamma - irradiation with doses of 4070 gy was studied in cancer patients with localized head and neck tumors .
the study of chromosomal abnormalities , the dna damages by the comet assay , and the micronuclei detection of the buccal cells revealed a statistically significant correlation between the initial cytogenetic indices in cancer patients and their dynamic changes during and after the radiation exposure .
in addition , the correlation was detected between the initial cytogenetic parameters and the functional stage of red blood system .
our results allow us to conclude that there is a need for further research to estimate the individual radiation risk to optimize and individualize the subsequent medical management of radiotherapy . |
the specific treatment of cyanide ( cn ) intoxication means the use of scavengers ( e.g. , methemoglobin former sodium nitrite ( sn ) or cobalt compounds or cyanohydrin formers , hydroxocabalamin ( cyanokit has been approved in the us ) , cobinamide , and/or the conversion of cn to the less toxic thiocyanate ( scn ) with exogenously administered sulfane sulfur and sulfurtransferase enzymes [ 24 ] .
rhodanese ( rh ) is the best characterized multifunctional , mitochondrial sulfurtransferase [ 58 ] catalyzing the transfer of a sulfane sulfur atom from a donor molecule to cyanide . determining the exact role of nitrite in cyanide antagonism
earlier studies were focusing on the methemoglobin - forming effect of nitrite that act as a scavenger by forming a relative stable complex of cyanomethemoglobin [ 3 , 4 ] .
very recent studies are focusing on the mitochondria - linked mechanism of nitrite as a nitric oxide donor [ 911 ] .
extensive researches are also focusing on developing effective sulfur - containing compounds serving as sulfur donors for reacting with cn with or without rh .
thiosulfate ( ts ) is the classical sulfur compound found to participate in the enzyme reaction [ 3 , 4 , 12 ] .
however , ts has limited ability to reach the endogenous rh enzyme because of a nearly exclusive extracellular distribution .
baskin et al . , reported results on the efficacy of various sulfur donors demonstrating that altering the chemical substituent of the longer chain sulfide modified the ability of the candidate molecule to protect against cn toxicity .
earlier investigations were focused on administration of free rh and the sulfur donor ( sd ) directly into the bloodstream [ 1518 ] .
unfortunately , the free rh enzyme was rapidly destroyed by the body 's immune system , which makes the efficacy of this approach quite limited . to overcome the limitations for the circulating free rh , micro- or nanosized carrier systems among others sterically stabilized unilamellar liposomes of ~100150 nm diameter are in the focus of recent encapsulation efforts .
the encapsulation of rh with a sulfur compound into liposomes the so - called coencapsulation can offer further advantages . over stability enhancement for rh
the coencapsulation can provide better overall conversion of cn , since the basis component for enzyme reaction , the sulfur donor no longer has to penetrate the liposome membrane .
the lipid composition has a significant impact on the encapsulation efficiency of the rh and/or sulfur compound and on the in vivo stability and antidotal effect of the carrier system .
thus , optimization of the liposomal composition is an inevitable step in the design of novel antidotal systems .
present work deals with a new lipophilic sulfur - containing compound , developed at the us army medical research institute of chemical defense , called dto . in order to achieve the highest cn antidotal protection ,
the objectives of this study are ( 1 ) optimization of the liposomal encapsulation for the new sulfur donor , dto , with superior sulfur donor reactivity to the present therapy ts ; ( 2 ) in vivo efficacy study of the coencapsulated dto with rh in combination with sodium nitrite on mice .
all chemicals employed were of the highest purity commercially available : potassium cyanide , ts , sodium nitrite , phosphate buffer components , ethanol , sodium chloride , concentrated hydrochloric acid , and sodium hydroxide were purchased from j. t. baker , ( phillipsburg , nj ) , formaldehyde and ferric nitrate were purchased from fisher scientific ( pittsburgh , pa ) . the liposome components ( 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine ( popc ) , 1 , 2-dioleoyl - sn - glycero-3-phosphocholine ( dopc ) , n-[1-(2,3-dioleoyloxy)propyl]-n ,
n , n - trimethylammonium chloride ( dotap ) , soy lecithin ( lec ) , cholesterol ( chol ) ) and rh were purchased from sigma - aldrich ( st . louis , mo ) , and 1,2-dipalmitoyl - sn - glycero-3-phosphoethanolamine - n-[methoxy(polyethylene glycol)-2000 ] ammonium salt ( peg - pe-2000 ) was purchased from avanti polar lipids ( alabaster , al ) .
bio - rad protein kit was purchased from bio - rad life sciences laboratories , hercules , ca .
male ( cd-1 ) mice ( charles river breeding laboratories , inc . , wilmington , ma ) weighing 1820 g were housed at 21c and in light - controlled rooms ( 12 h light / dark , full - spectrum lighting cycle with no twilight ) , and were furnished with water and 4% rodent chow ( teklad hsd , inc . , city , wi ) ad libitum .
all animal procedures were conducted in accordance with the guidelines by the guide for the care and use of laboratory animals ( national academic press , 1996 ) , accredited by aaalac ( american association for the assessment and accreditation of laboratory animal care ) . at the termination of the experiments
, surviving animals were euthanized in accordance with the 1986 report of the avma panel of euthanasia .
popc , dopc , dotap , lec , peg - pe-2000 , and chol dissolved in ethanol were applied in various molar ratios in order to determine the optimal lipid composition .
ph = 7.4 ; osmolarity = 290 mosm ) was added to the dry lipid films .
four different rh concentrations ( 0.25 mg / ml , 0.50 mg / ml , 1.00 mg / ml and 1.67 mg / ml ) , and four different dto concentrations ( 2.0 ; 10.0 ; 20.0 and 30.0 mm ) were investigated with various liposomal lipid compositions to evaluate the effects of these parameters on encapsulation efficiency for rh and/or dto .
the total lipid concentration ( lipids and chol together ) was 10.0 mg / ml . in order to obtain a homogenous population of small unilamellar vesicles ( suv ) , the multilamellar vesicles ( mlv ) were extruded through polycarbonate filters ( 100 and 400 nm ) with an extruder ( avanti polar lipids inc .
the formation of scn from cn was measured spectrophotometrically ( genesys 10uv , thermo electron corporation , waltham , ma ) by the method of westley , with minor modifications of petrikovics et al . .
one unit of rh was defined as the amount of enzyme that forms 1 mol of scn in 1 min .
formation of scn from cn with the investigated sulfur donors of ts and dto were determined spectrophotometrically by the method of westley with minor modifications of petrikovics et al . .
four different rh concentrations ( 0.25 mg / ml , 0.50 mg / ml , 1.00 mg / ml , 1.67 mg / ml ) were employed with a lipid composition of popc : chol : peg - pe-2000 with and without dotap .
optimal lipid composition for rh encapsulation was determined based on the highest enzyme activity achieved by the same encapsulation process with various lipid compositions .
unencapsulated rh was separated from sl - rh by gel filtration on a g-100 sephadex gel column ( 0.7 cm 10 cm ; ge healthcare biosciences ab , sweden ) .
( 1)encapsulation efficiency ( % ) = activity of sl - rhtotal rh activity 100 .
for the spectrophotometric assays ,
the encapsulation efficiency for the sulfur donor dto was determined by the rh assay described above with constant rh concentration . when rh concentration was constant , the rate of formation of scn was directly proportional to the sulfur donor concentration .
( 2)encapsulation efficiency ( % ) = concentration of encapsulated dtototal dto concentration 100 .
formation of scn by sl - rh - dto with various lipid compositions was measured spectrophotometrically as described above .
( 3)encapsulation efficiency ( % ) = scn formation by the given sl - rh - dtoscn formation by the original ( before encapsulation ) rh and dto concentration 100 .
experimental animals received kcn after pretreatment with antagonist(s ) ( sulfur donors and/or rh and/or sn ) .
freshly prepared sl - dto , sl - dto - ts , sl - dto - rh , and sl - dto - ts - rh were administered intravenously ( iv ) by tail vein injection to mice 10 min prior to receiving cn ( sc ) .
using 10 ml / kg doses of ( sl - dto - rh ; table 4 experiment 5 ) or ( sl - dto - ts - rh ; table 4 experiments 6 and 7 ) the animals received 1520 units of rh . employing 10 ml / kg injections of ( sl - dto ; table 4 experiment 2 ) or ( sl - dto - ts ; table 4 experiments 3 and 4 ) the dose for dto was 11.5 mg / kg and 14.2 mg / kg for the coencapsulated ts .
sn ( 100 mg / kg ; sc ) , was injected 45 min prior to cn ( sc ) injection ( table 4 experiments 4 and 7 ) .
the animals were evaluated 24 hours after cn exposure for mortality ; surviving animals were observed for an additional week for late - developing toxicity .
no toxic effects which could be attributed to sl - dto , sl - dto - ts , sl - dto - rh , sl - dto - ts - rh , ts , or sn ( when administered alone or in various combinations ) were noted in any of the mice at the doses applied .
ld50 values were determined by the dixon up and down method , using 818 mice for each ld50 determination .
( apr ) is expressed as a ratio of ld50 ( mean ) of cn with antagonists and ld50 ( mean ) of cn without antagonists .
these studies focused on the encapsulation optimization for new sulfur donor dto when encapsulated with rh and/or ts within sterically stabilized liposomes . the in
vitro sulfur donor reactivity comparison shows that dto reacts 15-times faster with cn at constant rh concentration than ts ( table 1 ) .
encapsulation efficiencies for both rh and dto were optimized as a function of rh - load , dto - load , and lipid composition .
when encapsulating rh alone , small amount of the cationic lipid dotap proved to be beneficial to enhance encapsulation efficiency ( table 2 ) .
the optimum rh concentration within the liposomes was 0.25 mg / ml ( table 2 ) .
for the encapsulation of dto with a concentration of 2 mm , six different liposomal compositions were examined to rule out the role of lipid composition ( table 3 ) .
each contained peg - pe-2000 in 5.1 mol% , lipid - to - chol ratio was 9 to 1 .
also , the cationic lipid , dotap in 3.0 mol% , was utilized to influence the charge of the liposomal surface in hopes that a positively charged surface would provide increased affinity for dto . on the basis of the results
it can be concluded that the presence of dotap leads to significant ( p < 0.05 ) higher encapsulation efficiency with dopc and popc as main lipid component .
lec liposomes demonstrated the least encapsulation efficiency for dto , and dotap appeared to provide little to no enhancement ( table 3 ) .
the role of dotap in enhancing the encapsulation efficiency can be explained with electrostatic interactions between dotap and dto at the ph value examined ( ph = 7.4 ) .
early experiments with the 60 : 40 ratio of dipalmitoyl - phosphatidylcholine ( dppc ) to chol composition provided very low encapsulation efficiency for encapsulation of dto ( data not shown here ) . as dto is a lipophilic sulfur donor ,
it can be expected that it is localized in the liposomal bilayer , more or less immersed in it .
the saturated bonds of dppc providing an ordered , relative rigid structure may inhibit the immersion of dto in the liposomal membrane .
contrarily , popc and dopc possessing double bonds and as a consequent of it a less ordered and more fluid membrane structure can promote the encapsulation of dto .
thus , instead of dppc , dopc , or popc were used for liposome preparation and ratios were adjusted to 90 : 10 lipid to chol .
the results shown in table 3 indicate that dopc liposomes containing 3 mol% dotap provided the highest encapsulation efficiency at 81.7 3.1% .
popc liposomes with 3% dotap were close behind with an encapsulation efficiency of 78.4 2.3% .
however , there was no significant difference between encapsulation efficiencies with dopc and popc .
the liposome compositions including dotap were used in further experiments due to the increase in encapsulation efficiency achieved by these films . the effect on encapsulation efficiency by the increase in dto concentration
was evaluated for dopc and popc containing liposome compositions with both sets of liposomes containing 3% dotap . in order to evaluate the role of dto concentration on the encapsulation efficiency
each set 's films were prepared with dto concentrations of 10 mm , 20 mm , and 30 mm .
the encapsulation efficiency remained high for each liposome formulation containing 3% dotap for each applied dto concentrations of 10 mm , 20 mm , and 30 mm .
the encapsulation efficiencies of dto for popc samples were 69.7 2.3% , 82.8 7.1% , 79.2 8.1% , while for the dopc samples dto encapsulation efficiencies of 74.2 2.0% , 86.2 3.9% , and 89.9 4.2% were determined , for 10 mm , 20 mm , and 30 mm dto concentrations , respectively .
for a given dto concentration there was no significant difference between the encapsulation efficiency values for dopc or popc liposomes ( p > 0.05 ) .
the optimal liposome composition for the encapsulation of rh was determined in earlier experiments to be the 60 : 40 ratio of popc to chol .
however , the prior experiments did not evaluate the dopc or the cationic lipid dotap .
furthermore , rh was added either in isotonic hepes buffer ( ph = 7.47.7 ) or in 5% ( w / w ) aqueous solution of glucose ( glu ; ph = 4.27.8 ) to the dry lipid films . for the purpose of coencapsulating dto with rh
, the rh encapsulation efficiency must be determined for the same lipid compositions and in the same hydrating systems as in the case of dto .
the optimal liposome composition for rh encapsulation was the 90 : 10 ratio of popc to chol with the use of dotap .
also , the 3.0 mol% dotap again increased the encapsulation efficiency for most of the different liposomal compositions ( table 2 ) . for the coencapsulation of dto and rh , the combination of popc , chol , peg - pe-2000 , and dotap ( with molar ratios of 82.7 : 9.2 : 5.1 : 3.0 ) was chosen as the most adequate liposome composition .
the mentioned composition of sterically stabilized , positively charged liposomes performed the best in the coencapsulation , with a coencapsulation efficiency for rh and dto of 88.6 17.1% ( with a rh load of 0.25 mg / ml and a dto concentration of 30 mm ) . as the coencapsulation efficiency was determined on the basis of scn formation by sl - rh - dto ; the given value represents the combined effect of rh and dto in cn conversion . for the sake of comparison , encapsulation efficiency for the coencapsulated rh alone with 0.25 mg / ml concentration was 74% , while for dto alone with 10 mm dto load was 57.7 8.1% . increasing the concentration of dto produced similar encapsulation efficiencies , than in case of 10 mm .
with dto loads of 20 mm and 30 mm for the coencapsulated dto encapsulation efficiencies of 55.6 4.0% and 61.6 17.6% were measured , respectively .
the conversion of cn to scn by the coencapsulation of 10 mm , 20 mm , and 30 mm dto with rh also proved to remain linear with an r value of 0.9930 .
the ability to co - encapsulate dto , or any other sulfur donor molecule with rh should provide better overall conversion of cn , since the sulfur donor no longer has to penetrate the liposome membrane .
in vivo evaluation of the optimized liposomal preparations made from dto/(dto + ts ) and/or rh were tested as cyanide antidotes on a mice model .
based on the above optimization efforts , for further in vivo evaluations we employed 3% dotap ; 0.25 mg / ml rh load , 30 mm dto load with the lipid composition of popc : chol : peg - pe-2000 : dotap = 82.7 : 9.2 : 5.1 : 3.0 .
the in vivo prophylactic treatment results with rh and dto / ts encapsulated within the optimized liposomal formulations are shown in table 4 .
this protection was enhanced ( apr = 4.8 ) when ts was coencapsulated with dto in a molar ratio of 1 : 1 ( table 4 experiment 3 ) .
coencapsulation of ts with dto is also believed to provide protection against product inhibition by sulphite ( zottolla , personal communication ) .
as it was expected , sn further enhanced the protection with the apr of 6.7 .
when dto was coencapsulated with rh ( table 4 experiment 5 ) , the apr was 3.9 .
when dto was coencapsulated with ts and rh , ( sl - dto - rh ) the apr was enhanced to 4.9 ( table 4 experiment 6 ) .
the highest protection ( apr = 15.3 ) was achieved with the combination of ( sl - dto - ts - rh ) and sn ( table 4 experiment 7 ) .
expressing the relative antidotal potency ratios ( rapr ) better indicates the differences in protection with two antidotal systems ( table 5 ) .
comparing experiment 2 and 3 ( rapr = 2.2 ) represents the effects of ts coencapsulation with dto . when comparing experiments 3 and 4 ( rapr = 1.4 ) or experiments 6 and 7 ( rapr = 3.1 ) the enhancement effects reached by sn are represented .
the significant enhancement by rh is expressed when comparing experiments 2 and 5 ( rapr = 1.8 ) and experiments 4 and 7 ( rapr = 2.3 ) confirming earlier studies with other types of encapsulation formulations for rh [ 23 , 26 , 27 ] .
table 6 shows the therapeutic antidotal protection with the ( sl - dto - ts - rh ) combinations with and without sn . at approximately 2 ld50 dose of kcn ( 15 mg / kg ) , all the 6 animals survived in each experiment ( table 6 experiments 1 , 2 , and 3 ) .
however , when the kcn dose was enhanced ( 20 mg / kg , approximately 3 ld50 ) the survival rate with ( sl - dto - ts ) + sn was 67% ( table 6 experiment 4 ) , while with ( sl - dto - ts ) without sn provided a 50% survival rate ( table 6 experiment 6 ) .
however the ( sl - dto - ts - rh ) antidotal system with and without sn also provided a 100% therapeutic protection ( table 6 experiments 5 and 7 ) .
the present experiments and results are confirming that the approach of utilizing externally administered , encapsulated , metabolizing rhodanese may have broad implications in cyanide antidotal therapy .
the application of this approach has been successfully tested in animal models . in summary , these studies are describing the prophylactic and therapeutic in vivo efficacy of the encapsulated rh and the new , reactive sulfur donor dto .
optimization efforts were attempted for the liposomal lipid compositions , rh - load , and coencapsulation of two sulfur donors ( ts and dto ) and rh to enhance the encapsulation efficiency for the given components .
optimization of the carrier systems is always a major part of these types of research efforts .
considering the high lipophilicity of dto , for further in vivo applications other introduction routes ( e.g. , intramuscular ) with further formulation optimizations are recommended . | present studies have focused on a novel cyanide antidotal system , on the coencapsulation of a new sulfur donor dto with rhodanese within sterically stabilized liposomes .
the optimal lipid composition for coencapsulation of dto with rhodanese is the combination of 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine , cholesterol , cationic lipid ( dotap ) , and 1,2-dipalmitoyl - sn - glycero-3-phosphoethanolamine - n-[methoxy(polyethylene glycol)-2000 ] ammonium salt ( with molar ratios of 82.7 : 9.2 : 3.0 : 5.1 ) . with the optimized compositions , prophylactic and therapeutic in vivo efficacy studies were carried out in a mice model .
when dto was coencapsulated with rhodanese and thiosulfate the prophylactic antidotal protection was 4.9 ld50 .
maximum antidotal protection against cyanide intoxication ( 15 ld50 ) was achieved with coencapsulated rhodanese and dto / thiosulfate in combination with sodium nitrite .
when applied therapeutically , 100% survival rate ( 6/6 ) was achieved at 20 mg / kg cyanide doses with the encapsulated dto - rhodanese - thiosulfate antidotal systems with and without sodium nitrite .
these data are indicating that the appropriately formulated dto is a promising sulfur donor for cyanide antagonism . |
the auditory portion includes the cochlea , which is involved in hearing , and the vestibular system which is involved in balance .
our ability to hear and our sense of balance are critically dependent on specialized sensory receptors called hair cells , these cells have structures called stereocilia , which sense sounds by bending back and forth , converting mechanical vibrations into electrical , or neural signals that are then passed to the brain through the auditory nerve ( 13 ) . finding ways to cure deafness represent a major scientific and clinical breakthrough .
recently , stem cells from the inner ear of adult mice have been identified ( 4 ) .
these adult stem cells are found in the utricle of the vestibular region of the inner ear .
they have the characteristic features of stem cells such as the capacity for self renewal and expansion ( they divide and multiply ) .
they form spheres , which begin to differentiate into new cell types termed as progenitor cells .
some progenitor cells differentiate into cells that express proteins and genes present in the developing inner ear and nervous system . under appropriate conditions ,
some cells differentiate into cells resembling hair cells , which have stereocilia ; hair bundles protruding from their surface and express specific hair cell marker proteins .
the discovery of such cells is a first step toward a promising line of treatment in restoring hearing and balance function ( 5 , 6 ) .
stem cells are unspecialized cells that have two defining properties : the ability to differentiate into other cells and the ability to self - regenerate ( 6 , 7 ) .
, embryonic stem cells can become all cell types ( pluripotent ) ( 811 ) .
adult stem cells are stem cells that can be derived from different parts of the body and , depending on where they are from , have different properties .
some studies have suggested that adult stem cells are very versatile and can develop into many different cell types ( 1223 ) . from a scientific perspective , scientists will first need to identify compounds and conditions that can increase the growth of stem cells and promote their differentiation into hair cells or supporting cells ( 24 ) . from a clinical perspective ,
surgical and technical procedures need to be developed to successfully transplant stem cells into the inner ear .
the critical question to be answered is whether transplanted stem cells can migrate to the correct location , differentiate into hair cells and restore hearing or balance ( 25 , 26 ) .
mobilization of endogenous stem cells provides an alternative way of replacing damaged inner ear hair cells , and correcting hearing loss ( 19 , 22 ) .
mobilizing host stem cells is less cumbersome than transplantation in that it avoids the logistical complexity associated with the use of embryonic as well as non embryonic stem cells , including supply , surgical trauma , and possibilities of graft rejection , uncontrolled graft cell proliferation and tumor formation .
the aim of this work was to assess the use of g - csf ( granulocyte colony stimulating factor ) to mobilize bone marrow stem cells to reach the inner ear of rats and its ability to repair damaged inner ear by amikacin sulfate .
thirty adult male sprague - dawley rats were supplied by the veterinary department of medical research institute , university of alexandria . they were housed and manipulated according to the work protocol approved by the research ethics committee , faculty of medicine , alexandria university .
all rats were examined under microscopic magnification for the tympanic membrane to exclude any ear abnormalities and then subjected to dpoaes for assessment of cochlear integrity and exclusion of rats with sensorineural deficits .
rats were randomly divided into two groups : group a consists of 15 rats which were dissected and the inner ear was histologically examined as a negative control for the other injected group .
group b consists of 15 rats which were subjected to the following : intra - tympanic injection under microscopic magnification with amikacin sulfate at starting dose of 5 mg / kg bilaterally in 10 rats and unilaterally in 5 rats .
dpoae of all injected rats ( group b ) was done to assess the hearing function 3weeks after first amikacin injection .
intra - tympanic amikacin injection was repeated until we had negative dpoae of all injected rats ( group b ) at 3 , 6 and 9 weeks after first injection .
subcutaneous injection of gcsf ( granulocyte colony stimulating factor ) at a dose of 200 microgram / kg / day for 5 successive days for 10 rats of the 15 rats injected with amikacin ( group b ) .
the other 5 rats were kept untreated with gcsf as positive control ( hearing damaged by amikacin but not treated with gcsf ) .
one month after gcsf treatment all rats were examined with dpoaes to assess the hearing function and the same was repeated every month till 6 months .
dissection of 2 rats was done every month one of each group and the inner ear was histologically examined . and after the follow up period of 6 months , all the remaining rats were dissected and the inner ear was histologically examind by : - light microscopic examination of paraffin sections using routine hematoxylin and eosin ( h&e ) stain ( 17 ) . - scanning electron microscopic examination of specimens ( sem ) by joel scanning electron microscope 525 kv , electron microscopy unit , faculty of science , university of alexandria ( 18 ) .
the onset for developing a negative dpoae record ( becoming deaf ) in experimented rats treated with amikacin sulfate showed individual variation .
six rats became dpoae negative three weeks after the first intratympanic injection , while another 6 rats following the second injection became deaf .
the remaining 3 rats became dpoae negative nine weeks later , and received a third dose with three weeks interval from the second amikacin injection .
eight rats ( out of ten ) survived to the end of the experiment among the group treated with gcsf simultaneously with amikacin sulfate .
they did not show any improvement in the dpoae records during the first two months of the experiment . by the end of the third month ,
2 rats revealed improvement of the sensorineural hearing functions as indicated by shift of their dpoae records from negative to positive .
four months from starting the experiment , another rat exhibited a positive shift in the dpoae assessment . by the end of the fifth month and the sixth month ,
three rats treated with amikacin sulfate without gcsf survived to the end of the experiment .
table 1 illustrates the statistical analysis of the 6 months follow up dpoaes assessment according to the number of experimented rats shows that 50% of the rats with amikacin sulfate induced hearing loss revealed statistically significant recovery of the sensorineural hearing function six months after treatment with gcsf ( p0.046 ) ( table 1 ) .
the negative control rats demonstrated a normal organ of corti with typical structural organization ( fig .
the sensory epithelium was formed of hair cells arranged in an inner and several outer rows ( fig .
the normal arrangement of the rows of inner and outer hair cells was demonstrated in the peculiar architecture of the reticular lamina of the cochlea as seen by the sem ( fig .
the intratympanic injection of amikacin sulphate caused almost complete destruction of the cellular elements forming the organ of corti as compared to normal structure ( fig .
in addition , there was an evident infiltration of the inner ear cavities with red blood corpuscle ( fig . 3b and 7c ) .
the vestibular structures of a normal control rat inner ear ( crista and macula ) histologically had no significant changes after amikacin injection ( fig .
4 ) and this indicates that its main effects were on the cochlear structures and 6 months after gcsf therapeutic regimen the stria vascularis ( fig .
5a ) reveals a complete sheet of intact superficial epithelium with residual interstitial vacuolation in the supporting connective tissue stroma .
histological examination of inner ear specimens from rats receiving gcsf therapeutic regimen confirmed the regeneration of the organ of corti that coincided with the positive shift of the dpoae records of these rats .
the earliest histological sign for the onset of regeneration of the organ of corti in the gcsf- treated rats was observed as appearance of aggregates of morphologically non specific cells on top of the intact basilar membrane ( fig .
after 2 months from receiving the gcsf therapeutic regimen , the scala media demonstrated a group of supporting - like cells that settled over the surface of the basilar membrane ( fig .
two months later ( 4 months from receiving gcsf ) , a primitive organ of corti could be depicted .
rows of inner and outer hair cells with the intervening tunnels could be recognized ( fig .
6c ) . by the end of the sixth month from receiving gcsf therapeutic regimen ,
most of the examined h&e sections revealed almost full reconstruction of the organ of corti .
the detailed structural components were identifiable , including a well formed tectorial membrane and a patent tunnel of corti supported by rigid , intact pillar cells ( fig . 6d ) .
the onset for developing a negative dpoae record ( becoming deaf ) in experimented rats treated with amikacin sulfate showed individual variation .
six rats became dpoae negative three weeks after the first intratympanic injection , while another 6 rats following the second injection became deaf .
the remaining 3 rats became dpoae negative nine weeks later , and received a third dose with three weeks interval from the second amikacin injection .
eight rats ( out of ten ) survived to the end of the experiment among the group treated with gcsf simultaneously with amikacin sulfate .
they did not show any improvement in the dpoae records during the first two months of the experiment . by the end of the third month ,
2 rats revealed improvement of the sensorineural hearing functions as indicated by shift of their dpoae records from negative to positive .
four months from starting the experiment , another rat exhibited a positive shift in the dpoae assessment . by the end of the fifth month and the sixth month ,
three rats treated with amikacin sulfate without gcsf survived to the end of the experiment .
table 1 illustrates the statistical analysis of the 6 months follow up dpoaes assessment according to the number of experimented rats shows that 50% of the rats with amikacin sulfate induced hearing loss revealed statistically significant recovery of the sensorineural hearing function six months after treatment with gcsf ( p0.046 ) ( table 1 ) .
the negative control rats demonstrated a normal organ of corti with typical structural organization ( fig .
1 ) . the sensory epithelium was formed of hair cells arranged in an inner and several outer rows ( fig .
the normal arrangement of the rows of inner and outer hair cells was demonstrated in the peculiar architecture of the reticular lamina of the cochlea as seen by the sem ( fig .
the intratympanic injection of amikacin sulphate caused almost complete destruction of the cellular elements forming the organ of corti as compared to normal structure ( fig .
in addition , there was an evident infiltration of the inner ear cavities with red blood corpuscle ( fig . 3b and 7c ) .
the vestibular structures of a normal control rat inner ear ( crista and macula ) histologically had no significant changes after amikacin injection ( fig .
4 ) and this indicates that its main effects were on the cochlear structures and 6 months after gcsf therapeutic regimen the stria vascularis ( fig .
5a ) reveals a complete sheet of intact superficial epithelium with residual interstitial vacuolation in the supporting connective tissue stroma .
histological examination of inner ear specimens from rats receiving gcsf therapeutic regimen confirmed the regeneration of the organ of corti that coincided with the positive shift of the dpoae records of these rats .
the earliest histological sign for the onset of regeneration of the organ of corti in the gcsf- treated rats was observed as appearance of aggregates of morphologically non specific cells on top of the intact basilar membrane ( fig .
after 2 months from receiving the gcsf therapeutic regimen , the scala media demonstrated a group of supporting - like cells that settled over the surface of the basilar membrane ( fig .
two months later ( 4 months from receiving gcsf ) , a primitive organ of corti could be depicted .
rows of inner and outer hair cells with the intervening tunnels could be recognized ( fig .
6c ) . by the end of the sixth month from receiving gcsf therapeutic regimen ,
most of the examined h&e sections revealed almost full reconstruction of the organ of corti .
the detailed structural components were identifiable , including a well formed tectorial membrane and a patent tunnel of corti supported by rigid , intact pillar cells ( fig .
the use of stem cells in the repair of damaged inner ear was first tried by ito et al . in 2001 using neural stem cells ( 19 ) . and also
in 2003 they studied the fate of neural stem cells grafted into injured inner ears of mice ( 20 ) . in 2003 li et al .
these adult stem cells are found in the utricle of the vestibular region of the inner ear ( 21 ) .
this discovery has opened the door for many other researchers to explore the utilization of stem cells in the inner ear repair and restoration of hearing function .
heller et al . in 2003 have studied the generation of hair cells by stepwise differentiation of embryonic stem cells ( 22 ) . and parker et al .
in 2004 studied the potential use of stem cells for cochlear repair ( 23 , 24 ) .
also many other stem cell studies have done in the next few years between 2003 and 2010 but most of these studies concentrated on the transplantation of stem cells into damaged inner ear or in vitro studies for generating hair cells from stem cells of different origins ( 25 , 26 ) .
the idea of mobilization of bone marrow stem cells is a new idea and it was proved to be successful in many other fields of medicine , in 2006 zohlnhfer et al .
also kloner et al . tried its use on human in 2006 in attempts to recruit stem cells for repair of acute myocardial infarction ( 27 , 28 ) .
mobilization of stem cells by gcsf also utilized in acute cerebral ischemia by schabitz et al . in 2003 and by shyu et al . in 2006 in acute ischemic stroke
also it was tried to use gcsf to mobilize bone marrow stem cells in retinal damaged photoreceptors by oishi et al . in 2008 and in a model of parkinson s disease by meuer
et al . in 2006 and in many other fields ( 31 , 32 ) . at the beginning of the study we had to make sure of the hearing ability of all rats because some genetic malformations may exist in rats similar to humans which is not uncommon , so we used electrophysiological study which is a portable oto - acoustic emission ( oae ) as a screening tool for hearing function and the rats which were negative were excluded before the start of the study .
we used aminoglycosides ( amikacin sulfate ) to cause cochlear damage to the rat s inner ear because it have a more cochlear side effect than vestibular unlike streptomycin and gentamicin which are the most commonly used drugs in the past studies but their effect is more vestibular than cochlear .
amikacin sulfate was injected via intra - tympanic approach not via systemic approach to avoid its systemic nephrotoxic effect which may have killed our rats before the follow up period of the study , and also to allow rapid toxic effect on the inner ears . and
amikacin injection was repeated until we had negative oaes of all injected rats and this was reached in all rats after 9 weeks of the first amikacin injection .
some rats were injected with amikacin bilaterally and others were injected unilaterally , in the rats with unilateral injection the contralateral ears were used as a control to have the same conditions and reduce the inter - animal variability .
we used a 5 day therapeutic regimen of subcutaneous injection of gcsf and the dose according to previous publications in literature in humans is 5~10 microgram / kg / day but in rats it is 100~200 microgram / kg / day ( 16 ) .
one month after gcsf treatment the rats were examined with dpoaes to assess any improvement in the hearing function and the same was done every month till 6 months .
after 6 months follow up we had 4 rats ( 5 ears ) which were negative oaes became positive oaes showing clinical improvement in hearing function .
the group which was bilaterally injected with amikacin we had 2 rats ( 3 ears ) improved after gcsf treatment after a total follow up period of 6 month while in the group which was unilaterally injected with amikacin we had improvement in 2 rats ( 2 ears ) .
the positive control group ( rats which were bilaterally injected with amikacin but did not treated with gcsf ) remains negative oaes after the same follow up period of 6 months and this indicates that there was no spontaneous improvement in hearing function over this period of time .
histological comparison was done between the inner ear of normal rats and the inner ear of amikacin injected rats and showed that the damage was more cochlear in the form of destruction of organ of corti and disappearance of the inner and outer supporting cells and hair cells . after treating amikacin injected rats with gcsf
, histological examination was done on variable durations and showed improvement in the arrangement of hair cells till we reached complete restoration of organ of corti after 6 months and this was consistent with the clinical results of oaes .
while histological examination of the positive control rats ( which were injected with amikacin and not treated with gcsf ) showed no improvement and this was also consistent with the clinical results .
in conclusion , knowledge of the structure , molecular biology and function of the inner ear enables the research on new modes of regenerative therapies .
the present work has achieved its primary aim in proving the possibility for recruitment of progenitor cell diffentiation into hair cells and supporting cells within the organ of corti by using granulocyte colony stimulating factor therapy .
the study of the histology of the obtained result in correlation to the oae testing proved the morphological and functional competence of the regenerated cells .
based upon the obtained results , it is recommended to expand research in this issue using different animal models to evaluate species dependent variations .
evaluation of the delayed systemic effects and possible hazards of gcsf therapy should be also investigated .
advanced investigation should be applied that allow quantitative evaluation of the recruited bone marrow derived stem cells together with application of specific techniques for tracing , characterization and monitoring of the fate of these cells . | background and objectivesthe utilization of the stem cells is widely used in the last few years in different fields of medicine , either by external transplantation or endogenous mobilization , most of these studies still experimental on animals ; few were tried on human as in the spinal cord injury or myocardial infarction . as regard its use in the inner ear ,
stem cell transplantation was examined in many previous studies , while the mobilization idea is a new method to be experimented in inner ear hair cell regeneration .
the present work assessed the possibility of mobilizing endogenous bone marrow derived stem cells ( scs ) in rats using granulocyte colony stimulating factor ( g - csf ) to induce regeneration and repair to experimentally damaged inner ear hair cells by amikacin injection.methodsthe study included thirty adult sprague dawley male rats .
experimental induction of inner ear damage was done by repeated intratympanic injection of amikacin sulfate .
mobilization of bone marrow scs was provoked by subcutaneous injection of gcsf .
cochlear integrity , induction of hearing loss and functional recovery of sensory hearing loss were assessed using distortion product otoacoustic emission ( dpoaes ) .
the morphological alteration and recovery of the organ of corti was assessed histologically using the light and scanning electron microscopes.resultsafter six month duration , there was improvement in 50% of the sensorineural dpoae results .
functional recovery coincided with the repair of structural components of organ of corti.conclusionsscs mobilization by g - csf is a promising alternative method for replacement therapy in sensorineural hearing loss . |
diastolic ( dys)function is of major clinical importance and is characterized by both elastic [ 14 ] and viscous properties [ 57 ] .
the giant elastic protein titin ( also known as connectin [ 8 , 9 ] ) is well known to be related to the elasticity of cardiac muscle impacting the end diastolic pressure volume relationship ( for current reviews see [ 1012 ] ) .
these elastic properties arise from the three extensible segments localized in the i - band of the sarcomere : the tandem ig segments , the proline - glutamic acid - lysine - valine rich pevk element , and the n2b unique sequence .
viscosity in cardiac tissues impacts diastolic filling by slowing ventricular pressure relaxation and causing a delayed relaxation phenotype during early filling [ 2 , 5 , 6 ] .
the ig domains that make up the tandem ig segments of titin 's spring region are a possible intrinsic source of viscosity .
individual ig domains were originally thought to unfold during stretch and refold during release causing a viscosity [ 14 , 15 ] , but single molecule and simulation studies suggest that the domain unfolding is unlikely to be a prominent process under physiologic conditions [ 1620 ]
. interactions between elements of the extensible i - band of titin and other structures in the sarcomere are another possible source of viscosity .
the interaction between titin and actin was hypothesized due to evidence of a viscoelastic interaction in the sarcomere , close proximity of the two filament types , and in vitro evidence of titin binding to actin [ 2225 ] .
first demonstrated by cosedimentation assays that the pevk region , but not the n2b element or ig domains , interacted with actin ; kulke et al .
both studies showed that f - actin sliding in the in vitro motility assays was slowed by the addition of recombinant pevk protein to the assay buffer suggestive that the pevk adds a viscous force that slows filament sliding .
mechanically , yamasaki found a reduction in the stress - response of rat cardiac myofibers using exogenously added recombinant pevk proteins .
kulke utilized gelsolin extraction of thin filaments to abolish native actin - pevk interaction in myofibrils and found that this reduced viscous stress relaxation ( although such extraction potentially releases near - z - disk titin that is normally firmly bound to actin ) . both studies confirmed in the fiber / fibril that a viscous response was due to pevk - actin interactions .
the development of a pevk ko mouse model provided a new tool to study pevk - actin interactions and its effect on viscosity .
utilizing skinned cells and fibers from this ko mouse , a 50% reduction was found in the viscosity in stress - relaxation and viscous moduli in sinusoidal experiments using protocols identical to the ones used in this study .
recently , using a pevk ko mouse , we quantified the contribution of pevk - actin interactions to viscosity in an ex vivo and in vivo whole heart preparation , and we showed a key physiologic consequence that pevk - actin interactions control 30% of viscosity during early rapid filling .
cardiac muscle of large mammals , including human , coexpresses both the stiffer n2b isoform and more compliant n2ba isoform unlike that of small rodents that is dominated by expression of the n2b cardiac isoform [ 10 , 12 ] .
the pevk element of the n2b isoform contains 7 exons with 5 ppak ( proline - rich ) motifs that are negatively charged and involved in actin binding [ 28 , 30 ] .
the pevk region of the cardiac n2ba isoform contains all of the elements expressed in the n2b isoform and additional pevk exons , including additional ppak repeats as well as two polye ( glutamic acid rich ) motifs .
the polye motifs appear to have a stronger binding affinity to actin than the ppak motifs . because of
these changes in pevk expression and the expression of additional ig domains in n2ba dominant tissues , direct extrapolations from the findings in small animals to humans are difficult .
thus , while characterization of the pevk - actin interaction shows physiologic relevance in small animals , actual application in large mammals including humans remains unclear . to study viscosity in tissues containing a high n2ba : n2b ratio , we utilized tissues from the pig .
viscous properties were probed using both stretch hold and frequency sweep perturbations to quantify viscosity and we utilized a competition assay with recombinant pevk proteins to disrupt the native pevk - actin interactions .
animal experiments were approved by the university of arizona institutional animal care and use committee and followed nih guidelines for using animals in intramural research . briefly , the heart was rapidly obtained from the pig < 5 minutes after sacrifice , washed with a calcium - free hepes - buffered solution ( [ in mm ] 10 hepes ; 133.5 nacl , 5 kcl , 1.2 nah2po4 ; 1.2 mgso4 , 11 glucose , 4 na - pyruvate with 2.5 u / l insulin , ph 7.44 ) , dissected into small strips , and placed in a skinning solution composed of relaxing solution ( [ in mm ] : 10 bes , 10 egta , 6.56 mgcl2 , 5.88 na - atp , 1 dtt , 46.35 potassium - propionate , 15 creatine phosphate ) with 1% triton x-100 ( thermo scientific , waltham , mass ) and a high concentration of protease inhibitors ( [ in mm ] 0.1 e64 , 0.4 leupeptin and 0.5 pmsf ) for 2448 hrs at 4c . after skinning ,
tissues were washed with triton - free relaxing solution with protease inhibitors then infiltrated with a 50% ( vol / vol ) solution of relaxing solution and glycerol at 4c then stored at 20c .
small fibers were dissected , glued to aluminum clips , and attached at either end to a force transducer ( ae801 , sensorone , sausalito , calif ) and length motor ( 308b , aurora scientific , aurora on , canada ) and washed with relaxing solution with inhibitors . cross - sectional area ( csa ) was measured ( 0.023 0.002 mm ) .
all fibers activated at 2.0 m sarcomere lengths ( sl ) using a pca 4.0 solution to confirm fiber quality , achieving tensions of 39.5 2.5 mn / mm .
length perturbations and force measurements were performed using a custom labview interface ( national instruments , austin , tex ) cardiac muscle fibers were stretched with two length perturbations to evaluate viscosity .
the fibers were stretched using a stretch - hold - release protocol from their slack sarcomere length to a sl of 2.15 m at 3 speeds : 0.1 , 1.0 , and 10.0 lengths / s .
after stretch , the fibers were held at their stretched length for 90 seconds , followed by a symmetric release .
a sinusoidal frequency sweep was applied at the end of hold of the 0.1 length / s stretch with frequencies from 0.1 to 400 hz at an amplitude of + /2% of the fiber length .
10 minutes of recovery time in between stretches was used . in order to probe the dependence on sarcomeric strain ,
frequency sweeps were also imparted at a sl of 2.25 m ( fibers were stretched from slack to a sl of 2.25 m at 0.1 lengths / sec ) . to test the magnitude of pevk - actin interactions ,
briefly , cdna fragments were amplified from human cdna with primer pairs corresponding to the human cardiac i27-pevk - i84 fragment with the flanking ig domains included to enhance the stability of the protein .
amplified cdna fragments were inserted into a petm11 vector , expressed in e. coli , and the obtained fragments were sequence verified . fragments were purified then dialyzed into pbs ( [ in mm ] 2.7 kcl , 1.5 kh2po4 , 137 nacl , 8 na2po4 ) and 10% glycerol , aliquoted , flash frozen , and stored at 80c . for use , proteins were thawed and dialyzed in relaxing solution with protein inhibitors .
pevk fragments were added to the pig fibers to a final concentration of 22 m in relaxing solution with protease inhibitors , allowed to incubate for > 1 hr , and probed with the mechanical protocol described above .
stress was calculated by dividing measured force by cross - sectional area . for stretch - hold experiments
, stress was measured at the end of the stretch to calculate the peak stress and again at the end of the hold ( 90 sec after peak ) to calculate a steady state stress .
the viscous stress is calculated as the peak minus steady - state stress during the hold .
the viscosity was calculated as the slope between the viscous stress and the log10 of the stretch speed ( in lengths / s ) .
the stress was used to first calculate the complex stiffness as the stress divided by the amplitude of the imposed length change ( normalized to fiber length ) .
phase delay was calculated as the phase difference between the stress and strain signals .
the viscous modulus was then calculated as complex stiffness times the sine of the phase delay ( see inset of figure 3(a ) ) at all frequencies .
student t - test was used to compare the viscosity ( slope and viscous modulus at a given frequency ) between groups .
using titin protein gels , we first established the titin isoform expression ratio of porcine cardiac lv tissues and found that the n2ba : n2b isoform ratio is ~1.0 ( figures 1(a ) and 1(b ) ) .
this expression ratio is much higher than that of the mouse lv ( ~0.2 ) and is slightly higher than that of normal human lv and similar to that of human lv of dcm patients ( figure 1(b ) ) . to quantify viscosity , we stretched skinned pig lv muscle from their slack sl ( ~1.9 m ) to 2.15 m and then held length constant for 90 sec to observe stress relaxation , followed by a release back to the slack length .
figure 1(c ) shows two examples of such stretch - hold - release experiments , one carried out in relaxing solution ( ctrl ) and the other in relaxing solution to which exogenous pevk had been added ( we confirmed the purity of the expressed pevk using gels , see inset of figure 1(c ) ) .
we measured viscous stress ( v ) from the stress relaxation during the hold phase of the protocol ( explained at top of figure 2(a ) ) .
viscous stress was measured at three stretch speeds encompassing subphysiologic ( 0.1 lengths / s ) to supraphysiologic speeds ( 10 lengths / s ) and the magnitude of the stress relaxation , defined by the peak minus steady - state stress ( figure 2(a ) , inset ) , was determined at each speed .
viscosity was quantified as the slope of stress relaxation versus ( log ) speed . at sl of 2.15 m , the viscosity ( slope )
was reduced by 35% ( p = 0.03 ) when fibers were incubated with recombinant pevk ( figure 2(b ) ) .
these results indicate that pevk - actin interactions are present and relevant in tissues expressing high levels of the compliant titin n2ba isoform .
we also measured the viscous moduli during a sinusoidal oscillation protocol at frequencies from 0.1 to 400 hz and using an amplitude of 2% of the fiber length and repeated the protocol in the absence and presence of recombinant pevk .
frequency analysis provides a robust method to quantify the viscous response of a tissue at a wide array of speeds [ 29 , 35 ] . to quantify the modulus , the ratio of the stress response and length perturbation was first used to calculate a complex stiffness and the phase delay between stress and length is used to calculate the viscous modulus ( figure 3(a ) ) .
the viscous modulus was reduced by 40% by the treatment with recombinant pevk at sl of 2.15 m ( figure 3(b ) ) .
we also performed experiments at a sl of 2.25 m and found that the viscous moduli were reduced by 50% at all frequencies ( figure 3(c ) ) .
importantly , the viscous modulus maintained a constant reduction at physiologic frequencies ( ~0.52 hz corresponding to the heart rate in the pig ) for both sl ranges .
these data indicate that the introduction of recombinant pevk does reduce the viscosity of the system at all frequencies , including frequencies corresponding to physiologic heart rates , and reveals that it does this in a sl - dependent manner .
in resisting the rapid expansion of the left ventricle , viscosity is an important modulator of diastolic function [ 2 , 57 ] .
while previous work has shown a contribution of pevk - actin interactions to viscosity both in vitro [ 13 , 26 , 36 , 37 ] and in vivo , such findings were largely obtained in small rodents that express titin that is stiffer than in larger mammals and often at speeds below [ 13 , 36 ] or above physiologic rates .
yet the relevance to human cardiac function and dysfunction is not direct , owing to the increased expression of the compliant n2ba isoform that contains additional ig domains and a longer pevk domain . to determine the viscous properties of titin in a large animal model with a human - like titin isoform expression ratio , we investigated pig myocardial tissues . additionally , we utilized recombinant pevk proteins to interrupt native pevk - actin interactions .
pevk - actin interactions in the pig were significantly reduced by competitive binding of recombinant pevk to the actin filament .
this reduced the viscous response to stretch by 35% and the viscous moduli by more than 40% at a sl of 2.15 m ( figures 2 and 3 ) . at longer sarcomere lengths ( 2.25 m ) that are likely to be reached during diastole in pigs , the viscous moduli obtained with the sinusoidal analysis increased to ~50% .
thus , pevk - actin interaction is a major source of viscosity in large mammals like the pig .
these novel findings are the first to quantify viscosity across physiologic speeds in tissues expressing high levels of compliant n2ba isoforms .
our findings indicate that pevk - actin interaction is likely to be physiologically important in humans where viscosity influences relaxation [ 2 , 5 , 6 ] .
the characterization of delayed relaxation has long been a clinical indicator of diastolic dysfunction in echocardiography .
recent advances in modeling further support that the delayed relaxation phenotype is caused by a viscous force .
recently , we used transmitral doppler echocardiography in a ko mouse deficient in pevk and found a significant reduction in viscosity during early rapid filling .
the ability to translate the echocardiographic findings in mice to humans could not be fully appreciated until the present results on pig myocardium that showed a clear presence of viscosity in tissues with a high n2ba : n2b ratio .
our work establishes that despite the longer pevk element and distinct sequence composition of the n2ba pevk , pevk - actin interactions are present in myocardial tissues with human - like titin isoform expression .
thus , this interaction is conserved across species , supporting the importance of these interactions to diastolic function .
the sl - dependence of the viscous modulus ( figure 3 ) was unexpected and may indicate that at longer sl where the pevk is stretched to a higher degree additional binding site for actin are available than at short sl or , alternatively , that the lattice compression that occurs at long sl enhances the interaction .
the increased viscosity at longer sl might reduce any potential sl overshoot during filling [ 36 , 40 ] and may allow for more precise end - diastolic sl control .
this incomplete inhibition is possibly due to the inability of the added pevk to compete off all native pevk - actin interactions .
alternatively , it indicates that there is an additional source of viscosity , a possibility that is consistent with results of the pevk ko mouse model that also show that in absence of the pevk element myocardial tissue continues to display ~50% of the viscosity found in wildtype myocardium .
stretch - hold and frequency sweep protocols in myofilament extracted mouse fibers have negligible viscosity suggesting that the extracellular matrix contributes little to viscosity of the myocardium .
thus , the non - pevk source of viscosity is likely to arise from interactions that involve the myofilaments . in our studies , in pevk ko mice , we evaluated microtubules , a known source of viscosity under pathological conditions [ 34 , 41 , 42 ] . however , colchicine treatment produced no effect on viscosity of the intact heart neither in wt nor in pevk ko mice , suggesting that , in normal hearts , there is little contribution of microtubules to viscosity .
thin - thick filament interaction is unlikely because this predicts that viscosity would be increased when the myofilament lattice is compressed in pevk ko tissue and this is not what we observed in our previous studies .
possibilities that require future testing are intermolecular interactions between adjacent titin molecules and interactions between intermediate filaments and any of the myofilament types . in summary , although additional sources of viscosity are likely to exist , the pevk - actin interaction is the only well - established molecular source of viscosity in cardiac tissues and contributes up to half of the total viscosity of skinned myocardium in relaxing solution .
it has previously been shown that pevk - actin interaction can be regulated by s100a1 , a calcium binding protein present in cardiac tissues ( for recent review see ) .
s100a1 is a member of the s100 calcium binding proteins that is preferentially found in cardiac tissues .
s100a1 normally targets to calcium handling proteins ( the sarcoplasmic reticulum , ryr2 ) , but also localizes in the i - band of the sarcomere .
when present in the sarcomere and activated by the presence of physiological calcium levels , s100a1 inhibits pevk - actin interactions [ 13 , 36 ] .
thus , pevk - actin interaction - based viscosity can be turned off during systole when high calcium levels are present in the cytosol and turned on again during diastole when calcium levels fall .
a second mechanism shown to influence pevk - actin interactions is protein kinase c - alpha ( pkc ) phosphorylation of titin .
pkc phosphorylation occurs at two sites in the pevk element that are conserved across a large number of mammalian species including the mouse , pig , and human . in both pig and mouse
skinned cardiac fibers [ 32 , 44 ] , pkc treatment increased viscosity by 2030% at physiologic speeds ; the effect was amplified by pretreating the fibers by protein phosphatase 1 ( pp1 ) to dephosphorylate titin .
these changes in viscosity indicate a potentially large dynamic range by which viscosity can be modulated .
the understanding of the functional effects of pkc phosphorylation and its regulation may aid in our understanding of the underlying dysfunction , for example , in heart failure where pkc is upregulated [ 45 , 46 ] .
this study establishes the presence of pevk - actin interactions in a large mammal , the pig , and is the first to conclusively determine with physiological stretch protocols the magnitude of pevk - actin interactions in hearts with a human - like titin isoform expression ratio .
competition assays with exogenous recombinant protein show that the magnitude of this contribution is up to 50% of the total viscosity seen in these tissues .
furthermore , we found that pevk - based viscosity is larger at longer sl , a property that might aid in preventing sl overshoot at the end of filling , and because of the influence of pevk - actin interactions on early rapid filling , modulation of these interactions are of potential clinical importance . | titin exhibits an interaction between its pevk segment and the actin filament resulting in viscosity , a speed dependent resistive force , which significantly influences diastolic filling in mice .
while diastolic disease is clinically pervasive , humans express a more compliant titin ( n2ba : n2b ratio ~0.51.0 ) than mice ( n2ba : n2b ratio ~0.2 ) . to examine pevk - actin based viscosity in compliant titin - tissues , we used pig cardiac tissue that expresses titin isoforms similar to that in humans .
stretch - hold experiments were performed at speeds from 0.1 to 10 lengths / s from slack sarcomere lengths ( sl ) to sl of 2.15 m .
viscosity was calculated from the slope of stress - relaxation vs stretch speed .
recombinant pevk was added to compete off native interactions and this found to reduce the slope by 35% , suggesting that pevk - actin interactions are a strong contributor of viscosity .
frequency sweeps were performed at frequencies of 0.1400 hz and recombinant protein reduced viscous moduli by 40% at 2.15 m and by 50% at 2.25 m , suggesting a sl - dependent nature of viscosity that might prevent sl overshoot at long diastolic sls .
this study is the first to show that viscosity is present at physiologic speeds in the pig and supports the physiologic relevance of pevk - actin interactions in humans in both health and disease . |
external or endoscopic dacryocystorhinostomy ( dcr ) is the standard treatment for nasolacrimal duct obstruction ( nldo ) , with or without a previous history of dacryocystitis .
the external approach is made through a skin incision near the lacrimal sac and the endoscopic approach is made through the nasal cavity with the aid of a nasal endoscope .
the primary benefits of the endoscopic approach are the lack of skin scarring ( there are no skin incisions ) and quicker healing time , and the primary benefits for the external approach are a faster and easier surgery to perform .
the surgical success rate was found to correlate with the duration of obstruction and patient 's age ; the surgical success rate was lower when the obstruction lasted longer and the patient was younger .
the success rates of the endoscopic approach in nldo are similar to those of the external approach and range between 80% and 96% [ 311 ] .
this variability in success rates is likely due to differences in surgical techniques , patient demographics , and lack of standardized outcome measures .
only a few earlier studies examined the effect of past dacryocystitis on the success rates of external dcr .
the success rates in those studies were lower than studies who examined dcr without dacryocystitis .
their reported success rates range from 69% to 88% [ 12 , 13 ] . according to our clinical experience ,
the success rates of dcr for patients with and without previous episodes of chronic or acute dacryocystitis should be similar .
our current study presents a series of patients with or without previous episodes of dacryocystitis who underwent external dcr for nldo and compares their preoperative characteristics , postoperative outcomes , and surgical success rates .
institutional review board ( irb ) approval for conducting this study was granted by the tel aviv sourasky medical center .
we retrospectively reviewed the medical files of all patients who were referred to our tertiary referral center and underwent external dcr between 2006 and 2011 .
the patients were divided into two groups : patients with and without previous episodes of chronic or acute dacryocystitis ( as determined by physical examination ) .
acute dacryocystitis was defined by the appearance of pain , erythema , or pus discharge and swelling of the lacrimal sac .
chronic dacryocystitis was defined by the appearance of epiphora and purulent discharge from the punctum . in the presence of dacryocystitis ,
the pus was drained and antibiotic treatment with oral amoxicillin and clavulanate ( 875 mg 2/d ) was started . only after resolution of the acute phase , an external dcr was performed . in some cases ,
if the main complaint was epiphora , lacrimal syringing was performed followed by external dcr in patients with nldo .
the extracted data included patients ' demographics , indication for surgery , background diseases ( systemic and ocular ) , duration of epiphora , side of obstruction , previous external dcr , early and late postoperative complications , duration of the stenting tube , and surgical outcome .
they all underwent external dcr and had a complete postoperative followup of at least 6 months .
excluded were patients who underwent endoscopic dcr , those with a functional obstruction , those aged less than 16 years , and those with inadequate followup .
success was determined by postoperative patency on syringing and resolution of epiphora at 6 months after surgery .
we use a standard technique based on a straight incision made medial to the angular vein at the level of the medial canthal ligament .
an osteotomy of 1.52 1.52 cm was created , and the lacrimal sac and mucosa were opened to form anterior and posterior flaps . a silicon tube ( bica , 2012 fci ophthalmics , marshfield hills , ma ) was inserted and tied , after which the anterior and posterior flaps are sutured with 6/0 vicryl sutures .
the wound is closed with absorbable sutures ( 5/0 vicryl rapide or 6/0 coated vicryl ) .
all patients were treated postoperatively with topical maxitrol ( dexamethasone , polymyxin b sulphate , and neomycin sulphate ) ointment on the skin incision and ofloxacin eye drops 3 times a day for 710 days .
if dacryocystitis was evident during the surgery ( pus in the lacrimal sac ) , treatment with oral amoxicillin and clavulanate ( 875 mg 2/d ) for 5 days was added .
the removal of the silicone tube took place only when there was a patency on syringing and resolution of epiphora and at least 6 weeks passed from the surgery .
fisher 's exact test and analysis were used for comparison between categorical variables , and a t - test was used for comparing continuous variables . for multivariate analyses ,
a logistic regression was used to calculate the odds ratio ( or ) which was used to predict the odds of developing dacryocystitis in the presence of glaucoma or past cataract surgery .
during the study period , 185 patients underwent external dcr and 152 of them met the inclusion criteria .
the participants included 100 females and 52 males ( mean age 67 15 years ) , of whom 60 were diagnosed with dacryocystitis ( acute or chronic ) and 92 were not .
the mean duration of epiphora prior to the first admission to the clinic was 33 months for patients without dacryocystitis and 28 months for patients with a previous episode of dacryocystitis ( p = 0.001 ) .
there was no difference in the percentage of laterality of the obstruction in the former group ( 27 ( 29.3% ) right , 29 ( 31.5% ) left , and 36 ( 39.1% ) bilateral ) .
in contrast , significantly fewer patients with a previous episode of dacryocystitis had bilateral obstruction ( 12 ( 20% ) , p = 0.028 ) , and significantly more of them had a left - side obstruction ( 29 ( 48.3% ) , p = 0.031 ) compared to the patients without dacryocystitis ( table 1 ) .
there was no significant association between hypertension , ischemic heart disease , hyperlipidemia , diabetes , hyperthyroidism , and anemia and the occurrence of dacryocystitis .
two ( 2.2% ) of the 92 patients without a previous episode of dacryocystitis were diagnosed with glaucoma , compared to 10 ( 16.7% ) of the 60 patients with dacryocystitis ( p = 0.002 ) .
the presence of glaucoma with epiphora duration of less than 11.5 months was significantly predictive of the development of dacryocystitis ( p = 0.019 ) ; the odds were 6.71 times greater than those for nonglaucomatous patients .
fourteen patients without dacryocystitis ( 15.2% ) and 17 patients with a previous episode of dacryocystitis ( 28.3% ) underwent cataract surgery in the past , and they had a tendency to develop dacryocystitis in the presence of nldo ( p = 0.064 ) ( table 2 ) .
the mean postoperative follow - up time was 19 months for patients without dacryocystitis and 20 months for patients with a previous episode of dacryocystitis ( p = 0.783 ) .
it occurred in 6 patients without dacryocystitis ( 6.5% ) and in 6 patients with a previous episode of dacryocystitis ( 10% ) ( p = 0.542 ) .
the silicone tube was removed after a mean duration of 86 days in patients without dacryocystitis and after a mean duration of 79 days in patients with a previous episode of dacryocystitis ( p = 0.333 ) .
after a minimal period of 6 months , patency on syringing and resolution of epiphora was documented in 80 patients without dacryocystitis ( 86.7% ) and in 56 patients with previous episode of dacryocystitis ( 94.4% ) ( p = 0.337 ) .
our comparisons of patients ' characteristics and surgical outcome results of external dcr in patients with nldo , with and without a past history of dacryocystitis , revealed no significant differences between the two groups . in the literature ,
the reported surgical success rates of external dcr in patients with no prior episodes of dacryocystitis ranged from 80% to 96% [ 311 ] .
only a few earlier studies examined the effect of past dacryocystitis on the success rates of external dcr and reported them as being lower than in cases without dacryocystitis .
follow - up assessments of 662 patients with chronic dacryocystitis who underwent external dcr included symptom evaluation and irrigation of the lacrimal passage .
surgical success was defined by being asymptomatic and having patent lacrimal passages , and it was achieved in 88.6% of their patients .
successful outcomes were defined as diminished epiphora or no observable reflux from the canaliculus during or after lacrimal irrigation .
the patients of both studies were followed up at 1 , 3 , and 6 months and one year after surgery .
additional predictive factors for lower external dcr success rates are the duration of obstruction and patient 's age .
noted that lower success rates are common in younger patients compared to older ones , while early operations offer greater success .
. found that patients who had longer - lasting symptoms of chronic dacryocystitis had lower success rates .
we found that , in patients with previous episodes of dacryocystitis , the obstruction was more common on the left side .
a suggested explanation is that the angle formed between the left nasolacrimal sac and duct is smaller than that on the right side , and this allows proliferation of bacteria [ 14 , 15 ] .
we found that a shorter mean duration of epiphora ( equivalent to a shorter duration of nldo ) correlated with previous episodes of dacryocystitis and that there was no difference in the surgical success rates in these patients .
we believe that the reason for similar surgical results in both groups is a shorter duration of epiphora in patients with dacryocystitis .
our results revealed that patients with glaucoma have higher odds ( 6.71-fold ) to develop dacryocystitis in the presence of nldo compared to patients without glaucoma . to the best of our knowledge , neither a direct correlation between glaucoma and nldo nor between glaucoma and dacryocystitis has been reported previously .
there are , however , a few reports on antihypertensive medications as a cause of nldo or lacrimal canalicular obstruction .
reported that chronic use of timolol - containing topical glaucoma therapy preparations in glaucoma patients is associated with an increased risk of developing nldo .
mcnab [ 17 , 18 ] demonstrated that the punctum and canaliculus are the main anatomical sites of lacrimal drainage system obstruction associated with topical antihypertensive medications .
although topical glaucoma therapy can cause nldo , we found no reports on a correlation between glaucoma / antihypertensive medications and dacryocystitis , nor any correlation between nldo caused by topical antihypertensive medications and the occurrence of dacryocystitis .
the correlation we observed between past cataract surgery and nldo with dacryocystitis did not reach a level of significance ( p = 0.064 ) .
we did not find any studies in the literature on such an association . according to our findings , patients with glaucoma or past cataract surgery who have nldo are prone to develop dacryocystitis .
we therefore suggest that these patients be warned about this complication and instructed to seek medical treatment at the first occurrence of epiphora .
in addition , the information on the patients ' diseases was taken from their medical charts , and the duration of epiphora was self - reported by the patients . in conclusion , we found no significant difference in the success rates of external dcr in patients with or without a previous episode of dacryocystitis .
our finding that patients with glaucoma and nldo have a higher risk of developing dacryocystitis should be confirmed in larger - scale studies . | objective . to compare pre- and postoperative characteristics and surgical success rates of patients with and without previous episodes of dacryocystitis , who underwent external dacryocystorhinostomy ( dcr ) for nasolacrimal duct obstruction ( nldo )
. methods .
the medical files of all patients who underwent external dcr between 2006 and 2011 in our institution were reviewed .
the retrieved data of patients with and without previous episodes of dacryocystitis were compared .
surgical success was determined by postoperative followup of at least 6 months .
results .
a total of 185 patients with nldo underwent external dcr of whom 152 ( 100 females and 52 males , mean age 67 15 years ) met the inclusion criteria .
sixty had previous episodes of dacryocystitis and 92 did not .
left - side obstruction was more common than right - side obstruction among patients with previous episodes of dacryocystitis ( 48.3% versus 31.7% , resp .
, p = 0.031 ) .
glaucoma patients were significantly more likely to develop dacryocystitis than patients without glaucoma ( p = 0.002 ) .
the success rate of external dcr was 94.4% for patients with previous episodes of dacryocystitis and 86.7% for patients without ( p = 0.337 ) .
conclusions .
the surgical outcomes of external dcr in patients with or without a previous episode of dacryocystitis were similar .
patients with glaucoma and nldo had a significantly higher risk of developing dacryocystitis . |
while substantial research has demonstrated the potential for preventing the adverse outcomes of type 2 diabetes , the increase in the number of people with diabetes ( pwd ) has outpaced the response of health systems [ 2 , 3 ] .
this incongruity is particularly marked in low and middle income countries ( lmic ) where 80% of deaths from diabetes occur .
the estimated adult prevalence of diabetes in guyana was 15.5% in 2011 and in 2008 diabetes was the fourth leading cause of death . prior to 2008 ,
diabetic foot complications were the most common admitting diagnosis to the surgical ward of the national referral and teaching hospital , georgetown public hospital corporation ( gphc ) , with 42% having a lower extremity amputation ( lea ) .
scoping visits by canadian expert team members participating in a recently introduced surgery residency program confirmed that diabetic foot complications were the most common reason for admission to the surgical units of gphc .
care was uncoordinated , with lack of a systems approach ( no screening for high risk feet / ulcers , practice in silos , overaggressive debridement without adequate vascular assessment , no plantar pressure redistribution , and narrow spectrum or missed antimicrobial doses ) .
the high burden of disease ( 30% inpatient population ) , prolonged stay , and frequent readmissions with poor patient outcomes resulted in staff demoralization .
surgical debridement took place either on poorly lit and unsanitary wards or had to compete with other surgical emergencies in the operating rooms .
local health care leaders were keen to address this problem and entered into a partnership with canadian surgeons and wound / foot care experts to develop the guyana diabetes and foot care project ( gdfp ) 20082013 .
the phase 1 goal was to create health system changes in evaluation and management to improve foot care in pwd and reduce diabetes - related lea at gphc , while phase 2 expanded this to 6 administrative regions , comprising 90% of the population , and added training in the management of diabetes and hypertension .
clinical activities resided within the guyanese public health system and staff and resource costs were paid by ministry of health ( moh ) .
multilevel knowledge to action ( k2a ) cycles was utilized to identify the challenges facing the guyanese health care system and develop the intervention . both process and clinical outcomes were monitored .
participants were all persons with type 2 diabetes presenting to the gphc or to regional facilities with personnel trained in the project , and the care provided to them was based on the decision of these personnel and patient wishes .
patient data was entered into a ministry approved database and identifiable personal information , apart from sex and age , was withheld from the authors . since this was a quality improvement ( qi ) project , run under the auspices of the ministry of health of guyana in public health facilities , approval of an ethics committee
project oversight and coordination was provided by steering committees , meeting regularly , with both canadian and guyanese members , including ministry of health officials .
the key interventions are detailed elsewhere . in brief , the intervention comprised development of a key opinion leader ( kol ) team , this following a train the trainer model .
the kols were trained using well established canadian training programs : the international interprofessional wound care course ( iiwcc ) and the international diabetes federation approved michener institute diabetes educator course .
health systems change was facilitated through networking with key stakeholders to establish foot care centres and embed practice change .
key opinion leaders ( kols ) attended these training programs ; then trained primary health care workers through iterative 3-day workshops on basic foot and wound care using the screening tool and referral criteria .
all trainings were interprofessional with doctors , nurses , medex ( doctor equivalents ) , and rehabilitation specialists learning together .
the canadian training programs were supplemented by on - site skills training and reflective practice to develop local expertise as well as supported by continued mentoring from canadian experts . given the limited local resources , it was important to allocate the available resources effectively , and this was facilitated by using clinical screening tools to recognize loss of protective sensation , and identification of the patient at high risk of ulceration or amputation .
the highest risk individuals were then referred to the national diabetic foot center ( dfc ) at the georgetown public hospital corporation ( gphc ) for more intensive surveillance , education on foot care foot wear and smoking cessation , debridement of callus linked to the use of protective footwear and orthotic devices , improved glycemic control , and the treatment of foot ulcers / complicating infections .
absence and cost of wound care products used for diabetic foot ulcer care in high income countries led to adaptation of more cost effective wound care practices , and commercially available ( darco ) wound care sandals were prescribed at a fraction of the price . in the absence of any foot specialists , principles of plantar pressure redistribution ( ppr ) therapy
were taught to rehabilitation assistants , orthotic , prosthetic , and cast room technicians . in phase 2 ,
the previously listed methods were applied to build capacity across the country , and the foot care program was expanded to 6 guyanese administrative regions .
a 3-day training program on diabetes and hypertension management was added with these components introduced throughout the project regions and hba1c testing introduced into the public system .
a project database was designed to capture the more complex project outcomes with clerks appointed and trained in data entry at each center .
targeted process outcomes were the establishment of a national centre of excellence in foot care at gphc and 7 regional foot care centers , project tools accepted and used by the moh , measurement of hba1c and blood pressure for people with diabetes , identification of the high risk foot using the simplified 60-second screening tool , and appropriate referral to regional or national dfcs . targeted clinical outcomes included reduction in major lea at gphc and measurement of the proportion of pwd with hba1c < 9% ( 75
diabetic foot admissions at gphc were determined using admission books on surgical wards and amputations from operating room records .
audits of the admissions book on surgical floors were undertaken to identify patients with diabetic foot complications from 2006 to 2010 because a review of chart coding based records found it to be inaccurate .
continuous variables were summarized using means ( sd ) and median ( iqr ) and tested using two sample t - tests and paired t - tests as appropriate .
although the t - test is robust to nonnormality , since some data was mildly skewed , we verified the results using a nonparametric wilcoxon test and found similar results .
categorical data was reported using frequency and percent and tested using the chi - square statistic .
odds ratios were also examined for comparisons between groups and the breslow - day statistic was used to test for the homogeneity of odds ratios .
time series analysis was employed to examine the effect of the intervention on the number of amputations after adjusting for autocorrelation and is presented in figure 1 .
autocorrelation and partial autocorrelation plots indicated a good fit . the augmented dickey - fuller test and ljung - box test indicated good fitting and no concern due to stationary or white noise [ 1517 ] .
key opinion leader ( kol ) team : a total of 16 trainees ( 7 doctors , 1 medex , 4 nurses , 3 rehabilitation specialists , and 1 diabetic foot care worker ) participated in the international interprofessional wound care course in 5 cohorts ; 14 completed the course and 10 are currently working in the kol team .
the kol team then trained a total of 340 other guyanese health care professionals ( f / m = 1.8 ) ( phase 1 : 65 hcp in 4 workshops ; phase 2 : 275 hcp in 18 workshops ) .
the simplified 60-second screening tool was developed in guyana and implemented to screen 3452 persons and 643 completed a follow - up screen .
a reliability study confirmed the utility of this tool , which was adopted by the moh to be used throughout guyana . from july 2010 to march 2013 , 7567 pwd were assessed with f / m = 2.09 .
as of march 2010 , there were a cumulative 6075 patient visits to the gphc foot center , an average of 13.6/day .
as of march 2013 , 1186 patients ( f / m = 1.60 ) with foot ulcers have been treated at regional dfcs ; there have been over 20,776 visits for dressing care .
hba1c testing was successfully introduced to the public system and a tool to ensure appropriate use of this limited resource was implemented . since april 2010 , 4062 pwd have had hba1c testing of whom 62% had an hba1c < 9% ( 75 mmol / mol ) .
the average hba1c was 8.56% ( sd 2.85 ) { 70 mmol / mol ; sd 28 mmol / mol } with women having significantly higher values than men 8.66 ( sd 2.89 ) { 71 mmol / mol ; sd 31.6 mmol / mol } versus 8.31 ( sd 2.77 ) { 67 mmol / mol ; sd 30.3 mmol / mol } ; p = 0.0001 .
mean hba1c was 13% higher in patients with foot complications with 44% having hba1c over 9% ( 75 mmol / mol ) .
the average blood pressure in 814 pwd was 134 mmhg systolic and 82 mmhg diastolic .
there was not enough power to detect a change in bp or hba1c over time as too few subjects had recurrent measures .
change in these outcomes is likely to be incremental rather than sudden . in the 42 months before the dfc opened , the mean monthly number of amputations was 7.95 ( sd 4.05 ) and this fell significantly to 3.89 ( sd 2.30 ) in the 54 months after the dfc opened through to december 2012 ( p < 0.0001 ) .
this represents a 51% decrease and translates to a saving of 219 limbs from july 2008 to december 2012 .
the time series analysis ( figure 1 ) demonstrated a significant decrease in the number of amputations ( 4.32/month ( 95% ci 2.40 , 6.24 ) ; p < 0.0001 ) coincident with the commencement of the project .
the opening of the dfc overlapped an ongoing postgraduate surgical training programme . while an apparent rise in major amputation numbers during months 2441
may have been associated with this increase in surgical capacity , this did not preclude the observed reduction in monthly amputations subsequent to dfc operation .
of even greater significance is the marked reduction in proportion of inpatients with diabetic foot complications subjected to major amputation ( table 1 ) despite a doubling of the rate of diabetic foot admissions at gphc .
the average monthly admissions rose from 21.2 before the dfc opened to 42 in the first 22 months of operation .
the proportion of inpatients subjected to a major amputation during this period fell from 41.4% to 11.9% ( p < 0.0001 ) .
a poisson regression model was used to examine the rate of amputations adjusted for patient volume and after intervention the risk of having an amputation decreased by 68.6% ( 95% ci 53.9% , 78.5% ) as compared to before intervention .
the number of specific types of major amputations , their means , and medians are shown in table 2 .
the sums are less than the total number of amputations reported because the type was not specified in some cases .
there was no significant difference ( p = 0.07 ) in the mean number of above knee amputations ( aka ) after intervention as compared to before , while there was a significant difference in below knee amputations ( bka ) ( p < 0.0001 ) .
the changes in the frequency of akas and bkas before and after the intervention give an indication of the limitations of this kind of project focused on primary care .
while bkas showed an 80% and significant reduction after the dfc was opened , akas showed no change .
currently there is no vascular surgical capacity in guyana to treat vascular insufficiency , a common comorbidity in diabetic foot complications .
we suggest that patients with both diabetic foot complications and uncorrected vascular insufficiency are more likely to require akas .
it would also speak to the need for developing a vascular surgical capacity in resource - constrained settings , if limb salvage in the diabetic foot is to be optimized .
figure 1 illustrates a plateau effect on amputations after intervention and it may be that this is the best that can be achieved without further resources ( e.g. , vascular surgery and renal dialysis ) .
we have already reported on the divergence from global averages of the sex ratios of type 2 diabetes in guyana and have estimated that the odds ratio for women compared to men is 2.486 ( 95% ci 2.442 , 2.531 , p
there were 544 major amputations ( 278 in women and 266 in men ) over 8 years with f / m of 1.05 .
since few regional hospitals in guyana have surgical capacity and most diabetic foot problems are referred to gphc , virtually all diabetes - related leas in guyana are being performed at that hospital . to calculate the sex - based relative risks we assumed that any amputations outside gphc follow the same distribution for sex and type and that the number of persons with diabetes remained constant over the 20052012 periods . since the estimated prevalence of diabetes in women is twice that in men , the odds ratio of amputations for males as compared to females during the entire study period is 2.16 ( 95% ci 1.83 , 2.56 ; p < 0.0001 ) .
the reasons for the increased risk of amputations in men are unknown but may be related to an increased risk of ulceration due to social factors ( occupational hazards , smoking ) or failure to seek medical attention .
the odds ratios for amputations in men compared to women increased from 1.86 ( 95% ci 1.50 , 2.31 ) before the intervention to 2.73 ( 95% ci 2.08 , 3.58 ) after the intervention ( p = 0.015 ) .
we tested whether aka and bka amputation rates differed between males and females before versus after the intervention and found that the odds ratios did not change before versus after intervention ( or = 0.9959 { 95% ci 0.6576 , 1.4682 } ) .
the moh embraced the model , which is described in detail elsewhere , and approved in the new moh strategic plan 20132020 : integrated prevention and control of non communicable disease in guyana . despite the many challenges facing the moh , a significant change in the approach to evaluation of the diabetic foot and diabetes management occurred
we demonstrated that it is possible to introduce the best practice methods to evaluate for the high risk foot in people with diabetes and achieve sustained improvements in evaluation and care of foot ulcers .
after the project began gphc achieved a marked and sustained reduction both in major amputation numbers and in the proportion of inpatients with diabetic foot complications requiring major amputation . that this reduction occurred almost immediately after project commencement suggests that surgeons embraced the importance of maintaining limb integrity .
change was likely sustained by provision of new alternate methods and dedicated clinic spaces for treatment based on context specific practice guidelines .
translating clinical guidelines and qi principles into practice , in both the developed and developing world is challenging . in low and middle income countries ( lmic )
one of our next steps is to investigate the transferability of our model to another limited resource setting . | background .
type 2 diabetes is the fourth leading cause of death in guyana , south america . a complex , interprofessional ,
quality improvement intervention to improve foot and diabetes care was rolled out in two phases .
methods & findings .
phase 1 : establishment of an interprofessional diabetic foot center ( dfc ) of excellence to improve foot care and reduce diabetes - related amputations at the national referral hospital .
phase 2 : regionalization to cover 90% of the guyanese population and expansion to include improved management of diabetes and hypertension .
fourteen key opinion leaders were educated and 340 health care professionals from 97 facilities trained .
eight centers for the evaluation and treatment of foot ulcers were established and 7567 people with diabetes evaluated .
3452 participants had foot screening and 48% were deemed high risk ; 10% of these had undocumented foot ulcers .
there was a 68% reduction in rate of major amputations ( p < 0.0001 ) ; below knee amputations were decreased by 80% , while above knee amputations were unchanged .
an increased association of diabetes with women ( f / m = 2.09 ) and increased risk of major amputation in men [ odds ratio 2.16 ( 95% ci 1.83 , 2.56 ) ] were documented .
conclusions .
this intervention improved foot care with reduction in major amputations sustained over 5 years . |
in general , the reaction of nucleophilic alkyl or aryl metal compounds with c = n groups yields the corresponding calkylated or arylated products.1 nevertheless , a number of cases are known in which this normal regioselectivity is suppressed in favor of an azaphilic addition ( umpolung).2 , 3 an early example of this unexpected addition mode was reported by kagan and fiaud in 1971 for the reaction of grignard reagents with iminoesters,4a and since then has been expanded into a viable route to amino esters.4 , 5 additionally , imino groups in special electronic environments , such as cyclopentadieneimines , have been found to follow similar reactive patterns.6
the observed azaphilic regioselectivity has been attributed , among other things , to the potentially chelating coordination of a lewis acidic metal cation ( mg for grignard reagents ) by the substrate ; this results in a conformation that favors naddition as well as polarization of the c = n bond due to the electronwithdrawing properties of the adjacent functional group ( in iminoesters ) .
another qualitative rationale refers to the similarity of this structural motif in iminoesters to the wellknown nucleophilic addition to vinylogous systems .
unfortunately , there is limited insight into this pattern of reactivity available from studies focusing on the underlying organometallic chemistry , experimental mechanistic work , or computational modeling .
herein , we demonstrate how the polarization and orientation of metalcoordinated ketimides may influence the regiochemistry of grignard additions to cn multiple bonds .
the focal point in the assembly is tetravalent titanium , which effects the fixation and orientation of the substrates , and thus controls the relative approach of key reagents.7 , 8 the presence of additional functional groups that act as ligating units may significantly alter the preferred coordination mode of the ketimide and , as will be demonstrated , the observed pattern of reactivity ( figure 1 ) .
ketimide compounds have been generated in situ by the group of de meijere and others through the reaction of grignard reagents with various nitriles , including heterocyclic derivatives ( 3 and 4pyridine , 2thiophene , 2furancarbonitrile , etc . ) to yield the corresponding trityl amine products.9 the starting point for our study was the observation that the reaction of 2pyridine carbonitrile with aryl grignard reagents under the same conditions as those described by de meijere et al .
, and subsequent hydrolytic workup gave narylated amines , and thus , the products of azaphilic addition ( scheme 1 ) .
we hypothesized that this unexpected regioselectivity was due to chelation control by the pyridyl ring , which generated reactive intermediates coordinated to the metal center that , in turn , could act as key species in metalcomplexinduced , multicomponent onepot reactions .
the critical role played by the nheterocyclic ring within the substrate for this reactive pattern was supported by control reactions ( see the supporting information ) as well as the isolation of a series of other naryl benzhydryl amines by the same titaniummediated route ( table 1 ) .
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please refer either to the publisher 's own online version of this product or the printed product where one exists .
significantly , we were able to separate the carbo and azaphilic arylation steps , and thus , selectively introduce different aryl substituents , by choosing the order in which the two equivalents of grignard reagent were added ( table 2 ) : the first equivalent of grignard reagent cleanly added to the nitrile carbon , whereas the second addition only took place in the presence of [ ti(oipr)4 ] and occurred at the nitrogen atom .
the reaction is not limited to grignard reagents nor to the presence of magnesium salts , since the corresponding lithium compounds are equally applicable in this transformation ( compounds 2 j and 2 k ) .
the publisher did not receive permission from the copyright owner to include this object in this version of this product .
please refer either to the publisher 's own online version of this product or the printed product where one exists . to obtain mechanistic insights into this intriguing transformation
, we aimed to isolate crystalline products of the individual reaction steps that might represent or be related to reaction intermediates .
first , grignard addition onto the nitrile moiety was investigated for the addition of 4fluorophenylmagnesium bromide to 2pyridine carbonitrile in thf at room temperature .
notably , and crucially , for the selectivity of this transformation , the magnesium imide generated in this reaction step is not susceptible to further reaction with excess grignard reagent . a singlecrystal xray structure analysis of the ketimido
magnesium compound 3 established the structural details of an octameric aggregate ( figure 2 ) , in which the 2pyridylimido unit acts as a chelating ligand for magnesium.10
top : in situ formation of oligomeric mg ketimido clusters .
hydrogen atoms and cocrystallized solvent molecules have been omitted for clarity . the central cube has been highlighted .
ketimido fragment , which exposes the imidon atom that acts as a bridging ligating unit to a neighboring magnesium center .
the observed reactivity in subsequent transformations indicates at least partial fragmentation of the octamer under the reaction conditions ( figure 2 ) .
the subsequent reaction step , transmetalation with the titanium reagent , was studied by reacting 3 with [ ti(oipr)4 ] and [ tibr(oipr)3 ] ( scheme 2 ) . in both cases , in situ h and c nmr spectroscopy as well as mass spectrometry results indicated the formation of a mixture of molecular species that defied characterization . however , with [ tibr(oipr)3 ] , we isolated a crystalline reaction product that could be characterized , inter alia , by xrd ( figure 3 ) .
the product was a ti / mg heterodinuclear complex 4 , in which the ti atom is coordinated by a bromide ligand , two remaining isopropoxido units and two chelating 2pyridylimido units , one of which bridges the titanium and magnesium centers . the coordination sphere of magnesium is completed by a halogenido ligand and a coordinated thf molecule . in situ formation of labile titanium and magnesiumcontaining heterometallic species .
hydrogen atoms and cocrystallized solvent molecules have been omitted for clarity . for selected bond lengths and angles ,
the 2pyridylimides act as bidentate chelates , and thus , feature bent ketimido metal units .
this form of ligation , which is significant for the subsequent transformations discussed below , should be seen in comparison with the large number of structurally characterized ketimides of the early transition metals , most of which adopt nearlinear c = n metal arrangements.11
the following key reaction step of the transformation , leading to the reaction products represented in table 1 and 2 , involves attack of the second grignard reagent and determines whether carbo or azaphilic coupling is favored .
given the complex equilibria of aggregates encountered in this type of chemistry , which precludes in situ characterization by spectroscopic means as well as systematic kinetic studies , additional insight had to be drawn from a computational study ( pbe0 calculations ) by employing an appropriately chosen model system ( see the supporting information for computational details ) . in the first stage , the simplest product of transmetalation between 3 and ti(oipr)4 ( or tibr(oipr)3 ) was considered .
this complex features three alkoxy ligands ( herein modeled by ome ) and one ketimide n=c(py)(ar ) ( py=2pyridine ; ar = parafluorophenyl ) with either coordination of the pyridine substituent ( acyc ) or lack of pyridine coordination ( a ) .
the two optimized geometries are shown in figure 4 and acyc is computed to be more stable than a by g=3 kcal mol .
o bonds ( 1.784 , 1.788 , and 1.802 ) and a significantly shortened ti
n distance of 1.865 , which is indicative of some double bond ( i.e. , azavinylidene ) character.12 upon pyridine coordination in acyc , the geometry becomes a trigonal bipyramidal with the apical pyridine unit and a methoxy ligand .
n bond is significantly elongated at 1.988 , whereas the n=c distance remains unchanged ( 1.269 , a ; 1.268 , acyc ) . computed structures of the ti complex fragments a and acyc and the ti mg heterodinuclear model complexes b and bcyc .
the optimized geometries are available in a single xyz file ( geom.xyz ) in the supporting information .
color code : green = ti , purple = br , orange = mg , yellow = f , red = o , blue = n , dark gray = c , light gray = h . a natural bonding orbital ( nbo ) analysis of the electronic structure of a and acyc highlighted significantly different electronic properties of the n=c bond in a and acyc .
the natural localized molecular orbital ( nlmo ) for the bond between n and c is delocalized on the ti center with overall weights of 2.7 , 57.1 , and 37.1 % on ti , n , and c , respectively .
for the same nlmo , the respective weights are 1.9 , 52.2 , and 42.1 % in acyc .
this indicates that the n=c bond is more polarized toward c in acyc than in a. therefore , the carbon atom is more electronrich in acyc than that in a , and thus , less prone to attack by a nucleophile .
this is also reflected in the natural population analysis ( npa ) charge for c , which is more positive in a than that in acyc ( 0.286 vs. 0.215 ) . in other words ,
coordination of mg(br)(ar ) ( ar = parafluorophenyl ) to either a or acyc is exergonic by g=7.7 and 17.5 kcal mol , respectively .
the geometries of adducts b and bcyc are shown in figure 4 . in adduct b ,
the mg cation is bridging between n and one methoxy ligand ( nmg=2.286 and mgo=2.122 ) . the ti
nc linkage deviates from linearity in adduct b with tinc=148. the n=c bond is slightly elongated ( 1.284 ) and the nlmo associated with the bond is more strongly polarized toward n in adduct b than that in the mononuclear reference system a ( 60.1 % n , 34.2 % c , 2.9 % ti ; charge on c in b : 0.334 ) .
coordination of mg(br)(ar ) has increased the reactivity of c toward a nucleophile . in bcyc ,
the mg cation is bridging between n and the apical methoxy ligand ( mgn=2.089 , mgo=2.027 ) .
the initial trigonal bipyramidal geometry of acyc is altered upon coordination of mgbr(ar ) to a pseudooctahedral geometry with creation of a tibr interaction ( 2.842 ) ; thus explaining the larger binding energy . as in the case of b ,
the geometry of bcyc is qualitatively similar to that observed for the isolated hetereobimetallic adduct ( figure 3 ) .
the transition state , tsbc ( figure 5 ) , corresponding to the arylation of c in b has been located on the potential energy surface with g
= 25.9 kcal mol . in tsbc ,
the forming cc bond length is 2.386 , whereas the n=c bond is elongated to 1.333 and the mg
ar bond length increases from 2.110 in b to a value of 2.185 in tsbc .
the carylation is exergonic with g=12.9 kcal mol and , in the product of carylation , c ( figure 5 ) , the mg cation is bridging n ( 2.105 ) and one methoxy unit ( 1.976 ) .
optimized geometries of the various extrema located along the pathway for direct carylation without ( left ) or with ( right ) pyridine coordination .
the optimized geometries are available in a single xyz file ( geom.xyz ) in the supporting information .
color code : green = ti , purple = br , orange = mg , yellow = f , red = o , blue = n , dark gray = c , light gray = h . in the transition state tsbccyc ( figure 5 ) , in which pyridine is coordinated to ti , the forming cc bond has an interatomic distance similar to that observed in tsbc ( 2.380 ) .
however , the n=c bond is less elongated ( 1.317 ) and the breaking mgar bond is longer ( 2.290 ) in tsbccyc .
this is in agreement with a greater reactivity of c toward nucleophilic attack in b than that in bcyc . as a matter of fact
, the activation barrier associated with tsbccyc is greater ( g
= 30.4 kcal mol ) than that for tsbc ( g
= 25.9 kcal mol ) .
there is thus a significant decrease in the carylation reactivity upon coordination of pyridine . from b or bcyc ,
no transitionstate structures associated with direct transfer of ar from mg to n could be located on the potential energy surface
. however , in both cases , a transitionstate structure associated with ar transfer from mg to ti could be located . in the case of tsbd ( figure 6 ) , when pyridine is not coordinated , the activation barrier is low , g
= 8.9 kcal mol , and the transformation is endergonic , g=6.9 kcal mol for d relative to b. the product of the reaction , d , features an aromatic ring bridging mg and ti ( mgc=2.247 and tic=2.353 , see figure 6 ) .
optimized geometries of the various extrema located along the pathway for narylation without ( top ) or with ( bottom ) pyridine coordination .
the optimized geometries are available in a single xyz file ( geom.xyz ) in the supporting information .
color code : green = ti , purple = br , orange = mg , yellow = f , red = o , blue = n , dark gray = c , light gray = h . with pyridine coordinated , the activation barrier associated with tsbdcyc is slightly lower ( g
= 4.8 kcal mol ) and the transformation is still endergonic , with dcyc lying at g=2.8 kcal mol above bcyc . here again , the transferring aromatic ring is bridging mg and ti in the product dcyc ( mgc=2.366 and tic=2.317 , see figure 6 ) . from intermediate d ,
narylation is effective through tsde with an activation barrier of g
= 20.8 kcal mol and leads to the narylated product e with g=12.0 kcal mol . in tsde , the migrating aryl group is bridging the ti
n bond ( tic=2.190 and nac=1.784 ) and the n=c bond is elongated to 1.342 .
the product of the reaction , e , features a ketimine moiety , n(ar)=c(py)(ar ) , in which the ti center interacts both with the nc ( tin=2.081 and tic=2.112 ) and car bonds ( tiar=2.482 ) , whereas the mg cation interacts with n ( 2149 ) and two methoxy groups .
interestingly , the energetics associated with the corresponding narylation from dcyc is similar , with tsdecyc lying at g
= 21.0 kcal mol above dcyc and ecyc lying at g=13.1 kcal mol below dcyc .
the migrating aromatic ring is further away from both ti ( tic=2.463 ) and n ( nc=1.930 ) in tsdecyc compared with tsde . only the n=c
bond exhibits a similar lengthening ( 1.344 ) . because of pyridine coordination , the ketimine moiety , n(ar)=c(py)(ar ) , in product ecyc only interacts with ti through the n=c bond ( tin=2.057 and tic=2.272 )
the mg cation is bridging n ( mgn=2.088 ) and one methoxy group . in figure 7
a , a comparison between the pathways for c and narylation when pyridine is not coordinated is shown ( decoord pathway ) .
although aromatic ring transfer is intrinsically easier from ti to n than it is from mg to c , the energy needed to transfer the aromatic ring from mg to ti overall destabilizes the narylation pathway .
a comparison between the pathways for c and narylation when pyridine is coordinated ( metallacycle pathway ) is shown in figure 7 b . although the product of carylation is preferred thermodynamically , the formation of the narylated product is preferred kinetically .
comparison between the pathways for c and narylation without ( a ) and with pyridine coordination ( b ) .
gibbs free energy values are expressed in kcal mol at 333 k relative to b ( path a ) or to bcyc ( path b ) ; bcyc is 12.7 kcal mol more stable than b. the kinetic preference for the narylation pathway is the result of two cooperative effects . as already explained , pyridine coordination reduces the intrinsic reactivity of c toward direct reaction with a nucleophile .
this leads to an increase in the energy of tsbccyc with respect to bcyc , compared with the energy of tsbc with respect to b. in addition , aromatic ring transfer from mg to ti is slightly favored when pyridine is coordinated ( by ca .
4 kcal mol ; compare tsbdcyc in figure 7 b with tsbd in figure 7 a ) .
consequently , the transition state of the ratedetermining step for the carylation pathway is destabilized , whereas that of the ratedetermining step for narylation is stabilized when pyridine is coordinated .
this eventually leads to inversion of the kinetically preferred pathway in favor of narylation when a substituent on the arylated ketimide can coordinate to titanium , as observed experimentally ( table 1 ) .
when such coordination is not possible , the expected carylation product will be obtained . in summary , the proposed mechanism of the azaphilic c
n coupling has revealed that the initial idea of an umpolung , in which a nucleophile is added onto the more electronegative nitrogen , has to be revised .
in fact , in the selectivitydetermining step , the choice is not between n and carylation , but between c and tiarylation ; the latter opens up a reaction pathway that allows further migration to the nitrogen atom .
the reaction product for the pathway involving azaphilic coupling , ecyc , represents a local minimum on the free energy hypersurface , and may rearrange to a pyridylligated titanaaziridine complex , fcyc , as depicted in scheme 3 .
complex fcyc is computed to be g=7.7 kcal mol more stable than ecyc .
it is therefore reasonable to assume that such a species represents a key intermediate for the subsequent conversions discussed below .
gibbs free energy values ( kcal mol , 333 k ) are expressed relative to bcyc . in recent work by mller , beckhaus and coworkers , aromatic aldimine and ketimine complexes of titanocene
were studied to highlight the accessibility and close structural relationship of three and fivemembered titanacycles ( figure 8) .
a structurally related example was reported by rosenthal et al.13b it was rationalized that steric factors determined the preferred coordination mode ( titanaaziridine vs. 1aza2titanacyclopent4ene species).13
structurally characterized titanaaziridine and azatitanacyclopentene complexes .
a key insight of the computational study described above has been the likely formation of the titanaaziridine species fcyc , which may thus be viewed as a key intermediate for subsequent reaction steps in the multicomponent transformations described herein .
the reactivity of metallacyclic aziridines of the group 4 metals has been studied previously by several research groups and has given rise to exciting examples of metalinduced transformations.14 the most common pattern of reactivity involves the coupling of the metalbonded carbon atom with various electrophilic reagents , including aldehydes or alkyl halides .
this opened up the possibility of developing the onepot formation of such species into multiplecomponent reactions for synthetic targets , in which quaternary carbon centers with four different substituents would be accessible in a chemoselective way.15
initially , we examined the addition of a range of different electrophiles to the reaction mixture prior to hydrolytic workup . upon the addition of an excess of paraformaldehyde , the oxazolidines 5a ,
b were isolated as the main products , depending on the electronic nature of the naryl substituent ( scheme 4 ) .
the oxazolidine is thought to be formed by a mannichtype mechanism from an initial aminoalcohol intermediate . in situ
hydrogen atoms in the molecular structure of 5 a have been omitted for clarity ; thermal ellipsoids are shown at the 50 % probability level .
subsequently , the reactive potential of the titanaaziridine intermediate towards various alkyl halides was investigated .
both allyl and benzyl bromides afforded the corresponding trisubstituted amines ( scheme 5 ) .
hydrogen atoms , except for n(1)h have been omitted for clarity ; thermal ellipsoids are shown at the 50 % probability level .
remarkably , and in contrast to the reaction involving formaldehyde , which had to be added in the final step of the onepot procedure , the halides were found to be inert under the reaction conditions chosen for the formation of the active titanium species , and therefore , could be added during the initial setup of the reaction , rendering this a true onepot , multicomponent coupling .
recently , micalizio and coworkers reported the reaction of in situ generated azatitanacyclopropanes ( generated from imines and the sato reagent16 ) with allylic or allenylic alkoxides to yield homoallylic amines or dienes.17a d this approach was used , inter alia , for the synthesis of complex natural products .
reacting allylic alkoxides with the titanium intermediate , we observed the formation of the corresponding homoallylic amines ( scheme 6 ) .
hydrogen atoms , except for n(1)h , have been omitted for clarity ; thermal ellipsoids are shown at the 50 % probability level .
a rationale for the observed e selectivity of these transformations based on an empirical model is depicted in the supporting information .
again , the allylic alkoxide could be added directly to the reaction mixture and did not interfere with the ti
n aryl transfer . using chiral allylic alkoxides as coupling partners , we obtained the corresponding enantioenriched reaction products , albeit with a slight erosion of chirality.18 chirality is thus transferred from an easily accessible chiral allylic alcohol to a metallacycle , thereby creating an enantioenriched quaternary carbon atom .
the selectivity observed in this transformation is consistent with an empirical model based on a formal metallorearrangement.17
finally , we exchanged the allylic alkoxides for allenylic alkoxides.19 instead of the expected dienecontaining product , the 1,5aminoalkohols 8a and 8b were isolated ( scheme 7 ) .
the results of a workup in d2o ( 8ad1 ) suggest a reaction mechanism through 1,2carbometalation of the terminal allene bond , in contrast to the previously reported formal metallorearrangement .
hydrogen atoms , except for n(1)h and o(1)h , have been omitted for clarity ; thermal ellipsoids are shown at the 50 % probability level .
we have developed a powerful new multicomponent strategy for the rapid convergent assembly of structural complexity starting from simple nheterocyclic nitriles , grignard reagents , and electrophilic coupling agents .
notably , this process relies on inexpensive , readily available starting materials , is highly modular , and yields environmentally benign and easily separable byproducts .
the range of products thus accessible comprises valuable nitrogencontaining heterocycles , enantioenriched quaternary carbon centers , and densely functionalized structural motifs of pharmaceutical and biomedical relevance , as exemplified by the class of cholesterylester transfer protein ( cetp ) inhibitors shown in figure 9.20 , 21
cetp inhibitors comprising an naryl tritylamine motif ( ms = mesyl ) . whereas the reaction products described herein are accessible through conventional approaches , namely , nucleophilic addition to diarylimines , these methods require multiple steps and tedious workup procedures.22 the reaction mechanism of the unexpected azaphilic addition of nucleophiles to ketimide titanium complexes was explored by dft methods , which revealed the formal umpolung of a c n group as the key step toward reactive intermediary metallacycles ( titanaziridines and azatitanacyclopentenes ) .
a central role is attributed to the tethered nitrogen donor moiety , which serves as a precoordinating directing group and , in conjunction with an ancillary system , as an electron reservoir .
finally , the reactivity profile of the titanacyclic intermediates was assessed by four mechanistically distinct followup reactions , namely , by insertion reactions with a c = o function , by nucleophilic substitution of halogenated alkanes , by allylic alkoxides featuring a formal metallorearrangement , and by allenic alkoxides resulting in a 1,2carbometalation reaction .
notably , these transformations show a high degree of regioselectivity , although the control of absolute stereochemistry in this process remains a challenge . moreover , the stepeconomic concept presented in this work shows the great utility of group 4 metals for mediating challenging c c and c n bond formations . | abstractconsecutive c and narylation of nheterocyclic nitriles is mediated by titanium(iv ) alkoxides .
the carbo and azaphilic arylation step may be separated by choosing the order in which the two equivalents of aryl transfer reagent are added . in the course of this transformation , the ancillary nheterocycle acts as both a directing anchor group and electron reservoir . in the selectivitydetermining step ,
the selectivity is governed by a choice between ( direct ) c and tiarylation ; the latter opens up a reaction pathway that allows further migration to the nitrogen atom .
the isolation of metalcontaining aggregates from the reaction mixture and computational studies gave insights into the reaction mechanism .
subsequently , a multicomponent onepot protocol was devised to rapidly access complex quaternary carbon centers . |
these patients usually have a short life span because of pulmonary embolism , heart failure , or cancer progression . and
therefore , early detection of intracavitary tumor thrombus and effective surgical resection are important factors of survival prolongation .
herein we report an echocardiographic detection of the right atrial mass in two cases of hepatocellular carcinoma .
a 61-year - old man , with a history of niddm ten years previously , was admitted due to epigastric pain , indigestion , and malaise . on admission
, he had a regular pulse rate , was normotensive and relatively well - looking .
abdominal ultrasonogram favored a hepatoma of the right lobe with a right hepatic vein thrombus(fig .
two - dimensional echocardiographic examination and transesophageal echocardiogram were performed , showing a large right atrial mass(fig .
the ivc had a tumor , which appeared to be continuous with the right atrial mass .
one year prior to admission , she had began suffer - ing from an abdominal fullness and weakness .
laboratory examinations manifested serum alt of 391iu / l , serum ast of 341iu / l , alkaline phosphatase of 279iu / l , gamma glutamyl transferase of 298iu / l , alpha - fetoprotein of more than 7000ng / ml and alpha 1-antitypsin of 410mg / dl .
abdominal computed tomography revealed an ill - definded large inhomogeneous mass filling the entire right lobe of the liver with underlying liver cirrhosis and tumor thrombus in the upper portion of the ivc and the right atrium ( fig .
a 61-year - old man , with a history of niddm ten years previously , was admitted due to epigastric pain , indigestion , and malaise . on admission
, he had a regular pulse rate , was normotensive and relatively well - looking .
abdominal ultrasonogram favored a hepatoma of the right lobe with a right hepatic vein thrombus(fig .
two - dimensional echocardiographic examination and transesophageal echocardiogram were performed , showing a large right atrial mass(fig .
the ivc had a tumor , which appeared to be continuous with the right atrial mass .
one year prior to admission , she had began suffer - ing from an abdominal fullness and weakness .
laboratory examinations manifested serum alt of 391iu / l , serum ast of 341iu / l , alkaline phosphatase of 279iu / l , gamma glutamyl transferase of 298iu / l , alpha - fetoprotein of more than 7000ng / ml and alpha 1-antitypsin of 410mg / dl .
abdominal computed tomography revealed an ill - definded large inhomogeneous mass filling the entire right lobe of the liver with underlying liver cirrhosis and tumor thrombus in the upper portion of the ivc and the right atrium ( fig .
hepatoma , testicular teratoma and smooth muscle sarcoma may cause the right atrial tumor growth through the inferior vena cava , . especially , hepatocellular carcinoma is known for its tendency to grow in the venous system .
but , the most common sites of metastasis are the regional lymph nodes and the lungs .
the incidence of metastatic involvement of the heart in hepatoma ranges from 1.5 to 18.3% .
however , such cardiac metastasis occur through the lymphatic system or by infiltrating from neighboring organs , such as the lung and breast . therefore , cardiac metastases occur mainly in the pericardium and myocardium .
intracavitary cardiac metastasis or tumor invasion is an uncommon form of secondary cardiac malignancy , and metastasis to the right atrium is even less common ( 0.674.8% ) .
a report by hanfling showed that intracardiac tumor thrombus occur from above the pulmonary vein to the left cardiac cavities , and from below the superior and inferior vena cava to the right cavities .
tumor implantation to the right ventricle without right atrial involvement and extensive myocardial invasion is unusual in hepatoma .
the left atrial involvement is probably related to tumor growth from the pulmonary veins following massive metastasis to the lung , direct invasion of the atrial septum or tumor implantation via subclinical right - to - left shunt through the patent foramen ovale .
lei et al . reported unusual patterns of cardiac metastasis in three cases of hepatocellular carcinoma : one patient was noted to have a large right ventricular tumor mass with intracavitary growth and myocardial invasion : the second had massive pulmonary and left atrial metastasis ; and the third patient had a right atrial tumor mass with concomitant right ventricular and left atrial involvement . in the past , antemortem diagnosis of right atrial tumor thrombi in patients with primary hepatoma was difficult , and such cases were reported by angiography .
okuda described the angiographic findings of the right atrial tumor thrombi from the liver;thread and streak sign . recently ,
ekg - gated magnetic resonance imaging(mri ) , computerized tomography(ct ) , transthoracic echocardiogrphy and transesophageal echocardiography are available to detect the right atrial tumor thrombus from hepatoma .
transthoracic echocardiography can be considered the first step in case of suspected cardiac masses , but it in sometimes limited by the thoracic conformation and not always discriminant . the resolutive diagnosis in possible thanks to mri .
therefore , mri can be considered the second step in the imaging of cardiac masses .
ct is limited by the only axial or para - axial scans with low quality reconstructions , worsened in these cases by respiratory and cardiac movements .
transesophageal echocardiography is semi - invasive , so esophageal varices were relative contraindication to transesophageal echocardiography .
chua and colleagues reported two cases of right atrial tumor associated with hepatoma diagnosed by two - dimensional echocardiography .
molinari et al . reported that mri compared to ct in the intracardiac spread of hepatocellular carcinoma .
therefore , abnormal cardiac sounds and murmurs subsequantly develop . when the tumor thrombus is mobile , this symptom
is termed the ball - valve thrombus syndrome . according to kato , 3 percent(5 of 156 ) of hepatocellular carcinoma cases had right atrial metastasis , and these patients had edema in the legs , venous dilatation in abdominal wall , ascites , and dyspnea;secondary budd - chiari syndrome . because of the high risk of sudden death from tricuspid valve obstruction in patients with mobile right atrial mass , prompt diagnosis and surgical intervention may prolong the patient s life . despite progress in liver surgery , hepatocellular carcinoma with a tumor thrombus extending into the right atrium has been considered beyond the reach of resection .
although sporadic reports of successful diagnosis have been published , surgical removal has rarely been reported .
similar procedures have been carried out by ehrlich , hill and hetzer , and miller .
these patients who have hepatoma and intracardiac extending mass usually die within a short period because of pulmonay embolism , heart failure or cancer progression .
there is ittle doubt that prompt diagnosis and surgical intervention relieved suffering and prolonged life . | hepatoma has a tendency to spread into the venous system , but intracavitary cardiac extension or metastasis of hepatocellular carcinoma is an uncommon form of cardiac malignancy.when the carcinoma grows from the hepatic vein into the right atrium , the right atrial tumor thrombis may hinder the blood flow .
therefore , these patients have the risk of sudden death.in the past , antemortem diagnosis of right atrial tumor thrombi in patients with primary hepatocellular carcinoma was difficult .
but , echocardiography allowed easy detection of the intracardiac tumor thrombi.we describe two cases of hepatocellular carcinoma with right atrial tumor thrombi . in these cases ,
the right atrial tumor thrombi was detected by two - dimensional echocardiography.recently , successful surgical removal of the right atrial tumor thrombi are reported in several cases.we advocate performing echocardiographic examination in patients with hepatoma who have cardiac symptoms and signs . |
ever since the first reference of an organism that could be considered a motile aeromonad in 1891 the taxonomy of the genus aeromonas , initiated in 1943 , is complex and continuously changing .
although historically the genus aeromonas was included in the family vibrionaceae , together with the genera vibrio , photobacterium , and plesiomonas , phylogenetic investigations indicated that they should form their own family : aeromonadaceae .
the family aeromonadaceae consists of gram - negative , facultative anaerobic , chemoorganotroph bacteria with an optimal growing temperature of about 22c to 28c .
generally they are motile by polar flagellation , able to reduce nitrates to nitrites and able to catabolize glucose and several carbohydrates while producing acids and often gases as well .
initially , in bergey 's manual of systematic bacteriology this family only included the genus aeromonas and was divided into two principal subgroups : the nonmotile and psycnrophilic species ( a. salmonicida ) and the motile and mesophilic species ( a. hydrophila , a. caviae , and a. sobria ) .
the current edition , list three genera in this family : aeromonas , oceanimonas , and tolumonas .
the first classifications within the aeromonas genus have been determined phenotypically ( phenospecies ) , based on growth characteristics and biochemical tests .
nevertheless , there is a great difficulty in identifying the different aeromonas strains on a species level by these characteristics , due to the phenotypical heterogeneity and growing number of known species .
one of the biggest steps forward in the taxonomic process was the introduction and continuous use of genotypical methods ( genospecies ) .
dna - hybridisation groups ( hg ) have been established , associated with the already described phenotypical species .
though some genospecies stay without an associated phenospecies , some phenospecies without associated genospecies and major problems occurred due to differences between the phenotypical and genotypical groups .
a number of molecular chronometers have been used to evaluate phylogenetic relationships and relatedness among aeromonas species .
the ribosomal gene 16s ( small subunit ) was very useful to classify the aeromonads , and a fast and low - cost method based on restriction fragment length polymorphisms ( rflp ) of the 16s rdna amplified by polymerase - chain - reaction ( pcr ) was developed [ 6 , 7 ] .
to better estimate the nucleotide substitution of the 16s - rdna gene , recent sequence analysis of housekeeping genes , like gyrb ( b subunit dna - gyrase ) and rpod ( factor ) , has been proposed [ 8 , 9 ] . according to the latest edition of bergey 's manual of systematic bacteriology , 17 species have been officially accepted within the genus aeromonas ( dna - hybridisation groups are indicated in parenthesis ) : a. hydrophila ( hg1 ) , a. bestiarum ( hg2 ) , a. salmonicida ( hg3 ) , a. caviae ( hg4 ) , a. media ( hg5 ) , a. eucrenophila ( hg6 ) , a. sobria ( hg7 ) , a. veronii [ ( bv . sobria ( hg8 ) and bv .
veronii ( hg10 ) ] , a. jandaei ( hg9 ) , a. schubertii ( hg12 ) , a. trota ( hg14 ) , a. allosaccharophila ( hg15 ) , a. encheleia ( hg16 ) , and a. popoffii ( hg17 ) ; recently three new species have been described : a. culicicola , a. simiae , and a. molluscorum .
( hg11 ) and aeromonas group 501 ( hg13 ; previously enteric group 501 ) , remain without association with an actual species .
they have been isolated from marine waters , rivers , lakes , swamps , sediments , chlorine water , water distribution systems , drinking water , and residual waters , especially during hot months in greater numbers .
the number of isolates from drinking water is generally low compared to its numbers found in food .
aeromonas strains have been found in different types of food , such as meat , fish , seafood , vegetables and processed foods .
potentially they could represent a serious problem in food , as many strains are able to grow at temperatures of a common refrigerator , at a ph of 410 and in presence of higher concentrations of salts .
furthermore it has been shown that they are able to produce exotoxins at low temperatures .
aeromonas strains are predominantly pathogenic to poikilothermic animals including amphibians , fish and reptiles , whereas they also can be found associated with infections of birds and mammals . in fish , they cause hemorrhagic septicemia that often leads to an elevated mortality and major economic losses in aquaculture . the psicrophilic a. salmonicida is considered an important pathogen among a variety of fishes , provoking systemic furunculosis in salmonidae .
mesophilic species ( a. hydrophila and a. veronii ) cause a similar assortment of diseases in fishes as carp , tilapia , perch , catfish , and salmon , and a. hydrophila and a. jandaei provoke aeromoniasis in eels . on the other hand
, a. hydrophila has been linked to major die - offs and fish kills around the globe over the past decade .
the mesophilic aeromonads are emerging as important pathogens in humans , causing a variety of extraintestinal and systemic infections , as well as gastrointestinal infections .
approximately 85% of the clinical isolates of the genus aeromonas consist of two species and a single biotype of a third species : a. hydrophila , a. caviae , and a. veronii sv .
caused extraintestinal infections include : septicemia , provoked by the dissemination of the organism , from the intestinal tract to systems of circulation , and in the majority observed in immunocompromised patients ; wound infections , mostly superficial cutaneous infections , but also infections of tendons , muscles , and bones ; infections of the respiratory tract , from epiglottitis to pneumonia ; and , less frequent , meningitis , peritonitis , eye infections and hemolytic uremic syndrome .
the most commonly described disease caused by aeromonas is the gastroenteritis that can appear in the form of a selflimiting liquid diarrhea to a more severe and invasive diarrhea , which specially is a problem for young children and infants . in the last couple of years also cases of travelling diarrhea caused by aeromonas
also , an increased isolation rate of aeromonas species was reported in the floodwater samples following hurricane katrina in new orleans , suggesting that this microbe could pose potential public health threats during natural disasters . despite the demonstration of the enterotoxic potential of some aeromonas strains ,
there is still a debate on its consideration as an etiological agent , as there were no big epidemical outbreaks described and no adequate animal model is available to reproduce the gastroenteritis caused by aeromonas .
microbes use their own strategies for survival and multiplication , fighting the defense mechanisms of the host 's immune system .
the observed clinical manifestations of aeromonas infections suggest that there could be a complex network of pathogenic mechanisms forming of a multifactorial process .
recent studies seem to strengthen this hypothesis as the virulence of this genus depends on the bacterial strain , the infection route , and the animal used as model organism . over the last years
there has been a big increase in the number of sequenced genomes of different bacteria in the databases .
this information permits a better understanding of the bacteria 's potential , though always within limits . to date five complete genomes of genus aeromonas
have been sequenced entirely , three of them made available to the public in publications : the strain a449 of aeromonas salmonicida , subspecies salmonicida , the strain atcc 7966 t of a. hydrophila , and the strain b565 of a. veronii .
this information is of great value , although there is a great diversity within the genus and some virulence factors will probably not be present in these strains or these strains show different mechanisms to infect the host than others .
the main pathogenic factors associated with aeromonas are surface polysaccharides ( capsule , lipopolysaccharide , and glucan ) , s - layers , iron - binding systems , exotoxins and extracellular enzymes , secretion systems , fimbriae and other nonfilamentous adhesins , motility and flagella .
the capsule ( cps ) is a structure composed of polysaccharides that usually covers the outer membrane of the bacterial cell .
it is highly hydrated ( approximately 95% is water ) and made up by repetitions of monosaccharides that are linked within each other by glycosidic bonds that can cause homo- or hetero - polymers .
the variety of capsule - forming monosaccharides , different linkage and possible modifications contribute to an additional elevated diversity and structural complexity .
this structure frequently forms the most outer layer of the bacterial cell and therefore participates in the bacteria interactions with the environment . in consequence
, capsules have been described as a major virulence factor of many pathogens , as they prevent phagocytosis , favor interactions to other bacteria and host tissue , and act as a barrier against hydrophobic toxins .
a. salmonicida , in vivo and in tsa medium ( tryptic soy agar ) , is able to form a capsular polysaccharide which is not detectable when growing in liquid tsb medium ( tryptic soy broth ) .
the reported o - chain polysaccharide of a. salmonicida produced in tsb and consisting of l - rhamnose , d - mannosamine and d - glucose which was also detected in the bacterial inoculums tsb culture used to prepare the in vivo growth chambers .
it could be established that the structure of the cps and lipopolysaccharide ( lps ) o - chain polysaccharide of a. salmonicida strain 80204 - 1 produced under in vitro growth conditions on tsa .
both polysaccharides were shown by composition , methylation analysis , nmr and ms methods to be composed of linear trisaccharide repeating units containing 3-linked2-acetamido-2-deoxy - d - quinovose , 4-linked 3-[(n - acetyl - l - alanyl)amido]-3-deoxy - d - quinovose and 2-acetamido-2-deoxy - d - galacturonic acid .
it has been confirmed by direct different chemical analysis that both cps and o - chain polysaccharide were also present in the in vivo - grown cells of a. salmonicida strain 80204 - 1 harvested at 72 h after implant surgery .
these polysaccharides were not detected in the in vitro - grown bacterial inoculum tsb culture used for the implants .
the role of this structure as a virulence factor was demonstrated , as it reduces opsonization by hindering phagocytosis [ 3335 ] and contributes to the invasion in cell lines of fish .
formation of capsular polysaccharide ( cps ) covering the a - layer has been reported to be produced during the in vivo culture of a. salmonicida in surgically implanted intraperitoneal culture chambers .
have reported that when grown under conditions promoting capsule formation , strains of a. salmonicida exhibited significantly higher ability to invade fish cell lines .
it suggests that , as with the a - layer and lps , cps is an important virulence factor , essential for host cell invasion and bacterial survival .
mesophilic aeromonas spp . , a. hydrophila ah-3 ( serogroup o : 34 ) , and a. veronii bv .
sobria ( serogroup o : 11 ) are also able to produce a capsule when grown in glucose - rich media .
the strains ppd134/91 and jcm3980 of a. hydrophila ( serogroup o : 18 ) also produce capsular polysaccharides , and it was the strain ppd134/91 where genes for biosynthesis and export of the capsule have been described for the first time within the genus . the genetic organization in three regions is similar to the group ii of capsular polysaccharides in other bacteria like escherichia coli [ 38 , 39 ] .
the cell envelope of some gram - negative bacteria display a form of subcellular differentiation in which peptidoglycan and outer membrane proteins at the cell poles remain stable for generations while material in the lateral walls is diluted by growth and turnover .
the outer membrane has a defined in vivo organization in which a subfraction of proteins and lipopolysaccharide ( lps ) are embedded in stable domains at the poles and along one or more helical ribbons that span the length of the gram - negative rod .
lps is a surface glycoconjugate unique to gram - negative bacteria and a key elicitor of innate immune responses , ranging from local inflammation to disseminated sepsis .
gram - negative bacteria have two membrane layers separated by a periplasmic space : an inner or plasma membrane and the outer membrane .
lps is a major component of the outer leaflet of the outer membrane and consists of lipid a , core oligosaccharide ( os ) , and o - specific polysaccharide or o antigen [ 41 , 42 ] . the o antigen , which is the most surface - exposed lps moiety , mediates pathogenicity by protecting infecting bacteria from serum complement killing and phagocytosis [ 4245 ] .
the chemical composition , structure , and antigenicity of the o antigens vary widely among gram - negative bacteria , giving rise to a large number of o - serogroups .
lps biosynthesis involves a large number of enzymes and assembly proteins encoded by more than 40 genes , recently reviewed in references [ 4648 ] .
it begins at the cytosolic or inner membrane , followed by the transit of the molecule to the outer leaflet of the outer membrane where it becomes surface - exposed .
the o antigen is synthesized as a lipid - linked glycan intermediate by a process that is remarkably similar to the biogenesis of lipid linked oss for protein n - glycosylation .
the lipid carrier in bacteria is undecaprenyl phosphate ( und - p ) , while eukaryotic cells and archaea utilize dolichyl phosphate ( dol - p ) .
the a. hydrophila ah-3 wecp represents a new class of udp - hexnac : polyprenol - p hexnac-1-p transferases ( ref wecp ) .
these transferase - catalyzed reactions involve a membrane - associated polyprenol phosphate acceptor and a cytoplasmic udp - d - n - acetylhexosamine sugar nucleotide as the donor substrate .
a. hydrophila ah-3 wecp transfers galnac to und - p and is unable to transfer glcnac to the same enzyme substrate .
furthermore , the wecp enzyme ( udp - galnac : polyprenol - p galnac-1-p transferase ) differs from weca ( udp - glcnac : polyprenol - p glcnac-1-p transferase ) in membrane topology ( ref wecp ) . the differences in substrate specificity and membrane topology between wecp and weca indicate a different phylogenetical branch .
the lipid a is a highly conserved structure and covalently linked to the polysaccharide complex .
it is the lipid component of lps and contains the hydrophobic , membrane - anchoring region of lps .
lipid a consists of a phosphorylated n - acetylglucosamine ( nag ) dimer with 6 or 7 saturated fatty acids ( fas ) attached .
some fas are attached directly to the nag dimer and others are esterified to the 3-hydroxy fatty acids that are characteristically present .
its biological activity appears to depend on a peculiar conformation that is determined by the glucosamine disaccharide , the po4 groups , the acyl chains , and also the 3-deoxy - d - manno - octulosonic acid ( kdo ) containing inner core .
the most prominent activity of lps is its immunostimulatory potency leading to the complex clinical syndrome of gram - negative sepsis when the initial host response to an infection becomes deregulated .
the clinical manifestation of sepsis is characterized by fever , hypotension , respiratory and renal failure , and intravascular disseminated coagulation .
these effects are not the result of lps toxicity , but are rather a consequence of cell activation by lps and a subsequent deregulation of the inflammatory host response .
the biological activity of lps is harbored in the lipid anchor of the molecule , termed lipid a or the endotoxic principle of lps .
the endotoxic lps properties derive from the release of lipid a of lysed bacteria which can provoke a major systemic inflammation known as septic or endotoxic shock .
salmonicida contained three major lipid a molecules differing in acylation patterns corresponding to tetra- , penta- and hexa - acylated lipid a species and comprising 4-monophosphorylated -2-amino-2-deoxy - d - glucopyranose-(16)-2-amino-2-deoxy - d - glucopyranose disaccharide , where the reducing end 2-amino-2-deoxy - d - glucose was present primarily in the -pyranose form .
the tetra - acylated lipid a structure containing 3-(dodecanoyloxy)tetradecanoic acid at n-2,3-hydroxytetradecanoic acid at n-2 and 3-hydroxytetradecanoic acid at o-3 , respectively , was found .
the penta - acylated lipid a molecule had a similar fatty acid distribution pattern and , additionally , carried 3-hydroxytetradecanoic acid at o-3. in the hexaacylated lipid a structure , 3-hydroxytetradecanoic acid at o-3 was esterified with a secondary 9-hexadecenoic acid . interestingly , lipid a of the in vivo rough isolate contained predominantly tetra- and pentaacylated lipid a species suggesting that the presence of the hexa - acylated lipid a was associated with the smooth - form lps .
the core can be subdivided into two regions , the inner and the outer core , based on their sugar composition .
the inner core is attached to the lipid a at the 6 position of one nag , and all known inner cores contain kdo or a derivative residue ( 3-glycero - d - talo - octulosonic acid ) .
in addition , the base structure of the inner core typically contains l - glycero - d - mannoheptose ( l , d - hep ) , but some bacteria contain d - glycero - d - mannoheptose ( d , d - hep ) alone or in combination with l - d - hep , while other lack heptoses entirely , like rhizobium . within a genus or family ,
in contrast , the outer core provides an attachment site to the o polysaccharide and shows more structural diversity , although variation within a given species , or even a genus , is still limited .
the complete lipid a - core os unit is translocated to the periplasmic face of the inner membrane by the msba transporter , which is a member of the glyco atp - binding cassette ( abc ) transporters superfamily requiring atp hydrolysis .
the chemical structure of the lps core of a. hydrophila o : 34 and a. salmonicida .
the complete genomics and proteomics of the lps core biosynthesis in a. hydrophila ah-3 ( serogroup o34 ) were achieved by the identification and characterization of three genomic regions . combining these data
together with the structure elucidation of the lps core in mutants in each gene from the three gene clusters enabled a presumptive assignment of all lps core biosynthesis gene functions .
salmonicida a450 and a. hydrophila ah-3 renders a great similarity in the inner and part of the outer lps core , but some differences in the distal part of the outer lps core .
the three genomic regions encoding lps core biosynthetic genes in a. salmonicida a450 were fully sequenced , being regions 2 and 3 with identical genes to a. hydrophila ah-3 .
a. salmonicida a450 region 1 showed seven genes , three of them identical to a. hydrophila ah-3 , three of them similar but not identical to a. hydrophila ah-3 and one of them without any homology to any well characterized gene . combining
the gene sequence and complementation test data with the structural data and phenotypic characterization of mutants , the complete genomics and proteomics of a. salmonicida were established . by hybridization studies with internal probes of the a. salmonicida specific genes using different a. salmonicida strains ( besides their subspecies or being atypical ) , was shown a unique or prevalent lps core type .
the o polysaccharide ( o antigen ) is usually attached to a terminal residue of the outer core and consists of repeating oligosaccharide subunits made up of 1 to 6 sugars .
the individual chains vary in length ranging up to 40 repeat units , which constitute the hydrophilic domain of the lps molecule , as well as a major antigenic determinant of the gram - negative cell wall .
the structural diversity of o - polysaccharides repeated units with more than 60 monosaccharides , different position and stereochemistry of the o - glycosidic linkage and presence or absence of different noncarbohydrate substituents least to great variability between species and even strains of gram - negative bacteria .
the variability of o - polysaccharides repeated units , particularly the terminal sugar , confer immunological specificity of the o - antigen .
the first useful scheme for the serogroup of aeromonas strains included 44 serogroups based on o antigens for a total of 307 a. hydrophila and a. caviae strains .
more than 60% of the septicemia cases are related to four of these serogroups : (o: 11 ; o : 16 ; o : 18 ; o : 34 ) .
serogroup o : 11 is associated with severe infections in humans , like septicemia , meningitis and peritonitis while serogroup o : 34 , the most common in mesophilic aeromonas , is associated with wound infections in humans and outbreaks of septicemia in fishes .
furthermore , the lps of serogroups o : 13 , o : 33 , o : 34 and o : 44 shows thermoadaptation .
thus , high growth temperatures ( 37c ) increase the levels of hydroxylated and saturated fatty acids in the lipid a of serogroup o : 34 and in serogroups o : 13 , o : 33 , o : 34 , and o : 44 , the s forms of lps predominate in growth conditions of 20c or 37c at higher osmolarity , while r forms predominate at 37c at lower osmolarity [ 65 , 66 ] .
salmonicida has been characterized , also the chemical structure of the o : 11 antigen of a. hydrophila ll1 with s - layer and aeromonas caviae atcc15468 .
furthermore , the o antigen biosynthesis genes in the a. hydrophila strain ppd134/91 ( serogroup o : 18 ) and ah-3 ( serogroup o : 34 ) have been described [ 38 , 71 ] . like in other polysaccharides
biosynthesis clusters , three classes of genes have been found : genes involved in the biosynthesis of activated sugars , genes that encode glycosyltransferases , and genes whose products are necessary for the o antigen translocation and polymerization .
the interaction of lps with cells of the innate immune system leads to the formation and release of endogenous mediators initiating inflammatory and immune responses essential for an antibacterial defense .
this primarily protective mechanism may become overshadowed by an acute pathophysiological response with the typical clinical symptoms of septic shock that frequently follows the release of inflammatory mediators , such as tumor necrosis factor ( tnf)-a during infection .
lps induces no degranulation in macrophages , but like allergens , it stimulates the de novo synthesis and release of cytokines in these cells .
activation of cells by lps is mediated by the toll - like receptor 4 ( tlr4 ) , a member of the highly conserved protein family of tlr , which is specialized in the recognition of microbial components . in mice , defects in tlr4 result in lps unresponsiveness . for functional interaction with lps
, tlr4 requires association with myeloid differentiation protein 2 ( md-2 ) . according to current consensus activation of tlr4
is preceded by the transfer of lps to membrane - bound or soluble cd14 by lps - binding protein ( lbp ) .
this mechanism is believed to be true for lps signaling generally . however , in a recent study showed that r - form lps and lipid a , but not s - form lps , are capable of inducing tnf- responses also in the absence of cd14 .
lpss from aeromonas are mainly high heterogeneous mixtures of s - form lps molecules containing 1 to over 50 repeating oligosaccharide units and contain ubiquitously a varying proportion of r - form molecules lacking the o - specific chain .
lpss are amphipathic molecules whose hydrophobicity decreases with increasing length of the sugar part . based upon these differences , s- and r - form lps show marked differences in the kinetics of their blood clearance and cellular uptake as well as in the ability to induce oxidative burst in human granulocytes and to activate the host complement system . in relation to the aeromonas ssp biological activities , like in other gram - negative bacteria , the lipid a induce the b cell polyclonal activation and the response to immunoglobulin m , both by a t mitogen - independent mechanism .
furthermore , different effects were observed after injection into animals : pyrogenicity , leucopenia followed by leucocytosis , septic shock , hemorrhagic necrosis of tumors , diarrhoea , and also death [ 80 , 81 ] . on the other hand ,
the s form of lps protects the bacteria from the bactericide effects of the nonimmune serum , since the complement component c3b binds to the long o antigen chains being far away from the membrane and unable to form the complement attack complex , and therefore avoids cell lysis .
the long o : 34 antigen chains increase hemolytic activity , virulence in fishes and mice , and adherence to human epithelial cells and can be considered an important in vivo colonization factor .
bacteria , such as escherichia coli , could show up to six distinct saccharide polymers simultaneously present within the glycocalyx . at present , the known components of the saccharide matrix include lps o - antigens , enterobacterial common antigen ( eca ) , capsular polysaccharides ( k - antigen ) , colanic acid ( ca or m - antigen ) , poly -1,6-n - acetyl - d - glucosamine ( pnag ) , and the -1,4-glucan bacterial cellulose .
the a. hydrophila ah-3 -glucan ( d - glucose linked 1 - 4 and sometimes branched in a 1 - 6 ) is a surface polysaccharide exported via the wecp which is also used by the o34-antigen lps , and ligated to the surface through the o34-antigen polysaccharide ligase ( waal ) . nevertheless , it is an independent polysaccharide versus the o34-antigen lps despite the common use of the export and ligation system , because we could find mutants devoid of either polysaccharide or both .
aeromonas surface glucan production may not have a significant role in cell adhesion but clearly has a role in biofilm formation .
some e. coli exopolysaccharides ( in particular ca and pnag ) are integral components of biofilms , acting as the cement , which holds together the various protein , lipid , and polysaccharide components .
several published reports indicate that the use of -glucans enhances aeromonas disease resistance in fish by potentiating innate immunity [ 91 , 92 ] .
the -glucans used are from yeast representing a heterogeneous group of glucose polymers , consisting of a backbone of -(13)-linked -d - glucopyranosyl units with -(16)-linked side chains of varying length and distribution .
however , in no case the authors were able to show the scientific reason for this aeromonas resistance .
the fact that aeromonas produces a surface -glucan may explain these results and also suggests that the use of -glucans instead of -glucans could be more helpful to enhance the fish resistance to aeromonas disease .
the s - layer is a surface protein layer of paracrystalline nature that is produced by a broad range of bacteria to form the outermost cell envelope .
chemical analysis showed that it is composed of a single protein or glycoprotein ( 40 - 200 kda ) and exhibits either oblique , square or hexagonal lattice symmetry with unit cell dimensions in the range of 3 to 30 nm .
s - layers are generally 5 to 10 nm thick and show pores of identical size ( diameter , 28 nm ) and morphology .
s - layers have been associated with a number of possible functions that relate to pathogenicity . due to its exposition on the cell surface ,
it plays a mayor role in diverse biological functions : adhesion , protection against complement and attack by phagocytes , antigenic properties , anchoring site for hydrolytic exoenzymes , bacteriophage receptor , and others . in 1981 ,
kay and coworkers identified a layer associated with virulence , initially called a - layer , outside the cell wall of a. salmonicida .
later , the constituting protein and the sequence of the encoding gene , vapa , were identified , and it was observed that this layer got lost after growth at temperatures above 25c , due to a deletion of the genetic material . in parallel , the s - layers of mesophilic aeromonas belonging to serogroup o : 11 , and
although these layers are similar to the one identified in a. salmonicida at a morphological level , they differ on the genetic and functional level and could therefore carry out a different role in pathogenicity .
more recently the presence of an s - layer was also described in pathogenic isolates of a. hydrophila belonging to serogroups o.14 and o : 81 .
the s - layer of aeromonas is composed by autoassembling subunits of a single protein that form a tetragonal complex that covers the entire bacterial cell and constitutes the predominant surface antigen .
the secretion of aeromonas s - layer subunits involves the cleavage of a signal peptide to be translocated across the plasma membrane , as well as different specific proteins , homologous to components of the type ii secretion system ( t2ss ) , to be transferred from the periplasm to the exterior .
the s - layer of a. salmonicida promotes association with extracellular matrix proteins and macrophages , binding to porphyrins and immunoglobulins , and protection against proteases and oxidative killing .
o : 11 increases their capacity of adherence which contributes to the colonization of intestinal mucosa as well as generating a major resistance to opsonophagocytosis which could facilitate systemic dissemination after invasion through the gastrointestinal mucosa . since the early days of s - layer glycoprotein research , it was evident that these cell surface components occur on archaea as well as on bacteria .
s - layer glycoproteins have been known for their occurrence among the major lineages of archaea . among bacteria , for a long time only gram - positive members of the bacillaceae family have been known to possess s - layer glycoproteins .
only very recently there were the first reports on the occurrence of glycosylated s - layer proteins in the gram - negative species .
evidence was obtained from biochemical analyses and so far nothing is known about either glycan structure or linkage of the glycans to the s - layer protein portion .
however , in contrast to the known s - layer glycoproteins from bacillaceae investigated these glycosylated s - layer proteins originate from potential pathogens and , therefore , might be of medical relevance . until today all s - layer aeromonas strains have one thing in common : a lipopolysaccharide ( lps ) that contains o - antigen polysaccharides of homogeneous chain length .
this fact leads to the speculation of the possible implication of the lps in linking the s - layer to the bacterial cell surface [ 100 , 106 ] , or to participate in s - layer glycosylation .
numerous environments contain less than 1 m of iron , which is considered the optimum for microbial growth .
the low availability of free iron makes bacterial growth and pathogenicity more difficult , but not impossible .
microorganisms developed a series of mechanisms to sequester iron from their hosts or from insoluble polymers of the environment , including reduction of ferric to ferrous iron , occupation of intracellular niches , utilization of host iron compounds , and production of siderophores .
while direct evidence that high - affinity mechanisms for iron acquisition function as bacterial virulence determinants has been provided in only a small number of cases , it is likely that many if not all such systems play a central role in the pathogenesis of infection .
the competition for iron between the host and the bacterial invader shows the advance of an invasion . due to the presence of iron - binding proteins in the host , such as hemoglobin , transferrin , lactoferrin , or ferritin ,
iron concentrations in the serum are far from the required minimum for growth during the infections of many bacteria . this capacity to deprive an essential nutrient of a microorganism
two high affinity mechanisms to acquire iron are known in aeromonas strains : siderophore - dependent and siderophore - independent mechanisms .
siderophores are low - molecular - weight peptides which present functional groups with elevated affinity and specificity towards iron ions .
these peptides need specific cell membrane bound receptors as well as a cell - associated apparatus to incorporate the metal into the bacterial metabolism .
the enterobactin is found in different gram - negative bacteria , while the amonabactin is only known in aeromonas ssp .
both siderophores are catecholates ( phenolates ) , as they have 2,3-dihydroxybenzoic - acid ( 2,3-dhb ) conjugated with aminoacids .
therefore , their biosynthesis in aeromonas spp . is encoded by two distinct gene groups : the amo genes , in strains that produce amonabactin , and the aeb genes ( aeromonad enterobactin biosynthesis ) , in enterobactin hydroxypyroridone producing strains .
furthermore , the amonabactin receptor of a. hydrophila shows low specificity , permitting the transport of an extraordinary ample range of siderophores , with various chelating groups like catecholate , hidroxamate and hydroxypyroridone .
recently , the a. salmonicida gene cluster involved in the catechol - type siderophore biosynthesis has been described . using a proteomic approach
, a recent study demonstrated that under iron - limited conditions a. salmonicida expresses three iron - regulated outer membrane receptors , and one of these receptors was proposed to be a putative heme receptor based on sequence homology .
heme uptake in gram negative bacteria usually involve outer membrane receptors as well as a tonb - dependent internalization process with two accessory proteins exbb and exbd , and this system is believed to transduce the energy of the proton motive force of the cytoplasmic membrane into transport energy required by the receptor .
subsequently , transport of heme across the cytoplasmic membrane is driven by atp hydrolysis , and an atp - binding cassette ( abc ) transporter is involved in this transport .
a. salmonicida siderophore - independent mechanisms consist of bacterial outer membrane proteins able to bind specific host iron- or heme - binding proteins without the intervention of siderophores [ 108 , 114 ] .
the acquisition of iron is recognized as one of the key steps in the survival of bacterial pathogens within their hosts , and contributes significantly to virulence .
the expression of genes involved in iron acquisition is tightly regulated by the ferric uptake regulator protein fur , which acts as an iron - responsive dna binding repressor . a genetic screening known as the fur titration assay on this bacterium
a screening of gene distribution demonstrated that all the analyzed strains shared genes for siderophore biosynthesis and transport and for heme utilization , indicating that these two systems of iron acquisition are a conserved trait .
iron - regulated a. salmonicida proteins have demonstrated to be protective antigens for fish and are good candidates for the improvement of vaccines .
however , all toxins described are not produced by all strains , although strains may possess their genes .
furthermore , some strains only express toxin genes in certain growth conditions . two main types of enterotoxins have been described in aeromonas spp .
cytotoxic enterotoxins , also known as cytolytic enterotoxins , provoke degeneration of crypts and villi of the small intestine and their producing strains are generally isolated from patients suffering diarrhea .
these toxins can lead to produce hemolysis , cytotoxicity and enterotoxicity [ 118 , 119 ] .
they are synthesized as a preprotein containing a signal peptide which separates after crossing the inner membrane .
the secreted protein is inactive and can be activated by proteolytic nicking near the c - terminus .
the active toxin binds to a glycoprotein on the surface of the target cell and oligomerizes forming pores in the host 's cell membrane that cause cell death . the cytotoxic enterotoxin act , from a. hydrophila ssu , plays an important role in aeromonas infections , since it induces early cell signaling in eukaryotic cells , which leads to the production of inflammation mediators in macrophages and in human epithelial cells .
detailed structural - functional studies of the act enterotoxin and two aerolysins from a. trota and a. bestiarum showed that these proteins are closely related .
however , some heterogeneity at the aminoacid level in some regions could lead to possible differences in folding of these molecules , resulting in differential neutralization of these toxins by specific monoclonal antibodies .
act is an aerolysin - related pore - forming toxin that is responsible for the hemolytic , cytotoxic , and enterotoxic activities of a. hydrophila , being its main virulence factor .
hemolysis involves pore formation in the membrane of the target cell and water entry from the external media , resulting in swelling of the cells and subsequent lysis .
the toxin interacts with the membranes of erythrocytes , inserts into the lipid bilayer as oligomers , and creates pores in the range of 1.14 to 2.8 nm .
cholesterol serves as the receptor for act , and the 3-oh group of this membrane constituent is important for the interaction .
once act has interacted with cholesterol on the cell membranes , the toxin is activated with subsequent oligomerization and pore formation [ 125 , 126 ] .
the toxin activity also includes tissue damage and high fluid secretion in intestinal epithelial cells , resulting from the induction of a proinflammatory response in the target cells .
act upregulates the production of proinflammatory cytokines such as tumor necrosis factor - alpha ( tnf- ) , interleukin-1 beta ( il-1 ) and il-6 in macrophages . tnf- and
il-1 stimulate the production of the inducible nitric oxide synthase ( inos ) that , through nitric oxide ( no ) production , is an essential element of antimicrobial immunity and host - induced tissue damage .
simultaneously , act has the ability to activate arachidonic acid ( aa ) metabolism in macrophages that leads to the production of eicosanoids ( e.g. , prostaglandin e2 [ pge2 ] ) coupled to cyclooxygenase-2 ( cox-2 ) pathway .
aa is a substrate for pge2 production , but is present at limited concentrations in cells .
act increases the amount of aa from phospholipids by inducing group v secretory phospholipase a2 ( spla2 ) , which acts in the membrane of eukaryotic cells .
act increases cyclic amp ( camp ) production in macrophages by indirect activation of adenylate cyclase by pge2 .
the a. hydrophyla toxin also induces the production of antiapoptotic protein bcl-2 in macrophages , preventing the occurrence of massive apoptosis resulting from the induction of the inflammatory response , which would be undesirable for the bacteria .
act also promotes an increased translocation of the nuclear factor kb ( nf - kb ) and camp - responsive element binding protein ( creb ) to the nucleus .
transcription factor nf - kb is important in a number of inflammation - related pathways .
the enhancer / promoter regions of some immunoregulatory cytokine genes , including the tnf- , il-1 , and il-6 , present binding elements for nf - kb and creb .
these transcription factors have also important regulatory functions in the transcription of cox-2 and are implicated in the induction of act cytotoxic activities .
it is secreted as an inactive precursor and undergoes processing at both the n- and c - terminal ends to demonstrate biological activity .
it has a leader sequence of 23 amino acids that allows the protein to transverse the inner membrane .
cytotonic enterotoxins do not produce degeneration of the epithelium and have mechanisms of action similar to those of the choleric toxin , since they increase the cyclic adenosine monophosphate ( camp ) levels and prostaglandins in intestinal epithelial cells .
aeromonas species produces cytotonic enterotoxins that show different molecular weights and variable reactivity to the choleric antitoxin [ 130 , 131 ] .
these enterotoxins have been divided into two groups : heat - labile ( 56c for 10 min . ) , without cross - reactivity with the choleric anti - toxin , and heat - stable ( 100c for 30 min . )
that react with the choleric antitoxin chopra and colleagues purified a heat - labile cytotoxic enterotoxin , alt , from a. hydrophila ssu that increased the camp levels and prostaglandins in rats intestinal mucosa .
the protein sequence shows similarity to the c - terminus of a. hydrophila phospholipase c ( plc ) .
cytotonic enterotoxin , ast , that provokes fluid secretion in rats small intestine and increases the camp levels in mucosal cells .
strains produce at least two other classes of hemolysins without enterotoxic properties : -hemolysins and -hemolysins .
the -hemolysins are synthesized in the stationary growth phase and lead to reversible cytotoxic effects and incomplete erythrocytes lysis .
the -hemolysins , on the other hand , are usually synthesized in the exponential growth phase .
they are thermostable ( 5 min . at 56c ) and pore forming toxins which lead to osmotic lysis and complete destruction of erythrocytes [ 17 , 132 ] .
secretes a wide range of extracellular enzymes , including proteases , lipases , amylases , chitinases , nucleases , and gelatinases .
although in many cases their role in pathogenicity is still to be determined , they represent a big potential to adapt to environmental changes .
extracellular proteases contribute to the metabolic versatility that allows aeromonas to persist in different habitats and that facilitate ecological interactions with other organism . in general , proteases can contribute to the pathogenicity promoting invasion by direct damage of host tissue or by proteolytic activation of toxins .
furthermore , they can also contribute to the establishment of infection overcoming the initial host defenses , for example , inactivating the complement system , or to providing nutrients for cell proliferation . in aeromonas spp .
three different types of proteases have been identified : a temperature - labile serine - protease and two metalloproteases , both temperature - stable but edta ( ethylenediaminetetraacetic acid)-sensitive and -insensitive , respectively .
additionally , aminopeptidases with a number of specific activities have been described : catabolism of exogenously supplied peptides , extracellular activation of aerolysine , or cleavage of amino - terminal methionine from newly synthesized peptide chains ( methionin aminopeptidase ) .
they may provide nutrients and constitute virulence factors by interacting with human leukocytes or by affecting several immune systems functions through free fatty acids generated by lipolytic activity . in a. hydrophila different lipases , such as the ah65 lipase / acyltransferase , h3 , apl1 and lip ,
have been described [ 134 , 135 ] , with the apl1 lipase showing phospholipase c activity . in aeromonas spp.serogroup o : 34 two lipases have been described : phospholipase a1 and c. the phospholipase c shows lecithinase and cytotoxic activities and its role as a virulence factor has been demonstrated .
furthermore , glycerophospholipid - cholesterol acyltransferases ( gcat ) , which digest erythrocytes membranes and leads their lysis , have been isolated from a. hydrophila and a. salmonicida .
gram - negative bacteria have a cytoplasmic membrane , a thin peptidoglycan layer , and an outer membrane containing lipopolysaccharide .
there is a space between the cytoplasmic membrane and the outer membrane called the periplasmic space . in order to transport proteins to the cell surface or
the extracellular space gram - negative bacteria had developed different secretion systems : secretion systems type i , ii , iii , iv , v , and vi .
this classification is based on proteins transport across the outer membrane and refers to the molecular nature of the transport machinery and the catalyzed reactions .
the mechanisms involved in protein transport across the cytoplasmic membrane , in all the above mentioned secretion systems , can be divided into two main groups : sec - dependent and sec - independent .
proteins secreted via the sec - dependent pathway contain an n - terminal signal peptide and utilize the sec translocase for transport across the cytoplasmic membrane .
the sec - dependent pathway includes the secretion system type ii and v. sec - independent pathways allow the export from the cytoplasm to the extracellular environment in one step and do not involve periplasmic intermediates .
these pathways include secretion systems type i , iii , iv , and vi , although type iv can also employ the sec - dependent pathway .
an alternative sec - independent pathway know as twin arginin translocation system ( tat - system ) , which recognize proteins containing two twin-arginine residues in the signal sequence , is employed to transport already folded proteins across the inner membrane .
have been documented to play a critical role in the virulence of many gram - negative bacteria , are often activated upon contact with target cells and deliver their toxin proteins , the so - called effectors , directly into the host cells cytosol .
the t3ss was first identified in pathogenic strains of yersinia spp . , and consist of a complex multicomponent system which transports bacterial proteins , frequently involved in pathogenicity , directly from the bacterial cytoplasm across the inner and outer membrane of the bacterial envelope to either the external medium or directly into the eukaryotic cells .
the t3ss contains three different types of proteins : ( a ) structural components that form needle - like structures , so called injectisomes ; ( b ) secretion substrates , so called effectors ; ( c ) chaperones that assist and protect structural and effector proteins during transport .
the injectisome consists of approximately 20 different proteins that assemble to form a needle - like structure with thin and rigid hollow needles that extend from the cell surface and are anchored to the envelope by basal structures resembling flagella basal bodies .
this structure is usually induced upon contact with the host cells and allows the translocation of the effectors into the eukaryotic cytosol .
the t3ss is independent of the sec system ; however the assembly of the secretion apparatus probably requires the sec machinery , since several components have the characteristic n - terminal signal sequences . the signal that allows effectors recognition and secretion or translocation into the host cells is unknown , although various theories have been suggested .
regardless the differences of these theories it seems that the region that codes the first 20 aminoacids , either in rna or peptide form , is essential for the effectors recognition and secretion . until today
the presence of a functioning t3ss has been described in a. salmonicida and in a. hydrophila strains ah-1 , ah-3 and ssu [ 145147 ] .
furthermore , four t3ss - effector proteins have been identified in a. salmonicida , aext , aopp , aopo and aoph [ 148150 ] , and one , aext and aext - like ( or aexu ) , in the a. hydrophila strains ah-3 and ssu , respectively [ 151 , 152 ] .
aext is a bifunctional toxin , homologous to the also bifunctional effectors exot / exos of p. aeruginosa , showing adp - ribosyltransferase and gap ( gtpase acting protein ) activities .
aopp belongs to the yopj family , a group of t3ss effectors that interferes with signaling pathways of mitogen - activated protein kinases ( mapk ) and/or the nuclear factor kappab ( nf-b ) .
the biological functions of aopo and aoph are unknown ; nevertheless they are homologues of the y. enterocolitica effectors aopo and yoph , respectively .
the t6ss was described by pukatzki and coworkers and seems to constitute a phage - tail - spike - like injectisome , which again serves the purpose of translocations effectors into host cells [ 153 , 154 ] .
it appears to be highly conserved and can be found in one or more copies in diverse gram - negative species , such as v. cholerae , p. aeruginosa , y. pestis , e. coli , s. enterica , agrobacterium tumefaciens , rhizobium leguminosarum , francisella tularensis , burkholderia spp , and edwardsiella spp .
however , the macromolecular structure of this system has not yet been resolved yet , and it is not known how t6ss machines assemble or deliver effectors .
a hallmark of all t6sss is the presence of hcp ( hemolysin coregulated protein ) and vgrg ( valine - glycine repeat protein g ) proteins in culture supernatants .
neither of these proteins is made with a signal peptide , and they are not proteolytically processed .
furthermore , they show structural similarities to components of viral injection machineries indicating that they do not act as classical secreted effectors but are rather surface exposed structural components that might be released in culture supernatants or into eukaryotic cells .
although necessary for e. tarda or francisella pathogenesis , t6ss are required for processes as different as resisting predation in v. cholerae , symbiosis in rhizobium leguminosarum , biofilm formation in enteroaggregative e. coli , killing of niche competitors in p. aeruginosa , burkholderia thailandensis , and v. cholerae , and stress sensing in v. anguillarum for a recent review .
several regulatory mechanisms controlling t6ss gene cluster expression have been identified in recent years : they are regulated at the transcriptional level by alternate sigma factors , two - component systems , or transcriptional factors .
several cases of regulation by quorum sensing have also been reported . because t6ss gene clusters are often found in pathogenicity islands or have been acquired by horizontal gene transfer , their gc content is sometimes different from the gc content of the core genome , and they are silenced by histone - like proteins .
t6ss subunit production is also regulated at the translational level through the action of small regulatory rna , and several t6ss need to be activated by posttranslational mechanisms .
recently , a functional t6ss has been described in a. hydrophila strain ssu and its involvement in virulence has been demonstrated .
t6sss gene clusters are also present in the genome of a. hydrophila ah-3 and atcc7966 , but their implication in virulence has not been proven . by bioinformatics approach we identified several aeromonas strains with clusters which possess typical 24/12 sequences , recognized by the alternate sigma factor 54 , which directs the rna polymerase to these promoters which requires the action of a bacterial enhancer binding protein ( bebp ) , which binds to cis - acting upstream activating sequences .
the importance of vash ( activator ) and vask of a. hydrophila ssu in the expression of the t6ss gene cluster and the secretion and translocation of t6ss associated effectors proteins and their crucial roles in evoking mouse lethality .
a vash isogenic mutant was unable to express and produce known t6ss proteins , such as hcp and vgrg2/3 , and vask isogenic mutant was able to express and translocate hcp into the host eukaryotic cell but unable to secrete it into the extracellular milieu .
the proteomics analysis indicated the existence of vgrg1 , with its gene localized outside the t6ss gene cluster .
this protein has a cooh - terminal extension containing a vegetative insecticidal protein-2 ( vip-2 ) domain , known for its actin adp - ribosylating activity ( 18 ) .
vgrg1 is an important virulence factor of a. hydrophila that is secreted and also translocated by the t6ss with actin adp - ribosylating activity .
the bacterial capacity to adhere and colonize the hosts ' mucosa is a critical step in the infection process .
two classes of adhesins which allow bacteria to bind to specific receptors on the eukaryotic cell surface have been described in aeromonas : those associated with filamentous structures and those associated with proteins of the outer membrane or other structures .
fimbriae / pili are filamentous structures on the bacterial surface , formed by subunits known as pilin .
although pili are often described as adhesive organelles , they have been implicated in other functions , such as phage binding , dna transfer , biofilm formation , cell aggregation , host cell invasion , and twitching motility .
the pili of gram - negative bacteria have been placed into four groups based on their assembly pathway : ( a ) pili assembled by the chaperone - usher pathway ; ( b ) the type iv pili ; ( c ) pili assembled by the nucleation / precipitation pathway ; ( d ) pili assembled by the alternative chaperon - usher pathway ( cs1 pili family ) . in clinical and environmental isolates of mesophilic aeromonas , two distinct types of fimbriae have been found based on their morphology : short , rigid fimbriae ( s / r ) that can be found in high numbers on the bacterial cell and long , wavy fimbriae ( l / w ) that can be found in smaller numbers .
the s / r fimbriae have a length of 0,6 to 2 m and are common epitopes in different analyzed species .
they are widely distributed ( more than 95% of strains ) and able to cause autoaggregation , but not hemagglutination or binding to intestinal cells .
furthermore , they are the predominant type in aeromonads with elevated pili number and in some clinical strains they can be induced under determined environmental conditions ( < 22c , in liquid media ) .
the l / w fimbriae are large , fine ( 47 nm ) , flexible , and considered hemagglutinins .
they are also the predominant type in strains isolated from fish which present a small number of pili ( < 10 per cell ) .
aminoacid sequence analysis indicates that they correspond to type iv pili , known as important structures for adhesion to epithelial cells and involved in biofilm formation and twitching motility .
two different type iv pili have been described in gastroenteritis - associated aeromonas species : the bundle - forming pili ( bfp ) and the type iv pili ( tap ) .
bfp pili are involved in adhesion to intestinal cells and exhibit n - terminal sequence homology with the mannose - sensitive hemagglutinin pilus of v. cholerae .
tap pili differ from bfp pili in their n - terminal sequences and molecular weights and exhibit highest homology with the type iv pili of pseudomonas and pathogenic neisseria species .
furthermore , one of the tap - family proteins ( tapd ) is essential for secretion of aerolysin and proteases , contributing to type ii secretion .
sobria adherence , by the study of a 22-kb locus encoding the bundle - forming that contained 17 pilus - related genes similar to the mannose - sensitive hemagglutinin ( msha ) of v. cholerae .
the bundle - forming pilus is required for a. veronii adherence and biofilm formation , and both the major and minor pilin proteins are essential for this process .
salmonicida , a bacterial pathogen of atlantic salmon , has no visible pili , yet its genome contains genes for three type iv pilus systems .
one system , tap , is similar to the p. aeruginosa pil system , and a second , flp , resembles the actinobacillus actinomycetemcomitans flp pilus .
the tap pili appeared to be polar , while the flp pili appeared to be peritrichous .
the tap pilus made a moderate contribution to virulence , while the flp pilus made little or no contribution . unlike the pili of other piliated bacterial pathogens , a. salmonicida subsp .
salmonicida type iv pili are not absolutely required for virulence in atlantic salmon . on the aeromonas spp .
surface there are also other macromolecules considered adhesins , like the s - layer monomers , the lipopolysaccharide and different outer membrane proteins . among the outer membrane proteins ,
the porins have been specially described to act like a lectin - type adhesins , binding the bacteria to carbohydrate - rich surfaces like erythrocytes and probably intestinal human cells .
depending on the environmental conditions , bacteria are able to move in a free individual manner or remain in the same place to form colony groups and colonize surfaces .
, bacteria can optimize growth and survival by the presence of different cell types that are able to perform specialized functions ; can have better access to nutrients ; better defense mechanisms for protection against unfavorable environmental conditions such as desiccation .
furthermore , bacteria in colonies secrete polysaccharides to form biofilms which enhance adhesion , survival , and movement .
different types of bacterial movement have been described : swimming , swarming , gliding , twitching , and sliding among others .
all of them are associated to movement over surfaces ; except for swimming , that is also used for motility in liquid media .
it has been shown that the twitching requires type iv pili , like several forms of gliding , whereas other forms of gliding remain to be explained .
only swimming and swarming are correlated with the presence of flagella . however , whereas swimming is an individual endeavour , swarming is the movement of a group of bacteria .
swimming in liquid media alternates between straight and tumbling movements . in bacteria with peritrichous flagella , such as e. coli , the counter - clockwise ( ccw ) flagella rotation results in the formation of a helical bundle that propels the cell forward in one direction in a smooth - swimming motion , a so - called run .
by contrast , the clockwise ( cw ) rotation causes unbundling of the helical bundle , allowing the bacterium to randomly reorient its direction , so - called tumbling . in bacteria with a single polar flagellum , such as aeromonas ,
ccw rotation propels the cell forward in a run , whereas cw rotation propels the cell backward with a concomitant random reorientation .
about 60% of mesophilic aeromonas strains showed swarming motility . within the gram - negative bacteria ,
the most studied model of flagellum has been that of e. coli and s. enterica sv .
the prokaryotic flagellum has been structurally divided into an external part , constituted by the filament and the hook , and an internal part embedded in the bacterial surface , the so - called basal body .
e. coli and salmonella express approximately 20.000 copies of a single flagellin ( flic ) .
however , v. parahaemolyticus is able to synthesize 6 different polar flagellins , a. caviae presents two polar and two lateral flagellins , and a. hydrophila presents two polar and only one lateral flagellin [ 172174 ] .
the rotor , composed of the flim , flin , and flig proteins that form the c ring structure at the base of the flagella basal body , and the stator , consisting of membrane - embedded proteins surrounding the ms - ring , that constitute proton or sodium ion channels and couple the flow of ions to flagella rotation . in the proton - driven motor of e. coli and s. enterica serovar typhimurium , the stator is composed of two integral membrane proteins , mota and motb polar flagella stator of vibrio species , such as v. alginolyticus and v. parahaemolyticus , require four proteins : poma , pomb , motx , and moty .
motp / poma and mots / pomb proteins are homologous to the proton - driven mota and motb , respectively .
motx and moty do not have paralogous proteins in e. coli and are components of the t - ring , which is located beneath the p - ring of the polar flagella basal body in vibrio species .
a. hydrophila poma2 and pomb2 are highly homologous to other sodium - conducting polar flagella stator motors .
aeromonas poma - b and poma2-b2 are redundant sets of proteins , as neither set on their own is essential for polar flagella motility either in aqueous or high - viscosity environments .
both poma - b and poma2-b2 are sodium - coupled stator complexes although poma2-b2 is more sensitive to low sodium concentrations than poma - b .
furthermore , the stator of the mesophilic aeromonas proton - driven lateral flagella motor ( motal [ laft ] and motbl [ lafu ] ) is additional to the polar flagella stators [ 169 , 177 ] . in bacteria with polar flagella as vibrio spp .
, mesophilic aeromonas and pseudomonas aeruginosa , genes are distributed in at least five chromosomal regions , but the majority is located in two of them ( figure 1 ) [ 171 , 174 ] .
region 1 is similar in vibrio and aeromonas , though vibrio possesses three more flagellin gene ( flacde ) .
organization of the fla genes in region 2 is also similar in vibrio and areomonas , with the difference that the fli genes of region 2 in vibrio are found in aeromonas ' region 3 .
however , vibrio also possesses one more flagellin gene ( flaf ) and a gene encoding a putative chaperone ( flai ) between flah and flaj .
furthermore , downstream of the aeromonas flaj lies a gene which encoded a homolog of the maf proteins reported in h. pylori , clostridium acetobutylicum , and campylobacter jejuni .
aeromonas polar flagella region 3 shows similar organization to the genes downstream of flam in vibrio region 2 with the absence of the motor genes .
no master regulatory genes encoding homologues of vibrio parahaemolyticus flak , flal and flam or p. aeruginosa fleq , fles , and fler , were found upstream of a. hydrophila flie .
in contrast , aeromonas have two genes ( poma and pomb ) which encode orthologues of the mota and motb , motor proteins of pseudomonas .
region 4 of vibrio and aeromonas includes a gene that encodes the sodium - driven motor protein motx which is involved with mota , motb and moty in torque generation of polar flagellum ; however in pseudomonas this region contains genes that encode the motor proteins motcd .
region 5 of vibrio and pseudomonas includes a gene that encodes the motor protein moty , however in aeromonas it contains the master regulatory genes ( flrabc ) .
recently , the aeromonas moty has been found in a different region with different behavior than the vibrio moty .
the complete set of genes involved in the formation of a functional and inducible lateral flagella system was described in two bacterial species : v. parahameolyticus and a. hydrophila .
these lateral flagella are encoded by 38 genes distributed in two discontinuous regions ( region 1 and 2 ) on chromosome ii in v. parahameolyticus and in one unique chromosomal region in a. hydrophila ( figure 2 ) . in these two bacterial species ,
a partial set of genes was described in other bacteria with functional dual flagella , such as a. brasilense , a. caviae , r centenum , and b. japonicum .
v. parahaemolyticus region
1 genes are divided into two divergent transcribed sets of genes : flgamnl and flgbcdefghijkll .
v. parahaemolyticus region 2 genes are arranged in four clusters : flimnpqrlflhbal , lafa and flidstklalmotabl transcribed in the same direction , and motyllafkfliefghijl transcribed in the opposite direction .
homologous a. hydrophila genes are transcribed in the same direction and do not contain any homologous gene to v. parahaemolyticus motyl .
furthermore , the a. hydrophila lateral flagella region contains , between flgll and lafa , a modification accessory factor gene , maf5 , which is independently transcribed .
the expression of flagellar genes is highly regulated , due to the energetic cost of biosynthesis and use of the flagellum for the bacteria .
flagellar systems are regulated by numerous environmental factors and global regulators of the bacteria . to ensure the maximum efficiency and precision of the biosynthesis , the bacteria use hierarchic regulatory networks that include transcriptional and posttranslational mechanisms to control expression of the flagellar components . in e. coli and s. enterica sv .
the early genes that correspond to the operon flhdc are controlled by class i promoters ( independent of ) and code for the transcriptional activator , flhd2c2 , for the next genes in the hierarchy .
the class ii genes ( also independent of ) code components of the exportation complex , the basal body , the hook , the flagellum specific sigma - factor ( flia ) and the anti- factor ( flgm ) .
flgm is a negative regulator that binds to and therefore inhibits its function . during flagellum biosynthesis ,
when the structure of the basal body is completed , flgm is secreted out of the cell by the flagellum exportation complex , resulting in flgm - depletion in the cytoplasm and release of to activate the late genes . the late genes ( class iii ) are activated by and include the system of chemotaxis , the motor , haps , and the flagellins .
there have also been described regulatory cascades with four transcriptional classes of bacteria with polar flagellation , like v. cholerae and p. aeruginosa .
the early genes express the protein flra in v. cholerae and fleq in p. aeruginosa , which binds to the factor and thus activates the class ii genes .
class ii genes include two - component - regulators , v. cholerae flrbc or p. aeruginosa flesr , and flia ( ) .
flrc and fler are regulators that also associated with the factor and activate class iii promoters , whereas factor activates class iv genes ( figure d ) . in v. parahaemolyticus possible regulatory cascades for its two flagellar systems have been described .
the v. cholerae and p. aeruginosa model was proposed for regulating the polar flagellum with the regulators flak , an early gene , and flalm , class ii genes . on the other hand ,
this transcriptional regulator , that shows similarity to flra and fleq , associates with the factor to activate the expression of class ii genes . within these genes
we find the gene that codes the factor , which activates the late genes . in this
species it was also observed that the loss of the flak - regulator of polar flagellation can be compensated by lafk , the lateral regulator .
a. hydrophila polar flagellum class i gene transcription is -dependent , which is consistent with the fact that a. hydrophila polar flagellum is constitutively expressed .
in contrast to other bacteria with dual flagella systems such as v. parahaemolyticus , the a. hydrophila lafk protein does not compensate for the lack of the polar flagella regulator flra ( v. parahaemolyticus flak homologue ) .
this is consistent with the fact that the a. hydrophila flra mutation abolishes polar flagella formation in liquid and on solid surfaces but does not affect inducible lateral flagella formation .
the results highlight that the polar and lateral flagella inter - connections and control networks are specific and that there are differences between the dual flagellar systems in a. hydrophila and v. parahaemolyticus .
furthermore , the a. hydrophila polar flagellum transcriptional hierarchy ( also in class ii , iii and iv genes ) shares not only some similarities , but also many important differences with those of v. cholerae and p. aeruginosa . comparative proposed a. hydrophila ,
v. cholerae , and p. aeruginosa polar flagellum gene transcription hierarchies are shown in figure 3 .
the flagella of pathogenic bacteria promote the colonization and invasion of the host 's mucosa .
once the bacteria reach the mucosa the flagellum structure becomes necessary for motility , adhesion and invasion .
this motility , coupled with chemotaxis , permits the pathogens to reach the target - tissue of the mucosa . in helicobacter pylori and
furthermore the flagella are reported to act like adhesins , most probably via its flagellin dominions d2 and d3 .
some enteropathogenic strains of e. coli adhere to the intestinal mucosa via a flagellum - dependent mechanism .
motility and presence of flagella is also associated with biofilm formation , which generally goes along with persistent infections .
the colonization of the mucosa provokes a proinflamatory response or inducible innate immune response , mainly stimulated by specific cells of the mucosa . in mammals tlr5 ( toll - like receptor 5 ) is implicated in flagellin recognition by its dominion d1 .
tlr5 stimulate the transcription of the nf-b ( nuclear factor - kappab ) and mapk ( mitogen - activated protein kinase ) dependent proinflamatory genes .
systemic injection of flagellins in mice induces the production of proinflammatory cytokines like tnf- ( tumor necrosis factor ) , il-6 ( interleukine 6 ) and nitric oxide . in epithelial cells flagellins of e. coli , s. enterica sv .
aeruginosa stimulate the secretion of il-8 , the essential chemokine to attract neutrophiles and macrophages to the infection site .
additionally , glycosylation of the flagellin protein was shown to play a role in the proinflammatory action of p. aeruginosa flagellin .
il-8 release from a549 cells stimulated with nonglycosylated flagellin was significantly reduced when compared to wildtype flagellin .
similar pathogenic mechanisms have been observed for aeromonas polar and lateral flagella ( , , and unpublished results ) .
glycans can be covalently attached to the amide nitrogen of asn residues ( n - glycosylation ) , to the hydroxyl oxygen of , typically , ser or thr residues ( o - glycosylation ) , and , in rare cases , to the indole c2 carbon of trp .
protein glycosylation was first demonstrated in the late 1930s and was long thought to exist only in eukaryotes . as more than two - thirds of eukaryotic proteins are predicted to be glycosylated and these modifications are essential for a multitude of cellular functions , it is not surprising that countless publications have been dedicated to this topic .
it took 40 years until the first bacterial and archaeal glycoproteins were discovered on the surface layers ( s - layers ) of the archaeon halobacterium salinarum and on the s - layers of two hyperthermophilic clostridium species [ 195 , 196 ] .
o - linked protein glycosylation occurs in all three domains of life , and the eukaryotic and bacterial pathways are well characterized . in this section ,
our understanding of bacterial flagellin- and pilin - specific o - glycosylation systems has also been growing , and general o - glycosylation pathways have been identified .
a distinguishing feature is that the o - linkages can be formed with ser , thr or tyr residues .
more than 20 years later , the first bacterial n - linked protein glycosylation ( pgl ) pathway was described in the bacterium c. jejuni .examples of surface - associated glycoproteins in gram - negative bacteria are the pilins of p. aeruginosa and neisseria spp .
, the adhesins tiba and aida-1 of e. coliand hmw1of haemophilus influenzae , and the flagellins of p. aeruginosa , h. pylori , clostridium botulinum , and c. jejuni / c . coli .
these species solely possess a polar flagellar system and the filaments are constituted by the two flagellins flaa and flab .
the structural characterization of the flagellins in this species led to identify the presence of pseudaminic acid ( pse5ac7ac ) decorated in o - linkage to serine ( ser ) or threonine ( thr ) residues in the central region of the primary sequence , which is predicted to be surface exposed in the assembled filament .
pseudaminic acid ( 5,7-diacetamido-3,5,7,9-tetradeoxy - l - glycero - l - manno - nonulosonate acid ) is a sugar of 9 carbon - atoms , similar to n - acetylneuroaminic acid or sialic acid ( neu5ac ) . in c. jejuni also
other residues that modify flagellins have been found , all of them deriving from pseudaminic acid , whereas in c. coli flagellin modification with pseudaminic acid and two derivates of the related nonulosonate sugar legionaminic acid ( 5,7-diacetamido-3,5,7,9-tetradeoxy - d - glycero - d - galacto - nonulosonic acid , leg5ac7ac ) was reported .
up to 19 glycosylation sites were found in both campylobacter flagellins and these modifications represent about 10% of the total protein mass in h. pylori , 7 glycosylation sites in flaa and 10 in flab were found .
the glycosylation sites do not seem to be related to a certain conserved peptide sequence , though a hydrophobic region next to the ser / thr residues was often described . at the genetic level ,
the gi of campylobacter appears to be one of the most variable loci in the genome , containing in between 25 and 50 genes , depending on the strain , that is situated close to those of the flagellins .
a number of genes of this locus encode proteins with homology to carbohydrate biosynthetic enzymes , including some ( neu - locus ) with homology to sialic acid biosynthesis enzymes .
extensive mutational analyses , in addition to novel metabolomic analyses and functional studies on recombinant enzymes , have defined the precise function of a number of gi genes in the pse5ac7ac biosynthetic pathway and provided preliminary evidence for the role of the ptm genes from this locus in the legionaminic acid biosynthetic pathway .
in addition to carbohydrate biosynthetic genes , the campylobacter gi contains multiple copies of hypothetical genes encoding proteins which belong to the motility accessory factor ( maf , cj1318 family ) of proteins .
insertional inactivation of individual copies of these genes has provided evidence for a role in motility or glycosylation although the precise function of each maf gene remains to be determined .
the functionality of glycosylated flagellins and the precise nature of glycosylation in pathogenic bacteria are still to be determined . in some species , like c. jejuni and h. pylori ,
glycosylation is necessary for filament assembly ; whereas the absence of glycosylation does not affect assembly nor motility in p. aeruginosa .
flagellar glycosylation reportedly plays an important role in intestine colonization of c. jejuni and the proinflammatory action of p. aeruginosa . in p.
syringae the flagellum - glycosylating residues determine the specificity to the host plant and also play a role in stabilization of the filament structure . in a. caviae , the polar flagellins , flaa and flab , were shown to be glycosylated , at six or seven sites , respectively , with a novel nonulosonate sugar derivate of 373 da mass , and glycosylation was required for flagellar assembly [ 207 , 208 ] . though bacterial adherence to hep-2 cells requires polar flagella , it is not yet known if the novel glycan plays a role in this process .
a. caviae sch3n possesses a small genomic island that is involved in both flagellin glycosylation and lps o - antigen biosynthesis .
this island appears to have been laterally acquired as it is flanked by insertion element - like sequences and has a much lower g - c content than the average aeromonad g - c content .
most of the gene products encoded by the island are orthologues of proteins that have been shown to be involved in pseudaminic acid biosynthesis and flagellin glycosylation .
two of the genes , lst and lsg , are lps specific as mutation of them results in the loss of only a band for the lps o - antigen .
the proteins encoded by flma , flmb , neua , flmd , and neub are thought to make up a pseudaminic acid biosynthetic pathway , and mutation of any of these genes resulted in the loss of motility , flagellar expression , and a band for the lps o - antigen .
studies on lateral flagella from a. hydrophila have indicated that their flagellins are also glycosylated , although the structure of the glycan has yet to be determined . of significance
is the identification of a homologue ( maf5 ) of the campylobacter maf family of genes , in the lateral flagella structural locus of a. hydrophila .
the product of this gene is required for lateral flagella production and provides the first preliminary evidence that glycosylation may also be required for lateral flagella assembly in this species .
a. hydrophila strain ah-3 in - frame deletion mutants of pseudaminic acid biosynthetic genes pseb and psef homologues resulted in abolition of polar and lateral flagella formation by posttranscriptional regulation of the flagellins , which was restored by complementation with wildtype pseb or f homologues or campylobacter pseb and f .
polar and lateral flagellin proteins from a. hydrophila strain ah-3 ( serogroup o34 ) were found to be glycosylated with different carbohydrate moieties .
the lateral flagellin was modified at three sites in o - linkage , with a single monosaccharide of 376 da , which we show to be a pseudaminic acid derivative .
the polar flagellin was modified with a heterogeneous glycan , comprised of a heptasaccharide , linked through the same 376 da sugar to the protein backbone , also in o - linkage . | the members of the aeromonas genus are ubiquitous , water - borne bacteria .
they have been isolated from marine waters , rivers , lakes , swamps , sediments , chlorine water , water distribution systems , drinking water and residual waters ; different types of food , such as meat , fish , seafood , vegetables , and processed foods .
aeromonas strains are predominantly pathogenic to poikilothermic animals , and the mesophilic strains are emerging as important pathogens in humans , causing a variety of extraintestinal and systemic infections as well as gastrointestinal infections . the most commonly described disease caused by aeromonas is the gastroenteritis ; however , no adequate animal model is available to reproduce this illness caused by aeromonas .
the main pathogenic factors associated with aeromonas are : surface polysaccharides ( capsule , lipopolysaccharide , and glucan ) , s - layers , iron - binding systems , exotoxins and extracellular enzymes , secretion systems , fimbriae and other nonfilamentous adhesins , motility and flagella . |
thirty - nine patients with plaque - type psoriasis ( 17 men , age : 47 10 years ) were recruited for the study at the department of dermatology , venereology , and allergology , wroclaw medical university , poland .
exclusion criteria included : ( i ) any acute or chronic illness that might influence iron metabolism ( including known malignancy , infection , severe chronic kidney disease requiring dialysis , chronic cardiovascular diseases , and haematological diseases ) and ( ii ) any treatment for anaemia or i d in the previous 12 months .
all patients received standard management for psoriasis ; severity of the disease was assessed with psoriasis area and severity index ( pasi).2,3 psoriatic arthritis was diagnosed according to the standard criteria.2,16 low body weight was defined as body mass index ( bmi ) below 24 kg / m .
baseline characteristics of patients with psoriasis and healthy subjects data are presented as mean standard deviation or percentage .
forty - four healthy subjects ( 30 men , age : 53 6 years ) were recruited among volunteers , relatives , and colleagues of the staff or patients at the centre for heart diseases , military hospital , wrocaw , poland .
the criteria for healthy subjects to be included in the study were the following : age 18 years , normal cardiovascular status , absence of any acute ( during the previous 6 months ) or chronic ( at any time in the past ) illness , and related therapy .
the study protocol was approved by the local ethics committee , and all subjects gave written informed consent .
the study was conducted in accordance with the declaration of helsinki . in all patients and control subjects ,
venous blood samples were taken in the morning following an overnight fast and after at least 15 min of supine rest .
after centrifuging , the plasma and serum were collected and frozen at 70c until further laboratory analyses .
the following haematinics were measured using an automatic system advia 120 ( siemens , healthcare diagnostics , deerfield , il , usa ) : haemoglobin concentration ( g / dl ) , haematocrit ( % ) , red blood cells ( t / l ) , mean corpuscular volume ( fl ) , mean corpuscular haemoglobin ( pg ) , and mean corpuscular haemoglobin concentration ( g / l ) .
anaemia was defined as haemoglobin level < 12 g / dl in women and < 13 g / dl in men.17 the following standard blood biomarkers reflecting iron metabolism were measured directly : serum concentrations of ferritin ( g / l ) , iron ( g / dl ) , and total iron binding capacity ( tibc , g / dl ) .
transferrin saturation ( tsat ) was calculated as the ratio of serum iron ( g / dl ) and tibc ( g / dl ) multiplied by 100 and expressed as a percentage .
serum ferritin was measured using an immunoassay based on electrochemiluminescence with the elecsys 2010 system ( roche diagnostics gmbh , mannheim , germany ) .
serum iron and tibc were assessed using a substrate method with feren s ( thermo fisher scientific , waltham , ma , usa ) .
circulating iron bound to transferrin ( expressed as tsat ) reflects the amount of iron available to metabolizing cells.9,18,19 the interpretation of circulating ferritin levels should always take into account its dual physiological role as the major iron storage molecule and as the acute phase protein , which expression is increased in response to inflammatory stimuli , regardless of the amount of stored iron.9,18,19 serum soluble transferrin receptor ( stfr , mg / l ) was measured using immunonephelometry ( siemens healthcare diagnostics inc . , deerfield , illinois , usa ) .
increased circulating stfr ( originating from all cells metabolizing iron ) is a sensitive indicator of i d and quantitatively reflects tissue iron demand along with the erythroid proliferation rate , but not body iron stores.20,21 serum hepcidin ( ng / ml ) was measured using a commercially available enzyme - linked immunosorbent assay ( elisa ) ( bachem ) .
this elisa method was validated with a gold standard for hepcidin assessment , namely liquid chromatography mass spectrometry developed at king s college london , confirming a strong correlation between the measurements performed using the liquid chromatography mass spectrometry and the bachem assay in patients with chronic kidney diseases and healthy subjects.22 increased serum hepcidin may be due to either inflammation or / and excessive iron stores12,13 ; low - circulating hepcidin reflects specifically depleted body iron stores with or without concomitant anaemia ; the lower the hepcidin , the more profoundly depleted iron stores in patients with chronic diseases.23,24 in this study , we also used the definition of i d applied in previous studies in patients with chronic diseases8,23,24 based on serum ferritin and tsat ( serum ferritin
. serum level of interleukin 6 ( il-6 , pg / ml ) was measured using a commercially available elisa ( r&d systems , minneapolis , minnesota , usa ) .
estimated glomerular filtration rate ( ml / min/1.73 m ) was calculated using the modification of diet in renal disease equation.25 the normality of the distributions of continuous variables was tested using the kolmogorov
the remaining continuous variables with a skewed distribution were expressed as medians with upper and lower quartiles .
for further analyses , these variables were log - transformed in order to normalize their distribution .
the statistical significance of differences between the groups was tested using student s t - test , mann whitney u test , or the test , where appropriate .
all statistical analyses were performed with statistica 10 ( statsoft , tulsa , ok , usa ) .
thirty - nine patients with plaque - type psoriasis ( 17 men , age : 47 10 years ) were recruited for the study at the department of dermatology , venereology , and allergology , wroclaw medical university , poland .
exclusion criteria included : ( i ) any acute or chronic illness that might influence iron metabolism ( including known malignancy , infection , severe chronic kidney disease requiring dialysis , chronic cardiovascular diseases , and haematological diseases ) and ( ii ) any treatment for anaemia or i d in the previous 12 months .
all patients received standard management for psoriasis ; severity of the disease was assessed with psoriasis area and severity index ( pasi).2,3 psoriatic arthritis was diagnosed according to the standard criteria.2,16 low body weight was defined as body mass index ( bmi ) below 24 kg / m .
baseline characteristics of patients with psoriasis and healthy subjects data are presented as mean standard deviation or percentage .
forty - four healthy subjects ( 30 men , age : 53 6 years ) were recruited among volunteers , relatives , and colleagues of the staff or patients at the centre for heart diseases , military hospital , wrocaw , poland .
the criteria for healthy subjects to be included in the study were the following : age 18 years , normal cardiovascular status , absence of any acute ( during the previous 6 months ) or chronic ( at any time in the past ) illness , and related therapy .
the study protocol was approved by the local ethics committee , and all subjects gave written informed consent .
in all patients and control subjects , venous blood samples were taken in the morning following an overnight fast and after at least 15 min of supine rest .
after centrifuging , the plasma and serum were collected and frozen at 70c until further laboratory analyses .
the following haematinics were measured using an automatic system advia 120 ( siemens , healthcare diagnostics , deerfield , il , usa ) : haemoglobin concentration ( g / dl ) , haematocrit ( % ) , red blood cells ( t / l ) , mean corpuscular volume ( fl ) , mean corpuscular haemoglobin ( pg ) , and mean corpuscular haemoglobin concentration ( g / l ) .
anaemia was defined as haemoglobin level < 12 g / dl in women and < 13 g / dl in men.17 the following standard blood biomarkers reflecting iron metabolism were measured directly : serum concentrations of ferritin ( g / l ) , iron ( g / dl ) , and total iron binding capacity ( tibc , g / dl ) .
transferrin saturation ( tsat ) was calculated as the ratio of serum iron ( g / dl ) and tibc ( g / dl ) multiplied by 100 and expressed as a percentage .
serum ferritin was measured using an immunoassay based on electrochemiluminescence with the elecsys 2010 system ( roche diagnostics gmbh , mannheim , germany ) .
serum iron and tibc were assessed using a substrate method with feren s ( thermo fisher scientific , waltham , ma , usa ) . circulating iron bound to transferrin ( expressed as tsat ) reflects the amount of iron available to metabolizing cells.9,18,19 the interpretation of circulating ferritin levels should always take into account its dual physiological role as the major iron storage molecule and as the acute phase protein , which expression is increased in response to inflammatory stimuli , regardless of the amount of stored iron.9,18,19 serum soluble transferrin receptor ( stfr , mg / l ) was measured using immunonephelometry ( siemens healthcare diagnostics inc . ,
deerfield , illinois , usa ) . increased circulating stfr ( originating from all cells metabolizing iron ) is a sensitive indicator of i d and quantitatively reflects tissue iron demand along with the erythroid proliferation rate , but not body iron stores.20,21 serum hepcidin ( ng / ml ) was measured using a commercially available enzyme - linked immunosorbent assay ( elisa ) ( bachem ) .
this elisa method was validated with a gold standard for hepcidin assessment , namely liquid chromatography mass spectrometry developed at king s college london , confirming a strong correlation between the measurements performed using the liquid chromatography mass spectrometry and the bachem assay in patients with chronic kidney diseases and healthy subjects.22 increased serum hepcidin may be due to either inflammation or / and excessive iron stores12,13 ; low - circulating hepcidin reflects specifically depleted body iron stores with or without concomitant anaemia ; the lower the hepcidin , the more profoundly depleted iron stores in patients with chronic diseases.23,24 in this study , we also used the definition of i d applied in previous studies in patients with chronic diseases8,23,24 based on serum ferritin and tsat ( serum ferritin
. serum level of interleukin 6 ( il-6 , pg / ml ) was measured using a commercially available elisa ( r&d systems , minneapolis , minnesota , usa ) .
estimated glomerular filtration rate ( ml / min/1.73 m ) was calculated using the modification of diet in renal disease equation.25 the normality of the distributions of continuous variables was tested using the kolmogorov
the remaining continuous variables with a skewed distribution were expressed as medians with upper and lower quartiles .
for further analyses , these variables were log - transformed in order to normalize their distribution .
the statistical significance of differences between the groups was tested using student s t - test , mann whitney u test , or the test , where appropriate .
all statistical analyses were performed with statistica 10 ( statsoft , tulsa , ok , usa ) .
the remaining continuous variables with a skewed distribution were expressed as medians with upper and lower quartiles . for further analyses ,
the statistical significance of differences between the groups was tested using student s t - test , mann whitney u test , or the test , where appropriate .
all statistical analyses were performed with statistica 10 ( statsoft , tulsa , ok , usa ) .
baseline clinical characteristics of patients with psoriasis and control subjects are shown in table
1 .
clinical severity of the disease was evaluated with the pasi , with baseline ranging from 2.1 to 19.8 ( mean : 9.3 4.4 ) ; 11 ( 28% ) patients had psoriatic arthritis , and 15 ( 38% ) received systemic treatment of the disease . compared with healthy controls , patients with psoriasis
had similar haematinics ( haemoglobin and haematocrit ) , slightly bigger mean corpuscular volume , and trends towards lower red blood count and mean corpuscular haemoglobin concentration ( table
2 ) . only two ( 5% ) patients had mild anaemia ( haemoglobin levels 9.9 and 11.9 g / dl , respectively ) .
comparison of haematological parameters , iron status biomarkers , and other laboratory parameters between healthy subjects and patients with psoriasis data are presented as mean standard deviation or median with lower and upper quartiles were appropriate ; p values for between - group comparison using mann
mean corpuscular haemoglobin ; mchc , mean corpuscular haemoglobin concentration tibc , total iron binding capacity ; mcv , mean corpuscular volume ; rbc , red blood cells ; stfr , soluble transferrin receptor ; tsat , transferrin saturation ; there was no difference in the ferritin levels between groups , but patients demonstrated significantly lower tsat ( p < 0.001 ) , elevated tibc ( p < 0.001 ) and stfr levels ( p = 0.01 ) , and markedly decreased hepcidin levels ( p < 0.0001 ) ( table
2 ) . applying
the definition of i d used in the previous studies in chronic diseases ( based on serum ferritin and tsat see the material and methods section ) , 20 ( 51% ) patients with psoriasis fulfilled this definition .
none of the iron - related biomarkers correlated with the severity of the disease assessed with pasi ( p > 0.1 for all correlatory coefficients ) .
also , patients receiving systemic treatment for psoriasis did not differ from not - treated patients in iron - related biomarkers ( p > 0.1 in all comparisons ) .
patients with psoriasis presented only slightly elevated levels of il-6 : mean 4.1 6.2 pg / ml , range 019 pg / ml . among iron - related biomarkers , hepcidin ( r = 0.54 , p < 0.001 ) and ferritin ( r = 0.44 , p = 0.001 ) significantly correlated with levels of il-6 .
eleven ( 28% ) patients had psoriatic arthritis complicating the natural course of the disease , and they demonstrated elevated levels of il-6 ( 6.3 5.6 vs. 2.4 5.4 pg / ml , patients with vs. without psoriatic arthritis , respectively , p < 0.05 ) .
neither haematologic parameters nor iron - related biomarkers were different between patients with and without psoriatic arthritis ( p > 0.2 in all comparisons ) .
body mass index significantly correlated with ferritin ( r = 0.61 , p < 0.001 ) and hepcidin ( r = 0.51 , p = 0.005 ) levels .
eleven ( 28% ) patients had low bmi ( i.e. bmi < 24 kg / m ) .
compared with patients with bmi 24 kg / m , lean patients demonstrated markedly deranged iron status reflecting iron deficiency it was evidenced by the following : low levels of ferritin ( p < 0.001 ) and hepcidin ( p = 0.03 ) and strong trend towards low tsat ( p = 0.06 ) and elevated stfr ( p = 0.06 ) ( table
3 ) .
comparison of iron status biomarkers between psoriatic patients with body mass index < 24 and 24.0 kg / m data are presented as mean standard deviation or median with lower and upper quartiles where appropriate ; p values for between - group comparison using mann
tibc , total iron binding capacity ; tsat , transferrin saturation ; stfr , soluble transferrin receptor .
applying definition of i d based on serum ferritin and tsat ( see the material and methods section ) , 9 ( 82% ) patients with low bmi had i d vs. 11 ( 39% ) with bmi 24 kg / m ( p = 0.02 ) ( figure
1 ) .
l with tsat < 20% in the whole group of patients with psoriasis and comparison between patients with body mass index ( bmi ) < 24 vs. 24 kg / m .
compared with healthy controls , patients with psoriasis had similar haematinics ( haemoglobin and haematocrit ) , slightly bigger mean corpuscular volume , and trends towards lower red blood count and mean corpuscular haemoglobin concentration ( table
2 ) .
only two ( 5% ) patients had mild anaemia ( haemoglobin levels 9.9 and 11.9 g / dl , respectively ) .
comparison of haematological parameters , iron status biomarkers , and other laboratory parameters between healthy subjects and patients with psoriasis data are presented as mean standard deviation or median with lower and upper quartiles were appropriate ; p values for between - group comparison using mann
mean corpuscular haemoglobin ; mchc , mean corpuscular haemoglobin concentration tibc , total iron binding capacity ; mcv , mean corpuscular volume ; rbc , red blood cells ; stfr , soluble transferrin receptor ; tsat , transferrin saturation ; there was no difference in the ferritin levels between groups , but patients demonstrated significantly lower tsat ( p < 0.001 ) , elevated tibc ( p < 0.001 ) and stfr levels ( p = 0.01 ) , and markedly decreased hepcidin levels ( p < 0.0001 ) ( table
2 ) . applying the definition of i
d used in the previous studies in chronic diseases ( based on serum ferritin and tsat see the material and methods section ) , 20 ( 51% ) patients with psoriasis fulfilled this definition .
none of the iron - related biomarkers correlated with the severity of the disease assessed with pasi ( p > 0.1 for all correlatory coefficients ) .
also , patients receiving systemic treatment for psoriasis did not differ from not - treated patients in iron - related biomarkers ( p > 0.1 in all comparisons ) .
patients with psoriasis presented only slightly elevated levels of il-6 : mean 4.1 6.2 pg / ml , range 019 pg / ml . among iron - related biomarkers , hepcidin ( r = 0.54 , p < 0.001 ) and ferritin ( r = 0.44 , p = 0.001 ) significantly correlated with levels of il-6 .
eleven ( 28% ) patients had psoriatic arthritis complicating the natural course of the disease , and they demonstrated elevated levels of il-6 ( 6.3 5.6 vs. 2.4 5.4 pg / ml , patients with vs. without psoriatic arthritis , respectively , p < 0.05 ) .
neither haematologic parameters nor iron - related biomarkers were different between patients with and without psoriatic arthritis ( p > 0.2 in all comparisons ) .
body mass index significantly correlated with ferritin ( r = 0.61 , p < 0.001 ) and hepcidin ( r = 0.51 , p = 0.005 ) levels .
eleven ( 28% ) patients had low bmi ( i.e. bmi < 24
kg / m ) . compared with patients with bmi 24 kg / m , lean patients demonstrated markedly deranged iron status reflecting iron deficiency it was evidenced by the following : low levels of ferritin ( p < 0.001 ) and hepcidin ( p = 0.03 ) and strong trend towards low tsat ( p = 0.06 ) and elevated stfr ( p = 0.06 ) ( table
3 ) .
comparison of iron status biomarkers between psoriatic patients with body mass index < 24 and 24.0 kg / m data are presented as mean standard deviation or median with lower and upper quartiles where appropriate ; p values for between - group comparison using mann
tibc , total iron binding capacity ; tsat , transferrin saturation ; stfr , soluble transferrin receptor .
applying definition of i d based on serum ferritin and tsat ( see the material and methods section ) , 9 ( 82% ) patients with low bmi had i d vs. 11 ( 39% ) with bmi 24 kg / m ( p = 0.02 ) ( figure
1 ) .
l with tsat < 20% in the whole group of patients with psoriasis and comparison between patients with body mass index ( bmi ) < 24 vs. 24 kg / m .
the major novel findings of this study are the following : ( i ) patients with psoriasis demonstrated deranged iron status , which can be characterized by decreased transferrin saturation and elevated stfr ( both reflecting negative tissue iron balance ) and low levels of hepcidin ( reflecting depleted iron stores ) , and such composition of iron - related biomarkers are typical for iron deficiency ; and ( ii ) iron deficiency in psoriatic patients was not related to severity of the disease , but predominantly determined by patients low body mass index .
this point is interesting , because no treatment is currently available for cachexia , that is , involuntary weight loss or a - priori - low body weight,26,27 but also , nutrition and rehabilitation offer only few advances at this stage.28,29 nevertheless , there are some promising new developments,30,31 and many new experimental therapies are in development.32 psoriasis represents an autoimmune disease where primary abnormalities are seen within the skin . however , there is increasing evidence that skin lesions constitute an element of a complex syndrome , and concomitant extra - cutaneous pathologies are crucial ( if not critical ) for disease progression , quality of life , and prognosis of these patients.1,2,4,5 impaired immune response is accompanied by marked inflammatory reaction with concomitant systemic pro - inflammatory milieu observed in patients with psoriasis.1,4 only recently , there is growing interest in iron status in chronic , immune - mediated disorders . however , in contrast to previous reports focusing mainly on potentially deleterious effects of iron overload and linking pathophysiology of several chronic diseases to iron excess,33,34 there is now mounting evidence placing iron deficiency with subsequent clinical consequences as the key abnormality of such diseases.8,9,23,24 it has been demonstrated that deranged iron status is a fundamental element underlying dysfunction of immune cells involved in innate immune response ( monocytes and derivates).6,7 interestingly , both innate immune response and processes of iron metabolism ( as well as an interplay between them ) belong to evolutionary conservative mechanisms , which as they tackle critically important for survival processes , have remained virtually unchanged during the phylogenesis .
additionally , patients with chronic diseases are prone to become iron deficient as a consequence of a depletion of iron stores but more frequently as a result of impaired iron metabolism in the course of underlying inflammatory processes.8,9,11 we have hypothesized that taking into consideration underlying systemic pathophysiological processes in psoriasis this chronic entity may be characterized by markedly deranged iron status . to test this , we investigated a group of patients presenting with fairly broad clinical spectrum of the disease ( including disease severity and medical management ) and applied novel biomarkers of iron status ( hepcidin and soluble transferrin receptors ) .
surprisingly , complex evaluation of iron status has never been performed is patients with psoriasis .
once we applied a previous definition of i d based on low ferritin levels and tsat,8,24 it appeared that half of our population tended to have i d . however , we are aware that the approach may well not be optimal .
serum ferritin reflects body iron stores , but either inflammation or oxidative stress ( both present in psoriasis ) may artificially increase its concentrations , regardless of actual iron status.18,19 importantly , high circulating ferritin provides no insight about the amount of intracellular iron available for metabolic ( including energetic ) needs .
tsat is a direct measure of the circulating iron pool , but it is unclear if this reflects the amount in peripheral tissues.18,19 thus , we used other biomarkers hepcidin and soluble transferrin receptor that we believe can better characterize iron status in psoriasis .
hepcidin , upon binding to the only known cellular iron export protein , ferroportin , results in a ferroportin degradation and the blockage of cellular iron egress with a decrease of duodenal iron absorption and retention of iron in the reticuloendothelial system.10,11 as a consequence , circulating iron concentrations and iron availability to target tissues are significantly reduced.10,11 inflammatory stimuli ( among which il-6 is the most potent ) are able to induce the hepatic expression of hepcidin , which seems to be the mechanism underlying the development of functional i d and anaemia in patients with chronic inflammatory diseases.8,10,11 interestingly , in our study , patients with psoriasis had markedly low levels of hepcidin . at the same time , they demonstrated correlation between il-6 and hepcidin levels , which indirectly confirms intact regulatory mechanisms mentioned before and forms the background to believe that low hepcidin levels reflecting depleted iron stores ( absolute i d ) in patients is not related to pro - inflammatory status .
it is worthy of noting that patients with psoriasis with low hepcidin did not demonstrate significantly impaired haematopoiesis as assessed based on haemoglobin level and red cell indices , which suggests that in psoriasis , depleted iron stores occur in patients without concomitant anaemia .
at the same time , psoriatic patients demonstrated elevated levels of stfr that did not correlate with the presence of anaemia nor with pro - inflammatory activation .
it strongly suggests that the major source of transferrin receptors shed into circulation is not the erythron ( as psoriatic patients had normal haematinics ) , but it reflects the extra - haematopoietic origin of circulating stfr and unmet iron needs within non - haematopoietic cells .
we have previously reported a similar pattern of i d is patients with heart failure that carried ominous clinical consequences.23,35 interestingly , deranged iron status was not related to disease severity evaluated with pasi score , and also once patients receiving systemic treatment and/or those who developed psoriatic arthritis were analysed separately .
the magnitude of changes reflecting iron deficiency was particularly strong in psoriatic patients with low bmi .
we did not study cachexia in the strictest sense , which is frequent in many chronic diseases36,37 and associated with high costs,38 but we observed that lean patients with psoriasis demonstrated features of depleted iron stores ( low circulating hepcidin and ferritin ) at the same time having unmet needs for iron at the periphery ( elevated levels of stfr ) .
additionally , the vast majority of them ( > 80% ) had i d defined according to the criteria used in the previous studies.23,24 this pattern was related neither to pro - inflammatory activation ( no significant difference in il-6 between patients with bmi < 24 and 24 kg / m data not shown ) nor to any other clinical variable we have analysed . taking into consideration the important role iron plays in numerous processes engaged into pathophysiology of psoriasis ,
it may well be hypothesized that profound i d seen in these patients may further deteriorate clinical status , leading to disease progression .
we are not aware of any previous report linking iron status to low body mass index in patients with chronic disorders . whereas there are studies showing that obese patients develop functional
i d associated with inflammatory response,39,40 the high prevalence of absolute i d in psoriatic patients is a novel and intriguing finding .
it may be a consequence of generalized malnourishment , but other unclear pathomechanisms may contribute here , and their elucidation seems to be of a particular importance .
our study was not designed to investigate the mechanisms that may underlie iron deficiency in psoriasis .
naturally , one can speculate that as in the other chronic diseases , pro - inflammatory activation may be the leading cause .
some previous studies reported accelerated loss of nutrients from the hyperproliferation and desquamation of the epidermal layer of skin in psoriatic patients , which may lead to id.41 as mentioned before , malnutrition may be involved in patients with low bmi .
further studies are needed to confirm iron deficiency in psoriasis and reveal underlying mechanisms . in conclusion
, psoriasis is associated with deranged iron status characterized by depleted iron stores with concomitant unmet cellular iron requirements .
the magnitude of these abnormalities is particularly evident in patients with low body mass index .
the deranged pattern of iron status seen in psoriatic patients reflects mainly absolute i d associated with very low circulating hepcidin and is not accompanied by augmented inflammation . | backgroundiron deficiency ( i d ) frequently complicates inflammatory - mediated chronic disorders , irrespective of anaemia .
psoriasis is a chronic , immune - mediated skin disease with systemic pro - inflammatory activation ; thus , these patients may be prone to develop i d .
i d adversely affects immune cells function , which can further contribute to disease progression .
this study investigates iron status in psoriasis.methodsserum concentrations of ferritin , transferrin saturation ( tsat ) , soluble transferrin receptor ( stfr ) , and hepcidin were assessed as the biomarkers of iron status in 39 patients with psoriasis ( 17 men , age : 47 10 years ) and 44 healthy subjects ( 30 men , age : 53 6 years).resultscompared with healthy controls , patients with psoriasis demonstrated similar haematologic status but deranged iron status as evidenced by decreased tsat and elevated stfr ( negative tissue iron balance ) and low levels of hepcidin ( depleted iron stores ) ( all p < 0.05 vs. controls ) . in patients , the levels of interleukin-6 ( level of pro - inflammatory activation ) significantly correlated with hepcidin ( r = 0.54 ) , but not with ferritin , tsat , and stfr .
biomarkers reflecting i d were not associated with the severity of the disease ( assessed with the psoriasis area and severity index ) but significantly correlated low body mass index ( bmi ) .
patients with bmi < 24 kg / m2 compared with those with bmi 24 kg / m2 demonstrated lower levels of ferritin ( 40 30 vs. 186 128 ng / ml , p < 0.001 ) and hepcidin ( 4.9 2.3 vs. 10.7 6.7 ng / ml , p = 0.03).conclusionpsoriasis is associated with deranged iron status characterized by depleted iron stores with concomitant unmet cellular iron requirements .
the magnitude of these abnormalities is particularly strong in patients with low body mass index . whether iron deficiency may become a therapeutic target in psoriasis needs to be investigated
. |
the author has not received any funding or benefits from industry or elsewhere to conduct this study . | this article describes a novel approach to giving dietary advice , which is called dietary advice on prescription ( dap ; matordning p recept [ mor ] in swedish ) .
it is the same principle as prescription on medicine and physical activity on prescription ( pap ; fysisk aktivitet p recept [ far ] in swedish ) .
the main idea is that a written prescription will strengthen the oral advice and emphasize certain aspects of the dietary recommendation .
the dap is on the brink of being tested in a planned study . |
sprains and ruptures of the ligaments supporting the ankle and knee joints as well as muscle strains occur quite often . in some cases , joint injuries are sustained through contact with another player .
a classic example occurs when one player collides with another , applying excessive force to the lateral side of the opponent 's knee .
this often results in damage to the medial collateral ligament , lateral meniscus , and the anterior cruciate ligament ( acl ) .
however , a substantial number of soccer injuries occur through noncontact mechanisms . in these cases
, an athlete may plant his or her foot then stop , cut , or turn . as the body changes direction while the foot is stationary , the knee and/or ankle experiences torque . as a result ,
these include intrinsic factors such as proprioception , muscular strength , ligament properties , and biomechanics as well as extrinsic factors such as the playing surface and other environmental conditions .
several studies have focused on this latter factor as important , specifically the use of artificial turf playing surfaces .
they suggest that the added friction between the shoe and the surface increases the torque experienced by the ankle and knee [ 1 , 2 ] .
in fact , early studies on the first - generation artificial turf ( at ) fields ( astroturf - type surfaces ) did show an increased injury risk compared to natural grass ( ng ) [ 3 , 4 ] .
the short - pile carpet laid over a thin pad has been replaced by a surface that contains long grass - like fibers that are embedded with granules of crushed rubber , sand , and/or silica and laid over a thick pad .
this results in a more compliant surface and one that results in a considerably lower shoe - surface coefficient of friction .
manufacturers of third - generation at surfaces argue that such a design may lower the risk of noncontact joint injury . on the other hand ,
many coaches , trainers , and athletes remain concerned that playing on at increases injury risk .
whether or not third - generation at alters the risk of noncontact injury remains unresolved .
several recent studies have focused on comparing injury incidence rates in soccer players who train and/or play matches on both at and ng .
unfortunately , the results are somewhat variable , with some studies showing reduced risk of some injuries on at and others showing no difference or slightly increased risk .
thus , the purpose of this investigation was to resolve this issue . to accomplish this , we used previous research and a meta - analytic approach to compare injury rates in soccer players performing on at and ng .
key search terms were soccer , injury , grass , synthetic turf , and artificial turf . to be included in the meta - analysis , studies met several criteria : ( 1 ) focused on competitive soccer players , ( 2 ) compared acute injuries sustained during soccer matches and/or training on ng and on third - generation at surfaces , ( 3 ) examined players who participated on both surfaces during the study period rather than teams that used one surface exclusively , ( 4 ) used the injury definition as described by fuller et al . , ( 5 ) reported both exposure times and injury occurrences for play on at and ng , and ( 6 ) written in english or an english translation .
the studies meeting our criteria were examined and exposure times , injury occurrences , and playing surface were recorded . in addition , the playing condition ( match or training ) , gender , and age ( youth or adult ) were noted .
chronic injuries were not considered as these could be attributed to both surfaces or to other causes .
acute knee injuries , ankle injuries , foot injuries , sprains , and muscle strains were recorded .
these specific locations and types of injuries were selected because they were the most consistently defined across all of the included studies .
crude injury rates were computed using the unweighted sum of total injuries and exposure times from all eight studies .
statistical analyses were performed on adjusted injury incidence rate ratios ( irrs , at / ng ) using the mantel - haenszel method for fixed effects .
the data were then categorized based on condition ( match or training ) , gender , and age ( youth or adult ) .
an initial literature search on pubmed returned more than 25 citations . of those , eight studies met our criteria .
these are shown in table 1 along with key characteristics of each study . as can be seen , individual studies varied in terms of the number of players and teams examined . in addition
, the period of investigation ranged from several months to several years resulting in varying exposure times .
three studies focused on either match or training injuries exclusively while five included both conditions .
five of the studies focused on adult players , generally college or professional players , whereas three studies focused on youth players that were 12 to 17 years of age .
of that , 571,196 hours were played on at ( 34.5% ) and 981,147 on ng ( 65.5% ) .
the amount of time spent in training and playing on at and ng varied between studies . in the study by soligard et al .
, players competed on at for less than 10% of the total time played . at the other end of the range , ekstrand et al .
studied players who trained and competed on at nearly three times as long as on ng .
matches accounted for 271,022 hours ( 18.1% ) of total exposure time whereas training accounted for 1,227,321 hours ( 81.9% ) .
of the total exposure time , males accounted for 949,568 hours ( 66.1% ) and females for 486,178 hours ( 33.9% ) .
did not distinguish between male and female players and was not included in the analysis of gender .
a total of 9737 injuries were recorded with 2670 occurring on at ( 27.4% ) and 7067 on ng ( 75.6% ) .
this resulted in crude injury incidence rates of 5.16 and 7.20 injuries per 1000 hours for at and ng , respectively .
crude incidence rates for matches were 20.26 and 24.45 injuries per 1000 hours for at and ng . for training injures on at and ng , the incidence rates were 2.91 and 2.68 injuries per 1000 hours .
figure 1 shows the injury irrs for each of the studies examined along with the overall adjusted irr of 0.86 ( 0.740.93 , p < 0.05 ) .
five of the eight studies showed irrs that were significantly lower than 1.0 , indicating a lower incidence rate on at [ 79 , 12 , 13 ] , while the other three showed nonsignificant differences .
the overall adjusted irr was statistically significant with a lower incidence rate on at .
ankle , and foot injuries as well as sprains , incidence rates were significantly lower on at compared to ng .
analyses of male , female , youth , and adult subcategories for all injuries showed irr values significantly lower than 1.0 . for knee injuries , incidence rates on at were lower for training injuries and adults .
for muscle strains , incidence rates were lower on at for match injuries , males , and both young and adult players . in no case was injury incidence increased on at .
for the entire data set , including all injuries as well as both conditions , gender and age ( figure 1 ) , the i value was 80% .
a portion of the heterogeneity was due to different injuries and locations . for specific injuries and sites ( figure 2 ) , i ranged between 43% and 74% . condition , gender , and age also contributed as i values for these categories ranged from 0 to 80% .
we found that variations in exposure to at accounted for some of the heterogeneity .
figure 3 shows a significant negative correlation between the relative amount of time spent on at ( at exposure / ng exposure ) and the irr for each study . as can be seen , the two studies in which the greater exposure time on at had the lowest irr .
this meta - analysis examined eight studies that compared soccer injury rates occurring on at and ng . in total , these studies report nearly 1.5 million hours of training and match play and almost 10,000 injuries .
the adjusted irr for all injures was significantly less than 1.0 indicating lower incidence rates for playing and training on at . for specific categories and specific injuries ,
thus , the overall results of our analyses do not support the idea that playing or training on at increases the risk of injury compared to ng .
in fact , our analyses suggest that at surfaces may reduce injury incidence for some types of injuries within specific categorizations .
first , despite the guidelines provided by fuller et al . , the eight studies varied in terms of their injury descriptions . for example , ekstrand et al . [ 8 , 9 ] reported the incidence of injuries based on location and type ( e.g. , knee sprains ) , whereas bjrneboe et al . , fuller et al .
[ 10 , 11 ] , soligard et al . , and steffen et al .
described injuries by location ( e.g. , knee ) or type ( e.g. , sprain ) . only ekstrand et al
. noted specific injuries such as anterior cruciate ligament tears . thus , our results are limited to the general classifications of location ( knee , ankle , and foot ) or type ( sprains and muscle strains ) .
second , in one study , exposure and incidence values for males and females were pooled .
third , the severity of injuries is categorized based on the number of training and match days missed .
for example , a minor or mild injury was defined as 17 days [ 7 , 10 , 11 , 13 ] , 47 days [ 8 , 12 ] , and 12 weeks missed .
given these discrepancies , we were not confident using severity as a subcategory for analysis . fourth , the studies failed to report environmental conditions such as temperature , wet / dry field conditions , or shoe type / cleat design . finally , only three of the studies differentiated between contact and noncontact injuries [ 10 , 11 , 13 ] .
this is an important limitation since contact injuries may or may not be directly attributable to playing surface .
our results do not provide an explanation for possible reductions in injury risk on at .
artificial turf surfaces provide a more consistent playing surface than ng , free of bare sports , ruts , and divots which could affect injury risk . however , the studies examined focused on teams competing at a high level , so it is likely that the ng surfaces were of high quality . traditionally , physical characteristics of at and frictional resistance at the shoe - surface interface have given rise to suggestions of greater injury risk on at . however , laboratory experiments yield conflicting results regarding this idea .
several studies show greater rotational torque between the shoe and at compared to ng [ 2 , 14 ] , whereas cawley et al .
further argue that stiffness ( the rate of change in torque ) may play a role in injury risk . yet
, greater peak torque on at does not result in increased stiffness . in human studies ,
ford et al . found different foot loading patterns during cutting maneuvers performed on at and ng . on the other hand ,
potthast reported no differences in rear foot and ankle movements of the plant foot when goal kicking between ng and rubber - filled at .
mcghie and ettema found that impact forces experienced during cutting maneuvers on at were not indicative of a hazardous condition . to further complicate this issue ,
torque and stiffness can be affected by the type of cleat worn on a particular surface as well as the temperature of the surface [ 2 , 14 ] .
thus , it is difficult to conclude with confidence which of the physical characteristics of at accounts for difference in injury incidence rates found in the present study .
match analyses show that the amount of time spent running at different intensities is similar between surfaces , but fewer slide tackles and shorter passes are executed on at .
the reduction in slide tackles may stem from the fear of skin abrasions but may limit the number of high risk
they feel that heat as well as speed of play on at requires greater physical effort compared to grass [ 18 , 19 ] .
however , these perceptions have been difficult to confirm as some controlled studies show increased energy expenditure while others show no differences in fatigue [ 2023 ] .
note that negative player perceptions of at are stronger in those who have limited exposure to at or are exposed to at later in their careers .
figure 3 suggests that more time spent in training and competing on at is associated with lower injury risk .
perhaps with increased exposure to at , players develop a less aggressive play with fewer slide tackles that reduces injury risk .
it should be pointed out , however , that this idea is speculative at this time .
clearly more research is needed regarding player movements , energy expenditure , and fatigue on at and ng surfaces .
this study highlights several areas that need to be addressed before firm conclusions can be drawn .
first , three of the eight studies focused only on male players [ 6 , 7 , 9 ] .
given the concern over the high incidence of acl injuries in women , it seems reasonable to suggest that more information is needed regarding playing surface as a potential risk factor in females .
fourth , more studies are needed on energy expenditure and fatigue on at and ng , especially using activities that replicate match play . given that fatigue can increase the risk of joint injury , it is important to understand if player perceptions of increased effort on at have a physiological basis .
fifth , while preliminary studies suggest that players adjust their style of play on at , it is not clear if this occurs across levels of play and across genders .
identifying specific movement patterns that either increase or decrease injury risk on at could impact player training .
as the use of at in club and school settings increases , a thorough understanding of safety and injury risks in youth and adolescent players is essential . in this investigation
, we found no evidence that playing matches or training on at raises the risk of soccer players sustaining injury .
in fact , the evidence suggests that the risk of some injuries and some subgroups might be lowered .
however , until more is known about how issues such as altered playing styles affect injury incidence , it is difficult to make firm conclusions regarding the direct and indirect roles of at in player safety . | the goal of this investigation was to determine if playing or training on third - generation artificial turf ( at ) surfaces increases the incidence rate of injuries compared to natural grass ( ng ) surfaces .
this was accomplished by a meta - analysis performed on previously published research .
eight studies met the criteria of competitive soccer players , participation on both surfaces , and presentation of both exposure time and injury occurrence .
exposure time and injury incidence values were used to generate injury rate ratios ( irrs , at / ng ) for all injuries as well as specific injuries .
subgroup analyses were also performed by condition ( match or training ) , gender , and age ( youth or adult ) .
the overall irr was 0.86 ( p < 0.05 ) suggesting a lower injury risk on at than ng .
however , there was considerable heterogeneity between studies .
analyses of individual injuries and subgroups found that in many cases irr values were significantly less than 1.0 . in no case was the irr significantly greater than 1.0 . based on this
, it appears that the risk of sustaining an injury on at under some conditions might be lowered compared to ng .
however , until more is known about how issues such as altered playing styles affect injury incidence , it is difficult to make firm conclusions regarding the influence of at on player safety . |
self - organized tio2 nanotube
layers have attracted remarkable
attention within the past 15 years due their unique architecture ,
high surface area , semiconductive properties , and biocompatibility .
in addition , they are produced by a low - cost electrochemical anodization
of ti substrates in suitable electrolytes containing fluorides .
all
these features enabled utilization of tio2 nanotube layers
in a wide number of applications as photocatalyst , anode
of dye - sensitized solar cells ( dssc ) and perovskite
solar cells ( psc ) , gas sensors , and biomedical materials , among others .
in all these applications ,
tio2 nanotube layers have shown
superior performance compared to other tio2 nanostructures .
in parallel , important efforts have been carried out to tune the nanotube
aspect ratio and to improve the nanotube ordering and crystallinity .
annealing treatment of amorphous as - synthesized tio2 nanotube layers leads to their crystallization into anatase
( > 280 c ) , a combination of anatase and rutile ( > 450 c ) ,
or rutile ( > 550 c ) .
the anatase nanotubular
structure has shown to be more favorable
than rutile for photoelectrochemically assisted applications , such
as photocatalysis and dssc .
thus , the stability of anatase nanotubular structure is highly desired ,
and numerous efforts have been focused on this target , especially
at high temperatures .
the introduction of alloying elements as nb or c was reported
to induce a shift of the anatase to rutile transition ( further noted
as art ) to higher temperature and increased thermal resistance against
collapse .
however , the main disadvantage of alloying is the formation
of undesired secondary impurity phases , e.g. , ti nb2o5 .
the art threshold depends
on whether the nanotube layers are attached or separated from the
ti substrate .
high temperature stability ( up to 700 c ) of tio2 nanotube arrays , preserving the nanotubular integrity and
anatase structure , was reported for free - standing tio2 nanotube
arrays . for tio2 nanotube
layers attached to ti substrate , anatase structure and no structural
collapse
is maintained either by a previous solvothermal
treatment or previous annealing at lower
temperature .
the highest published temperature
without nanotube collapse ( 1048 c ) was reached during
the flame annealing process . however ,
such flame high temperature processing led to undesired transition
to rutile structure and a significant uptake of carbon from the flame .
despite numerous efforts focused onto the high temperature stability
of tio2 nanotube layers , the temperature working window
is still restricted .
another constrain for applications of tio2 nanotube layers is their limited chemical stability in harsh
acidic environments , where nanotube layers undergo chemical dissolution .
the improvement of the thermal , chemical , and eventually also mechanical
properties of tio2 nanotube layers would enable their utilization
in previously nonimaginable working environments and surely interesting
expansion of their application range . in principle ,
addition of a
thin continuous coating of an appropriate secondary material ( with
excellent thermal and chemical stability ) within nanotubes should
significantly alter also their stabilities .
so far , however , no such
treatment has been shown . to date , the atomic layer deposition ( ald )
technique is the only method that enables homogeneous , continuous ,
and conformal coating of secondary materials into tio2 nanotube
layers .
deposition of al2o3 and zno coating by ald into tio2 nanotube layers has been reported
yielding interesting synergic effects .
the resulting composite heterostructures
revealed significant improvement of their photovoltaic and photocatalytic
performance due to enhanced charge separation induced by coatings
of secondary materials .
therefore , in the present work we investigated
the thermal , chemical ,
and mechanical properties of self - organized tio2 nanotube
layers uniformly coated with al2o3 layers of
different nominal thicknesses : 1 , 10 , and 42 nm .
these coatings were
carried out by atomic layer deposition ( ald ) using different number
of identical deposition cycles .
after the al2o3 coating , the tio2 nanotube layers were annealed at temperature
up to 870 c for 1 h to evaluate their thermal stability .
the
resulting crystal structure and composition were analyzed through
x - ray diffraction ( xrd ) and scanning transmission electron microscopy
( stem ) .
mechanical properties ( hardness ) were characterized by nanoindentation
measurements using an atomic force microscope .
the chemical stability
was tested by soaking the al2o3-coated tio2 nanotube layers into h3po4 solutions
of different concentrations for 48 h at laboratory temperature and
for an additional 8 h in solutions with temperature of 60 c .
self - organized
tio2 nanotube layers with a thickness
of 20 m and a nanotube diameter of 110 nm ( aspect
ratio 180 ) were fabricated via anodization of ti foils using
a previously published approach .
prior
to anodization , the ti foils ( sigma - aldrich , 0.127 mm thick , 99.7%
purity ) were degreased by sonication in isopropanol and acetone , then
rinsed with isopropanol , and dried in air . the electrochemical setup
consisted of a two - electrode configuration using a platinum foil as
the counter electrode , while ti foils ( working electrodes ) were pressed
against an o - ring of the electrochemical cell , leaving 1 cm open to the electrolyte .
a high - voltage potentiostat ( pgu-200 v ,
ips elektroniklabor gmbh ) was employed to carry out the electrochemical
experiments at room temperature .
ethylene glycol containing 1.5 vol
% deionized water and 176 mm nh4f was used as electrolyte .
electrolytes were aged before the first use for 15 h by anodization
of blank ti foils at 60 v under the same conditions for the anodization
experiments
ti foils were anodized for
4 h after sweeping the potential from 0 to 60 v with a sweeping rate
of 1 v / s .
the tio2 nanotube layers
by were coated with al2o3 by atomic layer deposition
tool ( thermal ald ,
tfs 200 , beneq ) .
this technique based on sequential and self - limiting
gas surface reactions allows conformal deposition of various
coatings within tio2 nanotube layers with a nanometer scale
accurate thickness , as shown previously .
. grade , 99.999+% ) and
deionized water ( 18 m ) were used as aluminum and oxygen precursors ,
respectively . under these deposition conditions ,
one growth ald cycle
was defined by the following sequence : tma pulse ( 1 s)n2 purge ( 3 s)h2o pulse ( 1 s)n2 purge ( 3 s ) .
all processes were carried out at a temperature
of 200 c and using n2 ( 99.9999% ) as carrier gas at
a flow rate of 400 standard cubic centimeters per minute ( sccm ) .
al2o3 deposition was carried out running 8 , 88 , and
366 ald cycles , leading to coatings of different nominal thicknesses :
1 , 10 , and 42 nm , respectively .
the number of cycles required for
the different al2o3 thicknesses was estimated
from the growth per cycle value of the al2o3 process at 200 c ( 1.1 / cycle ) .
the thicknesses
of al2o3 coatings were confirmed by variable
angle spectroscopic ellipsometry ( vase ellipsometer , j.a .
woollam ,
co. , inc . ) of al2o3 coatings on si wafers . upon the al2o3 coating process
the annealing process was carried out in a muffle oven in
an air atmosphere applying a heating rate of 15 c / min , until
the target temperature ( 870 c ) was reached .
the annealing process
proceeded at such temperature for 1 h. afterward , the layers were
allowed to naturally cool down .
the morphology of the tio2 nanotube layers was characterized
by a field - emission sem ( fe - sem jeol jsm 7500f ) and a scanning transmission
electron microscope ( stem , fei tecnai f20 x - twin ) fitted with a high
angle annular dark field ( haadf ) detector and operating at 200 kv .
the cross - sectional views were obtained from mechanically bent samples .
because of the rupture of the nanotube layers via this bending , it
was possible to visualize nanotubes within the layers and coatings
within nanotubes in various directions and nanotube layer depths .
average values and standard
deviations were calculated from at least three different locations
with a high number of measurements ( n > 100 ) .
diffraction analyses of the al2o3-coated
tio2 nanotube layers carried out using x - ray diffractometer
( xrd , d8 advance , bruker axe ) using cu k radiation with secondary
graphite monochromator and na(tl)i scintillation detector .
nanoindentation
measurements were performed to analyze the mechanical
properties ( hardness ) of the tio2 nanotube layers .
they
were determined by an atomic force microscope ( afm , solver next , nt
mdt ) equipped with a nanoindentation head ns01ntf and a berkovich
type of tip ( three - sided pyramid geometry with a parameter of static
stiffness , k = 10.2 0.3 kn / m ) .
the nanotube
layers were measured in longitudinal direction for compressive force
of 0.5 mn , loaded for 100 s. fused silica sio2 was used
as a calibration sample ( hardness , h = 9.5
0.5 gpa by iso 9450 - 76 ) .
the penetration depth of the tip was up to
a maximum 10% of the total thickness of the nanotube layer the hardness
was evaluated at 30 different areas of each nanotube layer
to ensure statistically relevant data set / significant results .
the chemical stability of al2o3-coated tio2 nanotube layers was analyzed by soaking in h3po4 solutions of different concentrations : 25 , 50 , 70 , and 85
wt % ( prepared from 85 wt % h3po4 , penta ) .
tio2 nanotube layers were soaked in these solutions for 48 h ,
including a thermal treatment for 8 h by which solutions were heated
up at 60 c to further study the nanotube chemical stability
under warm acidic conditions . before the subsequent sem analysis
highly ordered tio2 nanotube layers , with
a thickness of 20 m and an average diameter value of
110 nm ( aspect ratio 180 ) , were prepared by anodization
of ti foils as described in detail in the experimental
section . as - prepared amorphous tio2 nanotube layers
were coated with al2o3 of different nominal
thicknesses , namely 1 , 10 , and 42 nm by ald , as verified by sem and
ellipsometric measurements ( 1.1 0.2 , 10 0.5 , and 44
2.1 nm ) .
freshly coated nanotube layers were annealed at 870
c for 1 h along with reference uncoated tio2 nanotube
layers .
figure 1 shows
sem images of the tio2 nanotube layers with and without
al2o3 coating annealed at 870 c .
uncoated
tio2 nanotube layers ( figure 1a ) collapsed during the annealing process
into a pillar nanostructure ( figure 1b ) .
when coated , the nanotube layers were preserved
after the annealing process , regardless of the thickness of the al2o3 , as apparent for coatings of either 1 nm ( figure 1c , d ) , or 10 nm ( figure 1e , f ) .
it is quite
fascinating that even 1 nm thin al2o3 coating
can built a very thermally robust cage all over tio2 nanotubes
with some 2040 nm thick tube walls .
sem top - view images of
uncoated tio2 nanotube layer
( a ) before and ( b ) after annealing ; al2o3 coated
( 1 nm ) tio2 nanotube layer ( c ) before and ( d ) after annealing ;
al2o3 coated ( 10 nm ) tio2 nanotube
layer ( e ) before and ( f ) after annealing .
the annealing was carried
out at 870 c for 1 h. insets : magnification of the corresponding
sem images .
all the scale bars denote 100 nm . figure 2 shows
representative
stem - haadf images of the nanotube body ( separated from the annealed
al2o3 coated ( 10 nm ) tio2 nanotube
layer by mechanical bending of the layers followed by sonication in
methanol ) at a low ( a ) and at a high magnification ( b ) . especially
from figure 2b , the
interface between the tio2 wall and al2o3 coating is well distinguishable .
there are actually two interfaces
between the tio2 wall and al2o3 coating ,
as the al2o3 coating is deposited inside ( interior
coating ) and outside ( exterior coating ) the tio2 tube walls .
this feature is in accordance with our previous ald work , where we showed very good uniformity of al2o3 coatings on the amorphous tubes and absence
of any pinholes in the coating . as apparent from figure 2
, al2o3 coatings remained continuous and pinhole - free even after annealing ,
during which thermally induced crystallization of tio2 tube
walls occurred .
some delamination of the coating seen at the outer
and inner interface between tio2 wall and al2o3 coating ( especially at figure 2b ) stems most likely from the stress that
these layers are exposed to during the preparation of specimens for
sem and stem observation , which includes mechanical rupture of layers .
these roughening and delamination events have no detrimental effect
on coated nanotube layers that were not submitted for sem and stem
and that completely survived soaking in h3po4 solutions ( described later in text ) .
representative stem - haadf
images of ( a ) a fragment of al2o3 coated ( 10
nm ) tio2 nanotube and ( b ) the
corresponding higher magnification of the nanotube wall . interfaces
between individual parts of the tubes are distinguished by solid lines
and appropriate description . it is
generally accepted that the annealing process influences crystal structure ,
phase transition , and structural integrity of the tio2 nanotube
layers .
it has also been accepted that amorphous as - prepared
tio2 nanotube layers crystallize into anatase above 280
c in air .
the anatase to rutile
transition ( art ) has been reported at different temperatures , most
usually in the range of temperature between 500 and 600 c , depending
on the nanotube dimensions ( diameter , thickness , and composition ) .
annealing at temperatures higher 600 c leads to the
coexistence of anatase and rutile structures , while total conversion
to rutile structure takes place above 800 c .
figure 3a shows the
xrd pattern obtained for reference uncoated tio2 nanotube
layers annealed for 1 h at either 400 or 870 c , respectively .
in line with literature , the former exhibits pure anatase crystal
phase identified by typical anatase peaks associated with planes ( 101 ) ,
( 004 ) , ( 105 ) , and ( 211 ) , with a dominant orientation ( 101 ) .
the latter
reveals pure rutile crystal phase with well - defined diffraction peaks
of planes ( 110 ) , ( 011 ) , ( 111 ) , ( 211 ) , and ( 220 ) .
the intensity of
the peak at 2 = 27.4 indicates a preferred orientation
along the ( 110 ) direction ; no trace of anatase polymorphic phase is
detected .
xrd patterns of ( a ) uncoated tio2 nanotube layers annealed
at 870 and 400 c for 1 h ;
( b ) al2o3-coated
tio2 nanotube layers with different coating thicknesses
( 1 , 10 , and 42 nm ) annealed at 870 c for 1 h ; and ( c ) al2o3-coated ( 1 and 10 nm ) tio2 nanotube
layers preannealed ( 400 c , 1 h ) and second annealing at 870
c . a = anatase , r = rutile , and t = titanium substrate . the xrd patterns , obtained from
al2o3-coated
tio2 nanotube layers annealed at 870 c for 1 h , are
shown in figure 3b .
therein the coexistence of anatase and rutile structures can be clearly
seen , in contrast to the uncoated nanotube layers ( figure 3a ) where rutile was exclusively
formed at this temperature , in line with the previous work .
the incomplete art of coated tio2 nanotube layers annealed at 870 c stems from the hindered
surface reconstruction of the tio2 due to al2o3 coating and also the impact of al as the
phase transformation inhibitor . in contrast ,
for uncoated nanotube layers ( annealed at 870 c ) the surface s
reconstruction can easily take place ( no space constrains are present ) ,
allowing for the mass flow and rearrangements that yield complete
rutile conversion , expected at this temperature .
another evidence for limited reconstruction of coated tio2 nanotube layers annealed at 870 c is the fact that
they do not collapse ( sinter ) , which they would otherwise do without coating . in the case of thermally
stable coated nanotube layers , the coating acts in similar fashion
as it does for nanoparticles and nanorods that can be annealed , when
coated , at high temperatures without undergoing sintering events .
quantification of the content of each crystal phase and an average
of the corresponding crystallite size are given in table 1 .
the anatase : rutile ratio was found
to be dependent on the al2o3 coating .
the nanotube
layer with the thickest
al2o3 coating ( 42 nm ) exhibited dominant rutile
structure ( 62% ) with peaks corresponding to the planes ( 110 ) , ( 001 ) ,
( 111 ) , ( 211 ) , and ( 220 ) and only one minor anatase peak corresponding
to ( 101 ) plane .
in clear contrast , the anatase content for 10 and
1 nm thin al2o3 coating was found to increase
up to 73% and 83% , respectively , on account of rutile . in addition ,
identical rutile peaks were revealed for them as for the nanotube
layer with thickest al2o3 coating ( 42 nm ) .
it
is noteworthy that the 1 nm thin al2o3 coated
nanotube layer revealed anatase peaks with preferential orientation
along the ( 004 ) plane , instead of usual ( 101 ) plane .
who associated a particular thermal stability of the nanotube layers
to such anatase ( 004 ) plane . based on the xrd results in figure 3b ,
there is obvious
retardation of the anatase to rutile transition ( further noted as
art ) with decreasing thickness of al2o3 coatings
( 1 and 10 nm ) . in order to give a physical description of the
results , factors
affecting the art
first , we should consider
the influence of the number of oxygen vacancies within the tio2 on the art temperature .
reported that the larger is the number of oxygen vacancies within
tio2 , the lower is its art temperature , or the art does
not proceed at all and tio2 remains in anatase form .
the
defects ( in this case oxygen vacancies ) provide a low energy mass
transport route and lower such art temperature .
the number of oxygen vacancies within tio2 is
also strongly influenced by the annealing atmosphere .
previous works
clearly indicated that dry annealing atmospheres such as ar or co led to more
oxygen vacancies in tio2 than those performed in o2 or air .
thus , the art was enhanced in oxygen - free atmospheres
and resulted into larger rutile crystallites .
second , it is important to define the fundamental reasoning
behind
the art origin , which has been subject of controversy .
tio2 nanotube interface , where ti metal
would be directly thermally oxidized into rutile structure .
the presence
of oxygen vacancies in between the ti substrate and tio2 nanotube layer was believed to induce the art at such interface ,
spreading toward the whole nanotube walls in the course of time , as
reported by zhu et al . in contrast , yu
et al .
proposed that art does not stem
from metal ti , but from the anatase ( created at lower temperatures ,
while ramping up the temperature ) at the interface between tio2 nanotubes and ti substrate , which converts to rutile at temperatures
600 c .
in addition , works on annealing and crystallization
of free - standing nanotube layers ( i.e. , nanotubes
were detached from the ti substrate before annealing ) reported both
the preservation of the anatase structure in the nanotube walls at
temperatures higher than 600 c and much higher triggering art
temperature .
those results would point on a significant role of metal
ti substrate tio2 nanotube interface on the art .
there is a clear link between the experimental results obtained
in this work and the literature about factors affecting the art . according to figure 3 , the anatase : rutile ratio is clearly dependent on the al2o3 coating thickness .
that larger number of oxygen vacancies
promotes the art , it is clear that the al2o3 coating within our tio2 nanotube layers influences the
number of oxygen vacancies as it possesses a barrier against the oxygen
diffusion .
for example , the thickest
al2o3 coating ( 42 nm ) hinders the oxygen diffusion
most significantly from all used coatings in this work , leads to highest
number of oxygen vacancies within tio2 nanotubes , and boosts
the art process that ends up with the highest rutile content . in contrast
,
the oxygen diffusion into tio2 takes place more easily
through thinner al2o3 coatings ( 1 and 10 nm ) ,
resulting in a lower number of oxygen vacancies , retarding the art
within tio2 nanotubes .
in addition , the largest rutile
crystal size , calculated by the scherrer equation ( table 1 ) , corresponds to the thickest
al2o3 coating , which also corroborates previously
published findings on art and size of rutile crystals . to get a complete picture about the art
, we also fully explored
anatase tio2 nanotube layers ( annealed at 400 c for
1 h ) , shown in figure 3a , for al2o3 coating .
we coated these nanotube
layers with 1 and 10 nm of al2o3 by ald , before
undergoing a second thermal treatment at 870 c for 1 h. first ,
the figure 3c shows
that the al2o3-coated ( 10 nm ) tio2 nanotube layer consisted of 100% rutile , while in the al2o3-coated ( 1 nm ) tio2 nanotube layer rutile
content was only 26% .
these results confirm the active role of the
al2o3 coating for tio2 crystal structure
and are in line with the results and theories discussed in figure 3b .
second , the significantly
different crystal structure of the both investigated types of al2o3 ( 10 nm ) tio2 nanotube layer were
revealed . the formerly annealed al2o3 coated
( 10 nm ) tio2 nanotube layer ( fully anatase comprised ) underwent
a complete art and was 100% rutile comprised ( see figure 3c ) .
in contrast , the initially
amorphous tio2 nanotube layer revealed a predominant anatase
content of 74% ( see figure 3b ) .
this comparison clearly confirms that the tio2 structure influences the art and that it is clearly promoted
for the tio2 nanotube layers annealed to anatase before
art and ald coating . in other words , the lack of coating induces during
the thermal annealing to 400 c more oxygen vacancies in the
tio2 nanotube layers than it does when coatings are present
during this annealing step .
the mechanical
integrity of the
tio2 nanotube layers is of significant importance , especially
for synthesis of devices based on flow - through membranes utilizing
nanotube layers opened on both sides .
even though some nanoindentation analyses of the tio2 nanotube arrays were already carried out , nanotube layers modified with additional coatings , as in the present
case , have not yet been analyzed .
figure 4 shows hardness of tio2 nanotube
layers with al2o3 coatings of different thicknesses
as well as two reference nanotube layers .
if not denoted otherwise ,
all nanotube layers were annealed at 870 c for 1 h as the last
processing step .
first , the uncoated amorphous ( i.e. , did not undergo annealing ) tio2 nanotube layer displayed lower hardness value than the annealed
uncoated counterpart , fully rutile structure comprised . that was expected
as the crystal structure has ( as a rule of thumb ) higher hardness
than amorphous mass of the same compound .
second , the annealed al2o3-coated tio2 nanotube layers exhibited
larger hardness with the increasing al2o3 coating
thickness .
this can be ascribed to increasing content of rutile ( table 1 ) and to an increasing
al2o3 mass within the nanotubes .
,
all nanotube layers were annealed at 870 c for 1 h. even though rutile and anatase are similar in structure ,
the reason
to rutile to be more mechanically robust than anatase is that its
octahedra shares four edges instead four corners ( anatase case ) , which
leads to the formation of chains arranged subsequently in a 4-fold
arrangement .
this also explains why the al2o3-coated ( 1 nm ) tio2 coated layer ( which
has mainly anatase structure as shown in figure 3b ) has lower hardness than uncoated annealed
layers ( completely rutile based , also shown in figure 3a ) .
the hardness values presented here
for high aspect ratio ( 180 )
nanotube layers were larger than those found in the literature that
reports typical hardness in the range from 94 mpa to 3.5 gpa .
however , it is difficult to establish a comparison , principally
because the published reports show results for exclusively uncoated
and lower aspect ratio tio2 nanotubes layers with thicknesses
from 625 nm to 8.5 m ( compared to 20 m in the
present case ) .
moreover , the use of different indenter tips ( vickers
tips for microhardness , berkovitch tips for nanohardness , cube corner
tips for nanohardness , etc . ) entails different hardness values .
nevertheless ,
it can be concluded that both the annealing treatment and the al2o3 ald coating resulted in a substantial enhancement
of the mechanical properties of tio2 nanotube layer .
finally , the chemical stability
of al2o3-coated tio2 nanotube layers
was investigated in strongly acidic environment , namely in h3po4 solutions with different concentration .
such stability
is significant for the nanotube layers to sustain in environments ,
where up to now they could not preserve their morphological integrity .
for example , in various biological environments with low ph , the knowledge
about the stability threshold is important .
as it can be seen in figure 5 , even the thinnest
coating ( 1 nm al2o3 ) completely preserved the
tio2 nanotube layers from degradation in concentrated h3po4 . in line with that no degradation was observed
for any of the thicker al2o3 coatings : 10 and
42 nm ( data not shown here ) .
the soaking tests were performed on the
40 h time scale for all nanotube layers .
this expands the already wide range
of environments , where ald al2o3 coatings are
stable , in addition to published stability results of these coatings
in various acidic ( h2so4 , hno3 , hcl )
and alkaline ( koh ) environments and water . to make the h3po4 environment
even more harsh ,
the h3po4 solutions were heated
up to 60 c , and soaking was carried out for an additional 8
h ( in total 48 h ) . since again no visible changes were observed , soaking
experiments were terminated afterward .
reference uncoated layers ( namely
as - anodized amorphous and annealed ( 400 c for 1 h ) nanotube
layers ) did not survive these conditions . in order to determine the
chemical threshold conditions for these reference uncoated layers ,
lower h3po4 concentrations had to be used .
the
stability threshold was revealed to be 10 wt % ( figure 5e ) and 40 wt % ( figure 5f ) on the scale of 24 h for the amorphous
and annealed case , respectively , without any heating .
all in all ,
the results presented in figure 5 for al2o3-coated nanotube layers
confirm the outstanding enhancement of the chemical stability of tio2 nanotube layers provided by uniform al2o3 coatings .
sem top - view images of annealed al2o3-coated
( 1 nm ) tio2 nanotube layers before ( a ) and after soaking
in h3po4 solutions with different concentrations :
( b ) 50 wt % , ( c ) 70 wt % , and ( d ) 85 wt % in total for 48 h ( last
8 h at 60 c ) .
sem top - view images of reference uncoated amorphous
( e ) and anatase ( f ) tio2 nanotube layers after soaking
in h3po4 solutions of 10 and 40 wt % , respectively ,
for 24 h. all the scale bars denote 100 nm .
thus , the present results , especially for the thinnest al2o3 coating ( 1 nm ) , are very useful and promising
for practical
applications of the nanotube layers .
as - treated tio2 nanotube
layers : ( i ) maintain anatase structure ( more favorable for photovoltaics
and photocatalysis than rutile ) in the tubes over a very broad temperature
range , ( ii ) possess significantly improved charge separation on the
interface with various electrolytes ( especially because electrons
can tunnel to tio2 via al2o3 coatings
thinner than 2 nm ) , ( iii ) possess
strong mechanical integrity , and ( iv ) provide extremely good stability
in strongly acidic environments .
all these features pave favorable
way for the functionalization of tio2 nanotube layers by
secondary materials .
it is foreseen that additional materials , such
us various oxides , nitrides , sulfides , etc .
thermal
and chemical stability of al2o3-coated tio2 nanotube layers can extend the utilization of nanotube layers
for catalytic applications and sensing of gases ( such as co , nox , ch3ch2oh , h2 , and o2 ) at high temperatures and/or in harsh acidic
environment , so far unfeasible for uncoated tio2 nanotube
layers counterparts . in parallel , membranes composed of ultrahigh
aspect ratio tio2 nanotube layers that have been used for
photocatalytic or flow - through experiments may be prone to mechanical instabilities .
thus , they could greatly
benefit from a thin al2o3 coating to become
mechanically more robust .
in
this work , effects of al2o3 coating produced
by ald on the crystal structure , mechanical , and chemical properties
of tio2 nanotube layers were explored .
noteworthy improvement
of the thermal stability upon annealing in air was revealed up to
temperatures of 870 c , even with an extremely thin al2o3 coating ( 1 nm ) .
in contrast to uncoated tio2 nanotube layers ( 100 vol % rutile ) , a high fraction of anatase structure
( 83 vol % ) was determined for al2o3-coated ( 1
nm ) tio2 nanotube layers upon annealing at 870 c ,
which is highly desired due to its optical and electronic properties
for photovoltaic and photocatalytic applications .
an enhanced hardness
was revealed for al2o3-coated tio2 nanotube layers with a positive impact on the mechanical properties
of nanotube layers .
in addition , al2o3 coatings
provided to the tio2 nanotube layers extremely good stability
in extremely acidic environments of h3po4 solutions
with different concentrations .
all in all , self - organized tio2 nanotube layers coated with thin al2o3 coatings yield superior thermal , chemical , and mechanical stabilities
that will extend their application range to previously nonimaginable
working environments . | we report on a very
significant enhancement of the thermal , chemical ,
and mechanical stability of self - organized tio2 nanotubes
layers , provided by thin al2o3 coatings of different
thicknesses prepared by atomic layer deposition ( ald ) .
tio2 nanotube layers coated with al2o3 coatings
exhibit significantly improved thermal stability as illustrated by
the preservation of the nanotubular structure upon annealing treatment
at high temperatures ( 870 c ) .
in addition , a high anatase content
is preserved in the nanotube layers against expectation of the total
rutile conversion at such a high temperature .
hardness of the resulting
nanotube layers is investigated by nanoindentation measurements and
shows strongly improved values compared to uncoated counterparts .
finally , it is demonstrated that al2o3 coatings
guarantee unprecedented chemical stability of tio2 nanotube
layers in harsh environments of concentrated h3po4 solutions . |
metaplastic ossification within atypical lipomatous tumor / well - differentiated liposarcoma ( alt / wdlps ) is a rare occurrence , and when present is often associated with a dedifferentiated component .
we report the radiologic and pathologic features of a rare case of alt / wdlps with extensive metaplastic ossification , however without an element of dedifferentiated liposarcoma ( ddlps ) .
however , a large densely calcified mass was incidentally seen in the soft tissues overlying the right scapula ( fig .
the patient was unaware of the mass and denied trauma to the site , focal or asymmetric pain , weight loss , other constitutional symptoms , or known history of malignancy .
the possibilities of heterotopic ossification , myositis ossificans , tumoral calcinosis , and soft tissue sarcoma were raised ; the patient was thus referred for further clinical and radiographic evaluation .
figure 1radiograph of the right shoulder shows a large mass with extensive calcific density ( arrow ) overlying the right scapula .
radiograph of the right shoulder shows a large mass with extensive calcific density ( arrow ) overlying the right scapula .
a computed tomography ( ct ) scan of the chest , abdomen and pelvis , technetium-99 m - methylene diphosphonate [ tc]mdp ) bone scan , and magnetic resonance imaging ( mri ) were performed .
ct scan demonstrated a large 13 8 5 cm well - circumscribed mass with a thin capsule medially located in the right posterior shoulder , superficial to the scapula .
centrally the mass revealed dense , cloud - like calcification , which made up the bulk of the lesion .
this was surrounded by a rim of mostly simple appearing fat . associated thinning and irregularity of the underlying scapula were present , and were thought to be due to pressure erosion ( fig .
[ tc]mdp bone scan demonstrated a large amount of activity associated with the primary mass .
an additional focus of tracer accumulation in the right posterior seventh rib corresponded to a remote , healing rib fracture ( fig .
3 ) . mri demonstrated a large , lobulated , circumscribed mass adjacent to the rotator cuff musculature , displacing the infraspinatous and teres minor .
a large central area of hypointensity on both t1 and t2 sequences corresponded to areas of dense calcification seen on radiograph and ct .
a surrounding rim of fatty tissue showed minimal internal septation and hyperintense signal on t1-weighted imaging ( fig .
post - contrast t1 fat - saturated images revealed thin linear areas of enhancement traversing both the calcified and fatty component ( fig .
4b ) , with suppression of hyperintense signal of the fatty component .
figure 2axial ct scan of the right shoulder in ( a ) bone and ( b ) soft tissue windows reveals the mass in question .
the bone window reveals a central cloud - like area of high density , consistent with ossification ( arrow ) .
thinning and irregularity of the adjacent scapula , likely secondary to chronic pressure erosion ( arrow head ) is also present .
the soft tissue window reveals to advantage the encapsulated fatty component at the periphery of the mass ( arrow ) .
figure 3posterior view from a [ tc]mdp bone scan reveals intense tracer accumulation within the primary tumor , most intensely in the central area of ossification ( arrow ) .
a remote , healing seventh rib fracture was seen incidentally ( arrow head ) .
figure 4(a ) oblique coronal t1-weighted mri of the right shoulder reveals a large mass overlying the right scapula and displacing the infraspinatous and teres minor .
the central area of signal dropout corresponds to the ossified portion of the mass ( arrow ) , and the surrounding area of hyperintensity is consistent with fat ( arrowhead ) .
( b ) oblique coronal post - contrast t1-weighted mri with fat saturation demonstrates the area of signal drop out within the tumor corresponding to fat ( arrowhead ) .
minimal linear enhancement extending throughout the mass and faint enhancement of the central ossified portion ( arrow ) is seen .
( d ) axial post - contrast t1-weighted mri with fat saturation demonstrates minimal linear enhancement extending throughout the mass and faint enhancement of the central ossified portion ( arrows ) .
axial ct scan of the right shoulder in ( a ) bone and ( b ) soft tissue windows reveals the mass in question .
the bone window reveals a central cloud - like area of high density , consistent with ossification ( arrow ) .
thinning and irregularity of the adjacent scapula , likely secondary to chronic pressure erosion ( arrow head ) is also present .
the soft tissue window reveals to advantage the encapsulated fatty component at the periphery of the mass ( arrow ) .
posterior view from a [ tc]mdp bone scan reveals intense tracer accumulation within the primary tumor , most intensely in the central area of ossification ( arrow ) .
( a ) oblique coronal t1-weighted mri of the right shoulder reveals a large mass overlying the right scapula and displacing the infraspinatous and teres minor .
the central area of signal dropout corresponds to the ossified portion of the mass ( arrow ) , and the surrounding area of hyperintensity is consistent with fat ( arrowhead ) .
( b ) oblique coronal post - contrast t1-weighted mri with fat saturation demonstrates the area of signal drop out within the tumor corresponding to fat ( arrowhead ) .
minimal linear enhancement extending throughout the mass and faint enhancement of the central ossified portion ( arrow ) is seen .
( d ) axial post - contrast t1-weighted mri with fat saturation demonstrates minimal linear enhancement extending throughout the mass and faint enhancement of the central ossified portion ( arrows ) .
based on the imaging , the differential diagnosis for the mass included both benign and malignant entities .
benign entities included metastatic calcification , benign lipoma with calcified fat necrosis , variants of benign lipoma such as osteolipoma and chondrolipoma , parosteal lipoma , and myositis ossificans ; malignant entities included extraskeletal osteosarcoma and liposarcoma with osteosarcomatous dedifferentiation .
the patient was subsequently referred for percutaneous biopsy , and samples of both the calcified and non - calcified portions of the mass were obtained .
ct - guided core - needle biopsy of the right upper back mass was performed using a 13.5-gauge introducer , an 18-gauge 15-cm temno biopsy needle and lateral to medial approach .
four core biopsies were obtained through the soft tissue component of the mass ; a single core was obtained through the calcified portion of the mass using a bonopty drill advanced though the introducer .
tumor seeding along a needle path is sufficiently rare as not to be an issue in clinical practice for sarcomas .
histologically , the peripheral , non - calcified portion of the mass consisted of adipose tissue with slight variation in adipocyte size and occasional hyperchromatic stromal cells that were focally positive for mdm2 and cdk4 by immunohistochemistry ( fig . 5 ) .
alt / wdlps contains ring and giant marker chromosomes composed of amplified material from the long arm of chromosome 12 ( 12q1315 ) , which leads to overexpression of both mdm2 and cdk4 .
immunohistochemistry or fluorescence in situ hybridization ( fish ) can therefore be used to confirm the diagnosis , because benign lipomatous tumors do not harbor these genetic abnormalities .
( a ) non - ossified adipocytic component showing slight variation in adipocyte size , thickened fibrous septum , and occasional hyperchromatic cells .
( c ) immunohistochemical staining for mdm2 showed positive nuclear staining , confirming the diagnosis .
( a ) non - ossified adipocytic component showing slight variation in adipocyte size , thickened fibrous septum , and occasional hyperchromatic cells .
( c ) immunohistochemical staining for mdm2 showed positive nuclear staining , confirming the diagnosis . with the diagnosis of alt , the patient went on to full surgical excision for definitive treatment .
the lesion was easily excised from the surrounding soft tissues , and no bony invasion was seen intra - operatively .
gross examination revealed a mass that was over 95% ossified , with the remainder being composed of lobulated adipose tissue .
no adjuvant therapy was recommended , although due to the unusual finding of metaplastic ossification , the patient was closely followed clinically and radiographically . approximately 3 years after resection , the patient remains without evidence of local recurrence or distant metastasis .
lipogenic tumors , both benign and malignant , are common entities that have been reported throughout the body , accounting for nearly 50% of soft tissue neoplasms clinically encountered .
both benign and malignant lipogenic tumors have varying radiologic presentations with significant overlap in appearance ; final diagnosis is often made by genetic and histologic analysis .
for example , in a study by gaskin and helms in which 126 consecutive fatty masses were evaluated by mri , the positive predictive value was only 38% when the diagnosis of well - differentiated lps was suggested , with final pathologic diagnosis often being that of simple lipoma or a benign lipoma variant .
similarly , the presence of calcification or ossification within an adipocytic lesion can be a diagnostic dilemma for the radiologist .
benign entities such as lipoma should be considered , as trauma or ischemia can lead to central fat necrosis and subsequent calcification , although the extent of ossification and homogeneity seen in our case would be atypical .
myositis ossificans in the late stages ( more than 6 months ) presents as a densely ossified mass and can therefore appear similar , particularly on radiographs , although the presence of fat as in this case would be unusual .
history of trauma and various stages of maturation of myositis ossificans on imaging are important differentiating features .
moreover , zonal pattern of ossification toward the periphery on histology is a characteristic feature .
other rare , benign lipoma variants including chondrolipoma and osteolipoma can have grossly visible metaplastic bone formation .
chondrolipoma , furthermore , has a propensity to present in the proximal extremities and limb girdles .
parosteal lipoma , a benign lipoma adherent to the underlying bone , was a consideration in our case , as the scapula is a common location for this lesion and the mass itself can cause reactive changes in the associated bone .
malignant tumors should always be considered in the differential for adipocytic masses with calcification or ossification .
for example , rarely , metaplastic bone formation can be seen associated with well - differentiated lps , and ddlps not uncommonly shows metaplastic bone formation and heterologous elements , including osteosarcomatous differentiation .
ossification in the soft tissues without surrounding fatty tissue should also raise concern for extraskeletal osteosarcoma .
the term atypical lipomatous tumor and well - differentiated liposarcoma are indeed two names for the same entity .
histologically and genetically , the tumors are identical , consisting of well - differentiated adipocytic tissue with sometimes subtle histologic differences from benign lipoma ( including variation in adipocyte size , thickened fibrous septa , and atypical hyperchromatic stromal cells ) and ring or giant marker chromosomes containing amplified material from the long arm of chromosome 12 ( 12q1315 ) , resulting in mdm2 and cdk4 overexpression in most cases .
subtypes of benign lipomatous lesion are initially distinguished from well - differentiated liposarcoma by the image - guided percutaneous core - needle biopsies .
fish for mdm2 performed on core - needle biopsy material is reported to be more sensitive and specific than immunohistochemistry for well - differentiated liposarcoma ; however , fish and immunohistochemistry have similar results on resection specimens.the behavior of the tumors , furthermore , is the same in that they have no metastatic potential .
the true distinction , therefore , is solely anatomic , with tumors presenting in the extremities and subcutaneous tissues being labeled as alt and tumors in the mediastinum , retroperitoneum , mesentery , spermatic cord , or other locations being labeled as wdlps .
this developed from the idea that tumors presenting in the extremities or superficial tissues , the so - called atypical lipomatous tumors , have higher rates of complete surgical excision , lower rates of local recurrence , and lower risk of undergoing dedifferentiation compared with tumors arising in the mediastinum , retroperitoneum , or spermatic cord ; hence the term well - differentiated liposarcoma .
the entities of alt / wdlps and ddlps are often considered together due to their common genetic alterations , with the distinction of ddlps based on the histologic finding of a non - lipogenic component .
the histologic appearances of ddlps vary greatly , ranging from a relatively uniform spindle cell morphology to a highly pleomorphic undifferentiated appearance .
certain patterns , including a meningothelial - like whorling pattern of dedifferentiation , have been recently linked to bone formation within ddlps in some cases .
our case is rare in that it demonstrates extensive metaplastic ossification within an alt / wdlps not associated with meningothelial - like , osteosarcomatous , or other types of dedifferentiated tissue .
although a rare entity , alt / wdlps with metaplastic ossification should be considered in the differential diagnosis for a calcified soft tissue mass .
although our case represents an instance when no dedifferentiation was seen , it is important to be aware that metaplastic bone formation is often associated with tumors harboring a dedifferentiated component . | abstractthe presence of metaplastic ossification within atypical lipomatous tumor / well - differentiated liposarcoma ( alt / wdlps ) is a rare occurrence .
when present , bone formation is most often found in association with a dedifferentiated component arising within the primary tumor .
it is important for the radiologist not only to recognize the differential diagnosis of a calcified or ossified soft tissue mass but also be aware of the various soft tissue neoplasms , both aggressive and non - aggressive , that may show such features .
we report a case of alt / wdlps with unusual extensive metaplastic bone formation without an element of dedifferentiated liposarcoma . |
the number of women with newly diagnosed cervical cancer increases and the percentage of women with cervical cancer in all gynaecological cancers also increases annually in china1 .
radical hysterectomy with pelvic lymphadenectomy is the standard surgical treatment for patients with stage ia2-ib1 cervical cancer .
but the wide excision of parametrial tissues ( 3 cm ) increases the complications during and after the operation , especially the complications of the urinary system , which severely affect the life quality and the following adjuvant therapy2 , even in robotic sugeries3 .
can we decrease the complication by appropriately narrowing the scope of operation in selected patients without affecting the outcomes ?
data on this retrospective study was collected from the hospital of obstetrics & gynaecology , shanghai medical college , fudan university , shanghai , china from jan 2008 to dec 2011 , with the approval of ethics committee of hospital .
this hospital serves a diverse urban and rural population with over 1.3 million outpatients and 40,000 inpatients annually , and is a largest specialised referral obstetrics & gynaecology teaching hospital located in shanghai which is the largest city in china with 20 million populations .
the pathological and clinical data of patients with stage ia2-ib1 cervical cancer who received radical hysterectomy with pelvic lymphadenectomy in this ob / gyn hospital were collected and analyzed .
this retrospective study was exempt from irb approval because we only reviewed the clinical histories without revolving any information about patients ' private .
immunohistochemical examinations of d2 - 40 and cd31 were described as negative , positive and strong positive .
strong positive of d2 - 40 and cd31 meant d2 - 40 and cd31 positive and also with tumor thrombus .
those patients with diameter of tumor 2 cm , depth of cervical myometrial invasion<1/2 and without lymph vascular involvement formed the low - risk group , and the others formed the high - risk group .
both groups were further divided into two groups according to the resection width of parametrial tissues .
those patients with the resection width of parametrial tissues of both sides 3 cm formed group a , and those with the resection width of parametrial tissues of at least one side
<3 cm formed group b. the single factor analysis of the pathological and clinical data of patients was made with test .
the relationship of the resection width of parametrial tissues and the patients ' outcomes was analyzed with test and log - rank .
the single factor analysis of the pathological and clinical data of patients was made with test .
the relationship of the resection width of parametrial tissues and the patients ' outcomes was analyzed with test and log - rank .
there were 880 cases of stage ia2-ib1 cervical cancer received radical hysterectomy with pelvic lymphadenectomy in this hospital from jan 2008 to dec 2011 .
, there were 31 cases ( 6.0% ) recurred and 26 cases ( 5.1% ) died of cervical cancer during the follow - up period ( from 2 months to 66 months , averaged 39 months ) .
the single factor analysis showed that the diameter of tumor was separately related to the depth of cervical myometrial invasion ( p=0.000 ) , the diameter of tumor and the depth of cervical myometrial invasion were both related to lymph vascular involvement ( p=0.000 ) , the diameter of tumor , the depth of cervical myometrial invasion and lymph vascular involvement were all related to parametrial metastases ( p=0.000 ) , shown as table 2 .
immunohistochemical examinations of d2 - 40 were applied to 606 cases and examinations of cd31 were applied to 563 cases .
cd31 strong positive ( p=0.002 ) and d2 - 40 strong positive ( p=0.016 ) were also the high - risk factors of parametrial metastases ( shown as table 2 ) .
the test showed that parametrial metastases were related to the recurrence ( p=0.030 ) and the death ( p=0.019 ) of cervical cancer , but the immunohistochemical indexes were not related to the recurrence or the death of cervical cancer ( p>0.05 ) , shown as table 3 . the parametrial metastases rate and transfer rate of high - risk group were 4.1% and 9.5% , while those of low - risk group were 0% and 1.4% , shown as figure 1 .
the difference of progression free survival ( pfs , p=0.006 ) and overall survival ( os , p=0.004 ) ) of low - risk and high - risk groups was statistically significant .
the resection width of parametrial tissues ( varied mostly due to tumor size , performance of different surgeons and the objective conditions of patients ) had no statistically significant effect on the recurrence ( p=1.000 ) and the death ( p=1.000 ) of low - risk patients , shown as table 4 .
the resection width of parametrial tissues also had no statistically significant effect on the recurrence ( p=0.082 ) and the death ( p=0.147 ) of high - risk patients , shown as table 4 .
the resection width of parametrial tissues also had no statistically significant effect on the pfs ( p=0.610 ) and os ( p=0.608 ) of low - risk patients , shown as figure 2 .
according to the guideline of nccn 2014 , the standard surgical treatment for patients with stage ia2-ib1 cervical cancer is radical hysterectomy with pelvic lymphadenectomy , which demands wide excision of parametrial tissues ( cardinal ligament 3 - 4 cm , uterosacral ligament3 cm and all vaginal connective tissues ) .
the wide excision of parametrial tissues may block pelvic nerves which locate in cardinal ligament and uterosacral ligament , and increase the complications during and after the operation , especially the complications of the urinary system , which may severely affect the life quality and the following adjuvant therapy 2,3 .
studies of large samples have shown that the parametrial metastases rate of stage 1b1 cervical cancer is about 5.4 - 10% 4 - 5 .
the parametrial metastases rate of those patients with the diameter of tumor 2 cm , the depth of cervical myometrial invasion<10 mm and without lymph vascular involvement is about 0 - 1.1% 4 - 6 , which suggest that these factors can be considered as low - risk factors . on the other hand
, there was a report of 37 patients with stage 1a2 - 1b1 cervical cancer showing that the parametrial metastases rate was 33% for all patients and 4.5% for those with the diameter of tumor 2 cm and without lymph vascular involvement7 , but this result should be doubted because of the limited cases and the miss of considering the depth of cervical myometrial invasion . in our study , 880 patients with stage ia2-ib1 cervical cancer were analyzed and the total parametrial metastases rate was 2.4% .
the single factor analysis showed that the diameter of tumor > 2 cm , the depth of cervical myometrial invasion 1/2 and lymph vascular involvement were all independent high - risk factors of parametrial metastases .
there were no parametrial metastases in cases with the diameter of tumor 2 cm , the depth of cervical myometrial invasion<1/2 and without lymph vascular involvement at the same time , which suggested that over resection of parametrial tissues was not wise for these low - risk patients .
can we narrow the range of operation , decrease the possible complications and improve the life quality while without bad effect on the outcomes of these low - risk patients ?
the feasibility and safety of narrowing the scope of operation in selected patients are worthy of further discussion . among cases of stage ia2-ib1 cervical cancer which should have received radical hysterectomy with pelvic lymphadenectomy according to the guideline of nccn 2014 ,
there were 290 ( 56.5% ) cases whose actual resection width of parametrial tissues of at least one side < 3 cm , which is mostly varied mostly due to tumor size , performance of surgeons and the objective conditions of patients .
we analyzed these cases and found out that the recurrence rate was 1.4% for low - risk patients , which was lower than 9.5% for other patients . among all low - risk patients , the recurrence rate of those with the resection width of parametrial tissues of at least one side <3 cm ( group b ) was 1.8% , which had no statistically difference with that ( 0.1% ) of patients with the resection width of parametrial tissues of both sides 3 cm ( group a ) . among all high - risk patients ,
the recurrence rate of group b was 13.5% and also had no statistically difference with that of group a ( 6.3% ) , which may be due to the adjuvant chemotherapy , radiotherapy or both after the operation .
the pfs and os of high - risk patients , even with the help of adjuvant chemotherapy , radiotherapy or both after the operation , were still lower than those of low - risk patients .
the resection width of parametrial tissues had no statistically significant effect on the pfs and os of low - risk patients and further supported the feasibility and safety of narrowing the range of operation in low - risk patients , which was also supported by another paper 8 . but how can we accurately value those patients of cervical cancer before operation since the staging of cervical cancer is based on clinical characteristics
the magnetic resonance imaging ( mri ) and pelvic reconstruction can help with the accurate assessment of the diameter of tumor and the depth of cervical myometrial invasion .
so the accurate assessment of lymph vascular involvement becomes a key point of excluding the high - risk factors before operation .
the d2 - 40 antibody plays an important role in routine histopathologic diagnostics of normal lymphatic vessels and lymphaticderived tumors 9 .
platelet / endothelial cell adhesion molecule-1 ( cd31 ) is a cell adhesion and signaling molecule which plays in the biology of blood and vascular cells 10 .
so we used strong positive of d2 - 40 to stand by the lymphatic vessel invasion and strong positive of cd31 to stand by the vascular invasion of cervical cancer , and found out that strong positive of d2 - 40 and cd31 may suggest the parametrial metastases , although the expressive level of cd31 and d2 - 40 had no relationship with the recurrence and death of cervical cancer .
so we can make a fairly precise assessment of whether there is lymph vascular involvement or not by immunohistochemical examination of the expressive level of cd31 and d2 - 40 in the specimens of cervical biopsy or loop electrosurgical excision procedure of patients with cervical cancer .
the expression status of d2 - 40 and cd31 were not represented by the density of the lymphatic ducts and blood vessels . due to the length of the study and
the number of cases included , it was difficult to suggest the exact size of narrowing resection of parametrial tissues which is feasible and safe for low - risk patients . in summary , this retrospective analysis has shown that the diameter of tumor > 2 cm , the depth of cervical myometrial invasion 1/2 and lymph vascular involvement are all independent high - risk factors of parametrial metastases , and further highly related to the outcomes of stage ia2-ib1 cervical cancer .
low - risk patients without any of these three high - risk factors have fairly optimistic outcomes , and narrowing the range of operation , which means narrowing resection of parametrial tissues ( < 3 cm ) , is feasible and safe for low - risk patients .
mri , combined with the immunohistochemical examination of d2 - 40 and cd31 , can help with excluding the high - risk factors before operation . | background : radical hysterectomy with pelvic lymphadenectomy is the standard surgical treatment for patients with stage ia2-ib1 cervical cancer , but the wide excision increases the complications.objective : to analyze the feasibility of narrowing resection of parametrial tissues in stage ia2-ib1 cervical cancer.study design : retrospectively analyzed the pathological and clinical data of patients with stage ia2-ib1 cervical cancer who received radical hysterectomy with pelvic lymphadenectomy in ob / gyn hospital , fudan university , china from jan 2008 to dec 2011
. the affected factors of parametrial metastases and outcomes were discussed .
the single factor analysis was made with 2 test , and the relationship of the resection width of parametrial tissues and the patients ' outcomes was analyzed with 2 test and log - rank .
p - values < 0.05 were considered statistically significant.results : there were 31 cases recurred , 26 cases died of cervical cancer in 513 patients during the follow - up period ( from 2 months to 66 months , averaged 39 months ) .
the low - risk factors included diameter of tumor 2 cm , depth of cervical myometrial invasion<1/2 and without lymph vascular involvement .
there were no parametrial metastases in cases with all three low - risk factors . whether the resection width of parametrial tissues 3 cm or not had no statistically significant effect on progression free survival ( pfs ) or overall survival ( os ) of low - risk patients .
d2 - 40 and cd31 were related with parametrial metastases , but not with recurrence or outcomes.conclusions : the resection width of parametrial tissues has no effect on pfs and os of low - risk patients , and narrowing resection of parametrial tissues ( <3 cm ) is feasible . |
the study was approved by the institutional review board . written informed consent was signed by all study participants .
thirty central - southern italian patients with relapsing - remitting ms ( rrms ) were enrolled ( table ) by referral from the ms center , neurology unit , policlinico tor vergata , rome .
clinico - demographic characteristics of the studied sample patient eligibility was based on the following criteria : ( 1 ) clinically definite rrms , ( 2 ) remitting phase of disease , ( 3 ) expanded disability status scale ( edss ) score between 2 and 7 , and ( 4 ) presence of spasticity in the lower limbs .
disease onset was defined as the first episode of focal neurologic dysfunction indicative of ms .
all patients were in a remitting phase in the 3 months preceding recruitment and during the study .
remission was defined as the absence of new or recurrent neurologic symptoms not associated with fever or infection lasting for at least 24 hours .
all patients started disease - modifying drugs ( dmds ) at the time of diagnosis .
first - line treatment prescribed was glatiramer acetate ( 20 mg sc daily ) , interferon -1a ( 44 g sc 3 times weekly ) , interferon -1a ( 30 g i m ) , or interferon -1b ( 250 g sc every other day ) .
mitoxantrone ( 12 mg / m iv every 3 months with a lifetime maximum of 140 mg / m ) or natalizumab ( 300 mg iv every 4 weeks ) was prescribed as second - line treatment .
samples of peripheral blood from recruited patients were collected in bd vacutainer tubes with edta ( beckton dickinson , franklin lakes , nj ) .
genomic dna was isolated and extracted from human whole blood ( 200 l ) using a magna pure lc dna isolation kit and automated extractor ( roche diagnostics gmbh , mannheim , germany ) following manufacturer 's instructions .
one hundred fifty ng of genomic dna was used to amplify the cnr1 region , including the aat repeats through pcr .
pcr was carried out in a 25 l volume containing polymerase buffer , 1 mm mgcl2 , 0.2 mm of each dntp , 0.5 pmoles of each primer ( sense : 5-cacccctgggctgtaaaata-3 ; antisense : 5-gttgcagtgagccaagatca-3 ) , and 1.5 u taq dna polymerase ( invitrogen , madison , wi ) .
denaturation was achieved through a first step of 5 minutes at 94c followed by 35 cycles of 45 seconds at 95c , annealing for 1.5 minutes at 58c and elongation for 1 minute at 72c , and final elongation for 7 minutes at 72c . for sequencing analysis , 10 ng of pcr products
agencourt ampure pcr purification kit ( agencourt bioscience corporation , beverly , ma ) , 0.5 pmoles of the sequencing primer ( 5-acctccacccacaaatcaaa-3 ) , and the abi prism bigdye terminator v3.1 ready reaction cycle sequencing kit ( applied biosystems , foster city , ca ) were used .
sequencing reactions initiated with 1 minute denaturation at 96c and proceeded with 40 cycles of 10 seconds at 96c , 5 seconds at 50c , and 4 minutes at 60c .
product of sequencing was purified through a cleanseq dye terminal removal kit ( agencourt bioscience corporation ) and run on the applied biosystems 3730 dna analyzer instrument ( applied biosystems ) , complying with manufacturer 's instructions .
all patients were clinically assessed by expert neurologists who were blinded to laboratory and neurophysiology results .
edss score and a 010 numerical rating scale ( nrs ) score assessing self - reported severity of spasticity were recorded in all patients with ms before the first and immediately after the last pt session .
3 t mri scans consisted of a dual - echo turbo spin echo ( tse ) , a fluid - attenuated inversion recovery ( flair ) , and precontrast and postcontrast t1-weighted spin echo images .
t2 hyperintense lesions were determined by consensus of 2 investigators on proton density weighted short echo tse images .
lesions were outlined through semiautomated local thresholding contouring software ( jim 4.0 , xinapse system , leicester , uk ) .
presence of lesions within the brainstem , corpus callosum , posterior fossa , periventricular , and spinal cord was recorded for each patient to evaluate lesion distribution .
tms can activate neurons in a focal region of the cerebral cortex through electromagnetic induction .
delivered repetitively on the motor cortex , tms can induce plastic modifications of cortical excitability that can be measured by recording the motor evoked potentials ( meps ) from the muscles represented within the stimulated region . an increase or decrease of the mep amplitude lasting after the end of repetitive
tms indicates that ltp - like or long - term depression like plastic changes occurred , respectively .
electromyographic traces were recorded from the right first dorsal interosseus muscle ( fdi ) with surface electrodes , amplified ( digitimer , hertfordshire , uk ) , sampled at 5 khz , 20 hz2 khz bandpassed , and stored for offline analysis .
meps were evoked through a magstim 200 magnetic stimulator ( magstim company , whitland , wales , uk ) with a 70-mm diameter figure - of - eight coil . coil was positioned tangentially to the scalp over the left hemisphere to evoke meps from the right fdi ( hot spot ) , the handle pointing posterolaterally at 45. for itbs , a magstim rapid delivered 20 cycles of 10 bursts repeated at 5 hz and separated by 8-second pauses , with each burst composed of a triplet of tms pulses at 50 hz .
amt was defined as the minimum stimulation intensity required to evoke at least 5 mep , 200 v peak - to - peak amplitude out of 10 pulses during fdi 's voluntary contraction ( 10% of maximal ) . immediately before itbs and at 2 different time points ( 0 and 15 minutes ) after the end of itbs , we collected 35 meps evoked by a test stimulus ( ts ) with a stimulation intensity set to induce a stable mep of approximately 1 mv peak - to - peak amplitude in the relaxed fdi at baseline .
mep amplitudes were averaged at each time point and normalized to the mean baseline amplitude . short - interval intracortical inhibition ( sici ) and intracortical facilitation ( icf )
were tested using paired - pulse ( pp ) tms with a conditioning stimulus ( cs ) at 80% amt intensity , preceding the ts .
five conditions were randomly presented : ts alone and 4 pp conditions with cs preceding ts at 1 of 4 different interstimulus intervals ( isis ) ( 2 , 3 , 10 , and 15 msec ) .
short - interval intracortical facilitation ( sicf ) was tested using a cs at 90% of resting motor threshold ( rmt ) intensity .
six conditions were randomly presented : ts alone and 5 conditions with ts preceding cs at 1 of 5 different isis ( 1.5 , 2.1 , 2.7 , 3.7 , and 4.5 msec ) . long - interval intracortical inhibition ( lici ) , mediated by gabab ,
two conditions were presented in a random order : ts alone and cs preceding ts at an isi of 100 msec . for each experimental condition 10
mep changes at each isi were expressed as percentage of the mean unconditioned mep amplitude .
two to 4 weeks after tms evaluation , patients started the therapeutic exercise program at the physical rehabilitation department of the university hospital policlinico tor vergata in rome .
exercises were performed on a daily basis for 2 weeks and consisted of 1 hour on land followed by another hour in 2830c water in a 150-cm wide pool , as in a previous study .
the program was devised and coordinated by a physician specializing in physical medicine and rehabilitation and consisted of both passive and active therapeutic exercises specifically aimed at restoring or maintaining muscular flexibility , range of motion , balance , coordination of movements , postural passages and transfers , and ambulation .
different types of therapeutic exercises were scheduled according to the individual disability status and administered by qualified physiotherapists .
the exercises consisted of ( 1 ) repetition of different movements ( i.e. , tips and heels , 90 flexed hips and knees ) for ambulation and stair climbing ; ( 2 ) repetition of crossed patterns of movements for coordination ; ( 3 ) postural reactions while standing with eyes open and closed , including the use of oscillatory boards for balance ; ( 4 ) strengthening lower limb antigravitary muscles ( i.e. , gluteus minor and major , quadriceps femoralis ) ; and ( 5 ) low - intensity and long - duration static stretching of iliopsoas , rectus femoralis , hamstrings , triceps surae , and lumbar spinal muscles for muscular flexibility and range of motion . during each session patients performed either 2 or 3 sets per exercise type , with each set consisting of about 15 repetitions .
compensative pauses of time were included in relation to each patient 's tolerance to the exercise regimen , for proper restoring . as in previous studies ,
patients with ms were grouped according to the number of aat repeats of the cnr1 gene . in the short - aatn group patients had 1 or 2 alleles with 11 repeats of aat triplets , and in the long - aatn group patients had 2 alleles with 12 repeats .
student t test , mann whitney test , fisher exact test , and repeated - measures analysis of variance ( anova ) were used to analyze differences between groups , as appropriate .
type of dmd was entered as a covariate because of its effect as a potential confounder .
the study was approved by the institutional review board . written informed consent was signed by all study participants .
thirty central - southern italian patients with relapsing - remitting ms ( rrms ) were enrolled ( table ) by referral from the ms center , neurology unit , policlinico tor vergata , rome .
clinico - demographic characteristics of the studied sample patient eligibility was based on the following criteria : ( 1 ) clinically definite rrms , ( 2 ) remitting phase of disease , ( 3 ) expanded disability status scale ( edss ) score between 2 and 7 , and ( 4 ) presence of spasticity in the lower limbs .
disease onset was defined as the first episode of focal neurologic dysfunction indicative of ms .
all patients were in a remitting phase in the 3 months preceding recruitment and during the study .
remission was defined as the absence of new or recurrent neurologic symptoms not associated with fever or infection lasting for at least 24 hours .
all patients started disease - modifying drugs ( dmds ) at the time of diagnosis .
first - line treatment prescribed was glatiramer acetate ( 20 mg sc daily ) , interferon -1a ( 44 g sc 3 times weekly ) , interferon -1a ( 30 g i m ) , or interferon -1b ( 250 g sc every other day ) .
mitoxantrone ( 12 mg / m iv every 3 months with a lifetime maximum of 140 mg / m ) or natalizumab ( 300 mg iv every 4 weeks ) was prescribed as second - line treatment .
samples of peripheral blood from recruited patients were collected in bd vacutainer tubes with edta ( beckton dickinson , franklin lakes , nj ) .
genomic dna was isolated and extracted from human whole blood ( 200 l ) using a magna pure lc dna isolation kit and automated extractor ( roche diagnostics gmbh , mannheim , germany ) following manufacturer 's instructions .
one hundred fifty ng of genomic dna was used to amplify the cnr1 region , including the aat repeats through pcr .
pcr was carried out in a 25 l volume containing polymerase buffer , 1 mm mgcl2 , 0.2 mm of each dntp , 0.5 pmoles of each primer ( sense : 5-cacccctgggctgtaaaata-3 ; antisense : 5-gttgcagtgagccaagatca-3 ) , and 1.5 u taq dna polymerase ( invitrogen , madison , wi ) .
denaturation was achieved through a first step of 5 minutes at 94c followed by 35 cycles of 45 seconds at 95c , annealing for 1.5 minutes at 58c and elongation for 1 minute at 72c , and final elongation for 7 minutes at 72c . for sequencing analysis , 10 ng of pcr products
agencourt ampure pcr purification kit ( agencourt bioscience corporation , beverly , ma ) , 0.5 pmoles of the sequencing primer ( 5-acctccacccacaaatcaaa-3 ) , and the abi prism bigdye terminator v3.1 ready reaction cycle sequencing kit ( applied biosystems , foster city , ca ) were used .
sequencing reactions initiated with 1 minute denaturation at 96c and proceeded with 40 cycles of 10 seconds at 96c , 5 seconds at 50c , and 4 minutes at 60c .
product of sequencing was purified through a cleanseq dye terminal removal kit ( agencourt bioscience corporation ) and run on the applied biosystems 3730 dna analyzer instrument ( applied biosystems ) , complying with manufacturer 's instructions .
all patients were clinically assessed by expert neurologists who were blinded to laboratory and neurophysiology results .
edss score and a 010 numerical rating scale ( nrs ) score assessing self - reported severity of spasticity were recorded in all patients with ms before the first and immediately after the last pt session .
3 t mri scans consisted of a dual - echo turbo spin echo ( tse ) , a fluid - attenuated inversion recovery ( flair ) , and precontrast and postcontrast t1-weighted spin echo images .
t2 hyperintense lesions were determined by consensus of 2 investigators on proton density weighted short echo tse images .
lesions were outlined through semiautomated local thresholding contouring software ( jim 4.0 , xinapse system , leicester , uk ) .
presence of lesions within the brainstem , corpus callosum , posterior fossa , periventricular , and spinal cord was recorded for each patient to evaluate lesion distribution .
tms can activate neurons in a focal region of the cerebral cortex through electromagnetic induction .
delivered repetitively on the motor cortex , tms can induce plastic modifications of cortical excitability that can be measured by recording the motor evoked potentials ( meps ) from the muscles represented within the stimulated region . an increase or decrease of the mep amplitude lasting after the end of repetitive
tms indicates that ltp - like or long - term depression like plastic changes occurred , respectively .
electromyographic traces were recorded from the right first dorsal interosseus muscle ( fdi ) with surface electrodes , amplified ( digitimer , hertfordshire , uk ) , sampled at 5 khz , 20 hz2 khz bandpassed , and stored for offline analysis .
meps were evoked through a magstim 200 magnetic stimulator ( magstim company , whitland , wales , uk ) with a 70-mm diameter figure - of - eight coil .
coil was positioned tangentially to the scalp over the left hemisphere to evoke meps from the right fdi ( hot spot ) , the handle pointing posterolaterally at 45. for itbs , a magstim rapid delivered 20 cycles of 10 bursts repeated at 5 hz and separated by 8-second pauses , with each burst composed of a triplet of tms pulses at 50 hz .
amt was defined as the minimum stimulation intensity required to evoke at least 5 mep , 200 v peak - to - peak amplitude out of 10 pulses during fdi 's voluntary contraction ( 10% of maximal ) . immediately before itbs and at 2 different time points ( 0 and 15 minutes ) after the end of itbs , we collected 35 meps evoked by a test stimulus ( ts ) with a stimulation intensity set to induce a stable mep of approximately 1 mv peak - to - peak amplitude in the relaxed fdi at baseline .
mep amplitudes were averaged at each time point and normalized to the mean baseline amplitude . short - interval intracortical inhibition ( sici ) and
intracortical facilitation ( icf ) were tested using paired - pulse ( pp ) tms with a conditioning stimulus ( cs ) at 80% amt intensity , preceding the ts .
five conditions were randomly presented : ts alone and 4 pp conditions with cs preceding ts at 1 of 4 different interstimulus intervals ( isis ) ( 2 , 3 , 10 , and 15 msec ) .
short - interval intracortical facilitation ( sicf ) was tested using a cs at 90% of resting motor threshold ( rmt ) intensity .
six conditions were randomly presented : ts alone and 5 conditions with ts preceding cs at 1 of 5 different isis ( 1.5 , 2.1 , 2.7 , 3.7 , and 4.5 msec ) . long - interval intracortical inhibition ( lici ) , mediated by gabab ,
two conditions were presented in a random order : ts alone and cs preceding ts at an isi of 100 msec . for each experimental condition 10
mep changes at each isi were expressed as percentage of the mean unconditioned mep amplitude .
two to 4 weeks after tms evaluation , patients started the therapeutic exercise program at the physical rehabilitation department of the university hospital policlinico tor vergata in rome .
exercises were performed on a daily basis for 2 weeks and consisted of 1 hour on land followed by another hour in 2830c water in a 150-cm wide pool , as in a previous study .
the program was devised and coordinated by a physician specializing in physical medicine and rehabilitation and consisted of both passive and active therapeutic exercises specifically aimed at restoring or maintaining muscular flexibility , range of motion , balance , coordination of movements , postural passages and transfers , and ambulation .
different types of therapeutic exercises were scheduled according to the individual disability status and administered by qualified physiotherapists .
the exercises consisted of ( 1 ) repetition of different movements ( i.e. , tips and heels , 90 flexed hips and knees ) for ambulation and stair climbing ; ( 2 ) repetition of crossed patterns of movements for coordination ; ( 3 ) postural reactions while standing with eyes open and closed , including the use of oscillatory boards for balance ; ( 4 ) strengthening lower limb antigravitary muscles ( i.e. , gluteus minor and major , quadriceps femoralis ) ; and ( 5 ) low - intensity and long - duration static stretching of iliopsoas , rectus femoralis , hamstrings , triceps surae , and lumbar spinal muscles for muscular flexibility and range of motion . during each session patients performed either 2 or 3 sets per exercise type , with each set consisting of about 15 repetitions .
compensative pauses of time were included in relation to each patient 's tolerance to the exercise regimen , for proper restoring .
as in previous studies , patients with ms were grouped according to the number of aat repeats of the cnr1 gene . in the short - aatn group patients had 1 or 2 alleles with 11 repeats of aat triplets , and in the long - aatn group patients had 2 alleles with 12 repeats .
student t test , mann whitney test , fisher exact test , and repeated - measures analysis of variance ( anova ) were used to analyze differences between groups , as appropriate .
type of dmd was entered as a covariate because of its effect as a potential confounder .
the experimental procedures were well - tolerated by all study participants and no adverse reactions were reported .
long- and short - aatn cnr1 patients showed no clinico - demographic differences and no differences in mri lesion load ( table ) and distribution ( data not shown ) at baseline . for edss , repeated - measures anova with time ( baseline and post - pt ) as within - subjects and group ( short- and long - aatn ) as between - subjects main factors showed an effect of time ( df = 1 , f = 6.6 , p < 0.01 ) and time - group interaction ( df = 1 , f = 24.8 , p < 0.01 ) .
pyramidal functional system edss subscore showed an effect of time ( df = 1 , f = 35.9 , p < 0.001 ) and time - group interaction ( df = 1 , f = 37.8 , p < 0.001 ) , with a lower edss pyramidal subscore in the short - aatn group post - pt .
no differences emerged for the other edss functional system subscores ( visual , brainstem , cerebellar , sensory , bowel and bladder , and cerebral ) .
accordingly , the spasticity nrs showed an effect of time ( df = 1 , f = 4.2 , p < 0.05 ) and time - group interaction ( df = 1 , f = 6.4 , p < 0.05 ) , with nrs lower in the short - aatn group post - pt ( figure 1 ) .
dmd , entered as a covariate , had no effect on clinical response to pt .
after the 2-week physical therapy program , patients carrying a short number of aatn repeats ( aatn 11 in at least 1 allele ) show a better clinical response in both edss ( a ) and spasticity nrs ( b ) .
aatn = aat trinucleotide short tandem repeat ; edss = expanded disability status scale ; nrs = numerical rating scale . for itbs , repeated - measures anova with time ( baseline , 0 , and 15 minutes post - itbs ) as within - subjects and group ( short- and long - aatn ) as between - subjects main factors showed an effect of time ( mean se = 1.16 0.06 at 0 and 1.29 0.09 at 15 minutes post - itbs ; df = 1.78 , f = 4.38 , p < 0.05 ) , group ( mean se = 1.26 0.10 [ short - aatn ] and 1.05 0.09 [ long - aatn ] at 0 , and 1.39 0.16 [ short - aatn ] and 1.13 0.20 [ long - aatn ] at 15 minutes post - itbs ; df = 1 , f = 3.64 , p < 0.05 ) , and time - group interaction ( df = 1.78 , f = 6.43 , p < 0.05 ) . post hoc analysis revealed increased itbs in short - aatn at 15 minutes .
conversely , an effect of isi but not of group or isi - group interaction emerged for sici ( df = 2 , f = 8.96 , p < 0.001 ) , icf ( df = 2 , f = 3.28 , p < 0.05 ) , sicf ( df = 1.97 , f = 20.43 , p < 0.001 ) , and lici ( df = 1 , f = 76.88 , p < 0.001 ) ( figure 2 ) .
student t test did not reveal any between - group difference in baseline mep amplitudes ( short - aatn 1.46 0.18 mv vs long - aatn 1.36 0.22 mv ) , rmt ( short - aatn 46.7 8.2 vs long - aatn 47.2 8.8 ) , amt ( short - aatn 33.2 8.2 vs long - aatn 36.2 8.5 ) , and mep latency ( short - aatn 22.8 3.3 msec vs long - aatn 22.9 3.0 msec , all p > 0.1 ) .
dmd , entered as a covariate , had no effect on itbs , sici , icf , sicf , or lici .
multiple sclerosis patients with a long number of aatn repeats ( aatn 12 on both alleles ) on the cnr1 gene show defective ltp response to itbs in the motor cortex .
in contrast , patients with a short number of aatn repeats ( aatn 11 in at least 1 allele ) show a preserved ltp response after itbs ( a ) .
no differences were found between short- and long - aatn carriers in the sici / icf ( b ) , lici ( c ) , and sicf ( d ) paired - pulse tms protocols .
aatn = aat trinucleotide short tandem repeat ; icf = intracortical facilitation ; isi = interstimulus interval ; itbs = intermittent theta burst stimulation ; lici = long - interval intracortical inhibition ; ltp = long - term synaptic potentiation ; mep = motor evoked potential ; sicf = short - interval intracortical facilitation ; sici = short - interval intracortical inhibition ; tms = transcranial magnetic stimulation .
no correlation emerged between the edss score measured at baseline and itbs , sici , icf , sicf , or lici .
for edss , repeated - measures anova with time ( baseline and post - pt ) as within - subjects and group ( short- and long - aatn ) as between - subjects main factors showed an effect of time ( df = 1 , f = 6.6 , p < 0.01 ) and time - group interaction ( df = 1 , f = 24.8 , p < 0.01 ) . post hoc contrasts revealed lower edss in short - aatn post - pt .
pyramidal functional system edss subscore showed an effect of time ( df = 1 , f = 35.9 , p < 0.001 ) and time - group interaction ( df = 1 , f = 37.8 , p < 0.001 ) , with a lower edss pyramidal subscore in the short - aatn group post - pt .
no differences emerged for the other edss functional system subscores ( visual , brainstem , cerebellar , sensory , bowel and bladder , and cerebral ) .
accordingly , the spasticity nrs showed an effect of time ( df = 1 , f = 4.2 , p < 0.05 ) and time - group interaction ( df = 1 , f = 6.4 , p < 0.05 ) , with nrs lower in the short - aatn group post - pt ( figure 1 ) .
dmd , entered as a covariate , had no effect on clinical response to pt .
after the 2-week physical therapy program , patients carrying a short number of aatn repeats ( aatn 11 in at least 1 allele ) show a better clinical response in both edss ( a ) and spasticity nrs ( b ) .
aatn = aat trinucleotide short tandem repeat ; edss = expanded disability status scale ; nrs = numerical rating scale .
for itbs , repeated - measures anova with time ( baseline , 0 , and 15 minutes post - itbs ) as within - subjects and group ( short- and long - aatn ) as between - subjects main factors showed an effect of time ( mean se = 1.16 0.06 at 0 and 1.29 0.09 at 15 minutes post - itbs ; df = 1.78 , f = 4.38 , p < 0.05 ) , group ( mean se = 1.26 0.10 [ short - aatn ] and 1.05 0.09 [ long - aatn ] at 0 , and 1.39 0.16 [ short - aatn ] and 1.13 0.20 [ long - aatn ] at 15 minutes post - itbs ; df = 1 , f = 3.64 , p < 0.05 ) , and time - group interaction ( df = 1.78 , f = 6.43 , p < 0.05 ) .
conversely , an effect of isi but not of group or isi - group interaction emerged for sici ( df = 2 , f = 8.96 , p < 0.001 ) , icf ( df = 2 , f = 3.28 , p < 0.05 ) , sicf ( df = 1.97 , f = 20.43 , p < 0.001 ) , and lici ( df = 1 , f = 76.88 , p < 0.001 ) ( figure 2 )
. student t test did not reveal any between - group difference in baseline mep amplitudes ( short - aatn 1.46 0.18 mv vs long - aatn 1.36 0.22 mv ) , rmt ( short - aatn 46.7 8.2 vs long - aatn 47.2 8.8 ) , amt ( short - aatn 33.2 8.2 vs long - aatn 36.2 8.5 ) , and mep latency ( short - aatn 22.8 3.3 msec vs long - aatn 22.9 3.0 msec , all p > 0.1 ) .
dmd , entered as a covariate , had no effect on itbs , sici , icf , sicf , or lici .
multiple sclerosis patients with a long number of aatn repeats ( aatn 12 on both alleles ) on the cnr1 gene show defective ltp response to itbs in the motor cortex .
in contrast , patients with a short number of aatn repeats ( aatn 11 in at least 1 allele ) show a preserved ltp response after itbs ( a ) .
no differences were found between short- and long - aatn carriers in the sici / icf ( b ) , lici ( c ) , and sicf ( d ) paired - pulse tms protocols .
aatn = aat trinucleotide short tandem repeat ; icf = intracortical facilitation ; isi = interstimulus interval ; itbs = intermittent theta burst stimulation ; lici = long - interval intracortical inhibition ; ltp = long - term synaptic potentiation ; mep = motor evoked potential ; sicf = short - interval intracortical facilitation ; sici = short - interval intracortical inhibition ; tms = transcranial magnetic stimulation .
no correlation emerged between the edss score measured at baseline and itbs , sici , icf , sicf , or lici .
in the present study we observed that patients with ms carrying a genetic variant of the cb1r associated with reduced receptor expression are refractory to the effects of pt on motor function , and in parallel we found that motor cortex ltp is defective in these patients . after brain damage ,
pt boosts this compensatory plasticity , increasing activity - dependent synaptic potentiation and subsequent anatomical reorganization and driving axonal sprouting and synaptogenesis . in humans , cortical representation of the muscles involved in motor training enlarges and the activation threshold decreases .
ltp is considered the synaptic underpinning of exercise - induced plasticity , as this form of activity - dependent plasticity makes synapses more responsive to future stimulations and may thus potentially compensate for the loss of inputs caused by neuronal damage on surviving deafferented neurons . indeed , both in vitro ltp and exercise cause dendritic growth and new synapse formation , stimulating similar molecular pathways .
exercise increases endocannabinoid signaling in humans and attenuates clinical manifestations of experimental ms through increased cb1r sensitivity .
in contrast , genetic deletion of cb1r is associated with decreased improvement of motor performance induced by exercise and defective ltp in mice .
moreover , the cb1r seems to be involved in the control of spasticity both in eae and in patients with ms even though cannabinoid treatment showed mixed results , as several trials reported subjective benefit from patients whereas blinded assessments by investigators failed to show objective changes in spasticity .
spasticity derives from hyperactivity of the stretch reflex caused by damaged corticospinal tract function , and motor cortex ltp induced by itbs alleviates this symptom by increasing the excitability of the corticospinal tract . in this respect ,
the cb1r has been implicated in ltp in both animals and humans , as treatment with cannabis in ms favors tbs - induced ltp .
notably , itbs - induced effects originate cortically at impinging on pyramidal output cells , which are also particularly sensitive to cb1r modulation .
it is interesting that the brain - derived neurotrophic factor , an essential player in ltp induction and maintenance , is increased by exercise , and its genetic polymorphism can impair both tbs - induced ltp and exercise - induced cortical reorganization in humans .
the results of our investigation could be extended to pt effects in patients with diseases other than ms , such as stroke .
however , it is also possible that the specific mechanisms of brain damage in ms , which are greatly dependent on the release of inflammatory molecules by infiltrating immune cells , could exacerbate the negative influence of the genetically determined defective cb1r function on synaptic plasticity and motor recovery , as both eae and ms have been associated with reduced cb1r function . indeed , downregulation of cb1r caused by both genetic defect or subchronic tetrahydrocannabinol exposure causes activation of the microglia in mouse cerebellum .
this microglia activation causes deficits in cerebellar associative learning and motor coordination mediated by release of proinflammatory cytokines such as interleukin-1. accordingly , proinflammatory molecules ( namely interleukin-1 ) have been demonstrated to inhibit cb1rs in mice
. moreover , other cytokines that can be altered during inflammatory activity , such as the platelet - derived growth factor , have been shown to influence tbs - induced ltp and accumulation of disability in ms .
tbs aftereffects can last up to 1 hour ; however , in our study the recording was limited to the first 15 minutes only . moreover , because determination of amt requires voluntary activation of the target muscle , this may have caused metaplastic interactions with the subsequent itbs protocol . therefore our findings may be limited to early ltp induction and may be related to either itbs - induced ltp or its metaplastic interaction with prior voluntary activation .
finally , even though pt was administered following a detailed program , exercises differed among patients according to their level of impairment and disability . in addition , the effects of training on ambulation , balance , safety , and range of motion were not measured through specific scales .
because our pt program lasted only 2 weeks , we can not exclude the possibility that long - aatn cnr1 patients may still be responsive to a longer pt program .
these limitations may prevent the conclusion that exercise is more beneficial in patients with short - aatn .
further studies are needed to confirm our results and hypothesis and to test the effects of the cnr1 genetic variants both in healthy individuals and in other disease conditions and how they interact with other factors that may potentially influence tbs - induced ltp and effects of exercise .
our results provide the first evidence that genetic differences within the cb1r may determine at least in part the variable clinical response to pt and strengthen the proposed involvement of cb1rs in the control of spasticity .
defective plasticity mechanisms in long - aatn cnr1 patients with ms could also account for worse clinical evolution , as plasticity plays a key role in spontaneous compensation of brain damage , implying that defective plasticity reserve may contribute to clinical progression .
it is interesting that genome - wide association studies did not identify the genetic area containing the cnr1 gene as related to ms susceptibility ; however , long - aatn cnr1 ( 13 ) alleles are more frequent in patients with primary progressive ms , a condition characterized by progressive clinical worsening of disability with no periods of remission .
dr . mori : study concept and design , acquisition of data , analysis , interpretation , and manuscript preparation .
this study was sponsored by the fondazione italiana sclerosi multipla ( fism special project ) , by the italian national ministero dell'universit , and by fondazione baroni to d.c .
f. mori received travel funding from teva pharmaceuticals and merck - serono , received speaker honoraria from merck - serono , is an associate editor for bmc neurology , and consulted for grifols italia . c. ljoka , c.g .
s. rossi is on the advisory board for biogen idec , teva , and novartis ; has received speaker honoraria from novartis , teva , and biogen idec ; and has received travel funding from novartis , teva , merck - serono , and sanofi - aventis . c. foti is on the editorial board for bio med center med sport .
d. centonze is on the scientific advisory board for teva pharmaceutical industries ltd , merck - serono , bayer schering , and novartis ; has received speaker honoraria and travel funding from sanofi - aventis , merck - serono , serono symposia international foundation , bayer schering pharma , and biogen - dompe ag ; and has received research support from merck - serono , teva , novartis , bayer schering , sanofi - aventis , biogen idec , italian national ministry of education , and foundazione italiana sclerosi multipla ( fism ) . | objectives : therapeutic effects of physical therapy in neurologic disorders mostly rely on the promotion of use - dependent synaptic plasticity in damaged neuronal circuits . genetic differences affecting the efficiency of synaptic plasticity mechanisms could explain why some patients do not respond adequately to the treatment
. it is known that physical exercise activates the endocannabinoid system and that stimulation of cannabinoid cb1 receptors ( cb1rs ) promotes synaptic plasticity in both rodents and humans .
we thus tested whether cb1r genetic variants affect responsiveness to exercise therapy.methods:we evaluated the effect of a genetic variant of the cb1r associated with reduced receptor expression ( patients with long aat trinucleotide short tandem repeats in the cnr1 gene ) on long - term potentiation ( ltp)like cortical plasticity induced by transcranial magnetic theta burst stimulation ( tbs ) of the motor cortex and , in parallel , on clinical response to exercise therapy in patients with multiple sclerosis.results:we found that patients with long aat cnr1 repeats do not express tbs - induced ltp - like cortical plasticity and show poor clinical benefit after exercise therapy.conclusions:our results provide the first evidence that genetic differences within the cb1r may influence clinical responses to exercise therapy , and they strengthen the hypothesis that cb1rs are involved in the regulation of synaptic plasticity and in the control of spasticity in humans .
this information might be of great relevance for patient stratification and personalized rehabilitation treatment programs . |
the identification of replication origins will be helpful to reveal the regulatory mechanisms of the initiation step in dna replication ( 1,2 ) and discover new broad - spectrum antibacterial drugs ( 3 ) . based on the z - curve theory ( 4 ) ,
we have developed a web - based system ori - finder for finding orics in bacterial genomes with high accuracy and reliability ( 5 ) , and the predicted oric regions in bacterial genomes have been organized into an online database doric ( 6 ) .
based on the database , putative origins of replication in sorangium cellulosum , microcystis aeruginosa ( 7 ) and cyanothece 51142 ( 8) , which could not be determined by using standard gc skew , have been identified by taking advantage of comparative genomics .
the application of the proposed oric selection criteria and the comparison of different cyanobacterial strains may also gain insight into the replication origins in other cyanobacteria ( 9 ) .
as the database was constructed in 2007 , we noticed that the replication origins of anabaena sp .
pcc 7120 ( 10 ) , cytophaga hutchinsonii atcc 33406 ( 11 ) and synechococcus elongatus pcc 7942 ( 12 ) have been confirmed by experiments , which are all consistent with our predictions in doric .
because of continuous updates , our database has been widely used in the comparative genomics analysis .
for example , as a source of data , doric has been used in the study of the relationship between the functionality of essential genes and gene strand bias in bacterial genomes ( 13 ) , in the analysis of nucleotide compositional asymmetry between the leading and lagging strands of bacterial genomes ( 14 ) , in the investigation of the association between growth - related traits and minimal generation times ( 15 ) , in an algorithm for prediction of putative essential and core - essential genes in mycoplasma genomes ( 16 ) , in the research on coordination of spatiotemporal gene expression during the bacterial growth cycle ( 17 ) and in the study of the variation in terms of the percentage of leading strand genes across different bacteria ( 18 ) , etc .
it is expected that the new release of the database , doric 5.0 , will promote the study of orics in both bacteria and archaea .
in the current release , the database has been significantly improved compared with the initial release , and the main advances include ( i ) inclusion of orics in more bacterial genomes that increased from 435 to 1528 ; ( ii ) inclusion of orics in 81 archaeal genomes ; ( iii ) inclusion of detailed information about repeats in orics identified by reputer program ( 19 ) ; and ( iv ) addition of urls that link to ncbi map viewer ( 20 ) or ucsc archaeal genome browser ( 21 ) , which are useful to explore and discover the conserved features around the oric region .
consequently , the latest release of doric contains orics for > 1500 bacterial genomes and 81 archaeal genomes , which can be accessed from http://tubic.tju.edu.cn / doric/.
to identify oric regions of unannotated bacterial genomes , we have developed a web - based system , ori - finder , based on an integrated method comprising gene identification , analysis of base composition asymmetry using the z - curve method , distribution of dnaa boxes , occurrence of genes frequently close to orics and phylogenetic relationships .
consequently , the predicted oric regions have been organized into an online database , doric .
based on doric , the relationships between the conserved features associated with the oric regions , such as adjacent genes , dnaa boxes , etc . , and
for example , detailed analyses have shown that the consensus sequence of the dnaa boxes in oric regions and the distribution of genes around orics are strongly conserved among the bacteria in the phylum cyanobacteria ( 7,8 ) .
the feature that the oric is adjacent to dnan gene , which encodes the beta clamp processivity factor , has been found to be universal among the bacteria within the phylum cyanobacteria , and the species - specific dnaa box motif for the phylum cyanobacteria is
these strongly conserved features indicate that the in silico identified orics are reliable , as they have been confirmed by comparative genomics approaches .
this observation also shows that if the oric for one of the bacteria in the phylum cyanobacteria is confirmed experimentally , the orics for the other bacterial genomes in this phylum may be confirmed simultaneously . as we expected , the experimentally confirmed replication origins of anabaena sp .
pcc 7120 ( 10 ) and s. elongatus pcc 7942 ( 12 ) in the phylum cyanobacteria are all adjacent to the dnan gene , which encodes the beta clamp processivity factor .
therefore , the proposed rules may be helpful to predict the oric regions for some bacteria without complete genomes in the phylum cyanobacteria .
in addition , the application of the proposed rules derived from doric would speedup the experimental confirmation and functional analysis of orics in bacterial genomes .
because of the rapid growth in the number of sequenced bacterial genomes , the replication origins for those unsubmitted to genbank or not deposited in doric temporarily can be predicted by ori - finder firstly , which now has been used to analyze 30 newly sequenced bacterial genomes
. the z - curve analysis has been used to identify one replication origin in the genomes of methanocaldococcus jannaschii ( 22 ) and methanosarcina mazei ( 23 ) , two replication origins in the halobacterium species nrc-1 genome ( 24 ) , which have been confirmed by in vivo experiments ( 25,26 ) and three replication origins in the sulfolobus solfataricus p2 genome ( 24 ) , which have been later confirmed experimentally ( 27,28 ) . here , we collected the information of orics provided in the literature , such as the oric sequences , origin recognition boxes ( orb ) motifs , uncharacterized motif sequences , etc . , which were identified by in vivo experiments ( 2534 ) , as well as in silico analysis ( 4,2224,35 ) .
in addition , we also predicted some new replication origins by z - curve method , with the aid of homologous sequence search against the known replication origins , analysis of orb motifs and repeats , cdc6 gene location , etc .
consequently , oric regions in 81 archaeal genomes identified by in vivo experiments , as well as in silico analyses , have been added to our database .
the number of orics in archaea is correlated with the phylogeny , which has been summarized in detail in the
, it shows that there is one replication origin in the genomes within the order methanococcales ( 11 genomes ) and within the class thermococci ( 12 genomes ) , and three replication origins in sulfolobus species ( 13 genomes ) .
our results and the z - curves also show that the archaea within the crenarchaeota phylum contain multiple origins , although some origins could not be determined at the sequence level currently .
for example , pyrobaculum calidifontis has been experimentally characterized to contain four replication origins , which is the highest number detected in a prokaryotic organism ( 34 ) . however , only one origin can be determined at the sequence level ( 34 ) . during the course of the prediction
, we found that the location of some putative replication initiator gene besides cdc6 gene can be helpful to the oric prediction in some cases .
for example , in the genome of m. jannaschii , an orf ( mj0774 ) , annotated as a hypothetical protein , is a distant homolog of the cdc6 protein in fact ( 22 ) .
the name mc - prip for the putative replication initiator protein in methanococcales has been used here for mj0774 and related proteins to distinguish it from bona fide orthologous cdc6 .
we also found the genes , which encode mc - prip in other 10 genomes within the order methanococcales ( methanococcus aeolicus nankai-3 , methanocaldococcus fervens ag86 , methanococcus maripaludis c5 , m. maripaludis c6 , m. maripaludis c7 , m. maripaludis s2 , m. maripaludis x1 , methanococcus vannielii sb , methanococcus voltae a3 and methanocaldococcus vulcanius m7 ) , were annotated as
marr family transcriptional regulator , etc . based on the locations of these genes , the orics in the aforementioned genomes
were predicted reliably , which contains almost all the features of known replication origins in archaeal genomes .
urls that link to ncbi map viewer or ucsc archaeal genome browser ( if available ) are also provided , which will be useful to explore and discover the conserved features around the oric region . with the availability of an increasing number of archaeal genomes ,
the prediction will be more accurate and reliable , as the orb elements or genes frequently close to orics can also be analyzed by comparative genomics , and new rules for replication origins in archaeal genomes will also be extracted in the future with the continuous update of doric . here ,
motif - based sequence analysis tools , the multiple em for motif elicitation ( meme ) suite ( 36 ) , have been used to discover motifs in the replication origins of closely related species , e.g. the archaea from the order thermococcales .
consequently , orb motifs and some new uncharacterized motif sequences have been found by the meme suite and are also included in the database .
to identify oric regions of unannotated bacterial genomes , we have developed a web - based system , ori - finder , based on an integrated method comprising gene identification , analysis of base composition asymmetry using the z - curve method , distribution of dnaa boxes , occurrence of genes frequently close to orics and phylogenetic relationships .
consequently , the predicted oric regions have been organized into an online database , doric .
based on doric , the relationships between the conserved features associated with the oric regions , such as adjacent genes , dnaa boxes , etc . , and
for example , detailed analyses have shown that the consensus sequence of the dnaa boxes in oric regions and the distribution of genes around orics are strongly conserved among the bacteria in the phylum cyanobacteria ( 7,8 ) .
the feature that the oric is adjacent to dnan gene , which encodes the beta clamp processivity factor , has been found to be universal among the bacteria within the phylum cyanobacteria , and the species - specific dnaa box motif for the phylum cyanobacteria is
these strongly conserved features indicate that the in silico identified orics are reliable , as they have been confirmed by comparative genomics approaches .
this observation also shows that if the oric for one of the bacteria in the phylum cyanobacteria is confirmed experimentally , the orics for the other bacterial genomes in this phylum may be confirmed simultaneously . as we expected , the experimentally confirmed replication origins of anabaena sp .
pcc 7120 ( 10 ) and s. elongatus pcc 7942 ( 12 ) in the phylum cyanobacteria are all adjacent to the dnan gene , which encodes the beta clamp processivity factor .
therefore , the proposed rules may be helpful to predict the oric regions for some bacteria without complete genomes in the phylum cyanobacteria .
in addition , the application of the proposed rules derived from doric would speedup the experimental confirmation and functional analysis of orics in bacterial genomes .
because of the rapid growth in the number of sequenced bacterial genomes , the replication origins for those unsubmitted to genbank or not deposited in doric temporarily can be predicted by ori - finder firstly , which now has been used to analyze 30 newly sequenced bacterial genomes .
the z - curve analysis has been used to identify one replication origin in the genomes of methanocaldococcus jannaschii ( 22 ) and methanosarcina mazei ( 23 ) , two replication origins in the halobacterium species nrc-1 genome ( 24 ) , which have been confirmed by in vivo experiments ( 25,26 ) and three replication origins in the sulfolobus solfataricus p2 genome ( 24 ) , which have been later confirmed experimentally ( 27,28 ) . here , we collected the information of orics provided in the literature , such as the oric sequences , origin recognition boxes ( orb ) motifs , uncharacterized motif sequences , etc . , which were identified by in vivo experiments ( 2534 ) , as well as in silico analysis ( 4,2224,35 ) .
in addition , we also predicted some new replication origins by z - curve method , with the aid of homologous sequence search against the known replication origins , analysis of orb motifs and repeats , cdc6 gene location , etc .
consequently , oric regions in 81 archaeal genomes identified by in vivo experiments , as well as in silico analyses , have been added to our database .
the number of orics in archaea is correlated with the phylogeny , which has been summarized in detail in the
, it shows that there is one replication origin in the genomes within the order methanococcales ( 11 genomes ) and within the class thermococci ( 12 genomes ) , and three replication origins in sulfolobus species ( 13 genomes ) .
our results and the z - curves also show that the archaea within the crenarchaeota phylum contain multiple origins , although some origins could not be determined at the sequence level currently .
for example , pyrobaculum calidifontis has been experimentally characterized to contain four replication origins , which is the highest number detected in a prokaryotic organism ( 34 ) .
however , only one origin can be determined at the sequence level ( 34 ) . during the course of the prediction
, we found that the location of some putative replication initiator gene besides cdc6 gene can be helpful to the oric prediction in some cases .
for example , in the genome of m. jannaschii , an orf ( mj0774 ) , annotated as a hypothetical protein , is a distant homolog of the cdc6 protein in fact ( 22 ) .
the name mc - prip for the putative replication initiator protein in methanococcales has been used here for mj0774 and related proteins to distinguish it from bona fide orthologous cdc6 .
we also found the genes , which encode mc - prip in other 10 genomes within the order methanococcales ( methanococcus aeolicus nankai-3 , methanocaldococcus fervens ag86 , methanococcus maripaludis c5 , m. maripaludis c6 , m. maripaludis c7 , m. maripaludis s2 , m. maripaludis x1 , methanococcus vannielii sb , methanococcus voltae a3 and methanocaldococcus vulcanius m7 ) , were annotated as
marr family transcriptional regulator , etc . based on the locations of these genes , the orics in the aforementioned genomes
were predicted reliably , which contains almost all the features of known replication origins in archaeal genomes .
urls that link to ncbi map viewer or ucsc archaeal genome browser ( if available ) are also provided , which will be useful to explore and discover the conserved features around the oric region . with the availability of an increasing number of archaeal genomes ,
the prediction will be more accurate and reliable , as the orb elements or genes frequently close to orics can also be analyzed by comparative genomics , and new rules for replication origins in archaeal genomes will also be extracted in the future with the continuous update of doric . here ,
motif - based sequence analysis tools , the multiple em for motif elicitation ( meme ) suite ( 36 ) , have been used to discover motifs in the replication origins of closely related species , e.g. the archaea from the order thermococcales .
consequently , orb motifs and some new uncharacterized motif sequences have been found by the meme suite and are also included in the database .
with the increased availability of completely sequenced bacterial and archaeal genomes and experimental evidence , the database will become more useful because of including more information .
the application of the rules from the database will be helpful to develop new prediction algorithms of replication origins and speedup the experimental confirmation and functional analysis of orics in bacterial or archaeal genomes .
systematic and functional analysis of oric regions in bacteria and archaeal genomes will also be useful for the construction of the minimum genome and regulation of growth rate and generation time of bacteria and archaea , which play a key role in the emerging field of synthetic biology .
doric will be updated periodically to include more entries , and to integrate more information for each entry .
the national natural science foundation of china [ 31171238 , 30800642 and 10747150 ] . funding for open access charge : the national natural science foundation of china . | replication of chromosomes is one of the central events in the cell cycle .
chromosome replication begins at specific sites , called origins of replication ( orics ) , for all three domains of life .
however , the origins of replication still remain unknown in a considerably large number of bacterial and archaeal genomes completely sequenced so far .
the availability of increasing complete bacterial and archaeal genomes has created challenges and opportunities for identification of their orics in silico , as well as in vivo .
based on the z - curve theory , we have developed a web - based system ori - finder to predict orics in bacterial genomes with high accuracy and reliability by taking advantage of comparative genomics , and the predicted oric regions have been organized into an online database doric , which is publicly available at http://tubic.tju.edu.cn/doric/ since 2007 .
five years after we constructed doric , the database has significant advances over the number of bacterial genomes , increasing about 4-fold .
additionally , oric regions in archaeal genomes identified by in vivo experiments , as well as in silico analyses , have also been added to the database .
consequently , the latest release of doric contains orics for > 1500 bacterial genomes and 81 archaeal genomes , respectively . |
a method is described for isolating clara cells from the mouse lung that does not require the technique of elutriation .
mouse lungs totally perfused of blood are instilled with crystalline trypsin ( 0.25% ) and incubated for the optimum time of 15 min .
the lung tissue is chopped , mechanically agitated , and sequentially filtered to obtain a primary digest of 3 to 5 x 10(6 ) cells .
clara cells , identified routinely by histochemical localization of nadph diaphorase , using the stain nitrotetrazolium blue ( nbt ) , accounts for between 20 to 40% of the cells in the primary digest .
layering the cells of the primary digest on a discontinuous percoll gradient followed by centrifugation gives rise to a major band of cells , 52% that are clara cells ( 0.77 + /-
0.28 x 10(6)/mouse ) .
a second method was devised to purify the clara cells by simply centrifuging ( 32 g , 6 min , 10 degrees c ) the primary digest and discarding the supernatant that contained only a few nbt positive cells . when this process was repeated three times , the final pellet contained 68% clara cells realizing 0.55 + /-
0.16 x 10(6 ) cells / mouse .
the cells have typical clara cell morphology as confirmed by electron microscopy and have a high level of p-450 enzymes ( 7-ethoxycoumarin deethylase and coumarin hydroxylase ) .
furthermore , the primary digests and the purified isolates contain less than 1% alveolar type ii cells , although such cells , identified by the histochemical localization of alkaline phosphatase , can be obtained by a second , more extensive digestion procedure . the simple procedure described for the isolation of mouse clara cells could be further advanced if methods could be devised to prevent the loss of nadph diaphorase activity during enzymatic digestion and cell centrifugation.imagesfigure 1 .
1afigure 1 .
1bfigure 1 .
1cfigure 1 .
1dfigure 1 .
1efigure 1 .
1ffigure 2.figure 3.figure 4.figure 5.figure 6.figure 7.figure 8.figure 9.figure 10.figure 11.figure 12.figure 13 . | |
a 72-year - old woman presented for further examination of a right breast lump . according to the patient , the lump had been present for at least 10 years .
she complained of discomfort of recent onset in the right breast . on physical examination , a soft , mobile , 10 cm mass , which occupied nearly the entire right breast , was palpated . on mammography , a large circumscribed mass surrounded by a water - density capsule
the mass was a mixture of isodense and fat densities and had dystrophic calcifications in a branching pattern .
1a , b ) . on ultrasonography ( us ) , the mass was very heterogeneous and completely encompassed in a thin echogenic pseuduocapsule of compressed breast tissue , which was compatible with hamartoma .
however , a careful us examination revealed an irregular hypoechoic mass of 1.4 cm with a non - parallel orientation within the hamartoma ( fig .
1c ) . a retrospective review of the mammograms revealed focal asymmetry , which correlated with the suspicious mass on us ( fig .
1a , b ) . the lesion was early enhanced and a washout on enhanced mri scans ( fig .
surgical excision for entire mass was done and the diagnosis from the frozen specimen of the suspicious area was idc .
, there was no evidence of local recurrence of the idc or any distant metastasis .
mammary hamartomas , a term applied to breast tumors in 1971 by arrigoni et al .
( 13 ) , have also been referred to as lipofibroadenomas , adenolipofibromas , and fibroadenolipomas ( 1 ) .
hamartomas may present as tender or non - tender palpable lumps , but are often discovered incidentally during a screening mammography ( 1 , 14 ) .
the typical mammographic feature of hamartomas is a circumscribed fibrofatty mass ( 15 ) . on us ,
most mammary hamartomas have circumscribed margins , an oval shape , and heterogeneous internal echogenicity ( 16 , 17 ) .
the clinical , radiologic , and histologic findings of the previously described 14 cases of malignant hamartomas and the current case are summarized in table 1 .
the mean patient age was 56.3 years ( range , 25 - 78 years ) .
the size of the hamartomas range from 1.5 - 12.0 cm in diameter and the size of the associated carcinomas range from 0.3 - 3.5 cm in diameter . of the described 15 cases ,
mammography was obtained in 12 cases , of which 10 showed the typical appearance of hamartomas with suspicious features , such as clusters of microcalcifications , pleomorphic micocalcifications , and spiculated masses .
the remaining two cases had the typical appearance of a hamartoma with no suspicious features , thus co - existing malignancies were unexpected findings at the time of tumorectomy .
us findings were available in only six cases , of which four had suspicious masses with irregular margins , hypoechogenicity , or a non parallel orientation within the hamartomas ; and two cases were diagnosed pre - operatively as carcinomas by us - guided fine needle aspiration or core needle biopsies and underwent one - step curative surgery ( 6 , 7 ) . as stated above , the majority of cases had suspicious findings within the hamartoma on mammography or us . radiologists therefore need to pay careful attention in order to detect subtle suspicious findings , even though mammography or us may show typical hamartomas . among the 15 cases described here , 12 had carcinomas that were confined to the hamartomas and the remaining three cases had carcinomas that involved both the hamartomas and adjacent normal breast tissue . if carcinomas involve both the hamartoma and normal breast tissue , it is difficult to determine whether the carcinoma arises within the hamartoma or an isolated carcinoma which initiates growth nearby later extends into the hamartoma
. however , in the majority of cases described here , including the current case , the carcinomas were located within the hamartomas , ( 2 - 5 , 7 , 8 , 10 - 12 ) thus we believe that the malignancies arose within the hamartomas . in conclusion , breast hamartomas have generally been classified as rare , benign tumors , and carcinomas occur only rarely .
radiologists should recognize that malignancy may co - exist or develop in hamartomas and be alert to the presence of suspicious features within a hamartoma . | mammary hamartomas are typically a benign condition and rarely develop into malignant lesions .
only 14 cases of carcinomas associated with a hamartoma have been documented in the literature . in this case report
, we describe a case of invasive ductal carcinoma within a hamartoma in a 72-year - old woman .
mammography , ultrasonography , and magnetic resonance imaging showed the features of a typical hamartoma with a suspicious mass arising in it .
this case illustrates the importance of identification of unusual findings in a typical mammary hamartoma on radiologic examinations . |
traditionally , urinary catheter has been used in gynecological surgical practice to assess urinary output , to improve surgical exposure , reduce the possibility of injury to the urinary system and to prevent postoperative urinary retention .
however , catheterization is mainly required to keep the bladder out of the operative site and later on until patient becomes ambulatory .
shortest duration of the catheter is now a routine and unlike cesarean infection rate is much low even if kept for 24 - 48 h. although there seems to be no clinical uncertainty about the need for an empty bladder before and during surgery , controversy arises about the ideal time to remove the catheter postoperatively .
few studies reported significantly less pain and urinary tract infection ( uti ) in after immediate removal , whereas others showed that early removal urinary catheter after uncomplicated hysterectomy seem to decrease first ambulation time and hospital stay without posing any threats .
this prospective , randomized controlled trial was conducted in the department of obstetrics and gynecology of a tertiary care referral center from july 2008 to december 2009 .
the aim of this study was to compare the outcomes of immediate removal of urinary catheter versus removal on the first day after abdominal hysterectomy for benign gynecological disease .
informed consent was obtained from enrolled patients and protocol was approved by the institute 's ethical committee .
seventy patients who underwent an uneventful abdominal hysterectomy with or without salpingoophrectomy were included in this study .
patients with anticipated complicated surgical procedure requiring strict fluid replacement postoperatively , bladder suspension or colporraphy surgery , positive or unavailable preoperative urine culture report and co - morbid illness requiring strict intake output monitoring were excluded from the study .
group i immediate removal of the catheter in the operating room.group ii catheter removal after 24 h.
group i immediate removal of the catheter in the operating room .
group ii catheter removal after 24 h. randomization was performed by using a computer generated randomization table and allocation group was kept in sealed envelope .
the operating surgeon was made aware of randomization and accordingly the patient was assigned to one of the two groups . in all cases ,
all patients received one dose of antibiotic prophylaxis at the time of surgery and continued postoperatively as per department protocol .
a fresh catheter sample was taken for the urine microscopy and culture at the time of preoperative catheterization .
later , on the basis of study assignment , catheter was removed either in the operating room immediately or 24 h postoperative .
incidence of symptomatic utis , re - catheterization , subjective pain assessment and febrile morbidity was compared in both groups .
pain was assessed with a pictorial questionnaire that assed the level of pain and location of pain , that is , bladder or urethra versus surgical site .
febrile morbidity was defined as two consecutive oral temperatures of > 100.4f measured 6 h apart .
need of recatheterization was assessed by an inability to pass urine at the end of 12 h , or failure to void after two attempts .
the diagnosis of symptomatic uti was based on the presence of significant bacteriuria accompanied by at least one of the following symptoms : fever , dysuria , increased frequency of urination , urinary urgency , suprapubic pain , and burning micturition . a clear voided midstream urine specimen was obtained on .
a second clean voided midstream urine sample for culture and sensitivity was obtained at 2 week postoperative visit .
all the culture sensitivity reports were collected , recorded and patients with positive culture were treated according to the sensitivity .
group i immediate removal of the catheter in the operating room.group ii catheter removal after 24 h.
group i immediate removal of the catheter in the operating room .
group ii catheter removal after 24 h. randomization was performed by using a computer generated randomization table and allocation group was kept in sealed envelope .
the operating surgeon was made aware of randomization and accordingly the patient was assigned to one of the two groups . in all cases ,
all patients received one dose of antibiotic prophylaxis at the time of surgery and continued postoperatively as per department protocol .
a fresh catheter sample was taken for the urine microscopy and culture at the time of preoperative catheterization .
later , on the basis of study assignment , catheter was removed either in the operating room immediately or 24 h postoperative .
incidence of symptomatic utis , re - catheterization , subjective pain assessment and febrile morbidity was compared in both groups .
pain was assessed with a pictorial questionnaire that assed the level of pain and location of pain , that is , bladder or urethra versus surgical site .
febrile morbidity was defined as two consecutive oral temperatures of > 100.4f measured 6 h apart .
need of recatheterization was assessed by an inability to pass urine at the end of 12 h , or failure to void after two attempts .
the diagnosis of symptomatic uti was based on the presence of significant bacteriuria accompanied by at least one of the following symptoms : fever , dysuria , increased frequency of urination , urinary urgency , suprapubic pain , and burning micturition . a clear voided midstream urine specimen was obtained on . the second postoperative day for culture and sensitivity .
a second clean voided midstream urine sample for culture and sensitivity was obtained at 2 week postoperative visit .
all the culture sensitivity reports were collected , recorded and patients with positive culture were treated according to the sensitivity .
groups were compared by the chi - square test . a p < 0.05 was considered to be statistically significant .
a total of 70 women were enrolled in the study and equally assigned to two groups .
all women had similar clinical characteristics , that is , age , indication for hysterectomy and duration of surgery [ table 1 ] .
distribution according to clinical characteristics of 35 women only 3 ( 8.5% ) required recatheterization after immediate postoperative removal , while no one from late removal group needed to be recatheterized .
twelve women had positive culture at day 2 postoperative out of which nine were from late removal group as compared to three patients of early removal group ( p = 0.222 ) .
out of six culture positive cases at 2 weeks postoperative day , two women were from early removal group and four women from late removal group ( p = 0.532 ) .
pain perception was also higher in late removal group ( 45.7% ) as compared to early group ( 40% ) .
out of eight febrile patients on day 2 postoperative six patients were from group ii while the rest were from group i ( p = 0.133 ) and on day 3 postoperative day seven patient from late removal group were febrile when compared to three women of early removal group [ table 2 ] .
three women out of nine culture positive cases in late removal group had symptoms of uti and fever .
one women at 2 weeks who was positive for culture had a fever and other symptoms of uti .
abdominal hysterectomy for dysfunctional uterine bleeding is now becoming rare as vaginal hysterectomy and total laparoscopic hysterectomy or laparoscopy - assisted vaginal hysterectomy has taken a lead role .
utis comprise of all nosocomial infections and occur after urological instrumentation , usually urinary catheterization , in up to 75% of the patients .
the potential squeal of urinary catheterization include gram negative bacteremia , antimicrobial toxicity , chronic bacteriuria and chronic renal disease . with today 's heightened awareness of costs of medical care a nosocomial infection causing increased length of hospital stay would not only be a significantly morbid event , but also an economic consideration .
hemantha senanayka from colombo showed that elective cesarean in 344 women without catheterization does not compromise the safety or ease of surgery .
it reduces the risk of infection . a prospective randomized control trial conducted on 200 women by onile et al
. showed that immediate removal of catheter after cesarean is associated with a lower rate of uti .
various prospective trials have been conducted in other urogenital surgeries regarding early removal of the catheter . a prospective randomized study conducted on 90 women by sekhavat et al
. in anterior colporraphy showed that symptomatic uti was significantly lower in early catheter - removal group ; also patients in this group reported significantly less pain and voiding disturbances .
this study demonstrated that the immediate removal of an in - dwelling catheter is safe and not associated with adverse effects .
one interesting finding was that only three women in the early catheter - removal group required recatheterization after failing to void and none of these women had positive urine culture .
we are aware that a possible criticism to our findings may lie in the fact that number of patients was limited .
although our findings are strengthened by the fact the surgeon was made aware of the randomization only at the end of the operation , but , a limitation of our study may exist in the fact that the observer of outcome was not blinded to the randomization .
however , it seems unlikely that the parameters evaluated in the study ( rate of recatheterization , symptomatic utis , fever ) were biased by the observer , considering that they were determined on the basis of standardized criteria .
pain perception is a subjective measure , which could result in variations of perceived pain .
early catheter removal after abdominal hysterectomy was associated with a lower incidence of positive urine culture .
furthermore , there was no adverse outcome like significant rate of recatheterization and higher pain perception . | objective : indwelling transurethral catheter is frequently used after gynecological surgeries in order to prevent urinary retention .
there is controversy about the ideal time to remove the catheter after surgery .
this randomized controlled study was undertaken to determine whether the immediate removal of urinary catheter after abdominal hysterectomy affects the rate of symptomatic urinary tract infection ( uti ) , recatheterization , subjective pain perception and febrile morbidity.study design : this prospective randomized controlled trial included 70 women undergoing abdominal hysterectomy with or without salpingoophrectomy for benign diseases .
patients were divided into two equal groups on the basis of timing of removal of urinary catheter ( group i immediate removal after surgery , group ii removal after 24 h and evaluated for benefits versus risks of immediate catheter removal .
the results were compared by the chi - square test.results:recatheterization was required in three patients of immediate removal group and none in late removal group ( p = 0.07 ) .
higher incidence of positive urine cultures ( 25.9% ) and febrile morbidity ( 10% ) was found in group ii when compared to immediate removal group ( 8% ) .
pain perception was not statistically different in both groups ( p = 0.567).conclusions : the early removal of an indwelling catheter after surgery was not associated with an increased rate of febrile events , uti .
pain perception was also lower in early removal group .
although need of recatheterization was higher in early removal group , but not statistically significant . |
during nine months till the middle of december in 2014 , more than 19,000 people were infected by ebola virus ( fig . 1 ) and lots of the patients around 40% were dead by the ebola virus disease ( evd ) .
the outbreak through the year seems to keep increasing the number of cases and deaths unless efficient drugs or vaccines would not be supplied . also whole world
are nervously focusing on the upcoming situation in the ebola virus - affected countries in west africa day by day .
since the first case of evd was reported in march 2014 , the outbreak has continued and the total number of 19,065 patients was reported as the confirmed or suspected in the evd - affected countries , mostly happened in the three outbreak countries ( guinea , liberia , and sierra leone ) .
united states , spain , mali , and two other previously affected west african countries ( nigeria and senegal ) also have reported a few cases . among the cases ,
evd is an acute pathogenic symptom caused by expose to the ebola virus , one of four major hemorrhagic fever viruses ( hfvs ) generating harsh illness .
the rna viruses of these families causing hemorrhagic fever include flaviviridae , arenaviridae , bunyaviridae , and filoviridae ; yellow fever virus and dengue virus are included in flaviviridae , and they are usually transmitted by mosquito as a vector .
lassa virus and several south american hfvs transmitted by small rodent contact are well known pathogens of arenaviridae , and critical members of bunyaviridae may cause the rift valley fever ( rvf ) , crimean - congo hemorrhagic fever ( cchf ) , and hantaan virus is also a member of the family .
the most notorious hfvs are ebola virus and marburg virus which are classified as members of family filoviridae , first ebola virus was found in the area around ebola river in congo in 1976 , and marburg virus was discovered in the german city , marburg in 1967 . however , the ecology and the epidemiology of ebola virus are not clearly understood or natural host is not confirmed either even though they are considered one of the most highly dangerous pathogens and potential threatening to the humankind .
the viral isolates from the patients in guinea this year have high homology ( 98% ) with zaire strain of ebola virus ( zebov ) isolated from the patients of congo and gabon during 1994 - 1995 .
zebov is the most lethal pathogen among five reported ebola viral strains , and it was known to cause over the 90% of fatality in human and primates .
this virus may cause acute symptoms in infected patients from 2 to 21 days after exposed to the virus ; high fever , bleeding , disseminated intravascular coagulation , headache , abdominal pain , non - bloody diarrhea , myalgia , nausea , arthralgia , and malaise . as the diagnostic methods for ebola virus , enzyme - linked immunosorbent assay ( elisa ) and real - time reverse transcription - polymerase chain reaction ( rrt - pcr ) are generally used . while antigen or antibody ( igm / igg ) can be detected by elisa ,
rrt - pcr is the most common and useful diagnosis technique by which the existence of ebola virus - specific genes can be investigated after virus isolation . among these diagnostic methods ,
antibody - capture elisa is limited to be applied for the early diagnosis because antibodies usually do not appear within 1 or 2 week of illness .
virus isolation is also limited to apply for any situation because it requires a biosafety level 4 laboratories to perform this step .
after establishment of diagnostic methods as the first step for the preparedness against evd , therapeutic materials should be acquired to rescue the infected patients and suitable form of vaccine may be required to prevent the population form infection .
however , the development of therapeutics and vaccine for evd is still at the early stage and the proceeding is very slow because the most procedure during the research and development related to ebola virus is always highly risky and requires special protective facilities such as biosafety level 4 laboratories .
many scientists and companies can not perform the research related to selected agents such as ebola virus due to this limit of risk and facility even though they have willing to contribute to the progress of drug and vaccine development .
ebola outbreak in west africa this year asked world to reveal the potential to protect people from the dangerous pathogen , and now we realize there are some positively expected candidates of therapeutics and vaccine .
some of the therapeutics was already used for the treatment of patients in the united states and europe , and several vaccine candidates showing efficacy in animal tests were on the clinical tests in west africa now .
drug development for the evd treatment started around 2002 just after 911 in united states , and it was continuously supported by governmental institutes such as national institute of health ( nih ) , biomedical advanced research and development authority and defense threat reduction agency of united states , also public health agency in canada . based on the financial and technical support from governments , united states and canadian companies
have tried to develop some available forms of anti - ebola therapeutics ( table 1 ) .
first of all , well - known antibody therapeutics named zmapp was under development led by leaf biopharmaceutical inc .
zmapp is an antibody cocktail mixing the humanized mabs with the selected composition of c13c6 from mb-003 ( human - mouse chimeric mabs developed by mapp biopharmaceutical inc . located in san diego , ca , usa ) and c2g4 and c4g7 from zmab ( mouse mabs developed by defyrus located in toronto , canada ) .
both mb-003 and zmab are also cocktails of abs ; mb-003 is a mixture of human and human mouse chimeric mabs ( cl3c6 , h13f6 , and c6d8 ) , and zmab contains three mouse mabs ( m1h3 , m2g4 , and m4g7 ) .
the components of zmapp was produced in the tobacco plant ( nicotiana benthamiana ) at the farmyards using biopharming technology carried out by the kentucky bioprocessing after development funded by defense advanced research projects agency .
zmapp was designed and produced through two major steps : first of all , the mouse was inoculated with ebola virus , then the immunological memory on the mouse lymphocytes by the infection was cloned . as a second part
, cloned genes containing the memory related to ebola virus was transferred to the tobacco plants by transformation using bacterial carrier .
finally plants expressing anti - ebola mabs were cultured in the fields and the antibodies were purified after harvest .
produced zmapp showed enough efficacies to protect chimpanzees from ebola virus , however it was not tested yet before this outbreak in west africa .
zmapp was supplied and helped to rescue the first american ebola - infected patients this summer ; however , another patient in spain was not recovered even the zmapp was treated .
even it seems efficient material to rescue patients , zmapp still has critical limitation to be released to the clinical fields as a common ebola therapeutics due to its slow procedure of production .
in addition to zmapp , there are also several other well - recognized evd therapeutic candidates including tkm - ebola and favipiravir .
tkm - ebola developed by canadian company tekmira is a drug using rnai form designed to block the replication of the ebola virus . though it showed a good efficacy in animal tests and
also applied to clinical phase ii , the potential safety issues may be a limit to be quickly supplied to the clinical fields .
however , the tkm - ebola can be a one of important candidates to be applied for current west african outbreak because large volume of the rnai can be produced within comparatively short period and another product tkm - marburg also showed highly efficient protection in chimpanzee tests from infection of marburg virus .
as small molecules which can be easily synthesized by chemical reaction , favipiravir is considered as a top candidate to be produced for west africa .
favipiravir , also called as abigan of commercial name , is an rna polymerase inhibitor developed by japanese company toyama chemicals ( a subsidiary of fuji film ) and already under the clinical phase iii as a new influenza treatment .
this new drug was quickly focused as an efficient ebola hemorrhagic fever ( ehf ) treatment at the early stage of the outbreak in west africa because the role of rna polymerase also has the most important role during the replication of ebola virus similar to the replication of influenza viruses .
now favipiravir is under the test performed at the nih , and japanese company is ready to send the inhibitors to west africa if the efficacy for evd treatment would be confirmed and japanese government would approve to use .
in addition to the candidates , there is several reports that other antiviral drugs such lamivudine which is an anti - human immunodeficiency virus ( hiv ) drug may show efficacy to help the evd patients .
quick development of efficient drugs to be treated is the most urgent issue to rescue a lot of ebola - infected patients in west africa , and world health organization ( who ) is considering supplying some therapeutic candidates such as tkm - ebola or favipiravir to the ebola - affected countries in africa sooner or later to suppress the situation till the production of an effective vaccine .
because the ebola virus did not frequently generate outbreak , vaccine research and development was not eagerly preceded .
vaccine candidates were just tested at the preclinical levels using animals , and the development of manufacturing process was unpredictable step for the most potentially harmful pathogens such as hfvs including ebola virus .
however , the vaccine research has been continued for long history after virus identification in 1976 and some candidates were already confirmed about the efficacy in chimpanzees .
similar to the other vaccines , live attenuated vaccines was considered and studied as a first candidate ; efficacy and safety were tested using nonhuman primates as primary animal models .
however , the results did not discover any effective vaccine candidates even in the level of animal model .
even though there was ebola vaccine studies continued for last decade , the progress was not so fast or productive because the most procedure for the development of vaccines related to selected agents such as ebola virus has many limitations about the safety facilities and personal expertise .
it is very difficult for the private research groups or companies to handle the selected agents or maintain the high safety level facilities , and usually the most steps of research were performed by limited agencies .
recently , some vaccine approaches using dna vaccine type containing ebola antigen - coding genes announced positive results in animals .
vaccine research center at nih and developed an efficient candidate with adenovirus vector , and british research group at cambridge university studied another candidate for the veterinary vaccine in variable animal models to protect primates from ebola virus in africa while early animal study of live attenuated vaccine with guinea pigs were not satisfied to be effective form of vaccine .
dna vaccine as ebola virus vaccine candidate was tried during early 2000s and several positive results were suggested as possible direction . in dna vaccine type for evd prevention ,
inserted nucleoprotein or glycoprotein ( gp ) of ebola virus acts as an antigen to induce host immune responses and inserted antigen - delivering dna may boosts the immunization .
the dna vaccine candidate was continuously studied using animal models such as guinea pigs or chimpanzees with results of high protection efficacy . however
the efficacy in primates including human was not as high as the level of rodents though the plasmid containing gp maintained protective memory for long time in animals when ebola virus was challenged . therefore many research groups including nih vaccine research center tried to develop priming protocol to improve efficacy , and now there are two selected protocols based on the combination of viral vectors to remarkably enhance the immune responses in human and nonhuman primates ( table 1 ) .
after long time efforts related to develop vaccine platform using recombinant chimpanzee adenovirus vector for ebola vaccine , okairos ( acquired by gsk ) manufactured cad3 ebola vaccine ( recombinant chimpanzee adenovirus serotype 3 vectored ebola vaccine ) and this candidate has been tested for clinical phase i by gsk .
adenovirus vector has been considered as an effective platform for the dna vaccine for a wide range of infectious pathogens such as hiv and tuberculosis , and the vaccine research center of nih studied availability of cad3 ebola vaccine containing ebola gp gene to be expressed in hosts after vaccination .
chimpanzee adenovirus vector seems to be safe because the animal vector do not replicate in human hosts and the cad3 already showed hopeful result that protected all 16 animals from ehf after single vaccination .
there are two adenovirus vector - based ebola vaccine candidates ; monovalent vaccine against only zaire strain , and bivalent vaccine against zaire strain and sudan strain .
the monovalent vaccine is the candidate scheduled on september 2014 for the phase i clinical test in west africa .
recently , trial using vesicular stomatitis virus ( vsv ) platform designed as bivalent vaccine against ebola and marburg viruses revealed systemic immune responses protecting animals after injection .
this non - segmented , negative stranded rna virus is also considered as a promising candidate for the recombinant dna vaccine platform against many filoviruses because the virus is an animal pathogen which usually does not induce any severe symptoms in human , and the most advanced form using vsv for ebola vaccine is vsvg - zebov vaccine developed and sponsored by the public health agency of canada and newlink genetics corporation .
the vsvg - zebov vaccine contains highly attenuated recombinant vsv with substituted ebola virus zaire envelope gp and it can be cultured quickly for high titer .
the vsvg - zebov vaccine candidate also revealed the 100% protection efficacy in animals , furthermore this form of vaccine was administrated for a postexposure human patient injured by laboratory accident in german research group .
nih keeps the vaccine research for 10 years using various formats and announced clinical test in west africa based on these results last september . who and whole world may expect successful process by the 2015 for the ebola vaccine development .
the first clinical trial in ebola - affected area was scheduled in october 2014 and the results will be shown sooner or later . even though the evd is still in outbreak and keeps spreading in africa ,
there are no effective vaccines to protect people or no approved therapeutics to rescue the infected patients either .
it seems long way to stop the current outbreak in africa , also to clearly extinguish the threatening generated by the highly fatal pathogens such as ebola virus .
however , saving stockpile of vaccines after quick process of development will be the most effective way to prepare the crises related to the biological agents . as a warning of evd outbreak which has lethal fatality just after infection , we have to recognize the request for vaccine development with huge scope of potential pathogens which can be threatening in the future .
vaccination should be ultimate responsiveness against outbreak while the efficient therapeutics is urgently requested for the treatment of patients in the affected countries .
it is the best strategy to quickly make current vaccine development successful to prevent the world from spread of ebola virus . | since the first case of ebola virus disease ( evd ) in guinea was reported in march 2014 by world health organization ( who ) , the outbreak has continued through the year and the total number of 19,065 patients was reported as the confirmed or suspected in the evd - affected countries . among the cases , 7,388 patients were reported death by 19 december .
currently , available therapeutics to treat the infected patients or vaccines to prevent people from infection is not developed yet while viral diagnostic methods were already developed and firmly established in a lot of countries as a first step for the preparedness of ebola outbreak .
some potential therapeutic materials including zmapp were supplied and the treated people got over the evd .
several candidates of vaccines also were investigated their efficacy in animal models by national institute of health ( nih ) and department of defense , and they are processing of clinical tests in west africa aiming to finish the development by the 2015 . vaccine and therapeutic development is essential to stop the evd outbreak in west africa , also to protect the world from the risk which can be generated by potential spread of ebola virus . |
we previously studied generalization of odorants in the carpenter ant camponotus aethiops . by using an associative learning protocol known as the maxilla - labium extension response ( maler ) , we conditioned individual ants by training them to for example substance a , and afterwards tested their response to a mixture of a and a novel substance b. ants trained to substance a treated a mixture of a and b as similar , but surprisingly individuals trained to b did not generalize toward the mixture of a and b , indicating that generalization between stimuli is not only influenced by the similarity of molecular structure of the odorants involved , but also by the animal s previous experience .
while asymmetry in generalization itself has previously been found in many organisms , especially in the olfactory modality ( e.g. ) , most of these studies either used very few substances , or substances that greatly differed in their molecular structure or function ( e.g. , floral odour vs. alarm pheromone ) , making it impossible to draw conclusions about the molecular basis of the asymmetry . in order
to address this question , our experimental setup allowed us to describe how molecular structure systematically plays a role in predicting whether the ants would generalize or not .
the ants consistently generalized from molecules with a long carbon chain to mixtures containing this trained substance and a molecule with a shorter chain .
however , when trained to a molecule with a short chain , no generalization to a mixture containing this trained substance and a molecule with a longer chain was found . additionally , we elucidated a hierarchy of stimuli regarding functional groups , where ants trained to alcohols generalized to aldehydes and ketones of similar or shorter length . when trained to aldehydes
, ants would generalize to ketones of similar or shorter length , but not to alcohols .
lastly , when trained to ketones ants would not generalize to molecules with other functional groups at all .
this asymmetry thus follows an inclusion criterion ( a term adapted from guerrieri et al . ) , where substances with shorter chain lengths or certain functional groups are treated as if similar to the trained substance .
this raises the question about whether the inclusion criterion is a general phenomenon in the animal world , or whether it is exclusive to ants .
two pioneering studies by smith and menzel and guerrieri et al . in honeybees allowed for extensive analysis of asymmetry in generalization with regards to molecular structure , as both studies used different volatile floral odours varying in either chain length or functional group .
although asymmetry in generalization was found in these studies , it could not systematically be predicted by molecular structure , thus not supporting the inclusion criterion .
however , in these studies the test stimulus was always a single substance instead of a mixture of the conditioned stimulus and the test stimulus .
in these conditions , ants also show no evidence of the inclusion criterion . as generalization to a mixture after training to a single substance has , to my knowledge , not been studied in a systematic way in other animals , it is currently impossible to draw conclusions on how widespread the inclusion criterion is . in ants , a similar phenomenon of inclusion appears to come into play in nestmate recognition , where non - nestmates ( enemies ) are aggressed only if their cuticular chemical profile contains additional substances in comparison to the profile of the discriminating individual , but are not recognized when they have less substances .
indeed , some social parasites use this to their advantage , and are known to be chemically insignificant , having only a very small amount of substances on their cuticle , which ensures that they are not recognized by the host colony .
this implies that the inclusion criterion might be a general phenomenon , at least in ants .
now that we established that , at least in the carpenter ant c. aethiops , asymmetry in generalisation follows an inclusion criterion , the question remains about whether it arises from the perception or action component of communication .
if inclusion arises at the perception component , the receiving individual does not notice the difference between the trained odorant and the substance or mixture that it generalizes toward .
however , this does not necessarily mean the odour is not detected by the sensilla , as inclusion could potentially result from processes at different levels of the perception component .
intuitively , one could predict that these proteins might also bind smaller substances due to the size of their binding pocket ( while not binding larger molecules than the one they are tuned to ) . in turn , generalization toward a molecule smaller than the conditioned stimulus might be expected , as these proteins would bind to both to the trained substance , as well as smaller ones .
however , evidence points in the other direction , where sensory proteins tuned to a certain substance bind , next to the molecule they are tuned to , larger molecules better than smaller ones .
if larger molecules indeed activate proteins tuned to smaller molecules but not the other way around , a binary mixture containing a large and small molecule could activate the same sensory proteins as the large molecule alone , potentially explaining our results .
alternatively , not the binding pocket , but the chemical attributes of the odorants might cause asymmetry in generalization .
for example , there is a difference in the capability of alcohols and aldehydes / ketones to form hydrogen bonds .
the oxygen in all of these classes of molecules can build a hydrogen bond with the hydrogen atoms of the sensory protein , but only alcohols can also build a hydrogen bond with the protein s oxygen through the hydrogen in the alcohol s -oh group .
this might cause alcohols to bind better than aldehydes and ketones , possibly explaining the hierarchy of stimuli found in our study , where generalization occurred from alcohols to aldehydes and ketones , but not the other way around .
every odour elicits a unique pattern ( much like a barcode ) in the antennal lobe by activating a specific subset of glomeruli . if higher chain length molecules elicit the same pattern as their lower chain length counterparts , but activate some additional glomeruli in the process , this could give rise to asymmetry in generalisation , as the activation of additional compounds seems to enable discrimination , whereas the deactivation of many glomeruli does not .
however , smaller molecules do not necessarily activate less glomeruli , at least in the honey bee .
furthermore , mixtures are not always represented in the antennal lobe as the sum of their parts due to mixture interactions .
because of this , odour blends can acquire a unique quality , where sometimes not all information about the individual components is perceptually available anymore .
a map of the antennal lobe , including activation patterns of many different odorants and their mixtures would be required to test this hypothesis in ants .
unfortunately , due to the complex morphology of the ant antennal lobe , full mapping remains extremely challenging .
another potential cause for the inclusion criterion arising at the perception component could be inhibition due to overshadowing . overshadowing occurs when an individual encounters a mixture , but only perceives a subset of that mixture .
for example , if ants trained to e.g. , 2-hexanone are tested with a mixture of 2-hexanone and octanol , reaction might be inhibited if octanol overshadows 2-hexanone ( i.e. , the ant mainly perceives octanol , even though 2-hexanone is also present in the mixture ) . however , this seems to be an unlikely cause of our results , as we also conducted an overshadowing experiment by training ants to a mixture and afterwards testing their reaction to the individual components , which suggested that overshadowing is rare in c. aethiops . instead of the perception component , the inclusion criterion could arise at the action component .
this would imply that the animal receives all the information needed in order to discriminate between different substances , but follows certain decision rules resulting in asymmetrical generalisation .
our experiment showed that ants could perceive each tested substance , making it likely that the action component is the cause for the inclusion criterion .
however , as previous experience to a single substance can adjust processes in the perception component ( through for example sensory adaptation ) , more experimental data are needed in order to tease apart the two components from each other .
more species should be studied in order to find out whether our study species ( camponotus aethiops ) is unique with respect to the inclusion criterion or not . as learning paradigms
are well established for many different animals , a systematic investigation , including many substances of different chain - lengths and functional groups , of the reaction of individuals to a mixture of the conditioned stimulus and a novel odorant would elucidate whether the inclusion criterion is widespread or not . if the ant is indeed the exception and one of the only groups of animals that uses the inclusion criterion , this would suggest ( but not prove ) that it arises from the action component , as the olfactory pathway ( which is part of the perception component ) is well preserved between different groups of animals . whether the inclusion criterion is adaptive or not is not clear .
one potential benefit for ants could be during recognition of nestmates vs. non - nestmates .
as their cuticular chemical profile consists of many different substances , the inclusion criterion could
compact this profile into something simpler to interpret by treating multiple different substances as similar .
this hypothesis is supported by recent evidence in the argentine ant ( linipithema humile ) .
ants were exposed to a filter paper containing their own nestmate odour , to which they were not aggressive .
when one of eight synthetic hydrocarbons ( differing in branch position , chain length , or both ) was added to this filter paper , the ants started to be aggressive .
the authors found functional homologs , where hydrocarbons differing in chain length ( but not in the position of the functional group ) received equal levels aggression , whereas this was not the case when comparing aggression levels against hydrocarbons differing in the position of the functional group . | animals constantly face the challenge of extracting important information out of their environment , and for many animals much of this information is chemical in nature .
the ability to discriminate and generalize between chemical stimuli is extremely important and is commonly thought to depend mostly on the structural similarity between the different stimuli .
however , we previously provided evidence that in the carpenter ant camponotus aethiops , generalization not only depends on structural similarity , but also on the animal s previous training experience . when individual ants were conditioned to substance a , they generalized toward a mixture of a and b. however , when trained to substance b , they did not generalize toward this mixture , resulting in asymmetrical generalization .
this asymmetry followed an inclusion criterion , where the ants consistently generalized from a molecule with a long carbon chain to molecules with a shorter chain , but not the other way around . here
i will review the evidence for the inclusion criterion , describe possible proximate mechanisms underlying this phenomenon as well as discuss its potential adaptive significance . |
influenza data in africa is scarce , and only 18 countries among the 46 in the whoafro region are able to perform laboratory diagnosis of influenza as previously reported .
,
in april 2009 , a sentinel network for influenza surveillance was set up in niamey , the capital city of niger .
niger was one of the last countries affected by the recent a / h1n1 ( 2009 ) pandemic .
in temperate countries , influenza transmission is influenced by temperature and humidity that explains its seasonality .
in tropical countries ,
contrary to humid tropical countries , niger is a sahelian country where dryness predominates over a very short rainy season .
the effect of temperature at low values of humidity on influenza transmission is not clearly understood .
better knowledge of the role of climatic factors could help when forecasting the occurrence of influenza epidemics , so as to limit their health impact .
this work is therefore aimed at studying the link between some climatic factors , particularly temperature and humidity , and the occurrence of influenza in niamey .
a sentinel surveillance network was set up in niamey in april 2009 and comprised of 10 sentinel sites .
patients with influenza like illness ( ili ) or severe acute respiratory illness ( sari ) consulting at these sites were recruited between june 1 , 2009 and may 31 , 2010 . who clinical case definitions were used for ili and sari .
a nasopharyngeal swab was sampled from each recruited patient and collected into a standard virus transport medium .
detection and typing were performed according to the procedures of the national influenza centre , unit of molecular genetics of respiratory viruses , pasteur institute , paris .
in niger ,
the rainy season is from june to september , the hot season from october to november , the cold season from december to february and the very hot season from march to may .
time series of daily counts of influenza cases and climatic factors were linked using a generalized additive model .
minimal and maximal temperature , minimal and maximal relative humidity , wind speed and visibility routinely collected were analyzed ( visibility is defined as the maximal distance from which a fixed object or light can be clearly seen ) .
trend , seasonality , day of the week , holidays , religious festivals , and pilgrimage were taken into account in the model .
the package mgcv ( multifold generalized crossvalidation ) was used to control seasonal and longterm temporal trends and to minimize autocorrelation in the residuals .
,
the quality of the model adjustment was checked using partial autocorrelation function , residuals and predicted data versus observed data .
the choice of the final model was based on the akaike information criteria ( aic ) .
a total of 320 patients consulting at the sentinel sites were recruited . among them , 76 ( 23.8% ) were confirmed positive for influenza and consisted of 46 a / h1n1(2009 ) , 19 a / h3n2 and 11 influenza b , with the median age of 7 ( interquartile range = 216 years ) , 6 ( interquartile range = 214.5 years ) and 7 years ( interquartile range = 4.58 years ) , respectively .
the 244 negative patients had a median of age of 5.5 years ( interquartile range = 1.827.3 years ) .
thirtynine per cent ( 39% ) of the patients recruited during the cold season were confirmed positive for influenza as well as for 34.4% , 20.2% , and 15.8% during the rainy , the very hot , and the hot seasons respectively ( figure 1 ) .
all the a / h1n1 ( 2009 ) cases were detected between february 2 and april 8 , 2010 .
distribution of the different influenza viruses by season in niamey , niger ( 20092010 ) .
proportion of positive cases for influenza and description of climatic factors according to the seasons q1 , first quartile ; q3 , third quartile .
hot season : 1st october to 14 december 2009 . * * cold season : 15 december 2009 to 13 february 2010 . * * * very hot season : 14 february to 31 may 2010 .
influenza cases increased significantly with minimal temperatures between 13 and 22c ( = 3.99 , se = 1.74 , p = 0.022 ) .
increase in influenza cases occurred with visibility 9500 m , values encountered during the rainy season ( = 3.30 , se = 1.34 , p = 0.014 ) .
the preliminary results indicated that temperature and visibility were significantly correlated with the occurrence of influenza in niamey .
the majority of influenza cases were detected at low minimal temperatures during the cold season and the rainy season .
.
with guinea pig model infected by a / h3n2 virus ( influenza a / panama/2007/99 virus ) supports this result .
indeed , high values for maximal temperatures ( > 30c ) are observed almost all year around in niamey .
davidwest and cook observed that in nigeria , major outbreaks of ili and sari occurred at the lowest temperatures when the dry and dusty harmattan winds blow from november to march .
in this study , the transmission was efficient even at very low values of relative humidity observed during the cold season .
however , a study by hanley and borup showed that below 20% of relative humidity and irrespective of temperature , influenza transmission remains poorly known .
influenza was predominant during the rainy season when visibility was observed at its highest values and in climatic conditions closer to those which are observed in humid tropical countries .
visibility was found to be correlated with occurrence of influenza cases but was in fact probably more of a surrogate factor . indeed during rainy days , close contact favors virus transmission likely because people stay home for longer periods of time . in this work ,
relative humidity was not retrieved as a significant climatic factor contrary to studies on the incidence of influenza a virus in argentina and hong kong .
,
the role of humidity on influenza transmission efficiency is not well established in the tropics .
indeed , influenza generally leads to symptoms similar to malaria , and patients frequently treat themselves or consult the healthcare facilities only in case of serious disease .
second , the results of this analysis concerned only the main healthcare facilities within niamey , the capital city of niger .
the patients recruited in the study , however , came from different quarters of niamey and concerned all socioeconomic classes ( data not shown ) .
these results are in favor of a predominant seasonality of influenza during the cold and rainy seasons . during these seasons ,
positive patients were more frequently detected even though the influenza virus could circulate all year round in niamey .
this work provides a new insight into higher influenza transmission during low temperatures , even at the lowest values of relative humidity .
further work is ongoing in niger to provide more information regarding the link between climatic factors and influenza , as well as its seasonality . | please cite this paper as : jusot jfranois et al .
( 2011 ) influenza transmission during a oneyear period ( 20092010 ) in a sahelian city : low temperature plays a major role .
influenza and other respiratory viruses doi : 10.1111/j.17502659.2011.00286.x.this work aimed at studying the link between some climatic factors and the occurrence of influenza in niamey , niger .
patients with influenza like illness or severe acute respiratory illness were recruited through a sentinel network . a nasopharyngeal swab was sampled and tested for influenza viruses a and b by rtpcr .
time series of daily counts of influenza cases and climatic factors were linked using a generalized additive model . among the 320 patients recruited ,
76 were confirmed positive for influenza .
influenza cases increased significantly with minimal temperatures and high visibility .
this work brings some valuable explanation to the impact of low temperatures on influenza transmission . |
in a one - shot prisoner 's dilemma ( pd ) game , the two players have to choose between c and d ( to cooperate resp . to defect ) .
following the notation in rapoport and chammah ( 1965 ) , the payoff matrix is given by(1 ) in which the four payoff variables represent the reward for mutual cooperation r , the sucker 's payoff s , the temptation to defect t , and the punishment for mutual defection p. payoffs satisfy the inequalities t > r > p > s , such that defection is a dominant strategy , but mutual cooperation is preferred over mutual defection .
in addition to these inequalities , we shall also assume that(2)2r > t+s , such that mutual cooperation is unanimously preferred from a group perspective.1 in such cases , experimental evidence suggests that many players want to achieve conditional cooperation .
they are willing to play c , provided the co - player also plays c ( see , e.g. , fehr and fischbacher , 2003 ; yamagishi et al . , 2005 ) .
thus , players either have to trust their co - player , or else use their dominant strategy .
the situation is different for an iterated pd game ( ipd ) . diverse folk theorems state that any feasible and individually rational outcome can be sustained as an equilibrium if the probability of a further round is sufficiently large .
such outcomes can be enforced in various ways , and under a wide range of circumstances ( see , e.g. , friedman , 1971 ; aumann , 1981 ; aumann and shapley , 1994 ; fudenberg and maskin , 1986 ; kalai , 1990 ; myerson , 1991 ; mailath and olszewski , 2011 ) .
if subjects can make binding commitments ahead of the game , then analogous results can be obtained even for one - shot games ( kalai et al . , 2010 ) .
experimental research has uncovered considerable heterogeneity in human social preferences ( colman , 1995 ; kagel and roth , 1997 ; camerer , 2003 ) , and a similar variety can be found among the strategies that are played in the ipd ( milinski and wedekind , 1998 ; dal b and frchette , 2011 ; fudenberg et al . , 2012 ) .
players who in the one - shot game would opt for conditional cooperation should be willing to engage in partner strategies .
such strategies aim for an average payoff r per round , which necessarily provides the same payoff r for the co - player ; should the co - player not go along , however , then the co - player 's payoff will be less than r. thus , a partner strategy appeals to the co - player 's self - interest in order to further the own self - interest .
it is fair , and provides an incentive for the co - player to also be fair .
in contrast , some players tend to view their co - player as a rival , rather than a partner .
the main purpose , for such competitive players , is to do better , or at least as well as the other player .
a preference for dominating the co - player is particularly likely in the context of a game , which often has antagonistic connotations .
the aim of the present manuscript is two - fold : first , we are going to characterize all memory - one strategies which are either competitive , or partner strategies in the sense above .
we emphasize that players using such strategies can enforce their preferences against all comers , since we impose no restrictions on the strategies used by the co - players . for partner strategies , the corresponding results in the limiting case of an ipd without discounting
have been obtained within the last two years ( akin , 2013 ; stewart and plotkin , 2013 , 2014 ) . here
, we are going to extend the theory by allowing for discount factors 1 ( which may either be interpreted as the constant continuation probability of having another round , or as the players ' common discount rate on future payoff streams ) .
the recent progress was stimulated by the unexpected discovery of so - called zero - determinant ( zd ) strategies , a class of memory - one strategies enforcing a linear relationship between the payoffs of the two players , irrespective of the co - player 's strategy ( press and dyson , 2012 ) .
in particular , zd strategies can fix the co - player 's payoff to an arbitrary value between p and r ; or ensure that the own
surplus ( over the maximin value p ) is twice as large as the co - player 's surplus ; etc . also for zd strategies
, we are going to extend the theory to the case when future payoffs are discounted , and 1 .
our article does not focus on equilibrium behavior ( in particular , we do not aim to explore which payoffs rational players can achieve ) . instead , we define some interesting properties that a player 's strategy may have ( e.g. , being competitive ) ; and then we are going to characterize all memory - one strategies that have the respective property ( independent of whether such a strategy can be sustained as an equilibrium ) .
thereby , we do not make any assumptions on the behaviors of the co - players ( e.g. , we do not require them to play best responses , or to follow a predefined equilibrium path ) .
nevertheless , there are natural connections between several of the described strategy classes and equilibrium behavior , and in that case we will discuss these connections in detail . in the discussion , we will briefly review the previous development , and in particular the relevant findings in evolutionary game theory ( stewart and plotkin , 2012 , 2013 , 2014 ; adami and hintze , 2013 ; hilbe et al .
, 2013a , 2013b ; akin , 2013 ; szolnoki and perc , 2014a , 2014b ; wu and rong , 2014 ) .
in a nutshell , these findings say that in the context of populations of adapting players , partner strategies do well , whereas competitive strategies fare poorly .
we consider the standard setup of an ipd with perfect monitoring . in each round ,
that is , they choose an action from their respective action set ai={c , d } , with i{i , ii}. the outcome of a given round t can then be described by an action profile ata = aiaii .
after each round , both players observe the chosen action profile , and they receive the respective payoffs as specified in the payoff matrix ( 1 ) .
the round t history is a vector ht=(a0,a1, ,at1)at , and the set of possible histories is the union h=t=0at , with the initial history a0 being defined as the null set a0={}. a strategy for player i is a rule that tells the player how to act after any possible history ; that is , a strategy is a map i : h(ai ) , where (ai ) denotes the set of probability distributions over the action set ai.2 for given strategies of the two players , let va(t ) denote the probability that the resulting action profile played in round t is a{cc , cd , dc , dd}. for convenience , we use the following vector notation:(3)v(t)=(vcc(t),vcd(t),vdc(t),vdd(t))gi=(r , s , t , p)gii=(r , t , s , p ) using this notation , we can write the players ' expected payoffs in round t as i(t)=giv(t ) and ii(t)=giiv(t ) . for a discount factor <1 , the expected payoffs of the repeated game can then be defined by the abelian means(4)i=(1)t=0ti(t)=giv , and similarly ii = giiv , where v=(vcc , vcd , vdc , vdd ) refers to the ( abelian ) mean distribution(5)v=(1)t=0tv(t ) . in the limiting case =1 ,
the payoff per round is given by the cesaro mean(6)i = lim1+1t=0i(t ) ( if this limit exists ) , and a similar expression for ii.3 a theorem by frobenius states that if the cesaro mean exists , it is the limit of the abelian mean , for 1 . in the following , we will sometimes focus on players who only take the decisions in the previous round into account .
definition 1a strategy is a memory - one strategy if (ht)=(ht ) for all histories ht=(a0,
a strategy is a memory - one strategy if (ht)=(ht ) for all histories ht=(a0,
,at1 ) and ht=(a0, ,at1 ) with t , t1 and at1=at1 . for rounds
t1 , the move of a memory - one player is therefore solely determined by the action profile played in the previous round ( in particular , we note that such players do not condition their behavior on the round number , as sometimes considered in models of bounded recall , e.g. mailath and olszewski , 2011 ) .
such memory - one strategies can be written as a 5-tuple p=(pcc , pcd , pdc , pdd;p0 ) .
the continuation vector p:=(pcc , pcd , pdc , pdd ) denotes the conditional probabilities to cooperate in rounds t1 , depending on the outcome of the previous round ( slightly abusing notation , we let the first letter in the subscript refer to the player 's own action in the previous round , and the second letter to the co - player 's action . using this convention
, we ensure that the interpretation of a memory - one strategy does not depend on whether the player acts as player i or as player ii , see nowak and sigmund , 1995 ) .
examples of memory - one strategies include alld=(0,0,0,0;0 ) , tit for tat ( 1,0,1,0;1 ) , or win - stay , lose - shift ( 1,0,0,1;1 ) , see sigmund ( 2010 ) for a comprehensive discussion .
when both players apply a memory - one strategy , the resulting mean distribution v can be calculated explicitly ( nowak and sigmund , 1995 ) : if player i uses the memory - one strategy p=(pcc , pcd , pdc , pdd;p0 ) against a player ii with memory - one strategy
q=(qcc , qcd , qdc , qdd;q0 ) , then(7)v=(1)v(0)(i4m)1 , where v(0)=(p0q0 , p0(1q0 ) , ( 1p0)q0 , ( 1p0)(1q0 ) ) is the initial distribution , i4 is the 44 identity matrix , and m is the transition matrix of the markov chain,(8)m=(pccqccpcc(1qcc)(1pcc)qcc(1pcc)(1qcc)pcdqdcpcd(1qdc)(1pcd)qdc(1pcd)(1qdc)pdcqcdpdc(1qcd)(1pdc)qcd(1pdc)(1qcd)pddqddpdd(1qdd)(1pdd)qdd(1pdd)(1qdd ) ) .
but even if only one of the players is using a memory - one strategy p , there is still a powerful relationship between p and the resulting mean distribution v. lemma 1suppose player i applies a memory - one strategy
p , and let the strategy of player ii be arbitrary , but fixed.(i)in the case with discounting
( <1 ) , let
v
denote the mean distribution of the repeated game .
then(9)(pcc1)vcc+(pcd1)vcd+pdcvdc+pddvdd=(1)p0 ,
or in vector notation ,
( pg0)v=(1)p0 , where
g0=(1,1,0,0).(ii)in the case without discounting , we have(10)lim1+1t=0(pg0)v(t)=0 .
in particular , if the cesaro mean distribution
v
exists ,
( pg0)v=0
. suppose player i applies a memory - one strategy
p , and let the strategy of player ii be arbitrary , but fixed.(i)in the case with discounting
( <1 ) , let
v
denote the mean distribution of the repeated game .
then(9)(pcc1)vcc+(pcd1)vcd+pdcvdc+pddvdd=(1)p0 ,
or in vector notation ,
( pg0)v=(1)p0 , where
g0=(1,1,0,0).(ii)in the case without discounting , we have(10)lim1+1t=0(pg0)v(t)=0 .
in particular , if the cesaro mean distribution
v
exists ,
( pg0)v=0 . in the case with discounting
( <1 ) , let
v
denote the mean distribution of the repeated game .
then(9)(pcc1)vcc+(pcd1)vcd+pdcvdc+pddvdd=(1)p0 ,
or in vector notation ,
( pg0)v=(1)p0 , where
g0=(1,1,0,0 ) . in the case without discounting , we have(10)lim1+1t=0(pg0)v(t)=0 .
in particular , if the cesaro mean distribution
v
exists ,
( pg0)v=0
proofsuppose <1 , and let qi(t ) denote the probability that player i cooperates in round t. then qi(t)=g0v(t ) and qi(t+1)=pv(t ) .
it follows that w(t):=qi(t+1)qi(t ) is given by(11)w(t)=(pg0)v(t ) . multiplying each w(t ) by ( 1)t and summing up over t=0,
( 11),(13)(1)t=0tw(t)=(1)t=0t(pg0)v(t)(pg0)v as both limits need to coincide , we have confirmed eq . ( 9 ) . for the case without discounting , an analogous calculation as in eq . ( 12 ) yields(14)1+1t=0w(t)0 , whereas eq .
it follows that the limit of 1+1t=0(pg0).v(t ) for exists and equals zero .
suppose <1 , and let qi(t ) denote the probability that player i cooperates in round t. then qi(t)=g0v(t ) and qi(t+1)=pv(t ) .
it follows that w(t):=qi(t+1)qi(t ) is given by(11)w(t)=(pg0)v(t ) . multiplying each w(t ) by ( 1)t and summing up over t=0,
( 11),(13)(1)t=0tw(t)=(1)t=0t(pg0)v(t)(pg0)v as both limits need to coincide , we have confirmed eq . ( 9 ) . for the case without discounting , an analogous calculation as in eq . ( 12 ) yields(14)1+1t=0w(t)0 , whereas eq . ( 13 ) becomes(15)1+1t=0w(t)=1+1t=0(pg0)v(t ) .
it follows that the limit of 1+1t=0(pg0).v(t ) for exists and equals zero .
it is worthwhile to stress the generality of lemma 1 : it neither makes any assumption on the strategy used by the co - player , nor does it depend on the specific payoff constraints of a prisoner 's dilemma . in the limiting case =1 , lemma 1 allows
a geometric interpretation : the mean distribution v ( if it exists ) is orthogonal to pg0 ( see akin , 2013 ) .
definition 2a player 's strategy is nice , if the player is never the first to defect .
a player 's strategy is cautious if the player is never the first to cooperate .
a player 's strategy is nice , if the player is never the first to defect .
a player 's strategy is cautious if the player is never the first to cooperate . for memory - one strategies , nice strategies
, the strategy tft
( 1,0,1,0;1 ) is nice , whereas the defector 's strategy alld
( 0,0,0,0;0 ) is cautious .
lemma 2if
2r > t+s , then payoffs satisfy
i+ii2r
if and only if
i=ii = r
( which for
<1
holds if and only if both players are nice ) .
similarly , if
2p < t+s , then
i+ii2p
if and only if
i=ii = p
( which for
<1
is equivalent to both players being cautious ) .
if
2r > t+s , then payoffs satisfy
i+ii2r
if and only if
i=ii = r
( which for
<1
holds if and only if both players are nice ) .
similarly , if
2p < t+s , then
i+ii2p
if and only if
i=ii = p
( which for
<1
is equivalent to both players being cautious ) .
this requires both players to cooperate in every round ( if =1 , it only requires the players to cooperate in almost every round ) .
similarly , for a prisoner 's dilemma with 2p < t+s , the inequality i+ii2p implies vdd=1 .
this requires both players to cooperate in every round ( if =1 , it only requires the players to cooperate in almost every round ) .
similarly , for a prisoner 's dilemma with 2p < t+s , the inequality i+ii2p implies vdd=1 .
definition 3(i)a partner strategy for player i is a nice strategy such that , irrespective of the co - player 's strategy,(16)i < rii < r.(ii)a competitive strategy for player i is a strategy such that , irrespective of the co - player 's strategy,(17)iii .
( i)a partner strategy for player i is a nice strategy such that , irrespective of the co - player 's strategy,(16)i < rii < r.(ii)a competitive strategy for player i is a strategy such that , irrespective of the co - player 's strategy,(17)iii .
a partner strategy for player i is a nice strategy such that , irrespective of the co - player 's strategy,(16)i < rii < r .
a competitive strategy for player i is a strategy such that , irrespective of the co - player 's strategy,(17)iii .
the definition of partner strategies implies that these strategies are best replies to themselves , and thus they are nash equilibria . even more , because condition ( 16 ) is equivalent to ( iir)(ir ) , we can conclude due to lemma 2 that ( iir)(i=ii = r ) .
thus , no matter which best reply the co - player applies , a player with a partner strategy will always obtain the mutual cooperation payoff r. on the other hand , players with a competitive strategy always obtain at least the co - player 's payoff .
it is easy to see that for <1 a competitive strategy needs to be cautious ( otherwise the focal player would be outcompeted by an alld - player ) . in the limiting case =1 , competitiveness is closely related to the concept of being unbeatable , as introduced by duersch et al .
a strategy for player i is unbeatable , if against any co - player and for any number of rounds , the payoff differential t=0(ii(t)i(t ) ) is bounded from above ( in particular , if the average payoffs per round converge to i and ii , then iii ) .
proposition 1for a player i with a nice memory - one strategy
p , the following are equivalent:(i)p
is a partner strategy;(ii)if the co - player uses either alld or the strategy
( 0,1,1,1;0 ) , then
ii < r;(iii)the two inequalities
b1<0
and
b2<0
hold , with(18)b1=(tr)pdd(rp)(1pcd)+(1)(tr)b2=(tr)pdc(rs)(1pcd)+(1)(tr ) .
for a player i with a nice memory - one strategy
p , the following are equivalent:(i)p
is a partner strategy;(ii)if the co - player uses either alld or the strategy
( 0,1,1,1;0 ) , then
ii < r;(iii)the two inequalities
b1<0
and
b2<0
hold , with(18)b1=(tr)pdd(rp)(1pcd)+(1)(tr)b2=(tr)pdc(rs)(1pcd)+(1)(tr ) .
p
is a partner strategy ; if the co - player uses either alld or the strategy
( 0,1,1,1;0 ) , then
ii < r ; the two inequalities
b1<0
and
b2<0
hold , with(18)b1=(tr)pdd(rp)(1pcd)+(1)(tr)b2=(tr)pdc(rs)(1pcd)+(1)(tr ) .
then the definition of partner strategies implies that i=ii = r . since all players use memory - one strategies
, this would require that everyone cooperates after mutual cooperation , which is neither true for alld=(0,0,0,0;0 ) nor for the strategy ( 0,1,1,1;0).(ii ) ( iii)against a player using a nice memory - one strategy p ( with p0=pcc=1 ) , the payoff of an alld co - player is given by(19)ii=(1)t+pppcd+tpdd1+(pddpcd ) .
we note that this payoff is also defined when =1 , because pcd<1 ( otherwise p would satisfy p0=pcc = pcd=1 , and player i would always cooperate . in that case ,
an alld co - player would receive t > r , which is ruled out by ( ii ) ) .
elementary algebra yields(20)b1=(1+(pddpcd))(iir ) , in particular , b1 has the same sign as iir . on the other hand , if the co - player uses the strategy ( 0,1,1,1;0 ) , the co - player 's payoff is(21)ii=(1)t+s+((1)rs)pcd+(t+r)pdc1+2(pdcpcd)+pdc , and(22)b2=(1+2(pdcpcd)+pdc)(iir ) .
therefore , b2 has the same sign as iir.(iii ) ( i)suppose that b1<0 and b2<0 , and that iir . we need to show that ii=i = r . as iir=(giir1)v , with 1=(1,1,1,1 )
, we note that iir is equivalent to(23)(tr)vcd(rs)vdc(rp)vdd0 . using the linear equations 1v=1 and ( pg0)v=(1 ) ( lemma 1 with p0=pcc=1 , since the memory - one strategy is nice ) , we calculate vcd as a function of vdc and vdd:(24)vcd=(1(1pdc)))vdc+(1(1pdd))vdd(1pcd). the denominator of vcd is positive , as b1<0 implies pcd<1 . plugging ( 24 ) into ( 23 ) and multiplying both sides with
( 1pcd) shows that iir if and only if(25)b2vdc+b1vdd0 , with b1 and b2 as defined in ( 18 ) .
thus , the assumptions b1<0 and b2<0 indicate that vdc = vdd=0 , and by ( 24 ) that vcd=0 . we conclude that vcc=1 , and therefore i=ii = r , i.e.
then the definition of partner strategies implies that i=ii = r . since all players use memory - one strategies
, this would require that everyone cooperates after mutual cooperation , which is neither true for alld=(0,0,0,0;0 ) nor for the strategy ( 0,1,1,1;0).(ii ) ( iii)against a player using a nice memory - one strategy p ( with p0=pcc=1 ) , the payoff of an alld co - player is given by(19)ii=(1)t+pppcd+tpdd1+(pddpcd ) .
we note that this payoff is also defined when =1 , because pcd<1 ( otherwise p would satisfy p0=pcc = pcd=1 , and player i would always cooperate . in that case ,
an alld co - player would receive t > r , which is ruled out by ( ii ) ) . elementary algebra yields(20)b1=(1+(pddpcd))(iir )
, in particular , b1 has the same sign as iir . on the other hand , if the co - player uses the strategy ( 0,1,1,1;0 ) , the co - player 's payoff is(21)ii=(1)t+s+((1)rs)pcd+(t+r)pdc1+2(pdcpcd)+pdc , and(22)b2=(1+2(pdcpcd)+pdc)(iir ) .
therefore , b2 has the same sign as iir.(iii ) ( i)suppose that b1<0 and b2<0 , and that iir .
, we note that iir is equivalent to(23)(tr)vcd(rs)vdc(rp)vdd0 . using the linear equations 1v=1 and ( pg0)v=(1 ) ( lemma 1 with p0=pcc=1 , since the memory - one strategy is nice )
, we calculate vcd as a function of vdc and vdd:(24)vcd=(1(1pdc)))vdc+(1(1pdd))vdd(1pcd). the denominator of vcd is positive , as b1<0 implies pcd<1 . plugging ( 24 ) into ( 23 ) and multiplying both sides with
( 1pcd) shows that iir if and only if(25)b2vdc+b1vdd0 , with b1 and b2 as defined in ( 18 ) .
thus , the assumptions b1<0 and b2<0 indicate that vdc = vdd=0 , and by ( 24 ) that vcd=0 . we conclude that vcc=1 , and therefore i=ii = r , i.e.
then the definition of partner strategies implies that i=ii = r . since all players use memory - one strategies
, this would require that everyone cooperates after mutual cooperation , which is neither true for alld=(0,0,0,0;0 ) nor for the strategy ( 0,1,1,1;0 ) . against a player using a nice memory - one strategy p ( with p0=pcc=1 ) , the payoff of an alld co - player is given by(19)ii=(1)t+pppcd+tpdd1+(pddpcd ) .
we note that this payoff is also defined when =1 , because pcd<1 ( otherwise p would satisfy p0=pcc = pcd=1 , and player i would always cooperate . in that case ,
an alld co - player would receive t > r , which is ruled out by ( ii ) ) . elementary algebra yields(20)b1=(1+(pddpcd))(iir )
, in particular , b1 has the same sign as iir . on the other hand , if the co - player uses the strategy ( 0,1,1,1;0 ) , the co - player 's payoff is(21)ii=(1)t+s+((1)rs)pcd+(t+r)pdc1+2(pdcpcd)+pdc , and(22)b2=(1+2(pdcpcd)+pdc)(iir ) .
as iir=(giir1)v , with 1=(1,1,1,1 ) , we note that iir is equivalent to(23)(tr)vcd(rs)vdc(rp)vdd0 . using the linear equations 1v=1 and ( pg0)v=(1 ) ( lemma 1 with p0=pcc=1 , since the memory - one strategy is nice ) , we calculate vcd as a function of vdc and vdd:(24)vcd=(1(1pdc)))vdc+(1(1pdd))vdd(1pcd). the denominator of vcd is positive , as b1<0 implies pcd<1 . plugging ( 24 ) into ( 23 ) and multiplying both sides with (
1pcd) shows that iir if and only if(25)b2vdc+b1vdd0 , with b1 and b2 as defined in ( 18 ) .
thus , the assumptions b1<0 and b2<0 indicate that vdc = vdd=0 , and by ( 24 ) that vcd=0 . we conclude that vcc=1 , and therefore i=ii = r , i.e.
for example , tft is a partner strategy if and only if >trtp and >trrs , whereas wsls is a partner strategy if and only if >trrp and >trts , which is a sharper condition .
in analogy to the definition of partner strategies , one may define a mild partner strategy for player i as a nice strategy such that ir implies iir , irrespective of the co - player 's strategy.4 for memory - one strategies , the characterization of mild partner strategies is analogous to the characterization of partner strategies ( only the strict inequalities in proposition 1 need to be replaced by weak inequalities ) .
the existence of an equilibrium with individually rational payoffs ( i,ii ) in the ipd is typically shown by applying trigger strategies any deviation from the equilibrium path is punished with relentless defection ( as for example in friedman , 1971).5 the following corollary states that trigger strategies are , in some sense , the most effective means to enforce a cooperative equilibrium in the ipd .
corollary 1for a given prisoner 's dilemma and a given continuation probability , there exists a memory - one partner strategy if and only if the trigger strategy grim
= ( 1,0,0,0;1 )
is a partner strategy .
proofthe two quantities b1 and b2 in proposition 1 are minimal for pcd = pdc = pdd=0 .
thus , if there is a memory - one strategy that meets the inequalities b1<0 and b2<0 , then the corresponding inequalities are also met by grim .
for a given prisoner 's dilemma and a given continuation probability , there exists a memory - one partner strategy if and only if the trigger strategy grim
= ( 1,0,0,0;1 )
is a partner strategy .
the two quantities b1 and b2 in proposition 1 are minimal for pcd = pdc = pdd=0 .
thus , if there is a memory - one strategy that meets the inequalities b1<0 and b2<0 , then the corresponding inequalities are also met by grim .
from corollary 1 , we may also conclude that partner strategies exist if and only if >trtp ( this condition for the existence of fully cooperative equilibria has been previously derived by roth and murnighan , 1978 ; stahl , 1991 ) .
let us next give a characterization of competitive memory - one strategies : proposition 2suppose player i applies the memory - one strategy
p. then the following are equivalent:(i)p
is competitive.(ii)if the co - player uses either alld or the strategy
( 0,0,0,1;0 ) , then
iii.(iii)the entries of
p
satisfy
p0=pp=0
and
(pcd+pdc)1 .
suppose player i applies the memory - one strategy
p. then the following are equivalent:(i)p
is competitive.(ii)if the co - player uses either alld or the strategy
( 0,0,0,1;0 ) , then
iii.(iii)the entries of
p
satisfy
p0=pp=0
and
(pcd+pdc)1 . if the co - player uses either alld or the strategy
( 0,0,0,1;0 ) , then
iii .
proof(i ) ( ii)follows immediately from the definition , a competitive strategy yields iii against any co - player.(ii ) ( iii)if player ii applies alld , then an explicit calculation of payoffs yields(26)iii=(ts)((1)p0+pdd)1+(pddpcd ) , and thus iii implies p0=pdd=0 .
similarly , if player ii applies the strategy ( 0,0,0,1;0 ) , we obtain ( using p0=pdd=0):(27)iii=(ts)(1(pcd+pdc))1+(1(1+)pcd+pdc ) .
this is non - negative if and only if (pcd+pdc)1.(iii ) ( i)by lemma 1 and as pdd = p0=0,(28)pdcvdc=(1pcc)vcc+(1pcd)vcd . using the inequality pdc1pcd
( i ) ( ii)follows immediately from the definition , a competitive strategy yields iii against any co - player.(ii ) ( iii)if player ii applies alld , then an explicit calculation of payoffs yields(26)iii=(ts)((1)p0+pdd)1+(pddpcd ) , and thus iii implies p0=pdd=0 .
similarly , if player ii applies the strategy ( 0,0,0,1;0 ) , we obtain ( using p0=pdd=0):(27)iii=(ts)(1(pcd+pdc))1+(1(1+)pcd+pdc ) .
this is non - negative if and only if (pcd+pdc)1.(iii ) ( i)by lemma 1 and as pdd = p0=0,(28)pdcvdc=(1pcc)vcc+(1pcd)vcd . using the inequality pdc1pcd
follows immediately from the definition , a competitive strategy yields iii against any co - player . if player ii applies alld , then an explicit calculation of payoffs yields(26)iii=(ts)((1)p0+pdd)1+(pddpcd ) , and thus iii implies p0=pdd=0 .
similarly , if player ii applies the strategy ( 0,0,0,1;0 ) , we obtain ( using p0=pdd=0):(27)iii=(ts)(1(pcd+pdc))1+(1(1+)pcd+pdc ) .
this is non - negative if and only if (pcd+pdc)1 . by lemma 1 and as pdd
pdc1pcd , this leads to(29)(1pcd)vdc(1pcc)vcc+(1pcd)vcd , or equivalently ( 1pcd)(vdcvcd)(1pcc)vcc . this implies vdcvcd . as a consequence , iii=(gigii)v=(ts)(vdcvcd)0 .
2 shows the space of partner strategies ( and the space of competitive strategies ) as subsets of the nice memory - one strategies ( cautious memory - one strategies ) , respectively .
one can also define the dual properties , and derive the corresponding characterizations : a strategy for player i is said to be submissive if payoffs always satisfy iii , irrespective of the strategy of player ii ; and a cautious strategy for player i is said to be requiting if i > p implies ii > p ( see fig . 1 for a schematic representation of these strategy classes ) .
the corresponding characterizations are : proposition 3suppose player i applies the memory - one strategy
p. then the following are equivalent:(i)p
is submissive;(ii)if the co - player uses either allc or the strategy
( 0,1,1,1;1 ) , then
iii;(iii)the entries of
p
satisfy
p0=pcc=1
and
(1pcd)+(1pdc)1 .
suppose player i applies the memory - one strategy
p. then the following are equivalent:(i)p
is submissive;(ii)if the co - player uses either allc or the strategy
( 0,1,1,1;1 ) , then
iii;(iii)the entries of
p
satisfy
p0=pcc=1
and
(1pcd)+(1pdc)1 .
if the co - player uses either allc or the strategy
( 0,1,1,1;1 ) , then
iii ; the entries of
p
satisfy
p0=pcc=1
and
(1pcd)+(1pdc)1 .
then , for a player i with a cautious memory - one strategy
p , the following are equivalent:(i)p
is requiting;(ii)if the co - player uses either allc or the strategy
( 0,0,0,1;1 ) , then
ii > p;(iii)the two inequalities
b1>0
and
b2>0
hold , with(30)b1=(rp)pdc+(ps)pcc(ps),b2=(tp)pdc+(ps)pcd(ps ) .
then , for a player i with a cautious memory - one strategy
p , the following are equivalent:(i)p
is requiting;(ii)if the co - player uses either allc or the strategy
( 0,0,0,1;1 ) , then
ii > p;(iii)the two inequalities
b1>0
and
b2>0
hold , with(30)b1=(rp)pdc+(ps)pcc(ps),b2=(tp)pdc+(ps)pcd(ps ) . if the co - player uses either allc or the strategy
( 0,0,0,1;1 ) , then
ii > p ; the two inequalities
b1>0
and
b2>0
hold , with(30)b1=(rp)pdc+(ps)pcc(ps),b2=(tp)pdc+(ps)pcd(ps ) .
the previous results have highlighted how lemma 1 can be used to characterize several interesting strategy classes within the space of memory - one strategies ( for example , the strategies that allow a player to outcompete the opponent , or the strategies that provide incentives to reach the social optimum ) . in the following ,
we present another application of lemma 1 : there are strategies with which a player can unilaterally enforce a linear relationship between the players ' payoffs .
definition 4a memory - one strategy p is said to be a zd strategy if there exist constants ,, such that(31)p=gi+gii+((1)p0)1+g0 .
a memory - one strategy p is said to be a zd strategy if there exist constants ,, such that(31)p=gi+gii+((1)p0)1+g0 .
proposition 5let
<1 , and suppose player i applies a memory - one strategy
p
satisfying eq .
the same relation holds for
=1 , provided that the payoffs
i
and
ii
exist.6 let
<1 , and suppose player i applies a memory - one strategy
p
satisfying eq .
the same relation holds for
=1 , provided that the payoffs
i
and
ii
exist.6 proofthis follows directly from lemma 1 , using the identities i = giv , ii = giiv , and 1=1v .
this follows directly from lemma 1 , using the identities i = giv , ii = giiv , and 1=1v .
we proceed with a slightly different representation of zd strategies , using the parameter transformation = , = , and =(1).7 under this transformation , zd strategies take the form(33)p=[(1)(1gi)+(gigii)](1)p01+g0 , and the enforced payoff relationship according to ( 32 ) becomes(34)ii=(i ) .
( 34 ) implies that the payoffs lie on a line segment intersecting the diagonal at some value ( the payoff for the zd - strategy against itself ) and having a slope ( see fig .
3 ) . players can not use zd strategies to enforce arbitrary payoff relationships of the form ( 34 ) : since the entries of the continuation vector p correspond to conditional probabilities ( and hence need to be in the unit interval ) , the parameters , and need to obey certain restrictions .
definition 5for a given , we call a payoff relationship ( ,)r2
enforceable if there are r and p0 such that each entry of the continuation vector p according to eq .
we refer to the set of all enforceable payoff relationships as e. for a given , we call a payoff relationship ( ,)r2
enforceable if there are r and p0 such that each entry of the continuation vector p according to eq .
we refer to the set of all enforceable payoff relationships as e. proposition 6(i)the set of enforceable payoff relationships is monotonically increasing in the discount factor : if
, then
ee.(ii)there is a
<1
such that
( ,)e
if and only if
1<<1
and(35)max{p , st1}min{r , ts1 } ,
with at least one inequality in
( 35 )
being strict .
( i)the set of enforceable payoff relationships is monotonically increasing in the discount factor : if
, then
ee.(ii)there is a
<1
such that
( ,)e
if and only if
1<<1
and(35)max{p , st1}min{r , ts1 } ,
with at least one inequality in
( 35 )
being strict .
the set of enforceable payoff relationships is monotonically increasing in the discount factor : if
, then
ee. there is a
<1
such that
( ,)e
if and only if
1<<1
and(35)max{p , st1}min{r , ts1 } ,
with at least one inequality in
( 35 )
being strict .
proof(i)according to the definition , ( ,)e if and only if one can find r and p0 , such that the corresponding continuation vector p according to eq .
( 33 ) satisfies 0p1 , or equivalently,(36a)(1)(1p0)(1)(r)1(1)p0(36b)(1)(1p0)[(1)(s)+ts]1(1)p0(36c)(1)p0[(1)(t)+ts]+(1)p0(36d)(1)p0(1)(p)+(1)p0 we note that in ( 36a)(36d ) , the left hand side is monotonically decreasing in , whereas the right hand side is monotonically increasing in . in particular , if the conditions ( 36 ) are satisfied for some 1 they are also satisfied for any .(ii)()suppose ( ,)e , and therefore the conditions ( 36 ) hold for appropriate parameters and p0 . summing up the first inequality in ( 36a ) and the first inequality in ( 36d ) shows(37)1(1)(rp ) .
similarly , by taking the inequalities in ( 36b ) and ( 36c ) , we get(38)1(1+)(ts ) . in particular , 0<(1 ) and 0<(1+ ) , and therefore >0 and 1<<1 .
if none of the inequalities in ( 35 ) was strict , then ( 36a ) or ( 36b ) would require p0=1 , whereas ( 36c ) or ( 36d ) would require p0=0.()conversely , let 1<<1 , and suppose max{p , st1}<min{r , ts1}. then the inequalities ( 39 ) hold for any choice of >0 , with the first two inequalities being strict . in particular , we can choose a sufficiently small such that each term on the right hand 's side of ( 39 ) is bounded from above by 1/2 . by setting p0=0 and choosing a sufficiently close to one , it thus follows that all inequalities in ( 36 ) can be satisfied .
an analogous argument holds when =min{r , ts1 } , in which case one needs to set p0=1 .
( i)according to the definition , ( ,)e if and only if one can find r and p0 , such that the corresponding continuation vector p according to eq .
( 33 ) satisfies 0p1 , or equivalently,(36a)(1)(1p0)(1)(r)1(1)p0(36b)(1)(1p0)[(1)(s)+ts]1(1)p0(36c)(1)p0[(1)(t)+ts]+(1)p0(36d)(1)p0(1)(p)+(1)p0 we note that in ( 36a)(36d ) , the left hand side is monotonically decreasing in , whereas the right hand side is monotonically increasing in . in particular , if the conditions ( 36 ) are satisfied for some 1 they are also satisfied for any .(ii)()suppose ( ,)e , and therefore the conditions ( 36 ) hold for appropriate parameters and p0 . summing up the first inequality in ( 36a ) and the first inequality in ( 36d ) shows(37)1(1)(rp ) .
similarly , by taking the inequalities in ( 36b ) and ( 36c ) , we get(38)1(1+)(ts ) . in particular , 0<(1 ) and 0<(1+ ) , and therefore >0 and 1<<1 .
moreover , the conditions ( 36 ) imply(39)0(1)(r)0[(1)(s)+ts]0[(1)(t)+ts]0(1)(p ) . since >0 and <1 , these conditions are equivalent to condition ( 35 ) .
if none of the inequalities in ( 35 ) was strict , then ( 36a ) or ( 36b ) would require p0=1 , whereas ( 36c ) or ( 36d ) would require p0=0.()conversely , let 1<<1 , and suppose max{p , st1}<min{r , ts1}. then the inequalities ( 39 ) hold for any choice of >0 , with the first two inequalities being strict .
in particular , we can choose a sufficiently small such that each term on the right hand 's side of ( 39 ) is bounded from above by 1/2 . by setting p0=0 and choosing a sufficiently close to one , it thus follows that all inequalities in ( 36 ) can be satisfied .
an analogous argument holds when =min{r , ts1 } , in which case one needs to set p0=1 .
according to the definition , ( ,)e if and only if one can find r and p0 , such that the corresponding continuation vector p according to eq . ( 33 ) satisfies 0p1 , or equivalently,(36a)(1)(1p0)(1)(r)1(1)p0(36b)(1)(1p0)[(1)(s)+ts]1(1)p0(36c)(1)p0[(1)(t)+ts]+(1)p0(36d)(1)p0(1)(p)+(1)p0 we note that in ( 36a)(36d ) , the left hand side is monotonically decreasing in , whereas the right hand side is monotonically increasing in . in particular , if the conditions ( 36 ) are satisfied for some 1 they are also satisfied for any . ( )suppose ( ,)e , and therefore the conditions ( 36 ) hold for appropriate parameters and p0 .
summing up the first inequality in ( 36a ) and the first inequality in ( 36d ) shows(37)1(1)(rp ) .
similarly , by taking the inequalities in ( 36b ) and ( 36c ) , we get(38)1(1+)(ts ) . in particular , 0<(1 ) and 0<(1+ ) , and therefore >0 and 1<<1 .
moreover , the conditions ( 36 ) imply(39)0(1)(r)0[(1)(s)+ts]0[(1)(t)+ts]0(1)(p ) . since >0 and <1 , these conditions are equivalent to condition ( 35 ) .
if none of the inequalities in ( 35 ) was strict , then ( 36a ) or ( 36b ) would require p0=1 , whereas ( 36c ) or ( 36d ) would require p0=0.()conversely , let 1<<1 , and suppose max{p , st1}<min{r , ts1}. then the inequalities ( 39 ) hold for any choice of >0 , with the first two inequalities being strict . in particular , we can choose a sufficiently small such that each term on the right hand 's side of ( 39 ) is bounded from above by 1/2 . by setting p0=0 and choosing a sufficiently close to one , it thus follows that all inequalities in ( 36 ) can be satisfied .
an analogous argument holds when =min{r , ts1 } , in which case one needs to set p0=1 .
suppose ( ,)e , and therefore the conditions ( 36 ) hold for appropriate parameters and p0 . summing up the first inequality in ( 36a ) and the first inequality in ( 36d ) shows(37)1(1)(rp ) .
similarly , by taking the inequalities in ( 36b ) and ( 36c ) , we get(38)1(1+)(ts ) . in particular , 0<(1 ) and 0<(1+ ) , and therefore >0 and 1<<1 .
moreover , the conditions ( 36 ) imply(39)0(1)(r)0[(1)(s)+ts]0[(1)(t)+ts]0(1)(p ) . since >0 and <1 , these conditions are equivalent to condition ( 35 ) .
if none of the inequalities in ( 35 ) was strict , then ( 36a ) or ( 36b ) would require p0=1 , whereas ( 36c ) or ( 36d ) would require p0=0 .
conversely , let 1<<1 , and suppose max{p , st1}<min{r , ts1}. then the inequalities ( 39 ) hold for any choice of >0 , with the first two inequalities being strict . in particular
, we can choose a sufficiently small such that each term on the right hand 's side of ( 39 ) is bounded from above by 1/2 . by setting p0=0 and choosing a sufficiently close to one , it thus follows that all inequalities in ( 36 ) can be satisfied .
an analogous argument holds when =min{r , ts1 } , in which case one needs to set p0=1 .
the first part of proposition 6 shows that a given linear payoff relationship of the form ( 34 ) is easier to enforce when players are sufficiently patient . as 1 , the limiting set of enforceable payoff relationships ( , )
there are various remarkable subclasses of zd strategies ( as depicted in fig . 3 and fig .
, we encounter so - called equalizer strategies ( see boerlijst et al . , 1997 ; press and dyson , 2012 ) . by eq .
( 34 ) , player i can make use of such strategies to prescribe as payoff for player ii .
a player can thus determine the opponent 's payoff ( however , player i can not fix the own payoff , since this would require to be unbounded , which is ruled out by proposition 6 ) .
press and dyson ( 2012 ) also highlighted the class of extortion strategies ( with =p and 0<<1 ) .
extortion strategies guarantee that the own surplus over the minimax payoff p exceeds the opponent 's surplus by a factor of 1 . moreover , since >0 , the payoffs of the two players are positively related . hence , to maximize the own payoff , player ii needs to maximize player i 's payoff : the best response against an extortioner is to cooperate unconditionally . as a counterpart to extortioners , stewart and plotkin ( 2012 ) defined the class of generous strategies , which satisfy eq . ( 34 ) with =r and 0<<1 .
players using a generous strategy shoulder a larger burden of the loss ( with respect to the social optimum r ) than their co - player . since >0
, they also ensure that the payoffs of the two players are aligned , thereby motivating the co - player to cooperate .
finally , for games without discounting it was noted that strategies with =1 enforce i=ii ( for =1 , tft is an example of such a fair strategy , see press and dyson , 2012 ; hilbe et al . , 2014b ) . however , as proposition 6 shows , fair strategies cease to exist when future payoffs are discounted , and only approximately fair strategies ( with close to one ) may be feasible .
zd strategies can also be connected to the strategy classes discussed in the previous section .
generous strategies , for example , are exactly the zd strategies which are submissive partner strategies ( in particular it follows that every generous strategy is a nash equilibrium of the ipd ) . on the other hand , for stage games with 2p < t+s ( which ensures i+ii2p ) , extortion strategies are precisely those zd strategies which are requiting and competitive .
we note that herein , we have entirely focused on the repeated prisoner 's dilemma , due to the central role that this simple game situation takes in the literature on the evolution of cooperation ( rapoport and chammah , 1965 ; trivers , 1971 ; sugden , 1986 ; axelrod , 1984 ; sigmund , 2010 ) .
however , the proofs of lemma 1 and proposition 5 did not require any assumptions on the payoff values ( and in the proof of proposition 6 , we have only made use of the assumptions r > p and t > s ) .
moreover , for =1 , it was recently shown that similar results can also be obtained for stage games with 2 actions but n2 players ( hilbe et al . , 2014b ) .
thus , while we believe that our results are most intuitive in the context of a prisoner 's dilemma , the mathematics can be extended to more general strategic situations .
the recent development began with the paper of press and dyson ( 2012 ) introducing zd strategies for repeated games without discounting . in this context , press and dyson derived the linear relation ( 32 ) .
their proof was based on a neat formula for the payoffs achieved if both players use memory - one strategies .
press and dyson highlighted those zd strategies that fix the co - player 's payoff to a given value between p and r , as well as the sinister properties of extortion strategies .
they also stressed the fact that more complex strategies ( based on larger memories , for instance ) are not able to profit from their sophistication to gain the upper hand .
the intriguing aspects of zd strategies raised considerable attention ( see , e.g. , ball , 2012 ) . in the news section of the american mathematical society , it was stated that ' the world of game theory is currently on fire . ' a more skeptical view could be found among economists .
the well - known folk theorem for repeated games states that trigger strategies can induce a rational co - player to agree to any feasible payoff pair above the minimax level p , by threatening to switch to relentless defection otherwise ( see aumann , 1981 ; kalai , 1990 ; fudenberg and maskin , 1986 , 1990 ) . seen from this angle , the progress consisted merely in displaying memory - one strategies with a similar power to enforce specific payoff pairs .
however , there is a subtle difference : whereas the folk theorems are based on the assumption that players wish to maximize their payoffs , the results presented herein are independent of such an assumption .
interpreted in this way , we have explored how much control player i can exert on the resulting payoffs without being sure about the motives of player ii .
memory - one strategies able to fix the co - player 's payoff had already been derived in boerlijst et al .
( 1997 ) and sigmund ( 2010 ) , based on an approach different from that of press and dyson ( 2012 ) .
( 2013a ) to provide another derivation of ( 32 ) , not involving any determinants .
it was substantially extended by akin ( 2013 ) to yield a general equation for the mean distribution of memory - one strategies when =1 . in this case , the mean distribution is understood in the sense of cesaro , and need not always exist . in lemma 1 , we have extended this approach to cover the case <1 , from which akin 's result for =1 immediately follows .
the vector p consists of the conditional probabilities to play c in the next round ( is the probability that there is a next round ) , whereas g0 can be viewed as ' conditional probability ' to play c in the current round . in the limit of no discounting , lemma
1 states that no matter which strategy player ii is using , the limiting distribution v ( if it exists ) is on a hyperplane orthogonal to the difference of these two conditional probabilities .
it was also akin ( 2013 ) who extended the investigations beyond the case of zd - strategies , to characterize partner strategies for =1 ( calling them ' good ' strategies , a term we feel is too general ) . in a comments article ,
stewart and plotkin ( 2012 ) introduced an example of a generous strategy , and showed that in a round robin tournament conducted after the fashion of axelrod ( 1984 ) , this generous strategy emerged as winner .
stewart and plotkin also asked whether zd strategies were relevant for evolutionary game theory . in this context , one considers a population of players , each equipped with a strategy .
the players are then allowed to imitate other strategies , preferentially those with a higher payoff ( see , e.g. , weibull , 1995 ; samuelson , 1997 ; hofbauer and sigmund , 1998 ; nowak , 2006 ; sandholm , 2010 ) .
it is obvious that extortion strategies can not spread too much in such an evolutionary context ; if they become too common , they are likely to encounter their own , which bides ill .
if player i obtains twice the surplus of ii , and ii twice the surplus of i , each surplus is zero .
however , hilbe et al . ( 2013a ) showed that extortion strategies can pave the way for the emergence of cooperative strategies , similar to tft ( which , for =1 , can be regarded as a limiting case of an extortion strategy , press and dyson , 2012 ) .
this catalytic role of extortion strategies has also been confirmed for games on networks , in which players only interact within a small neighborhood ( szolnoki and perc , 2014a , 2014b ; wu and rong , 2014 ) .
overall , these studies confirm that extortionate strategies have problems to succeed within a population .
however , if the games are played between members of two distinct populations for instance , between hosts organisms and their symbionts then extortion strategies can emerge in whichever population is slower to adapt ( hilbe et al .
. the slower rate of evolution acts as a commitment device . in effect , the slowly evolving organism becomes the stackelberg leader in a sequential game , in which the slow player learns to adopt extortion strategies , whereas the faster evolving player learns to play the best response , and to cooperate unconditionally ( bergstrom and lachmann , 2003 ; damore and gore , 2011 ; gokhale and traulsen , 2012 ) . but even in a one - population setup , certain zd strategies prove successful : stewart and plotkin showed that evolutionary trajectories often visit the vicinity of generous strategies .
the dynamics leads from extortion to generosity ( the title of stewart and plotkin , 2013 ) .
this is also confirmed , by analytical means based on adaptive dynamics , by hilbe et al .
remarkably , stewart and plotkin ( 2013 , 2014 ) derived a characterization of all memory - one strategies which are robust in an evolutionary sense , for given population size n. this means that the replacement probability , as a resident strategy , is at most 1/n ( which is the probability to be replaced if the mutant is neutral ) . in the limit of weak selection , which roughly means
that the choice between two strategies is only marginally influenced by payoff ( see nowak et al .
, 2004 ) , all robust zd strategies need to be generous ( stewart and plotkin , 2013 ) .
these predictions have also been tested in a recent behavior experiment , in which human subjects played against various computer opponents ( hilbe et al . , 2014a ) .
although extortionate programs outcompeted their human co - players in every game , generous programs received , on average , higher payoffs against the human subjects than extortionate programs .
humans were hesitant to give in to extortion ; although unconditional cooperation would have been their best response in all treatments , they only became more cooperative over time if their co - player was generous .
intriguingly , if a player uses a generous strategy and the co - player does not go along , then the focal player will always shoulder a larger part of the loss ( with respect to the mutual cooperation payoff r ) . despite their forbearance ,
generous strategies do very well which is not the least of the surprises offered by the iterated prisoner 's dilemma game . | within the class of memory - one strategies for the iterated prisoner 's dilemma , we characterize partner strategies , competitive strategies and zero - determinant strategies .
if a player uses a partner strategy , both players can fairly share the social optimum ; but a co - player preferring an unfair solution will be penalized by obtaining a reduced payoff . a player using a competitive strategy never obtains less than the co - player .
a player using a zero - determinant strategy unilaterally enforces a linear relation between the two players ' payoffs .
these properties hold for every strategy used by the co - player , whether memory - one or not . |
hypothyroidism is among the common endocrine diseases accounting for 2 - 15% of diseases in the general population . in india
hypothyroidism in general is a prominent hypometabolic state and sympathetic activities are anticipated to be less in this condition as sympathetic activation is a common manifestation of hypermetabolic state such as hyperthyroidism .
however , sympathovagal imbalance ( svi ) due to increased sympathetic activity has been reported in hypothyroidism .
in addition , one report indicates that autonomic neuropathy in hypothyroidism is due to increased vagal tone that partly subsides with thyroxine therapy .
we have recently reported that svi in hypothyroidism in females is due to sympathetic activation and vagal withdrawal .
increased parasympathetic tone is beneficial for cardiac health and poor vagal tone is associated with increased cardiovascular morbidities . though cardiovascular morbidities are not uncommon in hypothyroidism , the pathophysiologic mechanisms of cardiovascular dysfunctions in hypothyroidism has not yet been fully elucidated .
chronic svi , inflammation , obesity and hyperlipidemia are reported to be associated with cardiovascular risks ( cvrs ) . though obesity is a common clinical feature of hypothyroidism and obesity
has been reported to induce autonomic imbalance , until date no study has assessed the link of obesity to svi in hypothyroidism . there is a report of increased cvrs in hypothyroidism and recently one report has suggested that insulin resistance and increased plasma level of insulin , c - peptide and lipoproteins in hypothyroid patients increases their risk for cardiovascular diseases .
though hyperlipidemia is a biochemical hallmark of hypothyroidism and there are reports of inflammation in subclinical hypothyroidism , to best of our knowledge no study has been conducted to date to assess the contribution of hyperlipidemia and inflammation to svi in the causation of cardiovascular morbidities in hypothyroidism . therefore ,
in the present study we have assessed the magnitude of svi in hypothyroidism and its plausible link to the cvrs in this condition .
in addition to the classical autonomic function tests ( cafts ) to assess sympathovagal balance , recently power spectral analysis of heart rate variability ( hrv ) has been documented as a sensitive measure of sympathovagal balance .
therefore , in the present study we have used hrv analysis to assess svi in hypothyroidism . as hypothyroidism is more common in females compared with males with the male - female ratio 1:6 - 8 , in the present study
we have assessed the contribution of obesity , dyslipidemia and inflammation to svi in female hypothyroid patients .
the present study was conducted in the department of physiology , jawaharlal institute of postgraduate medical education and research ( jipmer ) , pondicherry , india . after obtaining approval of the project plan from research and ethics committees of jipmer ,
104 female subjects ( 50 euthyroid subjects and 54 hypothyroid patients ) were recruited from the endocrinology clinic of jipmer hospital .
written informed consent was obtained from all the participants prior to initiation of the study .
female patients freshly diagnosed as primary hypothyroids , before initiation of the treatment were included for the study .
patients , who were already on treatment for hypothyroidism , known cases of diabetes mellitus , hypertension , heart diseases , autonomic failure or endocrine disorders and those receiving chronic medications were excluded from the study .
females receiving oral contraceptives , females in the perimenopausal age and who had attained menopause were also excluded from the study .
the subjects reported to polygraph laboratory at about 8 am without breakfast . height and weight
following 10 min of supine rest in polygraph laboratory ( room temperature maintained at 25c ) , the following recordings were done .
baseline hr and bp were recorded in the left arm after 10 min of rest in the supine position using automatic bp monitor ( omron healthcare co. ltd . , kyoto , japan ) .
for the recording of short - term hrv , the procedure as described earlier and recommendation of the task force on hrv was followed . for the purpose ,
electrocardiography ( ecg ) electrodes were connected and lead ii ecg was acquired at a rate of 250 samples / s during supine rest using bioharness 2 data acquisition system ( biopac inc . , goleta , ca , usa ) .
the data was transferred from bioharness to a windows - based pc with acqknowledge software version 4.1.0 .
hrv analysis was performed using the hrv analysis software version 1.1 ( bio - signal analysis group , kuopio , finland ) .
frequency domain indices such as total power ( tp ) , low - frequency power expressed in normalized unit ( lfnu ) , high - frequency power expressed in normalized unit ( hfnu ) , ratio of low - frequency to high - frequency power ( lf - hf ratio ) and time domain indices such as mean of the peak of r to r wave of ecg ( mean rr ) , square root of the mean squared differences of successive normal to normal intervals ( rmssd ) , standard deviation of normal to normal interval ( sdnn ) , the number of interval differences of successive nn intervals greater than 50 ms ( nn50 ) and the proportion derived by dividing nn50 by the total number of nn intervals ( pnn50 ) were recorded .
three cafts were performed following the standard procedures . in this test , hr and bp response to standing
the subject was instructed stand up in 3 s. the ecg was continuously recorded during the procedure .
the bp was recorded every 40 s by automatic bp monitor ( omron , sem-1 , kyoto , japan ) until 5 min .
30:15 ratio ( ratio of maximum rr interval at 30 beat to minimum rr interval at 15 beat following standing ) was calculated .
the subject in sitting posture , the hr and respiration monitoring was done from ecg recording and stethographic respiratory tracings recorded on the multichannel polygraph ( nihon - kohden , tokyo , japan ) .
a baseline recording of ecg and respiration was taken for 30 s. the subject was asked to take slow and deep inspiration followed by slow and deep expiration such that each breathing cycle lasted for 10 s , consisting of six breathing cycles per minute .
e : i ratio ( ratio of average rr interval during expiration to average rr interval during inspiration in six cycles of deep breathing ) was calculated from ecg tracing .
the subject was asked to press handgrip dynamometer at 30% of maximum voluntary contraction for 2 min .
the serum was separated from the blood samples of all the subjects for estimation of biochemical parameters .
free triiodothyronine 3 ( ft3 ) , free thyroxine ( ft4 ) and thyroid stimulating hormone ( tsh ) were assayed by chemiluminiscence method using the kits of siemens healthcare diagnostics inc .
, usa . lipid profile ( total cholesterol , triglycerides , high density lipoprotein [ hdl ] , low density lipoprotein [ ldl ] and very low density lipoprotein [ vldl ] ) were assessed using fully automated analyzer ( au400 , olympus , usa ) . anti - thyroperoxidase antibody ( anti - tpo ab ) , anti - thyroglobulin antibody ( anti - tg ab ) and immunoglobin e ( ige ) were estimated by indirect immunoenzymatic colorimetric method using enzyme - linked immunosorbent assay ( elisa ) kits ( dia metra , segrate , italy ) . the high - sensitive c - reactive protein ( hscrp )
was estimated by the enzyme immunoassay method using elisa kit ( dbc diagnostics biochem canada inc . ,
, the level of significance between the groups was tested by student 's unpaired t - test and for non - parametric data ; the welch 's corrected t - test was used .
the association between lf - hf and various parameters was assessed by pearson 's correlation analysis .
the independent contribution of various factors to lf - hf ratio was assessed by multiple regression analysis .
bivariate logistic regression was performed for the prediction of bp ( normotension ) status in control subjects and hypertension status in hypothyroid patients by lf - hf ratio .
female patients freshly diagnosed as primary hypothyroids , before initiation of the treatment were included for the study .
patients , who were already on treatment for hypothyroidism , known cases of diabetes mellitus , hypertension , heart diseases , autonomic failure or endocrine disorders and those receiving chronic medications were excluded from the study .
females receiving oral contraceptives , females in the perimenopausal age and who had attained menopause were also excluded from the study .
the subjects reported to polygraph laboratory at about 8 am without breakfast . height and weight were measured to calculate body mass index ( bmi ) . following 10 min of supine rest in polygraph laboratory ( room temperature maintained at 25c ) , the following recordings were done .
baseline hr and bp were recorded in the left arm after 10 min of rest in the supine position using automatic bp monitor ( omron healthcare co. ltd . ,
kyoto , japan ) . for the recording of short - term hrv , the procedure as described earlier and recommendation of the task force on hrv was followed . for the purpose ,
electrocardiography ( ecg ) electrodes were connected and lead ii ecg was acquired at a rate of 250 samples / s during supine rest using bioharness 2 data acquisition system ( biopac inc . , goleta , ca , usa ) .
the data was transferred from bioharness to a windows - based pc with acqknowledge software version 4.1.0 .
hrv analysis was performed using the hrv analysis software version 1.1 ( bio - signal analysis group , kuopio , finland ) .
frequency domain indices such as total power ( tp ) , low - frequency power expressed in normalized unit ( lfnu ) , high - frequency power expressed in normalized unit ( hfnu ) , ratio of low - frequency to high - frequency power ( lf - hf ratio ) and time domain indices such as mean of the peak of r to r wave of ecg ( mean rr ) , square root of the mean squared differences of successive normal to normal intervals ( rmssd ) , standard deviation of normal to normal interval ( sdnn ) , the number of interval differences of successive nn intervals greater than 50 ms ( nn50 ) and the proportion derived by dividing nn50 by the total number of nn intervals ( pnn50 ) were recorded .
three cafts were performed following the standard procedures . in this test , hr and bp response to standing was assessed . the bp and ecg were recorded in the supine position .
the subject was instructed stand up in 3 s. the ecg was continuously recorded during the procedure .
the bp was recorded every 40 s by automatic bp monitor ( omron , sem-1 , kyoto , japan ) until 5 min .
30:15 ratio ( ratio of maximum rr interval at 30 beat to minimum rr interval at 15 beat following standing ) was calculated .
the subject in sitting posture , the hr and respiration monitoring was done from ecg recording and stethographic respiratory tracings recorded on the multichannel polygraph ( nihon - kohden , tokyo , japan ) .
a baseline recording of ecg and respiration was taken for 30 s. the subject was asked to take slow and deep inspiration followed by slow and deep expiration such that each breathing cycle lasted for 10 s , consisting of six breathing cycles per minute .
e : i ratio ( ratio of average rr interval during expiration to average rr interval during inspiration in six cycles of deep breathing ) was calculated from ecg tracing .
the subject was asked to press handgrip dynamometer at 30% of maximum voluntary contraction for 2 min .
the subject was instructed stand up in 3 s. the ecg was continuously recorded during the procedure .
the bp was recorded every 40 s by automatic bp monitor ( omron , sem-1 , kyoto , japan ) until 5 min .
30:15 ratio ( ratio of maximum rr interval at 30 beat to minimum rr interval at 15 beat following standing ) was calculated .
the subject in sitting posture , the hr and respiration monitoring was done from ecg recording and stethographic respiratory tracings recorded on the multichannel polygraph ( nihon - kohden , tokyo , japan ) .
a baseline recording of ecg and respiration was taken for 30 s. the subject was asked to take slow and deep inspiration followed by slow and deep expiration such that each breathing cycle lasted for 10 s , consisting of six breathing cycles per minute .
e : i ratio ( ratio of average rr interval during expiration to average rr interval during inspiration in six cycles of deep breathing ) was calculated from ecg tracing .
the subject was asked to press handgrip dynamometer at 30% of maximum voluntary contraction for 2 min .
the serum was separated from the blood samples of all the subjects for estimation of biochemical parameters .
free triiodothyronine 3 ( ft3 ) , free thyroxine ( ft4 ) and thyroid stimulating hormone ( tsh ) were assayed by chemiluminiscence method using the kits of siemens healthcare diagnostics inc .
, usa . lipid profile ( total cholesterol , triglycerides , high density lipoprotein [ hdl ] , low density lipoprotein [ ldl ] and very low density lipoprotein [ vldl ] ) were assessed using fully automated analyzer ( au400 , olympus , usa ) . anti - thyroperoxidase antibody ( anti - tpo ab ) , anti - thyroglobulin antibody ( anti - tg ab ) and immunoglobin e ( ige ) were estimated by indirect immunoenzymatic colorimetric method using enzyme - linked immunosorbent assay ( elisa ) kits ( dia metra , segrate , italy ) . the high - sensitive c - reactive protein ( hscrp )
was estimated by the enzyme immunoassay method using elisa kit ( dbc diagnostics biochem canada inc . , canada ) .
, the level of significance between the groups was tested by student 's unpaired t - test and for non - parametric data ; the welch 's corrected t - test was used .
the association between lf - hf and various parameters was assessed by pearson 's correlation analysis .
the independent contribution of various factors to lf - hf ratio was assessed by multiple regression analysis .
bivariate logistic regression was performed for the prediction of bp ( normotension ) status in control subjects and hypertension status in hypothyroid patients by lf - hf ratio .
there was no significant difference in age of control and hypothyroid subjects [ table 1 ] .
bmi of hypothyroids was significantly more compared with that of controls ( p = 0.000 ) .
the basal heart rate of hypothyroid patients was significantly less ( p = 0.005 ) compared with that of euthyroid subjects .
though , there was no significant difference in systolic blood pressure ( sbp ) , the diastolic blood pressure ( dbp ) ( p = 0.000 ) and mean arterial pressure ( map ) ( p = 0.004 ) were significantly more in hypothyroid patients compared with the control subjects [ table 1 ] .
age , bmi , basal cv , hrv and caft parameters of control and hypothyroid subjects tp of hrv spectrum , hfnu were reduced significantly ( p = 0.000 ) and lfnu and lf - hf ratio were increased significantly ( p = 0.000 ) in hypothyroid group [ table 1 ] .
the time domain indices of hrv ( mean rr , rmssd , sdnn , nn50 , pnn50 ) were significantly decreased ( p = 0.000 ) in hypothyroid group compared with the control group .
the 30:15 ratio and dbpihg were significantly increased ( p = 0.000 ) and e : i ratio was significantly decreased ( p = 0.000 ) in hypothyroids compared to that of euthyroids [ table 1 ] .
there was a significant decrease ( p = 0.000 ) in ft3 and ft4 and increase in tsh ( p = 0.000 ) in hypothyroid group compared to the euthyroid group [ table 2 ] .
total cholesterol , triglyceride , ldl , vldl were increased ( p = 0.000 ) and hdl ( p = 0.000 ) was decreased in hypothyroid patients .
all the lipid risk factors were significantly high ( p = 0.000 ) in hypothyroid subjects .
levels of anti - tpo ab , anti - tg antibody and hscrp were increased ( p = 0.000 ) and the level of ige was not significantly altered in hypothyroid group compared to the control group [ table 2 ] .
thyroid profile , lipid profile , lipid risk factors , immunological and inflammatory markers of control and hypothyroid subjects lf - hf ratio was not significantly correlated with any of the parameter except ft3 ( p = 0.026 ) in the control group [ table 3 ] . in hypothyroid group
, there was a significant correlation of lf - hf ratio with all cardiovascular parameters , thyroid and lipid profile parameters , lipid risk factors and inflammatory and immunological markers except bmi , sbp and ige [ table 3 ] .
multiple regression analysis revealed independent contribution of map ( b 0.298 , p = 0.020 ) , hscrp ( b 0.578 , p = 0.009 ) , atherogenic index ( ai ) ( b 1.144 , p = 0.001 ) to lh - hf ratio [ table 4 ] .
bivariate logistic regression [ table 5 ] showed significant prediction of lf - hf to hypertension status ( odds ratio [ or ] 2.05 , confidence interval [ ci ] 1.110 - 5.352 , p = 0.008 ) in hypothyroid subjects , but the prediction was not significant in control subjects ( or 0.54 , ci 0.37 - 2.115 , p = 0.170 ) .
correlation of lh - hf with various parameters of control and hypothyroid subjects multiple regression analysis of lf - hf ratio ( as dependable variable ) with various other associated factors ( as independent variables ) in hypothyroid group bivariate logistic regression analysis of bp status or hypertension status ( as dependent variable ) with lf - hf ratio ( as independent variable ) in the control group and hypothyroid group after adjusting for age and bmi
in the present study , significant increase in lf - hf ratio in female hypothyroid patients compared to their age - matched controls [ table 1 ] indicates the presence of considerable svi in these patients , as lh - hf ratio is a sensitive marker of sympathovagal balance . increase in lf - hf ratio indicates increased sympathetic activity .
this was further supported by increase in lfnu ( p = 0.000 ) in hypothyroid group , as increased lfnu is an index of increased cardiac sympathetic drive . in hypothyroid subjects ,
significant decrease in hfnu ( p = 0.000 ) indicates decreased parasympathetic tone in these patients , as hfnu represents vagal drive to the heart .
this was supported by a significant reduction in tp of hrv in hypothyroid group , as tp in general indicates the vagal modulation of cardiac function .
moreover , all time - domain indices ( mean rr , rmssd , sdnn , nn50 , pnn50 ) were significantly less in hypothyroid subjects [ table 1 ] further indicating the decreased vagal tone in these subjects , as time - domain indices of hrv represent cardiac parasympathetic drive .
thus , it is evident from the present study that svi in hypothyroid patients is due to the concomitant increase in sympathetic activity and decrease in vagal activity , that corroborates with our earlier report and the report of cacciatori et al .
further , there was increased autonomic reactivity in these patients as evident from changes in caft parameters [ table 1 ] .
hr response to standing ( 30:15 ratio ) and deep breathing ( e : i ratio ) are parasympathetic function tests and bp response to isometric handgrip ( ddbpihg ) is sympathetic function test .
significantly high 30:15 ratio and decreased e : i ratio in hypothyroid subjects reflects lower vagal reactivity in hypothyroids . a heightened diastolic pressure response to isometric handgrip ( ddbpihg ) in these subjects
reflects increased sympathetic reactivity , as increase in dbp in handgrip test depends primarily on the vascular resistance that reflects sympathetic response .
thus , findings of the present study substantiate that the svi in hypothyroidism is due to increased sympathetic activity and reactivity , along with decreased parasympathetic activity and reactivity . though lf - hf ratio , the marker of sympathovagal balance , was correlated with t3 , t4 and tsh in hypothyroid subjects [ table 3 ] , multivariate regression did not show independent contribution of these parameters to lf - hf [ table 4 ] .
therefore , alteration in svi in hypothyroidism does not appear to be directly linked to the level of thyroid hormones and tsh . though svi is linked to the plasma level of thyroid hormones and tsh in hyperthyroidism ( state of thyroid excess )
, findings of the present study does not illustrate the association of autonomic imbalance with thyroid profile in hypothyroidism ( thyroid deficiency state ) .
one may suggest that significantly high bmi in hypothyroid subjects [ table 1 ] could be a major contributor to svi in this dysfunction , as increased adiposity has been reported to cause vagal inhibition and sympathetic overactivity .
however , obesity in hypothyroidism is not primarily due to excess adiposity as increase in bodyweight in thyroid deficiency state is mostly due to accumulation of water and mucopolysaccharides in subcutaneous tissues .
moreover , there was no significant correlation of lf - hf ratio with bmi in hypothyroid patients [ table 3 ] .
though the exact cause of svi in hypothyroidism can not be fully ascertained from the present study , it appears that svi is linked to the degree of hyperlipidemia .
not only there was significant dyslipidemia ( hypercholesterolemia , triglyceridemia , high ldl - hypercholesterolemia , high vldl - hypercholesterolemia and low hdl - cholesterolemia ) and increased lipid risk factors [ table 2 ] in hypothyroid subjects compared to the control subjects , but also all these factors were significantly correlated with lf - hf ratio [ table 3 ] .
moreover , the ai had significant independent contribution to lf - hf ratio [ table 4 ] . from among the lipid risk factors assessed in the present study , we selected ai for the regression model , as ai has recently been reported to be the better indicator of cv risk .
hyperlipidemia is very common in hypothyroidism and hypercholesterolemia has been reported to be associated with increased sympathetic activity .
thus , from findings of the present study it appears that chronic and profound hyperlipidemia could be a major contributor to svi in hypothyroidism . there is a report of low - grade immunological inflammation in thyroid deficient subjects . in the present study hscrp ,
anti - tpo ab and anti - tg ab were more in hypothyroid subjects compared to that of control subjects .
the hscrp was significantly correlated with lf - hf and had significant independent contribution to lf - hf ratio .
it has been reported that crp influences cardio - respiratory health through alteration in autonomic functions .
therefore , low - grade inflammation might play a significant role in the genesis of svi in hypothyroidism
. however , anti - tpo and anti - tg antibodies had no significant contribution to lf - hf ratio [ table 5 ] .
therefore , it is unlikely that immunological markers contribute to the genesis of svi in hypothyroidism .
there are reports of increased cvrs in conditions of svi , inflammation and hyperlipidemia . as such there are reports of increased cvrs in subclinical and overt hypothyroidism . in the present study ,
svi was associated with dyslipidemia , increased lipid risk factors and low - grade inflammation .
a report has suggested that lipid risk factors and crp are better markers of cardiovascular dysfunctions in females compared to the other predictors of adverse cardiovascular events such as apolipoproteins .
moreover , the reduction in hrv per se has been reported as an important cvr . in the present study ,
magnitude of hrv ( tp of hrv ) was grossly reduced in hypothyroid subjects predisposing them to cvr [ table 2 ] . furthermore , dbp and map had significant link to lf - hf ratio ( svi ) in hypothyroid subjects [ tables 3 and 4 ] .
recently we have reported the close association of svi with increased vascular tone and hypertension status that increases cvrs in prehypertensives . in the present study ,
lf - hf ratio had significant prediction for hypertension status in hypothyroid subject [ table 5 ] .
therefore , we presume that svi in hypothyroidism contributes to cvrs in hypothyroidism . in the present study ,
cvrs observed in hypothyroid patients are hypertension , decreased hrv , dyslipidemia and low - grade inflammation that are associated with svi .
it appears that dyslipidemia and inflammation contribute to the svi , which contributes to the hypertension status .
thus , considerable svi , sustained hyperlipidemia and chronic low - grade inflammation in hypothyroid patients predispose them to increased risk of cardiovascular morbidity and mortality . as
such hypothyroidism is a chronic disorder that takes months and years to achieve euthyroidism even after judicious treatment , especially in developing countries like india where patients compliance is poor . therefore , further research warrants the assessment of the efficacy of hypolipidemic and ant - inflammatory therapy on alleviation of svi in hypothyroidism .
hypothyroid subjects should also be encouraged to adapt non - pharmacological therapies such as pranayamic breathing exercises and yoga , as reports from our laboratory and others have documented reduction of sympathetic activity and improvement of vagal activity following practice of such life - style modification programs .
limitation of the present study is that we have not assessed cardiac dysfunctions by radio - imaging techniques and their possible correlation with svi in hypothyroid subjects .
in this study , hrv was found to be grossly reduced in hypothyroid patients predisposing them to cardiovascular morbidities .
svi in hypothyroid subjects was due to the concomitant increased sympathetic and decreased vagal activities , which was contributed by dyslipidemia and low - grade inflammation .
svi is linked to hypertension status in these patients . as chronic svi , hypertension , hyperlipidemia and inflammation
are known cvr factors , further research should be conducted to assess if improvement in sympathovagal homeostasis can improve the cardiovascular health in hypothyroid subjects . | background : cardiovascular morbidities have been reported in hypothyroidism.aims:the objective of this study is to investigate the link of sympathovagal imbalance ( svi ) to cardiovascular risks ( cvrs ) and the plausible mechanisms of cvr in hypothyroidism.materials and methods : age - matched 104 females ( 50 controls , 54 hypothyroids ) were recruited and their body mass index ( bmi ) , cardiovascular parameters , autonomic function tests by spectral analysis of heart rate variability ( hrv ) , heart rate response to standing , deep breathing and blood pressure response to isometric handgrip were studied .
thyroid profile , lipid profile , immunological and inflammatory markers were estimated and their association with low - frequency to the high - frequency ratio ( lf - hf ) of hrv , the marker of svi was assessed by multivariate regression.results:increased diastolic pressure , decreased hrv , increased lf - hf , dyslipidemia and increased high - sensitive c - reactive protein ( hscrp ) were observed in hypothyroid patients and all these parameters had significant correlation with lf - hf .
bmi had no significant association with lf - hf .
atherogenic index ( 1.144 , p = 0.001 ) and hscrp ( b 0.578 , p = 0.009 ) had independent contribution to lf - hf .
lf - hf could significantly predict hypertension status ( odds ratio 2.05 , confidence interval 1.110 - 5.352 , p = 0.008 ) in hypothyroid subjects.conclusions:svi due to sympathetic activation and vagal withdrawal occurs in hypothyroidism .
dyslipidemia and low - grade inflammation , but not obesity contribute to svi and svi contributes to cardiovascular risks . |
coronary artery embolism is an established cause of acute coronary syndrome , but paradoxical coronary artery embolism causing myocardial infarction is rare and requires a high degree of clinical suspicion for diagnosis .
recognition of this condition is important as these patients are at risk of future fatal embolic phenomena .
percutaneous device closure of interatrial communication either in the form of patent foramen ovale ( pfo ) or atrial septal defect ( asd ) should be considered to prevent future embolism .
a 64-year - old male with multiple myeloma was admitted for autologous stem cell transplantation .
two weeks previous he was diagnosed with a peripherally inserted central catheter ( picc)-related right basilic and axillary vein thrombosis [ figure 1a , arrows ] complicated by pulmonary embolism that was treated with low molecular weight heparin .
( b ) right coronary angiogram showing flush occlusion of posterior descending artery and posterolateral branch of right coronary artery .
( c ) transesophageal echocardiography ( tee ) with agitated saline showing bubbles crossing the patent foramen ovale ( pfo ) .
( e ) tee showing a thrombus in superior vena cava during an attempt at picc removal , the patient coughed and developed sudden severe left - sided chest pain .
an electrocardiogram showed st segment elevation in inferior ( ii , iii , and avf ) leads .
cardiac biomarkers were elevated ( troponin - t 2.230 ng / ml ( normal < 0.01 ng / ml ) and creatine kinase myocardial band fraction 65.1 ng / ml ( normal < 6.7 ng / ml ) ) .
coronary angiography revealed flush occlusion of the posterior descending artery and posterolateral branch of the right coronary artery [ figure 1b , arrows ] .
severe thrombocytopenia ( platelet count 37 109/l ) secondary to newly diagnosed heparin - induced thrombocytopenia precluded use of antiplatelet agents and hence , coronary intervention .
transesophageal echocardiography with agitated saline showed a small pfo ( figure 1c , arrow ) with bidirectional shunt seen on color doppler imaging [ figure 1d , asterix ] .
in addition , a large thrombus was seen in the superior vena cava [ figure 1e , arrow ] .
a diagnosis of st elevation myocardial infarction secondary to paradoxical embolism to the right coronary artery was entertained .
in addition to long - term treatment with fondaparinux for venous thrombosis , percutaneous pfo closure was recommended to prevent recurrent embolism .
paradoxical coronary embolism is rare and accounts for 1015% . of all paradoxical emboli , and 25% of acute coronary events in patients
our patient had a documented picc associated thrombus , and an episode of cough preceding the onset of chest pain caused transient elevation in right atrial pressure with right to left shunting and paradoxical embolism though a pfo .
the valsalva maneuver is routinely used to demonstrate shunting across the pfo during echocardiographic examination in individuals in whom spontaneous shunting is not seen .
paradoxical embolism was first reported by cohnheim in 1877 and is known to cause cerebral , peripheral arterial , and in rare instances coronary artery occlusion .
paradoxical coronary artery embolism should be suspected in patients who otherwise are at low risk for atherosclerotic coronary artery disease . in a given clinical scenario , demonstration of venoarterial communication ( most often at the atrial level )
together with identification of a venous source of embolus and lack of thrombi in the left heart fulfill the criteria for presumptive diagnosis of this condition .
a definite diagnosis requires demonstration of thrombus across the venoarterial communication either by echocardiography or contrast computed tomographic angiography of the chest .
many times paradoxical coronary artery embolism can only be made at the time of autopsy . when suspected clinically , a search for an underlying condition predisposing to venous thrombosis should be undertaken .
the management of acute coronary syndrome caused by paradoxical coronary artery embolism is similar to that occurring in the setting of atherosclerotic coronary artery disease .
manual aspiration thrombectomy with or without angioplasty and stenting followed by aggressive medical management including antiplatelet agents , is the standard of practice .
the management of patients with contraindication to antiplatelet therapy may be challenging as in this case .
aspiration and manual thrombectomy should be the main therapeutic intervention in such a situation unless the embolus is distal in a small coronary artery .
optimum therapeutic strategy in preventing recurrent embolic events in patients with pfo is not established .
a recent meta - analysis of three randomized clinical trials addressing the role of transcutaneous closure of pfo showed a benefit in preventing recurrent cerebrovascular ischemic events in patients with cryptogenic stroke when compared with medical therapy .
another study demonstrated lower recurrent neurological event rates with amplatzer compared with cardioseal - starflex and helex devices .
although percutaneous device closure has not been studied in paradoxical coronary embolism , it should be considered to prevent fatal recurrent events . in summary ,
in addition to acute management of acute coronary syndrome , pfo closure to prevent recurrent thromboembolism should be considered . | paradoxical coronary artery embolism is a rare , but often an underdiagnosed cause of acute myocardial infarction .
it should be considered in patient who presents with chest pain and otherwise having a low risk profile for atherosclerosis coronary artery disease .
we describe a case of paradoxical coronary artery embolism causing st segment elevation myocardial infarction in a patient with upper extremity venous thrombosis .
echocardiography demonstrated a patent foramen ovale ( pfo ) with bidirectional shunt .
in addition to treatment of acute coronary event closure of the pfo should be considered to prevent a recurrence . |
, governments , charities , and private companies have made large investments in this sector ( 1 ) .
studies in this area should be performed with high precision and confidence levels for both cost - benefit and cost - effectiveness purposes .
quantitative evaluation of scientific findings from research activities helps authorities and planners , allowing them to benefit from human and financial resources with less cost , which can optimize the economic and social structure of the country ( 2 ) .
the scientometric method is a quantitative evaluation method that properly balances budgets , economic costs , and increased research efficiency ( 3 ) . in this approach ,
various evaluation methods are used , and they deliver credible research outputs and knowledge production . reviewing the articles indexed in valid databases is the most efficient method of assessing scientific output and the overall credibility of studies . in these types of studies ,
quantitative measurement of scientific production may , to some extent , determine both the frequency and process of studies of any country , institution , discipline , and individual ( 4 ) . however , this method is not as precise as other methods , and it has many deficiencies .
for example , publications that are not cited also may be useful ( 5 ) , because many non - writer experts , e.g. , practitioners , master s students ( 6 ) , public people ( 7 ) , and instructors , read research papers ( 8) and use research publications in such activities as teaching ( 9 ) or business ( 10 ) .
for this reason , complementary methods have been added to the evaluation method in recent years , among which the concepts related to measures in scientific journals such as read , usage , and download ( 7 ) can be noted .
media or social networks provide tools that can provide the data used for this evaluation .
the tool can provide a broader image of scientific impact due to increasing popularity among people ( 11 ) .
bookmarking is one feature of these networks , which refers to the number of usages of a paper or document .
the number of bookmarkings of an article or document shows how many times that article or document was used ( 12 ) .
a by - product of the academic usage of a social website is that it determines the latest statistics on the popularity of articles on social websites .
this feature creates new online indexes for evaluating the effect of articles and a new category called altmetrics , which is a new approach for assessing and tracking the scientific impact of a social website ( 13 ) .
as mentioned earlier , one method can not be used for the evaluation of research output and the assessment of its effectiveness ; however , a combination of various methods may deliver useful data .
many studies have investigated the research production of different scholars in the field of medical sciences .
some studies have examined the scientific production related to such specific subjects as hiv ( 14 ) , stems cells ( 15 ) , and traditional medicine ( 16 ) .
some studies have also investigated the research production of scholars in various universities of medical sciences , such as isfahan university of medical sciences ( 2 ) , gilan university of medical sciences ( 17 ) , and iran university of medical sciences ( 18 ) . in most of these studies , scientific products indexed in the citation databases of web of science ( 19 ) , scopus ( 20 ) , or both databases ( 21 )
the authors did not found a study that investigated scientific outputs at the same time on citation databases and social networks , such as researchgate and mendeley .
for this reason , the authors decided to conduct a study of the scientific output of scholars at kashan university of medical sciences .
the present study aimed to evaluate their scientific output by the end of march 2014 based on scientometric measures of scopus , researchgate , and mendeley .
the specific objectives of the study were : to determine the number of indexed articles of scholars at kashan university of medical sciences in the scopus citation index in terms of year , type of article , journal , and citation . to determine the number of citations received by indexed articles of scholars at kashan university of medical sciences in the scopus citation index in terms of year and type of paper . to determine the coverage of social networks of researchgate and mendeley on articles from kashan university of medical sciences indexed in the scopus citation index . to determine the number of views of articles of scholars at kashan university of medical sciences in researchgate . to determine the number of articles of scholars at kashan university of medical sciences with the highest frequency of views and downloads in researchgate . to determine the frequency with which the articles of scholars at kashan university of medical sciences in mendeley were read . to determine the articles of scholars at kashan university of medical sciences with the highest reading frequency in mendeley . to determine the correlation between the number of views of articles of scholars at kashan university of medical sciences in researchgate and mendeley social networks with citations to the articles in scopus
no sampling procedure was conducted in this study , and all of the articles published in the scopus citation database by the scholars at kashan university of medical sciences by the end of march 2014 were investigated .
the required data were collected from the scopus citation index and two social networks , i.e. , researchgate and mendeley .
scopus was selected due to its relative homogeneity regarding all disciplines and delivery of better results .
this is unlike the science citation databases , which focus only on science and social science .
it should be stated that the two social networks are academic and scientific and provide social bookmarking and evaluation services ( 22 , 30 ) .
the authors followed two major steps to extract the required data as follows : the first step : data extraction from scopus articles the name , kashan university of medical sciences , was logged into the search section based on organizational affiliation in scopus 2014 in order to extract the titles .
all articles were reviewed due to the diversity of the writing styles of the authors and the need to determine the authors who were affiliated with the university .
in addition , information on each article was saved in an spss file to be analyzed in the final stage . the extracted information included title , authors , authors affiliations with kashan university of medical sciences , year of publication , type of article , name of the journal , and the number of citations of each article .
the second step : bookmark data extraction from researchgate and mendeley the titles of articles affiliated with kashan university of medical sciences that were indexed in the scopus citation databases were searched in two social networks , i.e. , researchgate and mendeley . in addition , bookmarking data on each article were recovered from the two social networks . in the case of availability of papers , reading frequency and full texts of every article were added to the data extracted in the first stage . for statistical analysis of the data , we used descriptive statistic for objectives ( 18 ) and , for the eighth objective , we used spearman s correlative test in spss version 16 ( spss , inc . , chicago , illinois , usa ) .
no sampling procedure was conducted in this study , and all of the articles published in the scopus citation database by the scholars at kashan university of medical sciences by the end of march 2014 were investigated .
the required data were collected from the scopus citation index and two social networks , i.e. , researchgate and mendeley .
scopus was selected due to its relative homogeneity regarding all disciplines and delivery of better results .
this is unlike the science citation databases , which focus only on science and social science .
it should be stated that the two social networks are academic and scientific and provide social bookmarking and evaluation services ( 22 , 30 ) .
the authors followed two major steps to extract the required data as follows : the first step : data extraction from scopus articles the name , kashan university of medical sciences , was logged into the search section based on organizational affiliation in scopus 2014 in order to extract the titles .
all articles were reviewed due to the diversity of the writing styles of the authors and the need to determine the authors who were affiliated with the university .
in addition , information on each article was saved in an spss file to be analyzed in the final stage .
the extracted information included title , authors , authors affiliations with kashan university of medical sciences , year of publication , type of article , name of the journal , and the number of citations of each article .
the second step : bookmark data extraction from researchgate and mendeley the titles of articles affiliated with kashan university of medical sciences that were indexed in the scopus citation databases were searched in two social networks , i.e. , researchgate and mendeley .
in addition , bookmarking data on each article were recovered from the two social networks . in the case of availability of papers , reading frequency and full texts of every article
for statistical analysis of the data , we used descriptive statistic for objectives ( 18 ) and , for the eighth objective , we used spearman s correlative test in spss version 16 ( spss , inc . , chicago , illinois , usa ) .
the findings showed that 533 articles from 1960 to 2014 were indexed in the scopus citation database in which at least one author was affiliated with kashan university of medical sciences .
in addition , 2,297 authors contributed to these articles , 1,263 of whom were affiliated with kashan university of medical sciences . in fact , an average of four authors were involved per article .
moreover , 35.4% of the articles were prepared by scholars at kashan university of medical sciences , and 65.4% of the articles were developed in collaboration with academics of other universities and research centers .
considering the type of articles , the results showed that 92.7% were research papers , and 7.3% were other types of articles .
approximately 20% of the citations were to seven articles , with 90 , 35 , 31 , 28 , 25 , 24 , and 22 citations ( table 1 ) .
table 2 shows the frequency distribution of citations of the articles based on the year of publication .
as shown in table 2 , only 21.2% of the articles were published prior to 2008 , and 78.8% of the articles were published after 2008 . however , 80% of the citations belonged to the articles published prior to 2011 .
table 3 shows the frequency distribution of then citations of articles based on the types of articles . as shown in table 3 ,
due to the ratio of research articles compared to review articles , review articles were cited more than research articles .
it was found out that 395 articles were indexed in researchgate among 533 articles indexed in scopus .
in addition , 395 indexed articles in researchgate were viewed 20,799 times and downloaded 2,324 times . moreover ,
all of these articles were viewed 901 times in this social network ( table 4 ) .
the articles were covered by researchgate ( 74% ) more than mendeley ( 44% ) . furthermore
, 98% of the articles indexed in researchgate and 92% of the articles indexed in mendeley were viewed at least once . as observed in table 4 , any article bookmarked in researchgate was viewed 54 times on average .
examining the frequency of viewing the articles in researchgate showed that 54% of the articles were viewed between
one to 50 times , 32% of the articles were viewed between 50 to 100 times , and 11% of the articles were viewed more than 100 times . only 2% of the articles
the results showed that five of the 29 articles ( 20% ) that were viewed the most in researchgate were review articles .
one article was downloaded 239 times which had highest rate of downloads . on average , any bookmarked article was downloaded six times in mendeley .
table 6 shows that an article entitled gastrointestinal parasites of stray cats in kashan , iran had the highest number of downloads ( 239 times , 10% of the downloading frequency ) .
also , two of the five articles that had the highest number of downloads in researchgate ( 20% ) were review articles .
examining the reading frequency of the articles in mendeley showed that 86% of the articles were read between 1 to 10 times , 5% of the articles was read between 11 and 20 times , and 2% of the articles were read more than 20 times . studied articles in mendeley were viewed 901 times .
table 7 shows that 20% of the articles had the highest number of views in mendeley .
two of the five articles maximally viewed ( 20% ) in mendeley were review articles .
the correlation between the number of views of articles in researchgate with citation number of the articles in scopus and reading frequency of the articles in mendeley with citation number in scopus were examined using the spearman correlation .
the results showed a significant relationship between the variables with 99% confidence level and an error level less than 1% . in this regard , the correlation coefficient between two variables of the number of views of articles in researchgate with citation number of the articles in scopus was 0.310 , which showed a positive relationship between the two variables .
it can be concluded that increased number of views of articles in researchgate increased citations to the articles .
the correlation coefficient between the two variables of reading frequency of the articles in mendeley with citation number of the articles in scopus was equal to 0.247 , which indicated a positive relationship between the two variables .
thus , the increased number of views of articles in mendeley increased the citations to the articles .
however , the correlation between the numbers of views of articles in researchgate was associated with higher citations of reading frequency of the articles in mendeley with the number of citations to the articles .
the findings showed that 533 articles from 1960 to 2014 were indexed in the scopus citation database in which at least one author was affiliated with kashan university of medical sciences .
in addition , 2,297 authors contributed to these articles , 1,263 of whom were affiliated with kashan university of medical sciences . in fact , an average of four authors were involved per article .
moreover , 35.4% of the articles were prepared by scholars at kashan university of medical sciences , and 65.4% of the articles were developed in collaboration with academics of other universities and research centers .
considering the type of articles , the results showed that 92.7% were research papers , and 7.3% were other types of articles .
approximately 20% of the citations were to seven articles , with 90 , 35 , 31 , 28 , 25 , 24 , and 22 citations ( table 1 ) .
table 2 shows the frequency distribution of citations of the articles based on the year of publication .
as shown in table 2 , only 21.2% of the articles were published prior to 2008 , and 78.8% of the articles were published after 2008 . however , 80% of the citations belonged to the articles published prior to 2011 .
table 3 shows the frequency distribution of then citations of articles based on the types of articles . as shown in table 3 ,
95.8% of the articles were research papers , and 3.4% were review articles . due to the ratio of research articles compared to review articles ,
it was found out that 395 articles were indexed in researchgate among 533 articles indexed in scopus .
in addition , 395 indexed articles in researchgate were viewed 20,799 times and downloaded 2,324 times . moreover ,
all of these articles were viewed 901 times in this social network ( table 4 ) .
the articles were covered by researchgate ( 74% ) more than mendeley ( 44% ) . furthermore
, 98% of the articles indexed in researchgate and 92% of the articles indexed in mendeley were viewed at least once . as observed in table 4 , any article bookmarked in researchgate was viewed 54 times on average .
examining the frequency of viewing the articles in researchgate showed that 54% of the articles were viewed between one to 50 times , 32% of the articles were viewed between 50 to 100 times , and 11% of the articles were viewed more than 100 times . only 2% of the articles were never viewed .
the results showed that five of the 29 articles ( 20% ) that were viewed the most in researchgate were review articles .
one article was downloaded 239 times which had highest rate of downloads . on average ,
table 6 shows that an article entitled gastrointestinal parasites of stray cats in kashan , iran had the highest number of downloads ( 239 times , 10% of the downloading frequency ) .
also , two of the five articles that had the highest number of downloads in researchgate ( 20% ) were review articles .
examining the reading frequency of the articles in mendeley showed that 86% of the articles were read between 1 to 10 times ,
5% of the articles was read between 11 and 20 times , and 2% of the articles were read more than 20 times .
table 7 shows that 20% of the articles had the highest number of views in mendeley .
two of the five articles maximally viewed ( 20% ) in mendeley were review articles .
the correlation between the number of views of articles in researchgate with citation number of the articles in scopus and reading frequency of the articles in mendeley with citation number in scopus were examined using the spearman correlation .
the results showed a significant relationship between the variables with 99% confidence level and an error level less than 1% . in this regard , the correlation coefficient between two variables of the number of views of articles in researchgate with citation number of the articles in scopus was 0.310 , which showed a positive relationship between the two variables .
it can be concluded that increased number of views of articles in researchgate increased citations to the articles .
the correlation coefficient between the two variables of reading frequency of the articles in mendeley with citation number of the articles in scopus was equal to 0.247 , which indicated a positive relationship between the two variables .
thus , the increased number of views of articles in mendeley increased the citations to the articles .
however , the correlation between the numbers of views of articles in researchgate was associated with higher citations of reading frequency of the articles in mendeley with the number of citations to the articles .
the results showed an increase in publication of articles in recent years . in terms of article type ,
92.7% covered original research , 2.6% encompassed review articles , and 4.7% was allocated to other types of articles .
investigating scientific products of shahre - kord university of medical sciences showed that 74.6% of scientific production in the web of science consisted of research papers ( 14 ) .
it can be observed that the nature of scientific production in the field of medical science significantly was the research format , i.e. , medical science produced mostly research papers .
examining the citations to articles showed that the highest number of citations to an article was 90 times .
in addition , 48% of the articles indexed in scopus were cited at least once .
examining 1,613 articles in nature and science journals in 2007 showed that all the articles were viewed at least once and a maximum of 1000 times in the science citation index ( 32 ) .
sotoudeh , mazarei and mirzabeygi investigated articles of 83 journals in the fields of librarianship and information science and showed that 22.17% of the articles were cited at least once in wos , and 22.6% of these papers were bookmarked in citeulike ( 26 ) .
coverage of articles and the number of bookmarking were higher in researchgate than in mendeley .
in addition , access to full text of the articles was higher in researchgate than in mendeley .
this showed the popularity of researchgate as a research scientific social network among the community of medical sciences .
this was because the correlation between all indices was reported on average or high levels ( 27 ) .
in most studies , the results showed excellent coverage of mendeley on articles of most scientific fields ( 28 , 25 ) .
thelwall and wilson ( 2015 ) reviewed the articles in the field of medical science indexed in scopus in 2009 and showed that 89% of the papers were saved in mendeley , 78% of the articles were at least read once in mendeley , and 73% of the articles were cited at least once in scopus ( 28 ) .
( 2015 ) studied the papers in four areas of clinical medicine , engineering and technology , social sciences , physics and chemistry , which were indexed in wos in 2008 .
another study showed that 80% of articles of public library of science were covered by mendeley , 31% were covered by citeulike , and 10% of the articles were covered by delicious ( 11 ) .
in addition , 355 studied articles were viewed 20,799 times in researchgate and 901 times in mendeley .
furthermore , 98% of the articles indexed in researchgate and 92% of the articles indexed in mendeley were viewed at least once .
any bookmarked articles in researchgate were viewed 54 times on average . any bookmarked article in mendeley was viewed four times on average .
the results showed that mean viewing number of the articles in researchgate was 10 times the mean number of citations to papers .
these findings indicated that the articles were read by many people but were rarely cited .
therefore , viewing number of the articles shows how many times the articles were used in other contexts , such as teaching and business .
( 2015 ) reported that the mean reading number of the articles in the field of social sciences was twice the mean number of citation to the articles in mendeley .
this was reversed in case of articles in the fields of chemistry , physics , and clinical medicine .
the mean reading number of the articles was equal to the mean number of citation to the articles in the field of engineering and technology ( 25 ) .
another study showed that the number of bookmarked works was higher than the mean number of citations in mendeley ( 3.32% bookmarking vs. 2.17% citations ) .
the highest number of bookmarked works was observed in the field of health and biomedical sciences ( 13.6% ) ( 29 ) .
mohammadi and thelwal ( 2015 ) showed that the mean number of saved articles in all disciplines , except psychology , was higher than the mean number of citations to articles in mendeley ( 24 ) .
these results are not in line with those obtained by sotoude , mazarei and mirzabeygi ( 2015 ) .
they showed that the number of bookmarked articles in citeulike was negligible compared to the number of citation to the articles in wos ( 26 ) .
although only 2.6% of the studied papers were review articles , these article covered part of 20% of the most visited and downloaded articles .
review articles are an important category of medical papers , which are mostly read and associated with high credibility due to importance and integrity .
editors are mostly interested in such articles because a review article written by a well - known author is widely welcomed , read more , and referred more , which increases the impact factor of the journal .
readers are also interested in this type of articles as the perfect solution to meet the increasing spread of knowledge and acquire up - to - date information .
the results indicated that the correlation coefficient between the two variables of viewing number of the articles in researchgate with citations to the article in scopus and the correlation coefficient between two variables of reading frequency of the articles in mendeley with the number of citations to articles in scopus were positive and significant .
it can be concluded that increased number of viewed articles in researchgate and mendeley increased the number of citations to articles .
2012 ) showed that 81.6% of works of 57 scholars in field of scientometrics indexed in scopus were covered by mendeley .
the results showed that the correlation coefficient between the number of citations to articles in scopus and reading frequency of the articles in mandeley was equal to 0.448 ( 33 ) .
mohammadi and thelwal ( 2013 ) showed a significant and positive correlation between the number of bookmarked articles in mendeley and number of citations to articles in studied fields ( 22 ) .
li and thelwal ( 2012 ) also showed a significant correlation between citations and altmetrics bookmarking index in mendeley for some genetic articles published in 2008 ( 34 ) .
thelwall and wilson ( 2015 ) showed a significant and positive correlation between frequency of bookmarking in mendeley and the number of citations in scopus in the field of medical sciences ( 28 ) .
although the correlation between altmetrics measures and the number of citations in citation indexes does not prove that the former leads to the latter , the study of this issue is the first step to rational evaluation of an altmetrics measure .
a positive correlation indicates that the two are not completely apart from each other and have common similarities .
a significant correlation between bookmarking and citation is not an evidence of causality but probably indicates that some readers have cited the papers .
the significant correlation between the two variables shows that bookmarking and citation reflect similar aspects of impact of an article .
however , the correlation between the two variables was not strong enough to conclude that the number of citations and bookmarking reflects the same image of significant impact of research .
it seems that frequency of bookmarking shows reading frequency of an article , which combines the two impacts of research and broad expertise .
this may be due to the fact that most researchgate users are working in the fields of medical sciences and biology .
high viewing number of articles in researchgate and mendeley suggests the high impact of the two social networks in increasing the visibility of scientific works .
it is recommended that medical scholars use this network as a means of self - archiving and finding information .
also , the ministry of health , universities , research institutions , and research centers should be aware of the importance of attendance and membership of scholars , faculty members , and even students in social networks , particularly higher education students .
this probably will be used as a criterion for scientific research assessment of scholars and academics in science policy - making . | introductionit is essential to evaluate the impact of scientific publications through citation analysis in citation indexes . in addition
, scientometric measures of social media also should be assessed .
these measures include how many times the publications were read , viewed , and downloaded .
the present study aimed to assess the scientific output of scholars at kashan university of medical sciences by the end of march 2014 based on scientometric measures of scopus , researchgate , and mendeley.methodsa survey method was used to study the articles published in scopus journals by scholars at kashan university of medical sciences by the end of march 2014 .
the required data were collected from scopus , researchgate , and mendeley .
the data were analyzed with descriptive statistics .
also , the spearman correlation was used between the number of views of articles in researchgate with citation number of the articles in scopus and reading frequency of the articles in mendeley with citation number in scopus were examined using the spearman correlation in spss 16.resultsfive-hundred and thirty - three articles were indexed in the scopus citation database by the end of march 2014 .
collectively , those articles were cited 1,315 times .
the articles were covered by researchgate ( 74% ) more than mendeley ( 44% ) .
in addition , 98% of the articles indexed in researchgate and 92% of the articles indexed in mendeley were viewed at least once .
the results showed that there was a positive correlation between the number of views of the articles in researchgate and mendeley and the number of citations of the articles in scopus.conclusioncoverage and the number of visitors were higher in researchgate than in mendeley .
the increase in the number of views of articles in researchgate and mendeley also increased the number of citations of the papers .
social networks , such as researchgate and mendeley , also can be used as tools for the evaluation of academics and scholars based on the scientific research they have conducted . |
arginine vasopressin ( avp ) is an antidiuretic hormone which is synthesized in the magnocellular neurons of the supraoptic ( son ) and paraventricular nuclei ( pvn ) in the hypothalamus .
avp is transported to the posterior pituitary through the axons , released into the systemic circulation , and plays a pivotal role in water balance by promoting reabsorption of free water through the v2 receptor in kidney .
the release and synthesis of avp are mainly regulated by plasma osmolality ( or serum na ) in physiological conditions .
the osmoregulation of the avp neuron system is so precise that only 12% increases in serum na levels significantly stimulate its release as well as the transcription of avp gene in the son and pvn .
serum na levels are thus maintained around 140 meq / l , the threshold level for avp release .
the deficiency of avp leads to hypotonic polyuria , a disorder called neurohypophysial or central diabetes insipidus ( hereafter , we use cdi as the abbreviation ) .
water intake also increases in order to maintain water balance in patients with cdi , as long as their thirst sensation remains intact . in this review ,
most causes of cdi are acquired , and its prevalence of cdi is reportedly 1:25,000 .
table 1 indicates the etiology of 165 japanese patients with cdi we reported previously .
the most frequent cause of cdi is the tumors in the central nervous system ( cns ) including craniopharyngioma and germ cell tumors , which could damage the avp neuron system .
cdi is also caused by inflammatory diseases such as lymphocytic infundibulo - neurohypophysitis ( linh ) and igg4-related disease .
, polyuria appears in the first 2 days after surgery and sometimes resolves spontaneously , although it could persist permanently if avp neurons are damaged substantially .
idiopathic cdi , 13% of total cdi in table 1 , is diagnosed when the causes of cdi are unclear .
it is of note that linh has been sometimes regarded as idiopathic cdi in some studies .
this is probably because linh is reported to be a possible underlying cause of idiopathic cdi .
this subclass of cdi is called familial neurohypophysial diabetes insipidus ( fndi ) , which is inherited mostly in an autosomal dominant mode .
more than 80 mutations that cause fndi have been reported so far , and they are mainly located in the coding region of neurophysin ii , a carrier protein of avp , in the gene locus ( table 2 ) .
mutations causing fndi * , mutations affecting the initiator atg are predicted to result in translation initiation at an alternative downstream atg ( codon 5 ) , causing a deletion of the first 4 amino acid residues . , mutations associated with autosomal - recessive inheritance of fndi .
, 10,396 base pair deletion involving the majority of the avp gene as well as its regulatory sequences in the intergenic region between the avp and the oxytocin gene .
if splicing of intron 2 is affected , the entire ( in - frame ) intron is inserted into the mrna leading to an insertion of 58 amino acids into the avp protein ( p. ? ) .
if splicing is not affected , this mutation causes to change codon 108 to a premature termination codon ( p. e108x ) .
cdi is characterized by hypotonic polyuria accompanied by polydipsia , as long as the thirst sensation is intact .
daily urine volumes exceed 3 liters in most cases . daily water intake , which could be estimated by asking the subjects how many cups of water they take every day , is almost equal to daily urine volumes .
if daily water intake seems to be less than 2 liters , the subjects are unlikely to have cdi .
it is also important to ask the subjects how often they urinate and drink water at night , because only daytime polydipsia and polyuria suggest psychologic polydipsia rather than cdi .
final diagnosis of cdi is made by confirming that avp release is hampered in response to increases in plasma osmolality or serum na levels .
for this reason , either injection of hypertonic saline or the water restriction test is performed . in the hypertonic saline examination , 5% nacl at the rate of 0.05 ml / kg body weight ( bw ) /min
is injected intravenously for 2 hours , and the serum na and plasma avp levels are measured before and every 30 minutes after starting the injection .
the serum na levels usually increase approximately by 10 meq / l , and plasma avp levels are increased in proportion to the increases in serum na levels in normal subjects .
in contrast , increases in avp release are blunted or even abolished in patients with cdi ( figure 1 ) . to perform this examination , it is essential to use a sensitive assay for plasma avp . plasma avp and serum na levels in response to hypertonic saline injection . in normal subjects ,
plasma avp levels increase in proportion to increases in serum na levels . in cdi patients ,
no sensitive assays for plasma avp are available , the water restriction test is performed .
the subjects are deprived of water and food for about 6 hours or until their bw is decreased by 3% . in normal subjects ,
in contrast , urine osmolality remains to be below 300 mosm / kg during the test in cdi patients .
injection of pitressin ( vasopressin ) at the end of the test could differentiate nephrogenic di from cdi : urine osmolality increases in response to pitressin in patients with cdi but not in those with nephrogenic di .
the high intensity in posterior pituitary in t1-weighted mri images , which reportedly reflects the stock of pituitary avp , is observed in normal but not cdi subjects .
however , as the avp stock in the posterior pituitary could be decreased under chronically dehydrated conditions , the lack of the high signal in t1-weighted pituitary mri images is not specific to cdi .
pituitary mri is also useful for the differential diagnosis of primary and secondary cdi . in case
there is no abnormality except for the lack of the high signal in t1-weighted images , the subjects are diagnosed as idiopathic cdi
. however , the follow - up of the imaging is necessary , particularly in children , since some abnormalities such as tumors in the cns may appear later on .
due to the lack of avp action on v2 receptors in kidney , patients with cdi show hypotonic polyuria and polydipsia .
the amount of daily urine volume as well as water intake could exceed 10 liters when avp release is almost completely abolished .
normal subjects feel thirsty when serum na levels exceed 145 meq / l , while the threshold of serum na levels for avp release is around 140
meq / l in cdi patients , in whom the action of avp is lacking .
thus , cdi patients consume water until their thirst sensation ceases , but they will soon feel thirsty because serum na levels exceed 145 meq / l again due to the lack of avp action . if the function of osmoreceptors in hypothalamus is also lost , the patients would not feel thirsty even if the serum na levels exceed 145 meq / l .
this is called masked di , and substitution of adrenocortical hormones leads to increases in urine volume in patients with cdi accompanied by adrenal insufficiency . while several mechanisms have been suggested for masked di , one possibility is that avp release , which is usually inhibited by adrenocortical hormones , is stimulated from the residual avp neurons in cdi patients with adrenal insufficiency .
however , it is still controversial whether or not glucocorticoid receptors are expressed in the avp neurons .
fndi patients are born with normal water balance , but manifest progressive polyuria and polydipsia several months or years after birth .
the analyses of animal models for fndi have demonstrated that accumulation of mutant avp precursors in the endoplasmic reticulum ( er ) causes er stress and dysfunction of the neurons , which finally lead to loss of avp neurons .
this is consistent with autopsy studies which showed that magnocellular neurons were lost in patients with fndi .
the desmopressin formula is either intranasal liquid , intranasal spray , or tablets . in japan , an oral disintegrating tablet ( odt ) was approved for the treatment of cdi in 2012 , and the qol of cdi patients has reportedly been improved by odt .
our study also showed that the incidence of hyponatremia in cdi patients treated with desmopressin is decreased after the switch from the nasal formula to odt .
this is probably due to the fact that the absorption and efficacy of odt are more stable than those of the nasal formula , making it easier to determine the required dose of desmopressin .
nevertheless , one should be aware that there is still a high incidence of hyponatremia in cdi patients treated with desmopressin , and it is safe to start the treatment with a low dose of desmopressin .
the ideal bw for water balance should be determined based on the serum na levels ( bw is considered to be ideal when serum na levels are within normal ranges ) , but this could vary over time and should be assessed periodically . the basal volume of water intake ( for example , 1 liter / day ) and the required dose of desmopressin ( for example , 60 g odt desmopressin twice a day ) to maintain the ideal bw should also be determined . however , one should recall that bw could substantially change from day to day in patients , and the scale of water intake must be set based on the bw changes .
for example , if the bw is 58 kg in adipsic cdi patients whose ideal bw has been determined to be 60 kg , 2 kg water has been lost . in this situation , 2 liters of water should be consumed in addition to the basal volume ( 1 liter ) of daily water intake ; that would mean that patients must drink 3 liters of water on the day , but it could be difficult for adipsic patients to consume such a volume of water , given that they do not sense thirst .
the prognosis of cdi due to tumors or inflammation depends on that of the primary diseases . on the other hand ,
the prognosis of idiopathic cdi patients is considered to be comparable to that of subjects without cdi , as long as the patients can drink water as necessary .
however , the adipsic cdi patients are associated with significant morbidity , and the incidence of serious infections requiring hospitalization and the risk of death have been shown to be higher in adipsic compared to non - adipsic cdi patients .
the etiology has been clarified in most cdi patients . while most cdi patients are well treated with desmopressin , controlling water balance in adipsic cdi patients
| abstractcentral diabetes insipidus ( cdi ) , characterized by polyuria and polydipsia , is caused by deficiency of arginine vasopressin ( avp ) , an antidiuretic hormone which acts on v2 receptors in kidney to promote reabsorption of free water .
cdi is classified into three subtypes ; idiopathic , secondary and familial .
a previous study suggests that infundibulo - neurohypophysitis might be an underlying cause of idiopathic cdi . among secondary cdi ,
the tumors in the central nervous system such as craniopharyngioma and germ cell tumors are the most frequent causes .
familial cdi is inherited mostly in an autosomal dominant mode , and
the number of causal mutations in the avp gene locus reported so far exceeds 80 .
cdi is treated with desmopressin , an analogue of vasopressin , and the tablet is preferred to the nasal form because it is easier to administer .
it is also shown that the oral disintegrating tablet formula increases qol and decreases the incidence of hyponatremia in cdi patients . in some cdi patients , the osmoreceptors in the hypothalamus do not function and patients do not sense thirst .
these adipsic cdi patients are treated with desmopressin and adjusting the amount of daily water intake based on body weight measurement ; but controlling the water balance is extremely difficult , and morbidity and mortality are shown to be high in these patients . |
loss of anterior teeth is a common form of injury , particularly in children and adolescents . on the other hand ,
elderly people , who are retaining their teeth for longer period of time , have often advanced caries or periodontal diseases which may lead to extraction of teeth .
patients with lost anterior teeth require immediate attention for restoration of esthetic and functional reasons .
the increased patient demand for tissue maintenance and esthetic , as well as the desire to reduce treatment costs , causes clinician to seek materials and techniques that enable minimally invasive and chairside ( direct ) fabrication on teeth replacement with fixed partial dentures ( fpds ) [ 1 , 2 ] . over the last few years
, the development of fiber - reinforced composite ( frc ) has offered the dental profession the possibility of fabricating resin - bonded , esthetically good and metal - free tooth restorations for single and multiple teeth replacement .
frc - fixed partial denture ( fpd ) could be an alternative to metal frame resin - bonded - fpd , and also to full - coverage - crown - retained fpd and implant supported crowns [ 3 , 4 ] .
frc , made of glass fibers , is the only existing esthetically acceptable material , which can be processed in mouth to the shape of a framework of a bridge , simultaneously adhere to the remaining tooth substance , and reach the adequate strength in terms of biting function of human .
many studies have focused on improvement of frc fpd 's strength [ 5 , 6 ] . the most accepted concept to fabricate frc fpds
is based on the use of continuous unidirectional glass ( bundle ) fibers in dimethacrylate - polymethylmethacrylate resin matrix as a substructure for the fpd . with the frc fpds
, there are two approaches on the use of the fibers : one is based on conventional tooth preparation and laboratory - made restorations , while the other is based on using the fibers in minimally invasive restoration ( conservative ) by direct or indirect fabrication .
frc systems enable the use of different retainer elements even in the same fpd ( hybrid - type ) .
for example , it is possible to create space for the occlusal support of the frc framework by removing old filling or to make completely surface - retained restorations when clinical conditions allow correct designing of the frc framework . in the dental literature ,
there are presently a limited number of clinical studies on the fiber - reinforced fpds ; however , based on those results , it is reasonable to expect frc prostheses to have good longevity , especially with those made by direct technique [ 4 , 8 , 9 ] .
this paper describes clinical cases of chairside-(directly ) made frc fpds , which was used according to the principles of minimal invasiveness .
thirty - sex - year - old female patient had a chief complaint of esthetics because of a gab of missing upper left lateral incisor and crown of root canal - treated canine ( figure 1 ) .
after discussion with the patient , it became clear that the placement of an implant for the replacement of missing teeth was not possible due to high costs of the treatment .
the fabrication of a conventional fixed partial denture was avoided and refused from patient in order to conserve the remaining tooth substance .
options for the conventional treatment with implants or crown - retained fpds were remained open for the future .
directly made frc fpds were chosen in order to provide good esthetics , preserve tooth substance , and postpone more invasive treatments .
there was free occlusal space on the palatal surface of central incisor for frc framework to be placed .
guttapercha root canal filling at the upper left canine was removed using gates glidden burs up to size 4 for the total length of 7 mm ( 4000 cycles min with water cooling ) .
the individually formed glass frc post ( everstick post , sticktech ltd , turku , finland ) was prepared following the manufacturer 's instructions ( figure 2 ) .
a bundle of preimpregnated glass fibers was cut to a length of 16 mm and spread from the ends for increasing the bonding surface area ( figure 3 ) .
the bundle was inserted in the canal and initially light polymerized with a hand light - curing unit ( optilux-501 ) for 20 s. then the post was removed from the canal and additionally light polymerized for 40 s. the surface of the frc post system was then wetted with resin ( stick resin ) and protected from any light source by a light proof box ( 3 m - espe , germany ) until cementation .
cement ( paracem universal , switzerland ) was placed on the post and the post was seated and extra cement was removed .
the frc framework was extended from the palatal surface of premolar to palatal surface of central incisor passing by the frc post of the canine .
after application of acid etching ( 37% phosphoric acid gel ) , the gel was rinsed thoroughly and gently air dried .
adhesive resins were applied according to the manufacturer 's instructions ( scotchbond multipurpose adhesive , 3 m espe , usa ) to tooth surface .
flowable composite resin ( stick flow , stickteck ltd , turku , finland ) was applied on the bonding surfaces prior placing the resin impregnated fibers ( everstick ) .
the flow composite was not light cured before fibers were pressed tightly against the tooth surface using a transparent silicone package ( mold ) of the fibers .
the purpose of the flow composite was to seal the space between the fibers and the enamel surface .
fiber framework was fully covered with a thin layer of flow composite resin , and pontic was built up layer by layer using hybrid - type particulate filler composite resin .
the shade of final veneered composite resin was selected using composite shade guide , and occlusion was carefully adjusted with articulating paper ( figures 5 and 6 ) .
the occlusion was adjusted carefully to avoid any primary or premature contacts or traumatic occlusal forces to the restored teeth .
the treatment outcome has been followed over three years without existence of any kind of serious problem .
fourteen - year - old male patient lost his upper central incisors due to trauma by an accident ( figure 7 ) .
after discussion with the patient 's father , it became clear that the placement of a single implant for the replacement of missing tooth was not possible due to patient age and high costs of the treatment . also , young age of the patient would have been a clear contraindication for an implant treatment .
the fabrication of a conventional fixed partial denture was avoided in order to conserve the tooth substance because of patient 's young age .
the missing teeth were planned to be replaced with an implant - retained crowns later on .
directly made frc fpds were chosen in order to provide good esthetics , preserve tooth substance , and postpone the final decision on the prosthetic treatment .
as described in the first clinical case , after etching and applying bonding agent , fibre - framework was extended between the palatal surfaces of lateral incisors ( figure 8) .
fiber - framework was covered with a thin layer of flow composite resin , and pontic was built up by using particulate filler composite resin .
the final step was the adjustment of occlusion and contouring and finishing the restoration ( figure 9 ) .
laboratory - made surface - retained resin - bonded prostheses made of metals are commonly supported and bonded from one end in order to reduce the number of debondings . in the case of surface - retained frc prostheses
, the framework can be supported from both ends because of better bonding characteristics and biomechanical flexibility of the frc framework .
the flexibility allows abutment teeth to move without stressing the cement - framework interface in function , and loosening the prostheses .
however , in the case of abutments with increased mobility , it is recommended to support also resin - bonded frc fpd from one end only . in the cantilever designs ,
special care has to be taken to ensure adequate design - based rigidity of the frc framework to resist bending forces by biting function .
fibers of the framework should cover as large surface as possible on the abutments and , in the anterior area , should be placed close to the incisal edge to eliminate the dislodging forces .
although resin - bonded frc fpds are most commonly used in the anterior and premolar regions , rather than molar region , recent laboratory investigations have suggested that optimally designed frc fpd made on nonprepared abutments can provide even higher load - bearing capacity for the fpd than conventional porcelain - fused to metal fpd can provide .
thus , the development of the frc materials and technologies may allow alternatives also for directly made molar replacements .
the frc framework is intended to be fully covered by veneering composite in order to obtain a polishable and tooth - coloured surface .
if the frc framework is not properly covered by veneering composite , the darkness of the oral cavity can be transmitted through the connectors and can cause esthetic problems .
the composition of the polymer matrix and fiber orientation has the major role in bonding ability and durability of veneered composite to the frc framework or resin luting cement .
it has been concluded that preimpregnation of the fibers with the light - polymerizable dimethacrylate resin system containing linear polymer phases is of importance to optimize the interfacial adhesion of frc framework to composite veneer . using a combination of
dimethacrylate monomer resin and linear polymer , which forms semi - interpenetrating polymer network ( semi - ipn ) after being polymerized , offers better bonding site for veneered composite by means of interdiffusion bonding [ 1214 ] .
recent laboratory studies showed that bond strength of directly fabricated frc fpd to the tooth surface is as good as particulate filler composite . from clinical point of view
however , the longitudinal studies reported general failure rates between 5% and 16% over periods up to 4 - 5 years [ 4 , 9 , 16 ] . these finding were demonstrated for prostheses with both extracoronal and intracoronal retainer designs , but only for patients who did not exhibit sever parafunctional habits .
van heumen et al . showed a survival rate of 64% after 5 years follow - up of 3-unit anterior frc prostheses made with the materials and techniques used in late 1990s .
the recent clinical data , on the semi - ipn resin matrix frc fpds made directly in patients mouth , suggest high survival percentages ( > 96% at five years ) , which reflects material development and learning of fabricating frc fpds .
most common failures in frc fpds reported in the earlier studies were delamination of veneering composite at pontic area , which are normally easy to be repaired in patients mouth .
the current designing principles enable to fabricate frc fpd to eliminate known risks for technical failures . as conclusion ,
the combination of filling composite veneer , adhesive system , and frc framework has introduced a new generation of metal - free direct teeth replacement .
the most recent fabrication principles need to be followed to ensure clinical success of the restorations . | missing anterior teeth is of serious concern in the social life of a patient in most of societies .
while conventional fixed partial dentures and implant - supported restorations may often be the treatment of choice , fiber - reinforced composite ( frc ) resins offer a conservative , fast , and cost - effective alternative for single and multiple teeth replacement .
this paper presents two cases where frc technology was successfully used to restore anterior edentulous areas in terms of esthetic values and functionality . |
vector - borne hemoparasites , including the apicomplexan protozoa babesia sp . and plasmodium sp .
, trypanosomes and filarial nematodes , are important pathogens in humans and domestic animals , causing babesiosis , malaria , sleeping sickness , lymphatic filariasis and canine heartworm disease .
endemic as well as introduced hemoparasites may also impact health and fitness of wildlife ( e.g. custer and pence , 1981 , atkinson et al .
, 2000 , garvin et al . , 2003 , donahoe et al . ,
furthermore , babesiosis is an emerging zoonosis worldwide , with wildlife reservoirs playing a particular role in its epidemiology ( gray et al . , 2010 , yabsley and shock , 2013 ) .
thus , characterizing hemoparasite infections in wildlife and understanding patterns of prevalence can potentially reveal emerging disease risks , especially in the light of ecological instabilities as a result of human encroachment into wildlife habitats ( daszak et al .
, 2001 , keesing et al . , 2010 ) and climate change ( daszak et al . ,
wild primate populations are of particular interest in this context , because nonhuman primates have played a major role in the emergence of human diseases , including malaria , in the past ( wolfe et al .
, 2007 , pedersen and davies , 2009 , pacheco et al . , 2011 ) .
age , sex or seasonal effects may have an influence on exposure and susceptibility to infection with many parasites .
age effects have been demonstrated for plasmodium sp . infections in chimpanzees and humans ( doolan et al .
infections in dogs and cattle ( boozer and macintire , 2003 , bock et al . , 2004 ) , with decreasing prevalence in older animals being most likely due to the development of an effective adaptive immune response ( frolich et al . , 2012 ) .
additionally , the innate immune system is an important controlling factor of apicomplexan parasite infectivity ( frolich et al . , 2012 ) and
sex differences in hemoparasite infections have been found in several vertebrates , including higher prevalences in male penguins ( merkel et al . , 2007 ) , lizards ( schall et al . , 2000 ) and
these sex differences could either be due to physiological differences , e.g. immunosuppression caused by higher testosterone levels in males ( klein , 2004 , roberts et al . , 2004 ) , or due to differences in parasite exposure .
for example , a sex - bias in vector feeding rates towards males was recently demonstrated in birds ( burkett - cadena et al . , 2014 ) .
although hemoparasite infections have been investigated in several wild primate species ( de thoisy et al .
, 2001 , maamun et al . , 2011 , de nys et al . , 2013 , thurber et al . , 2013 ) , no sex - differences in prevalence have been reported so far .
furthermore , it has been proposed that group - living may decrease the risk of infection with vector - borne pathogens by means of an encounter - dilution effect , analogous to a decrease of predation risk ( freeland , 1976 , mooring and hart , 1992 , kappeler et al . , 2015 ) .
( 2014 ) recently demonstrated that sentinel hosts caged inside large roosts of american robins seroconverted to west nile virus more slowly than those held outside of roosts , suggesting that exposure of individual hosts can indeed be reduced through group - formation .
however , larger groups may also be more conspicuous , and thus attract more vectors . in a comparative study ,
colonially - breeding bird species were shown to experience both higher prevalences and higher species diversity of blood parasites than solitarily breeding species ( tella , 2002 ) .
two studies on neotropical primates found that prevalence of plasmodium sp . increases with group size ( davies et al .
, 1991 , nunn and heymann , 2005 ) , but studies on the effect of within - species variability of group size on hemoparasite infections in primates are lacking .
furthermore , infections with vector - borne parasites are likely to covary with environmental conditions ( altizer et al . ,
rainfall and temperature affect mosquito abundance , biting rates and parasite development within mosquitoes ( altizer et al . , 2006 ,
additionally , dry , cold conditions as well as frequent temperature fluctuations can reduce the abundance of host - seeking ticks ( sutherst and bourne , 2006 , swai et al .
, 2006 , herrmann and gern , 2013 ) . despite this seasonality in vector biology , empirical evidence regarding seasonal variation in host infection rates is contradictory and mainly limited to malaria in humans ( smith et al . , 1993 , koram et al . , 2003 ,
mabaso et al . , 2007 ) . if transmission results in persistent infections , little seasonal variation in prevalence can be expected ( govender et al . ,
finally , in natural systems co - infections with multiple parasites are common , potentially resulting in complex interspecific interactions .
it has been shown that parasite community interactions may explain more variation in infection risk than the effects associated with host and environmental factors .
for example , in field voles ( microtus agrestis ) chronic infection with babesia microti reduces susceptibility to bartonella spp .
parasites may directly affect the fitness of co - infecting species through interference competition , they may compete for the same resources within a host , or affect each other 's abundance via interaction with the host 's immune system ( pedersen and fenton , 2006 ) .
cross - immunity between co - infecting parasite species might limit prevalence at the population level , whereas parasite - induced immunosuppression may lead to synergistic effects ( cox , 2001 , telfer et al . , 2008 ) .
we present the first study which systematically characterizes infections with several co - occurring hemoparasites in a population of malagasy primates , verreaux 's sifakas ( propithecus verreauxi ) .
madagascar faces acute risks of species extinctions as well as disease emergence in humans and wildlife due to intense human disruption of natural ecosystems ( harper et al . , 2007 , junge , 2007 , barrett et al . , 2013 , ratsimbazafy et al . , 2013 ,
verreaux 's sifakas are diurnal , sexually monomorphic lemurs which live in multi - male multi - female groups of varying size in seasonal habitats , in which mosquitoes are virtually absent for several months of the year , in western and southern madagascar ( kappeler and fichtel , 2012 ) .
the life expectancy of sifakas in the wild can exceed 20 years ( richard et al . , 2002 , kappeler and fichtel , 2012 ) .
we used samples collected from members of all age classes from 10 adjacent groups ranging in size from 2 to 7 individuals during annual captures to detect infections with plasmodium sp . , babesia sp . and filarial nematodes using a combination of a pcr - based approach and microscopical examination of blood smears .
these parasites have previously been reported to occur in blood samples of sifakas ( uilenberg et al . , 1972 , junge and louis , 2005 , duval et al . , 2010 , pacheco et al . , 2011 , rasambainarivo et al . , 2014 ) , but systematic investigations of infection patterns are lacking .
anopheline mosquitos and haemaphysaline ticks , are present in the study region ( davidson , 1966 , rodriguez et al . , 2012 ) , whereas the vectors for filarial parasites of lemurs are unknown ( irwin and raharison , 2009 ) . to illuminate natural drivers of infection patterns , we tested the influence of host age and sex , host group size and seasonality on individual infection status and hemoparasite species richness in our study population . to assess possible health consequences
, we also tested the influence of these infections on packed cell volume , to assess possible anemia , and total plasma protein as well as the neutrophil - lymphocyte ratio , which are usually increased during inflammatory processes ( thrall et al .
based on results of previous studies with other vertebrates , we predicted that infection probabilities would decrease with age and that males would harbor more hemoparasites than females .
furthermore , we predicted that group size would negatively affect the probability of testing positive for each hemoparasite , as well as individual hemoparasite species richness , in case of an encounter - dilution effect .
alternatively , we predicted that group size would have a positive effect on these measures if larger groups attract more vectors .
we also expected to find lower prevalences of hemoparasite infections during the dry season than during the hot , wet season .
the study was carried out in kirindy forest , western madagascar , located at approximately 4439e , 2003s .
the study area is part of a field site operated by the german primate center ( dpz ) since 1993 and is situated within a forestry concession managed by the centre national de formation , detudes et de recherche en environnement et foresterie ( cnferef ) .
kirindy forest is a dry deciduous forest and subject to pronounced seasonality , with a dry season from april to october and a hot , wet season from november to march ( kappeler and fichtel , 2012 ) . as part of an ongoing long - term study ( kappeler and fichtel , 2012 ) ,
several social groups of verreaux 's sifakas have been habituated to human observers and individually marked with microchips and unique collars .
a total of 45 blood samples were taken from 36 individual verreaux 's sifakas belonging to 10 social groups during routine immobilization procedures in april 2013 , august 2013 , march 2014 and april 2014 .
group sizes ranged from 2 to 7 individuals , and a minimum of 43% of individuals were sampled from each group .
seven animals were repeatedly sampled at intervals of 45 months , resulting in a maximum of 3 samples per individual ( table 1 ) .
packed cell volume was determined by centrifuging edta blood in a microhematocrit capillary using a sigma 114 centrifuge ( sigma laborzentrifugen gmbh , osterode am harz , germany ) and total plasma protein was estimated using a hand - held refractometer .
two to 3 blood smears were prepared per individual , air dried , stained ( diff quick stain , eberhard lehmann gmbh , berlin , germany ) and preserved with mounting medium ( eukitt , fluka analytics , sigma aldrich chemie gmbh , munich , germany ) and a cover slip .
blood smears were scanned for the presence of hemoparasites and used for a differential white blood cell count to assess the percentages of the following leukocyte categories : neutrophils , banded neutrophils , lymphocytes , monocytes , eosinophils and basophils .
photographs were taken with a zeiss axiocam erc 5s fitted to a zeiss primo star microscope ( carl zeiss ag , oberkochen , germany ) .
measurements were made using the zeiss zen lite 2012 software ( carl zeiss ag ) after calibration with a stage micrometer .
an aliquot of blood was mixed with the same amount of rnalater and frozen at 20 c until shipment to germany and further analysis .
all necessary research permits were obtained from the malagasy and german authorities and the study was approved by the ethics committee of the german primate center .
dna was extracted from rnalater - preserved blood samples using the qiamp blood mini kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions . to test for the presence of plasmodium spp . in blood samples ,
a semi - nested pcr was carried out targeting an approximately 1000 bp long fragment of the parasite 's cytochrome b gene . in the first amplification round , primers p.sp.cytb f1 ( 5-tgc cta gac gta ttc ctg att atc cag ; kaiser et al .
( 2010 ) ) and p.sp.cytb r1 ( 5- ctt gtg gta att gac atc cwa tcc ; kaiser et al .
( 2010 ) ) were used , followed by p.sp.cytb f2 ( 5-att ggd tca acw atg act tta ttt gg ) and p.sp.cytb r1 in the second round . the 25 l reaction mixture contained 1 l of dna - extract or pcr - product ( diluted 1:40 ) from the first round , respectively , 2.5 l 10x pcr buffer ( invitrogen , karlsruhe , germany ) , 2 l of 50 mm mgcl2 ( invitrogen , karlsruhe , germany ) , 2 l of 2.5 mm deoxynucleotide triphosphates ( thermo scientific fermentas , st . leon - rot , germany ) , 0.5 l of each primer ( 10 m ) and 0.2 l of platinum taq polymerase ( invitrogen , karlsruhe , germany ) .
the thermal profile was the same for both rounds , with an initial denaturation step at 95 c for 5 min followed by 40 cycles of 95 c for 30 s , 58 c for 45 s and 72 c for 60 s , and a final elongation step at 72 c for 10 min .
we tested for babesia spp . using primers bj1 ( 5-gtc ttg taa ttg gaa tga tgg-3 ) and bn2 ( 5-tag ttt atg gtt agg act acg-3 ) , targeting a 500 bp long fragment of the 18s rrna gene ( casati et al .
we used 5 l of dna - extract in a 25 l reaction mixture containing the same quantities of reagents as stated above .
the thermal profile consisted of an initial denaturation step at 94 c for 10 min followed by 40 cycles of 94 c for 60 s , 55 c for 60 s and 72 c for 2 min , and a final elongation step at 72 c for 5 min . to generate sequences for microfilaria observed in blood smears , we employed a nested pcr targeting an approximately 900 bp long fragment spanning part of the 18s rrna gene , the internal transcribed spacer 1 ( its1 ) and part of the 5.8s rrna gene .
primers used were nf1 ( 5-ggt ggt gca tgg ccg ttc tta gtt-3 ) ( porazinska et al . , 2009 ) and
nc2 ( 5-tta gtt tct ttt cct ccg ct-3 ) ( gasser et al . , 1993 , chilton et al . , 2003 ) in the first round and a modification of its1-f ( 5- ttg att acg tcc ctg ccc-3 ) ( vrain et al . , 1992 ,
2014 ) and the filaria - specific di5.8s - r ( 5-acc ctc aac cag acg tac-3 ) ( nuchprayoon et al . , 2003 , nuchprayoon et al . , 2005 ) in the second round . the 25 l reaction mixture contained 5 l of dna - extract in the first round and 1 l pcr - product ( diluted 1:40 ) in the second round , and the same quantities of reagents as in the other pcrs .
the thermal profile of the first round consisted of an initial denaturation step at 95 c for 5 min followed by 35 cycles of 94 c for 60 s , 58 c for 30 s and 72 c for 60 s , and a final elongation step at 72 c for 10 min .
the thermal profile of the second round consisted of an initial denaturation step at 94 c for 10 min followed by 35 cycles of 94 c for 30 s , 55 c for 30 s and 72 c for 60 s , and a final elongation step at 72 c for 10 min .
pcrs were run in a flexcycler thermal cycler ( analytic jena , jena , germany ) and amplification products were detected by electrophoresis on 1.5% agarose gels .
pcr products of the corresponding size were purified from 2% agarose gels using the jetquick gel extraction spin kit ( genomed , lhne , germany ) and sanger sequencing of both strands was performed by seqlab sequence laboratories gttingen gmbh ( gttingen , germany ) .
auckland , new zealand ) and compared to publicly available sequences using blast ( altschul et al . ,
new sequences were deposited in the embl nucleotide sequence database ( kulikova et al . , 2004 ) , under accession numbers ln869519 ln869522 . for plasmodium sequences
the two unique sequences identified in this study were put together with the representative sequences selected by pacheco et al .
( 2011 ) , which includes all sequences derived from lemur plasmodium available to this date .
we used jmodeltest v2.1.4 to identify the model of nucleotide substitution with the best fit to the data ( gtr + i + g4 ; ( darriba et al . , 2012 ) ) .
we then reconstructed a maximum likelihood tree under this model using phyml v3 ( guindon et al . , 2010 ) , as implemented on the phyml webserver ( guindon et al . , 2005 ) .
branch robustness was assessed through non - parametric bootstrapping ( 500 bootstrapped pseudo - replicates ; bp ) .
we used generalized linear - mixed models ( glmms ) with binomial error structure and logit link function to analyse which factors influenced the probability of testing positive for each parasite species .
we included sex , age , season ( wet or dry ) and size of the animal 's social group as fixed effects and individual identity nested in group as a random effect .
furthermore , we ran a glmm with poisson error structure and log link function to test influences on hemoparasite species richness , including the same fixed and random effects .
based on results of the glmms for plasmodium and babesia infections , we used another glmm with binomial error structure to test whether babesia infection status had an effect on plasmodium infection , again controlling for individual identity nested in group . to assess potential health impacts , we first tested whether age affected hematology values , using spearman rank correlation , and then used linear mixed models ( lmms ) to test influences of infection with each hemoparasite and hemoparasite species richness on log - transformed packed cell volume , total plasma protein and the log - transformed neutrophil : lymphocyte ratio , controlling for animal age and individual identity nested in group as a random effect .
we compared each full model to a null model comprising only the random effect in a likelihood ratio test using the r - function anova with the argument
statistical significance was inferred if the specific p - value of the factor as well as the p - value of the likelihood ratio test were < 0.05 .
seven individuals were tested more than once , and repeated sampling at different times of the year almost always gave identical results for these animals .
only results for one animal ( age 9 ) switched from negative to positive during the study period .
we did not observe any parasite stages in diff - quick - stained blood smears .
only two unique sequences were amplified in this study , which showed 95100% sequence identity to publicly available plasmodium sequences .
lld-2010 which has previously been found in verreaux 's sifakas ( duval et al . , 2010 ) .
ln869522 ) , which unambiguously nested within the clade of malaria parasites infecting lemurs ( bp 100 ; fig . 1 ) .
c - map-2012 ( which has been found in hapalemur griseus ( pacheco et al . , 2011 ) ) , although the statistical support for this association was moderate ( bp 59 ; fig . 1 ) .
the patristic distance between these two sequences was 0.21 substitution per site ( s.s ) , which is both higher than the 0.17 s s observed between the two most closely related lemur parasite sequences known to date ( plasmodium sp .
b - map.2012 and e - map-2011 ) and higher than the maximum 0.14 s s observed between p. reichenowii ( clade c1 , infecting chimpanzees ) and p. praefalciparum sequences ( clade g1 , infecting gorillas ; rayner et al . , 2011 ) .
test outcome , with older animals being more likely to test positive ( fig . 2 ) .
in fact , none of the animals up to the age of 5 years were infected ( n = 18 ) , 50% of animals between age 6 and 10 were infected ( n = 10 ) and all animals over the age of 10 tested positive ( n = 8) .
however , in the glmm including age as well as sex , season and group size as fixed factors , age only tended to have a statistically significant effect ( table 2 ) .
again , test results for only 1 individual ( age 7 ) changed from negative to positive during the study period . the babesia sequence amplified ( embl accession no .
ln869519 ) was the same in all samples and showed 94% identity to different babesia canis isolates .
we did not microscopically observe any babesia parasite stages in diff - quick - stained blood smears .
infections , young animals were more likely to test positive ( table 3 , fig . 2 ) .
only 3 animals ( 6 , 7 and 11 years old ) were co - infected with plasmodium sp . and babesia sp . based on this complementary age pattern , the low number of co - infections , and because neither age , sex , season nor group size influenced plasmodium spp . infections , we tested babesia infection status as a predictor of plasmodium infection , and found a highly significant negative effect ( table 4 ) .
two to 3 blood smears per sample were scanned for microfilaria and on average 4.2 microfilaria were measured per sample .
microfilaria of species 1 were 268 10.1 m long with the nerve ring located at 53 4.5 m from the anterior end .
microfilaria of species 2 were considerably shorter , 222 18.6 m , with the nerve ring located at 43 5.4 m .
species 1 produced a shorter band of about 850 bp and species 2 produced a longer band of about 900 bp .
ln869520 and ln869521 ) showed 99100% sequence identity to different filarial nematodes such as mansonella sp .
, dirofilaria sp . and onchocerca sp . in the conserved 18s and 5.8s regions , but
only 8490% similarity to other filarial nematodes in the variable its1 region . in 33 of 45 samples ,
the pcr results exactly confirmed the morphological diagnosis ( negative sample , presence of one or both species ) . in 7 cases ,
a species was morphologically identified but not detected by pcr , while in 5 cases an infection was revealed by pcr only .
thus , neither method seems to be 100% sensitive . for statistical analyses , we considered an infection present if it was detected by one of the methods .
prevalence of species 1 was 50% , while species 2 was found in 33.3% of animals .
probability of infection with species 1 significantly increased with age ( table 5 ) .
we did not find any significant influences on infection with the second species ( table 5 ) .
we tested whether host age , sex , season or group size affected the number of hemoparasite species found in each individual , but we did not find any statistically significant influences ( table 6 ) .
hematology results are summarized in table 7 packed cell volume and total plasma protein were significantly positively correlated with the animals age ( spearman rank correlation , n = 44 , = 0.44 , p = 0.003 , and n = 43 , = 0.4 , p = 0.008 , respectively ) and the ratio of neutrophils to lymphocytes tended to be positively correlated with age ( spearman rank correlation , n = 45 , = 0.26 , p = 0.08 ) .
controlling for age , we did not find any significant influences of infection with any single parasite species on packed cell volume ( pcv ; table 8) , and hemoparasite richness also did not influence pcv ( table 9 ) .
likewise , total plasma protein ( tp ) was neither significantly influenced by infection with any single parasite species ( table 8) , nor by hemoparasite species richness ( table 9 ) .
the ratio of neutrophils to lymphocytes ( nlr ) was 0.47 0.22 ( range : 0.151.25 ) .
no single infection influenced log - transformed nlr values , ( table 8) , and hemoparasite richness was also not a significant predictor ( table 9 ) .
the percentage of eosinophils was 0.96 0.91 ( range : 04.5 ) , which is in the range of published values for propithecus sp .
( bergeron and buettner - janusch , 1970 , junge and louis , 2005 , irwin et al . , 2010 ,
we identified 5 putative species of hemoparasites in verreaux 's sifakas in kirindy forest : 2 species of malaria parasites , only one of which has been previously identified in verreaux 's sifakas by means of pcr ( duval et al . , 2010 ) , the other one likely standing for a yet undetected and undescribed species ; one species of babesia ; and 2 species of filarial nematodes . as sequences of babesia parasites and filarial nematodes from madagascar
are not publicly available to date , we could not assign species names to our sequences .
morphological descriptions of microfilaria found in lemurs were ambiguous ( irwin and raharison , 2009 ) .
of the 4 species described in lemurs , which all belong to the family onchocercidae , species 1 could either be dipetalonema petteri or protofilaria furcata , while measurements for species 2 fall into the range described for paulianfilaria pauliani .
we did not recover any adult worms , as dissections of animals were not possible in this study , and the insufficient quality of blood smears did not allow us to use further microfilaria characteristics for identification . however , genetic analyses confirmed the presence of two different filarial nematode species in the population .
we found high prevalence of infection with babesia sp . in wild verreaux 's sifakas under the age of 5 , while infections with plasmodium sp . were absent from this age class , although most , but not all individuals of this age class were sampled during the wet season , when mosquitoes are abundant .
this is unusual , as it has been found in humans and chimpanzees that younger animals usually have higher plasmodium sp .
prevalences and that prevalence decreases with age , possibly due to naturally acquired immunity ( doolan et al . , 2009 , de nys et al . , 2013 ) .
the age - infection patterns we observed give no indication of age - related immunity to plasmodium sp .
in verreaux 's sifakas . in our study , animals older than approximately 10 years showed 100% plasmodium sp .
juveniles under the age of one year were never pcr - positive for filariasis , nor were microfilaria observed in their blood - smears .
however , we can not exclude the possibility that these animals were already infected , as the prepatent period of filarial nematodes , i.e. the period from infection to appearance of reproductive parasite stages in the blood , may span several months ( bowman and georgi , 2009 ) .
therefore , the result that age significantly affects filarial nematode infections should be treated with caution .
neither pcr nor microscopic examinations of blood smears seem to be sensitive enough to reliably detect filarial nematodes infections in all cases , so we combined the results of both methods . in cases of low parasitemia ,
pcrs produced some false - negatives although single microfilaria were identified in the corresponding blood smears , and species differentiation based on microfilaria morphology only is difficult , because the length ranges of both species may potentially overlap .
this could have led us to misdiagnose infections in some cases and we might have missed statistically significant effects on onchocercidae infections or hemoparasite species richness , as well as the effects of those on hematology values .
sex did not influence infection with any single species , nor species richness , indicating that males are neither more susceptible to infections nor more exposed .
although sex - biased patterns of parasitism , including the parasite taxa examined in this study , seem to exist in many mammals ( klein , 2004 ) , this effect does not seem to be universal ( kiffner et al . , 2013 ) .
specifically , sifakas do not display sexual size dimorphism ( kappeler , 1990 ) , which has been proposed to relate to sex differences in parasite susceptibility in general ( moore and wilson , 2002 ) , while larger bodied hosts may also attract more vectors ( davies et al . , 1991 ) .
furthermore , male and female sifakas , like other lemurs with female dominance , exhibitit smaller differences in androgen levels than other mammals ( von engelhard et al . , 2000 , drea , 2007 ) ,
we tested whether host social group size influenced infection with each single hemoparasite species as well as hemoparasite species richness , but we did not find any significant effect .
group sizes in this study ranged from 2 to 7 individuals , and this range may have been too small to detect any differences , although small group - size differences should have a more pronounced effect on individual infection risk in small groups .
thus , our data do not support the encounter - dilution hypothesis , nor the opposing prediction that larger groups attract more vectors , which was supported by two studies of plasmodium sp . prevalences across neotropical primates with group sizes ranging from 3 to 29 individuals ( davies et al .
infections nor on infections with any of the other parasites , although fewer mosquitoes are present during the dry season .
persistent infections as well as long developmental times of the parasite within the host , as might be the case for filarial nematodes , may mask seasonal effects . because of the opposite patterns of age - dependency of plasmodium and babesia infections , and because babesia sp .
infections were a significant negative predictor for plasmodium sp . infections , we suggest that infection with babesia sp . may be a natural protectant against plasmodium sp . in this lemur species . as sifakas in kirindy
give birth during the dry season , when almost no mosquitoes are present , juveniles likely acquire babesia sp .
infections via tick - bites , probably of haemaphysaline ticks , which occur in western madagascar ( rodriguez et al . , 2012 ) and have been detected on sifakas during animal captures , before they become exposed to plasmodium sp .
until babesiosis resolves , e.g. due to antibody - mediated immunity ( frolich et al . , 2012 )
the suppression of malaria infections in the course of ongoing babesiosis has been investigated in rhesus macaques ( macaca mulatta ) in laboratory settings ( van duivenvoorde et al . , 2010 ) , after it was found that a macaque infected with a b. microti - like parasite showed a suppressed plasmodium cynomolgi infection ( wel et al . ,
heterologous immunity between the two parasites has also been observed in mice ( cox , 1978 , zivkovic et al . , 1984 ) .
it has been proposed that the cross - protection is most likely due to immune responses such as the activation of a distinct class of pro - inflammatory monocytes , release of pro - inflammatory cytokines , and increased c - reactive protein levels , which potentially play an important protective role in malaria infections ( clark , 2001 , ansar et al . , 2006 ,
it has also been proposed that this heterologous immunity might be exploited for the development of malaria vaccines . however , an interaction between plasmodium and babesia could also arise due to resource competition within the host .
in fact , in humans it has been shown that co - infecting parasites mostly interact in this way ( griffiths et al . , 2014 ) .
both parasites invade and replicate in erythrocytes and both of them modify the erythrocytic membrane to achieve higher permeability , but do so using different mechanisms and at different time points after invasion ( alkhalil et al . , 2007 ) .
changes in membrane permeability induced by babesia infection may thus make subsequent infection with plasmodium sp . impossible and lead to a competitive advantage for babesia sp .
however , b. microti parasites suppressed p. cynomolgi parasitemia without altering the induction of initial anemia in rhesus macaques , suggesting the malaria parasites could enter erythrocytes during an infection with babesia ( van duivenvoorde et al . , 2010 ) .
the lack of positive microscopic observation of both babesia and plasmodium stages in the blood smears of infected individuals may partially result from technical limitations , but probably also indicates that parasitemia was generally low .
this may suggest that if competition occurs , the parasites only compete for a narrow erythrocyte subset . while the precise mechanism of interaction remains unclear , the pattern found in our study provides the first indication of a possible protective effect of babesiosis on malaria infections in a wild population of primates naturally infected with both parasites , thus adding external validity to the above observation .
finally , we did not find any signs of morbidity caused by the parasites tested in this study . only animal age affected hematology values , a common pattern also in primates ( mcpherson , 2013 ) .
packed cell volume was not significantly affected by infection with any single hemoparasite species nor by hemoparasite species richness , indicating that these parasites do not cause clinically relevant anemia .
the absence of anemia may be explained by the long coevolutionary history between hemoparasites and their hosts , leading to a complete dependence of the parasite on the host for survival , which favours long - lasting asymptomatic infections with low parasitemia ( chauvin et al . , 2009 ,
furthermore , evidence has been found that co - infection with filarial nematodes may attenuate anemia associated with clinical malaria in humans ( dolo et al . , 2012 ) .
total plasma protein and the neutrophil - lymphocyte ratio were not affected in a statistically significant way .
acute inflammation usually causes an increase in plasma globulin concentrations , which may reflect in increased total plasma protein levels , as well as an increase in neutrophilic granulocytes ( thrall et al . , 2006 ) .
however , the neutrophil - lymphocyte ratio may be affected in both directions by an excitement response with epinephrine release due to animal capture , as leucocytes are released from the marginal into the circulating pool ( thrall et al . , 2006 ) , which may have masked effects due to parasitism .
eosinophils typically show an increase as a response to helminth infection ( nutman , 2007 ) , and high values have been measured in the course of filarial infections in dogs and humans ( mackenzie , 1980 , niwetpathomwat et al . , 2007 ) .
elevations may also occur in the course of babesiosis ( vercammen et al . , 1997 ) .
the relative counts of eosinophils were not markedly elevated in any of the animals in this study , judging by comparison with previously reported values from both captive and wild propithecus sp .
populations ( bergeron and buettner - janusch , 1970 , junge and louis , 2005 , irwin et al . , 2010 , rasambainarivo et al . , 2014 ) .
however , this conclusion remains preliminary as we were not able to obtain absolute leucocyte counts due to restrictions of fieldwork . in conclusion ,
prevalences of hemoparasite infections in verreaux 's sifakas in kirindy forest , madagascar , are significantly related to host age , probably shaped by interaction between babesia sp . and plasmodium sp . , but independent of host sex , host group size and season . the possible cross - protection between babesia sp . and plasmodium sp .
is a prime example of how biodiversity may affect the ecology of infectious diseases . increasing evidence
has accumulated that biodiversity may have a buffering role on disease transmission , while biodiversity loss is thought to increase transmission , disease incidence and disease emergence ( daszak et al . , 2001 , ostfeld , 2009 , keesing et al .
, 2010 , vourch et al . , 2012 ) . in this case , the presence of tick - borne babesiosis seems to reduce plasmodium sp .
prevalence in a natural system , illustrating how within - host pathogen diversity may regulate the abundance of parasites .
infection status did not influence hematology values , suggesting that the parasites considered in this study do not cause clinically relevant anemia or inflammation , but that hosts and parasites are well - adapted to each other , resulting in persistent infections with low pathogenic potential . | hemoparasites can cause serious morbidity in humans and animals and often involve wildlife reservoirs .
understanding patterns of hemoparasite infections in natural populations can therefore inform about emerging disease risks , especially in the light of climate change and human disruption of natural ecosystems .
we investigated the effects of host age , sex , host group size and season on infection patterns of plasmodium sp .
, babesia sp . and
filarial nematodes in a population of wild malagasy primates , verreaux 's sifakas ( propithecus verreauxi ) , as well as the effects of these infections on hematological variables .
we tested 45 blood samples from 36 individuals and identified two species of plasmodium , one species of babesia and two species of filarial nematodes .
plasmodium spp . and babesia sp .
infections showed opposite patterns of age - dependency , with babesiosis being prevalent among young animals , while older animals were infected with plasmodium sp .
in addition , babesia sp .
infection was a statistically significant negative predictor of plasmodium sp .
infection .
these results suggest that plasmodium and babesia parasites may interact within the host , either through cross - immunity or via resource competition , so that plasmodium infections can only establish after babesiosis has resolved .
we found no effects of host sex , host group size and season on hemoparasite infections .
infections showed high prevalences and did not influence hematological variables .
this preliminary evidence supports the impression that the hosts and parasites considered in this study appear to be well - adapted to each other , resulting in persistent infections with low pathogenic and probably low zoonotic potential .
our results illustrate the crucial role of biodiversity in host - parasite relationships , specifically how within - host pathogen diversity may regulate the abundance of parasites . |
biliary complications are frequent after orthotopic liver transplantation ( olt ) , affecting 5 to 15% of patients after deceased olt and 28 to 32% after right - lobe living donor olt [ 14 ] .
post - olt bile leaks are reported in 10 to 15% of patients and are usually an early complication and represent a high morbidity condition for the patient .
endoscopic treatment is well recognized as first - line therapy in the management of post - olt biliary leaks or stricture [ 1 , 4 , 6 , 7 ] .
endoscopic retrograde cholangiography ( erc ) with biliary sphincterotomy and/or transpapillary plastic stent placement is typically offered as standard treatment [ 810 ] .
successful endoscopic therapy for postsurgical bile leaks has been reported in 88 to 97% of cases and up to 83.9% in those secondary to olt .
covered self - expandable metallic stents ( csemss ) have been increasingly used to treat benign biliary conditions and have been shown promising results for both biliary strictures and leakages [ 7 , 1214 ] .
their larger diameter , long - term patency , and proven removability have turned them into an appealing option to assess refractory and/or complex bile leaks [ 7 , 1315 ] .
although few studies have already demonstrated encouraging results for csems use in the treatment of postsurgical bile leaks [ 13 , 14 ] , their long - term safety and efficacy have not been established for this particular group of patients when compared to plastic stents . indeed in order for csems to replace plastic stents in this indication , not only they need to demonstrate efficacy but also long - term safety . therefore , the primary aim of this study was to review our experience with temporary placement of three different types of csems in the treatment of post - olt biliary leaks .
we retrospectively reviewed a prospectively established database to assess post - olt bile leaks treated with csems between december 2003 and december 2010 .
inclusion criteria were the same as previously defined and included patients with complex or high - grade leaks defined as those who failed endoscopic plastic stent therapy or with severe comorbidities that prevented multiple procedures who were referred to temporary placement of csems .
data was captured prospectively as each type of stent came on the market in chronological order ( first partially covered , then fully covered with fins , after that fully covered with flared ends ) .
our endpoints were long - term clinical success ( resolution of bile duct leak ) and safety .
our institutional review board approved the study , and written informed consent was obtained from all patients prior to erc .
all procedures were either performed or supervised by dedicated biliary endoscopists performing at least 500 erc yearly .
side - viewing endoscopes ( tjf-140 , tjf-160 and tjf-160vf , olympus america , center valley , pa , usa ) were used for all procedures .
three different types of csems were placed : partially csems ( pcsems ) ( wallstent , boston scientific corp , natick , ma , usa ) , fully csems ( fcsems ) with fins ( viabil , conmed , utica , ny , usa ) ( figure 1 ) , and fcsems with flared ends ( wallflex , boston scientific corp ; figure 2 ) . after selective biliary cannulation , biliary sphincterotomy was performed , and a retrieval balloon was used to perform an occlusion cholangiogram and locate the bile leak ( figure 3 ) . over the guidewire placed across the leak ,
the stent was placed , crossing the papilla for at least 1 cm after deployment .
successful csems placement was defined as deployment of the csems across the leak with resolution of the leak fluoroscopically .
proximal migration of the fcsems was defined as any migration of the csems into the bile duct .
distal migration was defined as migration of the stent into the duodenum from the transpapillary position .
spontaneous csems migration with resolution of the leak was recorded but was not considered a complication .
post - stent - removal biliary strictures were defined as a narrowing demonstrated on imaging associated with elevated liver function tests .
all patients were seen in our liver transplant clinic and digestive health center with consultation of nephrology , pathology , or infectious diseases whenever indicated .
short - term followup was obtained by a clinic visit , with cross - sectional imaging one month after the fcsems was removed .
laboratory values , including complete blood count and hepatic function panels , were also closely followed .
after leakage resolution , csems was removed by using the rat tooth and/or snare technique as previously described . in cases
where the csems had foreshortened or migrated within the bile duct , balloon dilation was used to disimpact the csems with subsequent rat tooth removal .
this was particularly important when dealing with the partially csems due to the development of tissue overgrowth at the proximal portion , imbedding the csems or when removing the fcsems with fins , which anchor the csems within the bile duct .
the fcsems has a loop at its distal end , which permits extraction using a rat tooth , and , unless the loop was imbedded within the ampullary tissue , this technique was used preferentially .
choledochoscopy was performed in patients whenever a lesion or a stricture was suspected on fluoroscopy following csems removal and used the single operator system ( spyglass , boston scientific ) .
clinical response to csems placement and procedure - related morbidity and mortality rates were analyzed .
descriptive data were expressed in means , medians , and standard deviations ( sds ) .
fischer 's exact tests were conducted to observe any significant differences between stent groups , with the statistical significance set at p < 0.05 .
a choledochocholedochostomy was performed in 428 patients ( 95% ) while 23 patients received a roux anastomosis ( 5% ) .
posttransplant leak was observed in 54 ( 12% ) patients and ischemic cholangitis in 23 ( 5% ) patients .
thirty - one patients underwent csems placement for bile leak treatment after deceased liver transplant , three in the pcsems group , eighteen in the fcsems with fins group , and ten in the fcsems with flare ends group .
there was no evidence of significant differences among them regarding age , gender , leak site , time interval between olt , and procedure to csems deployment and previous treatment with plastic stent .
all patients had a single duct - to - duct anastomosis , and there was no reference to complex arterial reconstruction among patients treated in this series .
partially csemss ( 80 mm length ) were temporary placed in three patients for a median time of 89 days ( range 55 to 110 ) . after a median followup of 1,353 days ( range 1,348 to 2,208 ) , clinical success was achieved in all patients .
there was one case of spontaneous migration , even though the patient had the leak resolved .
another patient developed a late - onset hilar stricture , refractory to both endoscopic plastic stenting therapy and underwent hepaticojejunostomy .
choledochoscopy was performed for that patient prior to surgery confirming tissue overgrowth at the hilum . in this group ,
18 patients underwent temporary placement of the fcsems with fins ( 80 and 100 mm length ) for a median of 102 days ( range 35 to 427 ) and were followed after removal for a median of 849 days ( range 323 to 1,111 ) .
long - term leakage control was achieved in 14 patients , 77.8% in an intention to treat analysis .
one patient died from unrelated cause , and 2 underwent liver retransplantation due to hepatic artery thrombosis ; by excluding them from the long - term analysis , the clinical success rate would be 93.3% ( 14/15 ) .
one patient presented cholangitis after stent deployment and was treated with repeated erc and plastic stent placement .
post - stent - removal complications included six clinically significant hilar biliary strictures that required biliary drainage ( 6 plastic with plastic stents , 2 with csems , and 1 underwent a surgical hepaticojejunostomy ) .
choledochoscopy was performed in five patients and demonstrated ulcerations ( figure 5 ) in 4 patients ( 80% ) that were managed conservatively and one ( 20% ) hilar stricture ( figure 6 ) that was treated with plastic stenting .
the csems was kept in place for a median time of 98 days ( range 96 to 139 ) , and 3 patients still have the stent in situ .
median followup after stent removal was 203 days ( range 95 to 305 ) , and so far 70% ( 7/10 ) of patients presented clinical resolution of biliary leakage .
one patient ( 10% ) developed a hilar stricture after csems removal and received plastic stenting .
four patients underwent choledochoscopy during stent removal , which revealed inflammation in two patients ( 20% ) and ulceration in one ( 10% ) , but no hyperplasia .
fischer 's exact test was conducted to analyze for differences in clinical success rates ( leak resolution ) among the fcsems groups .
there was no evidence of significant differences in the 2 stent groups with regards to clinical success , age , gender , leak location ( anastomotic or non - anastomotic ) , previous treatment with plastic stent , stent indwelling , postplacement , and post - removal complications ( all p > 0.05 ) .
followup was significantly longer in the fcsems with fins group ( p < 0.01 ) .
biliary leaks occur in 10 to 15% of patients after olt and usually present with clinical symptoms earlier than strictures in the postoperative course .
endoscopy stands as a first - line treatment for post - olt biliary leaks [ 11 , 18 ] .
however , many anastomotic leaks may require surgical repair and , therefore , have been defined as a complex leak [ 13 , 14 ] .
the main principle of endoscopic therapy for biliary leakage is to reduce transpapillary pressure gradient via transpapillary stenting with or without biliary sphincterotomy ; this is conventionally performed using plastic stents [ 8 , 9 , 11 ] . with the large diameter provided by metal stent and the ability to remove covered metal stents ,
this was initially described by baron and poterucha , when they reported 3 cases of complex bile leaks successfully treated with csems .
promising results of csems have also been further reported by our team with long - term leak control obtained in 87% ( 14/16 ) of patients with post - surgical bile leaks ; however , long - term results were not available yet .
csemss have mainly been used as a rescue therapy for patients who failed standard endoscopic therapy with plastic stenting .
however , more recently has been used as a first - line measure in patients with complex and high - grade leaks [ 19 , 20 ] . the rationale to deploy a sems through a leak is to grant the larger diameter possible allowing the patient to have the faster recovery time , with fewer sessions , and prevent further complications associated with the bile leak .
the median indwelling of csems in this study was 189 , 102 , and 98 days , respectively , for the pcsems , the fcsems with fins , and the fcsems with flare ends , respectively .
the use of csems for the treatment of bile leak has the theoretical advantage of decreasing the number of procedures needed to control the leakage in patients with complex or high - grade fistulas when compared to plastic stents .
it is presumed that the initial increased cost associated with the use of csems will be compensated by the reduction of sessions required as well as the days of hospitalization .
all patients in the partially csems group had previously failed plastic stenting ( i.e. , persistent leak after plastic stent placement ) , and , after placement of the metal stent , leak control was achieved in all of them ( tables 1 and 2 ) .
wallstent was the first pcsems commercially available in the us market but led to mucosal hyperplasia at its uncovered portions and migration [ 12 , 13 ] .
proximal migration is especially problematic since it can be associated with hyperplasia and secondary stricture after stent removal .
studied both radial ( rf ) and axial forces ( af ) of wallstent and viabil .
the results demonstrated wallstent to have high af , possibly related to biliary wall damage , kinking , and sludge formation and migration . in the present study ,
distal migration was noted in one patient ( 33.3% ) from pcsems and one ( 10% ) from fcsems with flare ends group .
this migration rate has been reported in other studies [ 20 , 22 ] and might be related to the respective force of the 2 csemss , which are similar ( unpublished data from isayama ) . the fcsems with anchoring fins positioned at opposite ends
study , what might cause an excessive high pressure to the biliary wall , resulting in increased tissue injury and eventual stricture . in this study ,
tissue injury was found in 4/5 ( 80% ) patients when choledochoscopy was performed in this group and an increased number of post - stent - removal strictures ( 35% ) that could be a consequence of biliary compression and ischemia . even though migration rate was lower ( 5.6% ) ,
a discussion is raised if the incidence of postremoval strictures was related to the high radial force or to the presence of fins that could stimulate tissue reaction and scarring .
finally , fcsems with flare ends was recently released as fully covered sems , coated with premalume .
although the leakage controlled has been encouraging in this study , we are still dealing with spontaneous distal migration ( 10% ) and postremoval stricture ( 10% ) .
interestingly there no statistically significant difference in either group in term of final outcome ; this might be related to a type 2 error , which can be overcome by increasing the number of patients in each sample ; however the complications ' rate associated with all three csems does not justify such a study .
it probably needs to be fully covered with an inert and resistant coating and has no fins , which seem to be associated to significant tissue reaction .
further csems soon invading the market might offer those characteristics and need to be carefully evaluated . in conclusion ,
however , postremoval biliary stricture requiring further endoscopic treatment was seen especially with the fcsems with fins group . at the present time , csems can not be recommended in this patient population until major design changes have been made . | objectives . endoscopic management of bile leak after orthotopic liver transplant ( olt ) is widely accepted .
preliminary studies demonstrated encouraging results for covered self - expandable metal stents ( csems ) in complex bile leaks .
methods .
thirty - one patients with post - olt bile leaks underwent endoscopic temporary placement of csems ( 3 partially csems , 18 fully csems with fins and 10 fully csems with flare ends ) between december 2003 and december 2010 .
long - term clinical success and safety were evaluated .
results .
median stent indwelling and follow - up were 89 and 1,353 days for pcsems , 102 and 849 for fcsems with fins and 98 and 203 for fcsems with flare ends .
clinical success was achieved in 100% , 77.8% , and 70% , respectively .
postplacement complications : cholangitis ( 1 ) and proximal migration ( 1 ) , both in the fcsems with fins .
postremoval complications were biliary strictures requiring drainage : pcsems ( 1 ) , fcsems with fins ( 6 ) and with flare ends ( 1 ) .
there was no significant differences in the fcsems groups regarding clinical success , age , gender , leak location , previous treatment , stent indwelling , and complications .
conclusion
. temporary placement of csems is effective to treat post - olt biliary leaks .
however , a high number of post removal biliary strictures occurred especially in the fcsems with fins .
csems can not be recommended in this patient population . |
immune responses against pathogens determine the course of
infection as well as the pathogenesis of diseases caused by the
pathogens . during the responses , immune cells , such as dendritic
cells , macrophages , and t and b lymphocytes ,
for
instance , during infections with some protozoa , such as
leishmania major or trypanosoma cruzi , the
importance of ifn- has been proven not only experimentally
but also clinically [ 1 , 2 ] .
ifn- is produced mainly
by helper t cell ( th ) 1-differentiated cd4 t cells , a
population induced by il-12 , although other types of cells , such
as nk / nkt cells and cd8 t cells are also known to produce
ifn-. while being critically important for the defense
against pathogens , cytokines are sometimes notorious as causative
agents for the development of immunopathology and inflammation ,
which may result in organ / tissue damage .
tnf- , a cytokine
produced by th1-type cd4 t cells and activated macrophages
for defense against intracellular pathogens , may induce cell
apoptosis or necrosis depending on situation .
therefore , it is
essential that one should mount proper immune responses against
pathogens , making sure the response is not too weak to eliminate
pathogens or too strong to damage the host .
recently , two new
cytokines , il-23 and il-27 have been identified as members of the
il-12 cytokine family . with il-12 , a cytokine known as the most
potent th1-inducing cytokine
, the il-12 cytokine family has been
shown to be involved in various diseases such as infection ,
autoimmune diseases , and inflammations .
this review will focus on
the complicated roles of il-27 , as well as those of related
cytokines , and their potential importance during some protozoan infections .
when stimulated through t - cell receptors , cd4 t cells
proliferate and differentiate into either th1 or th2 cells , two
functionally distinct subsets that produce characteristic
cytokines , respectively .
th1 cytokines , especially
ifn- , are critical for the macrophage activation and
nitric oxide production required for eliminating intracellular
pathogens such as l. major . in contrast , th2 cytokines such as il-4 , il-5 , and il-13 are important for inducing
humoral immunity required to expel helminth from the digestive
canal . th1 and th2 cells develop from the same th precursor ( thp or th0 ) cells , but which subset differentiates in a
given situation is driven by factors , particularly cytokines , in
the surrounding microenvironment .
il-12 promotes ifn-
production and th1 development , whereas il-4 binding to the
il-4 receptor promotes il-4 production and th2 development
.
developments of th1 and th2 are mutually exclusive and differentiation of one suppresses that of the other by various
mechanisms .
in addition to these two subsets , recent lines of
evidence have shown that there is another th subset , called th17
or thil-17 [ 79 ] ( figure 1 ) .
th17
cells produce il-17 for induction of various inflammatory
responses and for defense against some pathogens [ 10 , 11 ] .
the differentiation of th17 cells requires il-6 plus tgf- for commitment and il-23 for development , while other cytokines such as il-1 and tnf- also support the development of th17 [ 1214 ] .
il-12 was originally identified as a potent inducer of
ifn- production by t , nk , and other types of lymphocytes
and was shown later to be a potent inducer of th1 differentiation
of cd4 t cells . until recently
, il-12 has been the only known heterodimeric cytokine , composed of two subunits , p35
and p40 .
the p35 subunit is homologous to il-6 and g - csf with a
four--helix bundle structure , while the p40 subunit is
homologous to the extracellular portion of il-6r and
related cytokine receptors .
thus , it was proposed that il-12
evolved from a cytokine of the il-6 family , which is
covalently bound to the extracellular portion of its primordial
chain receptor .
recently , il-23 and il-27 were identified as heterodimeric cytokines functionally and
structurally related to il-12 [ 16 , 17 ] ( see [ 18 , 19 ] for review ) .
now along with two other cytokines , clc / soluble cntfr and clc / clf-1 , il-12 , il-23 , and il-27 compose a family of
heterodimeric cytokines [ 18 , 20 ] .
il-23 , a heterodimeric cytokine composed of the il-12p40 subunit and the il-12p35-related molecule p19 , preferentially acts on memory th1 cd4 t cells for their proliferation , although il-23 has recently been shown to promote th17 differentiation .
il-27 , another
heterodimeric cytokine composed of the il-12p40-related protein
ebi-3 plus the il-12p35-related protein p28 , acts on nave cd4 t cells to induce the expression of the
il-12r2 gene and make the cells responsive to il-12 . while
il-12 is the most potent inducer of th1 differentiation and
ifn- production acting on effector th1 cells ,
chronologically differential roles and differential usage of
il-12 , il-23 , and il-27
first , il-27 commits
nave cd4 t cells to differentiate into th1 cells by
inducing il-12r2 , then il-12 acts on committed effector
th1 cells for ifn- production , followed by il-23
mediating the proliferation of memory th1 cells ( figure 2 ) . while the th1 immune response is pivotal for the host defense against pathogens including some bacteria and
parasites such as listeria monocytogenes ,
mycobacterium tuberculosis , and l. major
( reviewed by romani et al . in ) , as well as
against tumors , excess of th1 responses may cause some autoimmune diseases including rheumatoid arthritis and multiple
sclerosis [ 24 , 25 ] .
it is of note that recent lines of evidence have shown that some of these th1-mediated autoimmune diseases are actually mediated by il-23-driven th17 cell populations but not by il-12-driven th1 cell populations , as has
been shown for experimental autoimmune encephalomyelitis , an
experimental model for multiple sclerosis [ 7 , 8 ] ( see also [ 26 , 27 ] for review ) .
nonetheless , proper initiation ,
reinforcement , and maintenance of the th1 response seems to be
ensured , and at the same time tightly regulated by related , but
distinct , cytokines .
sprecher et al . cloned an orphan cytokine receptor and named it
wsx-1 ( what is now called il-27r chain
; also known as
tccr ) after the wsxws motif , a characteristic feature of cytokine
receptors , in its extracytoplasmic portion .
wsx-1 binds to the gp130 ( of il-6r ) to conform to a fully functional il-27
receptor complex , downstream of which , stat1 and stat3 are
activated by ligand binding [ 30 , 31 ] .
although the function of
wsx-1 was not clear in the original cloning paper , two groups
including ours independently generated strains of mice with
wsx-1 ( or tccr ) gene disruption and demonstrated
that wsx-1 is critical for induction of th1 responses and
ifn- production .
chen et al . demonstrated that wsx-1 mice showed impaired ifn-
production and remarkable susceptibility to l. monocytogenes .
in the report by yoshida et al . , an impairment of th1 development and ifn- production was also demonstrated in
wsx-1 mice infected with l. major .
interestingly , impaired production of ifn- as only observed at early phases of l. major infection , and the
ifn- production in wsx-1 mice was restored to the wild - type level at later phases of infection . in accordance with this in vivo observation , t cells from
wsx-1 mice produced much less ifn- than wild - type cells during primary stimulation in the presence of il-12 for th1 differentiation , while fully differentiated
wsx-1 th1 cells produced a comparable level of ifn- upon secondary stimulation . these data , both in vivo and in vitro , indicated that wsx-1 is required for the initial mounting of the th1 response by nave cd4 t cells , but its role is later mitigated presumably by the il-12
system in fully activated and differentiated th1 cells .
the
importance of wsx-1 in th1 differentiation was further
substantiated by the discovery of il-27 , a ligand for wsx-1 , as
another th1-inducing composite cytokine .
early expression of p28 , a component of il-27 , prior to that of il-12
components , by activated dendritic cells also supported the idea
that il-27 was involved in the early phase of th1 differentiation
. a report on the role of ebi-3 ,
a subunit of il-27 , in which ebi-3-deficient mice showed susceptibility to l.
major infection with impaired th1 response also supported the
importance of il-27/wsx-1 in th1 differentiation .
( see table 1 for comparison . ) while resistance against l. major exclusively depends on
proper th1 responses , expulsion of trichuris muris , a
gastrointestinal nematode from digestive canals largely depends on
th2 responses . in our recent t. muris infection
experiments
[ 35 , 36 ] , wsx-1 mice successfully
expelled the worm and failed to harbor chronic infection in
contrast to wild - type mice .
wsx-1 lymphocytes produced significantly less ifn- and much more il-4 and il-13 than wild - type cells in response to the worm antigen ,
reiterating that wsx-1 is important for the initial mounting of
proper th1 responses during infection .
another interpretation of
the results , however , is also possible for the impairment of th1
responses against l. major and t. muris
infection .
stat1 was shown to bind to the cytoplasmic portion of wsx-1 in a tyrosine
phosphorylation - dependent manner and following stat1 activation by
il-27 stimulation , t - bet was activated to promote th1
differentiation .
the binding of stat1 to wsx-1 is reasonable ,
since wsx-1 has pyekhf motif at the conserved tyrosine residue ,
which shares amino acid similarities to the stat1 docking site of
the ifn-r , pydkph [ 38 , 39 ] .
stat1 activation per se
by il-27 stimulation has no direct effect on ifn-
production but stat1-mediated t - bet activation followed by
il-12r2 expression was one of the critical events for th1
differentiation .
other mechanisms , such as mhc class i
augmentation , icam-1 expression , and suppression of the expression
of gata-3 , a transcription factor pivotal for th2 differentiation ,
were also involved for il-27-mediated th1 differentiation
[ 4042 ] .
it is now clear that the molecular basis of
il-27/wsx-1-mediated th1 initiation is the induction of t - bet ,
which is reminiscent of the finding that the ifn-
stimulation of cells induces t - bet in a stat-1-dependent fashion
[ 43 , 44 ] . interestingly , even in the absence of wsx-1 , t - bet was induced at later time points after t cell stimulation ( our unpublished observation ) , presumably due to the small amount of
ifn- produced or a stat1-independent mechanism yet to be
determined .
this delayed induction of t - bet may explain the restoration of ifn- production and il-12
responsiveness of wsx-1 t cells at later time points .
in sharp contrast to the finding that il-27 is important for th1
promotion , recent reports also demonstrated that il-27 plays a
regulatory role in immune responses .
wsx-1 mice , when infected with t. cruzi or
toxoplasma gondii , showed remarkable sensitivity to the
infection [ 45 , 46 ] . this high sensitivity to infection was not due to impaired th1 differentiation or ifn- production .
unexpectedly , over production of various proinflammatory cytokines including inf- , tnf- , and il-6 was observed in infected wsx-1 mice , followed by lethal liver
necrosis .
rosas et al . also reported severe liver immunopathology
in tccr- ( another name for wsx-1 ) deficient mice infected with
leishmania donovani .
sources of the cytokines were not limited to t cells , but other types of cells such as
nk / nkt cells and macrophages also produced various cytokines . in
addition , wsx-1 cd4 t cells were more activated than wild - type cells in terms of cell cycle progression
and expression of surface activation makers .
similarly , wsx-1 nkt cells produced more of the proinflammatory cytokines , inducing lethal liver damages in con
a - induced liver damage experiment , which is an experimental model
of viral or autoimmune hepatitis .
although the
development of nkt cells were normal in wsx-1 mice , these wsx-1 nkt cells produced more ifn- and il-4 than wild - type nkt cells in response to con a or -galactosyl ceramide stimulation in vitro . when infected with m. tuberculosis , wsx-1
mice also suffered from liver damages and cachexia due to over
production of inflammatory cytokines , such as tnf-
.
interestingly , wsx-1 mice harbored significantly lower bacterial loads over wild - type mice due to higher amounts of tnf- and ifn-. these results remind us of the fact that immunity against pathogens is a double - edged sword ; it kills the pathogens in one way but may hurt
the host in another way . beside the observations described above ,
il-27 actually suppressed cytokine production - activated cd4
t cells in vitro .
il-27 also suppressed cytokine
production such as tnf- and il-12 by macrophages
[ 49 , 50 ] . in these reports , il-27 stimulation induced stat3 activation and concurrently suppressed cytokine production by lps - stimulated macrophages .
interestingly , il-27 stimulation
appeared less effective in macrophage suppression than il-4 or
il-10 .
stimulation of macrophages with il-4 , il-10 , or il-27
resulted in differential gene induction , suggesting distinct
suppression mechanisms by respective cytokines .
wirtz et al . also reported the suppressive effect of il-27 on monocytes
and granulocytes reactive oxygen intermediate ( roi ) production
.
administration of recombinant il-27 to mice in the septic peritonitis model resulted in higher mortality than
untreated mice due to reduced bacterial clearance by the down
regulation of granulocyte / monocyte function .
collectively , these
data revealed the novel role of il-27/wsx-1 as an attenuator of
proinflammatory cytokine production and cell activation of t cells
as well as macrophages , to suppress excess of inflammation and/or
to cease immune responses . in this regard , a distinct ( but not mutually exclusive ) hypothesis
on the impairment of th1 responses in the wsx-1 mice should be described .
artis et al . reported an overproduction of il-4 in l. major - infected wsx-1 mice and suggested that the overproduction of th2 cytokines at early phases of infection reciprocally suppressed the following
ifn- production and th1 development .
similarly , artis et al . also reported that upregulation of th2 responses is independent from th1 impairment in t. muris - infected
wsx-1 mice , claiming that primary role of il-27 is the suppression of immune responses ( including th2 responses )
but may not be the induction of th1 differentiation ( see and see review by hunter et al . ) .
although reconciliation of the two seemingly conflicting ideas awaits further analyses of
the role of il-27 in various settings , a recent report by our
group sheds light on this issue . in this report ,
yoshimura et al
. demonstrated that il-27 acts on nave t cells
for ifn- production while the same cytokine suppresses
cytokine production by affecting fully activated cells .
therefore ,
the activation status of the cells may be the key determinant for
the effects of il-27 .
the underlying mechanisms for this immune / inflammation suppression
by il-27 are not fully elucidated .
it has been reported that il-27
inhibited il-2 production by cd4 t cells thereby regulating
an excess of cell activation [ 54 , 55 ] . in wsx-1 mice ( or cells ) , however , not only il-2 production but also the production of various types of cytokines were augmented .
additionally , the source of cytokine production was not limited to cd4 t cells , but other types of cells were also affected by wsx-1 deficiency .
il-10 production
was unexpectedly augmented similarly by wsx-1 deficiency
[ 45 , 46 ] and the so - called regulatory t cells ( treg ) did not appear to be affected by the receptor deficiency .
a
mechanism that collectively regulates cytokine production and/or
cell activation should be taken into consideration .
il-23 , another il-12 cytokine family member , has recently been
described to induce the differentiation of a new subset of th
cells , namely , th17 [ 7 , 8 , 57 ] .
th17 cells differentiate from
the same precursor cells as th1 or th2 cells , during which il-6
and tgf- plus il-23 are required .
th17 cells exclusively
produce il-17 , an inflammatogenic cytokine , but not ifn- or il-4 .
il-17 plays a pivotal role in the induction
of some inflammatory diseases such as experimental autoimmune
encephalomyelitis [ 7 , 59 ] , inflammatory bowel diseases , and rheumatoid arthritis .
it has also been
reported that il-17 is critically involved in some forms of
infection , either by induction of inflammatory cytokines such as
tnf- or by recruiting neutrophils [ 10 , 11 ] , although
not much is known about the role of il-17 on the defense against
parasite infection at this moment . a recent report by stumhofer et al . revealed an intriguing function of il-27 in view
of its immune suppression . in their report , stumhofer et al . revealed that wsx-1-deficient mice chronically infected with
t. gondii developed severe neuroinflammation with a
prominent il-17 response
we also reported that il-27
receptor - deficient mice infected with t. cruzi produced
more il-17 in the sera than wild - type mice .
although the precise role of il-17 in pathogenesis of encephalomyelitis and
infection - induced inflammation has yet to be elucidated ,
therefore , il-17 produced in response to pathogens appears at
least partially responsible for tissue damages induced by
inflammation . in both reports and one by
it is interesting that
il-23 , a member of il-12 cytokine family , induces the development
of th17 for the induction of inflammatory responses while il-27 ,
another member of the family , inhibits the th17 differentiation to
suppress inflammation ( figure 3 ) .
availability of il-12
family cytokines plus other cytokines , such as il-6 and tgf- in the surrounding milieu , may determine the direction
of immune responses toward either augmentation or regression .
il-27 thus may be a potential target for treating inflammatory
diseases of infection and autoimmune origin mediated by il-17 .
the
molecular basis for the inhibition of th17 differentiation is
still unclear . while stumhofer et al . and batten et al .
showed that
the il-27-meidated th17 inhibition is dependent on stat1 by taking
advantage of stat1-deficient mice , we demonstrated the requirement
of stat3 for this inhibition by using stat3-deficient t cells .
although further elucidation of the molecular mechanisms for the
il-27-mediated th17 inhibition is required , it would be reasonable
to assume that appropriate balance of stat1 and stat3 activation
downstream of the receptor determines the function of il-27
signaling , as implicated by a transgenic mice study or by examination of surface wsx-1 expression on nave and
activated t cells .
since its discovery , il-12 has been the only th1-promoting
cytokine for more than 10 years .
it has been well recognized that
the balance between il-12-induced th1 responses versus
il-4-mediated th2 responses determines the immune responses
against pathogens .
discoveries of two other il-12-related
cytokines along with the new th cell population have been
challenging and changing the paradigm .
as discussed in this
review , the il-12 family members play differential roles in
induction and maintenance of th1 responses during the time course
of infection / immune responses .
il-27 is critical in the commitment
of th precursor cells towards th1 differentiation and il-27
receptor deficiency resulted in impaired th1 responses with high
susceptibility to l. major infection . while having some
overlapping functions , the members of the family also have
distinct roles during immune responses .
il-23 , one of the new
comers to the family , augments inflammation by induction of th17
cells , while il-27 , the other new comer , attenuates inflammation
by suppressing cytokine production .
these findings remind us of
the intriguing fact that immune responses are elaborately
regulated along the time course of infection to fight the
dangerous pathogens and to simultaneously avoid hazardous damage
to oneself . in this view , the multifaceted role of il-27 is
particularly notable .
although the induction of th1
differentiation and suppression of cytokine production are
seemingly conflicting , these distinct functions are quite
reasonable when different target cells ( nave versus
activated ) and/or different time points ( early versus late phase
of activation ) are taken into consideration
. it would be premature
to discuss the possibility of using il-27 for treatment of disease
because of the complexity of its role . however , further
elucidation of the molecular mechanisms underlying the two
distinct functions of il-27 signaling as well as clarification of
the situations where the two roles are differentially used will
ensure that it will eventually be possible to use il-27 either to
reinforce defense against infectious agents or to treat
infection - induced immunopathology and inflammatory diseases . | cytokine - mediated immunity is crucial in the defense against pathogens .
recently , il-23 and il-27 were identified , which along with il-12 belong to
the il-12 cytokine family .
il-27 is pivotal for the induction of helper t
cell ( th ) 1 responses while il-23 is important for the proliferation of
memory type th1 cells .
recent studies revealed that il-27 also has an
anti - inflammatory property . in some protozoan infection , various
proinflammatory cytokines were over produced causing lethal inflammatory
responses in il-27 receptor - deficient mice .
the anti - inflammatory effect of
il-27 depends , at least partly , on inhibition of the development of th17
cells , a newly identified th population that is induced by il-23 and is
characterized by the production of the inflammatogenic cytokine , il-17 .
il-27 thus has a double identity as an initiator and as an attenuator of
immune responses and inflammation . with the discoveries of the new
il-12-related cytokines and th17 cells ,
th development is facing a new
paradigm . |
the
structural mechanism by which hsp70-type chaperones interact
with hsp40-type co - chaperones has been of great interest , yet still
remains a matter of debate . here , we used solution nmr spectroscopy
to investigate the atp-/adp - dependent interactions between escherichia coli hsca and hscb , the specialized hsp70/hsp40
molecular chaperones that mediate iron sulfur cluster transfer .
we observed that nmr signals assigned to amino acid residues in the
j - domain and its hpd motif of hscb broadened severely
upon the addition of atp - bound hsca , but these signals were not similarly
broadened by adp - bound hsca or the isolated nucleotide binding domain
of hsca complexed with either atp or adp .
an hscb variant with an
altered hpd motif , hscb(h32a , p33a , d34a ) , failed to manifest wt - like
nmr signal perturbations and also abolished wt - like stimulation of
atp hydrolysis by hsca .
in addition , residues 153171 in the
c - terminal region of hscb exhibited nmr signal perturbations upon
interaction with hsca , alone or complexed with adp or atp .
these results
demonstrate that the hpd motif in the j - domain of hscb directly interacts
with atp - bound hsca and suggest that a second , less nucleotide - dependent
binding site for hsca resides in the c - terminal region of hscb . | |
limbic signs dominate the clinical picture in the former whereas a paralysis of lower motor neuron type dominates the latter .
the diagnosis of rabies is not difficult if it presents with classical symptoms of excitations or phobias .
however , it poses a diagnostic challenge when presented as acute flaccid paralysis ( afp ) .
we report a case of paralytic rabies following intradermal anti - rabies vaccine , following cat scratch .
a 15-year - old girl was brought with the complaints of fever of 5 days , headache and vomiting of 4 days , weakness of bilateral lower limbs , followed by upper limbs , change in voice and nasal regurgitation of feeds since 2 days .
gag reflex was absent with pooling of secretions and absence of movements of soft palate and uvula .
power in bilateral upper and lower limbs was 1/5 ( medical research council grade ) .
she was immunized with a complete course of intradermal cell culture anti - rabies vaccine first dose started on day 2 of cat scratch .
updated thai red cross schedule ( 2 - 2 - 2 - 2 - 2 ) was used for intra dermal vaccination . cat scratch was not cleaned .
provisional diagnosis of paralytic rabies was made on the basis of paralysis with history of cat scratch .
although rare with the modern cell culture vaccines , the possibility of vaccine - induced gbs was also considered .
laboratory investigations revealed normal complete blood counts , serum electrolytes , liver and kidney function tests .
cerebrospinal fluid ( csf ) analysis showed 412 cells with 10% neutrophils and 90% lymphocytes with sugar of 62 mg / dl .
rabies virus rna was detected in csf and skin biopsy samples by reverse transcription polymerase chain reaction .
neutralizing antibodies to rabies virus were detected in both serum and csf by the rapid fluorescent focus inhibition test .
conservative management was started , but rapid progression with respiratory paralysis occurred over the next 2 days .
there was no improvement and the patient succumbed on the 10 day of onset of the disease .
about 55,000 human deaths occur due to rabies annually worldwide ; about 36% of these deaths occur in india .
the disease infects domestic and wild animals , and is spread to people through close contact with infected saliva via bites or scratches .
there are two forms of human rabies : ( 1 ) the well - known encephalitic ( furious ) and ( 2 ) the paralytic ( dumb ) rabies . the encephalitic form starts with fever , malaise , pharyngitis , and paraesthesia at the site of the bite , followed by the classical neurological symptoms of hydrophobia , aerophobia , agitation , hypersalivation , and seizures .
this is followed by paralysis and coma ; death is usually due to respiratory failure .
the second clinical form of rabies , paralytic ( dumb ) or guillain - barre - like , is characterized by progressive paralysis without an initial furious phase . even though , the paralytic rabies is unfamiliar to health care providers , 20 - 30% of rabies victims present in this manner .
paralytic rabies is more common after rabid vampire bat bites and in persons who have received post - exposure vaccination .
studies conducted in the united states by the center for disease control and prevention have documented that a regimen of one dose of rig and five doses of the human diploid cell culture vaccine ( hdcv ) over a period of 28 days was safe and induced an excellent antibody response in all recipients .
clinical trials with rabies vaccine adsorbed and purified chick embryo cell vaccine have also demonstrated immunogenicity equivalent to that of hdcv .
many individuals did not receive immunoglobulin where indicated , and some of them received the vaccine in the gluteal region instead of in the deltoid . although our patient had grade iii cat scratch for which rig is strongly recommended , she only received the vaccine in the deltoid region without rig .
neurological reactions following newer vaccine administration have been extremely rare . after millions of vaccinations worldwide , three guillain - barre type paralytic reactions have been described , and all cases recovered completely .
rare findings in our case are paralytic rabies following cat scratch and development rabies after administration of intradermal rabies vaccine .
the limitation of our report was that we were not able to do magnetic resonance imaging of brain and nerve conduction studies .
paralytic rabies should be considered if any child presenting with afp with brain stem symptoms and signs , even if they had already received anti - rabies vaccination and also following cat scratch .
most rapidly immunogenic vaccine regimen should be used in patients at high risk , especially if rig has not been given . | only few reports of failure of intradermal postexposure prophylaxis for rabies following cat scratch exist in the published literature .
we are reporting such a case in a 15-year - old girl .
the child had category iii cat scratch on her face .
she presented with progressive paralysis , finally developing quadriplegia and respiratory paralysis .
typical hydrophobia and aerophobia were absent .
she received intra - dermal anti - rabies cell culture vaccine .
she did not receive anti - rabies immunoglobulin .
the girl succumbed on the 10th day of weakness .
diagnosis of rabies was confirmed by isolation of rabies virus rna in cerebrospinal fluid and skin biopsy sample by reverse transcription polymerase chain reaction . |
her medical history included maffucci syndrome with multiple enchondromas of several phalanges and metatarsal bones of the left hand ( fig.1a , b ) , combined with hemangiomas of soft tissue in the right foot ( fig.1c ) .
she had skeletal deformities caused by enchondromas of the phalanges of the left fingers , particularly her index finger , appearing at 10 years of age , and hemangiomas on her right foot , appearing at 16 years of age .
the left - hand deformities gradually worsened , and she underwent surgical excision of the proximal phalanx of the left index finger and complete removal of the hemangiomas in her right foot at 21 years of age .
( b ) plain x - ray image showing multiple cartilaginous tumors in many phalanges and metacarpal bones of the left hand . ( c )
motor function was almost normal , and her remaining cranial nerve function was otherwise intact .
there were no abnormal findings on the laboratory blood tests . computed tomography ( ct ) and mri showed a 40 30-mm mass located in the posterior fossa , which showed a low signal on t1-weighted images ( fig.1e ) and a high signal on t2-weighted images ( fig.1f , g ) .
the tumor extended from the middle cerebellar peduncle to the cerebellar vermis , and the intraoperative pathological examination revealed diffuse astrocytoma ; therefore , partial removal was performed to avoid cerebellar ataxia or cerebellar mutism .
pathological examination by he staining demonstrated a mild focal increase in cellularity and a lack of vascular proliferation and necrosis within the sampled tissue ( fig.1h ) .
the final diagnosis was anaplastic astrocytoma . given the patient 's consent , the relative lack of symptoms and the diffuse nature of the pathology , the patient was administered local radiotherapy with a radiation boost to the residual tumor ( 50 gy in total ) .
the patient underwent endoscopic tumor resection , followed by stereotactic radiotherapy ( peripheral dose 2 gy , total 20 gy ) and temozolomide ( tmz ) .
tmz was prescribed at a dosage of 150 mg / m / day for 5 days every 28 days , but the patient died at 31 years of age .
genomic dna was extracted from formalin - fixed , paraffin - embedded tissue sections extracted by surgery using mightyamp for ffpe ( takara bio , shiga , japan ) according to the manufacturer 's instructions and with the informed consent of the patient .
we performed mutation analyses of idh1 , idh2 and tp53 genes ( data s1 ) .
we identified the idh2 mutation ( c.516g > t encoding r172s ) in the enchondroma , hemangioma and anaplastic astrocytoma tissues , and the same idh2-r172 mutation was detected in all these tissues ( fig.2a ) .
subsequently , the pcr products were subcloned into pcr4-topo vectors , and 14 clones ( enchondroma ) , 17 clones ( hemangioma ) and 69 clones ( astrocytoma ) were sequenced to confirm the idh2-r172s mutations ( data s1 ) . as a result , 28.6% ( 4/14 ) , 11.8% ( 2/17 ) and 2.9% ( 2/69 ) of each tissue , respectively , were shown to carry the idh2-r172s mutation , although these percentages do not necessarily imply those of idh2-r172s - possessing tumor cells ( fig.2b ) . furthermore
, missense mutations ( 562c > a encoding l188 m and 1118a > g encoding k373r ) of the tp53 gene were identified in the astrocytoma tissues , but not in the enchondroma or hemangioma tissues ( table1 , fig .
( a ) direct dna sequencing of idh2 was performed in the enchondroma , hemangioma and astrocytoma tissues .
( b ) pcr products of idh2 were subcloned into pcr4-topo vectors , and each clone was sequenced to confirm the heterogeneous mutation of idh2 .
we identified the same idh2 ( c.516g > t encoding r172s ) mutation in enchondroma , hemangioma and anaplastic astrocytoma tissues and tp53 mutation in astrocytoma taken from a patient with maffucci syndrome .
idh mutations may be a common genetic background in both gliomas and other tumors , and tp53 mutation may have triggered gliomagenesis in our patient with maffucci syndrome .
report idh1 mutations in a case of low grade glioma with ollier disease ; however , they did not perform mutation analyses of tp53.9 in contrast , our case suggests that an additional acquired tp53 mutation might induce the malignant transformation in patients with maffucci syndrome .
it is interesting to speculate that our patient with maffucci syndrome , who developed an anaplastic astrocytoma and other benign tumors with an idh2 mutation , represents a case of early post - zygotic genetic events , which may have initiated the disease process .
appropriate diagnoses of ollier disease and maffucci syndrome are crucial because these diseases are associated with a significant risk of brain tumors , as in our case , and other malignant tumors.10,11 patients with ollier disease and maffucci syndrome have better prognoses if the patients are diagnosed with and treated for malignant tumors in the early stages .
recently , we revealed that an anti - idh1/2 monoclonal antibody ( mab , clone msmab-1 ) , which was developed against idh1-r132 g , cross - reacts with idh2-r172s.1214 using msmab-1 , we performed immunohistochemistry against anaplastic astrocytoma tissues .
. there might be several problems about the low detection level of idh2-r172s in immunohistochemistry : ( i ) very low sensitivity of msmab-1 ; ( ii ) low frequency of the idh2-r172s expression ; or ( iii ) idh - r172s protein degradation in a paraffin section .
although further studies are required to determine the molecular pathogenesis of astrocytic tumors , our case is unique and supports the idh - dependent genetic pathway and second - hit model for gliomagenesis .
platform for drug discovery , informatics , and structural life science from the ministry of education , culture , sports , science and technology ( mext ) of japan . regional innovation strategy support program from mext .
kakenhi ( 25462242 ) : grant - in - aid for scientific research ( c ) from mext .
additional supporting information may be found in the online version of this article : materials and methods . mutational analysis of tp53 .
direct dna sequencings of tp53 exon5 ( a ) and exon10 ( b ) were performed in the astrocytoma tissues .
immunohistochemical analyses against anaplastic astrocytoma tissues using msmab-1 . mutated idh2-r172s protein expression was determined immunohistochemically in paraffin - embedded tumor specimens .
then , they were autoclaved in citrate buffer ( ph 6.0 ; dako ) for 20 min .
sections were incubated with 5 g / ml of msmab-1 ( a ) or control ( b ) overnight at 4c , followed by treatment with an lsab kit ( dako ) .
color was developed using 3 , 3-diaminobenzidine tetrahydrochloride ( dab ; dako ) for 10 min , and counterstained with hematoxylin . | we report on a 24-year - old woman who was diagnosed as having maffucci syndrome with anaplastic astrocytoma .
we analyzed the idh1 and idh2 mutations of enchondroma , hemangioma and anaplastic astrocytoma tissues and the same somatic mosaic mutation in idh2 gene was identified in all these tissues . in addition , we identified additional mutation of the tp53 gene in anaplastic astrocytoma tissue but not in other benign tumors .
this is the first report of the detection of an identical idh2 mutation in multiple tissues and tp53 mutation in anaplastic astrocytoma in a patient with maffucci syndrome .
this case is unique and supports the idh2-dependent genetic pathway and second - hit model for gliomagenesis . |
singlet exciton fission
is the quantum mechanical process by which
a singlet exciton splits into two distinct spin triplet excitons .
the triplets are initially coupled into an overall singlet state ,
conserving spin and allowing for extremely fast and highly efficient
triplet formation .
this phenomenon has recently become the object of
intense study due to its proposed use for carrier multiplication in
solar cells .
singlet fission has already
been successfully harnessed in devices , in some exceptional cases with an internal quantum efficiency near
200% .
the record external quantum efficiency in
these systems of 135% is the highest achieved for any photovoltaic
technology , demonstrating the great potential of singlet fission devices .
further practical advances will demand a more thorough understanding
of the underlying mechanism of singlet fission , as well as how it
relates to intermolecular structure .
for example , it has been shown
in amorphous films of diphenyltetracene that long - range order is not
required for efficient triplet formation , but other studies on disordered systems highlight the importance
of specific local interactions .
these local interactions ,
and particularly the strength of intermolecular coupling , have been
proposed to play a central role in determining the rate and dominant
mechanism of singlet fission nonadiabatic or adiabatic in
a new model covering all acenes .
this significant progress has occurred almost exclusively within
a relatively narrow class of materials : tetracene , pentacene , and
their derivatives .
property
relationships and singlet fission mechanisms needed to develop new
materials and broaden the library of candidate chromophores .
one class
of materials with significant promise for singlet fission is the polyenes , which are characterized by a low - lying ( dark )
excited state of the same ag symmetry as the ground state .
the theoretical basis for fission in these materials lies in the symmetry
of this state , which has an equivalent description as a weakly coupled
pair of triplet excitons , suggesting the possibility of intramolecular
singlet fission .
however , as we have previously shown in poly(3-dodecylthienylenevinylene ) ,
intramolecular singlet fission does not actually proceed via the expected
2ag state but occurs directly from the initially photoexcited
1bu exciton . in carotenoids ,
too , singlet fission has been observed ; these polyenes are evidently
too short to support intramolecular triplet pair formation but undergo
singlet fission in biological complexes with proteins or in self - assembled aggregates .
it is not clear how
the original 2ag mediated mechanism can be extended to
these materials , as the model is fundamentally intramolecular in nature .
wang and tauber have suggested that singlet
fission in carotenoid aggregates does not involve the 2ag state , but no studies have been made with sufficient time resolution
to confirm this hypothesis .
here we use broadband transient
absorption spectroscopy from the
tens of femtoseconds to microseconds time scales to investigate the
singlet fission process in aggregates of the carotenoid astaxanthin
( figure 1a ) .
we study a series of five distinct
aggregates to determine both the role of intermolecular structure
and the interplay between triplet formation and internal conversion
to the 2ag state .
direct formation from
1bu ( figure 1d)observed
in the intramolecular polymer system reported previously .
the initial rate of triplet formation is seen
to only weakly correlate with the strength of intermolecular coupling
with a fastest time constant of only 65 fs , among the fastest of any
reported system .
these results raise important questions about the
electronic structure and intermolecular interactions of the polyenes
and also point the way toward a universal mechanism for ultrafast
singlet fission .
( b ) normalized
uv vis absorption of five stable aggregates of axt in 9:1 water : dmso
( i and ii ) or 4:1 water : acetone ( iii v ) as well as monomeric
axt in acetone ( dashed ) .
ultrafast internal conversion from 1bu to 2ag is followed by fast nonradiative decay to the ground state .
( d )
model of singlet fission and triplet annihilation in carotenoid aggregates ,
as measured here . upon aggregation , the 1bu state no longer
couples to 2ag .
the 2ag state is lower in energy
than the triplet pair and enables efficient recombination back to
the ground state .
type - a
gelatin and c60 fullerene were purchased from sigma - aldrich .
enantiomerically pure 3s,3s- , 3s,3r- , and 3r,3r - astaxanthin for circular dichroism
measurements were purchased from carotenature .
axt monomer solutions
were prepared at a concentration of 100 m
in acetone or dmso and heated at 50 c until clear .
the series
of five distinct , stable aggregate solutions ( figure 1a ) was prepared as follows .
for aggregate i a 420 m
solution of axt in dmso at room temperature was mixed in a 1:9 ratio
with water at 5 c .
aggregate ii was prepared by mixing a 1000
m solution of axt in dmso at 80 c in a 1:9 ratio with
water at 80 c .
the three red - shifted aggregates were prepared
instead from acetone stock solutions heated to 65 c , and to
impart long - term colloidal stability , type - a gelatin aqueous solutions
at 50 c were used instead of pure water .
the organic : aqueous mixing ratio for all three species was 1:4 .
aggregate iii used an axt concentration of 250 m and a 5 mg / ml
gelatin solution .
the axt stock concentration for aggregate iv was
375 m , with a gelatin concentration of 2.5 mg / ml .
aggregate
v was prepared with the same acetone stock as aggregate iv and a higher
gelatin content of 10 mg / ml , and the solution was heated at 65 c
for an additional 10 min after mixing .
time - resolved measurements
were only performed on aggregates that were stable for > 1 week .
sub - picosecond transient absorption ( ta ) measurements were performed
on a previously reported setup with slight
modifications .
narrow - band ( 10 nm ) excitation pulses were
generated in an optical parametric amplifier ( topas , light conversion
ltd . ) coupled to the output of a 1 khz regenerative amplifier ( spectra - physics
solstice ) . to ensure that ta spectra reflected specific aggregate
types rather than mixtures of species , pump photon energies
were scanned
across the aggregate absorption bands ( vertical lines in figure 1a ) , with typical pump fluences below 4 10 photons / pulse - cm . because excessive pump fluence
results in spurious ultrafast effects , all measurements were also
performed at lower fluence , and the fluence dependence was studied
in detail for all samples ( see below ) .
the sample transmission was
probed using broadband pulses generated in two home - built non - collinear
optical parametric amplifiers ( nopas ) roughly spanning 0.752.5
ev . the probe beam was split to provide a reference
signal not affected by the pump to mitigate any laser fluctuation
effects , and both were dispersed in a spectrometer ( andor , shamrock
sr-303i ) and detected using a pair of linear image sensors ( hamamatsu ,
g11608 ) driven and read out at the full laser repetition rate by a
custom - built board from stresing entwicklungsbro .
the differential
transmission ( t / t ) was then
measured as a function of probe photon energy and pump probe
delay .
this setup afforded a temporal resolution of approximately
120 fs . the same setup was employed for nanosecond ta , using the frequency - doubled
( 2.33 ev ) nanosecond output of a q - switched nd : yvo4 laser as the excitation
source .
high - time - resolution measurements were also performed on a
similar setup , in which both the pump and probe beams were generated
by home - built nopas generating sub-30 fs ( pump ) and sub-10 fs ( probe )
pulses using chirped mirror compression .
the spectra in figure 1a demonstrate the
great potential of astaxanthin ( axt ) aggregates for a study of structure
this system is noteworthy both for the
large number of distinct aggregates formed and the wide range over
which the primary absorption band can be tuned approximately
1.2 ev through simple control of water content , temperature ,
and gelatin stabilization .
axt also appears to be unique in having
two blue - shifted structures and the strongly red - shifted aggregate
v , which is unlike any other reported carotenoid aggregates .
we consider that the uv vis absorption spectra shown here
represent distinct aggregate types , rather than mixed populations .
because these aggregates form through gradual transitions from one
species to the next ( see supporting information ,
figures s1 and s2 ) , it is easy to generate mixtures of species
through slight changes in preparation conditions . in all cases , the
uv vis spectra of such samples can be decomposed into the species
shown in figure 1a . to control for the possibility
of aggregate mixtures , we performed ta measurements with selective
excitation across the absorption band ( see below and supporting information , figures s6s10 ) . only aggregate
the determination of the structure of carotenoid aggregates is
a long - standing problem , and the diversity of intermolecular packing attained here highlights
its complexity . from the uv vis absorption
spectra alone , little
can be determined about the relevant intermolecular parameters . in
the absence of vibronic structure at room temperature
likewise , the degree of red- or blue - shift , a gauge of
the strength of exciton coupling , can not be determined precisely due
to significant solvatochromism ( see supporting
information , figure s3 ) .
aggregate i is strongly blue - shifted and exhibits significant
narrowing of the absorption band
aggregate ii exhibits a weaker blue - shift
as well as a change in shape that would be consistent with a decrease
in the 00/01 peak ratio , making it most likely a weakly
coupled h - aggregate .
aggregate iii is a borderline case which would
typically be described as a j - aggregate , but on the basis of spano s
analysis of lutein aggregates , it may
equally be a very weakly coupled h aggregate .
the appearance of a
pronounced 00 peak and strong red - shift in aggregates iv and
v is characteristic of j - aggregation , which is presumed to be the
primary interaction in these species .
aggregates similar to i are almost exclusively formed by carotenoids
with at least one hydroxyl group on the terminal rings , pointing to
the importance of intermolecular hydrogen bonding .
the observed sensitivity of the formation of i to ph is consistent
with this motif .
furthermore , the absorption spectrum of crustochrin ,
a carotenoprotein predominant in yellow lobster carapace , is almost
identical to that of aggregate i , and the two share similar raman
signatures .
however , that protein has
never been crystallized , and little is known about the orientation
or coupling of the axt molecules in this material .
aggregates such
as ii are preferentially formed by carotenoids with carbonyl but no
hydroxyl groups ; it is likely that in
this species the supramolecular structure is primarily mediated through
the c
ho motif described by bartalucci et
al . for crystals of axt and canthaxanthin .
strongly red - shifted axt aggregates such as iv and v have long been
studied in the context of lobster coloration .
the primary carotenoprotein
in lobster carapace , crustacyanin , exhibits a similarly pronounced
red shift .
recent studies attribute the bulk of this effect
to planarization of the axt molecules within the protein so that the
terminal rings lie in trans conformation , with a consequent extension
of the conjugation length .
indeed , the
spectra of iv and v each closely match different portions of the reported
absorption of a crystal of an all - trans axt derivative .
we thus consider species iv and v to contain
all - trans axt , with packing similar to the reported crystal structure .
the fact that each aggregate agrees so well with a separate section
of the crystal spectrum indicates the presence of two polymorphs in
the solid state .
the uv vis absorption and circular dichroism
data ( supporting information , figure s4 ) at hand are
insufficient to reliably determine the intermolecular packing in these
aggregates , and further structural characterization is needed . for
our purposes here , it is sufficient to rank them by approximate strength
of intermolecular coupling : i > v > iv >
, we first examine the excited
state processes of monomeric axt in pure organic solution . because
the carotenoids exhibit pronounced solvatochromism ( suporting information , figure s3 ) , monomer solutions were
prepared in both acetone and dmso .
the key results of ta measurements
on axt monomers excited at the absorption maximum are presented in
figure 2 .
our ta results are presented throughout
in units of t / t , in which
the absorption of photogenerated states appears negative .
positive
features can reflect either increased transmission of the probe through
the sample due to bleaching of the ground state or probe amplification
arising from stimulated emission .
the results for the two solvents were almost identical save for a
slight red - shift of all photoinduced absorption ( pia ) features in
dmso ( figure s3 ) , so only the acetone data
will be addressed here .
axt shows the characteristic polyene behavior
illustrated in figure 1c and can be described
in terms of just two states .
( a ) selected sub - ps
ta timeslices
following excitation at 2.6 ev with 26 j / cm .
the initial
1bu pia can only be clearly observed within the pump pulse
duration ( black ) , and it decays to form the well - known 2ag pia in the visible region .
the region below 1.1 ev is magnified
10 to show the weak 2ag 1bu pia .
( b ) the corresponding decay kinetics ( lines ) show direct , instrument - limited
conversion from 1bu to 2ag and rapid decay to
the ground state .
these processes are completely independent of pump
fluence over nearly 2 orders of magnitude ( symbols , data normalized
with respect to pump intensity ) the data for 0.78 ev are magnified
by a factor of 4 for clarity . within the instrument response ( black trace ) ,
a sharp pia
can be
detected between 1.0 and 1.5 ev , accompanied by a positive feature
around 2.25 ev which agrees well with steady - state photoluminescence
spectra and can be assigned to stimulated emission .
these are the
signatures of the initial 1bu exciton , which decays with
instrument - limited kinetics to the dark 2ag state ( green
traces ) .
this state shows a pronounced pia peaked at 2.05 ev as well
as a weak pia tail at the low - energy edge of the available spectral
range , assigned to the 2ag 1bu transition .
as in other polyenes , after fast thermalization
the 2ag state decays uniformly and nonradiatively back
to the ground state through efficient coupling to the vibrational
manifold .
we thus treat the monomer decay as following the simple
model of 1bu hot 2ag 2ag 1ag ( ground state ) .
the entire
spectral range can be fitted well with three time constants :
an instrument - limited 120 fs constant describing the internal conversion
from 1bu , a 200 fs thermalization within the 2ag manifold and a 5 ps final decay to the ground state .
this behavior
is completely independent of pump fluence for all spectral features
over nearly 2 orders of magnitude ( figure 2b ) .
there is no sign of or apparent need for intermediate states
between 1bu and 2ag to explain these results ,
as confirmed with sub-30 fs ta measurements ( see below ) .
it should
be stressed that there is also no sign of triplet formation by singlet
fission or any other process at any pump photon energy ; individual
carotenoid molecules are evidently too small to support triplet pair
states .
ta measurements were
performed on all five aggregates at four or more pump photon energies .
due to the overall similarity of the aggregate ta data , only the representative
aggregates ii ( weak h - aggregate ) and iv ( strong j - aggregate ) will
be discussed in detail here .
the spectra of other aggregates and other
pump photon energies can be found in the supporting
information , figures s5s10 .
comparison of the ta spectra
for aggregate ii in figure 3a , b with the monomer
data in figure 2 reveals two immediate differences :
the pia bands are broader and much less pronounced in ii , and the
final decay is significantly slower .
as will be shown below in figure 4 , the final state in this aggregate is the triplet .
its pia is dominant well before 3 ps ( see below ) , and the spectrum
decays without further evolution out to the s time scale .
the
only spectral changes that can be clearly discerned occur on an ultrafast
instrument - limited time scale : much like in monomeric axt the initial
excited state absorbs in the nir , seen as a broad , weak pia in the
earliest ta spectrum .
as shown in figure 3c ,
the decay of the nir band is matched by the decay of the slight ground - state
bleach ( gsb ) observed around 2.4 ev .
this kinetic reflects the sub - ps
formation of triplet pia , which strongly overlaps with the ground - state
absorption and results in a negative overall signal .
( a ) selected timeslices
from sub - ps ta of ii excited at 2.6 ev .
the prompt decay of the broad
nir pia is the only significant spectral change in the entire measurement
range and reflects internal conversion from the initial 1bu state .
( b ) timeslices from ns ta show continued uniform decay of
the state formed within 3 ps .
( c ) integrated decay kinetics from sub - ps
ta reveal instrument - limited decay of the gsb and nir pia ( solid lines )
to form the final state .
( d ) equivalent sub - ps measurement of iv excited
at 2.3 ev , showing similar rapid decay of the initial band in the
nir and formation of the final state within 3 ps , which ( e ) uniformly
decays on the s time scale .
( f ) integrated decay kinetics from
sub - ps ta of iv show an instrument - limited internal conversion from
the initial state ( thin solid line ) similar to ii .
the slower rise
at 1.94 ev reflects a gradual shift of the crossing point between
gsb and pia , related to the generation of vibrationally excited ground
states following nonradiative decay of triplet pairs .
( a ) integrated decay
kinetics from all
species , at the peak of pia , following excitation at 2.3 ev .
all aggregates
( solid ) show an enhancement of the final state lifetime of 5 orders
of magnitude , relative to the monomer ( dashed ) .
( b ) comparison of
the ta signal at 100 ns for all five aggregates reveals the same pia
signature in each , combined with a gsb related to the uv
( c )
comparison of the absorption edge ( dashed ) and long - lived ta signal
( solid ) of monomer axt following triplet sensitization ( green ) and
aggregate ii .
the similarity of shape confirms the assignment of the
long - lived aggregate state to triplets , produced via singlet fission . on first inspection the ta results
for iv in figure 3d
the most prominent difference is the sharp positive t / t band above 2.0 ev .
this signal perfectly
matches the position of the vibronic peaks observed in steady - state
absorption ( figure 1a ) and can be assigned
to gsb of the aggregate . as in figure 3a , an
ultrafast decay from the initial 1bu state can be inferred
from the instrument - limited loss of the pia in the nir along with
a matching rise of pia in the visible and a slight blue - shift of the
gsb edge .
the final state in this aggregate again formed on
ultrafast time scales is the same as in aggregate ii , namely
triplet excitons .
indeed , all five aggregates form triplets
on ultrafast time scales ,
with most spectral evolution complete by 1 ps . in a system such as
aggregated axt , with no heavy atoms and no significant intersystem
crossing yield in free solution ,
we detect none of the signatures
of 2ag , such as 2ag 1bu pia
below 1 ev , or of any other excited state within the 120 fs
temporal resolution of this measurement .
we thus propose that intermolecular
singlet fission in this polyene system occurs directly from the 1bu exciton , the same mechanism we observed for intramolecular
singlet fission in poly(3-thienylenevinylene ) .
figure 4 compares the final state observed
in each aggregate following excitation at 2.33 ev .
we first highlight
the dramatic enhancement of the final state lifetime upon aggregation ,
by roughly 5 orders of magnitude .
only thermal effects or the formation of charged species or triplet
excitons would be expected to yield such long lifetimes .
the only
long - lived features are found in the visible spectral region ( figure 4b ) .
the agreement of the pia bands on the low - energy
side of the spectrum is striking , and the differences at higher energies
can be accounted for as different gsb contributions , related to the
shape of the ground - state absorption spectra .
this comparison not
only confirms that the same long - lived species is present in every
sample but also ensures that it is an excited state of the primary
aggregate type observed in uv vis absorption rather than a
minority population .
the close spectral overlap of the pia bands allows
us to rule out pump - induced thermal modulation , as it is highly unlikely
for this effect to produce the same red - absorbing species in five
widely varied aggregate structures with absorption edges varying by
1.5 ev . we can also rule out charge formation , as the characteristic
nir absorption bands of carotenoid anions and cations are absent in
all cases .
these considerations already enable an assignment of the terminal
long - lived state to triplet excitons , and for further confirmation
we use the triplet sensitization technique of sasaki et al .
briefly , a mixed solution of axt and c60 in toluene was excited at 2.33 ev with the long - delay ta setup .
within the pump pulse duration
, we observe the decay of axt singlets
and formation of triplet excitons on c60 ( supporting information , figure s11 ) . on a hundreds of picoseconds
time
scale , the triplet excitons undergo triplet energy transfer to
axt , resulting in the sharp characteristic pia of carotenoid triplets
( figures 4c , green , and s11 ) .
comparison with our long - delay spectra , using only aggregate
ii for clarity , shows a similar overall shape .
however , the long - lived
signal in aggregates is slightly broader and red - shifted by 0.2
ev . a small red - shift for carotenoid triplet absorption in aggregates
or complexes has been previously observed and can be partially attributed to the change in dielectric environment
upon aggregation .
moreover , the shift is comparable to that observed
in the ground state absorption edge ( dashed lines ) , which may indicate
that gsb also contributes to the apparent red shift .
as for
the spectral broadening , it is actually a consequence of
singlet fission .
the same phenomenon has been observed in poly(3-dodecylthienylenevinylene ) and in polydiacetylene . in the latter work
it was proposed that the initial geminate triplet
pairs still interact following singlet fission , though they are no
longer fully coupled into a singlet state .
these interactions perturb
the triplet energy levels , leading to a broadening and further red - shift
of the pia .
the agreement of our results with a similar system and the reasonable match between the long - lived spectrum and the
triplet reference are sufficient to conclude that the final state
in all axt aggregates is triplet excitons .
the match of this
triplet pia across the entire aggregate series
is striking and merits further consideration .
we recall that optically
allowed transition varies by 1 ev between aggregates i and
v , demonstrating the strong effect of aggregation on the singlet excited
state . on the other hand ,
the most significant effect on the t1tn absorption band in figure 4 is the mere fact of aggregation : all aggregates share approximately
the same redshift of this band relative to the sensitized monomer
triplet .
this result is a likely consequence of the highly localized
nature of carotenoid triplets . whereas the precise nature of the nearest - neighbor
coupling within the aggregates can strongly influence singlet absorption ,
the triplet only reflects bulk - type dielectric effects . on the basis
of this behavior
, we can be confident that the energy of the triplet
exciton does not change through aggregation .
( a ) integrated decay kinetics of ii following sub - ps excitation at
2.6 ev .
the same multiexponential function fits the decay at all fluences ,
requiring the recombination process to be entirely geminate .
lifetimes
( 0.9 , 6 , and 49 ps ) are not assigned to individual processes but rather
reflect the wide distribution of triplet pair decay rates .
( b ) integrated
decay kinetics of ii on longer time scales , following ns excitation
at 2.3 ev , show strong fluence dependence indicative of non - geminate
tta .
though all of the aggregates undergo
the same process of singlet fission , the kinetics in figure 4a reveal clear differences in the subsequent fate
of the triplets . to better understand this behavior , we begin by evaluating
the dependence of these kinetics on pump fluence in detail for aggregate
ii ( see supporting information , figures s13 and
s14 for qualitatively similar results for the other aggregates ) .
we first consider the ps - ns regime ( figure 5a ) , during which the triplet pia spectrum decays uniformly with at
most a slight narrowing ( figure 3a ) .
the multiexponential
decay we observe on these time scales is far too short for the intrinsic
triplet lifetime .
instead , in agreement with studies of zeaxanthin
aggregates we attribute this behavior to
annihilation processes , specifically triplet triplet annihilation
( tta ) .
this is a highly efficient process though some fraction
of the triplets survives out to s time scales , at least 90%
of the excited population annihilates between 1 ps and 1 ns . at the
same time
, this initial tta regime is completely independent of pump
fluence and must thus correspond to the annihilation of geminate triplet
pairs .
these facts enable an important insight into the energetics
of singlet fission in carotenoids . according to the conventional
energy scheme 1bu > 2ag
> 2t1 > t2 , only one pathway for tta
is permitted : t1 + t1 t2 + s0 t1 + s0 .
such a model
could potentially explain a prompt loss of 50% of the triplet population
through geminate tta , but any further losses would depend on intrinsically
slow triplet diffusion
. this latter process would be strongly dependent
on excitation density , which can not be reconciled with the fast , fluence - independent
decay observed here .
the usual treatment of
tta assumes a randomized population of triplets , such that pairs of
triplets can couple and annihilate into an overall spin singlet or
spin triplet state .
the triplet pair produced through fission is
by definition coupled into a spin singlet , so tta is only possible
back to a singlet state until the coherence is broken by spin
thus , fast geminate
recombination of the type t1 + t1 t2 + s0 should not be possible , even if it is energetically
favorable .
instead , the only available pathway for geminate
tta that passes
through another electronic state is t1 + t1
s1 ( i.e. , 2ag ) + s0 . this process
would be completely independent of excitation density and
could proceed
on a range of time scales , governed by the couplings in the distribution
of triplet pair states formed . in short ,
this model of tta very closely
matches the results presented in figures 5a
and s13 on axt aggregates .
it
is generally accepted that 1bu > 2ag >
2t1 > t2 , with a relatively small gap between
2ag and 2t1 . however , triplet energies are notoriously
difficult to measure , particularly in the absence of phosphorescence ,
and if the t1 energy were even slightly higher , the state
ordering would become 1bu > 2t1 > 2ag . on the longer time
scales shown in figure 5b , the relatively few triplets that survive the
initial tta regime
are stabilized and do not appear to undergo geminate recombination .
instead , they are free to diffuse and decay with a strong fluence
dependence , a clear indicator of non - geminate tta . at lower fluences
the final decay still converges well to a single exponential decay
of 1.25 s , which is reasonable as the intrinsic triplet
lifetime , but at the highest excitation density the triplet decay
is distinctly nonexponential over the entire range .
it is important
to note here that the fluence behavior is more complicated than in
standard bimolecular recombination , as the ability of the triplets
to annihilate depends not only on the rate of triplet diffusion but
also on the distribution of aggregate sizes , both quantities unknown
but likely to vary across the aggregate series . with this understanding
of tta in axt aggregates
the fastest initial
decay is observed for the aggregates i and v that exhibit the strongest
intermolecular coupling as would be anticipated , the triplet
pairs formed by singlet fission are similarly more strongly coupled
and annihilate quickly .
conversely , the
decay over the first 1 ns is slowest in the relatively weakly coupled
aggregate ii .
the dependence on excitation density and strong variation
in absorption cross - section between aggregates preclude direct comparison
of the longer - time decay kinetics , though all exhibit a similar characteristic
lifetime of order 1 s .
as noted above , the process
of singlet fission identified in figure 3 is
largely complete within the instrument response of the sub - ps ta experiment .
to fully resolve the triplet formation
, we performed sub-30 fs ta
measurements on the full set of aggregates as well as monomeric axt
in acetone and dmso . in dilute solution ( figure 6 ) we find that the monomer reproduces the behavior observed previously
using sub - ps ta , namely direct conversion of 1bu into a
hot 2ag state which rapidly cools .
the higher time resolution
enables a clean determination of the 1bu 2ag internal conversion time constant , which we find to vary
with solvent from 105 fs in dmso to 125 fs in acetone with no discernible
intermediate states .
it is unclear what environment is most appropriate
for comparison to axt molecules embedded in ( or at the surface of )
aggregates , but this measurement establishes an approximate 100 fs
time scale with which singlet fission must compete to proceed efficiently .
( a ) transient absorption timeslices from sub-30 fs ta on monomeric
axt in dmso , showing smooth transition from 1bu ( pia below
1.5 ev ) to 2ag ( pia above 1.8 ev ) .
the corresponding decay kinetics ( symbols ) in ( b ) dmso and ( c ) acetone
can be well described with an exponential time constant ( lines ) of
105 or 125 fs , respectively .
iv , for which the fission
process can be most clearly distinguished , are presented in figure 7 .
data for aggregates i and v can be found in the supporting information . in all cases ,
the spectra
agree well with the sub - ps ta measurements on the same aggregates .
the initial state in all species has a pia in the nir , in the same
region where the 1bu state absorbs in monomeric axt .
this
band rapidly decays , resulting in the same triplet signature identified
in figure 4b . to capture the time scale of
this process ,
the decay kinetics for each aggregate were globally
fitted with a simple biexponential function , revealing two distinct
temporal regimes : a sub-100 fs process that describes the formation
of triplet pia , and a slower ( hundreds of femtoseconds ) redshift consistent
with thermalization of the hot triplet pair state .
these fits give
a singlet fission time constant of 85 , 90 , and 65 fs in aggregates
ii , iii , and iv , respectively , distinctly faster than the ultrafast
1bu 2ag internal conversion detected
in the monomer . to confirm the assignments from these decay
kinetics ,
the data
were analyzed with a spectral decomposition technique based on a genetic
algorithm . in aggregates ii
iv ,
the transition was best modeled with only two species ( figure 7d f ) showing the characteristic features
of 1bu and the triplet described above .
we observe singlet
fission directly from 1bu on time scales consistent with
the raw kinetic fitting and find no evidence of any other excited
states .
the rates of triplet formation here are among the fastest
reported
for any intermolecular singlet fission system .
this rapid fission
can not be explained solely by the energetic difference between singlet
and triplet pair states : the triplet energy should not change with
aggregation , while the singlet varies by over 1.2 ev .
using typical
values , we may even expect the singlet
and triplet pair states to be nearly degenerate in aggregate v. the
two weakly coupled aggregates ii and iii are the slowest , forming
triplets with roughly the same time constant 90 fs .
aggregate
iv exhibits even faster singlet fission , which we propose is related
to its stronger intermolecular coupling . indeed , in the two most strongly
coupled aggregates i and v we are unable to distinguish any state
prior to triplets within the temporal resolution of the measurement ,
which was restricted in those systems to 70 fs due to scattering
and a strong coherent solvent response .
unlike the initial rate
of tta , we find the rate of triplet formation
is only weakly dependent on the strength of intermolecular coupling .
indeed
, the mechanism of singlet fission in these aggregates must
be remarkably robust : variation of the band gap over a range of 1.2
ev , with concomitant changes in the energetic driving force for triplet
pair formation , yields fission time constants that span the narrow
range 50100 fs . even in the most weakly bound aggregates
ii and iii , where singlet fission is only slightly faster than the
expected 1bu 2ag time scale , we detect
no signs of any other excited states , and we thus consider that this
initial step is highly efficient in all aggregates . the surprising
result that the ultrafast 1bu
2ag internal conversion channel of the monomer is completely
deactivated within the aggregates merits closer consideration .
we
note that this internal conversion is generally accepted to proceed
via a conical intersection and is thus driven by nuclear motion .
it is conceivable that the same crossing is
approached in the aggregates , with the important distinction that
the strong exciton coupling ensures a delocalized state .
the predicted
triplet pair character of 2ag results in a short - lived
singlet state when it is confined to a single polyene chain , but delocalization
over multiple chromophores may allow sufficient separation between
the constituent triplets to give the state more triplet - like character .
while such a model could explain the early time transient absorption
behavior , it is inconsistent with the energetic structure evident
from the analysis of tta kinetics .
it may nonetheless be instructive
to consider in relation to other possible triplet pair states .
we
propose instead that aggregation alters the vibrational landscape
and distorts the 1bu potential energy surface , such that
a crossing with the triplet pair state is strongly favored over that
with 2ag .
full clarification of this intriguing behavior
will likely call for both extensive new computational studies of the
electronic structure of coupled polyenes and a detailed exploration
of vibronic coupling in the aggregates .
( a ) sub-30 fs ta measurements of aggregate
ii in the visible and nir spectral regions show a direct transition
from the initial singlet ( solid ) to triplets , already evident by 150
fs .
equivalent
measurements on aggregates ( b ) iii and ( c ) iv reveal similar behavior .
( d f )
species extracted with a genetic algorithm using a two - state model
agree well with the characteristics of 1bu and triplet
excitons , and no additional states are needed to describe the data .
( g i ) integrated decay kinetics for each aggregate type ( symbols )
can all be globally fitted with biexponential functions ( lines ) capturing
the initial singlet fission process and subsequent relaxation of the
triplet pair . the singlet fission time constant varies with aggregate
structure , from 85 and 90 fs for ii and iii to 65 fs for iv .
we
have isolated a system of five distinct aggregates of astaxanthin ,
allowing adjustment of the intermolecular coupling while maintaining
the same monomer chemical and electronic structure .
our transient
absorption measurements demonstrated that ultrafast singlet fission
occurs in all aggregate types .
sub - ps ta data revealed the presence
of only two excitonic states the bright 1bu singlet
exciton and the triplet produced via singlet fission suggesting
a mechanism of singlet fission directly from 1bu rather
than via 2ag .
this model is consistent with our previous
high - time - resolution measurements of intramolecular singlet fission
in polyenes , and also agrees with the
proposal of wang et al .
we confirmed
this two - state model with sub-30 fs ta , demonstrating for the first
time in a polyene system that intermolecular singlet fission proceeds
directly from the initial 1bu state .
furthermore , the purely
geminate tta dynamics immediately following singlet fission indicate
that the triplet pair energy is in fact greater than that of 2ag , which instead functions merely as an efficient decay pathway
back to a vibrationally excited ground state . in other words ,
the
mechanisms of singlet fission and subsequent tta in polyenes are strikingly
similar , whether the fission event is intramolecular like in poly(3-dodecylthienylenevinylene ) or intermolecular like in axt aggregates .
these
findings raise important questions about the role of low - lying 2ag states .
the existence of such a state appears to be a useful
sign of the ability to undergo singlet fission , but in polymers and
aggregates the state not only does not participate in triplet formation
but actually harms the overall yield . further theoretical work
is required to determine how the triplet
pair state forms directly from 1bu , a process beyond the
scope of current models .
turning to recent high - level calculations
of polyene excited states , we note that another triplet pair state ,
1bu , is predicted to lie just below
the initial allowed 1bu transition .
the coupling between these states would likely be strong ,
with consequently fast relaxation into the triplet pair .
comparison
with the more established polyacenes sheds some light onto the mechanism
of singlet fission discussed here . in the most strongly coupled acenes
( pentacene and tips - pentacene ) ,
singlet fission is described as adiabatic ,
with a rate independent of intermolecular coupling strength . for the
majority of the more
weakly coupled acenes , however , triplet formation
appears to be highly dependent on intermolecular coupling .
by contrast , in astaxanthin similar sub-100
fs singlet fission kinetics are observed in strongly ( i , v ) and weakly
( ii , iii ) coupled systems with large ( i ) and small ( v ) energetic driving
force .
this suggests that singlet fission remains in the adiabatic
regime in all aggregates and may reflect a unique property of singlet
fission in polyenes .
this is perhaps not surprising given the widely
accepted role of conical intersections in carotenoid photophysics ,
and it will be important to investigate how vibrational dynamics are
implicated in singlet fission within the aggregates . a recent study
of tips - pentacene , for example , has revealed that ultrafast triplet
formation is mediated by vibronic coupling between s1 and
the triplet pair state , and a similar
mechanism may apply here . our results also allow important insight
into the processes following
triplet generation .
the observation of two distinct regimes of tta fast
geminate and slow bimolecular highlights the unique quantum
mechanical properties of triplet pairs generated through singlet fission ,
which are initially entangled into an overall singlet state .
the conversion
from geminate to bimolecular recombination reflects a combination
of the time scales for triplet pair separation and de - coherence and
triplet diffusion , affording a window to study these processes with
multipulse techniques or under applied fields and build a model of
how and why triplet pairs break apart .
more broadly , this system of
carotenoid aggregates can serve as a platform to investigate tta and
how it can be controlled through material properties , which is of
great interest in the context of triplet - based light - emitting diodes .
an understanding of such annihilation processes
and their interrelation with intermolecular coupling and triplet generation
will be essential to realize the full potential of singlet fission
in solar energy harvesting . | singlet exciton fission allows the
fast and efficient generation
of two spin triplet states from one photoexcited singlet .
it has the
potential to improve organic photovoltaics , enabling efficient coupling
to the blue to ultraviolet region of the solar spectrum to capture
the energy generally lost as waste heat .
however , many questions remain
about the underlying fission mechanism . the relation between intermolecular
geometry and singlet fission rate and yield
is poorly understood and
remains one of the most significant barriers to the design of new
singlet fission sensitizers . here
we explore the structure
property
relationship and examine the mechanism of singlet fission in aggregates
of astaxanthin , a small polyene .
we isolate five distinct supramolecular
structures of astaxanthin generated through self - assembly in solution .
each is capable of undergoing intermolecular singlet fission , with
rates of triplet generation and annihilation that can be correlated
with intermolecular coupling strength .
in contrast with the conventional
model of singlet fission in linear molecules , we demonstrate that
no intermediate states are involved in the triplet formation : instead ,
singlet fission occurs directly from the initial 1bu photoexcited
state on ultrafast time scales .
this result demands a re - evaluation
of current theories of polyene photophysics and highlights the robustness
of carotenoid singlet fission . |
all multiple pregnancies are at an increased risk of antepartum complications , relative to singletons . in monochorionic ( mc )
multiples , the perinatal morbidity and mortality risk is significantly higher than dichorionic ( dc ) twins , which are the commonest type of twins.13 approximately one in 80 births in australia is twins , and of these , around 30% are monozygotic ( identical ) .
mc diamniotic ( mcda ) twins are identical twins who share a placenta but not an amniotic sac , with this architecture arising in around two - thirds of monozygotic twins,1,4,5 or around one in 400 of all pregnancies .
mcda pregnancies are associated with three to six times increased risk of complications compared to dc twins , where each fetus has its own placenta.69 due to the presence of placental vascular anastomoses , various complications can occur including twin twin transfusion syndrome ( ttts ) , twin anemia polycythemia sequence ( taps ) , twin reversed arterial perfusion ( trap ) , and selective intrauterine growth restriction ( siugr ) .
in addition , severe discordant congenital anomalies occur more frequently in mcda pregnancies and are associated with an increased risk of morbidity and mortality.10 in the case of spontaneous intrauterine demise of one fetus , its co - twin may subsequently die ( 15% ) or suffer from severe cerebral injury ( 34% ) due to acute exsanguinations into the circulation of the demised twin through the placental vascular anastomoses.11 although long - term outcome has been studied extensively in cohorts of children delivered following specific mcda complications , including ttts12 and co - twin death survivors,13 only a few studies have investigated the long - term outcomes in mc and dc twin cohorts in general.1416 in a study comparing the neurological outcomes of mcda and dc diamniotic ( dcda ) twins , adegbite et al15 documented the rates of neuromorbidity to be 23% and 4% , respectively .
advances in the treatment of ttts have decreased its influence on these outcomes ; however , the neuromorbidity rates of more recent studies by acosta - rojas et al6 ( 3% complicated ,
0.5% uncomplicated ) and ortibus et al17 ( 10% complicated , 7% uncomplicated ) continue to provide a wide range of risk estimates .
hack et al14 compared neurodevelopmental outcomes in 140 mcda and 142 dcda children recruited across nine sites at age 22 months , using the health status classification system preschool children ( hscs - ps ) questionnaire completed by parents , the findings of which were in con - cordance with those of the griffiths mental development scales .
the authors reported that the majority of 2-year - old twins had normal developmental status , and that there were no significant differences between mc and dc twins , apart from a slight delay in hearing and language development in mc twins .
birth weight discordancy did not appear to influence long - term outcome , although there was a trend toward lower griffith developmental quotient ( dq ) scores in the smaller twin than in its larger co - twin .
there was , however , a significant difference in development between boys and girls , whereby boys had lower dq scores and the proportion of mildly delayed development was higher amongst male infants .
moreover , infants born before 32 weeks of gestation and/or with a birth weight < 1,500 g had significantly lower dq scores than infants born after 32 weeks and/or with a birth weight of 1,500 g. the study showed that across both the mc and dc groups , the largest proportion of identified developmental concerns on the griffith scales was within the locomotor domain ( ie , 26.5% of the mc group ) .
the authors noted that follow - up at an older child age would be preferable as difficulties relating to neurological handicaps and intellectual disability may only become evident at that time .
nonetheless , differences in selection / exclusion criteria , criteria for neurodevelopmental delay , and typically small study cohorts make comparison of the available studies difficult .
it is also difficult to ascertain factors influencing uncomplicated mcda twins specifically , given the mixed cohorts which have included the full range of complications including ttts , co - twin death , and premature birth .
critically , studies have tended to focus on mortality and core neurodevelopmental status , while not exploring the impacts upon socio - emotional and behavioral development .
thus , there do not appear to be any studies available where the effects of uncomplicated mcda pregnancy on long - term neurodevelopmental outcome are examined .
these are important questions because such pregnancies form the majority of cases , and the structural challenges of shared placental anatomy do conceivably place the fetus at risk for mild neurodevelopmental difficulties as well as socio - emotional and behavioral challenges , which may be evident only later in the child s life .
further , the literature suggests that mcda pregnancies that appear uncomplicated using standard screening procedures may indeed carry significant undetected complications , sometimes leading to fetal death.18 the aim of the study was to examine longer - term neurodevelopmental outcomes in a cohort of antenatally uncomplicated mcda twins delivered in the third trimester .
the study had ethical approval from the human research ethics committees of the south western sydney local health district and university of new south wales .
the study was undertaken at the feto - maternal unit ( fmu ) at liverpool hospital in sydney .
liverpool hospital is a tertiary referral center in the south western sydney local health district , which covers a total of seven local government areas and an estimated population of 820,000.19 the fmu provides a tertiary prenatal scanning , testing , and counseling service for approximately 3,500 pregnancies out of a total obstetric population of 12,000 per year as a subunit of the division of women s and children s health at the university of new south wales .
retrospective data were extracted from the existing fmu database in the form of an initial patient list , by selecting for mcda pregnancies that were uncomplicated and undelivered before 28 weeks between the 1st of january 2007 and 31st of december 2009 .
prospective data were collected in the form of three parent - completed questionnaires that were sent and received by mail .
there were 97 cases of mcda twins with a gestational age > 28 weeks during the study period between 2007 and 2009 .
twenty - seven cases were deemed unsuitable for the study due to complicated status and/or wrongful inclusion by the data extraction process , and were therefore excluded . of the remaining 70 cases ,
eight were listed as requiring an interpreter and were excluded from the final list due to the unavailability of an interpreter to the study due to resourcing , leaving 62 eligible cases .
a further 16 of these cases were uncontactable , leaving a final pool of 46 eligible cases .
consent to participate in the study was obtained via an initial phone call in 44 cases , with two patients declining to participate .
study packs , which included a study information and consent form for patients to sign , were sent to these 44 , and follow - up phone calls were made if packs were not returned within 3 weeks .
twenty - two packs were ultimately returned within the allotted time period for data collection of 2 months , providing a response rate of 50% ( 22 of 44 ) .
information about the pregnancy and birth details of the child were obtained from the fmu internal database and new south wales electronic medical records .
these included gestational age at birth , birth weight and estimated fetal weight and their percentiles , mode of delivery , apgar scores , and ultrasound doppler studies .
chorionicity was determined by ultrasound in accordance with accepted criteria.4,5 all pregnancies for which this was not conclusive were treated as mc until chorionicity was confirmed antenatally by placental histology . the term uncomplicated was used to define those pregnancies unaffected by mc - specific conditions and identifiable discordant growth patterns up to 28-week gestational age .
pregnancies complicated by major congenital malformations , aneuploidy , and the intrauterine demise of a co - twin were also excluded .
the mc - specific conditions , ttts , taps , and trap were identified sonographically.2022 comparison with an uncomplicated dc control group was not possible as only mc twins are offered routine surveillance every 2 weeks until delivery in the fmu .
participant study packs comprised the child behaviour checklist ( cbcl),23 child development chart ( cdc),24 and a parent questionnaire .
the cbcl23 has strong psychometric properties and was scored using the associated achenbach system of empirically based assessment scoring profile.25 cbcl results were converted into norm - referenced percentiles for the six behavior domains : anxious / depressed , withdrawn , sleep problems , somatic problems , aggressive behavior , and destructive behavior .
percentile scores were categorized as normal ( < 95th percentile ) , borderline ( 95th98th percentile ) , or clinical ( > 98th percentile ) .
these behavior domains were collapsed into composite internalizing ( anxious / depressed and withdrawn ) , externalizing ( aggressive and destructive behavior ) , and total domain score categories , which were reported as a norm - referenced
score out of 100 , with categories of normal ( t < 60 ) , borderline ( t = 6063 ) , and clinical ( t > 63 ) . the cdc24 was scored by calculating the dq ( that is ,
( developmental agechronological age)100 ) of the child in the domains of social , self - help , gross motor , fine motor , and language , as well as an overall dq , which was calculated as the average of the five domain scores .
quotients were classified as normal ( > 85 ) , borderline ( 8570 ) , and clinical ( < 70).26 the cdc chart did not test beyond 58 months , so parents with children aged > 58 months were asked to complete the chart from their memory of the child at that age ; hence , chronological age used in this scenario was 58 months .
there is a long history of research supporting the accuracy of parent self - report of child development when using structured inventories.27,28 for example , in their 2013 review , bedford et al reported that
measures completed by parents have been shown to be as accurate as those administered by professionals in identifying children with developmental problems ( p. 59).29
measures completed by parents have been shown to be as accurate as those administered by professionals in identifying children with developmental problems ( p. 59).29
the parent questionnaire was constructed for the purposes of the study to obtain specific information regarding demographics of the child , diagnosed developmental issues , and his / her family background .
data were analyzed using spss software . in light of the small sample size recruited for this study ,
independent t - tests were used in a broad comparison of all developmental and behavioral problems with birth weight and gestational age .
the cohen s d metric was used to calculate effect sizes and was calculated by dividing the mean difference between groups by the pooled standard deviation ( sd ) .
it is widely accepted that cohen s d values of 0.20.49 denote small - sized effects , 0.50.79 denote medium - sized effects , and > 0.8 denote large effect sizes .
the study was undertaken at the feto - maternal unit ( fmu ) at liverpool hospital in sydney .
liverpool hospital is a tertiary referral center in the south western sydney local health district , which covers a total of seven local government areas and an estimated population of 820,000.19 the fmu provides a tertiary prenatal scanning , testing , and counseling service for approximately 3,500 pregnancies out of a total obstetric population of 12,000 per year as a subunit of the division of women s and children s health at the university of new south wales .
retrospective data were extracted from the existing fmu database in the form of an initial patient list , by selecting for mcda pregnancies that were uncomplicated and undelivered before 28 weeks between the 1st of january 2007 and 31st of december 2009 .
prospective data were collected in the form of three parent - completed questionnaires that were sent and received by mail .
there were 97 cases of mcda twins with a gestational age > 28 weeks during the study period between 2007 and 2009 .
twenty - seven cases were deemed unsuitable for the study due to complicated status and/or wrongful inclusion by the data extraction process , and were therefore excluded . of the remaining 70 cases ,
eight were listed as requiring an interpreter and were excluded from the final list due to the unavailability of an interpreter to the study due to resourcing , leaving 62 eligible cases .
a further 16 of these cases were uncontactable , leaving a final pool of 46 eligible cases .
consent to participate in the study was obtained via an initial phone call in 44 cases , with two patients declining to participate .
study packs , which included a study information and consent form for patients to sign , were sent to these 44 , and follow - up phone calls were made if packs were not returned within 3 weeks .
twenty - two packs were ultimately returned within the allotted time period for data collection of 2 months , providing a response rate of 50% ( 22 of 44 ) .
information about the pregnancy and birth details of the child were obtained from the fmu internal database and new south wales electronic medical records .
these included gestational age at birth , birth weight and estimated fetal weight and their percentiles , mode of delivery , apgar scores , and ultrasound doppler studies .
chorionicity was determined by ultrasound in accordance with accepted criteria.4,5 all pregnancies for which this was not conclusive were treated as mc until chorionicity was confirmed antenatally by placental histology . the term uncomplicated was used to define those pregnancies unaffected by mc - specific conditions and identifiable discordant growth patterns up to 28-week gestational age .
pregnancies complicated by major congenital malformations , aneuploidy , and the intrauterine demise of a co - twin were also excluded .
the mc - specific conditions , ttts , taps , and trap were identified sonographically.2022 comparison with an uncomplicated dc control group was not possible as only mc twins are offered routine surveillance every 2 weeks until delivery in the fmu .
participant study packs comprised the child behaviour checklist ( cbcl),23 child development chart ( cdc),24 and a parent questionnaire .
the cbcl23 has strong psychometric properties and was scored using the associated achenbach system of empirically based assessment scoring profile.25 cbcl results were converted into norm - referenced percentiles for the six behavior domains : anxious / depressed , withdrawn , sleep problems , somatic problems , aggressive behavior , and destructive behavior .
percentile scores were categorized as normal ( < 95th percentile ) , borderline ( 95th98th percentile ) , or clinical ( > 98th percentile ) .
these behavior domains were collapsed into composite internalizing ( anxious / depressed and withdrawn ) , externalizing ( aggressive and destructive behavior ) , and total domain score categories , which were reported as a norm - referenced
score out of 100 , with categories of normal ( t < 60 ) , borderline ( t = 6063 ) , and clinical ( t > 63 ) .
the cdc24 was scored by calculating the dq ( that is ,
( developmental agechronological age)100 ) of the child in the domains of social , self - help , gross motor , fine motor , and language , as well as an overall dq , which was calculated as the average of the five domain scores .
quotients were classified as normal ( > 85 ) , borderline ( 8570 ) , and clinical ( < 70).26 the cdc chart did not test beyond 58 months , so parents with children aged > 58 months were asked to complete the chart from their memory of the child at that age ; hence , chronological age used in this scenario was 58 months .
there is a long history of research supporting the accuracy of parent self - report of child development when using structured inventories.27,28 for example , in their 2013 review , bedford et al reported that
measures completed by parents have been shown to be as accurate as those administered by professionals in identifying children with developmental problems ( p. 59).29
measures completed by parents have been shown to be as accurate as those administered by professionals in identifying children with developmental problems ( p. 59).29
the parent questionnaire was constructed for the purposes of the study to obtain specific information regarding demographics of the child , diagnosed developmental issues , and his / her family background .
data were analyzed using spss software . in light of the small sample size recruited for this study ,
independent t - tests were used in a broad comparison of all developmental and behavioral problems with birth weight and gestational age .
the cohen s d metric was used to calculate effect sizes and was calculated by dividing the mean difference between groups by the pooled standard deviation ( sd ) .
it is widely accepted that cohen s d values of 0.20.49 denote small - sized effects , 0.50.79 denote medium - sized effects , and > 0.8 denote large effect sizes .
of the 22 pairs of twins included in the study , the mean age was 4.3 years ( sd = 0.65 ) .
the cohort was split evenly by sex , with 11 male and 11 female twin pairs .
conception was assisted in three pregnancies , by either in vitro fertilization ( ivf ) ( n=2 ) or intracytoplasmic sperm injection ( n=1 ) , and was spontaneous in the remaining 19 pregnancies .
twenty - four of the deliveries were by elective cesarean section ( 54.6% ) . in two cases ,
an emergency cesarean section was performed following the vaginal birth of the first twin due to birth complications ( placental abruption and uncorrectable malpresentation ) .
mean gestational age was 35 weeks and 5 days , with a range from 29 to 38 weeks .
the distribution of gestational ages was relatively normal with no evidence of bimodality : only two cases were born at < 32 weeks ( 29 weeks 3 days in both the cases ) .
mean birth weight was 2,306 g , and three sets of twins presented with significant birth weight discordances of 26.7% , 27% , and 33.1% . as these three sets of twins were not identified using the antenatal screening methodology employed in the study , they were retained in the sample .
preeclampsia was diagnosed in one primiparous pregnancy , resulting in an overall prevalence of 4.5% which is lower than the recorded figures for nulliparous ( 26.7% ) and primi-/multiparous ( 17.1% ) mc pregnancies.30 full pregnancy and birth details can be found in table 2 .
developmental and behavioral data in the form of the parent survey and cbcl were received for all the children . of the conditions specifically screened for in the parent survey , cases of speech delay requiring speech pathology ( n=6 , 14% children 8 , 9 , 10 , 11 , 17 , and 18 in table 3 ) , anxiety ( n=2 , 4.5% children 6 and 7 in table 3 ) , and cerebral palsy ( cp ) ( n=1 , 2.3% child 12 in table 3 ) were reported .
in addition , other medical issues were reported as follows : asthma ( n=3 , 6.8% ) , eczema ( n=3 , 6.8% ) , and vision problems ( n=2 , 4.5% ) ; however , these were not considered in subsequent analyses .
cbcl results are presented in table 4 and are organized according to normal , borderline , and clinical categories .
the average frequency of normal scores in the six cbcl baseline categories per child was 95.9% , indicating a largely problem - free population .
consistent with this , the average t score ( sd , range ) for the internalizing composite was 43.2 ( 10.5 , 3067 ) , externalizing composite 41.9 ( 9.0 , 3068 ) , and overall domain 42.2 ( 10.1 , 2770 ) .
borderline scores were identified in all of the nine categories , and clinical scores were identified in three ; however , all of the borderline and clinical scores were attributable to only six children .
therefore , six of 44 ( 13.6% ) children were found to have some indication of behavioral / emotional problems by the cbcl .
four of these children comprised two female twin pairs , with the scores of one pair of twins ( children 1 and 2 in table 3 ) indicating externalizing behavioral difficulties , while those of the other ( children 6 and 7 in table 3 ) reflected internalizing difficulties .
the remaining two children , from separate twin pairs , had vastly different developmental pro - files .
one ( child 13 in table 3 ) , a female twin with a normal sibling , obtained a borderline score in only one category ( anxious / depressed ) ; however , the final child ( child 14 in table 3 ) , a male twin with a normal sibling , received borderline scores in four categories and clinical scores in two . taken together with the reports of two children experiencing anxiety from parent questionnaire data , the overall rate of behavioral / emotional difficulties was 18% ( eight of 44 ) .
five of 36 ( 13.9% ) children received a borderline score in the gross motor category , while two ( 5.6% ) received a borderline score in the fine motor category .
therefore , seven of 36 ( 19.4% ) of the overall cohort showed some indication of motor delay .
of these cases , five children were identified as having borderline scores in the absence of any other developmental concerns as reported by the parents .
these cases comprised two twin pairs and a child with a normally scoring sibling ( children 6 and 7 , 15 and 16 , and 5 in table 3 ) .
the two remaining children with borderline gross motor dqs ( both 82.6 ) were the female twin pair identified with externalizing difficulties on the cbcl ( children 1 and 2 in table 3 ) . with respect to mean dq scores ( sd , range ) across the cohort , these were as follows : social dq 99.9 ( 7.5 , 88113 ) , self - help dq 102.1 ( 8.1 , 89126 ) , gross motor dq 96.2 ( 10.6 , 71119 ) , fine motor dq 97.7 ( 8.3 , 84120 ) , language dq 100.0 ( 6.3 , 90111 ) , and overall dq 99.2 ( 6.5 , 86113 ) . taking into account the presence of overlap between results from the different screening methods , 11 cases were identified as meeting the criteria for developmental concerns on the questionnaire measures used . in addition ,
seven children were identified by parent report as having previously existing developmental or behavioral problems , namely speech delay requiring speech pathology ( n=6 ) , anxiety ( n=2 ) , and cp ( n=1 ) . however , none of the children with previously indicated problems were identified in either the cbcl or the cdc tests .
thus , the overall rate of developmental concerns in this cohort was 18 of 44 children or 41% of the sample .
detailed information regarding these 18 cases in relation to pregnancy - related factors is summarized in table 3 .
statistical analyses were conducted to explore whether there were any significant relationships between the presence of developmental concerns and a number of the specific pregnancy / birth factors outlined in table 3 .
the comparison of pregnancy / birth factors between these groups is summarized in table 6 .
with alpha set at 0.05 , an independent t - test indicated that there was a difference that approached statistical significance between the developmental concerns and no developmental concerns groups with respect to birth weight ( t(42 ) = 1.9 , p=0.06 ) .
there was a mean difference of 303 g between the developmental concerns cohort ( 2,127 g ) and no developmental concerns cohort ( 2,430 g ) which reflected a cohen s d effect size of 0.59 which is of medium size .
gestational age and birth weight were shown to be highly correlated ( pearson s r(44 ) = 0.76 ) , which is unsurprising given the well - understood relationship between the two variables .
interestingly , however , gestational age was not significantly different between the two groups ( t(42 ) = 1.5 , p=0.13 , cohen s d=0.49 ) , which is approaching medium size .
the relatively small size of the cohort prevented meaningful statistical comparison between the rates of assisted conception or the presence of birth weight discordance > 25% .
results did , however , indicate that a higher proportion of the cohort with developmental concerns were conceived via assisted conception ( 17% vs 12% ) .
similarly , males were overrepresented in the group with developmental concerns relative to those without such concerns ( 61% vs 42% ) .
further , it was observed that there were a number of variables on the cdc where boys scored lower than girls overall , namely social dq ( t(32 ) = 2.45 , p=0.02 ; boys mean = 96.8 [ 5.9 ] , girls mean = 102.7 [ 7.8 ] ) , self - help dq ( t(32 ) = 3.6 , p<0.001 ; boys mean = 97.6 [ 5.1 ] , girls mean = 106.2 [ 8.2 ] ) , fine motor dq ( t(32 ) = 2.23 , p=0.03 ; boys mean = 94.6 [ 5.9 ] , girls mean = 100.6 [ 9.2 ] ) , language dq ( t(32 ) = 4.34 , p<0.001 ; boys mean = 96.0 [ 4.8 ] , girls mean = 103.6 [ 5.3 ] ) , and also the overall dq ( t(32 ) = 3.43 , p=0.002 ; boys mean = 95.7 [ 5.3 ] , girls mean = 102.3 [ 5.9 ] ) .
there were no sex differences on gross motor dq or on any aspects of the cbcl , however .
conception was assisted in three pregnancies , by either in vitro fertilization ( ivf ) ( n=2 ) or intracytoplasmic sperm injection ( n=1 ) , and was spontaneous in the remaining 19 pregnancies .
twenty - four of the deliveries were by elective cesarean section ( 54.6% ) . in two cases ,
an emergency cesarean section was performed following the vaginal birth of the first twin due to birth complications ( placental abruption and uncorrectable malpresentation ) .
mean gestational age was 35 weeks and 5 days , with a range from 29 to 38 weeks .
the distribution of gestational ages was relatively normal with no evidence of bimodality : only two cases were born at < 32 weeks ( 29 weeks 3 days in both the cases ) .
mean birth weight was 2,306 g , and three sets of twins presented with significant birth weight discordances of 26.7% , 27% , and 33.1% . as these three sets of twins were not identified using the antenatal screening methodology employed in the study , they were retained in the sample .
preeclampsia was diagnosed in one primiparous pregnancy , resulting in an overall prevalence of 4.5% which is lower than the recorded figures for nulliparous ( 26.7% ) and primi-/multiparous ( 17.1% ) mc pregnancies.30 full pregnancy and birth details can be found in table 2 .
developmental and behavioral data in the form of the parent survey and cbcl were received for all the children . of the conditions specifically screened for in the parent survey ,
cases of speech delay requiring speech pathology ( n=6 , 14% children 8 , 9 , 10 , 11 , 17 , and 18 in table 3 ) , anxiety ( n=2 , 4.5% children 6 and 7 in table 3 ) , and cerebral palsy ( cp ) ( n=1 , 2.3% child 12 in table 3 ) were reported .
in addition , other medical issues were reported as follows : asthma ( n=3 , 6.8% ) , eczema ( n=3 , 6.8% ) , and vision problems ( n=2 , 4.5% ) ; however , these were not considered in subsequent analyses .
cbcl results are presented in table 4 and are organized according to normal , borderline , and clinical categories .
the average frequency of normal scores in the six cbcl baseline categories per child was 95.9% , indicating a largely problem - free population .
consistent with this , the average t score ( sd , range ) for the internalizing composite was 43.2 ( 10.5 , 3067 ) , externalizing composite 41.9 ( 9.0 , 3068 ) , and overall domain 42.2 ( 10.1 , 2770 ) .
borderline scores were identified in all of the nine categories , and clinical scores were identified in three ; however , all of the borderline and clinical scores were attributable to only six children .
therefore , six of 44 ( 13.6% ) children were found to have some indication of behavioral / emotional problems by the cbcl .
four of these children comprised two female twin pairs , with the scores of one pair of twins ( children 1 and 2 in table 3 ) indicating externalizing behavioral difficulties , while those of the other ( children 6 and 7 in table 3 ) reflected internalizing difficulties .
the remaining two children , from separate twin pairs , had vastly different developmental pro - files .
one ( child 13 in table 3 ) , a female twin with a normal sibling , obtained a borderline score in only one category ( anxious / depressed ) ; however , the final child ( child 14 in table 3 ) , a male twin with a normal sibling , received borderline scores in four categories and clinical scores in two . taken together with the reports of two children experiencing anxiety from parent questionnaire data , the overall rate of behavioral / emotional difficulties was 18% ( eight of 44 ) .
five of 36 ( 13.9% ) children received a borderline score in the gross motor category , while two ( 5.6% ) received a borderline score in the fine motor category .
therefore , seven of 36 ( 19.4% ) of the overall cohort showed some indication of motor delay . of these cases ,
five children were identified as having borderline scores in the absence of any other developmental concerns as reported by the parents .
these cases comprised two twin pairs and a child with a normally scoring sibling ( children 6 and 7 , 15 and 16 , and 5 in table 3 ) .
the two remaining children with borderline gross motor dqs ( both 82.6 ) were the female twin pair identified with externalizing difficulties on the cbcl ( children 1 and 2 in table 3 ) . with respect to mean dq scores ( sd , range ) across the cohort , these were as follows : social dq 99.9 ( 7.5 , 88113 ) , self - help dq 102.1 ( 8.1 , 89126 ) , gross motor dq 96.2 ( 10.6 , 71119 ) , fine motor dq 97.7 ( 8.3 , 84120 ) , language dq 100.0 ( 6.3 , 90111 ) , and overall dq 99.2 ( 6.5 , 86113 ) .
taking into account the presence of overlap between results from the different screening methods , 11 cases were identified as meeting the criteria for developmental concerns on the questionnaire measures used . in addition , seven children were identified by parent report as having previously existing developmental or behavioral problems , namely speech delay requiring speech pathology ( n=6 ) , anxiety ( n=2 ) , and cp ( n=1 ) . however , none of the children with previously indicated problems were identified in either the cbcl or the cdc tests .
thus , the overall rate of developmental concerns in this cohort was 18 of 44 children or 41% of the sample .
detailed information regarding these 18 cases in relation to pregnancy - related factors is summarized in table 3 .
statistical analyses were conducted to explore whether there were any significant relationships between the presence of developmental concerns and a number of the specific pregnancy / birth factors outlined in table 3 . the comparison of pregnancy / birth factors between these groups is summarized in table 6 . with alpha set at 0.05
, an independent t - test indicated that there was a difference that approached statistical significance between the developmental concerns and no developmental concerns groups with respect to birth weight ( t(42 ) = 1.9 , p=0.06 ) .
there was a mean difference of 303 g between the developmental concerns cohort ( 2,127 g ) and no developmental concerns cohort ( 2,430 g ) which reflected a cohen s d effect size of 0.59 which is of medium size .
gestational age and birth weight were shown to be highly correlated ( pearson s r(44 ) = 0.76 ) , which is unsurprising given the well - understood relationship between the two variables .
interestingly , however , gestational age was not significantly different between the two groups ( t(42 ) = 1.5 , p=0.13 , cohen s d=0.49 ) , which is approaching medium size .
the relatively small size of the cohort prevented meaningful statistical comparison between the rates of assisted conception or the presence of birth weight discordance > 25% .
results did , however , indicate that a higher proportion of the cohort with developmental concerns were conceived via assisted conception ( 17% vs 12% ) .
similarly , males were overrepresented in the group with developmental concerns relative to those without such concerns ( 61% vs 42% ) .
further , it was observed that there were a number of variables on the cdc where boys scored lower than girls overall , namely social dq ( t(32 ) = 2.45 , p=0.02 ; boys mean = 96.8 [ 5.9 ] , girls mean = 102.7 [ 7.8 ] ) , self - help dq ( t(32 ) = 3.6 , p<0.001 ; boys mean = 97.6 [ 5.1 ] , girls mean = 106.2 [ 8.2 ] ) , fine motor dq ( t(32 ) = 2.23 , p=0.03 ; boys mean = 94.6 [ 5.9 ] , girls mean = 100.6 [ 9.2 ] ) , language dq ( t(32 ) = 4.34 , p<0.001 ; boys mean = 96.0 [ 4.8 ] , girls mean = 103.6 [ 5.3 ] ) , and also the overall dq ( t(32 ) = 3.43 , p=0.002 ; boys mean = 95.7 [ 5.3 ] , girls mean = 102.3 [ 5.9 ] ) .
there were no sex differences on gross motor dq or on any aspects of the cbcl , however .
higher rates of neuromorbidity in twins are attributed mainly to prematurity , very low birth weight , and complications related to chorionicity.31 this cohort of 44 uncomplicated mcda twins delivered with a mean gestational age of 35 weeks 5 days and a mean birth weight of 2,306 g , amongst the first such uncomplicated cohorts studied , reduces the influence of these factors .
the rate of 41% identified for developmental or behavioral concerns in this study is much higher than previous neuromorbidity estimates of 0.5% and 7% for similar cohorts;15,17 however , vast differences in sample sizes and negative outcome inclusion criteria as well as the use of different measures mean that comparisons are problematic .
the present study has also included a range of markers of mild - to - moderate developmental concern .
the results of this study may be considered relative to population estimates of developmental delay , which refers to chronic conditions originating in childhood.32 a previous australian research using the parents evaluation of developmental status33 has reported that 9% of children were classified as being at high risk of disabilities and 19% were classified as being at medium risk.34 this is similar in international estimates and rates reported in other studies using other techniques , such as the 16.6% rate reported by hamilton.35 thus , neurodevelopmental and behavioral concerns in mcda pregnancies would appear to occur at a rate higher , potentially around double , than that observed in the general population .
the most prevalent developmental problem in this cohort was borderline motor delay , identified in seven of 36 ( 19.4% ) children with no other abnormal dq scores .
this finding is similar to that of hack et al,14 who reported higher rates of locomotor difficulties , relative to other areas of development , in a cohort of mcda twins assessed at 22 months of age using the griffiths mental development scales . in this
regard , twinning has been suggested to be an independent risk factor for developmental delay with genetic factors contributing strongly to certain motor development outcomes.36 in a study of 84 pairs of mostly dc twins and 72 singletons from the gambia , the mean age of achieving motor milestones , such as maintaining head stability , sitting without support , and walking , was found to be significantly higher in twins after adjustment for confounders , with over 90% of population variance attributed to genetic factors rather than environmental factors.36 parent - reported language difficulties occurred within the cohort at a rate of 14% .
hack et al14 also reported a slight increase in rates of language and hearing difficulties in mc relative to dc twins .
more broadly , it is well documented that twins have more language difficulties than singletons , with the rationale for this placed not only on physiological differences.37 twins are thought to be spoken to as an individual less frequently and for shorter periods of time by parents , given the temptation to address both together.38 a relationship has also been observed between the presence of a secret language between twins and language delay.39 despite these twin - specific environmental and social influences , speech and language delay is common , with an estimated prevalence of 2.3%29% in children aged 27 years .
more detailed understanding of the factors influencing on the development of language difficulties in this cohort is limited by the screening nature of the questionnaires used , although none of the children showed any indication of delay in the cdc .
consistent with findings from the available literature regarding neurodevelopmental difficulties such as autism spectrum disorder and attention - deficit hyperactivity disorder , where males are affected at higher rates than females , several domain scores on the cdc were significantly lower amongst males than females , namely social dq , self - help dq , fine motor dq , and language dq .
the overall dq score was also significantly lower in boys , and males were overrepresented in the cohort of children in the study ultimately identified with developmental concerns , relative to those without such concerns ( 61% vs 42% ) .
these findings are reminiscent of twin data reported by hack et al,14 and suggest that being male may confer an additional risk factor for neurodevelopmental difficulties .
internalizing and externalizing problems are thought to affect up to 25% of youth in australia.40 current available estimates of high internalizing scores in normal singleton populations at age 5 suggest rates of 8% ( borderline and clinical scores ) and 4.6% ( clinical scores only).40,41 a rate of 11.3% for cbcl - identified and parent report of diagnosed internalizing problems in the current cohort therefore does appear higher than would normally occur .
it is difficult to determine the extent to which removal of extreme prematurity , low birth weight , and mc conditions from this cohort decreases the risk of developmental delay , given the small sample size of this study ; however , it could be reasoned that for these behavioral problems , environmental factors may have played a role .
for example , the relationship between parental anxiety and children s outcomes is well documented.42,43 while the collection of relatively limited personal data in this study means that environmental factors can only be speculated , a twin pregnancy , even when uncomplicated , and subsequent care of two infants can be considered a stressful event.38 similarly , ivf conception may have added further to the stress of several of the parents in this cohort .
clinical cbcl scores for externalizing problems were identified in three children , producing a rate of 6.8% in this cohort .
rates reported by bayer et al40 and potijk et al41 of 5.5% ( borderline or clinical scores ) and 5.2% ( clinical only ) , respectively , indicate that the present results are broadly consistent with population norms , if slightly higher ; however , the influence of environmental factors on these rates would again require further investigation .
the above - noted modest developmental concerns regarding motor development , speech and language development , and socio - emotional development are amenable , in the main , to early intervention provided by allied professionals such as occupational therapists , speech pathologists , and psychologists .
it would appear that one direct implication of this study would be for mcda twins to have regular developmental checks with a pediatrician during the first several years of life so as to identify any emerging concerns quickly and obtain a referral for appropriate intervention . in australia
, there is a process for universal developmental surveillance , and parents could be explicitly encouraged and supported to adhere to attending regular developmental checks through until school - age , rather than dropping out as often occurs after the infant is aged around 12 months.44 one child presented with previously diagnosed cp , resulting in a prevalence of 2.3% within this population .
twins are known to have a four times greater rate of cp than singletons ( 0.59% vs 0.14% ) , with a further increased risk of mcda pregnancies.45 studies by ortibus et al17 and hack et al14 have reported rates of 2% and 2.2% , respectively , in complicated mcda cohorts ; however , ortibus et al17 reported a cp rate in uncomplicated mcda twins of only 0.63% , which is more comparable with overall twin estimates .
a number of risk factors can increase the likelihood of cp in twins , including discordant growth and preterm birth , with 20% of cp cases in twins occurring in children born between 32 and 36 weeks of gestation.46 the child with cp in the current sample had a gestational age of 35 weeks 2 days and a high birth weight discordancy of 22.5% .
assisted reproductive technology ( art ) is also considered to increase the likelihood of cp ; however , this is likely to be attributable to higher rates of twinning and resultant complications , such as prematurity.46 art was found to be responsible for higher rates of neuromorbidity in mcda twins;17 however , these findings were based on a cohort of both complicated and uncomplicated cases , making comparison with the current sample difficult .
birth weight and gestational age were the only pregnancy / birth factors able to be analyzed for statistical significance in this study .
the developmental concerns group tended to have a lower birth weight ( p=0.06 ) of medium effect size .
gestational age was not significantly different between the groups ; however , the correlation between birth weight and gestational age was high at r=0.76 .
this expected finding adds additional validity to the measures used in the study to determine neurodevelopmental and behavioral concerns , and when considered alongside evidence from the literature regarding the timing of delivery,47 this also appears to further support the argument for later delivery.48 while the majority of extant research has focused on outcomes of mcda pregnancies with abnormal clinical episodes , these cases may be considered the more extreme end of a spectrum that extends from mcda twins with no anastomoses and equal share , to various imbalances of blood volume distribution associated with the different types of anastomoses for varying periods of time .
for example , it is widely accepted that during a pregnancy with no intervention for ttts or siugr , one may see significant amniotic fluid discordance commonly which usually resolves.49 furthermore , in uncomplicated mcda twins managed with scan surveillance every 2 weeks who have reached the third trimester of pregnancy , unexpected fetal loss risk as high as 4.3% at 32 weeks of gestation has been reported,18 or a comparable magnitude ( 3.8% ) of pregnancies with infants having severe neurodevelopmental delay following the prenatal diagnosis of unexpected acute - onset complications.50 this demonstrates that mcda fetal development is exposed to significantly greater changes in physiology than dc twins , which in turn may negatively influence neurodevelopmental outcomes .
the mechanisms underlying this require further research including mcda cases nominally described as uncomplicated . as well as small cohort size
the questionnaires used to obtain data in this study were parent - completed screening tests , as opposed to the direct pediatric developmental evaluations as used in several available studies.14,15,17 nonetheless , parent report of child development using structured tools is thought to be accurate relative to observational measures,2729 and many aspects of the present results , including lower developmental scores across many domains for boys , and relatively high rates of motor difficulties , are consistent with the findings of hack et al,14 who used the griffiths mental development scales .
furthermore , in that study , parent report using the hscs - ps was also found to be commensurate with the griffiths results .
screening tests are designed to identify groups at sufficient risk to warrant a more in - depth evaluation , rather than providing a quantitative assessment or diagnosis and therefore potentially have a higher rate of positive results.51 furthermore , it has been suggested that this problem is more pronounced for twins , where parents must remember two separate developmental trajectories and are likely to compare and contrast the two children , potentially leading to an overestimation or under - estimation of differences between the two.52 in any event , the observed differences between parent - structured questionnaire report of difficulties ( eg , cdc , cbcl ) and parent qualitative report of previously diagnosed difficulties merit investigation in future studies .
data were initially extracted from the fmu by selecting for mc status and then further condensing the cohort manually based on date of birth and gestational age ; however , more detailed cross - referencing with hospital databases revealed that a number of patients included did not truly fit the mcda inclusion criteria .
a total of 27 of 90 patients were revealed as inappropriate , meaning that patients were correctly identified at a rate of 70% .
while these cases did not affect the final outcome of this study , they do cast doubt on the specificity and reproducibility of the method . the size , timing , and differences in liverpool fmu protocol for the screening and care of dc twins also meant that comparison with dc data from within the same selection population was not possible . comparison with a control
would have better contextualized the results , and the contrast between the two groups may have highlighted trends not as obvious in this study .
one of the elements of this study that we would like to have further explored is the specific details of chorionicity .
problems with determining chorionicity sonographically would most likely not have affected our results ; however , more detailed analysis of placental histology may have highlighted relationships unable to be tested in this study .
placental angioarchitecture in relation to clinical outcome is an interesting and fast - developing area of research , and while the size and resources of this study did not allow for further investigation , it is an area that will hopefully be researched by this facility in the future . in any event
, longer - term follow - up studies would appear to be strongly indicated with mcda cohorts .
the most prevalent developmental problem in this cohort was borderline motor delay , identified in seven of 36 ( 19.4% ) children with no other abnormal dq scores .
this finding is similar to that of hack et al,14 who reported higher rates of locomotor difficulties , relative to other areas of development , in a cohort of mcda twins assessed at 22 months of age using the griffiths mental development scales . in this
regard , twinning has been suggested to be an independent risk factor for developmental delay with genetic factors contributing strongly to certain motor development outcomes.36 in a study of 84 pairs of mostly dc twins and 72 singletons from the gambia , the mean age of achieving motor milestones , such as maintaining head stability , sitting without support , and walking , was found to be significantly higher in twins after adjustment for confounders , with over 90% of population variance attributed to genetic factors rather than environmental factors.36 parent - reported language difficulties occurred within the cohort at a rate of 14% .
hack et al14 also reported a slight increase in rates of language and hearing difficulties in mc relative to dc twins .
more broadly , it is well documented that twins have more language difficulties than singletons , with the rationale for this placed not only on physiological differences.37 twins are thought to be spoken to as an individual less frequently and for shorter periods of time by parents , given the temptation to address both together.38 a relationship has also been observed between the presence of a secret language between twins and language delay.39 despite these twin - specific environmental and social influences , speech and language delay is common , with an estimated prevalence of 2.3%29% in children aged 27 years
. more detailed understanding of the factors influencing on the development of language difficulties in this cohort is limited by the screening nature of the questionnaires used , although none of the children showed any indication of delay in the cdc .
consistent with findings from the available literature regarding neurodevelopmental difficulties such as autism spectrum disorder and attention - deficit hyperactivity disorder , where males are affected at higher rates than females , several domain scores on the cdc were significantly lower amongst males than females , namely social dq , self - help dq , fine motor dq , and language dq .
the overall dq score was also significantly lower in boys , and males were overrepresented in the cohort of children in the study ultimately identified with developmental concerns , relative to those without such concerns ( 61% vs 42% ) .
these findings are reminiscent of twin data reported by hack et al,14 and suggest that being male may confer an additional risk factor for neurodevelopmental difficulties .
internalizing and externalizing problems are thought to affect up to 25% of youth in australia.40 current available estimates of high internalizing scores in normal singleton populations at age 5 suggest rates of 8% ( borderline and clinical scores ) and 4.6% ( clinical scores only).40,41 a rate of 11.3% for cbcl - identified and parent report of diagnosed internalizing problems in the current cohort therefore does appear higher than would normally occur .
it is difficult to determine the extent to which removal of extreme prematurity , low birth weight , and mc conditions from this cohort decreases the risk of developmental delay , given the small sample size of this study ; however , it could be reasoned that for these behavioral problems , environmental factors may have played a role .
for example , the relationship between parental anxiety and children s outcomes is well documented.42,43 while the collection of relatively limited personal data in this study means that environmental factors can only be speculated , a twin pregnancy , even when uncomplicated , and subsequent care of two infants can be considered a stressful event.38 similarly , ivf conception may have added further to the stress of several of the parents in this cohort .
clinical cbcl scores for externalizing problems were identified in three children , producing a rate of 6.8% in this cohort .
rates reported by bayer et al40 and potijk et al41 of 5.5% ( borderline or clinical scores ) and 5.2% ( clinical only ) , respectively , indicate that the present results are broadly consistent with population norms , if slightly higher ; however , the influence of environmental factors on these rates would again require further investigation .
the above - noted modest developmental concerns regarding motor development , speech and language development , and socio - emotional development are amenable , in the main , to early intervention provided by allied professionals such as occupational therapists , speech pathologists , and psychologists .
it would appear that one direct implication of this study would be for mcda twins to have regular developmental checks with a pediatrician during the first several years of life so as to identify any emerging concerns quickly and obtain a referral for appropriate intervention . in australia
, there is a process for universal developmental surveillance , and parents could be explicitly encouraged and supported to adhere to attending regular developmental checks through until school - age , rather than dropping out as often occurs after the infant is aged around 12 months.44 one child presented with previously diagnosed cp , resulting in a prevalence of 2.3% within this population .
twins are known to have a four times greater rate of cp than singletons ( 0.59% vs 0.14% ) , with a further increased risk of mcda pregnancies.45 studies by ortibus et al17 and hack et al14 have reported rates of 2% and 2.2% , respectively , in complicated mcda cohorts ; however , ortibus et al17 reported a cp rate in uncomplicated mcda twins of only 0.63% , which is more comparable with overall twin estimates .
a number of risk factors can increase the likelihood of cp in twins , including discordant growth and preterm birth , with 20% of cp cases in twins occurring in children born between 32 and 36 weeks of gestation.46 the child with cp in the current sample had a gestational age of 35 weeks 2 days and a high birth weight discordancy of 22.5% .
assisted reproductive technology ( art ) is also considered to increase the likelihood of cp ; however , this is likely to be attributable to higher rates of twinning and resultant complications , such as prematurity.46 art was found to be responsible for higher rates of neuromorbidity in mcda twins;17 however , these findings were based on a cohort of both complicated and uncomplicated cases , making comparison with the current sample difficult .
birth weight and gestational age were the only pregnancy / birth factors able to be analyzed for statistical significance in this study .
the developmental concerns group tended to have a lower birth weight ( p=0.06 ) of medium effect size .
gestational age was not significantly different between the groups ; however , the correlation between birth weight and gestational age was high at r=0.76 .
this expected finding adds additional validity to the measures used in the study to determine neurodevelopmental and behavioral concerns , and when considered alongside evidence from the literature regarding the timing of delivery,47 this also appears to further support the argument for later delivery.48 while the majority of extant research has focused on outcomes of mcda pregnancies with abnormal clinical episodes , these cases may be considered the more extreme end of a spectrum that extends from mcda twins with no anastomoses and equal share , to various imbalances of blood volume distribution associated with the different types of anastomoses for varying periods of time . for example
, it is widely accepted that during a pregnancy with no intervention for ttts or siugr , one may see significant amniotic fluid discordance commonly which usually resolves.49 furthermore , in uncomplicated mcda twins managed with scan surveillance every 2 weeks who have reached the third trimester of pregnancy , unexpected fetal loss risk as high as 4.3% at 32 weeks of gestation has been reported,18 or a comparable magnitude ( 3.8% ) of pregnancies with infants having severe neurodevelopmental delay following the prenatal diagnosis of unexpected acute - onset complications.50 this demonstrates that mcda fetal development is exposed to significantly greater changes in physiology than dc twins , which in turn may negatively influence neurodevelopmental outcomes . the mechanisms underlying this require further research including mcda cases nominally described as uncomplicated .
as well as small cohort size , aspects of the methodology may have influenced the outcomes of this study .
the questionnaires used to obtain data in this study were parent - completed screening tests , as opposed to the direct pediatric developmental evaluations as used in several available studies.14,15,17 nonetheless , parent report of child development using structured tools is thought to be accurate relative to observational measures,2729 and many aspects of the present results , including lower developmental scores across many domains for boys , and relatively high rates of motor difficulties , are consistent with the findings of hack et al,14 who used the griffiths mental development scales .
furthermore , in that study , parent report using the hscs - ps was also found to be commensurate with the griffiths results .
screening tests are designed to identify groups at sufficient risk to warrant a more in - depth evaluation , rather than providing a quantitative assessment or diagnosis and therefore potentially have a higher rate of positive results.51 furthermore , it has been suggested that this problem is more pronounced for twins , where parents must remember two separate developmental trajectories and are likely to compare and contrast the two children , potentially leading to an overestimation or under - estimation of differences between the two.52 in any event , the observed differences between parent - structured questionnaire report of difficulties ( eg , cdc , cbcl ) and parent qualitative report of previously diagnosed difficulties merit investigation in future studies .
data were initially extracted from the fmu by selecting for mc status and then further condensing the cohort manually based on date of birth and gestational age ; however , more detailed cross - referencing with hospital databases revealed that a number of patients included did not truly fit the mcda inclusion criteria .
a total of 27 of 90 patients were revealed as inappropriate , meaning that patients were correctly identified at a rate of 70% .
while these cases did not affect the final outcome of this study , they do cast doubt on the specificity and reproducibility of the method .
the size , timing , and differences in liverpool fmu protocol for the screening and care of dc twins also meant that comparison with dc data from within the same selection population was not possible .
comparison with a control would have better contextualized the results , and the contrast between the two groups may have highlighted trends not as obvious in this study .
one of the elements of this study that we would like to have further explored is the specific details of chorionicity .
problems with determining chorionicity sonographically would most likely not have affected our results ; however , more detailed analysis of placental histology may have highlighted relationships unable to be tested in this study .
placental angioarchitecture in relation to clinical outcome is an interesting and fast - developing area of research , and while the size and resources of this study did not allow for further investigation , it is an area that will hopefully be researched by this facility in the future . in any event
, longer - term follow - up studies would appear to be strongly indicated with mcda cohorts .
within this cohort of preschool - aged mcda twins , we observed a high rate of neurodevelopmental concerns ( 41% ) of mild - to - moderate severity . while these encompassed a wide range of concerns , lower average scores for motor ( 19% ) and speech and language ( 14% ) development , as well as internalizing difficulties ( 11% ) predominated .
consistent with the existing literature , the presence of developmental concerns was associated with a lower birth weight . however , further studies are needed to elucidate whether twinning in itself may increase the risk for developmental delay , and in particular motor development , and the unique contributions of genetic factors including on placental development in addition to perinatal factors such as low birth weight and prematurity
. further research with an increased sample size and a refined methodology are required to understand the possible mechanisms and to give more clinical significance to these conclusions . | backgroundrelatively little is known about the neurodevelopmental and behavioral outcomes of monochorionic diamniotic ( mcda ) twin pregnancies where there are no antenatal complications peculiar to monochorionicity or prematurity.methodstwenty-two mcda twins ( 44 children ) with an average age of 4.3 years , and with no antenatal complications detected by 28 weeks of gestation , were recruited from a feto - maternal unit database .
parents completed a battery of neurodevelopmental and behavioral assessment questionnaires.resultseighteen children ( 41% ) were identified as having developmental or behavioral concerns , predominantly of mild severity , which in turn were associated with a lower birth weight of medium effect size ( cohen s d=0.59).conclusionmcda twins delivered in the third trimester with no antenatal monochorionic complications in the first two trimesters appear to be at risk for subtle neurodevelopmental difficulties , associated with a lower birth weight . ongoing developmental surveillance of these children during preschool - age is indicated for early identification and intervention . |
a 72-year - old female patient with corneal edema due to fuchs corneal dystrophy , after 2 years , uneventful phacoemulsification surgery with intraocular lens implantation was scheduled for dsaek .
the following eye had visual acuity of 20/25 ( correction + 1,0 dsph ) and underwent descemet 's stripping endothelial keratoplasty ( dsek ) combined with phacoemulsification surgery 3 years earlier .
spectral - domain optical coherence tomography ( sd oct ) examination revealed corneal edema with descemet membrane folds on posterior corneal surface and fluid spaces under the corneal epithelium .
the donor cornea was dissected with a moria microkeratome ( 350 m keratome plate ) . after fashioning the clear corneal incision ,
the surgery was discontinued and the optical coherence tomography ( oct ) exam with commercially available sd oct device ( ivue ; optovue , fremont ; usa ) was performed .
acquiring of the image was gained by pressing the foot pedal after focusing the anterior eye segment structures within the oct scan [ fig . 1 ] . after making sure that all of descemet membrane was removed ,
the air was injected into the anterior chamber with following eye surface sweeping in order to remove fluid from the graft interface .
the graft seemed to be attached when analyzing in the surgical microscope . after performing sd oct ,
multiple fluid micro - spaces were revealed in the area of the graft interface [ fig . 2 ] .
due to that , we performed corneal stab incisions in the four main clock hours .
after incisions , sd oct revealed a proper graft adhesion to the posterior corneal surface [ fig . 3 ] . at the end of the surgery ,
the whole procedure lasted for 35 min , which increased the time of the surgery by approximately 15 min .
there is a border on posterior corneal surface with and without descemet membrane spectral - domain optical coherence tomography scan performed after donor disc implantation .
there is an inaccurate adhesion of a graft with accompanying fluid spaces noticeable spectral - domain optical coherence tomography scan performed after corneal stab incisions with a surface sweeping .
assessment of the intraoperative donor disc attachment could be performed routinely with the use of a slit - lamp attached to the surgical microscope or alternatively by observing the donor lenticule border using very high microscope magnification .
however , none of those methods gives such a certainty of proper graft attachment as performing intraoperative oct .
intraoperative oct analysis by revealing the residual interface space has a potential to reduce the post - operative disc dislocation rate .
optical coherence tomography is a high speed , high resolution , non - contact optical imaging technique developed for noninvasive cross - sectional imaging in biologic systems .
after dsek surgery , oct was used to monitor cornea and graft morphometric dynamics and its influence on refractive changes after the surgery .
the first paper describing the use of handhold oct ( bioptigen sd oct ) during the dsaek to assess the donor - host interface was published in 2010 .
this is the first report presenting the intraoperative commercially available oct device . in our opinion , there are two main problems regarding the intraoperative use of oct : the surgery during the sd oct exam is interrupted and there is a necessity of the use additional surgical drapes to prevent the possible touching of the operating field .
this inconvenience could be removed by using c - arm allowing the oct exam in the controlled manner .
the ideal way would be to use integrated oct and surgical microscope , similar to those used in the vitreoretinal surgery .
to conclude : oct assisted dsek allows the assessment of the accuracy of the descemet stripping and donor disc attachment . | optical coherence tomography has already been proven to be useful for pre- and post - surgical anterior eye segment assessment , especially in lamellar keratoplasty procedures .
there is no evidence for intraoperative usefulness of optical coherence tomography ( oct ) .
we present a case report of the intraoperative donor disc attachment assessment with spectral - domain optical coherence tomography in case of descemet stripping automated endothelial keratoplasty ( dsaek ) surgery combined with corneal incisions .
the effectiveness of the performed corneal stab incisions was visualized directly by oct scan analysis .
oct assisted dsaek allows the assessment of the accuracy of the descemet stripping and donor disc attachment . |
the last several years have seen substantial theoretical developments related to the hypothesized behavioral functions of nucleus accumbens dopamine ( da ) .
it has become evident to many investigators that there are conceptual limitations and empirical problems with the traditional da hypothesis of reward ( baldo and kelley , 2007 ; barbano and cador , 2007 ; salamone et al . , 1997 , 2005 , 2007 ;
even the use of the term reward itself often is problematic ( cannon and bseikri , 2004 ; salamone et al .
, 2005 ; salamone , 2006 ; sanchis - segura and spanagel , 2006 ; yin et al . , 2008 ) .
researchers rarely define what they mean by reward when they are using it to describe a psychological process ; some use it as though it were a synonym for reinforcement , or in reference to
appetite or primary motivation , while still others employ it as a code word to mean pleasure . in some papers
, the word reward seems to be used as a rather monolithic , all - encompassing term that refers to any and all aspects of appetitive learning , motivation and emotion , whether conditioned or unconditioned . used in this way , the term reward is a rather blunt instrument .
these problems are not merely semantic , as it is difficult to test a hypothesis which maintains that a neurotransmitter mediates such an ill - defined set of functions . it has been suggested that it is advantageous to maintain the distinction between the terms reward and reinforcement ; with this usage , reinforcement refers more directly to instrumental learning mechanisms ( wise , 2004 ; sanchis - segura and spanagel , 2006 ) , while reward connotes the primary motivational and emotional effects of reinforcing stimuli ( everitt and robbins , 2005 ; salamone et al .
, 2005 , 2007 ) . against the backdrop of these conceptual and terminological issues ,
there is a tremendous weight of empirical evidence that has built up against the various iterations of the da hypothesis of reward .
it is somewhat ironic that the processes most directly linked to the use of the term reward ( i.e. , primary motivation , subjective pleasure ) are the ones that have proven to be most problematic in terms of demonstrating the involvement of mesolimbic da ( salamone et al . , 2007 ) .
for example , low doses of da antagonists and depletions of nucleus accumbens da have been shown to produce effects that do not closely resemble extinction ( salamone , 1986 ; salamone et al .
, 1995 , 1997 ; rick et al . , 2006 ) , pre - feeding ( salamone et al . , 1991 ; aberman and salamone , 1999 ) , or appetite suppressant drugs ( cousins et al . , 1994 ; salamone et al . , 2002 ;
although it is well known that whole forebrain da depletions can produce aphagia ( i.e. , lack of eating ) , it is da depletions in the lateral or ventrolateral caudate / putamen , rather than the nucleus accumbens , which have most conclusively been linked to this effect ( ungerstedt , 1971 ; dunnett and iversen , 1982 ; salamone et al . , 1993a ) .
it has been shown repeatedly that nucleus accumbens da depletions or antagonism do not substantially impair appetite for food , or produce a general disruption of primary food motivation ( ungerstedt , 1971 ; koob et al . , 1978 ; bakshi and kelley , 1991 ; salamone et al .
da deficient mice , restoration of da production in caudate putamen , but not nucleus accumbens , was able to rescue feeding behavior ( szczypka et al . , 2001 ) .
in summarizing their findings that injections of da d1 or d2 family antagonists into either the core or the shell subregions of nucleus accumbens impaired locomotion and rearing , but did not suppress food intake , baldo et al .
( 2002 ) stated that da receptor blockade did not abolish the primary motivation to eat
furthermore , the idea that nucleus accumbens da mediates the pleasure associated with positive reinforcers has been strongly challenged ( berridge , 2007 ; salamone et al . , 2007 ; berridge and kringlebach , 2008 ) .
interference with accumbens da transmission does not impair appetitive taste reactivity for sucrose ( berridge , 2007 ; berridge and kringlebach , 2008 ) .
several studies in humans have reported that da antagonists did not blunt the subjective euphoria produced by drugs of abuse ( gawin , 1986 ; brauer and de wit , 1997 ; haney et al .
, 2001 ; nann - vernotica et al . , 2001 ; wachtel et al . ,
moreover , the potential role of da systems in instrumental behavior or learning is not limited to situations involving appetitive motivation .
there is considerable evidence that striatal mechanisms in general , and mesolimbic da in particular , also participate in aspects of aversive learning and aversive motivation ( salamone , 1994 ; munro and kokkinidis , 1997 ; blazquez et al . , 2002 ; pezze and feldon , 2004 ; delgado et al . , 2008 ; faure et al . , 2008
although imaging studies often are used to support the idea that nucleus accumbens mediates pleasure ( e.g. , sarchiapone et al . , 2006 ; wacker et al . , 2009 ) , this appears to be oversimplified ; indeed , research employing various imaging methods has demonstrated that the human nucleus accumbens also responds to stress , aversion and hyperarousal / irritability ( liberzon et al . , 1999 ; jensen et al .
, 2003 ; pavic , 2003 ; phan et al . , 2004 ; pruessner et al . , 2004 ; levita et al . ,
physiological and neurochemical studies in animals clearly indicate that da neuron activity is not simply tied to the delivery of primary reinforcers or pleasurable stimuli . rather , vta neuron activity and da release can be activated by a number of different appetitive and aversive conditions ( mccullough and salamone , 1992 ; mccullough et al . , 1993 ;
, 2004 ; young , 2004 ; anstrom and woodward , 2005 ; broom and yamamoto , 2005 ; marinelli et al .
, 2005 ; schultz , 2007a , b ; brischoux et al . , 2009 ) , with changes seen across varying time scales , including tonic , slow phasic and fast phasic signals ( salamone , 1996 ; salamone et al .
, 2007 ; schultz , 2007a , b ; salamone , in press ; see also lapish et al .
, 2007 for a discussion of various time scales associated with the postsynaptic effects of da release and da receptor stimulation ) . of course , one would not want to throw the baby out with the bathwater .
it is apparent that mesolimbic da participates in several complex functions related to aspects of instrumental behavior , learning and incentive motivation , and pavlovian / instrumental interactions ( wise , 2004 ; everitt and robbins , 2005 ; kelley et al .
berridge , 2007 ; robbins and everitt , 2007 ; redgrave et al . , 2008 ; yin et al . ,
the more difficult aspect of research and theory in this area is to ask which specific aspects ?
exploration of these diverse areas of dopaminergic function has become a rich and fruitful area of inquiry .
indeed , this literature is so extensive that a thorough review of the behavioral functions of nucleus accumbens da is beyond the scope of the present article ( see salamone et al . , 2007 ) .
for the purposes of this special issue , the present review will focus upon the role of nucleus accumbens da in effort - related processes , with a special emphasis on effort - based choice behavior that depends upon cost / benefit analyses .
even as the popularity of the da hypothesis of reward was growing during the 1980s , it was becoming apparent that there were alternative conceptual frameworks available for organizing what was known about the behavioral functions of da systems , particularly mesolimbic da .
( 1980 ) suggested that nucleus accumbens acted as a functional interface between the limbic system and the motor system , facilitating the ability of information related to emotion and motivation to impinge upon the neural systems involved in the instigation of action .
it had been emphasized for several decades that behavioral activation , i.e. , the vigor , persistence and effort seen in the pursuit of motivational stimuli , and the heightened activity induced by conditioned stimuli that predict reinforcers , was a fundamental aspect of motivation ( e.g. , cofer and appley , 1964 ) .
da antagonists or accumbens da depletions were shown to suppress the activities such as excessive drinking , wheel running , and locomotion that are induced by scheduled presentation of food ( robbins and koob , 1980 ; wallace et al . , 1983 ; salamone , 1986 , 1988 ) .
it also was reported that the effects of da antagonists on reinforced behavior interacted powerfully with the kinetic requirements of the instrumental response . for example , doses of da antagonists that suppressed reinforced lever pressing had minimal effects on reinforced nose poking behavior ( ettenberg et al . , 1981 ; mekarski , 1988 ) .
although 0.1 mg / kg haloperidol severely reduced responding on a fixed ratio ( fr ) 20 schedule of lever pressing , a dose four times that size had no effect on the reinforced response of simply being in proximity to the food dish on a fixed interval 30 s schedule ( salamone , 1986 ) .
as this research was being reported , investigators began to employ economic concepts , such as exertion of effort and cost - benefit analyses , to describe the behavioral functions of accumbens da .
neill and justice ( 1981 ) hypothesized that injection of amphetamine into nucleus accumbens could be increasing the willingness of rats to exert effort to obtain a given level of reinforcement . in a contemporary review of the behavioral functions of da systems ( salamone , 1987 ) ,
it was noted that da in nucleus accumbens could be involved in the exertion of effort , and it was suggested that future experiments could offer animals choices between various reinforcers that are associated with operants of varying difficulty ( p. 602 ) so that researchers could determine if the allocation of behavioral resources could be biased toward or away from more or less effortful responses by administration of dopaminergic drugs .
this recognition of dopaminergic involvement in the exertion of effort , and effort - based choices related to cost benefit analyses , fit nicely with an emerging emphasis in the behavioral literature on work , response costs or constraints , and economic models of operant behavior .
several behavioral investigators have emphasized how response costs or constraints affect operant response output ( staddon , 1979 ; kaufman , 1980 ; kaufman et al . , 1980 ;
collier and colleagues studied how work requirements , such as the number of lever presses necessary for obtaining food , could serve as determinants of response output and affect consumption parameters ( collier and jennings , 1969 ; johnson and collier , 1987 ) .
economic models of operant behavior have emphasized how a number of factors , including not only reinforcement value , but also conditions related to the characteristics of the instrumental response , can determine behavioral output ( lea , 1978 ; allison , 1981 , 1993 ; bickel et al . , 2000 ) .
( 1988 ) suggested that , in terms of behavioral economics , the price of food reinforcement as a commodity is a cost / benefit ratio expressed as the effort expended per unit of food value consumed .
optimal foraging theory was proposed to account for the observation that the amount of effort or time expended to obtain motivational stimuli was an important determinant of foraging choice ( krebs , 1977 ) , an idea that is still very influential in the ethology research today ( e.g. , hengeveld et al . ,
, several lines of evidence have converged to strengthen the original observation that the effects of interference with da transmission interact powerfully with the work requirements of an instrumental task .
one of the ways of controlling work requirements in an operant schedule is to vary the ratio requirement ( i.e. , the number of times the animal must press the lever to receive a unit of reinforcement ) .
the effects of the da antagonist haloperidol on food - reinforced behavior were shown to be dependent upon the particular ratio schedule that was used [ i.e. , fr1 vs. progressive ratio ; caul and brindle ( 2001 ) ] .
accumbens da depletions also produce effects that interact powerfully with the ratio requirement of the schedule employed .
2004 ) found that accumbens da depletions that substantially impaired fr5 lever pressing had no significant effect on fr1 performance .
aberman and salamone ( 1999 ) systematically studied a wide range of ratio schedules ( fr1 , 4 , 16 , and 64 ) to assess the effects of accumbens da depletions . while fr1 performance was not affected by da depletion , and fr4 responding was only transiently and mildly suppressed , the schedules with large ratio requirements ( i.e. , fr16 and fr64 ) were severely impaired ( figure 1a ) .
in fact , da depleted animals responding on the fr64 schedule showed significantly fewer responses than those responding on the fr16 schedule ( aberman and salamone , 1999 ) .
this pattern indicates that accumbens da depletions exacerbate an effect known as ratio strain . in untreated animals ,
the overall relation between ratio size and response output is inverted - u shaped . up to a point ,
as ratio requirements get larger , animals adjust to this challenge by increasing response output .
however , if the ratio requirement is high enough ( i.e. , if the cost is too high ) , the animal reaches the point at which additional responses being required actually tend to suppress responding . for normal rats , responding at levels of fr64 , fr100 or higher ,
even if there is only one 45 mg food pellet being delivered , does not seem to be problematic .
a completely different function is shown by rats with accumbens da depletions , which are much more sensitive to the size of the ratio requirement . in behavioral economic terms
, this pattern can be described as reflecting an increase in the elasticity of demand for food reinforcement ( salamone et al . , 1997 ; aberman and salamone , 1999 ; see figure 1b ) .
the term elasticity is widely used in economics , but price elasticity of demand refers to the sensitivity of consumption to changes in price ( vuchinich and heather , 2003 ) .
thus , if the ratio requirement is analogous to the price of the commodity ( in this case , reinforcement pellets ) , it appears that rats with accumbens da depletions are more sensitive than control animals to the price of the food reinforcers .
of course , rats do not use currency to purchase operant pellets ; rather , it has been argued that an operant procedure is more of a barter system , in which the rat trades its work ( or reductions in leisure ) for a commodity ( rachlin , 2003 ) .
thus , another way of describing this effect of impaired da transmission is to say that rats with accumbens da depletions are more sensitive than control animals to work - related response costs , or that they are less likely to trade high levels of work for food . in another study ( salamone et al . , 2001 ) , the increased effects of accumbens da depletions with increasing ratio requirements were observed when rats were tested across a broader range of ratio schedules as high as fr300 , even when the overall relation between lever pressing and food delivered per lever press was kept constant ( i.e. , fr50 , one pellet every 50 responses ; fr100 , two pellets every 100 responses ; fr200 , four pellets every 200 responses ; fr300 , six pellets every 300 responses ; salamone et al .
thus , both the magnitude and the organization of the ratio requirement appear to be critical determinants of the sensitivity of an operant schedule to the effects of accumbens da depletions .
this figure shows the effect of ratio requirement on the number of lever presses emitted and operant pellets consumed in rats with accumbens da depletions compared to rats in the vehicle control group .
figure ( a ) is re - drawn based upon aberman and salamone ( 1999 ) ; these data are depicted in terms of number of responses , as in the original article . for ( b ) , the data are represented as number of operant pellets consumed . each data point shown is the mean value from each group at each ratio level .
although comparable levels of consumption in da depleted and control groups were seen with the fr1 schedule , da - depleted rats showed markedly reduced consumption relative to the control group at higher ratio levels . in order to be sure that these results reflected the influence of ratio size , as opposed to other variables such as time , additional studies examined the effects of accumbens da depletions on tandem schedules , in which a ratio requirement was attached to an interval requirement . in a conventional variable interval ( vi ) schedule ,
a time interval must elapse before the first response is reinforced , and the particular time interval varies around an average value .
a tandem vi / fr schedule has an additional ratio requirement attached to the interval .
for example , with a tandem vi 30 s / fr5 schedule , the animal is reinforced for the fifth response after the interval elapses , rather than the first . in this way
, one can vary the ratio requirement of a schedule while keeping the programmed time intervals the same .
research employing tandem vi / fr schedules with varying combinations ( e.g. , vi 30 s / fr5 , vi 60 s / fr10 , vi 120 s / fr10 ) has yielded a consistent pattern ; accumbens da depletions do not impair overall response output in rats responding on the conventional vi schedules ( i.e. , those requiring only one response after the interval ) , but do substantially reduce responding on the corresponding vi schedule with the higher ratio requirement attached ( correa et al .
these results are consistent with research showing that accumbens da antagonism did not impair performance on a progressive interval task ( wakabayashi et al . , 2004 ) , and suggest that interval requirements per se do not pose a severe constraint to rats with compromised da transmission in nucleus accumbens .
this serves to underscore the critical importance of ratio requirements as providing a work - related challenge to rats with accumbens da depletions or antagonism . in summarizing these results ,
salamone and correa ( 2002 ) stated that nucleus accumbens da depletions appear to have two major effects : ( 1 ) they reduce the response - enhancing effects that moderate - size ratio requirements have on operant responding ( i.e. , the ascending limb of the function relating ratio requirement to response output ) , and ( 2 ) they enhance the response - suppressing effects that very large ratios have on operant responding ( i.e. , the descending limb of the function , enhancing ratio strain ) . furthermore , finer grained analyses of detailed patterns of responding reveal more insights into the behavioral manifestations of accumbens da depletions . accumbens da
depletions produce a slight reduction in the local rate of responding , as indicated by the distribution of inter - response times ( salamone et al .
the latter may indicate a fragmentation in the pattern of responding ( mingote et al .
, 2005 ) , a reduction in the ability to sustain uninterrupted response output , or a lack of engagement in the task ( nicola , 2007 ) .
recently , computational approaches have been used to analyze these effects of accumbens da depletions on response rate ( e.g. , niv et al .
this relation between response output and da function has been interpreted to mean that da release in nucleus accumbens could provide a window of opportunistic drive during which the threshold cost expenditure to obtain the reward is decreased ( phillips et al . , 2007 ) .
in discussing the effects of dopaminergic manipulations on ratio performance , it is useful to mention the term reinforcement efficacy , which is sometimes used to describe the effects of drug manipulations on progressive ratio performance .
with progressive ratio schedules , the ratio requirement increases as successive ratios are completed , and the break point is said to occur at the point at which the animal essentially ceases to respond .
one can operationally define reinforcement efficacy in terms of the break point in a progressive ratio schedule ( and also by measuring ratio strain in rats responding across different fr schedules ) .
the determination of reinforcement efficacy can be a very useful tool for characterizing some of the fundamental reinforcing actions of drugs that are self - administered , and for comparing self - administration behavior across different substances or classes of substances ( e.g. , richardson and roberts , 1996 ; marinelli et al .
reinforcement efficacy is essentially being employed as an empirical descriptor of a particular behavioral outcome .
nevertheless , given the terminological problems mentioned above , it is worth emphasizing that the term reinforcement efficacy should not be used simply as a replacement for reward , nor should progressive ratio breakpoints be viewed as necessarily providing some direct and unambiguous measure related to the subjective pleasure produced by the stimulus ( salamone , 2006 ) .
changes in progressive ratio break points can reflect more than just changes in the appetitive motivational properties of a reinforcing stimulus ( richardson and roberts , 1996 ; hamill et al . , 1999 ) .
for example , changing the kinetic requirements of the instrumental response ( e.g. , increasing the height of the lever ) was shown to decrease progressive ratio break points ( skjoldager et al .
although some researchers have maintained that the break point provides a direct measure of the appetitive motivational characteristics of a stimulus , it is , as explicitly stated in a classic review by stewart ( 1974 ) , more directly a measure of how much work the organism will do in order to obtain that stimulus .
progressive ratio break points and measures of ratio strain are essentially outcomes that result from effort - related decision making processes .
the animal is making a cost / benefit choice about whether or not to continue to respond , based partly on factors related to the reinforce itself , but also upon the work - related response costs and time constraints imposed by the ratio schedule . for these reasons , interpretations of the actions of drugs or lesions on progressive ratio break points
should be done with caution , as should be the case for any individual task .
a drug that alters the break point could do so for many different reasons ; it may be affecting functions related to the processing of reward value , or alternatively it could be affecting exertion of effort , or decision making processes .
the ability to exert effort , sustain work , overcome obstacles , and attain access to motivationally relevant stimuli is necessary for survival .
but it is only part of the story . in a complex environment , which affords many opportunities for obtaining significant stimuli , and multiple paths for accessing them
the variables that need to be evaluated to make these decisions are complex and multidimensional , but among the most important are those involving cost / benefit assessments based upon effort and reinforcement value ( salamone and correa , 2002 ; salamone et al . , 2003 , 2005 , 2007 ; van den bos et al . , 2006 ; walton et al . , 2006 ) .
considerable evidence indicates that nucleus accumbens da , along with other transmitters and structures , participates in the overall circuitry that regulates effort - based choice behavior ( salamone et al . , 2003 , 2005 , 2007 ; floresco et al . , 2008a ; hauber and sommer , 2009 ) .
one of the procedures that has been used to assess the contribution of accumbens da to response allocation and effort - related choice behavior is a task that offers rats the option of either lever pressing to obtain a relatively preferred food ( e.g. , bioserve pellets ; usually obtained on a fr5 schedule ) , or approaching and consuming a less preferred food ( lab chow ) that is concurrently available in the chamber .
well trained rats under baseline conditions typically get most of their food by lever pressing , and consume only small quantities of chow ( salamone et al . , 1991 ) .
low - to - moderate doses of da antagonists , which block either d1 or d2 family receptor subtypes , produce a substantial alteration of response allocation in rats performing on this task .
the da antagonists cis - flupenthixol , haloperidol , raclopride , eticlopride , sch 23390 , skf83566 , and ecopipam all decreased lever pressing for food but substantially increased intake of the concurrently available chow ( salamone et al . , 1991 , 1996 , 2002 ; cousins et al . , 1994 ; sink et al . , 2008 ; worden et al . ,
the use of this task for assessing effort - related choice behavior has been validated in many ways .
for example , the low dose of haloperidol that produced the shift from lever pressing to chow intake ( 0.1 mg / kg ) did not affect total food intake or alter preference between these two specific foods in free - feeding choice tests ( salamone et al . , 1991 ) . although da antagonists have been shown to reduce fr5 lever pressing and increase chow intake , appetite suppressants from different classes , including amphetamine ( cousins et al . ,
1994 ) , fenfluramine ( salamone et al . , 2002 ) , and cannabinoid cb1 antagonists ( sink et al . , 2008 ) , failed to increase chow intake at doses that suppressed lever pressing .
similarly , pre - feeding to reduce food motivation was shown to suppress both lever pressing and chow intake ( salamone et al . , 1991 ) .
furthermore , attachment of higher ratio requirements ( up to fr20 ) caused animals that were not drug treated to shift from lever pressing to chow intake ( salamone et al .
together with other results , these findings demonstrate that interference with da transmission does not simply reduce appetite , but does act to alter response allocation between alternative sources of food that can be obtained through different instrumental responses . the shift from lever pressing to chow intake in rats performing on this task
is associated with da depletions in nucleus accumbens , but not the neostriatum . although it has been suggested that caudate / putamen da may have some types of motivational functions related to feeding ( palmiter , 2007 ) , da depletions in anteroventromedial neostriatum , which is dorsal to nucleus accumbens , had no behavioral effect , while ventrolateral neostriatal da depletions produced severe motor impairments that merely decreased both lever pressing and feeding ( cousins et al . , 1993 ) .
in contrast , decreases in lever pressing and increases in chow intake occur as a result of accumbens da depletions , as well as intra - accumbens injections of d1 or d2 antagonists ( salamone et al .
, 1991 ; cousins et al . , 1993 ; cousins and salamone , 1994 ; sokolowski and salamone , 1998 ; koch et al . , 2000
the shift from lever pressing to chow intake on this task has been shown to occur in rats if d1 or d2 family antagonist are injected into the medial core , lateral core , or dorsal shell subregions of the accumbens ( salamone et al . , 1991 ; nowend et al . ,
thus , although lever pressing is decreased by accumbens da antagonism or depletions , the rats show a compensatory reallocation of behavior and select a new path to an alternative food source .
consistent with these effects observed in rats that have impaired da transmission , da transporter knockdown mice , which have enhanced da transmission , show increased selection of lever pressing relative to chow intake when tested with this task ( cagniard et al . , 2006 ) .
( 1994 ) also developed a t - maze procedure in order to assess the effects of da antagonists and accumbens da depletions on effort - related decision making . with this procedure
, the two choice arms of the maze can have different reinforcement densities ( e.g. , four vs. two food pellets , or four vs. zero food pellets ) , and under some conditions a 44-cm barrier can be placed in the arm with the higher density of food reinforcement to present an effort - related challenge to the rat . when no barrier is placed in the arm with the high reinforcement density , rats mostly choose that arm , and neither haloperidol nor accumbens da depletion alters their response choice ( salamone , 1994 ) . when the arm with the barrier contained four pellets , but the other arm contained no pellets , rats with accumbens da depletions were very slow , but still managed to choose the high density arm , climb the barrier , and consume the pellets ( cousins et al . , 1996 ) . yet
accumbens da depletions dramatically altered choice behavior when the high density arm ( four pellets ) had the barrier in position , and the arm without the barrier contained an alternative food source ( two pellets ) . in this case , da depletions or antagonism decreased choice for the high density arm , and increased choice for the low density arm ( salamone , 1994 ; cousins et al . , 1996 ; denk et al . , 2005 ; mott et al . ,
, the t - maze task for measuring effort - based choice behavior also has undergone considerable behavioral validation and evaluation ( salamone , 1994 ; cousins et al . , 1996 ;
for example , in a recent t - maze choice study with mice , it was confirmed that haloperidol reduced choice of the arm with the barrier , and it also was demonstrated that haloperidol had no effect on choice when both arms had a barrier in place ( correa et al . , 2009 ) .
thus , dopaminergic manipulations did not alter the preference for the high density of food reward over the low density , and did not affect discrimination or memory processes related to arm preference . over the last several years
, variants of this task have been used by several laboratories to characterize the effects of brain lesions or drug manipulations ( salamone , 1994 ; walton et al . , 2003 ; denk et al . , 2005 ; schweimer and hauber , 2005 ; van den bos et al . , 2006 ; bardgett et al . , 2009 ;
the results of the t - maze studies in rodents , together with the findings from the operant concurrent choice studies reviewed above , indicate that low doses of da antagonists and accumbens da depletions cause animals to reallocate their instrumental response selection based upon the response requirements of the task , and select lower effort alternatives for obtaining rewards ( see reviews by salamone et al .
( 2008b ) investigated the effects of dopaminergic and glutamatergic drugs on both effort and delay discounting .
the da antagonist haloperidol altered effort discounting even when the effects of time delay were controlled for ( floresco et al .
a t - maze effort discounting task was recently developed ( bardgett et al . , 2009 ) , in which the amount of food in the high density arm of the maze was diminished each trial on which the rats selected that arm ( i.e. , an adjusting - amount discounting variant of the t - maze procedures , which allows for the determination an indifference point for each rat ) .
administration of both the d1 family antagonist sch23390 and the d2 family antagonist haloperidol altered effort discounting , making it more likely that rats would choose the arm with the smaller reward .
increasing da transmission by administration of amphetamine blocked the effects of sch23390 and haloperidol , and also biased rats toward choosing the high reward / high cost arm , which is consistent with operant choice studies using da transporter knockdown mice ( cagniard et al . , 2006 ) . together with other results , the findings reported by bardgett et al .
( 2008b ) support the suggestion that , across a variety of conditions , da transmission exerts a bidirectional influence over effort - related decision making .
as reviewed above , considerable research has demonstrated that da antagonists and accumbens da depletions affect behavioral activation , instrumental response output , response allocation , and effort - related processes ( salamone et al . , 1991 , 2007 ; salamone and correa , 2002 ; phillips et al . , 2007 ; robbins and everitt , 2007 ;
clearly , da does not participate in effort - related processes in isolation , and for that reason it is important to review how other brain areas and neurotransmitters interact with dopaminergic mechanisms . within the last few years , considerable emphasis has been placed upon interactions between da and adenosine .
caffeine and other methylxanthines , which act as minor stimulants , are non - selective adenosine antagonists ( ferr et al . , 2008 ) .
recently , there has been a rapid growth of research on adenosine receptor neurochemistry and pharmacology , particularly concerning the a2a subtype of adenosine receptor .
da - rich striatal areas , including both the caudate / putamen ( neostriatum ) and the nucleus accumbens , have a very high degree of adenosine a2a receptor expression ( schiffmann et al . , 1991 ; demet and chicz - demet , 2002 ;
there is considerable evidence of a functional interaction between striatal da d2 and adenosine a2a receptors ( fink et al .
, 1992 ; ferr , 1997 ; hillion et al . , 2002 ; fuxe et al . ,
this interaction frequently has been studied in regard to neostriatal motor functions that are related to parkinsonism ( ferr et al .
, 1997 , 2001 ; hauber and munkel , 1997 ; svenningsson et al . , 1999 ;
, 2001 ; wardas et al . , 2001 ; morelli and pinna , 2002 ; correa et al . , 2004 ; jenner , 2005 ; pinna et al . , 2005 ; ishiwari et al . , 2007 ;
several reports also have characterized aspects of adenosine a2a receptor function related to cognitive processes ( takahashi et al . , 2008 ) ,
anxiety ( correa and font , 2008 ) , and motivation ( salamone et al .
adenosine a2a receptors also are involved in aspects of behavioral activation and effort - related processes ( farrar et al .
, 2007 ; font et al . , 2008 ; mingote et al . , 2008 ; mott et al . ,
injections of the adenosine a2a agonist cgs 21680 directly into the accumbens can produce effects that resemble those of accumbens da depletions or antagonism .
intra - accumbens injections of cgs 21680 were shown to reduce locomotor activity ( barraco et al . , 1993 ) .
local infusions of cgs 21680 into the accumbens reduced responding on a vi 60 s schedule with a fr10 requirement attached , but did not impair performance on a conventional vi 60 s schedule ( mingote et al . , 2008 ) ; this pattern is similar to that previously shown with accumbens da depletions ( mingote et al . , 2005 ) .
in rats responding on the operant fr5/chow feeding concurrent choice procedure , injections of cgs 21680 into the accumbens decreased lever pressing and increased chow intake ( font et al . , 2008 ) , a pattern of effects similar to that produced by accumbens da depletions and antagonism .
consistent with the observation that an adenosine a2a agonist could produce actions similar to those resulting from da depletion or blockade , it also has been reported the locomotor suppression induced by the da antagonist haloperidol was reduced by injections of the adenosine a2a antagonist msx-3 into nucleus accumbens core , but not into the shell or the ventrolateral neostriatum ( ishiwari et al . , 2007 ) .
furthermore , it has been demonstrated that adenosine a2a receptor antagonists can reverse the effects of da d2 antagonists on both the operant concurrent choice task ( farrar et al .
2009 ) and the t - maze choice procedure ( correa et al . , 2009 ; mott et al . , 2009 ) .
recently , studies with intracranial injections revealed that systemic or intra - accumbens injections of the adenosine a2a antagonist msx-3 were able to block the effects of intra - accumbens injections of the d2 antagonist eticlopride in rats responding on the operant concurrent choice task ( farrar , 2009 , unpublished doctoral dissertation , university of connecticut ) .
these studies afford an interesting opportunity to assess the overall interaction between da and adenosine receptor subtypes .
adenosine a2a receptor antagonists msx-3 and kw 6002 reliably attenuate the effects of d2 antagonists such as haloperidol and eticlopride in rats responding on the operant concurrent choice procedure ( farrar et al . , 2007 ;
in contrast , msx-3 was relatively ineffective at reducing the effects of the d1 antagonist ecopipam ( sch 39166 ; worden et al . , 2009 ) on this task .
although the non - selective adenosine antagonist caffeine was able to partially reverse the effects of haloperidol on the concurrent choice task , dpcpx , which is highly selective for the adenosine a1 receptor subtype , was ineffective ( salamone et al . ,
similar results were obtained with rats and mice responding on the t - maze barrier choice task . although msx-3 was able to reverse the effect of haloperidol on selection of the arm with the barrier , the a1 antagonists dpcpx and cpt were not ( correa et al . , 2009 ; mott et al . , 2009 ) .
the results described above demonstrate that there is a relatively selective interaction between da d2 and adenosine a2a receptor subtypes ( table 1 ) . based upon anatomical studies , it appears that this is likely to be due to the pattern of cellular localization of adenosine a1 and a2a receptors in striatal areas , including the nucleus accumbens ( ferr , 1997 ) .
adenosine a2a receptors are typically co - localized on striatal and accumbens enkephalin - positive medium spiny neurons with da d2 family receptors , and these receptors converge onto the same signal transduction pathways and show the capacity for forming heteromeric complexes ( fink et al .
, 1992 ; ferr , 1997 ; svenningsson et al . , 1999 ; hillion et al . ,
thus , adenosine a2a receptor antagonists appear to be so effective in reversing the effort - related actions of d2 antagonists because of direct interactions between da d2 and adenosine a2a receptors located on the same neurons ( figure 2 ) . on the other hand ,
da d1 receptors are more likely to be co - localized with adenosine a1 receptors ( ferr , 1997 ) , which could help to explain why it is more difficult for adenosine a1 receptor antagonists to reverse the effects of d2 receptor blockade .
interestingly , despite the fact that d1 and a1 receptors tend to be co - localized on the same neurons , the a1 antagonists dpcpx and cpt were unable to reverse the effects of the d1 antagonist ecopipam in rats responding on the concurrent choice operant procedure ( nunes et al . , 2009 ) .
this suggests that a2a antagonists exert an overall greater effect than a1 antagonists on effort - related functions of nucleus accumbens .
there was a mild increase in lever pressing in ecopipam - treated rats that received the a2a antagonist msx-3 , but no reversal of the chow intake effect of the d1 antagonist .
data from pardo ( 2009 ) , unpublished masters thesis , university of jaume i. anatomical diagram depicting the pattern of da and adenosine receptor localization in nucleus accumbens .
mgp , medial globus pallidus ; epn , entopeduncular nucleus ; s. nigra , substantia nigra ; vta , ventral tegmental area .
research on the brain mechanisms involved in effort - related processes may lead to new ways of thinking about behavioral analysis and theory in behavioral economics .
one of the contributions that behavioral neuroscience can make to behavioral theory is to use manipulations ( e.g. , drugs , lesions ) that dissociate complex behavioral processes into component parts ( salamone et al . , 2007 ) .
in this regard , it is useful to consider that a given parameter that is generated from curve - fitting analyses , when viewed in terms of its biological characteristics , has many factors that contribute to it .
a good example of this is the ed50 , which is used in pharmacology to provide a measure of the potency of a drug based upon dose - response analysis . empirically , the ed50 is the dose that produces an effect that is 50% of the maximal effect . although the ed50 is expressed as one number , that simplicity is deceptive because many biochemical factors contribute to it , including the affinity of a drug for a receptor , duration of action , drug metabolism , and penetration into the target tissue .
a useful example of this principle from the behavioral neuroscience literature is the progressive ratio break point ; as discussed above , this measure also has many factors that can contribute to it .
another case in which this point is important to consider is threshold measures used in intracranial self - stimulation studies .
such measures often are viewed as providing rate - free indices of reinforcement value , nevertheless , they are influenced by lever pressing ratio requirements as well as the electrical current level ( fouriezos et al . ,
some research related to behavioral economics , reinforcer value , and the functions of da systems has used response - reinforcement matching methods ( e.g. , heyman and monaghan , 1987 ; aparicio , 2007 ) .
matching equations have been employed to describe the results of studies with both conventional and concurrent vi schedules , and one of the parameters ( re ) can be used to represent reinforcement value ( e.g. , herrnstein , 1974 ; see equation below for single - lever conventional vi schedules , in which b represents response rate , r represents reinforcement density , k is the constant for maximal responding , and re represents the reinforcement level that generates 50% of maximum responding ) .
however , used in this way , re does not selectively represent only the reinforcement value of food per se ; actually , it reflects the relative value of lever pressing for and consuming the food reinforcer compared to the reinforcing value of all other stimuli and responses available ( salamone et al . , 1997 ) .
several factors can contribute to this composite measure , which is one of the reasons why other matching equations have been developed that account for deviations from matching by allowing for estimates of reinforcer sensitivity , as well as response preference or bias ( baum , 1974 ; williams , 1988 ; aparicio , 2001 ) . clearly , a drug or lesion manipulation could yield apparent effects on reinforcement value that actually reflect changes in response bias ( salamone , 1987 ; salamone et al .
, 1997 ) . for these reasons , it may be useful to think more deeply about how terms such as value are used in neuroeconomics research .
the aggregate reinforcement value of an instrumental activity ( e.g. , lever pressing for and consuming food ) should perhaps be viewed as a composite measure that includes both the reinforcing value of the reinforcer itself , plus any net value or costs associated with the instrumental response that is required to obtain the reinforcer .
viewed in this way , the effects of dopaminergic manipulations on effort - related choice behavior could be described in terms of actions upon the response costs associated with the particular reinforcer , rather than the reinforcing value of the food stimulus itself .
although the effects of haloperidol on bias may be minimal when two levers that are relatively similar are used ( e.g. , aparicio , 2007 ) , they may be much larger when very different responses are compared ( e.g. , lever pressing vs. sniffing ; lever pressing vs. unrestricted access to food ; barrier climbing vs. locomotion ) .
future research will determine if measures of bias based upon the matching equations , or some other type of mathematical formulation , would be the best way to capture these drug effects quantitatively .
in summary , da and adenosine in the nucleus accumbens interact to regulate effort - related functions .
additional research has shown that a number of components of the cortico - striato - pallidal loop system also are involved ( walton et al . , 2006 ; floresco and ghods - sharifi , 2007 ; farrar et al . , 2008 ; mingote et al . , 2008 ; hauber and sommer , 2009 ) .
disconnection studies have revealed that serial connections between basolateral amygdala , anterior cingulate cortex , nucleus accumbens , and ventral pallidum are involved in the exertion of effort and effort - related choice behavior ( floresco and ghods - sharifi , 2007 ; farrar et al . , 2008 ; mingote et al . , 2008 ; hauber and sommer , 2009 ) .
within the last few years , there has been considerable progress in characterizing the functional anatomy underlying this important aspect of motivation and decision making .
several transmitters across multiple brain regions are involved in effort - related functions , and researchers are only beginning to piece together the complex puzzle of all the potential brain systems that are involved . presently , the specific way in which each structure contributes to the overall function of the system is unclear .
it is uncertain which brain areas are involved in the exertion of effort , or the perception of effort , vs. the actual decision making process itself .
for example , it is possible that nucleus accumbens is involved in the actual decision making processes , but it also is possible that it is mainly involved in regulating energy output , or setting effort - related constraints or feedback that in turn influences decisions made at other levels in the system . if the latter is true , then it is possible that the decision making effects of drug or lesion manipulations of nucleus accumbens are an outcome reflecting the constraints that are set after compromised da function in accumbens , rather than a direct effect upon decision making processes per se .
future research will be necessary to tease apart these distinct aspects of effort - related function .
in addition to providing insights into aspects of animal behavior and natural motivation , research on effort - related processes also has clinical implications . within the last few years
, there has been a greater emphasis upon effort - related functions involved in drug self - administration ( e.g. , vezina et al . ,
addicts will go to great lengths to obtain their preferred drug , overcoming numerous obstacles and constraints , both behavioral and economic .
furthermore , addiction is characterized not only by a re - organization of the preference structure of the person , but also by a dramatic change in the allocation of behavioral resources toward the addictive substance ; there is a heightened emphasis upon drug seeking and drug taking , typically at the expense of other motivational activities .
as well as being related to aspects of drug taking and addiction , research on behavioral activation and effort has implications for understanding the neural basis of psychiatric symptoms such as psychomotor slowing , anergia , fatigue and apathy , which are seen in depression as well as other psychiatric or neurological conditions ( salamone et al . , 2006 , 2007 ) . these motivational symptoms , which can have devastating behavioral manifestations ( stahl , 2002 ;
. , 2005 ) , represent impairments in aspects of behavioral activation and effort that can lead to problems in the workplace , as well as limitations in terms of life function , interaction with the environment , and responsiveness to treatment .
there is considerable overlap between the neural circuitry involved in effort - related functions in animals and the brain systems that have been implicated in psychomotor slowing and anergia in depression ( salamone et al . , 2006 ) .
thus , research on effort - related behavioral processes , and their neural regulation , could have substantial impact on clinical research related to addiction , depression , and other disorders .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . | there are numerous problems with the hypothesis that brain dopamine ( da ) systems , particularly in the nucleus accumbens , directly mediate the rewarding or primary motivational characteristics of natural stimuli such as food .
research and theory related to the functions of mesolimbic da are undergoing a substantial conceptual restructuring , with the traditional emphasis on hedonia and primary reward yielding to other concepts and lines of inquiry .
the present review is focused upon the involvement of nucleus accumbens da in behavioral activation and effort - related processes .
viewed from the framework of behavioral economics , the effects of accumbens da depletions and antagonism on food - reinforced behavior are highly dependent upon the work requirements of the instrumental task , and da depleted rats are more sensitive to increases in response costs ( i.e. , ratio requirements ) .
moreover , interference with accumbens da transmission exerts a powerful influence over effort - related choice behavior .
rats with accumbens da depletions or antagonism reallocate their instrumental behavior away from food - reinforced tasks that have high response requirements , and instead these rats select a less - effortful type of food - seeking behavior .
nucleus accumbens da and adenosine interact in the regulation of effort - related functions , and other brain structures ( anterior cingulate cortex , amygdala , ventral pallidum ) also are involved .
studies of the brain systems regulating effort - based processes may have implications for understanding drug abuse , as well as energy - related disorders such as psychomotor slowing , fatigue or anergia in depression and other neurological disorders . |
the laparoscopic extirpation of normal - sized spleens has become more common in the last few years .
since the first attempts less than a decade ago , several reports have described the challenges associated with this type of surgery .
contraindications to a laparoscopic approach include severe portal hypertension , uncorrectable coagulopathy , severe ascites , and most traumatic injuries to the spleen .
spleen size is expressed in terms of the maximum interpole length and is generally classified into 3 categories : normal spleen size ( < 11 cm ) ; moderate splenomegaly ( 11 cm to 20 cm ) ; and severe or massive splenomegaly ( > 20 cm ) .
extremely large spleens present special technical problems that test the current limits of laparoscopic surgery .
however , as laparoscopic techniques , surgical skills , and instrumentation have improved , so have the safety and efficacy of this procedure even in the presence of splenomegaly .
since june 1997 , 82 laparoscopic splenectomies have been performed for hematologic pathologies including malignancies .
starting in september 2003 , we attempted a new approach in cases of severe splenomegaly .
the purpose of the study was to examine the safety and efficacy of minimal - access splenectomy ( mas ) in the setting of massive splenomegaly .
between september 2002 and october 2003 , 9 patients were identified as having massive splenomegaly potentially treatable through a minimal access approach .
six male and 3 female patients underwent mas for the following disorders : non - hodgkin 's lymphoma in 5 cases and idiopathic myelofibrosis in 4 .
mean age and bmi ( body mass index ) of the patients were respectively 58.8 years ( range , 41 to 72 ) and 24.3 ( range , 21.4 to 26 ) .
patient and disease characteristics preoperative ultrasonography performed to determine the size of the spleen showed a median interpole length of 27.9 cm ( range , 23 to 32 ) .
the indications for splenectomy included persistent thrombocytopenia and anemia due to hypersplenism , as well as continuous steroid medication .
the subjective symptoms of all the patients included abdominal distension and fullness , difficulty in lying on the left side and left - sided upper quadrant pain .
preoperatively , the patients were categorized as american association of anesthesiology ( asa ) class ii ( 5 patients ) and class iii ( 4 patients ) .
only 1 patient had a normal platelet count ; the others had thrombocytopenia , with an average count of 65 400/mm ( range , 8000 to 140 000/mm ) .
the patients were placed in the supine decubitus position with a sandbag under the left flank .
this step afforded access to the gastrosplenic ligament and the opening of the lesser sac in its lateral portion .
next , the short gastric vessels were divided by using the ultrasonic dissector or eventually over clips ( figure 1 ) . the uppermost vessels of the gastrosplenic ligament are very short and enlarged in splenomegaly , and they are located in the immediate vicinity of the diaphragm . access to these vessels required combined dislocation of the stomach to the right and gentle retraction of the upper splenic pole to the left .
then the dissection reached the hilum and the vessels could be isolated from the pancreas and the surrounding tissues by combining a careful digital dissection with the use of the ultrasonic dissector .
if a distributed anatomy was present , the splenic branches were usually dissected and divided between silk ligatures or clipped . in case of magistral anatomy , after providing that the tail of the pancreas is identified and dissected away , we performed separate silk ligation of the splenic vessels with a delayed ligature of the vein , to achieve a volumetric reduction of the spleen .
then the splenic flexure was partially mobilized by incising the splenocolic ligament , the lower part of the phrenicocolic ligament , and the sustentaculum lienis .
finally , the phrenicocolic ligament was incised all the way to the left crus of the diaphragm by the ultrasonic dissector .
this bag was introduced into the abdominal cavity , and the spleen was manually slipped inside to prevent splenosis during the subsequent manipulations .
grasping forceps were used to hold the edges of the bag and to effect partial closure . at the end of the procedure ,
a nasogastric tube and an abdominal drain in the left hypochondrium were left in place .
between september 2002 and october 2003 , 9 patients were identified as having massive splenomegaly potentially treatable through a minimal access approach .
six male and 3 female patients underwent mas for the following disorders : non - hodgkin 's lymphoma in 5 cases and idiopathic myelofibrosis in 4 .
mean age and bmi ( body mass index ) of the patients were respectively 58.8 years ( range , 41 to 72 ) and 24.3 ( range , 21.4 to 26 ) .
patient and disease characteristics preoperative ultrasonography performed to determine the size of the spleen showed a median interpole length of 27.9 cm ( range , 23 to 32 ) .
the indications for splenectomy included persistent thrombocytopenia and anemia due to hypersplenism , as well as continuous steroid medication .
the subjective symptoms of all the patients included abdominal distension and fullness , difficulty in lying on the left side and left - sided upper quadrant pain .
preoperatively , the patients were categorized as american association of anesthesiology ( asa ) class ii ( 5 patients ) and class iii ( 4 patients ) .
only 1 patient had a normal platelet count ; the others had thrombocytopenia , with an average count of 65 400/mm ( range , 8000 to 140 000/mm ) .
the patients were placed in the supine decubitus position with a sandbag under the left flank .
this step afforded access to the gastrosplenic ligament and the opening of the lesser sac in its lateral portion .
next , the short gastric vessels were divided by using the ultrasonic dissector or eventually over clips ( figure 1 ) .
the uppermost vessels of the gastrosplenic ligament are very short and enlarged in splenomegaly , and they are located in the immediate vicinity of the diaphragm .
access to these vessels required combined dislocation of the stomach to the right and gentle retraction of the upper splenic pole to the left .
then the dissection reached the hilum and the vessels could be isolated from the pancreas and the surrounding tissues by combining a careful digital dissection with the use of the ultrasonic dissector .
if a distributed anatomy was present , the splenic branches were usually dissected and divided between silk ligatures or clipped . in case of magistral anatomy , after providing that the tail of the pancreas is identified and dissected away , we performed separate silk ligation of the splenic vessels with a delayed ligature of the vein , to achieve a volumetric reduction of the spleen .
then the splenic flexure was partially mobilized by incising the splenocolic ligament , the lower part of the phrenicocolic ligament , and the sustentaculum lienis .
finally , the phrenicocolic ligament was incised all the way to the left crus of the diaphragm by the ultrasonic dissector .
this bag was introduced into the abdominal cavity , and the spleen was manually slipped inside to prevent splenosis during the subsequent manipulations .
grasping forceps were used to hold the edges of the bag and to effect partial closure . at the end of the procedure ,
a nasogastric tube and an abdominal drain in the left hypochondrium were left in place .
the operative time ranged from 75 minutes to 165 minutes ( mean , 124 ) .
the average weight of the spleen was 2500 g ( range , 1700 to 3200 ) .
the average intraoperative blood loss was 572 ml ( range , 150 to 1000 ) .
five patients received an average of 3u of packed red cells , and 4 of the splenectomies were performed without such a transfusion . in all patients ,
oral intake was assured from the second to the sixth postoperative day ( mean , 3 ) .
postoperative complications occurred in 2 cases : one patient had pleural effusion and another patient developed pneumonia , which were treated medically .
one perioperative death occurred 15 days after the operation in a patient with idiopathic myelofibrosis as a consequence of a secondary blast crisis .
perioperative features of patients who underwent minimal - access splenectomy ( n=9 ) mean postoperative hospital stay was 9.1 days ( range , 6 to 15 ) .
the average preoperative platelet count was 108 911/mm ( range , 8000 to 457 000 ) .
the average postoperative platelet count was 479 111/mm ( range , 6,000 to 1 262 000 ) .
massive splenectomy virtually always relates to hematological malignancies . in these patients , the local discomfort from massive splenomegaly and
open splenectomy has been associated with substantial morbidity and mortality , and the rates may be higher in patients with splenomegaly , patients with myeloproliferative disorders , and the elderly .
we consider all patients with massive splenomegaly as potential candidates for laparoscopic splenectomy , and the laparoscopic approach has been used since june 1997 .
starting in september 2002 , in cases of massive splenomegaly , we have adopted the mas technique for 2 reasons .
first , in the laparoscopic approach , a service mini - laparotomy is always performed when pathologic examination of the surgical specimen is required to document the hematological disease .
second , laparoscopic - assisted removal of the entire spleen via an accessory mini - laparotomy does not affect the duration of surgery , rate of complications , or length of hospital stay .
its use is advocated in every step of the procedure : from the dissection of the splenocolic to the gastrosplenic ligament , from the dissection of the hilar vessels to the spleno - diaphragmatic ligament .
the use of a clip applier or a vascular stapler was necessary in only a few cases .
separate ligation of the hilar vessels was performed whenever possible . once the lesser sac is opened , the next step is the identification and ligation of the splenic artery .
volumetric reduction of the spleen after this manoeuvre is important , thus enabling the surgeon to perform the next steps of the dissection more easily .
manual handling succeeds in creating a working space that would be difficult to achieve with the simple manipulation of the laparoscopic instruments .
furthermore , hand assistance , by regaining tactile feedback , also increases the safety of the procedure , because it enables the surgeon to rapidly identify vascular structures and , in the case of accidental bleeding , allows immediate hemostatic control by digital compression .
postoperative morbidity was low ; no pancreatitis was observed because dissection in the mas technique tends to identify and dissect away the tail of the pancreas .
a severe blast crisis not directly related to surgery was responsible for the only perioperative death that occurred in a patient affected by idiopathic myelofibrosis .
median length of hospital stay for the rest of the patients was 7.5 days . in regard to massive splenomegaly in malignancies , this value is comparable to that reported in the literature and to that of our historic patients treated with a fully laparoscopic approach .
we believe that experience with laparoscopic splenectomy is necessary to perform mas ; in fact , the laparoscopic skills of the surgeon greatly facilitate dissection with this approach .
mas in patients with severe splenomegaly represents a viable option , because it is feasible and effective .
we found mas to be an attractive alternative to laparoscopy in cases of massive splenomegaly .
it combines the advantages of hand assistance , shorter operative times , and increased safety of the procedure with the classical benefits of minimally invasive surgery . | background : laparoscopic splenectomy of normal - sized spleens or in moderate splenomegaly is performed with increasing frequency . by using a modification of the open laparotomy ,
minimal - access splenectomy is an attractive alternative in severe splenomegaly.methods:between september 2002 and october 2003 , 9 patients ( mean age , 58.8 years ; range , 41 to 72 ) with severe splenomegaly ( mean length , 27.9 cm ; range , 23 to 32 ) underwent minimal - access splenectomy .
indications for splenectomy were non - hodgkin 's lymphoma in 5 cases and idiopathic myelofibrosis in 4.results:minimal-access splenectomy was successfully completed in all patients .
mean operative time was 124 minutes ( range , 75 to 165 ) .
postoperative complications occurred in 2 cases ; one perioperative death occurred in a patient with idiopathic myelofibrosis as a consequence of a secondary blast crisis .
median postoperative hospital stay was 9.1 days ( range , 6 to 15).conclusions : minimal - access splenectomy seems to be a viable alternative to laparoscopic splenectomy in cases of severe splenomegaly .
it combines the advantages of hand assistance like shorter operative times and increased safety of the procedure to the classical benefits of minimally invasive surgery . |
segregation distortion is a phenomenon that the genotypic frequency array of a locus does not follow a typical mendelian ratio .
depending on the population under investigation , mendelian ratio of a locus varies from 1 : 1 for a backcross to 1 : 2 : 1 for an f2 and to 1 : 1 : 1 : 1 for a four - way cross .
, the effects of distorted markers on the result of qtl mapping were not known .
for the reason of precaution , people simply discarded all the distorted markers in qtl mapping .
recently , we found that distorted markers can be safely used for qtl mapping with no detrimental effect on the result of qtl mapping .
this finding can help qtl mappers save tremendous resources by using all available markers , regardless whether they are mendelian or not .
we also found that if distorted markers are handled properly , they can be beneficial to qtl mapping .
the reason behind the distortion is due to one or more segregation distortion loci ( sdl ) .
these loci are subject to gametic selection , zygotic selection , or both and their ( unobservable ) distorted segregation causes the observed markers to deviate from the mendelian ratio .
several investigators [ 411 ] have attempted to map these segregation distortion loci using molecular markers .
agricultural scientists are interested in mapping qtl for economically important traits while evolutionary biologists are interested in mapping sdl that respond to natural selection .
since the theory of segregation distortion has been introduced and discussed in previous studies [ 7 , 8 ] and our own research , this study only presents the em ( expectation - maximization ) implementation of the statistical method .
the variance - covariance matrix of estimated parameters under the em algorithm is also derived and presented in appendix a for interested readers .
we only investigate interval mapping where a model contains a single qtl at a time and the entire genome is scanned through repeated calling of the same program for different locations of the genome . the technical difference between the joint
mapping and qtl mapping occurs only in one place . in the traditional interval mapping of qtl ,
the conditional probabilities of genotypes for a qtl are calculated using flanking marker genotypes with the prior probabilities of qtl genotypes being substituted by the mendelian ratio . for the joint mapping , the genotypic frequencies ( segregation ratios ) are treated as unknown parameters that are subject to estimation .
let m and n be the left and right flanking markers bracketing the qtl ( denoted by g for short ) .
the interval of the genome carrying the three loci is labeled by a segment mgn .
the three genotypes of the qtl are denoted by g1g1 , g1g2 , and g2g2 , respectively .
let r1 and r2 be the recombination fractions for segments mg and gn , respectively .
the joint distribution of the marker genotypes conditional on the qtl genotype can be derived using the markov chain property under the assumption of no interference between consecutive loci in segregation .
let us denote the three ordered genotypes , g1g1 , g1g2 , and g2g2 , by genotypes 1 , 2 , and 3 , respectively .
if individual j takes the th genotype for the qtl , we denote the event by gj = , for all = 1 , 2 , 3 .
the joint probability of the two markers conditional on the genotype of the qtl is
( 1)pr(mj=,nj= gj= ) = pr(mj= gj=)pr(nj= gj= )
for all , , = 1 , 2 , 3 , where pr(mj = gj = ) = 1( , ) and pr(nj =
we use i( , ) to denote the th row and the th column of the following transition matrix :
( 2)i=[(1ri)22ri(1ri)ri2ri(1ri)(1ri)2+ri2ri(1ri)ri22ri(1ri)(1ri)2 ] , i=1,2 .
for example ,
( 3)pr(mj=1,nj=2 gj=3 ) = pr(mj=1 gj=3)pr(nj=2 gj=3 ) = 1(3,1)2(3,2)=2r12r2(1r2 ) .
let = pr(g = ) , for all = 1 , 2 , 3 , be the probability that a randomly sampled individual from the f2 family has a genotype . we use a generic notation p for probability so that p(gj = ) represents pr(gj = ) and p(mj , nj gj = ) stands for pr(mj , nj gj = ) .
the log likelihood function of the flanking marker genotypes in the f2 population is
( 4)l( m)=j=1nln [ =13p(gj=)p(mj , nj gj=)]=j=1nln [ =131(,mj)2(,nj ) ] ,
where = [ 1 2 3 ] is a vector of parameters with constraint =1 = 1 , where m in l( m ) stands for marker information . note that without any prior information , p(gj = ) = , for all j = 1 , , n. under the assumption of mendelian segregation , = where = [ 1 2 3 ] = [ 1/4 1/2 1/4 ] .
because we are dealing with the genotypic frequencies , the segregation distortion is called zygotic distortion
we postulate that deviation of from causes a marker linked to locus g to show distorted segregation .
let yj be the phenotypic value of a quantitative trait measured from individual j. the probability density of yj conditional on the genotype of individual j is normal with mean
( 5)=xj+h
and variance , that is ,
( 6)p(yj gj=)=122exp [ 122(yjxjh)2 ] ,
where h is the th row of matrix h and
( 7)h=[+110111 ] .
this h matrix can be defined in a different scale , for example ,
( 8)h=[+100110 ] ,
which does not affect the significance test .
the advantage of choosing the scale in ( 7 ) is that the expectation of the dominance indicator is zero .
the design matrix xj and the regression coefficients capture non - qtl effects , for example , field location effects , year effects , and so on .
the likelihood function of the phenotypic values in the f2 population is
( 9)l(,,2, y ) = j=1nln [ =13p(gj=)p(yj gj= ) ] = n2ln ( 2)+j=1nln { =13exp [ 122(yj)2 ] } ,
where letter y in l( , , , y ) stands for the phenotype .
this likelihood function is the one used in segregation analysis of quantitative traits because no marker information is required .
let = [ ] be a vector of all parameters in the joint analysis .
the likelihood function is obtained by combining ( 4 ) and ( 9 ) :
( 10)l( m , y)=j=1nln [ =13p(gj=)p(yj gj=)p(mj , nj gj=)]=j=1nln { =13exp [ 122(yj)2]1(,mj)2(,nj ) } n2ln ( 2 ) .
for qtl mapping under segregation distortion
the previous two likelihood functions ( for markers and for phenotypes ) were presented as background information to introduce this joint log likelihood function .
the mle ( maximum likelihood estimate ) of the parameters is solved via an em algorithm .
let us define a delta function as
( 11)(gj,)={1if gj=,0if gj ,
if the genotypes of all individuals are known , that is , given (gj , ) for all j = 1 , ,
n and = 1 , 2 , 3 , the complete - data log likelihood is
( 12)l(,)=j=1nln [ p(yj gj)p(mj , nj gj)p(gj ) ] ,
where
( 13)p(yj gj)=122exp [ 122=13(gj,)(yj)2],p(mj , nj gj)=k=13p(mj , nj gj=)(gj,)=k=13[1(,mj)2(,nj)](gj,),p(gj)==13(gj, ) .
therefore , we need to take expectation of the log likelihood with respect to . the expected complete - data log likelihood function is
( 14)l( (t))=e[l(, ) (t)]=0+1()+2( ) .
note that e[l( , ) ] stands for the expectation of l( , ) with respect to conditional on parameters at the current state and the data ( the symbol for data is suppressed for simplicity ) .
the three components of ( 14 ) are
( 15)0=j=1n =13e[(gj, ) (t ) ] [ln 1(,mj)+ln 2(,nj)],1()=n2ln ( 2 ) 122j=1n =13e[(gj, ) (t)](yj)2,2()=j=1n =13e[(gj, ) (t)]ln .
the first component 0 is a function of but not a function of . therefore , it is considered as a constant .
the expectation step of the em algorithm requires computing the expectation of conditional on the data and . because is a bernoulli variable , the expectation is simply the probability of = 1 , that is ,
( 16)e[(gj, ) (t)]=pr[(gj,)=1 (t),m , y]=p(gj=)p(yj gj=)p(mj , nj gj=)=13p(gj=)p(yj gj=)p(mj , nj gj=)=exp [ (1/22)(yj)2]1(,mj)2(,nj)=13exp [ (1/22)(yj)2]1(,mj)2(,nj ) .
the maximization step of the em algorithm requires taking the partial derivatives of l( ) with respect to , setting the partial derivatives equal to zero , and solving for the parameters :
( 17)l( (t))=1()+2()=0 .
the solutions of the parameters are
( 18)=[j=1nxjtxj]1[j=1n=13e[(gj,)]xjt(yjh)],=[j=1n =13e[(gj,)](hth)]1 [j=1n =13e[(gj,)]ht(yjxj)],2=1nj=1n =13e[(gj,)](yjxjh)2,=1nj=1ne[(gj, ) ] , =1,2,3 .
although the wald test can be performed for testing the presence of qtl , such a test is not justified for testing the null hypothesis of mendelian segregation .
therefore , the likelihood ratio tests are more justifiable . regardless what hypothesis is tested , the full model joint log likelihood function given in ( 10 ) is required .
let us reintroduce this joint log likelihood function using a different notation so that different likelihood ratio tests are easily interpreted .
the joint likelihood is rewritten as
( 19)lqs(,)=n2log ( 2)+j=1nln { =13exp [ 122(yj)2]1(,mj)2(,nj ) } ,
where represents qtl effects and stands for non - mendelian segregation .
the null hypothesis for qtl detection is hqtl : = 0 while the null hypothesis for detecting segregation distortion is hsdl : = . the null hypothesis is hqtl : = 0 .
the likelihood ratio test statistic is
( 20)qtl=2[ls(0,^)lqs(^,^ ) ] ,
where ls(0,^ ) is the log likelihood value under the null model = 0 , which is
( 21)ls(0,^)=l(^,^2 y)+l(^ m ) ,
where
( 22)l(^,^2 y)=n2ln ( ^2)12^2j=1n(yjxj^)2,l(^ m)=j=1nln [ =13^1(,mj)2(,nj ) ] .
the estimated parameters in ( 22 )
are obtained by maximizing the corresponding likelihood functions ( see appendix b for the estimation ) .
the null hypothesis is hsdl : = . the likelihood ratio test statistic is
( 23)sdl=2[lq(^,)lqs(^,^ ) ] ,
where
( 24)lq(^,)=n2ln ( ^2)+j=1nln { =13exp [ 12^2(yj^)2]1(,mj)2(,nj)}.
again , the mles of the parameters in ( 24 ) are obtained by maximizing this likelihood function ( see appendix b for the estimation of parameters under the null model ) .
the null hypothesis is h0 : = 0 and = . the likelihood ratio test statistic is
( 25)qs=2[l(0,)lqs(^,^ ) ] ,
where
( 26)l(0,)=l(^,^2 y)+l(
m ) .
the two components of ( 26 ) are
( 27)l(^,^2 y)=n2ln ( ^2)12^2j=1n(yjxj^)2,l( m)=j=1nln [ =131(,mj)2(,nj ) ] .
the parameters involved in these log likelihood functions are estimated using formulas given in appendix b. this hypothesis is rejected if either 0 or or both inequalities hold .
suppose that , for some reason , we know for sure that the population from which the sample is drawn is a mendelian population . the sample drawn from this population is selected based on extreme phenotypes ( selective genotyping ) .
the sample is then non - mendelian regarding the qtl that control the trait subject to phenotypic selection .
the reason is that segregation distortion in the sample is solely caused by selective genotyping .
this example demonstrates the application of the method to joint mapping of qtl and sdl in a wheat qtl mapping experiment .
the experiment was conducted by dou et al . who made the data available to us for this analysis .
a female sterile line xnd126 and an elite cultivar gaocheng 8901 with normal fertility were crossed for genetic analysis of female sterility measured as a ratio of the number of seeded spikelets to the total number of spikelets per plant . the parents and their f1 and f2 progeny
were planted in the huaian experimental station in china for the 2006 - 2007 growing season under the normal autumn sowing condition .
the sterility trait was transformed using the angular sine transformation , y = arcsin(x ) , where x is the phenotypic value expressed as ratio .
these markers covered five chromosomes of the wheat genome with an average genome marker density of 15.5 cm per marker interval .
the five chromosomes are only part of the wheat genome . the model for the female sterility is
( 28)yj = xj+zj11+zj22+j ,
where xj = 1 for all j , is the intercept , zj1 = { + 1,0 , 1 } is the genotype indicator variable for the additive effect 1 = a , and zj2 = { -1,1 , 1 } is the genotype indicator variable for the dominance effect 2 = d. we used an interval mapping approach to scanning the entire genome .
therefore , the model contains one qtl at a time . with the interval mapping , zj = [ zj1 zj2 ] is missing and
can take one of three values denoted by h for = 1 , 2 , 3 ( see definition of h ) in section 2 .
the likelihood ratio test statistics were divided by 4.61 to obtain their corresponding lod scores .
the top panel ( a ) shows the lod profile for qtl detection regardless whether there is segregation distortion or not .
figure 3(b ) shows the lod profile for testing segregation distortion , regardless whether the qtl is present or absent .
we used the quick method of piepho to calculate the genome wide critical value of the lod for significance test .
we found that the genome wide critical value was slightly less than lod = 3 criterion ( data not shown ) .
in addition to the two major loci , several other regions of the genome also showed significant peaks of the lod score profile .
overall we detected eight loci , four qtls and four sdls . among the detected qtl ,
each explains from 10% to 47% of the phenotypic variance ( listed as heritability denoted by h in table 1 ) . among the four sdl detected , all showed bias in favor of the xnd126 parent ,
that is , the homozygote of xnd126 allele was over represented at the cost of low representation of the other parent .
the frequency of the heterozygote was close to the mendelian frequency of 0.5 , but the homozygote of gaocheng 8901 allele was almost wiped out .
the estimated genotypic frequencies are plotted against the genome location as shown in figure 2 .
one of the major theoretical contributions of this study is the development of the variance - covariance matrix of the estimated qtl - sdl parameters .
the covariances between pairs of estimated parameters are not of interest , but the variances of the estimated parameters are important .
we reported the standard errors for two selected loci , locus 4 ( qtl ) and locus 7 ( sdl ) .
the standard errors are the square roots of the variances obtained from the em algorithm .
the variance - covariance matrix of the estimated parameters takes the inverse of the information matrix . as a result
we also performed a bootstrap analysis ( 1000 bootstrap samples ) to provide more accurate estimation of the variance .
the results of the bootstrap estimates of the standard errors for the two loci are also listed in table 2 for comparison .
the approximate standard errors from the em algorithm are indeed biased downward , especially for locus 4 ( qtl ) .
the bootstraps method is recommended for obtaining more accurate estimates of the standard errors if the sample size is small .
methods also are available for mapping viability loci or segregation distortion loci when markers do not segregate in a typical mendelian ratio [ 46 , 911 ] .
however , qtl mapping and sdl mapping have never been combined in a single analysis .
this study is the first attempt to combine the two seemingly different analyses into a joint one .
when qtl and sdl are loosely linked or not linked , the joint analysis does not offer much advantage over the separate analyses .
when they do overlap , a phenomenon called pleiotropy , joint mapping does offer some advantage .
unfortunately , the wheat experiment introduced here is not a good example to demonstrate the advantage of joint analysis because the qtl and sdl detected do not overlap .
an obvious situation where the joint analysis can be more powerful is qtl mapping with selective genotyping . in most designed selective genotyping experiments ,
directional ( one - tailed ) selection is rarely used for selective genotyping because it artificially reduces the variation of the trait and thus reduces the statistical power of qtl detection . however , with the joint analysis , the power can increase under directional selection .
we simulated 500 f2 individuals for a single chromosome of 300 cm long .
we placed 16 markers evenly over the chromosome with 20 cm per marker interval .
we selected 300 smallest individuals out of the 500 ( one - tailed selection ) for mapping .
figure 3(a ) shows the test statistic for qtl regardless whether segregation is distorted or not .
the panel in the middle ( b ) shows the lod test statistic for segregation distortion , regardless whether a qtl is present or not .
the panel at the bottom ( c ) shows the lod score profile of the joint analysis where the null model is no qtl and no sdl .
if lod = 3 is the threshold value , neither of the separate analyses is significant .
however , the joint analysis has a lod score as high as 4.5 , indicating a significant qtl in the middle of the chromosome .
our model was developed under zygotic selection because we are dealing with the genotypic frequencies . however ,
if the true cause of segregation is gametic selection , we can still detect segregation distortion as long as the gametic selection leads to the genotypic frequencies deviating from the expected mendelian ratio .
a model particularly handling gametic selection has not been developed yet , but it is not difficult .
let us take the f2 population as an example to show the gametic selection model .
denote the frequencies of g1 and g2 alleles from the female parent by 1 and 2 , respectively , for 1 + 2 = 1 , and the corresponding allele frequencies from the male parent .
when gametic selection occurs , we treat 1 and 1 as unknown parameters for estimation .
the genotypic frequencies are simply functions of the two unknown parameters , as given by 1 = 11 , 2 = 12 + 21 and 3 = 22 .
qtl parameters are estimated using the same algorithm as described in the zygotic selection model because they depend only on the genotypic frequencies . estimating parameters 1 and 1 requires a modified algorithm . under the gametic selection model
, we can test whether the segregation distortion is caused by the distortion of female gametes , male gametes , or both .
the joint analysis developed in this study only applies to line crossing data where the marker linkage phases are known .
application of the method to pedigrees warrants further investigation and it is not obvious to us at this moment .
we have already extended the method to bc ( backcrosses ) , ril ( recombinant inbred lines ) , dh ( double haploids ) , and fw ( four way crosses ) and incorporated them into our qtl mapping program that is described in the next paragraph .
however , interval mapping is still the quickest method of qtl mapping , even though multiple qtl mapping programs are available .
compared with traditional qtl interval mapping , this joint analysis involves one additional step of updating the genotypic frequencies .
this additional step presents a complication where the conditional genotypic frequencies given flanking marker genotypes can not be calculated prior to qtl mapping .
fortunately , we have incorporated the joint qtl / sdl mapping into our qtl mapping program .
the method = ml option in the proc qtl statement must be turned on with an additional suboption /distortion to invoke the joint mapping procedure . without the /distortion option
proc qtl is available on our website ( http://www.statgen.ucr.edu/software.html ) and users can download the program with no charge . the program is also accompanied with a detailed user manual .
let us define the individual - wise complete - data log likelihood for plant j as
( a.1)lj(,)=[12ln ( 2)122=13(gj,)(yjxjh)2]+=13(gj,)[ln ( t1(,mj))+ln ( t2(,nj))]+=13(gj,)ln ,
where 3 = 1 1 2 so that 3 is excluded from the parameter vector . to derive the variance covariance matrix of the estimated parameters , we need to define the score vector sj( , ) and the hessian matrix hj( , ) for the individual - wise complete - data log likelihood function .
the louis information matrix of the parameters under the em algorithm is
( a.2)i(^)=j=1ne[hj(^,)]j=1nvar[sj(^, ) ] ,
where the expectation and variance are taken with respect to the missing values . once the information matrix is define , the variance matrix of the estimated parameters simply takes
( a.3)var(^)i1(^ ) .
the standard error of each parameter takes the square root of each diagonal element of the above matrix . the approximation performs better for large sample sizes .
the score vector is denoted by sj( , ) = lj( , )/ , which consists of five blocks , as follows :
( a.4)sj(,)=lj(,)=12k=13(gj,)xjt(yjxjh),sj(,)=lj(,)=12=13(gj,)ht(yjxjh),sj(2,)=lj(,)2=122 + 124=13(gj,)(yjxjh)2,sj(1,)=lj(,)1=(gj,1)11(gj,3)1112,sj(2,)=lj(,)2=(gj,2)12(gj,3)1112 .
concatenating the above five scores vertically
the hessian matrix is denoted by hj( , ) = lj( , )/ , which is block diagonal with non - zero blocks given as follows :
( a.6)hj()=2lj(,)t=12xjtxj , hj()=2lj(,)t=12=13(gj,)xjth,hj(2)=2lj(,)2=14=13(gj,)xjt(yjxjh),hj()=2lj(,)t=12=13(gj,)hth,hj(2)=2lj(,)2=14=13(gj,)ht(yjxjh),hj(22)=2lj(,)22=12416=13(gj,)(yjxjh)2,hj(11)=2lj(,)11=(gj,1)112(gj,3)132,hj(12)=2lj(,)12=(gj,3)132,hj(22)=2lj(,)22=(gj,2)122(gj,3)132 .
the expectation of the hessian matrix e[hj( , )]and the variance matrix of the score vector var[sj( , ) ] can be expressed explicitly because both the hessian matrix and the score vector are linear functions of the missing value
( a.8)j=[(gj,1)(gj,2)(gj,3)]t .
therefore , e[hj( , ) ] and var[sj( , ) ] can eventually be expressed as functions of the expectation and variance of j , which have simple expressions because j is a multinomial variable .
the hessian matrix hj( , ) is already expressed in linear function of j and thus the expectation can be obtained straightforwardly by replacing j by e(j ) .
let us define the following matrices :
( a.9)cj()=[12xjt(yjxjh1)12xjt(yjxjh2)12xjt(yjxjh3)],cj()=[12h1t(yjxjh1)12h2t(yjxjh2)12h3t(yjxjh3)],cj(2)=[124(yjxjh1)2124(yjxjh2)2124(yjxjh3)2],cj(1)=[11013 ] , cj(2)=[01213 ] .
the score vector in matrix notation is
( a.10)sj(,)=lj(,)=[cjt()cjt()cjt(2)cjt(1)cjt(2)]j+[0012200]=cjj+d .
as a result ,
( a.11)var[sj(,)]=cjvar(j)cjt ,
where
( a.12)var(j)=diag [ e(j)]e(j)e(jt )
is the variance - covariance matrix of the multinomial variable j .
this appendix provides methods for estimating parameters under various null models that are required for constructing likelihood ratio test statistics . the log likelihood for the null model is
( b.1)ls(0,^)=l(^,^2 y)+l(^m ) ,
where
( b.2)l(^,^2 y)=n2ln ( ^2)12^2j=1n(yjxj^)2,l(^ m)=j=1nln [ =13^1(,mj)2(,nj ) ] .
the mles of the population parameters are obtained explicitly without iteration :
( b.3)^=[j=1nxjtxj]1[j=1nxjtyj],^2=1nj=1n(yjxj^)2 .
the estimated genotypic frequencies , however , require the following iterations :
( b.4)(t+1)=1nj=1ne[(gj, ) ] , =1,2,3 ,
where
( b.5)e[(gj,)]=(t)1(,mj)2(,nj)=13(t)1(,mj)2(,nj ) .
the log likelihood function for the null model is
( b.6)lq(^,)=n2ln ( ^2)+j=1nln { =13exp [ 12^2(yj^)2]1(,mj)2(,nj)}.
finding the solution of the parameters requires iterations as given as foolows :
( b.7)(t+1)=[j=1nxjtxj]1 [j=1n =13e[(gj,)]xjt(yjh(t))],(t+1)=[j=1n =13e[(gj,)](hth)]1 [j=1n =13e[(gj,)]ht(yjxj(t))],2(t+1)=1nj=1n =13e[(gj,)](yjxj(t)h(t))2 ,
where
( b.8)e[(gj,)]=exp [ (1/22)(yj)2]1(,mj)2(,nj)=13exp [ (1/22)(yj)2]1(,mj)2(,nj ) .
the mendelian frequencies 's are constants , not parameters , as 1 = 3 = ( 1/2)2 = 1/4 .
the log likelihood function for the null model is
( b.9)l(0,)=l(^,^2 y)+l( m ) ,
where
( b.10)l(^,^2 y)=n2ln ( ^2)12^2j=1n(yjxj^)2,l(m)=j=1nln [ =131(,mj)2(,nj ) ] . | quantitative trait locus ( qtl ) mapping is usually performed using markers that follow a mendelian segregation ratio .
we developed a new method of qtl mapping that can use markers with segregation distortion ( non - mendelian markers ) .
an em ( expectation - maximization ) algorithm is used to estimate qtl and sdl ( segregation distortion loci ) parameters .
the joint analysis of qtl and sdl is particularly useful for selective genotyping .
application of the joint analysis is demonstrated using a real life data from a wheat qtl mapping experiment . |
recent national surveys have indicated that staff providing care for people with intellectual disabilities faces a high risk of exposure to violence at work .
violence and threats are common and 50% of all staff report being exposed to one or more episodes during the last year .
the complex and demanding management of violent service users often provokes adverse feelings and contributes to a negative working experience .
people with intellectual disabilities ( i d ) who are also offenders are some of the most difficult health service users to treat and historically they have also received little attention from researchers or the wider society . the research that has been conducted
offenders with a moderate or more severe level of i d seldom enter the criminal justice system ( cjs ) , as they are diverted into mental health services , i d services , or forensic mental health services . as a result
, there is a dearth of studies exploring the needs and living conditions of offenders with moderate i d compared with offenders with mild i d .
offenders with i d have many characteristics in common with offenders in the general population .
they tend to be young and male and have experienced social disadvantage , unstable environments , and financial instability .
there is little research on how the characteristics of people with i d who are labeled offenders may differ from those with i d who do not offend .
proposed that two groups of offenders with i d can be distinguished according to whether they are known to the i d services . amongst the smaller group who are known to services the term offence
the norwegian system has quite restrictive policies regarding diversion of offenders with i d from the criminal justice system .
there has been some focus on intellectual impairment among offenders , but the government s priorities are focused elsewhere on , for instance , building more prison accommodation , encouraging multidisciplinary cooperation in preventing recidivism , developing alternatives to imprisonment alongside better rehabilitation services and evidence - based research .
norway s new penal code from 2002 , mandatory care established stringent criteria for bringing people with i d into the forensic services .
these include the commission of a serious or life - threatening crime by a person defined as non - responsible due to i d with a level of intellectual functioning corresponding to moderate or severe i d ( iq<55 )
. the risk of reoffending must also be regarded as significant before a sentence can be imposed .
offenders who do not fulfill these criteria are given standard prison sentences with no access to i d specific services .
work - related stress may occur when the perceived demands of the job exceed an individual s resources to cope and do the job .
the stressors typically reported by intellectual disability support staff include challenging behavior , interpersonal issues with colleagues and organizational concerns ( e.g. inadequate staffing ) .
while the incidence of developing problems associated with the negative aspects of providing care seems to be low , they can be serious and can affect the individual , their close relationships , the care that they provide , and their organizations .
burnout is a psychological syndrome that can occur in response to chronic , uncontrollable work demands when providing a service to people in some way .
it is most common in workers who give of themselves emotionally and especially when they give out more than they get back .
the quality of services with fewer positive interactions and less staff contact has been found to be correlated to staff burnout symptoms .
staff in services for adults with intellectual disabilities seem to be at no higher risk of burnout compared to staff in other caring services according to a systematic review .
however , those staff most vulnerable to developing burnout tend to be the workers most valued by the services and burnout often indicates poor organizational support .
resilience ( hardiness ) is a personality trait which may moderate the relationship between work demands and work related quality of life .
it has been described as a set of personality characteristics that function as resources to draw upon when encountering stressful demands .
research with social workers has suggested that older individuals may well have a more effective and mature repertoire of coping styles . in this study
, we describe the self - reported experience of work related quality of life and resilience among caring staff in the national norwegian i d offender services .
local community based services are compared with their counterparts who are institutionally based . while the institutional staff emphasize the assessment and initial treatment issues during the acute period , the local community staff have to cope with lasting and more pervasive needs alongside the challenges of social integration . given these differences ,
the purpose of this study therefore was to compare the staff in i d offender institutional services to the staff in similar local community services in terms of professional quality of life and resilience .
work - related stress may occur when the perceived demands of the job exceed an individual s resources to cope and do the job .
the stressors typically reported by intellectual disability support staff include challenging behavior , interpersonal issues with colleagues and organizational concerns ( e.g. inadequate staffing ) .
while the incidence of developing problems associated with the negative aspects of providing care seems to be low , they can be serious and can affect the individual , their close relationships , the care that they provide , and their organizations .
burnout is a psychological syndrome that can occur in response to chronic , uncontrollable work demands when providing a service to people in some way .
it is most common in workers who give of themselves emotionally and especially when they give out more than they get back .
the quality of services with fewer positive interactions and less staff contact has been found to be correlated to staff burnout symptoms .
staff in services for adults with intellectual disabilities seem to be at no higher risk of burnout compared to staff in other caring services according to a systematic review
. however , those staff most vulnerable to developing burnout tend to be the workers most valued by the services and burnout often indicates poor organizational support .
resilience ( hardiness ) is a personality trait which may moderate the relationship between work demands and work related quality of life .
it has been described as a set of personality characteristics that function as resources to draw upon when encountering stressful demands .
research with social workers has suggested that older individuals may well have a more effective and mature repertoire of coping styles . in this study
, we describe the self - reported experience of work related quality of life and resilience among caring staff in the national norwegian i d offender services .
local community based services are compared with their counterparts who are institutionally based . while the institutional staff emphasize the assessment and initial treatment issues during the acute period , the local community staff have to cope with lasting and more pervasive needs alongside the challenges of social integration .
given these differences , the purpose of this study therefore was to compare the staff in i d offender institutional services to the staff in similar local community services in terms of professional quality of life and resilience .
the study was conducted in 2012 among staff in the norwegian national unit for mandatory care .
the penal code was set in 2002 , and at the time of data collection , 10 offenders with i d were serving a sentence .
nine of these had a local community placement and one was incarcerated at the institution for assessment and treatment planning .
the institutional setting also serves as a custody unit for i d offenders / alleged offenders .
a total number of 106 staff members were given a set of questionnaires covering professional quality of life , and resilience .
the response rate was high ( 80% ) with 85 completed questionnaires returned ; 69 were from the locally based services and 16 from the institutional setting .
the participants ( 36 women , 49 men ) had a modal age category of 40 - 45 .
there were 38 registered nurses ( 45% of the sample ) , 15 licensed practical nurses ( 18% ) , and 25 unqualified staff ( 29% ) .
the mean length of work experience within i d forensic services was 2.56 years ( standard deviation=1.0 ) .
these demographic data was based on aggregated information from each setting rather than individual data in order to maintain confidentiality .
a short registration form to obtain some basic demographic and occupational data was also distributed with these questionnaires .
assessments of professional quality of life were made using the professional quality of life scale ( proqol ) .
proqol is a 30 item self - report measure designed to assess the following dimensions : compassion satisfaction , burn - out and compassion fatigue .
the compassion satisfaction dimension ( cs ) measures the degree of pleasure derived from being able to do one s work well .
the burnout dimension ( bo ) in this scale is associated with feelings of hopelessness and difficulties in dealing with work demands .
the compassion fatigue dimension ( cf ) relates to secondary exposure to extremely stressful work - related events ( e.g. experiencing the trauma of someone one helped , even to the extent of avoiding activities to avoid reminders of the trauma ) .
the alpha reliabilities for the scales of the proqol have been found to be high : =0.88 ( cs ) , 0.75 ( bo ) and 0.81 ( cf ) . the construct validity is good with over 200 published papers .
resilience was measured using a norwegian version of the dispositional resiliency scale ( drs ) .
the drs is recognized as the best available measure of hardiness , and the norwegian version is based on bartone s short 15-item version ( drs-15 ) .
the drs-15 consists of 15 statements requiring respondents to indicate agreement on a 4-point scale ( not at all true to completely true ) . to create hardiness scores ,
in addition to a total score , three subscale scores can be created by adding the relevant five items for each of the facets : commitment , challenge , and control .
the alpha reliability of the total resilience score of the norwegian version have been found to be =0.79 .
all executives in the local units and the institution were informed about the study and gave consent for their service to be accessed .
the questionnaires were then sent to the executives for onward distribution to their staff members .
the regional committee for medical and health research ethics approved the study ( reference 2011/1321 ) .
the study was conducted in 2012 among staff in the norwegian national unit for mandatory care .
the penal code was set in 2002 , and at the time of data collection , 10 offenders with i d were serving a sentence .
nine of these had a local community placement and one was incarcerated at the institution for assessment and treatment planning .
the institutional setting also serves as a custody unit for i d offenders / alleged offenders .
a total number of 106 staff members were given a set of questionnaires covering professional quality of life , and resilience .
the response rate was high ( 80% ) with 85 completed questionnaires returned ; 69 were from the locally based services and 16 from the institutional setting .
the participants ( 36 women , 49 men ) had a modal age category of 40 - 45 .
there were 38 registered nurses ( 45% of the sample ) , 15 licensed practical nurses ( 18% ) , and 25 unqualified staff ( 29% ) .
the mean length of work experience within i d forensic services was 2.56 years ( standard deviation=1.0 ) .
these demographic data was based on aggregated information from each setting rather than individual data in order to maintain confidentiality .
a short registration form to obtain some basic demographic and occupational data was also distributed with these questionnaires .
assessments of professional quality of life were made using the professional quality of life scale ( proqol ) .
proqol is a 30 item self - report measure designed to assess the following dimensions : compassion satisfaction , burn - out and compassion fatigue .
the compassion satisfaction dimension ( cs ) measures the degree of pleasure derived from being able to do one s work well .
the burnout dimension ( bo ) in this scale is associated with feelings of hopelessness and difficulties in dealing with work demands .
the compassion fatigue dimension ( cf ) relates to secondary exposure to extremely stressful work - related events ( e.g. experiencing the trauma of someone one helped , even to the extent of avoiding activities to avoid reminders of the trauma ) .
the alpha reliabilities for the scales of the proqol have been found to be high : =0.88 ( cs ) , 0.75 ( bo ) and 0.81 ( cf ) . the construct validity is good with over 200 published papers .
resilience was measured using a norwegian version of the dispositional resiliency scale ( drs ) .
the drs is recognized as the best available measure of hardiness , and the norwegian version is based on bartone s short 15-item version ( drs-15 ) .
the drs-15 consists of 15 statements requiring respondents to indicate agreement on a 4-point scale ( not at all true to completely true ) . to create hardiness scores ,
in addition to a total score , three subscale scores can be created by adding the relevant five items for each of the facets : commitment , challenge , and control .
the alpha reliability of the total resilience score of the norwegian version have been found to be =0.79 .
all executives in the local units and the institution were informed about the study and gave consent for their service to be accessed .
the questionnaires were then sent to the executives for onward distribution to their staff members .
the regional committee for medical and health research ethics approved the study ( reference 2011/1321 ) .
the sample was divided into two comparative groups ; staff in the local community services ( n=69 ) and staff in the institutional services ( n=16 ) .
the institutional group tended to be slightly younger with higher proportions of male and qualified staff ( table 1 ) . in both groups the respondents scored very similarly to the instrument norms for all three proqol subscales ( figures 1 - 3 ) .
the total score of the dsr is also presented in a box - plot , although no norms were found in the literature ( figure 4 ) .
independent t - tests were conducted to compare scores on the measures in the two groups ( table 2 ) .
no significant differences were found for any of the proqol subscales but overall resilience and perceived control were significantly higher in the community staff group .
the internal consistency of the proqol subscales and total resilience ( drs-15 ) was high ( cronbach s alpha : cs=0.83 , bo=0.70 cf=0.74 for cf , drs=0.74 ) .
the staff participating in this study were compared according to the nature of their relationship with the i d offender .
the institutional and the local community services are interlinked but they are distinct in several respects as described in the introduction . there were no significant differences between the local community staff and the institutional staff on any of the proqol subscales .
this may be due to low statistical power , especially with regard to the small group of institutional staff .
the scores were close to the normative data derived from extensive studies of 1187 people who worked in various helping professions .
although not significant , the results suggested somewhat greater compassion satisfaction alongside lower burnout symptoms and fatigue among the institutional staff .
local community staff have a more stable relationship with the offender and his or her natural environment . their capacity to focus on
the more technical and procedural approach to treatment in the institutional setting may set up a more cognitive , less emotional relationship .
different ways of working in the two settings may also explain the differences in resilience ( drs-15 ) , some of which were statistically significant .
the control subscale indicated a significantly greater sense of control in the community group , and this may reflect greater independence in working practices among local community staff .
institutions are often more focused on routines , rules and hierarchical systems compared to local services where emphasis is more directed to the service users .
the community staff consequently would be expected more often to trust his / her own evaluations .
the normative data for the norwegian dsr-15 was based on a sample of military cadets and indicated a mean of 30.03 , ( standard deviation=4.42 ) . both groups in the present study scored within these norms suggesting unremarkable levels of resilience .
looking specifically at the community staff , they are older , less educated and had higher proportions of women . the higher scores on resilient control in this group fit with previous findings about age .
however the possible impact of gender or education on resilience in health care services has not been studied previously .
the response rate was very high for this sort of survey indicating that the sample is quite representative of the population of staff working with i d offenders .
on the other hand , the relatively small institutional sample weakened the analysis and there was also comparison across groups unbalanced in size .
the homogeneity of one institutional staff group compared to nine smaller and more differentiated community groups may also be a confounder .
this study was conducted in a sample of staff employed in the forensic services for people with i d .
further research should look at other groups working with challenging behavior in people with i d , or staff in forensic mental health services . a comparison between such larger and more defined groups
could also been expedient . in conclusion , a significant difference was found between staff in institutional and local settings in terms of overall resilience and the control subscale .
it may be worth interviewing community staff about their coping strategies which might underpin such greater resilience and then conveying these strategies to their colleagues in the institutional setting thus potentially improving job satisfaction and care effectiveness . | staff in forensic services for people with intellectual disabilities ( i d ) are expected to deal with a wide range of emotional challenges when providing care .
the potential impact of this demanding work has not been systematically explored previously .
this article explores the professional quality of life ( qol ) and the resilience ( hardiness ) of the staff in this setting . the professional qol questionnaire and the disposional resilience scale were completed by staff ( n=85 , 80% response rate ) in the norwegian forensic service for i d offenders .
responses from staff working in institutional settings were compared to those from staff in local community services .
staff in the local community services had higher resilience scores compared to the staff in the institutional setting , ( t=2.19 ; p<0.05 ) . however in the other qol and resilience domains
there were no differences between the staff in the two settings .
the greater sense of resilient control among community staff may be a function of both the number of service users they work with and the institutional demands they face .
even though these participants worked with relatively high risk clients , they did not report significantly impaired quality of life compared to other occupations . |
with the help of yellow pages of puducherry and indian dental association , puducherry branch approximately 150 general dentist were found to be practicing in puducherry , and sample size was calculated with sample size calculator software by keeping confidence level as 95 and confidence interval as 5 . with the help of monthly outpatient statistics in department of obstetrics and gynecology of mahatma gandhi medical college and research institute ,
approximately 800 pregnant women were recorded as new patients every month . keeping confidence level as 95 and confidence interval as 5
the first questionnaire was on the attitude and behavior among private practitioners in and around puducherry regarding pregnant patient 's oral health , which was framed from the reference articles .
the second questionnaire was on pregnant women perception toward their oral health , which was again framed from the reference articles .
the questionnaires were given to a number of 10 dentists and 10 pregnant women and reliability , and the validity of the questionnaires were assessed .
the attitude and behavior among private practitioners in and around puducherry regarding pregnant patient 's oral health were assessed with a pretested questionnaire . another pretested questionnaire on perception toward their oral health
was given to pregnant women reporting to department of obstetrics and gynecology according to the inclusion criteria for the study .
initial assessment of the dental practitioner and the pregnant women were done , and data were collected in a structured proforma .
this was followed by the assessment of attitude and practice behaviors using the formatted questionnaire form comprising a comprehensive set of questions .
they were instructed to point to an answer on each question that describes the best how they felt .
data collected from the assessment form were well documented and statistically analyzed by frequency and percentage distribution , following which the results were calculated .
the first questionnaire was on the attitude and behavior among private practitioners in and around puducherry regarding pregnant patient 's oral health , which was framed from the reference articles .
the second questionnaire was on pregnant women perception toward their oral health , which was again framed from the reference articles .
the questionnaires were given to a number of 10 dentists and 10 pregnant women and reliability , and the validity of the questionnaires were assessed .
the attitude and behavior among private practitioners in and around puducherry regarding pregnant patient 's oral health were assessed with a pretested questionnaire . another pretested questionnaire on perception toward their oral health
was given to pregnant women reporting to department of obstetrics and gynecology according to the inclusion criteria for the study .
initial assessment of the dental practitioner and the pregnant women were done , and data were collected in a structured proforma .
this was followed by the assessment of attitude and practice behaviors using the formatted questionnaire form comprising a comprehensive set of questions .
they were instructed to point to an answer on each question that describes the best how they felt .
data collected from the assessment form were well documented and statistically analyzed by frequency and percentage distribution , following which the results were calculated .
only 17.6 % felt safe to perform dental care to pregnant women [ figure 1 ]
only 27.8 % have received any specific information on dental care to pregnant women [ figure 1 ]
63.9 % dentist only felt that emergency care be performed throughout pregnancy [ figure 1 ]
50 % of dentist only consider consulting patient 's obstetrician a must [ figure 2 ]
10.2 % consider dental imaging , a safe procedure throughout pregnancy [ figure 2 ]
30.6 % agree that delay in dental treatment would affect both the mother and child [ figure 2 ]
41.7 % of dentist only consider the legal risk associated with negative outcomes as a barrier for inappropriate oral health care of pregnant patients [ figure 3 ]
16.7 % of dentist only have participated in continuing education courses on management of pregnant patients during your professional period [ figure 3 ] .
only 17.6 % felt safe to perform dental care to pregnant women [ figure 1 ]
only 27.8 % have received any specific information on dental care to pregnant women [ figure 1 ]
63.9 % dentist only felt that emergency care be performed throughout pregnancy [ figure 1 ]
50 % of dentist only consider consulting patient 's obstetrician a must [ figure 2 ]
10.2 % consider dental imaging , a safe procedure throughout pregnancy [ figure 2 ]
30.6 % agree that delay in dental treatment would affect both the mother and child [ figure 2 ]
41.7 % of dentist only consider the legal risk associated with negative outcomes as a barrier for inappropriate oral health care of pregnant patients [ figure 3 ]
16.7 % of dentist only have participated in continuing education courses on management of pregnant patients during your professional period [ figure 3 ] .
questionnaire on the attitude and practise behaviours among private practitioners questionnaire on the attitude and practise behaviours among private practitioners questionnaire on the attitude and practise behaviours among private practitioners
78.1% feel that oral health check up should be a routine component of monitoring pregnant women [ figure 4 ]
only 11.9 % agree that poor oral health could lead to premature birth [ figure 4 ]
91.2 % feel dental treatment during pregnancy is unsafe for the unborn baby [ figure 4 ]
only 14.6 % feel dental treatment can be done during pregnancy [ figure 5 ]
93.5 % feel routine dental check up will facilitate the pregnant women with an access to treatment at an early stage [ figure 5 ]
71.2 % feel dental treatment during pregnancy will transmit any infection to the fetus [ figure 5 ]
88.1 % feel getting dental treatment done during pregnancy is emotionally stressful [ figure 6 ]
84.2 % agree undergoing dental check up during pregnancy does not eliminate current or future risk of dental caries / problems [ figure 6 ]
91.9 % feel further research and education is required regarding the dental treatment on pregnant women [ figure 6 ] .
78.1% feel that oral health check up should be a routine component of monitoring pregnant women [ figure 4 ]
only 11.9 % agree that poor oral health could lead to premature birth [ figure 4 ]
91.2 % feel dental treatment during pregnancy is unsafe for the unborn baby [ figure 4 ]
only 14.6 % feel dental treatment can be done during pregnancy [ figure 5 ]
93.5 % feel routine dental check up will facilitate the pregnant women with an access to treatment at an early stage [ figure 5 ]
71.2 % feel dental treatment during pregnancy will transmit any infection to the fetus [ figure 5 ]
88.1 % feel getting dental treatment done during pregnancy is emotionally stressful [ figure 6 ]
84.2 % agree undergoing dental check up during pregnancy does not eliminate current or future risk of dental caries / problems [ figure 6 ]
91.9 % feel further research and education is required regarding the dental treatment on pregnant women [ figure 6 ] .
questionnaire on the perception towards oral health by pregnant women questionnaire on the attitude and behavior among private practitioners in and around pondicherry regarding pregnant patient 's oral health questionnaire on the perception towards oral health by pregnant women in and around pondicherry
only 17.6 % felt safe to perform dental care to pregnant women [ figure 1 ]
only 27.8 % have received any specific information on dental care to pregnant women [ figure 1 ]
63.9 % dentist only felt that emergency care be performed throughout pregnancy [ figure 1 ]
50 % of dentist only consider consulting patient 's obstetrician a must [ figure 2 ]
10.2 % consider dental imaging , a safe procedure throughout pregnancy [ figure 2 ]
30.6 % agree that delay in dental treatment would affect both the mother and child [ figure 2 ]
41.7 % of dentist only consider the legal risk associated with negative outcomes as a barrier for inappropriate oral health care of pregnant patients [ figure 3 ]
16.7 % of dentist only have participated in continuing education courses on management of pregnant patients during your professional period [ figure 3 ] .
only 17.6 % felt safe to perform dental care to pregnant women [ figure 1 ]
only 27.8 % have received any specific information on dental care to pregnant women [ figure 1 ]
63.9 % dentist only felt that emergency care be performed throughout pregnancy [ figure 1 ]
50 % of dentist only consider consulting patient 's obstetrician a must [ figure 2 ]
10.2 % consider dental imaging , a safe procedure throughout pregnancy [ figure 2 ]
30.6 % agree that delay in dental treatment would affect both the mother and child [ figure 2 ]
41.7 % of dentist only consider the legal risk associated with negative outcomes as a barrier for inappropriate oral health care of pregnant patients [ figure 3 ]
16.7 % of dentist only have participated in continuing education courses on management of pregnant patients during your professional period [ figure 3 ] .
questionnaire on the attitude and practise behaviours among private practitioners questionnaire on the attitude and practise behaviours among private practitioners questionnaire on the attitude and practise behaviours among private practitioners
78.1% feel that oral health check up should be a routine component of monitoring pregnant women [ figure 4 ]
only 11.9 % agree that poor oral health could lead to premature birth [ figure 4 ]
91.2 % feel dental treatment during pregnancy is unsafe for the unborn baby [ figure 4 ]
only 14.6 % feel dental treatment can be done during pregnancy [ figure 5 ]
93.5 % feel routine dental check up will facilitate the pregnant women with an access to treatment at an early stage [ figure 5 ]
71.2 % feel dental treatment during pregnancy will transmit any infection to the fetus [ figure 5 ]
88.1 % feel getting dental treatment done during pregnancy is emotionally stressful [ figure 6 ]
84.2 % agree undergoing dental check up during pregnancy does not eliminate current or future risk of dental caries / problems [ figure 6 ]
91.9 % feel further research and education is required regarding the dental treatment on pregnant women [ figure 6 ] .
78.1% feel that oral health check up should be a routine component of monitoring pregnant women [ figure 4 ]
only 11.9 % agree that poor oral health could lead to premature birth [ figure 4 ]
91.2 % feel dental treatment during pregnancy is unsafe for the unborn baby [ figure 4 ]
only 14.6 % feel dental treatment can be done during pregnancy [ figure 5 ]
93.5 % feel routine dental check up will facilitate the pregnant women with an access to treatment at an early stage [ figure 5 ]
71.2 % feel dental treatment during pregnancy will transmit any infection to the fetus [ figure 5 ]
88.1 % feel getting dental treatment done during pregnancy is emotionally stressful [ figure 6 ]
84.2 % agree undergoing dental check up during pregnancy does not eliminate current or future risk of dental caries / problems [ figure 6 ]
91.9 % feel further research and education is required regarding the dental treatment on pregnant women [ figure 6 ] .
questionnaire on the attitude and behavior among private practitioners in and around pondicherry regarding pregnant patient 's oral health
pregnancy is a unique complex period with various physiologic changes that support the formation and maturation of a new life .
oral cavity is the mirror of the human body as it reflects and influences systemic health .
prenatal health is very much influenced by maternal oral health , and hence consistent and timely dental care is mandatory in a pregnant patient .
poor oral health can affect the pregnant patient leading to complications such as gestational diabetes , preeclampsia .
most predominant oral manifestation during pregnancy is the occurrence of pregnancy gingivitis and periodontitis due to the influence of increased levels of sex hormones such as estrogen and progesterone which are associated with increased permeability of oral vasculature thereby increasing the severity of inflammation in oral tissue to any stimuli .
it is pertinent to note that numerous studies have revealed the relationship between periodontitis and preterm delivery , low birth weight , and occasionally stillbirth .
considering the impact of maternal oral health on the newborn , it is very essential that the pregnant patient be educated and motivated to consult dentists without any fear or myth regularly . in an attempt to sensitize this valuable platform ,
a questionnaire study was planned and conducted which has revealed that the majority of pregnant patient 's perception toward oral health is poor .
it is an astonishing fact that there exists a dearth of attitude and practice behavior among dental practitioners toward pregnant patients oral health as part of this study .
there is a very significant dilemma existing among dentists in performing dental treatment during pregnancy and very prominently with respect to the use of x - rays in diagnosis .
however , well - collimated beams , lead aprons , and very minimal doses as low as 10 rads ( 100 mgy ) cause less impact on the fetus .
ultimately , the realization and recognition of lack of attitude and practice behavior among general dental practitioners and poor perception of oral health by pregnant patients is a matter of concern for both health of the woman and the future children , which can be professionally protected and by raising awareness among both the dentists and patients for successful outcome .
every gestational woman should be encouraged to seek medical and dental care during pregnancy , as a failure in treating the developing problems affects the health of both the mother and the fetus .
the dentist should attain a basic understanding of the underlying physiological changes of pregnancy , the influences , which are related to the use of medications during gestation and how these may interact with the delivery of dental care .
the realization of this fact helps in the formulation of the treatment plan and the delivery of the necessary medical , nutritional , and dental care as well as it prepares the professionals for counseling their pregnant patients . | background : this study aims to evaluate the knowledge , attitude , practice behaviors among general dental practitioners and assess the perception toward oral health by pregnant patients in and around puducherry.methodology:a self - designed and structured questionnaire was used to obtain information from the dental practitioner and the pregnant patients.results:the majority of the dental practitioners had a lack of knowledge , attitude , practice behaviors regarding pregnant patient 's oral health and similarly majority of pregnant patient 's perception toward oral health was poor.conclusions:drowning and dilemmatic attitude and practice behavior of dentists to be streamlined to render right care to the pregnant women at the right time . perplexing perception toward oral health care by pregnant women to be overcome to orient them to understand the impact of oral health on their general systemic health . |
osteoporosis is among the ten most important and prevalent chronic diseases [ 1 , 2 ] affecting approximately 200 million people worldwide .
osteoporosis is characterized by a progressive decrease in bone mass and microarchitectural deterioration , resulting in increased bone fragility and ultimately an increased risk of fracture [ 46 ] .
osteoporotic fractures , especially hip and vertebral fractures , are associated with significant morbidity and mortality in the elderly and also pose a significant burden on the health - care system . as the population worldwide continues to age
, the prevalence of osteoporosis will continue to climb , with a growing need for effective therapies to prevent fractures [ 1 , 2 ] .
bisphosphonates have been shown to effectively reduce the risk of vertebral and hip fractures in osteoporotic patients .
unfortunately , several large studies have found that the majority of postmenopausal women stop bisphosphonate therapy within 1 year of beginning treatment [ 79 ] .
reasons for poor persistence include conflicting patient beliefs , patient preferences , financial limitations , and previous adverse effects including rash , myalgia , nausea , and diarrhea [ 7 , 10 , 11 ] .
impaired compliance and persistence to long - term bisphosphonate treatment can ultimately lead to an increased risk of fracture .
denosumab is a fully human monoclonal antibody against rank ligand used in the treatment of osteoporosis . in osteoporosis , the thinning and increased porosity of cortical bone and disruption of cortical and trabecular architecture
is the result of an imbalance in bone remodeling where the rate of bone resorption exceeds bone formation via changes in the rank / rank ligand pathway , an important regulator of osteoclast activity [ 1217 ] . in postmenopausal women , increases in rank ligand production have been associated with an increase in osteoclast activity and overall net bone resorption [ 18 , 19 ] .
denosumab has an affinity and specificity for rank ligand and is responsible for inhibiting the proliferation and maturation of osteoclast precursors and functioning of mature osteoclasts .
denosumab has been found to increase bone mineral density ( bmd ) annually and decrease bone turnover markers for at least six months after injection .
clinical trials have demonstrated that denosumab treatment given subcutaneously once every six months is well tolerated and results in significant decreases in hip , nonvertebral , and vertebral fracture risk [ 21 , 22 ] .
the objective of this study is to assess the effectiveness of denosumab therapy in the real world setting and the impact that noncompliance with the regular dosing regimen has on bmd ( measured at the lumbar spine [ ls ] and femoral neck [ fn ] ) compared to patients who receive their scheduled dosing regimen .
all patients from the charlton centre for specialized treatments in hamilton , ontario , canada , on denosumab were screened for eligibility in the review .
a retrospective cohort study was performed from august 2012 to december 2013 for all osteoporotic patients who received a minimum of two subcutaneous injections of denosumab since may 2010 .
patients were eligible for this inclusion if they were above 50 years of age with or without prevalent fractures , received at least two subcutaneous injections of denosumab , and had at least two sequential bmd measurements performed one year apart .
patients were excluded if they had a subsequent bmd measurement performed more than one year before or after the initial injection of denosumab , a change in bmd dxa - measurement machine on subsequent bmd measurements , or if they had a prevalent fracture in the femoral neck and/or lumbar spine during the review period , thus making the results incomparable .
included patients were treatment nave ( 46 ) or have previously been treated with oral bisphosphonates [ risedronate ( 210 ) , etidronate ( 4 ) , and alendronate ( 95 ) ] , the intravenous bisphosphonate [ zoledronic acid ( 29 ) ] , raloxifene ( 29 ) , or teriparatide ( 23 ) .
patients were allocated to one of three groups based on their compliance to the product monograph of injections given every 6 months : subsequent injection of denosumab ( 1 ) less than five months , ( 2 ) between five and seven months , and ( 3 ) more than seven months after their initial subcutaneous injection .
compliance was determined by calculating time elapsed between the initial and subsequent injection of denosumab .
patients were considered to be compliant if the two injections of denosumab were 6 months + / 4 weeks apart .
fractures were considered to be incident if they occurred during the time frame of this retrospective review .
multivariable regression analyses were performed to evaluate the differences among the three prespecified groups in bmd change ( g / cm ) after one year of denosumab therapy at both ls and fn .
the group with subsequent injection between five and seven months was considered the reference group . all multivariable regression analyses were adjusted for baseline bmd values , age of the patient , proton pump inhibitors ( yes / no ) , selective serotonin reuptake inhibitors ( yes / no ) , serotonin - norepinephrine reuptake inhibitors ( yes / no ) , tricyclic antidepressants ( yes / no ) , and the number of months between the first and second bmd measurements . all statistical analyses were performed using the sas / stat ( version 9.2 ; sas institute , cary , nc , usa ) software package running on windows xp professional .
out of the 924 charts reviewed , 403 female ( 92.4% ) and 33 male ( 7.6% ) with osteoporosis and on denosumab therapy were eligible .
the mean age and standard deviation ( sd ) of patients were 67.8 ( 10.7 ) years .
the baseline bmd at the lumbar spine and femoral neck was 0.803 ( 0.128 ) and 0.622 ( 0.099 ) g / cm .
a total of 54.13% of patients had a history of at least one nonvertebral fracture while 23.17% had a history of a vertebral fracture prior to denosumab therapy . upon completion of the analysis
, patients were allocated to one of three groups based on their compliance to denosumab therapy ( subsequent injection of denosumab ( 1 ) less than five months , ( 2 ) between five and seven months , and ( 3 ) more than seven months after their initial subcutaneous injection ) .
twelve patients received a subsequent injection of denosumab less than 5 months , 365 patients between five and seven months , and 59 patients greater than seven months .
baseline bmd results are presented for each of the defined groups along with the history of nonvertebral and vertebral fractures .
moreover , the frequency of proton pump inhibitors , selective serotonin reuptake inhibitors , serotonin - norepinephrine reuptake inhibitors , and tricyclic antidepressants for each group are reported .
descriptive statistics are provided along with the bmd values after one year of denosumab treatment .
the mean duration and standard deviation ( sd ) of treatment from baseline injection were 3.03 months ( 1.18 ) , 6.05 months ( 0.38 ) , and 9.43 months ( 3.06 ) for patients who received a subsequent injection of denosumab less than 5 months , between 5 and 7 months , and greater than 7 months , respectively . to compare the change in bmd after one year of denosumab therapy
, multivariable regressions were performed for the lumbar spine and femoral neck . using the group receiving a subsequent injection between 5 and 7 months as a reference , the groups receiving a subsequent injection
results are provided in table 3 . the difference in lumbar spine bmd change ( 95% confidence interval ) after one year was 0.00035 g / cm ( 0.0124 , 0.0131 ) for patients receiving a subsequent injection greater than 7 months and 0.020 g / cm ( 0.0058 , 0.0479 ) for patients receiving a subsequent injection less than 5 months , as compared with the reference group receiving an injection between 5 and 7 months after their initial injection , respectively .
the difference in femoral neck bmd change ( 95% confidence interval ) after one year was 0.0054 g / cm ( 0.0171 , 0.0063 ) for patients receiving a subsequent injection greater than 7 months and 0.0079 g / cm ( 0.0165 , 0.0324 ) for patients receiving a subsequent injection less than 5 months , as compared with the reference group receiving an injection between 5 and 7 months after their initial injection , respectively .
the relationship between drug administration and change in bmd was not clinically or statistically significant ( p > 0.05 ) .
the efficacy of a treatment depends on both the effectiveness of the therapy and the adherence to recommended dosage regimens .
osteoporosis , like other chronic medical conditions , must have strategies to maintain patient compliance to therapy .
noncompliance to long - term bisphosphonate or denosumab treatment can result in the decline of bmd values and ultimately result in an increased risk of fracture .
it is a fully human monoclonal antibody that binds to the receptor activator for the rank ligand , leading to increased bone mineral density and decreased risk of fractures . while the pharmacology of denosumab is ideal , the success of the drug as therapy for osteoporosis hinges on patient adherence to established clinical guidelines
this highlights the importance of identifying reasons for poor adherence and adapting clinical guidelines accordingly .
this study explores the extent to which noncompliance to denosumab can affect the changes in bmd . in this retrospective cohort study
, the multivariable regressions revealed that there were no statistically significant differences in the bmd values between patients who received a subsequent subcutaneous injection of denosumab less than 5 months , between 5 and 7 months , and greater than 7 months .
however , our study does not suggest that patients and clinicians can extend the duration between subsequent injections of denosumab , since there is a steady decline in drug levels in circulation 6 months following administration , with complete elimination of the drug at the 9-month mark .
this study does suggest that , only under unforeseen circumstances , when a patient is otherwise unable to receive a subsequent injection of denosumab in a timely manner , then a delay may be acceptable .
otherwise , all patients should continue to receive their subsequent doses every 6 months , as per the product monograph .
there are factors inherent to the nature and administration of prolia , as well as factors unique to this patient population , that may explain improved compliance rates .
firstly , it must be acknowledged that denosumab can only be administered through subcutaneous injection by a healthcare professional .
therefore , physicians have significant control over the adherence to denosumab , being able to establish a dosing course , directly monitor adherence to the treatment , and communicate the importance of adherence to patients when doses are missed . furthermore , there are factors that may explain the higher adherence rates in this study compared to other observational studies .
one such reason is that the subjects of the study are recommended to use the provital program , a support program aimed at helping patients stay on their dosing schedule .
patients receive a call from a nurse a week before their next scheduled injection as a reminder .
additionally , at our academic center , a specialty pharmacy exists , which also reminds patients about their appointment one week in advance .
together , these two factors have contributed to the high compliance rate for patients in this study and may be significantly higher than many community practices .
the limited number of patients in the less than 5 months and greater than 7 month groups makes it difficult to draw definitive conclusions .
we are also unable to comment on differences in fracture rates between the groups given the small number of events owing to the small size of the cohort and short follow - up duration .
a longer follow - up duration in a larger cohort may in fact show a difference in bmd or even fracture rates in patients not compliant to therapy .
furthermore , there is uncontrolled variability among patients in each group as additional medications can influence bmd , and this was not accounted for in the multivariable analysis .
finally , the patients excluded due to a fracture in the femoral neck and lumbar spine during the review period along with a change in dxa machine could affect the results of the investigation .
patient compliance is a simple and economic way for patients to benefit from drug efficacy .
no matter how effective a drug is , without the proper adherence to the recommended dosing regimens , the patient may not fully benefit from the course of therapy . in summary , our study found that there were no statistically significant differences in bmd values between patients who received a subsequent injection of denosumab less than five months , between five and seven months , and greater than seven months .
these findings indicate that clinicians may have some flexibility for administering denosumab if there are extenuating circumstances for which patients must delay their subcutaneous injection .
however , the short follow - up duration and small sample size do not allow us to make such a conclusion .
patients should continue to receive their denosumab therapy every six months if this is possible .
a follow - up study with a larger sample size and longer follow - up duration will allow for a more in - depth characterization of this relationship .
furthermore , our study suggests that utilizing different patient adherence programs either at a local or at a national level is an effective method to maintain a high level of patient compliance , thus ultimately improving the real - world effectiveness of this therapy . | although denosumab ( prolia ) has been shown to be a safe and efficacious therapy for osteoporotic patients in numerous clinical trials , few studies have determined its effectiveness in real world clinical practice .
a retrospective review of patients prescribed prolia assessing the impact that noncompliance from the regular dosing regimen of six months for denosumab has on bone mineral density ( bmd ) was performed .
924 patient records were reviewed between august 2012 and september 2013 with 436 patients meeting the eligibility criteria . patients were divided into three groups : subsequent injection of denosumab ( 1 ) less than five months , ( 2 ) between five and seven months , and ( 3 ) more than seven months after their initial subcutaneous injection .
a multivariable regression analysis was conducted comparing the differences among the three prespecified groups in bmd change ( g / cm2 ) after one year of denosumab therapy at both the lumbar spine ( ls ) and femoral neck ( fn ) .
the differences in ls and fn bmd have shown that the relationship between the timing of drug administration in these three groups and change in bmd over 1 year was not clinically or statistically significant ( p > 0.05 ) . a follow - up study with a larger sample size and longer follow - up duration
is required to further characterize this relationship . |
archived dna from all samples received for typing during january 2007december 2008 that showed c. hominis cowp pcr - rflp rsai profiles were retested by single - round small subunit ( ssu ) rrna pcr - rflp using sspi , which generates a pattern unique to c. cuniculus ( 4 ) .
an exception occurred during the outbreak period ( july and august 2008 ) when all cryptosporidium spp.positive stool samples were tested by a pan - genus nested pcr specific for the ssu rrna gene and products digested with sspi and vspi ( 4 ) .
although differentiating more species / genotypes than the cowp pcr - rflp ( 7 ) , this assay is unsustainable for typing large numbers of samples on a routine basis . c. cuniculus was confirmed by sequencing 830 bp of the ssu rrna gene and 850 bp of the 60-kda glycoprotein ( gp60 ) gene by using nested pcr protocols ( 4 ) .
data were analyzed in epi info version 6 ( centers for disease control and prevention atlanta , ga , usa ) .
incomplete data for northern ireland were excluded , but no c. cuniculus cases were identified there .
case - patients with sporadic c. cuniculus , c. parvum , and c. hominis infections were compared by age using the mann - whitney 2-sample test , by sex using the mantel - haenszel version of the test , by month of specimen submission , and by government office region ( england and wales ) or health board ( scotland ) of the primary diagnostic laboratory . in total , 37 ( 1.2% ) of 3,030 infections were caused by c. cuniculus : 23 in 2007 and 14 in 2008 ( table a1 ) .
twenty - five were in patients from england and wales , and 12 patients were from scotland .
other cryptosporidia detected were c. parvum ( n = 1,506 , 49.7% ) , c. hominis ( n = 1,383 , 45.6% ) , c. meleagridis ( n = 26 ) , c. felis ( n = 8) , cervine genotype ( n = 8) , co - infection c. hominis and c. parvum ( n = 5 ) , novel or unidentified genotypes ( n = 5 ) , and c. hominis monkey genotype ( n = 1 ) ; 88 did not amplify with the pcr primers .
substitution of routine typing with the ssu rrna nested pcr - rflp during the outbreak did not increase the number of unusual cryptosporidia , apart from c. cuniculus , indicating the routine cowp pcr - rflp is otherwise appropriate for typing for epidemiologic purposes in the united kingdom .
the age range of patients with sporadic c. cuniculus infection was 174 years ( mean 29 years ; median 31 years ) , significantly older than c. hominis case - patients ( range 083 years ; mean 19 years ; median 13 years ) ( mann - whitney 2-sample test value = 11.12 , df = 1 , p = 0.0009 ) and c. parvum case - patients ( range 086 years ; mean 17 years ; median 29 years ) ( mann - whitney 2-sample test value = 15.24 , df = 1 , p = 0.00009 ) ( figure 1 ) .
the sex distribution was 14 ( 37% ) female and 22 ( 58% ) male patients , with the sex of 1 patient not known , compared with sporadic c. parvum ( 781 [ 51.9% ] female ) and c. hominis ( 736 [ 53.2% ] female ) cases , although the difference was not significant ( = 4.01 , df = 2 , p = 0.13 ) .
age distribution of patients with sporadic cases of cryptosporidium cuniculus , c. hominis , and c. parvum infection in england , wales , and scotland , 20072008 .
more c. cuniculus cases were detected in the late summer and autumn than in the winter and spring , similar to infections with c. hominis but not c. parvum ( figure 2 ) .
three c. cuniculus cases were identified in the east midlands , the outbreak affected region , but none were found in northamptonshire , the outbreak - affected area . most cases ( 24% ) were in the eastern region of england .
two case - patients had traveled outside the uk , one to spain and the other destination not known , during the incubation period .
monthly distribution of sporadic cases of cryptosporidium cuniculus , c. hominis , and c. parvum infection in england , wales , and scotland , 20072008 .
occupational and environmental exposure data were available for 14 c. cuniculus case - patients from patient questionnaires administered by local environmental health departments .
one patient ( a 9-year - old boy ) reported direct contact with rabbits ( a pet ) , and 2 patients had potential environmental contact ( a 63-year - old woman played golf , and a 36-year - old man sat on grass during a walking holiday ) . two case - patients reported diarrhea in other residential contacts .
none of the patients reported links with ( lived in , had visited or received visitors from ) the outbreak - affected area .
one c. cuniculus case - patient was an immunosuppressed child who had received a kidney transplant .
two gp60 subtype families , va ( n = 18 ) and vb ( n = 19 ) , were detected in sporadic c. cuniculus isolates , linked to patient sex ; 10/14 ( 71.4% ) female patients had va subtype , compared with 7/22 ( 31.8% ) male patients ( = 5.24 , df = 1 , p = 0.022 ) .
no significant difference in age or regional distribution was found , but cases with va occurred only in august through december , while vb cases occurred all year but mostly in august ( figure 2 ) .
representative sequences have been deposited into genbank : gu971631gu971650 ( gp60 ) and gu971628gu971630 ( ssu dna ) .
the latter are identical to those deposited previously ( eu437413 , fj262724fj262726 ) ( 1,4 ) and are c. cuniculus ( 5 ) .
c. cuniculus was first identified as a human pathogen during a waterborne outbreak , and its epidemiology has now been described for sporadic cases in the united kingdom . although the numbers are small , and the data need to be interpreted with caution , it was the third most commonly identified cryptosporidium species in patients with diarrhea during the study period , after c. parvum and c. hominis .
all c. cuniculus isolates identified by pcr - rflp were confirmed by sequence analysis , indicating the reliability of the test algorithm used , although the development of specific probes will enhance testing capability .
all ages were infected with little age delimitation < 50 years , after which numbers declined .
contrast this to c. parvum and c. hominis , which are both linked to young age .
c. cuniculus distribution is seasonal , peaking in august through november , and differences in seasonal distribution of gp60 subtypes were marked .
rabbits are the natural hosts for c. cuniculus ( 5 ) , and seasonal distribution and variation in humans may reflect rabbit breeding seasons and infections , although good epidemiologic studies of cryptosporidium spp . in wild rabbits are lacking ( 7 ) .
studies of farmed and wild rabbits so far indicate that gp60 subtype vb predominates ( 79 ) , although the outbreak was caused by va ( 4 ) .
unlike with c. hominis , seasonal distribution of cases was not linked to foreign travel .
the distribution of sporadic cases was the opposite of the outbreak in which more case - patients were female ( 6 ) .
a small number of case - patients had possible exposure risks , although this requires further investigation .
the only known hosts of c. cuniculus are humans and european rabbits ( oryctolagus cuniculus ) ( 5,79 ) , and until population - based studies of rabbits are undertaken , the risks can not be fully evaluated , nor the human epidemiology fully explained . | to investigate sporadic human cryptosporidiosis trends in the united kingdom , we tested 3,030 cryptosporidium spp.positive fecal samples , submitted for routine typing in 20072008 , for c. cuniculus . c. cuniculus prevalence was 1.2% ; cases were mostly indigenous and occurred across all age groups .
most occurred during august october and may be linked to exposure opportunities . |
a lip switch flap is an axial - pattern flap using the inferior labial artery as a feeding blood vessel .
its morphology is appropriate to improve the upper lip morphology in the presence of severe cleft lip and nose deformity .
it is capable of simultaneously reconstructing the philtrum and extending the bridge of the nose , and was reported to be very useful and be associated with fewer complications in many cases.[14 ] cutting et al .
described it was very important to proceed in secondary bilateral cleft lip reconstruction with lip switch flap only after the midface and nose repair done . on the other hand ,
the vascular pedicle of this flap is very thin and circulatory disorder occurs due to 180 rotation of the pedicle , being a risk for flap necrosis .
the grafted flap was returned to the donor site to improve blood circulation of the flap , followed by its re - transplantation , which prevented necrosis of the flap . here ,
severe cleft lip nose deformity , i.e. a short bridge and flat apex of the nose , were observed . under general anesthesia
, the nasal morphology was improved using cartilage of the nasal septum , and the lip deformity was improved by forming a tubercle of the upper lip using a lip switch flap with left inferior labial arterial and venous pedicles , the philtrum and bridge of the nose .
the lower lip flap was designed to have a 10-mm width and a 17-mm length and transplanted to the upper lip .
its color immediately after surgery was favorable ( figure 1(a ) ) , but marked swelling of the flap and surrounding tissue was noted 6 h after surgery , with which the flap developed venous congestion ( figure 1(b ) ) . to release pressure due to the swelling on the vascular pedicle
, sutures around the pedicle were removed , but blood circulation of the flap did not improve and congestion aggravated .
since flap necrosis was predicted , the flap that had been transplanted to the upper lip was returned to the lower lip 8 h after surgery to improve its blood circulation and prevent necrosis . under venous sedation
, the flap transplanted to the upper lip was released to a free state so that the mouth could be opened , oropharyngeal intubation was applied and sedation was switched to general anesthesia .
hematoma around the flap was removed by washing well with saline . after confirming blood flow from the flap stump ,
the flap was returned to the lower lip and fixed by sutures , and the transplantation site was simply sutured ( figure 1(c ) ) .
( a ) at the completion of surgery , ( b ) 6 h after surgery and ( c ) the flap was returned to the lower lip .
after surgery , the flap was covered with heparinized saline - soaked gauze to further reduce congestion .
congestion was improved 7 h after surgery ( figure 2(a ) ) , and the flap returned to the donor site had almost completely recovered 1 week after surgery ( figure 2(b ) ) .
the flap was re - transplanted with sufficient tissue around the pedicle under local anesthesia ( figure 2(c ) ) .
as of 2 years after surgery ( figure 2(d ) ) , the cleft lip nose deformity had improved with a favorable course .
( a ) seven hours after returning the flap to the donor site , ( b ) 1 week after surgery , ( c ) re - transplantation of the lip switch flap and ( d ) 1 week after surgery .
in cleft lip patients with upper lip tissue defects , a complication of a lip switch flap , flap necrosis , results in failure of upper lip reconstruction .
the feeding artery of a lip switch flap , the inferior labial artery , is distributed within 15 mm from the upper end of the lower lip .
it is very thin , and 180 rotation of the vascular pedicle is likely to cause ischemia due to twisting and pressure . since the vein distributing along with the inferior labial artery is unclear , venous perfusion by the surrounding oral mucosa
but , when oral mucosa is too thick , there is the problem that the shape of upper lip comes to worse .
similarly , when pressure is loaded on the vascular pedicle and surrounding tissue by tension induced by flap rotation and swelling after surgery , venous congestion readily occurs , being a risk for partial or total necrosis .
since the vascular pedicle of the flap is very thin , the risk should be fully considered , and we encountered it in 2 of 32 patients treated over the last 10 years . in the present patients , venous congestion was treated by removing sutures around the vascular pedicle to release tension loaded by the surrounding tissue , such as mucosa and muscle , and phlebotomy was also applied , but these were ineffective , and congestion of the flap rapidly progressed , being likely to result in total necrosis .
we considered the application of medical leeches to improve congestion , but this was abandoned because the patient rejected it and the progression of congestion was rapid due to the small size of the flap .
when severe venous congestion develops in a lip switch flap , like this case , it is likely to rapidly progress and result in total necrosis .
thus , the early discovery of congestion by careful observation of the flap after surgery and rapid treatment upon suspecting congestion are very important
. the flap could be re - transplanted after it was returned to the donor site to sufficiently recover circulation in tissue around the vascular pedicle , and it engrafted and achieved a favorable upper lip morphology , reconfirming that , when congestion is suspected , the flap should be treated without hesitation .
various modifications have recently been added to the previous lip switch flap , and the vascular pedicle was further thinned to improve mobility in some modifications . in these two cases ,
attention should be paid to venous congestion , and preparation for its treatment may be important .
the authors report no conflicts of interest . the authors alone are responsible for the content and writing of this article . | abstractin a 21-year - old male with bilateral cleft lip who developed marked venous congestion of a lip switch flap , we returned the grafted flap to the donor site to improve blood circulation of the flap and then re - transplanted it , which prevented flap necrosis . here ,
we report the procedure and case . |
fine needle aspiration cytology ( fnac ) is the first - line diagnostic test for evaluating thyroid nodules .
it is a simple , rapid , and cost - effective test that can effectively distinguish between neoplastic and non - neoplastic lesions of the thyroid .
fnac can effectively triage patients with neoplastic thyroid nodules as to who require surgery and who do not .
however , due to the lack of a standardized system of reporting , pathologists have been using different terminologies and diagnostic criteria , thereby creating confusion amongst referring clinicians in the interpretation of the cytopathology report , ultimately hindering a definitive clinical management . to overcome this issue ,
multiple organizations have proposed diagnostic guidelines for reporting thyroid fnac results , including the papanicolaou society of cytopathology task force and the american thyroid association , although none have been universally accepted . in the year 2007
, the national cancer institute ( nci ) , bethesda , maryland , united states , organized the nci thyroid fine needle aspiration state - of - the - science conference , and an initiative was undertaken to publish an atlas and guidelines using a standardized nomenclature for the interpretation of thyroid fine needle aspirates ( fnas ) , known as the bethesda system for reporting thyroid cytopathology .
the atlas describes six diagnostic categories of lesions : non - diagnostic / unsatisfactory , benign , atypical follicular lesion of undetermined significance ( aflus ) , suspicious for follicular neoplasm ( sfn ) , suspicious for malignancy ( sm ) , and malignant .
the six diagnostic categories of the bethesda system have individual implied risks of malignancy that influence management paradigms .
this study was undertaken to elucidate the usefulness of the bethesda system in reporting thyroid fnas .
in our institute , it is the cytopathologist who performs the procedure of fnac and procures the aspirate , be it under ultrasound guidance or without guidance .
the smears thus prepared are stained by leishman - giemsa and papanicolaou stains and reporting is done within 24 hours . in the present study ,
we retrospectively collected thyroid fnac slides reported in our institute between april 2009 and march 2012 and reviewed them by three cytopathologists as per the current recommended bethesda nomenclature .
a smear was categorized as non - diagnostic if it did not fulfill the adequacy criteria laid down by the bethesda system . for a solid nodule ,
a specimen was considered adequate if it contained at least six well - preserved and well - stained follicular groups , containing at least ten cells .
in contrast , abundant thick colloid , as found in a colloid nodule , did not have a requirement for a minimum number of follicular cells .
thyroid cysts containing histiocytes but with little or no follicular cells were interpreted as non - diagnostic .
an aspirate smear containing significant cytological atypia , was never considered inadequate , regardless of cellularity .
cases were interpreted as benign if they showed the cytomorphological features of colloid goiter / adenomatoid goiter , hashimoto 's thyroiditis , thyrotoxicosis , de quervain 's thyroiditis , or granulomatous thyroiditis due to koch 's [ figure 1a ] .
( a ) microphotograph showing hashimoto 's thyroiditis with thyroid follicular cells showing hurthle cell change and lymphocytes in the background , categorized into the benign category ( pap , 400 ) , ( b ) microphotograph of a smear categorized as aflus , showing predominantly benign thyroid follicular cells in sheets , with some cells showing anisonucleosis and forming microfollicles ( pap , 400 ) as per the guidelines of the bethesda system , aspirates which were considered adequate , had some features of atypia but could not be categorized definitely into either of the benign , sfn , sm , or malignancy categories were grouped under this category . for example , a smear that showed cytological features of high cellularity , tiny follicular cells arranged in sheets , clusters or singly , with occasional occurrence of multinucleated giant cells , and focal occurrence of hurthle cells was described as aflus [ figure 1b ]
. aspirates with cytomorphologic features of moderate to high cellularity , scant or absent colloid , with predominantly microfollicular or trabecular configuration of follicular cells in repetitive pattern were grouped under this category .
aspirates that had cytological features suggestive of , but not definitive of , papillary carcinoma , medullary carcinoma , or lymphoma were placed in this category .
for example , an aspirate that had cytological features of high cellularity , comprised elongated oblong cells with occasional plasmacytoid appearance with some cells showing hurthle cell change and possessed a matrix different from colloid , possibly amyloid , was classified as suspicious for medullary carcinoma .
( a ) thyroid follicular cells arranged in papillae with anatomical borders ( pap , 400 ) , ( b ) nuclear grooving and pseudoinclusion ( arrow ) ( pap 400 ) , ( c ) cyst macrophages in the background ( lg , 400 ) , ( d ) multinucleated giant cells ( lg 400 ) we could pursue follow - up histology for 323 cases . we compared the diagnoses offered in fnac in the light of the bethesda system , with the diagnoses obtained on final histopathologic examination ( hpe ) , for these 323 cases . thereby , we could calculate the malignancy risk for each category and compared it with that in other studies .
incidental papillary carcinomas ( < 1 cm ) on resection were not considered malignant , except when prior cytologic interpretation was sm or malignant .
a smear was categorized as non - diagnostic if it did not fulfill the adequacy criteria laid down by the bethesda system . for a solid nodule ,
a specimen was considered adequate if it contained at least six well - preserved and well - stained follicular groups , containing at least ten cells . in contrast , abundant thick colloid , as found in a colloid nodule , did not have a requirement for a minimum number of follicular cells .
thyroid cysts containing histiocytes but with little or no follicular cells were interpreted as non - diagnostic .
an aspirate smear containing significant cytological atypia , was never considered inadequate , regardless of cellularity .
cases were interpreted as benign if they showed the cytomorphological features of colloid goiter / adenomatoid goiter , hashimoto 's thyroiditis , thyrotoxicosis , de quervain 's thyroiditis , or granulomatous thyroiditis due to koch 's [ figure 1a ] .
( a ) microphotograph showing hashimoto 's thyroiditis with thyroid follicular cells showing hurthle cell change and lymphocytes in the background , categorized into the benign category ( pap , 400 ) , ( b ) microphotograph of a smear categorized as aflus , showing predominantly benign thyroid follicular cells in sheets , with some cells showing anisonucleosis and forming microfollicles ( pap , 400 )
as per the guidelines of the bethesda system , aspirates which were considered adequate , had some features of atypia but could not be categorized definitely into either of the benign , sfn , sm , or malignancy categories were grouped under this category . for example , a smear that showed cytological features of high cellularity , tiny follicular cells arranged in sheets , clusters or singly , with occasional occurrence of multinucleated giant cells , and focal occurrence of hurthle cells was described as aflus [ figure 1b ] .
aspirates with cytomorphologic features of moderate to high cellularity , scant or absent colloid , with predominantly microfollicular or trabecular configuration of follicular cells in repetitive pattern were grouped under this category .
aspirates that had cytological features suggestive of , but not definitive of , papillary carcinoma , medullary carcinoma , or lymphoma were placed in this category .
for example , an aspirate that had cytological features of high cellularity , comprised elongated oblong cells with occasional plasmacytoid appearance with some cells showing hurthle cell change and possessed a matrix different from colloid , possibly amyloid , was classified as suspicious for medullary carcinoma .
microphotograph of a smear categorized as papillary carcinoma . ( a ) thyroid follicular cells arranged in papillae with anatomical borders ( pap , 400 ) , ( b ) nuclear grooving and pseudoinclusion ( arrow ) ( pap 400 ) , ( c ) cyst macrophages in the background ( lg , 400 ) , ( d ) multinucleated giant cells ( lg 400 )
we compared the diagnoses offered in fnac in the light of the bethesda system , with the diagnoses obtained on final histopathologic examination ( hpe ) , for these 323 cases .
thereby , we could calculate the malignancy risk for each category and compared it with that in other studies .
incidental papillary carcinomas ( < 1 cm ) on resection were not considered malignant , except when prior cytologic interpretation was sm or malignant .
of the 1020 cases who underwent fnac during the period from april 2009 to march 2012 , initially , 69 cases ( 6.8% ) turned out to be non - diagnostic , 847 ( 83% ) benign , 9 ( 0.9% ) aflus , 42 ( 4.1% ) sfn , 14 ( 1.4% ) sm , and 39 ( 3.8% ) malignant .
also , 69 cases who were initially non - diagnostic , were re - aspirated , out of which , 39 cases were re - aspirated under ultrasound guidance . after re - aspiration , 12 cases ( 1.2% ) out of 1020 patients remained non - diagnostic .
the distribution of cases in the other bethesda categories following re - aspiration were benign 893 cases ( 87.5% ) , aflus 10 cases ( 1% ) , sfn 43 cases ( 4.2% ) including 7 hurthle cell types , sm 14 cases ( 1.4% ) , and malignancy 48 cases ( 4.7% ) [ table 1 ] .
distribution of cases in the bethesda categories as per our study ( n=1020 ) out of 1020 cases , 323 cases were available for follow - up histopathology .
out of these 323 cases , 3 cases had original fna diagnoses as non - diagnostic , 222 cases as benign , 5 cases as aflus , 36 cases as sfn , 12 cases as sm , and 45 cases as malignant .
we compared the original fna diagnoses of these 323 cases with the diagnoses obtained on hpe and calculated the malignancy risk for each category [ table 2 ] .
comparison of pre - operative fnac diagnoses with the diagnoses on hpe after surgical resection , and calculation of malignancy risk for each bethesda category accordingly , none of the three cases who had original fna diagnoses as non - diagnostic were reported to be malignant on follow - up hpe .
out of the 893 benign cases , specimens of 222 cases were available for follow - up hpe , among which , 10 cases were found to be malignant .
five out of the 10 cases reported as aflus could be followed - up for hpe .
one was found to be malignant , giving a malignancy risk of 20% in this category .
thirty - six out of the 43 cases of sfn were available for hpe ; 11 out of which were found to be malignant , giving a malignancy risk of 30.6% in this category .
nine out of the 12 surgically resected cases of sm were found to be malignant by hpe in the follow - up period , giving a malignancy risk of 75% in this category .
forty - five out of the 48 patients in the malignancy category were available for hpe and 44 cases turned out to be actually malignant , giving a malignancy risk of 97.8% in this category .
while reviewing the fnacs in our study , it was noted that many of the descriptions and diagnoses that were offered previously seemed like a vague and complicated jargon of pathological terminologies , which would be confusing and of no clinical significance to the clinicians .
however , when we recorded the diagnoses as per the criteria laid down in the standardized nomenclature of the bethesda system , it seemed more simplified , systematic , with greater clarity ; thus , it would prove to be more useful in guiding clinicians towards the management of thyroid nodules .
we compared the results obtained in our study with the studies of jo et al .
, yassa et al . , yang et al . , and nayar and ivanovic [ tables 3 and 4 ] .
comparison of the percentages of distribution of fine needle aspiration diagnoses of present study with other studies comparison of the percentages of follow - up malignancy of present study with other studies it was seen that the distribution of cases as per the six - tier bethesda system in our study differed from that in the above mentioned studies , with the percentage of cases in the benign category being higher and that in the non - diagnostic and aflus categories being lower .
the reason for the number of cases in the benign category being higher can be attributed to the fact that , our institute , despite being a tertiary care centre , not only caters to the needs of patients on a referral basis , but also patients come here directly without referral .
so a large population , representative of the general population , is encountered in our institute .
therefore , the proportion of benign cases that is a lot higher in the general population , is reflected proportionately in our study . according to jo et al .
most of the above mentioned studies have been done in tertiary care centers , where patients come only on a referral basis and , hence , is not exactly representative of the general population .
a second reason for our institute serving a large representative population is the fact that the procedure of fnac is easily accessible to the economically backward sections of the society for whom the test can be performed free of cost .
the reason for the lower percentage in the non - diagnostic and aflus categories can be attributed to the fact that , in our institute , usually an ultrasound - guided fnac is performed for small nodules or nodules that appear heterogeneous on palpation , so that the aspirate can be procured from the exact pathological site .
another important point is that , as the cytopathologist himself performs the procedure of fnac in our institute , a better quality and adequate aspirate can be ensured .
these probably have led to a reduction in the non - diagnostic or indeterminate diagnoses , thereby allowing a more specific cytological diagnosis .
the malignancy risk for the different categories in our study , as seen by follow - up hpe , has corroborated well with the implied risks mentioned in the bethesda system and also with the studies of jo et al .
, yassa et al . , yang et al . , and nayar and ivanovic though few differences have been noted .
first of all , the malignancy risk for the non - diagnostic category is 0% as compared to the higher rates in the other studies .
secondly , the malignancy risk for the aflus category is 20% as compared to 6% in the study of nayar and ivanovic .
both findings can be explained by the fact that , in our study , we have a smaller denominator population for the non - diagnostic and aflus categories , as a result of which the malignancy risk of the non - diagnostic category with other studies and malignancy risk of the aflus category with the study of nayar and ivanovic can not be accurately compared . as we were doing a retrospective study ,
clinical , biochemical , and radiological correlation was not available for few cases , due to which we faced a slight difficulty in categorizing those aspirates .
secondly , few slides , especially those older than two years , had a faded quality of staining , which even after re - staining did not gain the quality of a freshly stained smear and was thus found difficult to report .
the bethesda system is very useful for a standardized system of reporting thyroid cytopathology , improving communication between cytopathologists and clinicians , and inter - laboratory agreement , leading to more consistent management approaches .
the high malignancy risk for the aflus , sm , and malignancy categories reflects the importance of these categories in the six - tier bethesda system .
however , a prospective study over a larger population would lend more insight into the merits and demerits of the proposed system . | background : the bethesda system for reporting thyroid cytopathology represents a major step towards standardization , reproducibility , improved clinical significance , and greater predictive value of thyroid fine needle aspirates ( fnas).aims : to elucidate the utility of the bethesda system in reporting thyroid fnas.materials and methods : we retrospectively reviewed thyroid fnas between april 2009 and march 2012 , classified them using the bethesda system , found out the distribution of cases in each bethesda category , and calculated the malignancy risk for each category by follow - up histopathology.results:of the 1020 fnas , 1.2% were non - diagnostic , 87.5% were benign , 1% were atypical follicular lesion of undetermined significance ( aflus ) , 4.2% were suspicious for follicular neoplasm ( sfn ) , 1.4% were suspicious for malignancy ( sm ) , and 4.7% malignant .
of 69 cases originally interpreted as non - diagnostic , 12 remained non - diagnostic after re - aspiration . in 323 cases ,
data of follow - up histopathologic examination ( hpe ) were available .
rates of malignancy reported on follow - up hpe were non - diagnostic 0% , benign 4.5% , aflus 20% , sfn 30.6% , sm 75% , and malignant 97.8%.conclusions : reviewing the thyroid fnas with the bethesda system allowed a more specific cytological diagnosis . in this study ,
the distribution of cases in the bethesda categories differed from some studies , with the number of benign cases being higher and the number of non - diagnostic and aflus cases being lower .
the malignancy risk for each category correlated well with other studies .
the bethesda system thus allows standardization in reporting , improves perceptions of diagnostic terminology between cytopathologists and clinicians , and leads to more consistent management approaches . |
in canada and many other developed countries , over 50% of deaths each year take place in hospital . among healthcare professionals ,
nurses are the most responsible for furthering the possibility of good deaths in hospital .
hospital nurses are not only directly accountable for providing hands - on care and for advocating for appropriate end - of - life ( eol ) care as staff nurses and as clinical nurse specialists or nurse practitioners , but nurse managers have additional responsibilities to ensure that high - quality eol care is consistently provided .
many recent health system and other changes could have impacted the care of dying persons in hospital [ 24 ] . the shift from inpatient - based to ambulatory or outpatient care and the many technological advancements to date
are of particular note , as these have contributed to shorter and more acute inpatient episodes , changes that could threaten the opportunity to recognize active dying processes and devise appropriate eol care plans .
unfortunately , a relatively recent study revealed that dying hospital patients have higher rates of unmet needs as compared to those who die at home .
an understanding of current eol care needs and care practices in hospital is thus needed for hospital quality assurance and improvement purposes . to address this need ,
a descriptive - comparative study was undertaken in two accessible full - service western - canadian acute care hospitals .
all charts for patients who died from august 1 , 2008 through july 31 , 2009 were examined using a standardized ( post - death ) review process .
although the findings of this study can not be generalized to other hospitals in canada or elsewhere , this study provides current evidence for broad - based reflection .
reflection on trends is also possible as the findings of this study are compared to a similar mid-1990s canadian study of eol care needs and provided care .
most deaths in developed countries occur now in advanced old age and typically at the end of a noticeable decline in health with one or more chronic incurable illnesses .
it is not surprising then that 8095% of all deaths in canada and other developed countries are now considered expected or anticipated because of a clearly evident and irreversible declining health state .
only a small proportion of deaths now are due to sudden catastrophic illnesses or accidents , with these unexpected deaths providing much less opportunity to plan or prepare for and potentially enable good deaths .
good deaths have become a major research focus , with a rapidly growing number of studies seeking to determine the factors that contribute to good deaths or that define a good death . to date , many factors have been identified as important or potentially important , including dying at home or in your home community .
an enduring concern exists about the overuse of life - prolonging technologies and treatments in hospital .
it is imperative that hospital eol care be as humane as possible , as life - extending care could be in direct opposition to the care needs and care preferences of terminally ill or dying hospital patients .
despite some data issues and other limitations , chart reviews have been a common method for many years of acquiring information that is useful for healthcare policy and practice changes .
chart reviews allow for many data variables to be studied over the routinely collected minimum hospital dataset information .
chart reviews are also common in palliative and eol research because concurrent studies could burden dying persons and their caregivers . as indicated above , a chart review in two full - service hospitals was conducted in one canadian city .
the charts of all persons who died in these two hospitals over a recent 12-month period were reviewed following research ethics approval from a university 's health research ethics committee , hospital administrative approval , and the receipt of grant funding to permit data collection .
what care was needed and provided before death in hospital and did this care differ in relation to select patient characteristics ?
what proportion of decedents had an expected death , and was care appropriate for expected versus unexpected deaths ? as outlined in the appended data variables , a wide range of socio - demographic , care needs and care data were of interest .
the data variables listed were decided upon by the principal investigator and co - investigator after a review of the literature and after a search of five charts to determine what data existed and were thus possible to collect from the charts . following this ,
the data were collected by a research assistant who was directly supervised by the principal investigator . to ensure that correct data were collected , the principal investigator and the research assistant first independently collected data from the same five charts and then compared their findings .
any differences were discussed and rectified , using the charts as reference points . following this , ongoing monitoring of the data collection process and
data were maintained with the data collected also assessed by the co - investigator for completeness and possible data errors .
all chart data were entered into a computer spreadsheet and analyzed using spss ( version 18 ) .
statistical testing primarily involved basic descriptive statistics and bivariate tests , as comparisons were made ( chi - square [ ] primarily , as appropriate for the level of variables ) .
a total of 1,018 persons died in the two hospitals , with all 1,018 charts reviewed . the majority ( 78.9% )
were admitted to hospital via the emergency room ( er ) and 92 ( 9.0% ) died in the er . hospital stays averaged 19.9 days , with stays ranging from 1 to 367 days ( median = 9 , sd = 32.0 ) ; however , the most common stay was 1 day ( 11.5% ) , followed by 2 days ( 7.8% ) , 3 days ( 6.6% ) , and 4 days ( 6.0% ) .
most ( 88.4% ) patients had no surgery performed . in 2/3 cases ( 68.6% ) , a family member was present at the time of death .
deaths were unevenly distributed over the year , with more occurring in the winter months and fewer in the summer months , as illustrated by a high of 10.8% in march and low of 6.4% in august .
friday had the highest share of deaths ( 16.6% ) and sunday the lowest share ( 12.6% ) .
the mean age of decedents was 72.5 ( range = birth 101 , median = 77 ) , with the majority aged 65 + ( 73.8% ) and most having been urbanites ( 79.5% ) who resided in the city where the two hospitals were located .
the most common primary diagnosis was cancer ( 36.2% ) , followed by cardiovascular disease ( 23.0% ) and respiratory disease ( 18.8% ) .
comorbidities were common ( mean = 5.9 , median = 5 , range = 025 ) . among all 1,018 decedents ,
13.8% had cpr performed at some point during this hospitalization , with only 89 patients ( 8.8% ) having cpr performed immediately prior to death being pronounced .
in addition , 16.5% received care in a critical care unit ( ccu ) , while 21.7% died on a palliative care unit ( pcu ) and 25.7% had a palliative care referral .
in contrast , 89.0% had oxygen and 89.0% had an intravenous ( iv ) infusion in use at the time of death ; 97.3% had one or more technologies in use at the eol .
only 7.0% had one or more technologies withdrawn before death , with these withdrawn at various stages throughout the hospital stays .
as shown in table 1 , considerable differences existed in care needs and provided care among the decedents . of particular note , patients dying of cardiovascular causes , younger patients , males , and rural patients were more likely to have had cpr performed , both at the end of life and at any time during this hospitalization .
similarly , younger persons , males , rural residents , and those dying of cardiovascular disease were more likely to have received ccu care .
younger patients , rural persons , and those dying of cardiovascular diseases were also the most likely to have one or more technologies withdrawn before death whereas patients dying of cancer were the least likely to have had any technologies withdrawn .
patients who died on a pcu and also those who received a palliative care referral were more often younger , married , and a suburbanite .
patients with a respiratory disorder were more likely to have oxygen in use at the eol and those with a cardiovascular disorder were more likely to have an iv in use at the eol .
in addition , 77.0% had pain recorded as present on the day of death , with younger patients and patients diagnosed with cancer more likely to be in pain on the day of death .
among all patients who indicated or showed that they had pain on the day of death , 98.6% received analgesia . in the few cases where analgesia was not given ,
analgesic use was only recorded when potent analgesics , such as opioids , were given .
younger patients and patients with cancer were more likely to receive analgesia on the last day of life .
as shown in table 2 , care needs and the care provided differed in relation to the ability to mobilize and perform activities of daily living ( adl ) .
males and patients dying of cardiovascular causes were more likely to be able to walk unassisted and to perform self care .
younger patients were also more likely to be able to perform self care without assistance .
in contrast , older patients , female patients , and patients suffering from all disorders other than cardiovascular illnesses were more likely to be bedridden and/or to need complete or partial adl care .
patient deaths were determined to be expected if a dnr ( do - not - resuscitate ) order was written before death ; 87.5% of all 1,018 patients had this order written in their chart .
hospital policy requires dnr orders to be written when indicated by the clinical state of the patient , and after the patient and/or family have been consulted ( whenever possible ) or whenever they have indicated that they do not want resuscitation to be attempted . dnr orders were written 1 to 367 days in advance of death occurring , with a mean of 11.9 days and median of 7 days .
most dnr orders were written close to death ; however , with 11.9% written within 1 day of death , 11.4% within 2 days , 8.7% within 3 days , 7.3% within 4 days , and 5.0% within 5 days .
as shown in table 3 , patients with a dnr order were more likely to receive care in a pcu and/or receive a palliative care referral .
similarly , patients with a dnr order were less likely to die with an iv infusing , but they were also less likely to have any technologies withdrawn .
patients with dnr orders were considerably more likely to have pain and to receive analgesia as compared to patients without a dnr order .
patients with dnr orders were also much more likely to be bedridden or needing assistance to walk , and they also were more likely to need total adl care or to require some assistance with adl as compared to the patients who died without a dnr order .
although chart reviews do not capture all relevant eol data , this retrospective descriptive - comparative chart review conducted at two full - service hospitals in a canadian city revealed much about the eol care needs and care provided to 1,018 patients who died over a one - year period .
the following outlines these findings and compares them to other studies , although primarily wilson 's study , as it was a similar study conducted in 1994 in canada .
that study was confined , however , to the use of cpr , and the presence of dnr orders and technologies at the end of life , with minimal data collected on the patient 's age and gender , and their ability to walk and care for self near the end of life .
more deaths occurred in winter than summer and in the middle of the night shift , findings that likely confirm what many practicing hospital nurses have noticed over the years .
the decedents were mostly older , another unsurprising finding , as death typically takes place in old age now in developed countries .
as also expected , given population mortality trends [ 6 , 12 , 13 ] , the most common diagnosis was cancer , followed by cardiovascular and respiratory - related deaths , and with comorbidities common .
these findings help to understand the high proportion ( 1/3 ) of patients who provided an advance directive , with these directives potentially demonstrating their personal and likely family awareness of impending death and/or their consideration of the inappropriateness of resuscitation efforts .
death has previously been noted among older persons and persons suffering from end - stage illnesses , with this desire now thought to be leading to more deaths taking place outside of hospital . a very high proportion ( 87.5% )
these findings collectively show hospital nurses and physicians are increasingly engaging in decisions to withhold cpr whenever resuscitation is not indicated , appropriate , or desired by the patient and/or their family .
although some may have concerns that treatment withdrawing or withholding occurs too often now , and that all patients should have resuscitation routinely attempted , this study found that only 7.0% had any technologies stopped over the course of this final hospitalization .
most deaths in these two hospitals arguably were not due to treatment withdrawal but instead to uninterrupted dying processes . this conclusion is supported by the finding that only 8.8% had cpr performed immediately prior to death and only 13.8% had cpr performed at some point during this hospitalization .
the finding that dnr orders were more often written for patients who were bedridden or needing assistance with mobility and for patients who needed full or partial assistance to complete basic adls helps to demonstrate that some basic indicators could be signaling impending and irrevocable death , and thus necessitating actions to prepare for this eventuality
. there could be many indicators , however , that contribute to dnr orders being written , including the evidence - based awareness that cpr and other resuscitative measures would have a high probability of either failing completely to sustain life , or only serving to extend a dying process and thus potentially increasing pain and suffering near death .
further reading of these charts could perhaps also reveal that a serious and noticeable decline in health had occurred , with this decline being a chief factor for dnr orders in hospital and for advance directives to have been developed by patients before this hospitalization . in canada , where all citizens have universal access to health services , insurance status and personal income have little bearing on hospital admission rates and
is therefore defined by past customs and also by practices arising from an increasing evidence base .
it was not surprising to find that younger patients , those diagnosed with cardiovascular diseases , and rural patients were less likely to have a written dnr order and more likely to have had cpr performed .
younger patients would tend to have more acute and shorter - term illnesses and greater potential for recovery as compared to older patients .
cardiovascular diseases are marked by acute episodes and they are more unpredictable in terms of treatment response , as compared to cancer where death often occurs after all possible treatments have failed .
respiratory - related deaths are also more predictable than cardiovascular deaths , as respiratory failure typically occurs now after all available treatments are no longer effective at maintaining life .
rural people who receive care in city hospitals are also more likely to be suffering from potentially treatable conditions , as this health state would be responsible for their transfer from smaller rural hospitals and for direct er admissions . as such ,
cpr was not an uncommon form of care provided in these two hospitals , but it was more often reserved for persons who would likely benefit from it essentially younger and rural patients , and those diagnosed with cardiovascular illnesses .
similarly , the patients who died in a pcu or who received a palliative care referral were more often younger .
however , these patients were typically diagnosed with cancer , and cancer has long been known to have severe and difficult to manage pain and other symptoms . as such , it is not surprising that younger persons would be the most likely not only to have cpr performed but also to receive specialized palliative care services before death . at the same time , these findings raise concern for older patients dying of cancer and other causes , as they too could have a high burden of pain and other symptoms .
although specialized palliative care services were oriented to younger persons and those suffering from cancer , basic technologies were a common form of eol care for all patients everywhere .
most decedents had an iv infusing ( 89.0% ) and/or oxygen in use ( 89.0% ) when dying .
nearly all ( 97.3% ) had one or more technologies in use at the time of death .
this eol practice routine is very similar to wilson 's finding that 94.2% of dying hospital patients had one or more continuous technologies ( mainly oxygen and iv fluids ) in use at the time of death .
although it is possible that these life - extending technologies were used to deliver analgesics and to otherwise ease the dying process , their use is highly controversial [ 16 , 17 ] .
oxygen and iv fluids can extend the dying process , and they can also exacerbate or increase eol suffering [ 16 , 17 ] .
oxygen , for instance , is uncomfortable when provided at high flow rates and when masks are placed tightly over the nose and mouth .
oxygen masks can prevent or reduce communication with loved ones and nursing staff , and oxygen masks can mar the facial features and thus final memories of loved ones .
ivs can contribute to restlessness and wet beds as renal output often continues well into the dying process .
pulmonary edema can occur or be exacerbated by iv therapy , with restlessness , falls , and other issues as a result .
ivs also have the problem that they need to be restarted at times , with all starts painful , and with restraints needed at times to ensure that the iv remains in place .
practicing nurses could possibly identify other concerns with the routine use of ivs and oxygen at the eol , but they may also identify some value in their use . nursing research into eol care practices is obviously needed , and particularly as this study found that a large proportion of dying patients were bedridden or needing assistance to mobilize , and a large proportion of dying patients were either needing complete or partial adl assistance . as such , it is clear that dying patients typically require a great deal of basic nursing care , and this care has not been acknowledged in hospital staffing patterns or staff requirements . instead , eol care appears to be routinely technologically based , with concerns then that opportunities for a more individualized and humanized process leading to death are lost .
this concern about technologically based eol care could be supported by the finding that the cpr rate has apparently increased since 1994 , from 2.9% then to 8.8% now at the time of death , and with 13.8% of current patients receiving cpr at some point during their hospital stay .
although this apparent change may suggest that more people are being aggressively treated in hospital to prevent death , it should be remembered that many more people are dying at home or in nursing homes now .
the shift of death and dying out of hospital , or at least expected deaths out of hospital , is illustrated by a comparison of the current study 's findings with wilson 's study which found that no patients in 1994 provided an advance directive and only 13.1% of patients then verbalized no - cpr preferences . in the current study ,
these comparative findings illustrate an increasing realization and acceptance among patients and likely families of imminent and irrevocable death .
the current study 's finding of a very high rate of dnr orders and the finding that only 16.5% of patients received care in a ccu at some point during their entire hospital stay are also highly important for indicating that hospital nurses and physicians now are increasingly realizing and accepting states of imminent and irrevocable death .
wilson 's previous study found that 27.0% of hospital decedents died in a ccu , while the current study found that only 16.5% had received ccu care at some point during their hospital stay .
another key current finding is the exceptionally high provision of analgesia ( 98.7% ) on the last day of life .
this finding is in contrast to wilson 's previous study which found that analgesia was given infrequently in the last 13 days of life . as such
, the findings of the current study suggest that some improvements in eol care have occurred .
although technologies still appear to be normal practice , and cpr is increasingly used in hospital , there are several indications that impending death and active dying are more often recognized in hospital now .
in addition , with this recognition , it would appear now that care measures considered appropriate for the eol are routinely provided .
this study , although confined to two canadian hospitals and thus with findings that can not be generalized across canada or outside of canada , helps to focus attention to death and dying in hospital . in canada and
other countries , a considerable portion of deaths each year occur in hospital , with these deaths expected or unexpected .
differences in care needs and in provided care were found when death was expected versus unexpected .
the findings of this study also show , as compared to those reported in a similar mid-1990s canadian study , that impending death is more often openly recognized now by hospital nurses and physicians , as well as terminally ill or dying patients and/or their families .
technologies continue to be routinely but controversially used , as they can extend life and it is not certain that these are effective eol comfort measures
. the increased rate of cpr from 2.9% previously to 8.8% could reflect the 1994 + shift of ( primarily expected ) deaths out of hospital , with hospital - based eol care being more complex now that there is a need for a rapid determination that death is not only inevitable but also imminent .
this chart review also identified some areas for research attention and/or improvements in eol care .
given the nature of nursing work , nurses are charged with these future developments to ensure that eol care is comforting , and that letting dying patients die is not only increasingly possible but these patients also are well supported in hospitals and all other places .
chart number ( starting with 1 ) residence ( rural , urban , suburban ) admit type ( er or planned admission ) living will present , yes or no dnr order present , yes or not number of days between dnr order and death withdrawal of technology , yes or no sudden death , yes or no level of consciousness near death ( alert , semiconscious , unconscious ) expected death , yes or no mobility on day of death ( walking independently , walking with assistance , or not walking ) adl requirement ( independent adl , assisted adl , or total adl assistance ) pain 3 days prior to death , yes or no pain on day of death , yes or no surgery performed during hospitalization , yes or no icu / ccu care provided during hospitalization , yes or no palliative care unit care provided during hospitalization , yes or no palliative care referral during hospitalization , yes or no technologies in use ( total number and types at time of death ) cpr at end of life , yes or no cpr prior to end of life , yes or no cpr during entire stay , yes or no analgesic on day of death , yes and types or no analgesic 3 days prior to death , yes and types or no oxygen in use at time of death , yes or no iv infusing at time of death , yes or no family present at death , yes or no . | a descriptive - comparative study was undertaken to examine current end - of - life care needs and practices in hospital .
a chart review for all 1,018 persons who died from august 1 , 2008 through july 31 , 2009 in two full - service canadian hospitals was conducted .
most decedents were elderly ( 73.8% ) and urbanite ( 79.5% ) , and cancer was the most common diagnosis ( 36.2% ) .
only 13.8% had cpr performed at some point during this hospitalization and 8.8% had cpr immediately preceding death , with 87.5% having a dnr order and 30.8% providing an advance directive .
most ( 97.3% ) had one or more life - sustaining technologies in use at the time of death .
these figures indicate , when compared to those in a similar mid-1990s canadian study , that impending death is more often openly recognized and addressed .
technologies continue to be routinely but controversially used .
the increased rate of end - stage cpr from 2.9% to 8.8% could reflect a 1994 + shift of expected deaths out of hospital . |
the long - range atmospheric transport of
particulate matter ( pm)-bound
polycyclic aromatic hydrocarbons ( pahs ) and nitro - pahs ( npahs ) to
remote sites , including mountains in france , norway , sweden , czech republic , and the canadian arctic , as well as trans - pacific transport to the olympic
peninsula of washington and to oregon has
been documented .
once emitted from combustion sources , some pahs undergo
reaction with oh radicals , no3 radicals , n2o5 , and o3 , converting the parent pahs into more
polar species , including npahs .
the transformation of pahs presumably
can occur locally ( i.e. , near emission sources ) and/or enroute to
downwind receptor sites .
since some pah derivatives exhibit higher
direct acting mutagenicity than their parent pahs , human
exposure to pah derivatives , including npahs , is of interest and these
pah derivatives have been detected at many sites throughout the world .
some npahs are classified as probable or possible human
carcinogens and have been identified
as major contributors to the overall direct - acting mutagenicity of
ambient pm despite being present at much lower concentrations than
those of their parent pahs .
the
reactivity of pm - bound pahs varies , to some extent , with the
composition and the microenvironment of the particles .
the mineral content , organic and black carbon concentrations , water
content , physical state of the organic layer surrounding the core
of the particles , and surface coverage of parent pah may all influence
the reactivity of pm - bound pahs .
various artificial substrates , including silica , graphite , diesel
soot , fly ash , wood smoke , and kerosene soot , have been used in laboratory
experiments to simulate pm - bound pah reactions and derive heterogeneous
rate coefficients .
we recently reported that pahs with more than 4 rings sorbed to
quartz fiber filters are transformed to npahs by reaction with no3/n2o5 .
however ,
few laboratory studies have been conducted on the transformation of
pahs and npahs on ambient pm , which can provide important
information needed in extrapolating model studies to ambient conditions .
we concluded from our recent study of the formation of npah from
reactions of ambient particles with no3/n2o5 that pah in pm quickly become deactivated
or unavailable for nitration .
the evidence
included little formation of 1-nitropyrene ( 1-np ) from no3/n2o5 exposures of daytime ambient pm samples ,
compared to nighttime samples with similar pyrene concentration , and
relatively little formation of 1-np in both daytime and nighttime
samples from downwind sites ( riverside and banning , ca ) upon exposure
to no3/n2o5 , relative to exposure
of a downtown los angeles , ca , nighttime pm sample .
we speculated that daytime gas - phase reactions of volatile
organic compounds ( vocs ) with oh radicals and/or o3 producing
products , which then adsorbed onto the pm , were responsible for the
apparent deactivation of the pm - bound pahs .
in addition ,
2-nitrofluoranthene ( 2-nf ) , the most abundant npah in the unexposed
ambient pm samples , was not formed from the heterogeneous exposure
of the pm to no3/n2o5 .
the lack of
heterogeneous formation of 2-nf is consistent with 2-nf being only
formed during gas - phase radical - initiated reactions of fluoranthene
and confirms its usefulness as a marker of atmospheric aging
( ref ( 33 ) and references
therein ) . in this earlier work ,
ambient 24 h samples from beijing ,
china ,
and a los angeles nighttime sample were the most reactive samples
in terms of 1-np formation .
the objectives of our present research
were to ( 1 ) confirm the reactivity differences for no3/n2o5 exposure between samples collected at an urban
site , namely , beijing , china , and the downwind site of riverside ,
ca .
beijing pm has very high pah and npah concentrations resulting
from strong primary emissions , while
riverside can be strongly influenced by chemically aged pm transported
from upwind areas in the los angeles air basin , in addition to local
primary emissions ; ( 2 ) extend the study of the reactivity
of pm - bound pahs and npahs to include heterogeneous exposures to oh
radicals and o3 ; and ( 3 ) examine differences in bacterial
mutagenicity of the pm extracts from beijing prior to and after heterogeneous
exposures .
the pah and npah measurements were carried out using gc / ms ,
and the salmonella mutagenicity assay was conducted with and without
microsomal activation . to simulate long - range atmospheric transport
conditions in our environmental chamber , the average concentrations
of no3 radicals , oh radicals , and o3 over the
8 h chamber exposure periods were 420 ppt , 0.8
ppt , and 800 ppb , respectively .
these concentrations were
equivalent to 7 days exposure to ambient concentrations of
no3 radicals , oh radicals , and o3 ( see supporting information for further discussion ) ,
noting that the n2o5 concentrations in the chamber
no3/n2o5 exposures were at least
2 orders of magnitude higher than average ambient concentration .
the
23 parent pahs and 20 npahs measured
( and their abbreviations ) are listed in table 1 . deuterium - labeled pahs and npahs were purchased from cdn isotopes
( point - claire , quebec , canada ) and cambridge isotope laboratories
( andover , ma ) .
the isotopically labeled pah and npah surrogates used
as recovery controls included fluorene - d10 , phenanthrene - d10 , pyrene - d10 , triphenylene - d12 , benzo[a]pyrene - d12 , benzo[ghi]perylene - d12 , 1-nitronaphthalene - d7 , 5-nitroacenaphthene - d9 , 9-nitroanthracene - d9 , 3-nitrofluoranthene - d9 , 1-nitropyrene - d9 , and 6-nitrochrysene - d11 .
the labeled pah and npah internal standards included acenaphthene - d10 , fluoranthene - d10 , benzo[k]fluoranthene - d12 , 2-nitrobiphenyl - d9 , and 2-nitrofluorene - d9 .
purchased from
accustandard ( new
haven , ct ) and chem service ( west chester , pa ) . purchased from chiron as ( norway ) ,
accustandard ( new haven , ct ) , chem service ( west chester , pa ) , and
sigma - aldrich corp .
the beijing sampling site was located
on the roof of the 7-story ( about 25 m above ground ) geology building
on the peking university campus ( pku ) .
this site
is located in northwestern beijing and is primarily a residential
and commercial area .
dominant pah emission sources near the site include
vehicular traffic and fuel combustion for cooking .
pm2.5 and pm10 were collected on prebaked ( 350 c ) quartz
fiber filters ( no.1851 - 865 , tisch environmental , cleves , oh ) using
a high volume cascade impactor ( series 230 , tisch environmental , cleves ,
oh ) .
pm was collected continuously over 24 h periods , with the sampler
being changed over in the late morning .
pm10 and
pm2.5 samples were collected from may 2009 to february
2010 and in april 2011 , respectively ( table si.1 , supporting information ) .
briefly , sampling in riverside
was conducted at a site in the agricultural operations area at the
university of california , riverside , campus , approximately 90 km downwind
of los angeles .
pm2.5 samples were
collected during october 1997 using an ultrahigh volume particulate
sampler , containing four teflon - impregnated glass fiber ( tigf ) filters
( each 40.6 cm 50.8 cm ) .
after collection , the filters were
stored at 20 c . for this study ,
three 20.3 cm
25.4 cm portions were cut out from 40.6 cm 50.8 cm filters
( table si.1 , supporting information ) .
the average flow rate for each cut - out filter portion was 1
m min .
beijing pm pah and
npah concentrations vary significantly from day - to - day . in order to measure changes in the pah and npah
concentrations with and without exposure in the chamber for the chemical
study ,
single 24 h 20.3 cm 25.4 cm filter samples were cut
into six equal portions of 8.5 cm 10.2 cm ( figure si.1 , supporting information ) .
three 8.5 cm
10.2 cm portions were exposed in the chamber , and the remaining three
8.5 cm 10.2 cm portions of single 24 h filter samples were
used as unexposed controls . in order to measure changes in the mutagenicity
of the pm with and without exposure in the chamber for the mutagenicity
study ,
single 24 h 20.3 cm 25.4 cm filter samples were cut
into four equal portions of 10.2 cm 12.7 cm because the salmonella
assay did not adequately measure the mutagenicity of the 8.5 cm
10.2 cm portions ( figure si.1 , supporting information ) .
two 10.2 cm 12.7 cm portions of the single 24 h filter
samples were exposed in the chamber , and the remaining two 10.2 cm
12.7 cm portions were used as unexposed controls .
the pah and
npah concentrations of the exposed and unexposed 10.2 cm 12.7
cm portions used for the mutagenicity study were also measured and
directly compared to the results of the salmonella assay .
overall ,
six pku and three riverside 20.3 cm 25.4 cm filter samples
were tested in each exposure .
the pm2.5 and pm10 filters were exposed to no3/n2o5 , oh radicals , and o3 in a 7000
l indoor collapsible
teflon film chamber equipped with two parallel banks of blacklamps
( used for the oh radical exposures ) and a teflon - coated fan at room
temperature ( 296 k ) and 735 torr pressure .
the filters were placed within the teflon chamber as shown , for
example , in figure si.2 , supporting information . for all exposure experiments , blank ,
clean
filters were also placed in the teflon chamber to test for background
contamination in the chemistry analyses and mutagenicity assays .
the
details of the no3/n2o5 , oh radical ,
and o3 exposures are given in the supporting information and have been previously reported .
details of the sample
extraction and analysis have been previously described and are given
in the supporting information .
the
basic methodology
followed that reported by maron and ames and used salmonella typhimurium strain ta98 .
the positive control doses were 2 g of 2-aminoanthracene
( 2-aa ) and 20 g of 4-nitro-1,2-phenylenediamine ( npd ) for assays
with and without metabolic activation ( rat s9 mix ) , respectively .
all filter extracts
were tested in triplicate . on the basis of preliminary studies
and the limit of detection in the salmonella assay , only the beijing
pm samples were tested for mutagenic activity ( table si.1 , supporting information ) .
positive controls
of npd and 2-aa gave mean revertant counts of 3500/plate and
1000/plate , respectively .
the revertant counts
for the control blanks were comparable to the background revertant
count , indicating no interference from the purified air in the chamber .
it should be noted that different sets of filters were used for the
mutagenicity and chemical studies ( figure si.1 , supporting information ) and that the pah and npah concentrations
of the pm samples used for the mutagenicity testing were measured
and directly compared to the results from the salmonella assay .
the masses of individual pahs and
npahs on each 24 h filter sample cut - out ( shown in figure si.1 , supporting information ) were measured for the
exposed filters ( pahexposed and npahexposed )
and unexposed filters ( pahunexposed and npahunexposed ) .
the amount of individual pah or npah degraded ( or formed ) after
exposure to no3/n2o5 , oh radicals ,
or o3 was calculated by dividing the exposed mass by the
unexposed mass ( a ratio close to 1.0 indicates no net degradation
or formation of a given individual pah or npah after exposure to the
various oxidants ) .
the pahexposed / pahunexposed ratios and the npahexposed / npahunexposed ratios
for the beijing pm after exposure to no3/n2o5 , oh radicals , and o3 are shown in figure 1a , b , respectively .
an asterisk indicates a statistically
significant difference in mass after exposure to the various oxidants
( p - value < 0.05 , according to the paired student s t test ) .
the means and standard errors of pah and npah masses ,
measured in the beijing filters before and after exposure to the various
oxidants , are given in tables si.2si.4 , supporting information .
the 3- to 4-ring pahs exist in both
the gas and particulate phases in the atmosphere at ambient temperatures . while it is possible that a portion of the pah
initially sorbed to the pm desorbed from the pm into the gas phase
of the chamber during the experiments , it is expected that pah available
for volatilization would have done so in the atmosphere and/or during
collection of the pm . therefore , we believe that volatilization of
these compounds from the pm , during the course of the experiments ,
was minimal .
the temperature in the chamber was maintained at room
temperature ( 296 k ) and held constant to 2 k ; no formation
of npahs due to gas - phase reactions was observed ( see below ) , and
the blank filters installed in the reactor at the same time as the
pm filter samples had pah concentrations below the detection limit . (
a ) pahexposed / pahunexposed and ( b ) npahexposed / npahunexposed of beijing pm filters ( n = 9 for no3/n2o5 and
oh radical exposures , and n = 8 for o3 exposure ) used for the chemical study .
an asterisk denotes the statistically
significant difference between the unexposed and exposed masses ( n.d .
=
as shown in figure 1a and in tables si.2si.4 , supporting information , all of the pahs underwent
some degradation upon exposure to no3/n2o5 , oh radicals , and o3 , apart from chr + tri exposed
to oh radicals .
the data denoted by asterisks in figure 1a identify those pahs which had statistically significant
losses .
the highest percent removal was 57% for ant exposed to no3/n2o5 , and a number of other pahs ( including
pyr , bcflu , baa , and bap ) had up to 50% removal from one or
more of the exposures ( figure 1a ) .
noteworthy
is the observation that none of the pahs were completely
removed from exposures to no3/n2o5 , oh radicals , or o3 .
our data from the o3 exposure
are consistent with a previous study where ambient pm was exposed
to 4 ppm of o3 for 60 min , with the pyr and baa concentrations being observed to plateau at
70% of their initial concentrations following exposure to
o3 .
in contrast , a study examining
what can be considered fully available pyr ( coated
on silica particles ) found that the pyr reacted completely , within
10 min , after exposure to 1.3 ppm of o3 in a flow
reactor .
there have now been a number
of studies that indicate that the reactivity of particle - bound pahs is substrate - specific
and can be inhibited by the decreased availability of these pahs in
the ambient pm . of the pahs containing more than 4 rings measured
on the beijing
pm
, bap exhibited the greatest relative degradation upon exposure
to no3/n2o5 , oh radicals , and o3 ( figure 1a ) .
this is consistent with
the high previously reported photochemical reactivity of bap and a study of ambient pm collected in paris , france , which identified
bap as the most reactive pah toward o3 , oh radicals , and
no2/o3 .
the
means and standard errors of npah masses measured on the beijing pm
samples , before and after exposure to the various oxidants , are given
in tables si.2si.4 , supporting information .
2-nf and 3-nf were chromatographically separated and quantified
using a 30 m db-17 gc column in all beijing pm samples that were exposed
to no3/n2o5 , as well as in one - third
of the beijing pm samples that were exposed to oh radicals and o3 .
the 3-nf concentrations in the beijing pm samples , both
before and after exposure to oh radicals and o3 , were below
the detection limit , suggesting no marked increase in concentration
upon exposure . in all of the beijing pm exposures , there was no significant
increase in the masses of 2-nf and 2-np , which are products of gas - phase
radical - initiated reactions .
this provides
further evidence that there was no significant volatilization of 4-ring
pahs from the ambient pm into the gas phase in the chamber and implies
that the increase in the mass of other nitro - pahs during the exposure
experiments was likely due to heterogeneous formation .
a number
of the npahs were formed to a statistically significant
degree during the no3/n2o5 exposure ,
with the npahexposed / npahunexposed ratios reaching
49 for 6-nch ( figure 1b and table si.2 , supporting information ) . while 1-np and 6-nbap
were formed from both the no3/n2o5 and oh radical exposures , 9-nph , 3-nf , 7-nbaa , 1-ntr , 6-nch , 2-ntr ,
and 1,8-dnp were only formed during the no3/n2o5 exposure ( figure 1b ) .
our measured
formation of 3-nf and 1-np is consistent with results from previous
studies of the reaction of fla and pyr adsorbed on tigf filters with
gaseous n2o5 .
these previous
studies also showed that the major npah isomers formed from the heterogeneous
reactions of fla and pyr with n2o5 ( 3- , 8- ,
7- , and 1-nf and 1-np ) are distinct from the 2-nf and 2-np formed
from gas - phase reactions of no3 radicals with fla and pyr .
while 2-np is formed in ambient air from the
oh radical - initiated
reaction of pyr , it is only formed from the no3 radical - initiated
reaction at no2 concentrations much higher than those observed
in ambient air .
the major npah isomers formed
by heterogeneous nitration have the no2 group added to
the highest electron density position .
the radical - initiated reactions of fla and pyr are expected to follow
a different mechanism where the oh or no3 radical attacks
fla or pyr at the highest electron density position , followed by no2 addition at the ortho position and subsequent
loss of h2o or hno3 , yielding 2-nf and 2-np
as the major npah products .
in addition , the n2o5 concentration and the n2o5/no2 ratio in the photochemical chamber during our no3/n2o5 exposure experiments were at least 2
orders of magnitude higher than the average ambient concentration , suggesting a higher potential for heterogeneous
reactions with n2o5 .
the heterogeneous
and gas - phase reactions of tri were previously
reported to both form 1- and 2-ntr , and
in this study , 1-ntr and 2-ntr were formed in equal amounts upon exposure
to no3/n2o5 ( figure 1b ) .
our measured formation of 7-nbaa , 6-nch , and 6-nbap ( figure 1b ) was in agreement with previous results showing
their formation when pahs associated with diesel soot were reacted
with n2o5 .
ratios of 1-npexposed /1-npunexposed ( mean
of 4.8 ) and 6-nbapexposed /6-nbapunexposed ( mean
of 6.5 ) were measured when the beijing pm was exposed to oh radicals
( figure 1b ) .
nitration of pyr by the radical - initiated
reaction discussed above would be expected to lead to 2-np .
therefore ,
the formation of 1-np and 6-nbap is attributed to heterogeneous nitration
of parent pahs by hno3/no2 . during the oh radical exposure experiments ,
no2 was formed from the reaction of ho2 and organic peroxy
radicals with no after the photolysis of methyl nitrite ( the oh radical
precursor ) and reaction of no2 with oh radicals leads to
formation of hno3 .
in contrast to 1-np and 6-nbap , 3-nph
and 2 + 3-nf , and especially 9-nan and 7-nbaa , were degraded on the
beijing pm during the oh radical exposure ( figure 1b ) . because these same npahs were not as significantly degraded
during the dark exposure with o3 ( figure 1b ) , it is possible that the degradation observed during the
oh radical exposure could be due , in part , to direct photolysis .
previously ,
pitts et al . observed the direct photolysis
of 9-nan adsorbed on silica gel and identified quinones as degradation
products . in another study ,
the direct photolysis of 7-nbaa resulted
in increased direct - acting mutagenicity to ta98 in the salmonella
assay with increasing irradiation time .
the orientation of the nitro group has been related to the photochemical
stability of npah , and both 9-nan and
7-nbaa have nitro group orientations that are out of the aromatic
plane , reducing the steric effects exerted by two peri - hydrogens .
this structure makes these pahs less photochemically stable and may
explain their significant degradation during the oh radical exposure
( figure 1b ) .
although
6-nbap also has a structure where the nitro group is out of the aromatic
plane and is therefore expected to be photolabile , there was still
a net formation after exposure of beijing pm to oh radicals ( mean
nbapexposed / nbapunexposed = 6.5 ) ( figure 1b ) . as expected , exposure of the beijing pm to o3 did not lead to significant npah formation ( figure 1b and table si.4 , supporting
information ) and , except for the degradation of 7-nbaa , the
npahs sorbed to beijing pm were not significantly degraded during
o3 exposure ( figure 1b ) .
the pahexposed / pahunexposed ratios and the npahexposed / npahunexposed ratios
for the riverside pm samples after exposure to no3/n2o5 , oh radicals , and o3 are shown
in figures si.3a and si.3b , respectively , supporting
information .
the means and standard errors of pah and npah
masses measured in the riverside filters , before and after exposure
to the various oxidants , are given in tables si.5si.7 , supporting information .
compared to the beijing
pm samples , a smaller number of individual pah and npah in the riverside
pm samples was present at concentrations above their detection limits .
pahexposed / pahunexposed ratios slightly above
1.0 and statistically significant ( p - value < 0.05 )
were measured for 2-mphe , 1-mphe , fla , pyr , and chr + tri upon exposure
of riverside pm to oh radicals and similarly for phe upon exposure
to no3/n2o5 ( ratio = 2.9 ) and oh
radicals ( ratio = 3.3 ) ( figure si.3a , supporting
information ) . however , these small changes in mass ( tables
si.5 and si.6 , supporting information ) are attributed to experimental uncertainty ( for example , possible
inhomogeneity in the particles across the filter ) , since pah formation
in these experiments was not possible .
compared to the beijing pm
samples , the higher - ring pahs in the riverside pm samples were more
resistant to degradation during the no3/n2o5 and oh radical exposures ( figures 1a and si.3a , supporting information ) .
exposure of the riverside pm samples to o3 did not result
in significant degradation of pahs , including the higher - ring pahs
( figure si.3a , supporting information ) .
consistent with the absence of significant pah degradation on
the riverside pm , there was no significant formation of npahs in any
of the riverside pm exposures , including the no3/n2o5 exposure ( figure si.3b , supporting information ) .
a small , but statistically significant
( p - value < 0.05 ) , reduction in mass was measured
for 2 + 3-nf after exposure to oh radicals and for 3-nph , 2 + 3-nf ,
and 1-np after exposure to o3 ( figure si.3b , supporting information ) .
although artifacts
during filter collection may occur , it has previously
been shown that 2-nf is not formed during collection and the artifactual
formation of 1-np is minimal ; therefore ,
heterogeneous formation of these npah can be examined using pm - bound
pahs on filters .
furthermore , our recent study utilizing 16 samples
collected during a 1997 photochemical pollution episode showed an
excellent correlation between the naphthalene and other volatile pahs
analyzed immediately after collection and pah measured on the archived
filters from this study . for example
( see figure s10 in ref ( 33 ) ) , the highest naphthalene , bghip , and bep concentrations occurred
in the morning ( 612 h ) and evening ( 1806 h ) los angeles
samples and the concentrations decreased monotonically at the increasingly
downwind sites of azusa , riverside , and banning .
because 1-np has been
consistently reported to form from heterogeneous reactions of pyr
adsorbed to a surface or particles , we used 1-np
formation as a measure of the reactivity of pm to npah formation .
the percent reactivity of each of the beijing pm samples was calculated
as1where [1-np ] = ( [ 1-np]exposed [ 1-np]unexposed ) and [ pyr]0 = [ pyr]unexposed .
we also calculated
the [ 2-nf]/[bep ] ratio of the unexposed pm samples to indicate the
degree to which the pm had undergone atmospheric processing ( or aging )
in the atmosphere prior to collection .
2-nf is formed from the gas - phase
reaction of fla with oh and no3 radicals in the presence
of no2 , while bep is a relatively stable
pah .
a larger [ 2-nf]/[bep ] ratio therefore
indicates a greater contribution of photochemical atmospheric aging
in comparison to direct emissions .
the relationship between the reactivity
toward nitration upon no3/n2o5 exposure
and the degree of aging has been previously discussed in zimmermann
et al . a plot of the reactivity
versus [ 2-nf]/[bep ]
is shown in figure si.4 , supporting information , for the present study , and the reactivity
decreases monotonically as the atmospheric aging of the pm increases ,
as measured by the [ 2-nf]/[bep ] ratio .
beijing pm samples with lower
reactivity had higher [ 2-nf]/[bep ] ratios , suggesting that the pahs
sorbed to beijing pm that had undergone more aging in the atmosphere
were less available for reaction with no3/n2o5 . the lack of reactivity of the riverside pm samples
has been explained by a higher extent of aging in the atmosphere prior
to sample collection . although
the riverside samples were archived filters , we found
a similar lack of reactivity to no3/n2o5 for riverside samples collected in 2012 with minimal storage
times ( < 4 days ) , and we also encountered a reactive riverside nighttime
sample ( presumably with local pah emissions and little aging , based
on its 2-nf / bep ratio )
. additionally ,
a set of 16 archived daytime / nighttime pm samples , collected from
four sites along the los angeles air basin during a photochemical
pollution episode , showed a range of reactivities , with the nighttime
urban la sample having the highest reactivity .
therefore , we have no evidence that cold - storage of the
pm on filters affects the pah reactivity toward heterogeneous nitration .
most
npahs are known to
be direct - acting mutagens , requiring no exogenous bioactivation to
convert them into the active form .
the
direct - acting mutagenic activity ( without addition of s9 ) of the paired
beijing pm samples ( with and without exposure to no3/n2o5 , oh radical , and o3 ) is shown in
figure 2 .
figure si.5 , supporting information , shows the pahexposed / pahunexposed ratios and the npahexposed / npahunexposed ratios for the beijing pm used in the mutagenicity assay after exposure
to no3/n2o5 , oh radicals , and o3 . in general , the results were comparable to the beijing pm
used in the chemical study in that there was significant formation
of npahs upon exposure of the beijing pm - bound pahs to no3/n2o5 ( figures 1b and
si.5b , supporting information ) .
direct - acting
and indirect - acting mutagen densities ( rev / m ) of exposed
and unexposed beijing pm extracts and the associated
total pah and npah concentrations ( pg / m ) . lower direct - acting mutagenic activity was measured
in the two
unexposed beijing extracts with lower npah concentrations ( pku-3 and
pku-4 ) , compared to the unexposed beijing pm extract with higher npah
concentrations ( pku-5 ) ( figure 2 and table
si.8 , supporting information ) .
this is
consistent with a previous study from our laboratory that showed significant
daily variation in the direct - acting mutagenicity , and in the corresponding
npah concentrations , of beijing pm .
after
no3/n2o5 exposure , the direct - acting
mutagenicity of the beijing pm increased 2- to 26-fold ( figure 2 ) .
of the beijing pm samples exposed to no3/n2o5 , the largest increase in direct - acting
mutagenicity ( 26-fold ) was measured in sample pku-4 , which also had
the largest increases in 1,3- , 1,6- , and 1,8-dnp masses after exposure
to no3/n2o5 ( table si.8 , supporting information ) . among these dnps ,
the greatest increase in mass was for 1,8-dnp ( 10.2 ng ) ( table si.8 , supporting information ) .
the 1,8-dnp concentrations
measured in the other two mutagenicity study samples exposed to no3/n2o5 ( pku-3 and pku-5 ) were either
below the detection limit ( pku-3 ) or three times less than that measured
in the pku-4 sample ( pku-5 ) . in previous mutagenicity assay studies ,
dnps , especially 1,8-dnp , were found to be powerful direct - acting
mutagens .
small amounts of dnps have
been shown to contribute significantly to the total direct - acting
mutagenicity of diesel particles .
for the oh radical exposure in the direct - acting mutagenicity study ,
two of the beijing pm samples had no 1-np formation ( and hence zero
reactivity according to eq 1 ) ( pku-8 and pku-9 )
and one sample had only 4.1% reactivity ( pku-7 ) ( figure 2 and table si.9 , supporting information ) .
overall , the increase in direct - acting mutagenicity after oh radical
exposure ( mean of 1.7-fold ) was lower than the increase in direct - acting
mutagenicity after no3/n2o5 exposure
( mean of 11-fold ) .
the npahexposed /npahunexposed profiles , from both the mutagenicity and chemical studies , showed
the formation of 1-np and 6-nbap from oh radical exposure ( figures 1b and si.5b , supporting information ) . given the significant 6-nbap formation in all of the beijing pm
samples from exposure to oh radicals , resulting from the formation
of hno3 in the chamber ( figures 1b and si.5b , supporting information ) ,
and the minimal corresponding increase in direct - acting mutagenicity
of these same samples , 6-nbap does not appear to contribute significantly
to the overall direct - acting mutagenicity of the beijing pm .
this
is consistent with the structure of 6-nbap which has a no2 group nearly perpendicular to the aromatic ring that does not allow
for favorable nitro - reduction .
however ,
an increase in direct - acting mutagenicity and a decrease in total
npah mass , as well as a decrease in mutagenic 1-np mass , was observed
after pku-8 and pku-9 were exposed to oh radicals ( figure 2 ) .
this suggests that other mutagenic degradation
products , not measured in this study or below the detection limit
of our analytical method , may have contributed to the enhanced direct - acting
mutagenicity of the beijing pm after exposure to oh radicals . for the beijing
pm samples , figure si.5b , supporting
information , shows that 9-nan , 3-nph , 2 + 3-nf ,
1-np , and 2-np were degraded significantly during the o3 exposure in the mutagenicity study , while only 7-nbaa was significantly
degraded during the o3 exposure in the chemical study ( figures 1b and si.5b , supporting information ) .
only pku-14 showed a significant decrease in direct - acting mutagenicity
of the beijing pm after exposure to o3 , which is consistent
with the corresponding decrease in the total npah mass ( figure 2 and table si.10 , supporting
information ) .
some
parent pahs and npahs ,
including 6-nbap and 1-nitrocoronene , contribute to the indirect - acting
mutagenicity of pm . on average
, there
was a 7-fold and 1.4-fold increase in the indirect - acting
mutagenicity of the beijing pm after exposure to no3/n2o5 and oh radicals , respectively ( figure 2 ) . because most parent pahs were degraded after
exposure to no3/n2o5 and oh radicals
( figure 2 and tables si.8 and si.9 , supporting information ) , it is possible that
the npahs formed contributed to the increase in indirect - acting mutagenicity
.
increased indirect - acting mutagenicity after parent pah exposure to
no3/n2o5 and oh radicals was observed
in our previous studies .
. found that the exposure of wood soot to no2 and o3 resulted in an increase in both direct-
and indirect - acting mutagenicity of the npah fraction .
in the same
study , the most polar fraction made the largest contribution to the
total indirect - acting mutagenicity .
this suggests that other , more
polar , transformation products may contribute significantly to the
indirect - acting mutagenicity of the extracts .
moreover , the high molecular
weight pahs ( mw 302 ) , including dibenzo[a , l]pyrene which is 30 times
more toxic than bap , may also play a
significant role in the indirect - acting mutagenicity and were shown
to be a significant contributor to the inhalation cancer risk in beijing
air .
the results of our previous study
on the heterogeneous nitration of dibenzo[a , l]pyrene adsorbed on filters
showed that the indirect - acting mutagenicity increased 2.5-fold after
the no3/n2o5 exposure and that 6-nitrodibenzo[a , l]pyrene
was the only nitro product identified .
for the o3 exposure , the pahexposed / pahunexposed profiles of beijing pm samples were comparable in
the mutagenicity and chemical studies ( figures 1a and si.5a , supporting information ) .
the reduction in the mutagenicity ( 33% ) of the two beijing samples
exposed to o3 ( pku-12 and pku-14 )
may be associated with
the degradation of the total parent pah and npah masses ( figure 2 ) .
however , there was no significant change in the
indirect - acting mutagenicity in pku-13 when total pah and npah masses
decreased ( figure 2 ) .
our present and previous data suggest that the riverside pm had undergone
more aging prior to collection and was less reactive than the beijing
pm .
the decreased reactivity of the riverside pm , compared to the
beijing pm , may be because the riverside sampling site is located
downwind of los angeles and receives photochemically aged
moreover , we previously observed that a night - time pm sample
collected from downtown los angeles had a similar reactivity and [ 2-nf]/[bep ]
ratio as some of the beijing pm samples .
in contrast to riverside ( but like downtown los angeles at night ) ,
the beijing sampling site is located within a major urban source region
and appears to receive air masses that are not as photochemically
aged and more influenced by direct emissions .
one can therefore conclude
that aged particles exhibit decreased reactivity , most probably due
to a lower availability of the pahs for reaction and due at least
in part to the accumulation of atmospheric reaction products on the
surface of ambient pm . on the basis of the observed decrease in reactivity
of pm collected along an upwind downwind trajectory in the
los angeles air basin
, it appears that ,
at least in that air basin at that time , photochemical aging occurred
rapidly , on a time - scale of a few hours during daytime , and that little
or no additional heterogeneous reactions would occur for additional
atmospheric transport times .
the extent of pah transformation
observed in this study may be limited by the multilayer coverage of
the ambient pm on the filters . in reality
, the transformation of pahs
on pm may be more significant in the atmosphere , where the pahs are
present on individual particles in the atmosphere rather than on pm
layered onto filters .
however , the resistance of the riverside pm
samples to chemical reaction suggests that secondary pollutant formation
plays an important role in making pm - bound pahs less available for
chemical reactions .
overall , our present and previous results suggest that heterogeneous pah degradation and
concurrent npah formation on ambient pm occurs mainly , or only , for
particles with minimal photochemical aging and that photochemically
aged particles are nonreactive toward heterogeneous reactions of pahs . | the
heterogeneous reactions of ambient particulate matter ( pm)-bound
polycyclic aromatic hydrocarbons ( pahs ) and nitro - pahs ( npahs ) with
no3/n2o5 , oh radicals , and o3 were studied in a laboratory photochemical chamber .
ambient
pm2.5 and pm10 samples were collected from beijing ,
china , and riverside , california , and exposed under simulated atmospheric
long - range transport conditions for o3 and oh and no3 radicals . changes in the masses of 23 pahs and 20 npahs ,
as well as the direct and indirect - acting mutagenicity of the pm ( determined
using the salmonella mutagenicity assay with ta98 strain ) , were measured
prior to and after exposure to no3/n2o5 , oh radicals , and o3 . in general ,
o3 exposure
resulted in the highest relative degradation of pm - bound pahs with
more than four rings ( benzo[a]pyrene was degraded equally well by
o3 and no3/n2o5 ) .
however ,
npahs were most effectively formed during the beijing pm exposure
to no3/n2o5 . in ambient air , 2-nitrofluoranthene
( 2-nf )
is formed from the gas - phase no3 radical- and oh
radical - initiated reactions of fluoranthene , and 2-nitropyrene ( 2-np )
is formed from the gas - phase oh radical - initiated reaction of pyrene .
there was no formation of 2-nf or 2-np in any of the heterogeneous
exposures , suggesting that gas - phase formation of npahs did not play
an important role during chamber exposures .
exposure of beijing pm
to no3/n2o5 resulted in an increase
in direct - acting mutagenic activity which was associated with the
formation of mutagenic npahs .
no npah formation was observed in any
of the exposures of the riverside pm .
this was likely due to the accumulation
of atmospheric degradation products from gas - phase reactions of volatile
species onto the surface of pm collected in riverside prior to exposure
in the chamber , thus decreasing the availability of pahs for reaction . |
eosinophils are potent effector cells implicated in allergic responses and helminth infections and have cytotoxic granules containing major basic protein ( mbp ) , eosinophil cationic protein ( ecp ) , eosinophil peroxidase ( epo ) , and eosinophil - derived neurotoxin ( edn ) ( 1 ) . responding to stimuli such as an allergen , helminth infection , or tissue injury , they release their granule - derived cytotoxic proteins and are involved in inflammatory processes . however , the intestine ( constantly in contact with the gut microbiota ) is a major site where eosinophils are abundantly present under homeostatic conditions .
intestinal eosinophils rarely undergo degranulation and have a long lifespan as compared with eosinophils in the blood and lungs ( 23 ) .
a possible reason is that inhibitory receptor signal regulatory protein ( sirp)/cd172a , highly expressed on the intestinal eosinophils , inhibits degranulation of eosinophils and promotes their survival ( 2 ) . in support of this notion ,
the ligand for sirp/cd172a , cd47 , is expressed in the intestine ( 2 ) .
in addition , intestinal eosinophils highly express common -chain , which is an integral part of the cytokine receptors for il-2 , il-4 , il-7 , il-9 , il-15 , and il-21 ; intestinal eosinophils are severely reduced in number in the common -chain - deficient mice unlike eosinophils in blood and lungs ; therefore , common -chain - dependent signals seem to play an important role in selective survival of intestinal eosinophils ( 3 ) .
intestinal eosinophils are produced from hematopoietic stem cells in bone marrow and migrate into the intestine under the influence of chemokine eotaxin-1 ( 14 ) . during the development and survival of eosinophils , signaling of the common -receptor chain shared by il-3 , il-5 , and gm - csf is required ( 4 ) , and among these cytokines , il-5 is particularly important ; overproduction of il-5 in transgenic mice results in eosinophilia ( 5 ) whereas deletion of il-5 abrogates eosinophilia induced by an aeroallergen ( 6 ) . according to a recent report
, il-5 is mainly produced by type 2 innate lymphoid ( ilc2 ) cells , and its production in ilc2 cells is enhanced by vasoactive intestinal peptide ( 7 ) .
additionally , transcription factor gata-1 is crucial for the development of eosinophils ; deletion of the palindromic double gata high - affinity binding sites in the gata-1 promoter results in eosinophil - deficient mice ( called dbl gata-1 mice ) ( 8) .
phil mice , which express diphtheria toxin a chain under the control of the eosinophil - specific epo promoter , are also eosinophil - deficient mice ( 9 ) . in recent studies on these eosinophil - deficient mice
, it has been revealed why eosinophils are abundant in the intestine without inflammation and what their physiological roles are under homeostatic conditions .
some papers have shown that intestinal eosinophils promote formation and maintenance of iga - expressing plasma cells ( 10 ) and abundantly produce il-1 receptor antagonist ( il-1ra ) ( 11 ) , contributing to intestinal immune homeostasis .
several lines of evidence have shown that eosinophils also exist in adipose tissue and are involved in metabolic homeostasis ( 1213 ) .
therefore , in this review , we summarize recent findings regarding eosinophils and discuss the homeostatic functions of these cells .
recently , it has been reported that small - intestinal eosinophils produce large amounts of il-1ra ( 11 ) . the latter competes with il-1 for receptor binding and
meanwhile , il-1 is primarily produced by activated macrophages and functions as a key mediator in various inflammatory diseases including inflammatory bowel disease and rheumatoid arthritis ( 14 ) . in this context , a decrease in the il-1ra to il-1 ratio is linked to inflammatory bowel disease in humans ( 15 ) .
il-1ra - deficient mice spontaneously develop arthritis with a marked increase in the number of th17 cells ( 1617 ) . in line with this finding
, it has been reported that microbiota - induced il-1 , but not il-6 , plays an important role in the development of th17 cells in the intestine ( 18 ) .
therefore , small - intestinal eosinophils suppress the differentiation and maintenance of th17 cells by constantly secreting large amounts of il-1ra , thus contributing to intestinal immune homeostasis ( 11 ) ( fig .
intestinal eosinophils also inhibit differentiation and/or proliferation of th1 cells in the lamina propria ( lp ) though less strongly in comparison with the effects on th17 cells ( 11 ) .
however , because il-1ra - deficient mice do not show a significant increase in the number of th1 cells , intestinal eosinophils may suppress differentiation and/or proliferation of th1 cells by secreting other unidentified cytokines .
unlike th1 and th17 cells , small - intestinal eosinophils do not affect frequencies of foxp3 regulatory t ( treg ) cells and th2 cells in the lp ( 11 ) .
particularly in food allergy , intestinal eosinophils activate dendritic cells ( dcs ) through a release of epo and promote their migration from the intestine to draining lymph nodes , thus initiating a primary th2 immune response ( 19 ) . in contradiction to these findings , one group reported that the numbers of cd103 dcs and cd103 cd4 t cells including foxp3 treg cells are reduced in the lp of eosinophil - deficient mice , although they did not explain the mechanism in the paper ( 10 ) , and this phenomenon should be confirmed later . because small - intestinal eosinophils , but not eosinophils in blood and bone marrow , produce a large amount of il-1ra under homeostatic conditions , we can hypothesize that gut microbiota and its metabolites may be involved in il-1ra production . on the other hand , small - intestinal eosinophils in both spf and
gf mice produce similar levels of il-1ra , and metabolites such as retinoic acid and short - chain fatty acids do not induce il-1ra production in bone marrow eosinophils ( 11 ) .
instead , gm - csf , which is known to be involved in the development and survival of eosinophils , performs an important function in il-1ra production ( 11 ) . in this regard ,
the high basal concentration of gm - csf in the small intestine and high expression of gm - csf receptor in small - intestinal eosinophils seem to explain the unique ability of small - intestinal eosinophils to continuously secrete a large amount of il-1ra under steady - state conditions .
in addition , because th17 cells can produce gm - csf in an il-1- and il-23-dependent manner ( 20 ) , it is plausible that gm - csf produced by th17 cells endows eosinophils with the superior capacity for il-1ra production and regulates th17 cell homeostasis via a negative feedback loop .
in contrast , ironically , large - intestinal eosinophils , when activated by gm - csf produced by th17 cells , secret epo as well as proinflammatory cytokines tnf and il-13 , and aggravate colitis ( 21 ) .
these findings imply that small - intestinal eosinophils are different from large - intestinal ones . in support of this notion ,
large - intestinal eosinophils produce much smaller amounts of il-1ra and show a distinctive expression pattern of surface proteins including cd11c , st2 , and ly6c as compared with small - intestinal ones ( 19 ) .
in addition , although eosinophils are mainly present throughout the intestine , large - intestinal eosinophils are scarce in a steady state ( 1119 ) , and only under intestinal inflammatory conditions , does their frequency dramatically increase ( 21 ) .
accordingly , large - intestinal eosinophils seem to be proinflammatory unlike their small - intestinal counterparts .
most of the intestinal plasma cells are located in the lp and mostly produce iga ( 22 ) .
iga is secreted into the intestinal lumen and functions as the first line of defense through neutralization and clearance of enteric pathogens .
nevertheless , iga - producing b cells are generally formed in peyer 's patches ( pps ) and migrate into the lp ( 22 ) . in pps , antigen - experienced cd4 t cells migrate into b - cell follicles via cxcr5 and activate b cells ( 22 ) .
these specialized cd4 t cells are called follicular b helper t ( tfh ) cells and are different from other cd4 t cell lineages .
they highly express costimulatory molecules for their development and function , e.g. , cd40l , ox-40 , and programmed cell death 1 ( pd-1 ) rather than cytokines and transcription factors characteristic of th1 , th2 , and th17 cells ( 22 ) .
transcription factor b cell lymphoma 6 ( bcl-6 ) plays an important role in tfh differentiation , whereas transcription factor b lymphocyte maturation protein 1 ( blimp-1 ) inhibits it ( 22 ) .
the activated b cells induce activation - induced cytidine deaminase ( aid ) and provoke class switch recombination ( 22 ) . because tgf-1 , a key cytokine for iga switching , is expressed by many cells in pps , activated b cells preferentially switch their isotype from igm to iga ( 2223 ) .
moreover , il-21 secreted by tfh cells enhances the proliferation and differentiation of iga plasma cell precursors in synergy with tgf-1 ( 24 ) .
meanwhile , igm b cells in the lp are activated by polyclonal stimuli or by antigens presented by dcs in the lp ( lp - dcs ) and differentiate into iga - producing plasma cells without the help of tfh cells ( 22 ) . in this t cell - independent formation of iga - producing b cells ,
important functions are performed by tgf-1 , b - cell activating factor of the tumor - necrosis factor family ( baff ) , and a proliferation - inducing ligand ( april ) produced by dcs and stromal cells in the lp ( 22 ) . in vitro ,
cytokines such as il-6 , tnf , il-10 , il-4 , and il-5 , also prolong the survival of plasma cells ( 25 ) . in this context , bone marrow eosinophils have been reported to support the survival of plasma cells by secreting april and il-6 and contribute to the long - term maintenance of plasma cells in bone marrow ( 26 ) .
although intestinal eosinophils are mainly in the lp , not in pps , they are involved in the formation and maintenance of iga - producing b cells in pps as well as in the lp ( 10 ) .
intestinal eosinophils express baff , tgf-1 , and matrix metalloproteases ( that are involved in the release of tgf-1 from the large latent complex ) as well as april and il-6 at the mrna level ( 10 ) . therefore , intestinal eosinophils appear to influence t - cell - independent formation and maintenance of iga - producing b cells via production of these cytokines and enzymes .
accordingly , the amount of iga is dramatically reduced in the intestine of eosinophil - deficient mice ; this change increases bacterial load in the gut and affects composition of the microbiota there ( 10 ) .
therefore , it seems likely that eosinophils indirectly exert their effects on t - cell - dependent formation of iga - producing b cells in pps .
recently , one paper revealed that eosinophils can be involved in iga production through il-1 ( 27 ) .
moreover , eosinophils can release mitochondrial dna in a catapult - like manner and kill bacteria ( 28 ) . nevertheless ,
the release of mitochondrial dna does not cause cell death of eosinophils . for the release of mitochondrial dna , eosinophils should be activated by lipopolysaccharide under conditions where they are primed with il-5 or ifn- ( 28 ) . in the intestinal environment
, gut bacteria can exert their effects on immune cells through bacteria - derived molecules , and immune - cell populations producing ifn- ( th1 ) and il-5 ( ilc2 ) exist ; therefore , it seems plausible that intestinal eosinophils reduce the bacterial load in the gut via the release of mitochondrial dna under homeostatic conditions .
accordingly , intestinal eosinophils prevent the host from bacterial invasion and contribute to homeostasis of intestinal immunity .
obesity induces chronic low - grade inflammation , consequently causing insulin resistance and type 2 diabetes ( 2930 ) . in the obese state ,
a large number of monocytes migrate into adipose tissue and differentiate into classically activated ( m1 ) macrophages expressing tnf- and inducible nitric oxide synthase , which triggers inflammation and results in metabolic diseases ( 2930 ) .
by contrast , resident adipose - tissue macrophages in the lean state express il-10 and arginase 1 , and show an anti - inflammatory phenotype of alternatively activated ( m2 ) macrophages ( 2930 ) .
activation of the anti - inflammatory m2 macrophages requires th2 cytokines such as il-4 and il-13 ( fig .
1 ) , whereas activation of m1 macrophages requires th1 cytokine ifn- ( 2930 ) .
eosinophils are a major il-4-expressing cell population in adipose tissue and help to sustain m2 macrophages ( 12 ) ( fig .
1 ) . high - fat diet - induced obesity significantly reduces the number of eosinophils in adipose tissue ( 12 ) .
therefore , eosinophil - deficient mice show aggravation of obesity - induced metabolic diseases via a dramatic reduction in the number of m2 macrophages in adipose tissue , indicating that eosinophils are intimately involved in metabolic homeostasis ( 12 ) . besides , ilc2 cells promote accumulation of eosinophils and activate m2 macrophages in adipose tissue through production of il-5 and il-13 and are implicated in metabolic homeostasis ( 3132 ) .
the latter represents clusters of uncoupling protein 1 ( ucp-1)-expressing adipocytes in white adipose tissue and functions in heat production under conditions of increased energy expenditure , such as cooling and exercise ( 3435 ) .
thus , beige adipocytes contribute to suppression of obesity and alleviation of metabolic diseases . by contrast
, white adipocytes do not express ucp-1 and are not involved in thermogenesis ; instead , they participate in fat storage , contributing to obesity - induced metabolic diseases ( 35 ) .
il-4 released by eosinophils induces the tyrosine hydroxylase expression of m2 macrophages under cold conditions and drives these macrophages to produce catecholamines such as norepinephrine ( 13 ) . the macrophage - derived catecholamines trigger expression of the ucp-1 gene in subcutaneous white adipose tissue and are involved in the development of beige fat ( 13 ) .
in addition , meteorinlike ( metrnl ) , which is a hormone induced in muscle after exercise and in adipose tissue upon cold exposure , exerts its effect on eosinophils and increases their expression levels of th2 cytokines such as il-4 and il-13 , consequently promoting the development of ( and thermogenesis in ) beige fat ( 36 ) . in particular , ilc2 cells contribute to the biogenesis of beige fat through production of methionine - enkephalin peptide as well as via activation by il-33 receptor signaling ( 32 ) .
increasing evidence suggests that obesity and other metabolic syndrome are associated with altered composition of the gut microbiota ( 37 ) . in agreement with this notion ,
the transfer of gut microbiota from obese to germ - free mice increases obesity more than gut microbiota from lean mice does ( 38 ) .
microbiota depletion either by antibiotic treatment or in germ - free mice promotes beige fat biogenesis and alleviates obesity and other metabolic syndrome ; these effects are mediated by eosinophil accumulation , enhanced th2 cytokine signaling , and m2 macrophage polarization in white adipose tissue of microbiota - depleted mice ( 39 ) . in this
regard , intestinal eosinophils may inhibit obesity and other metabolic syndrome through a reduction of bacterial burden via iga production ( 1027 ) .
in addition , because obesity - induced metabolic diseases show elevated levels of il-1 ( 29 ) , intestinal eosinophils may alleviate inflammation by producing large amounts of il-1ra and thus alleviate metabolic diseases .
according to recent reports , eosinophils are a heterogeneous cell population and have different characteristics depending on the site of residence ( 3111921 ) . although eosinophils are mainly distributed in the intestine , the phenotype of large - intestinal eosinophils is different from that of small - intestinal ones , and the number of large - intestinal eosinophils in a steady state is much smaller than that of their small - intestinal counterparts ( 111921 ) .
moreover , small - intestinal eosinophils in response to gm - csf produce large amounts of il-1ra and exert an anti - inflammatory function ( 11 ) , whereas large - intestinal eosinophils increase production of proinflammatory cytokines tnf- and il-13 and promote colitis ( 21 ) .
these observations mean that eosinophils do not all have an identical function , but rather their function is likely controlled by local milieu .
thus , if anti - inflammatory eosinophils can be upregulated or inflammatory eosinophils can be converted into anti - inflammatory ones , then these modalities should help to combat inflammatory diseases such as allergies , inflammatory bowel disease , and obesity - related metabolic diseases . to this end
, it would be worthwhile to identify the surface markers that can distinguish homeostatic and inflammatory eosinophils .
japonica nakai increases the number of small - intestinal eosinophils and suppresses differentiation and/or proliferation of th1 and th17 cells ( 40 ) .
regulatory eosinophil - recruiting prebiotics or probiotics may be useful for prevention of inflammatory and metabolic diseases .
more studies should be conducted in this field for successful development of eosinophil - targeting treatments . | eosinophils are potent effector cells implicated in allergic responses and helminth infections . responding to stimuli , they release their granule - derived cytotoxic proteins and are involved in inflammatory processes .
however , under homeostatic conditions , eosinophils are abundantly present in the intestine and are constantly in contact with the gut microbiota and maintain the balance of immune responses without inflammation .
this situation indicates that intestinal eosinophils have an anti - inflammatory function unlike allergic eosinophils . in support of this notion
, some papers have shown that eosinophils have different phenotypes depending on the site of residence and are a heterogeneous cell population .
recently , it was reported that eosinophils in the small intestine and adipose tissue , respectively , contribute to homeostasis of intestinal immune responses and metabolism .
accordingly , in this review , we summarize new functions of eosinophils demonstrated in recent studies and discuss their homeostatic functions . |
several species of spotted fever group ( sfg ) rickettsiae have been isolated from ticks in the united states ; however , the only species considered to cause human disease in maryland is rickettsia rickettsii , the causative agent of rocky mountain spotted fever ( rmsf ) .
the potential pathogenicity of rickettsial organisms is most often predicted by the ability of the species to cause disease in guinea pigs .
the reliability of this method has been debated , and researchers have suggested that " every rickettsial species may have pathogenic potential , provided that its reservoir arthropod is capable of biting humans " ( 1,2 ) . the prevalence of sfg rickettsia infection in dermacentor variabilis , the primary vector of r. rickettsii in the eastern united states , has been estimated in several studies .
prevalences from 0.2% in ohio ( 3 ) to 8.6% in ( table 1 ) maryland ( 4 ) have been reported .
many studies have implied that these infections were r. rickettsii , but few have confirmed these identities ( 5 ) .
numerous sfg - rickettsial species have been isolated or partially characterized from molecular evidence in the eastern united states ; these species include r. rickettsii , r. rhipicephali , r. montanensis ( = r .
these species have been identified , either together or separately , in areas where rmsf is endemic ( table 2 ) .
as the distributions of different sfg - species in disease - endemic areas become better understood , determining the relationship between the rickettsiae involved in human disease and those isolated from vector ticks and mammal and tick reservoirs may be necessary .
ci , confidence interval ; sfg , spotted fever group . fisher exact p values
. ticks with unknown month of collection were excluded from the statistical analyses for this characteristic .
ci , confidence interval . differentiating the tick - borne sfg rickettsia before the 1990s depended largely on culture and epitope recognition techniques , such as immunoflourescence and agglutination tests and mouse serotyping with monoclonal antibodies .
genotypic studies of rickettsiae conducted during the 1990s led to two rickettsial genes that can be used to identify rickettsial infections : citrate synthase ( glta ) and rompa ( 9 ) .
citrate synthase encodes the first enzyme of the tricarboxylic acid cycle and is highly conserved among all rickettsia species , serving as a polymerase chain reaction ( pcr ) target to identify any rickettsial infection .
rompa encodes a surface - expressed protein of sfg - rickettsiae that is important for adhesion to host cells ( 10 ) .
only sfg rickettsia contain the rompa gene ( 11 ) , making it an ideal pcr target to identify sfg rickettsia infections .
approximately 35 cases of rmsf are reported annually in maryland . from 1994 through 1998 , maryland ranked 8th nationally , reporting 112 cases .
these cases , confirmed by the maryland department of health and mental hygiene , meet the centers for disease control and prevention ( cdc ) case definition , yet not much information exists to characterize the infection rate of sfg rickettsiae in d. variabilis in the state .
this cross - sectional study examined the prevalence and composition of sfg rickettsia in d. variabilis in maryland . in 2002 , genomic dna was extracted from 392 adult d. variabilis collected by flagging in anne arundel , baltimore , calvert , charles , prince george 's , and st .
quality of the modified hexadecyltrimethylammonium bromide ( ctab ) dna extractions was verified by amplifying a tick 16s mtdna fragment ( 12 ) . modifying the existing extraction procedure involved an additional phenol : chloroform : isoamyl alcohol ( 25:24:1 ) extraction step to further stabilize the extracted dna .
tick extractions were screened by pcr for evidence of infection with rickettsia by using primers specific to the rickettsia citrate synthase gene ( 9 ) .
the rickettsia infection rate was 6.1% ( 24/392 , 95% confidence interval [ ci ] 4.0%9.0% ) .
all rickettsia - positive tick extractions were subsequently screened by pcr for sfg rickettsia by using primers for the rompa gene of sfg - rickettsia ( 9 ) .
the prevalence of sfg rickettsia infection was 3.8% ( 15/392 , 95% ci 2.2%6.2% ) .
single strand conformational polymorphism ( sscp ) banding patterns were identical for all tick - derived rompa pcr amplicons .
similarly , sscp banding patterns of the tick - derived citrate synthase amplicons for the sfg - rickettsia positive samples were monomorphic .
these results suggest that these tick infections represent a single sfg rickettsia taxon ( 13 ) .
citrate synthase and rompa pcr products from three ticks were sequenced with the citrate synthase and shortened rompa pcr primers , respectively .
sequences of each respective gene fragment derived from these ticks were identical and confirm the sscp findings ( genbank accession no .
the derived sequences were also compared to rickettsiae sequences in the public domain and were identical to those derived from r. montanensis from d. andersoni ( genbank accession no .
prevalence estimates were reported as percentages with exact 95% ci based on the binomial distribution .
fisher exact test was used to compare infection prevalence across the strata of selected characteristics .
the association between each characteristic and the prevalence of infection was quantified as odds ratios ( or ) , calculated with logistic regression or exact methods for categorical data when the data were highly unbalanced .
all statistical analyses were performed with stata ( version 7.0 ; stata corporation , college station , tx ) or statxact ( version 5.0.3 ; cytel software corporation , cambridge , ma ) .
the variation in prevalence of rickettsia - positive ticks across all counties was marginally significant ( p = 0.052 ) , with a higher prevalence in st .
mary 's county compared to all other counties ( or 5.1 , 95% ci 0.527.2 , p value = 0.08 ) .
in contrast to the equivocal results for the geographic distribution of rickettsia - positive ticks , temporal heterogeneity was evident , as the prevalence of rickettsia - positive ticks varied significantly with month of collection ( p = 0.007 ) .
risk for infection was significantly elevated for any rickettsia organism in ticks collected in july or august ( or 4.1 , 95% ci 1.511.5 ) compared to those collected in april .
further analyses combining the data from the spring and early summer months showed that the risk for infection with any rickettsia organism in july or august was even higher ( or 4.7 , 95% ci 2.011.3 ) .
the risk for infection with r. montanensis with the late summer months , compared to the spring and early summer months , was somewhat less but still approached statistical significance ( or 3.0 , 95% ci 0.810.2 , p value = 0.06 )
. this observation may be an artifact of diminishing tick abundance later in the summer months .
the prevalence of sfg rickettsia in d. variabilis estimated from this study ( 3.8% ) was lower than that in previous reports from maryland .
however , in regions where rmsf is observed annually , prevalence estimates range widely , from 2% in connecticut to 10% in alabama , with intermediate prevalences in new york , kentucky , tennessee , and arkansas ( 5 ) . in addition , r. montanensis had not been previously recognized in maryland .
most earlier studies of sfg rickettsia infection prevalence did not identify the rickettsia to the species level , although the sfg - positive samples were sometimes assumed to represent r. rickettsii .
one study in maryland in which 26 rickettsia isolates were obtained from d. variabilis determined the species composition of the rickettsiae .
two isolates were r. rickettsii , 1 isolate was r. bellii ( non - sfg ) , and 23 ( 88% ) were identified as wb-8 - 2 , a then - unnamed sfg - rickettsia ( 5 ) .
weller et al . performed a phylogenetic analysis and found wb-8 - 2 ( " r . amblyommii " ) to be closely related to r. montanensis ( 14 ) , although they can be differentiated by serotyping .
r. montanensis has been isolated from ticks in other eastern states . during the 1980s , feng et al .
reported that r. montanensis represented 41 ( 91% ) of 45 of the sfg isolates from d. variabilis collected in cape cod , massachusetts ( 7 ) .
anderson et al . reported isolation of r. montanensis from d. variabilis in connecticut ( 6 ) , and in 1990 , pretzman et al .
reported that most sfg rickettsia isolated from dermacentor ticks throughout ohio was r. montanensis ( 3 ) .
further , these researchers noted that r. rickettsii were not isolated from ticks collected in several ohio counties where rmsf was considered endemic .
these studies illustrate that the rickettsial composition and dynamics within the rmsf - endemic areas are complex and need to be addressed with greater scrutiny .
the role of sfg rickettsia in human health is largely unknown , and many are considered to be nonpathogenic either because the bacteria have not been isolated from humans or they do not demonstrate pathogenicity in animal models .
for example , r. montanensis is avirulent in guinea pigs but virulent in voles ( 15 ) .
r. montanensis and other " nonpathogenic " sfg rickettsia infected ticks may also benefit human health by decreasing r. rickettsii in tick populations as a result of the " interference " phenomenon ( 15 ) .
the findings of this study and others raise important questions . in 2000 , a total of 495 cases of rmsf were reported to cdc and 4 deaths were attributed to spotted fever caused by rickettsia rickettsii .
the extent to which r. rickettsii is the agent responsible for reported cases of rmsf should be reevaluated , considering the number of studies completed in rmsf - endemic regions , including this one , that have found non
( u50/ccu319554 ) and niehs training awards ( t32es07141 ) to j.m.a . and k.i.s . | three - hundred ninety - two adult dermacentor variabilis were collected from six maryland counties during the spring , summer , and fall of 2002 .
infection prevalence for spotted fever group rickettsia was 3.8% , as determined by polymerase chain reaction .
single strand conformational polymorphism ( sscp ) analysis followed by sequencing indicated that all infections represented a single rickettsial taxon , rickettsia montanensis . |
according to the world health organization , there are over 500 million obese individuals worldwide .
bariatric surgery has been shown to be an effective treatment strategy to produce marked weight loss in patients with moderate to severe obesity ( bmi > 35 kg / m ) .
specifically , vertical banded gastroplasty ( vbg ) , a primarily restrictive bariatric surgical procedure , was first described by mason in 1982 . despite initial optimism with vgb ,
only 20% of patients maintained 50% excess weight loss ( ewl ) at 10-year followup .
weight regain following vbg may be related to staple - line dehiscence and stomal pouch dilation .
in addition , reoperation and revision of vbg are needed in 20% to 30% of patients [ 4 , 6 ] .
roux - en - y gastric bypass ( rygb ) remains the most common revisional procedure following failed vgb .
rygb had been shown to produce marked weight loss as a revisional procedure for previously failed restrictive bariatric surgical procedures [ 79 ] . however , some studies have suggested increased complication rates with revisional bariatric operations as high as 12% to 41% ( schwartz rw , obes surg 2002 ; .
recently , an endoscopic treatment strategy involving the stomaphyx device ( endogastric solutions inc . , redmond , wa , usa ) has been used to revise gastric pouch dilatations .
endoscopic revision via the stomaphyx has been reported in patients following vbg and rygb successfully [ 12 , 13 ] .
our objective was to retrospectively compare weight loss and complication rates , following revision of failed vgb with either the stomaphyx device or formal surgical revision to rygb at our institution .
a retrospective review was completed for all patients with a previous vbg presenting to a comprehensive adult weight management clinic ( weight wise ) with weight regain between 2003 and 2010 .
the multidisciplinary team at weight wise including physicians , nurses , physiotherapy , and dieticians assessed these patients .
patients with previous vgb that presented with persistent weight gain despite conservative measures and meeting the canadian guidelines for surgical intervention were considered for revision by stomaphyx or open conversion to rygb .
vbg revision endoscopically via the stomaphyx device was performed as a part of a clinical trial , with the results previously reported by manouchehri et al . .
stomaphyx is an endoluminal device that has recently been developed as an alternative to revisional surgery .
a gastroscope is inserted through the internal lumen of the device and full - thickness gastric tissue is suctioned into the device allowing for the application of a polypropylene fastener which if repeated circumferentially creates a circular pleat thus downsizing the gastric pouch .
the stomaphyx device has since been used and studied at our institution as a minimally invasive revisional option for patients with failed vbg .
patient demographics were collected retrospectively , including age , sex , and mean preoperative weight and body mass index ( bmi ) following the initial vbg .
this included perioperative and postoperative complications such as anastomotic leakage , intra - abdominal abscess , dehiscence , respiratory or cardiac complications , and incisional hernia .
also , the operative time to complete either vbg revision with stomaphyx or formal conversion to rygb was recorded , along with length of hospital stay ( los ) .
comparison of pre- and postbariatric procedure outcomes was performed using a paired student t - test .
there were a total of thirty - seven patients from 2003 to 2010 that were included in the analysis .
twenty - three patients were identified that had previously failed vbg and conversion to rygb .
the preoperative bmi in the rygb group was 47.7 7 kg / m compared to 43 10 kg / m in the stomaphyx group . as seen in figure 1 , patients with previously failed vbg had a significant decrease in bmi following rygb ( 47.7 7 kg / m to 35 7 kg / m at 24-month followup , p = 0.0007 ) . in patients following stomaphyx endoscopic revision , there was also a significant decrease in bmi ( 43 10 kg / m to 40 9 kg / m at 6-month followup , p = 0.0007 ) .
the only complications observed in the stomaphyx group were short - term nausea and headache ( table 2 ) . on the other hand
major complications in the rygb group include anastomotic leak ( 8.7% ) , incisional hernia ( 13% ) , fistula ( 8.7% ) , respiratory failure ( 8.7% ) , and perforation ( 4.4% ) .
the median los following rygb was six days compared to 1.5 0.5 days following stomaphyx endoscopic revision .
this study retrospectively compares the use of two distinct treatment strategies in patients with failed vbg at a single institution .
formal conversion of the vbg to a rygb in morbidly obese patients results in marked weight loss , however , with considerable morbidity .
on the other hand , endoscopic revision with the stomaphyx device in morbidly obese patient produced less weight loss , with minimal morbidity .
reported that 38% of patients converted to rygb had either early or late complications , including anastomotic leak , abscess , stricture , bleeding , and respiratory failure .
other studies have also suggested increased complication rates ranging from 12% to 41% , following revisional surgery in previously failed restrictive bariatric procedures [ 10 , 11 ] .
endoscopic treatment avoids the need for operative revision and related intra - abdominal complications , such as anastomotic leakage .
two common reasons for vbg failure are enlargement of the gastric pouch and staple line dehiscence .
the stomaphyx device is used to decrease the size of the gastric pouch by approximating and immobilizing two or more serosal surfaces through tissue fastening .
mikami et al . reported successful use of the stomaphyx device in 39 patients to reduce the size of the gastric pouch following rygb .
these authors reported a 10 kg weight loss at one - year followup with no major complications and no mortalities .
leitman et al . also reported a 7.3 kg weight loss at one - year followup and no major complications , following endoscopic revision with the stomaphyx device .
the two main limitations of our study are duration of followup and a small sample size .
there are also other potentially viable options such as revision with an adjustable gastric band that were not included . with these major limitations
it does , however , present an interesting debate about the relative importance of less complications versus more weight loss .
in conclusion , endoscopic revision via the stomaphyx device is a safe revisional treatment strategy in morbidly obese patients that have failed vbg . in addition
, endoscopic revision may be a reasonable initial approach to failed vbg , with low complication rates .
further studies are needed to clarify the role of the stomaphyx pouch reduction in patients with failed vbg . | background . weight regain secondary to vbg pouch dilation is a typical referral for bariatric surgeons . in this study
we compare an endoluminal pouch reduction ( stomaphyx ) to rygb for revision . methods .
a retrospective review was completed for patients with a previous vbg presenting with weight regain between 20032010 .
results .
thirty patients were identified for study 23 rygb , 14 stomaphyx .
significant post procedure bmi loss was seen in each cohort ( rygb , 47.7 7 kg / m2 to 35 7 kg / m2 ; stomaphyx 43 10 kg / m2 to 40 9 kg / m2 , p = 0.0007 ) . whereas nausea and headache were the only complications observed in stomaphyx patients , the rygb group had a 43.5% complication rate and 1 mortality .
complications following rygb include : incisional hernia ( 13% ) , anastomotic leak ( 8.7% ) , respiratory failure ( 8.7% ) , fistula ( 8.7% ) , and perforation ( 4.35% ) .
the median length of stay following rygb was 6 days compared to 1.5 0.5 days following stomaphyx .
conclusion .
this study suggests that while rygb revision may achieve greater weight loss , the complication rates and severity is discouraging .
stomaphyx may be a safe alternative .
further technical modifications of the device and longer follow - up may clarify the role of this approach . |
thin is a longitudinal primary care medical records database of over 9 million patients in total , which currently covers around 6% of u.k .
thin database contains individual patient information recorded by pcps as part of their routine clinical care such as demographic factors , pcp consultations , referrals , hospitalizations , laboratory test results , and prescriptions written by pcps .
letters from specialist visits and hospital admissions ( i.e. , discharge letters ) are also available .
prescriptions written by pcps are generated and recorded automatically in the database using a coded drug dictionary ( multilex ) .
several validation studies have been conducted in thin database reporting high confirmation rates of recorded diagnoses and completeness of data ( 1316 ) .
this primary care database has already been used for studies of diabetes ( 1719 ) .
the study research protocol was approved by the uk research ethics committee ( 09/h0305/64 ) .
the source population was made up of all individuals aged 184 years between january 2000 and december 2007 who were enrolled at least 2 years with a pcp , with 1 year or more elapsed since the first recorded prescription and with some recorded health care contact in the previous 2 years . only individuals with an enrollment status of permanent ( currently enrolled with the pcp ) or
start date was the date when an individual met all above eligibility criteria . to select the study population of diabetic patients , all members from the source population
were followed from start date until the earliest occurrence of first record of diabetes or antidiabetic treatment , death , or 31 december 2007 .
we then excluded all women with gestational diabetes ( because of the specific idiosyncrasy of this diabetes ) , patients with type 1 diabetes without antidiabetic treatment ( considered as possibly misclassified ) , and individuals with a first - ever diagnosis of diabetes within 30 days of death and no treatment ever recorded ( considered as having incomplete information ) .
finally , we excluded all those with any diagnosis code for retinopathy or maculopathy recorded until the first diagnosis of diabetes resulting in a final study cohort of 121,834 diabetic patients ( 55% of them were men ) .
distribution of this cohort by sex , age , and diabetes type is shown in table 1 .
diabetes cohort distribution by sex , age , and diabetes type we followed our study population of diabetic patients from the date of first record of diabetes or antidiabetic treatment in the study period until first recording of dr or dmp including dmo .
two separate follow - ups were performed : 1 ) until the earliest occurrence of one of the following end points : dr , 85 years of age , death , or 31 december 2008 ; 2 ) until the earliest occurrence of one of the following end points : dmp , 85 years of age , death , or 31 december 2008 . for dr , we identified patients with a recorded code suggesting an incident diagnosis of retinopathy related to diabetes ( table 2 ) .
diagnoses related to age - related macular degeneration or to any other cause of retinopathy not related to diabetes were excluded . read codes used to identify dr and dmp nos , not otherwise specified ; o / e , on examination . for , dmp and dmo , we identified patients with a recorded code suggesting any incident diagnosis of maculopathy related to diabetes including macular edema , exudative maculopathy , or any other nonspecific maculopathy code ( table 2 ) .
a two - step validation strategy was used for both outcomes : dr and dmp ( including dmo ) .
we identified 20,838 dr patients ( 49.5% had only background / nonproliferative dr codes within the first month , 2.3% had a proliferative dr code , and 48.2% had only unspecific dr codes ) and 4,064 dmp patients , based on automated diagnosis codes .
we then requested from thin all anonymized free - text comments recorded by the pcps between 1 month before and 2 months after the recorded diagnosis for a random sample of 500 ( 2.4% ) of dr patients ( automated random numbers were generated to obtain the sample ) and for all dmp patients . in the free - text entries , pcps can provide additional information derived from referral letters , diagnostic procedures , and test results such as visual acuity , intraocular pressure , and fundoscopy , among others .
we performed a manual review of computer profiles of patients with dr and assigned the event as probable , possible , doubtful , and non - dr case .
we defined probable dr case subjects as patients with an objective diagnosis of retinopathy related to diabetes describing the grade of retinopathy or site affected .
possible dr case subjects were all patients with a specific diagnosis but no mention of grade of retinopathy or site affected .
doubtful dr case subjects were patients with only screening appointments or conflicting entries between procedure results and recorded diagnosis .
non - dr case subjects were patients where the diagnosis was explicitly excluded or the date of first dr diagnosis occurred before the date of diabetes ascertained in the study period . similarly , we reviewed the computerized profiles of patients with dmp and assigned the event as probable , possible , and non - dmp case .
we defined probable dmp case subjects as patients with a diagnosis of maculopathy related to diabetes and recording of the type of maculopathy or site affected .
when the diagnosis was not specifically mentioned in the read code or confirmed in free text , we categorized them as possible dmp .
noncase subjects were patients with an unspecific code and/or confirmation of the absence of maculopathy .
we defined the subgroup of probable dmo case subjects as patients with a read code mentioning macular edema or an indication in the free - text comments of the site , the grade of edema , or retinal thickening .
then , in a second step , a questionnaire was sent to the pcps of 200 patients randomly sampled from the subgroup of the 500 dr patients whose computer profiles were manually reviewed and to the pcps of a random sample of 200 patients of the 4,064 manually reviewed dmp patients .
pcps were requested to confirm the diagnosis of dr or dmp ( and in particular dmo ) and were also asked to send a copy of all records related to the event of interest , including referral letters , diagnostic procedures , test results such as visual acuity , intraocular pressure , or fundoscopy results .
the researchers did not contact the practices directly , but did so via the additional information services ( ais ) , which is part of the thin organization . once contacted and having received the questionnaires , each pcp sent the supplementary information and the response to the questionnaire to ais , and ais ensured that all personal data were removed before forwarding the information to the researchers .
after the review of all this supplementary information , a final case status of dr or dmp was assigned for the random sample of dr and all dmp patients . additionally , the percentage of confirmed case subjects in each of the categories defined for dr and dmp ( i.e. , probable , possible , and noncase ) was determined .
the date of first diagnosis of dr or dmp also was ascertained based on all information available , including that provided by the pcps in their responses to questionnaires and copies of ophthalmology records provided .
the confirmation rate of dr and dmp assigned in our first step ( review of computerized patient profiles including free - text comments ) was computed using as numerator the number of final confirmed case subjects based on all information provided by the pcps ( gold standard ) and as denominator the total number of patients considered case subjects after manual review of patient profiles .
thin is a longitudinal primary care medical records database of over 9 million patients in total , which currently covers around 6% of u.k .
thin database contains individual patient information recorded by pcps as part of their routine clinical care such as demographic factors , pcp consultations , referrals , hospitalizations , laboratory test results , and prescriptions written by pcps .
letters from specialist visits and hospital admissions ( i.e. , discharge letters ) are also available .
prescriptions written by pcps are generated and recorded automatically in the database using a coded drug dictionary ( multilex ) .
several validation studies have been conducted in thin database reporting high confirmation rates of recorded diagnoses and completeness of data ( 1316 ) .
this primary care database has already been used for studies of diabetes ( 1719 ) .
the study research protocol was approved by the uk research ethics committee ( 09/h0305/64 ) .
the source population was made up of all individuals aged 184 years between january 2000 and december 2007 who were enrolled at least 2 years with a pcp , with 1 year or more elapsed since the first recorded prescription and with some recorded health care contact in the previous 2 years . only individuals with an enrollment status of permanent ( currently enrolled with the pcp ) or
start date was the date when an individual met all above eligibility criteria . to select the study population of diabetic patients , all members from the source population
were followed from start date until the earliest occurrence of first record of diabetes or antidiabetic treatment , death , or 31 december 2007 .
we then excluded all women with gestational diabetes ( because of the specific idiosyncrasy of this diabetes ) , patients with type 1 diabetes without antidiabetic treatment ( considered as possibly misclassified ) , and individuals with a first - ever diagnosis of diabetes within 30 days of death and no treatment ever recorded ( considered as having incomplete information ) .
finally , we excluded all those with any diagnosis code for retinopathy or maculopathy recorded until the first diagnosis of diabetes resulting in a final study cohort of 121,834 diabetic patients ( 55% of them were men ) .
distribution of this cohort by sex , age , and diabetes type is shown in table 1 .
we followed our study population of diabetic patients from the date of first record of diabetes or antidiabetic treatment in the study period until first recording of dr or dmp including dmo .
two separate follow - ups were performed : 1 ) until the earliest occurrence of one of the following end points : dr , 85 years of age , death , or 31 december 2008 ; 2 ) until the earliest occurrence of one of the following end points : dmp , 85 years of age , death , or 31 december 2008 .
for dr , we identified patients with a recorded code suggesting an incident diagnosis of retinopathy related to diabetes ( table 2 ) . diagnoses related to age - related macular degeneration or to any other cause of retinopathy not related to diabetes were excluded .
read codes used to identify dr and dmp nos , not otherwise specified ; o / e , on examination . for , dmp and dmo
, we identified patients with a recorded code suggesting any incident diagnosis of maculopathy related to diabetes including macular edema , exudative maculopathy , or any other nonspecific maculopathy code ( table 2 ) .
a two - step validation strategy was used for both outcomes : dr and dmp ( including dmo ) .
we identified 20,838 dr patients ( 49.5% had only background / nonproliferative dr codes within the first month , 2.3% had a proliferative dr code , and 48.2% had only unspecific dr codes ) and 4,064 dmp patients , based on automated diagnosis codes .
we then requested from thin all anonymized free - text comments recorded by the pcps between 1 month before and 2 months after the recorded diagnosis for a random sample of 500 ( 2.4% ) of dr patients ( automated random numbers were generated to obtain the sample ) and for all dmp patients . in the free - text entries
, pcps can provide additional information derived from referral letters , diagnostic procedures , and test results such as visual acuity , intraocular pressure , and fundoscopy , among others .
we performed a manual review of computer profiles of patients with dr and assigned the event as probable , possible , doubtful , and non - dr case .
we defined probable dr case subjects as patients with an objective diagnosis of retinopathy related to diabetes describing the grade of retinopathy or site affected .
possible dr case subjects were all patients with a specific diagnosis but no mention of grade of retinopathy or site affected .
doubtful dr case subjects were patients with only screening appointments or conflicting entries between procedure results and recorded diagnosis .
non - dr case subjects were patients where the diagnosis was explicitly excluded or the date of first dr diagnosis occurred before the date of diabetes ascertained in the study period .
similarly , we reviewed the computerized profiles of patients with dmp and assigned the event as probable , possible , and non - dmp case .
we defined probable dmp case subjects as patients with a diagnosis of maculopathy related to diabetes and recording of the type of maculopathy or site affected .
when the diagnosis was not specifically mentioned in the read code or confirmed in free text , we categorized them as possible dmp .
noncase subjects were patients with an unspecific code and/or confirmation of the absence of maculopathy .
we defined the subgroup of probable dmo case subjects as patients with a read code mentioning macular edema or an indication in the free - text comments of the site , the grade of edema , or retinal thickening .
then , in a second step , a questionnaire was sent to the pcps of 200 patients randomly sampled from the subgroup of the 500 dr patients whose computer profiles were manually reviewed and to the pcps of a random sample of 200 patients of the 4,064 manually reviewed dmp patients .
pcps were requested to confirm the diagnosis of dr or dmp ( and in particular dmo ) and were also asked to send a copy of all records related to the event of interest , including referral letters , diagnostic procedures , test results such as visual acuity , intraocular pressure , or fundoscopy results .
the researchers did not contact the practices directly , but did so via the additional information services ( ais ) , which is part of the thin organization . once contacted and having received the questionnaires , each pcp sent the supplementary information and the response to the questionnaire to ais , and ais ensured that all personal data were removed before forwarding the information to the researchers .
after the review of all this supplementary information , a final case status of dr or dmp was assigned for the random sample of dr and all dmp patients . additionally , the percentage of confirmed case subjects in each of the categories defined for dr and dmp ( i.e. , probable , possible , and noncase ) was determined .
the date of first diagnosis of dr or dmp also was ascertained based on all information available , including that provided by the pcps in their responses to questionnaires and copies of ophthalmology records provided .
the confirmation rate of dr and dmp assigned in our first step ( review of computerized patient profiles including free - text comments ) was computed using as numerator the number of final confirmed case subjects based on all information provided by the pcps ( gold standard ) and as denominator the total number of patients considered case subjects after manual review of patient profiles .
five hundred dr computerized patient profiles with free - text comments were manually reviewed by one of the authors .
we categorized 331 ( 66.2% ) as probable dr and 87 ( 17.4% ) as possible dr . in addition , 2.8% ( n = 14 ) were categorized as
doubtful case subjects and 13.6% ( n = 68 ) as noncase subjects .
figure 1 shows the results of the review process and resulting categorization of the sample of dr patients reviewed . in the second step
, we received 176 valid questionnaires of the 200 requested ( pcp response rate was 88% ) : a majority of them ( 71.5% ) included copies of anonymized records related to the retinopathy . among these ,
150 had been classified as probable or possible dr case subjects in the first step and 131 of them were finally assigned a diagnosis of dr based on the questionnaires and additional information received ( confirmation rate of 87.3% ) . among the 131 confirmed dr , 73.3% were background / nonproliferative dr , 2.3% were proliferative dr , and 24.4% had no specification on dr grading .
we also received questionnaires for 26 diabetic patients who were originally classified as doubtful or noncase subjects , and the diagnosis of dr was finally confirmed in 30.8% : 50% of doubtful and 25.0% of noncase subjects , respectively .
validation chart of dr detected codes during the years 2000 to 2008 in thin database .
the final estimated confirmation rate among all initially computer - detected patients was 78.0% , resulting from the confirmation rate weighted by the corresponding percentage in each category ( 87.3% confirmed dr weighted by 83.6% categorized as
probable dr and possible dr plus 30.8% confirmed dr weighted by 16.4% categorized as
the confirmation rates for patients with background / nonproliferative dr , proliferative dr , and unspecified retinopathy grade within 1 month were 92.0 , 100 , and 65.1% , respectively .
we reviewed all 4,064 computerized patient profiles with free - text comments of patients automatically detected with a code of dmp .
of these , 2,460 ( 60.5% ) were categorized as probable dmp , 1,216 ( 29.9% ) as possible dmp , and 388 ( 9.5% ) as noncases .
similar to the process of validation of dr , 200 patients were randomly selected in a second step , and questionnaires were sent to their pcps . of these ,
176 valid questionnaires were returned ( response rate of 88% ) , and 73.2% of them enclosed copies of anonymized records related to the maculopathy . among
the valid questionnaires returned , there were 172 who had been considered probable or possible case subjects of dmp after the manual review of patient profiles .
in this subgroup , 150 patients were assigned a final diagnosis of dmp based on the questionnaires , resulting in a confirmation rate of 87.2% ( 91.9% among probable dmp and 78.7% among possible dmp , respectively ) .
we also received questionnaires for four diabetic patients originally considered as noncase subjects , and all of them were confirmed as noncase subjects .
validation chart of dmp ( and edema type ) detected codes during the years 2000 to 2008 in thin database .
the final estimated confirmation rate among all computer - detected patients was 78.8% , resulting from the confirmation rate weighted by the corresponding percentage in each category ( 87.2% confirmed dmp weighted by 90.4% of categorized as
we also performed the validation of dmo . among the 4,064 maculopathies reviewed in the first step ,
17.5% ( n = 711 ) were considered as probable dmo . in the second step , 36 patients were randomly sampled and questionnaires were sent to the pcps . of these ,
31 valid questionnaires were returned ( 86% ) , and 81% of them enclosed copies of anonymized records related to the maculopathy .
computer codes that specifically included the term oedema in their literal presented a confirmation rate of 86.2% .
there also were 33 patients registered as maculopathies without any other specification of type in their code or in free - text comments who were finally confirmed as macular edema in the second step , representing 54% of all finally confirmed dmo case subjects .
five hundred dr computerized patient profiles with free - text comments were manually reviewed by one of the authors .
we categorized 331 ( 66.2% ) as probable dr and 87 ( 17.4% ) as possible dr . in addition , 2.8% ( n = 14 ) were categorized as
doubtful case subjects and 13.6% ( n = 68 ) as noncase subjects .
figure 1 shows the results of the review process and resulting categorization of the sample of dr patients reviewed . in the second step
, we received 176 valid questionnaires of the 200 requested ( pcp response rate was 88% ) : a majority of them ( 71.5% ) included copies of anonymized records related to the retinopathy . among these ,
150 had been classified as probable or possible dr case subjects in the first step and 131 of them were finally assigned a diagnosis of dr based on the questionnaires and additional information received ( confirmation rate of 87.3% ) . among the 131 confirmed dr , 73.3% were background / nonproliferative dr , 2.3% were proliferative dr , and 24.4% had no specification on dr grading .
we also received questionnaires for 26 diabetic patients who were originally classified as doubtful or noncase subjects , and the diagnosis of dr was finally confirmed in 30.8% : 50% of doubtful and 25.0% of noncase subjects , respectively .
validation chart of dr detected codes during the years 2000 to 2008 in thin database .
the final estimated confirmation rate among all initially computer - detected patients was 78.0% , resulting from the confirmation rate weighted by the corresponding percentage in each category ( 87.3% confirmed dr weighted by 83.6% categorized as
probable dr and possible dr plus 30.8% confirmed dr weighted by 16.4% categorized as
the confirmation rates for patients with background / nonproliferative dr , proliferative dr , and unspecified retinopathy grade within 1 month were 92.0 , 100 , and 65.1% , respectively .
we reviewed all 4,064 computerized patient profiles with free - text comments of patients automatically detected with a code of dmp .
of these , 2,460 ( 60.5% ) were categorized as probable dmp , 1,216 ( 29.9% ) as possible dmp , and 388 ( 9.5% ) as noncases .
similar to the process of validation of dr , 200 patients were randomly selected in a second step , and questionnaires were sent to their pcps . of these ,
176 valid questionnaires were returned ( response rate of 88% ) , and 73.2% of them enclosed copies of anonymized records related to the maculopathy . among
the valid questionnaires returned , there were 172 who had been considered probable or possible case subjects of dmp after the manual review of patient profiles . in this subgroup ,
150 patients were assigned a final diagnosis of dmp based on the questionnaires , resulting in a confirmation rate of 87.2% ( 91.9% among probable dmp and 78.7% among possible dmp , respectively ) .
we also received questionnaires for four diabetic patients originally considered as noncase subjects , and all of them were confirmed as noncase subjects .
validation chart of dmp ( and edema type ) detected codes during the years 2000 to 2008 in thin database .
the final estimated confirmation rate among all computer - detected patients was 78.8% , resulting from the confirmation rate weighted by the corresponding percentage in each category ( 87.2% confirmed dmp weighted by 90.4% of categorized as probable dmp or
we also performed the validation of dmo . among the 4,064 maculopathies reviewed in the first step , 17.5% ( n = 711 )
in the second step , 36 patients were randomly sampled and questionnaires were sent to the pcps . of these , 31 valid questionnaires were returned ( 86% ) , and 81% of them enclosed copies of anonymized records related to the maculopathy . the pcps confirmed 28 as incident dmo ( 90.3% ) .
computer codes that specifically included the term oedema in their literal presented a confirmation rate of 86.2% . there also were 33 patients registered as maculopathies without any other specification of type in their code or in free - text comments who were finally confirmed as macular edema in the second step , representing 54% of all finally confirmed dmo case subjects .
this is the first study to validate dr and dmp diagnoses in a primary care database .
the validation strategy was successful thanks to the high proportion of pcps who returned completed validation questionnaires ( i.e. , 90% ) .
furthermore , most pcps provided with the completed questionnaire copies original clinical records that usually included specialist notes after ophthalmologic assessment , which was instrumental for the validation of diagnoses .
the validation of a sample of automatically identified dr patients showed that the proposed set of computer codes was moderately accurate ( confirmation rate of 78% ) in predicting the actual diagnosis . however , the confirmation rate ( 87% ) increased after the inclusion of free - text comments from the pcp in the computerized patients profiles .
the selected set of computer codes predicted the diagnosis with moderate accuracy ( confirmation rate of 79% ) , and this increased to 87% when reviewing the patients profiles after incorporating free - text comments .
we showed that specific codes of dmo have a high confirmation rate ( 86% ) .
however , a high proportion of true dmo case subjects would not be identified based on the computer codes alone or with the addition of free - text comments ( 54% ) . to ascertain all dmo case subjects , additional information from the pcps
prior studies conducted using claims data have also assessed recorded diagnosis of diabetic eye complications .
one study reported good agreement between incidences of dr estimated using claims data versus population - based studies ( 20 ) , and another one assessed the validity of the diagnoses of dmo and found high sensitivity ( 88% ) and specificity ( 96% ) ( 20,21 ) . nevertheless , our study is the first one to test the validity of registration of ophthalmological complications of diabetes in a primary care database .
similar to what has been observed in a previous study with thin database , the validity of the studied outcome increased with the addition of free - text comments to computerized patient profiles ( 15 ) .
first , we relied on the existing coding system ( i.e. , read ) in thin . yet , the read dictionary in some instances may not be specific enough to enable the recording of the outcomes of interest , such as the severity scale for retinopathy or the type of maculopathy .
however , there were specific codes of dr , dmp , and dmo that pcps could enter to record the specific grade of diagnosis without having to resort to more unspecific ones ( shown in table 2 )
. some diabetic patients also could consult optician practices without having to first go through their pcp , and consequently pcps would not have recorded the relevant information systematically .
however , this potential limitation should have had a limited impact in our study because ophthalmologic screening care is offered to all u.k .
diabetic patients since it was shown to be more cost - effective to detect cases of sight threatening dr than incidental screening ( 7 ) .
all diabetic patients are invited for diabetic eye complication screening , a national screening program .
pcps participating in thin database also are encouraged to record results from screening programs as well as information resulting from ophthalmologist referrals , as part of diabetic quality outcomes framework .
according to a recent study , around 3% of all patients screened for dr in the national diabetic retinopathy screening committee require referral to ophthalmology department because of positive results and 79% of them are referred for maculopathy ( 22 ) .
it is our opinion that this system guarantees that thin database is a rich data source for studies on ophthalmological diseases in patients with diabetes . for feasibility and practical reasons we were not able to validate all dr potential case subjects (
however , it appears sensible to infer the results from our manual review of a random sample to the whole dr computer - detected population . as shown by the current study , recording of read codes for dr , dm , and dmo
the validity of these conditions improved with the incorporation of free - text comments to the patient s computerized profile when contrasted with additional information directly provided by the pcp .
thin database has proved to be a valuable resource to perform studies of ophthalmological diabetes complications in the general population when supplemented with information present in free - text notes . | objectiveto describe the validity of recorded diabetic retinopathy ( dr ) and diabetic maculopathy ( dmp ) diagnoses , including edema ( dmo ) in the health improvement network ( thin ) database.research design and methodsin two independent computer searches , we detected 20,838 patients with diabetes aged 184 years with a first dr computer read entry in 20002008 and 4,064 with a first dmp entry .
a two - step strategy was used to validate both outcomes as follows : 1 ) review of patient profiles including free - text comments from primary care practitioners ( pcps ) ( containing referral information and test results ) of a random sample of 500 dr and all dmp computer - detected patients .
we classified them in probable , possible , and noncase according to the diagnosis plausibility based on the manual review of the computerized information ; and 2 ) review of questionnaires sent by pcps and medical records in a random sample ( n = 200 for each outcome including 36 diabetic macular edema [ dmo ] ) .
gold standard was pcps confirmation.resultsafter profiles review , we categorized 418 as probable / possible dr .
in addition , 3,676 dmp were categorized as probable / possible ( including 711 dmo ) .
after review of information sent by pcps , confirmation rates were 87.3 and 87.2% , respectively ( 90.3% for dmo ) . when we applied them to the whole sample of computer - detected patients , the weighted confirmation rate was 78.0% for dr and 78.8% for dmp ( 86.2% for dmo).conclusionsread codes for dr , dm , and dmo are moderately accurate in identifying incident case subjects of these ophthalmologic complications .
the validity improved when incorporating pcps text comments to the patient s profile .
thin database proved to be a valuable resource to study ophthalmological diabetes complications . |
however , the possibility of using a folk theory for predicting others should be taken more seriously in social psychology .
folk psychology consists of an array of lay theories or platitudes about how the mind works , which are believed more or less uncritically .
mundane examples for widely held lay theories are : people who work hard succeed ; the longer an event lies in the past , the worse it is remembered ; people can not change their personality , moral character , or intelligence ; opposites attract ; or that happiness is a matter of wealth . lay theories
also include information about typical everyday settings , typical characters , and how people are expected to behave in certain contexts ( karniol , 2003 ) .
the answer to krueger s question where such lay theories come from is that they are a compendium of day - to - day psychological wisdom based on various personal experiences .
it takes time to develop these theories , and , like scientific theories , they are refutable and revisable ( e.g. , harris , 1992 ) . if our original article can be interpreted as saying that the basis of these lay theories is an inference from well - known people to people in general , we apologize for this misunderstanding .
lay theories come from everyday experiences , irrespective of whether well - known or unknown people are involved .
of course , lay theories can be enriched with information about specific people if such information exists ( nickerson , 1999 ) .
for example , a social - cognitive variant of theory theory , namely karniol s ( 2003 ) self - as - distinct model , holds that general ( generic , in karniol s terminology ) knowledge can be used in predictions .
specific knowledge , that is applicable only to a certain individual , can lead to an activation of the self , but this is not equal to using the self as a basis for a prediction . in sum , there is an alternative to the projection view that is theoretically plausible and empirically substantiated .
our example of a choice between carrion and apples as food is only a far - fetched example to illustrate that the use of a theory can also lead to a prediction that entirely overlaps with one s own choice .
most people would ( correctly ) predict a preference for cooked beef , but some people ( sometimes ) prefer raw beef ( otherwise there would be no beef tartar ) .
however , for predicting this , there is no need to project your own preference .
rather , you can apply the simple rule that people prefer cooked over raw meat . if you do so , a high correlation between own and predicted choice follows .
krueger ( 2008 , 2012 ) correctly points out that there are ways to identify biases based on projection by using corrected correlations ( see below ) , but most of the evidence for social projection still is based on uncorrected correlations .
knowledge used in predictions can be wrong , and it does not necessarily have to do anything with oneself knowledge can happen to lead to a prediction that is similar to one s own point of view , but it needs not to .
therefore , correlations are hard to interpret and are unreliable indicators of social projection . however , correlations varying widely in magnitude all have been interpreted as providing evidence for social projection .
krueger points out that by sampling error alone , self - other correlations will vary , and that variation is a general property of empirical evidence .
meta - analysis is a suitable method to aggregate effect sizes over studies and to thereby wash away the error .
while effect sizes do vary , this variation is treated differently in meta - analyses ( cf .
borenstein , hedges , higgins , & rothstein , 2009 ) . under the fixed - effect model ,
the assumption is that there is only one true effect which underlies all studies , and differences in the effect sizes are due to sampling error only .
in contrast , under the random - effects model , the assumption is that there is an array of true effects depending on the characteristics of the studies , and therefore , that there are different effect sizes that underlie the effect size variations in various studies .
variations in the effect sizes are due to sampling error , in addition to true variation in effect sizes .
therefore , depending on the approach , variation in the magnitude of correlations can be interpreted as a variation around a fixed - effect ( indicating the same process , i.e. , projection ) , but also as a variation that indicates different processes ( e.g. , the application of different theories ) .
meta - analysis by itself does not tell you whether the effect is fixed or random ; rather this is up to the researcher to decide .
a related point is which magnitude of correlation should be used as a benchmark for demonstrating projection .
krueger points out that our proposal of using self - reliability has a problem : it sets a ceiling and ensures that only a bias of underprojection can be discovered
social projection can be justified and be statistically appropriate ( dawes , 1989 ; hoch , 1987 ) , and there are measures that capture whether a truly false consensus effect ( krueger & zeiger , 1993 ) exists , by correlating item endorsements with the difference between estimated and actual consensus .
a truly false consensus effect tells us that people are getting their predictions wrong , but again , not what the source of this bias is ( i.e. , simulation vs. theory ) .
the other aspect is conceptual : from the perspective of simulation theory , one can not use the self too much .
either you simulate or you do nt , but this is not a matter that is related to the correctness of the prediction .
simulation means that you use your own system in exactly the same way for predicting thoughts , actions , and feelings of another person as you would use it if you thought , acted , or felt yourself .
krueger s observation thus is true ; by using self - reliability you could only detect underprojection , because overprojection is logically impossible . if social cognition were to use this benchmark , the conclusions probably would be quite different : that there are many cases of underprojection . taken together , additional methods that go beyond simple correlations are necessary , for example , the use of empathy gaps ( loewenstein , 1996 ) , the method of juxtaposed versus independent predictions by perner , gschaider , khberger , and schrofner ( 1999 ) , or the use of process tracing methods in addition to input output measures ( schulte - mecklenbeck , khberger , & ranyard , 2011 ) . in addition , even self - reports can be successfully used in studies on social projection ( dunning & hayes , 1996 ) . in sum , it is not that correlations should be abandoned altogether , but they should be interpreted with caution and supported by additional methods . | in a commentary to our article on the role of theory and simulation in social predictions , krueger ( 2012 ) argues that the role of theory is neglected in social psychology for a good reason .
he considers evidence indicating that people readily generalize from themselves to others . in response
, we stress the role of theoretical knowledge in predicting other people s behavior .
importantly , prediction by simulation and prediction by theory can lead to high as well as to low correlations between own and predicted behavior .
this renders correlations largely useless for identifying the prediction strategy .
we argue that prediction by theory is a serious alternative to prediction by simulation , and that reliance on correlation has led to a bias toward simulation . |
telemedicine is defined by the world health organization as , the delivery of health care services , where distance is a critical factor , by all health care professionals using information and communication technologies for the exchange of valid information for diagnosis , treatment and prevention of disease and injuries , research and evaluation , and for the continuing education of health care providers , all in the interests of advancing the health of individuals and their communities .
telemedicine may play an important role improving access to subspecialty expertise for patients in locations without access to specialists or for patients requiring time - sensitive care .
for example , the use of telemedicine in adult stroke care has been shown to permit regional hospitals to retain patients and reduce costs while also improving thrombolysis decisions .
many regional hospital neonatal intensive care units also provide care without immediate access to subspecialty consultation , forcing neonatologists to decide between forgoing subspecialty involvement and transferring critically ill patients to sometimes distant hospitals .
previous research has demonstrated that clinical agreement between neonatologists is good to excellent with telemedicine , and that clinicians had a positive impression of telemedicine use .
however , studies of telemedicine to perform detailed dermatologic evaluations suggest difficulty with some features , cautioning that the use of telemedicine may need to be evaluated for each subspecialty indication .
studies have not evaluated the use of telemedicine for neurologic or dysmorphology evaluations in the neonatal intensive care unit .
we performed a prospective study comparing in - person and telemedicine dysmorphology and neurologic examinations of subjects in the neonatal intensive care unit .
we aimed to establish the accuracy of telemedicine examinations in this setting and to identify measures to optimize telemedicine examinations .
the study was approved by the institutional review board and informed consent was obtained from each subject s parent(s ) .
patients in the neonatal intensive care unit were eligible for participation if they had received : 1 ) consultation by the genetics service for dysmorphic features or ; 2 ) consultation by the neurology service for encephalopathy .
each subject underwent telemedicine examination using an intouch health rp - lite remote presence system ( chelmsford , ma ) and a amd-2500 general examination camera with a 50x zoom lens ( chelmsford , ma ) .
figure 1 presents a procedure schematic . a remote consultant physician ( geneticist or neurologist ) in a remote location evaluated each subject with the assistance of a bedside clinician ( geneticist or neurologist ) .
the bedside clinician followed the instructions of the remote consultant physician ( e.g. positioning of the infant , demonstrating reflexes ) to optimize visualization .
the examination components did not need to be performed in any particular order , and order was often determined by initial state and cooperation of the infant with the examination .
the bedside clinician noted any discrepancies between what they saw in person and what was displayed on the screen , but the bedside clinician did not communicate any of these observations to the remote consultant physician .
the in - person bedside clinician examination was considered the accurate gold - standard to which the remote bedside physician s examinations were compared .
examination accuracy was determined by calculating the percent agreement between abnormal physical examination findings noted by the bedside clinician examination ( gold standard ) and the remote consultant physician examinations .
examination components identified accurately > 90% of the time were considered easy to identify .
examination components deemed as not achievable were categorized as those unable to be appreciated without an in - person examination or those that were unidentifiable with the clinical resolution of the tested equipment . after completing the telemedicine assessment ,
the remote consultant physician came to the bedside for the second phase of the study .
the bedside clinician and the remote consultant physician discussed any discrepancies between the telemedicine and in - person examinations and attempted technique adjustments to achieve optimal visualization of those examination components .
a remote consultant physician ( geneticist or neurologist ) in a remote location evaluated each subject with the assistance of a bedside clinician ( geneticist or neurologist ) .
the bedside clinician followed the instructions of the remote consultant physician ( e.g. positioning of the infant , demonstrating reflexes ) to optimize visualization .
the examination components did not need to be performed in any particular order , and order was often determined by initial state and cooperation of the infant with the examination .
the bedside clinician noted any discrepancies between what they saw in person and what was displayed on the screen , but the bedside clinician did not communicate any of these observations to the remote consultant physician .
the in - person bedside clinician examination was considered the accurate gold - standard to which the remote bedside physician s examinations were compared .
examination accuracy was determined by calculating the percent agreement between abnormal physical examination findings noted by the bedside clinician examination ( gold standard ) and the remote consultant physician examinations .
examination components identified accurately > 90% of the time were considered easy to identify .
examination components deemed as not achievable were categorized as those unable to be appreciated without an in - person examination or those that were unidentifiable with the clinical resolution of the tested equipment .
after completing the telemedicine assessment , the remote consultant physician came to the bedside for the second phase of the study . the bedside clinician and the remote consultant physician discussed any discrepancies between the telemedicine and in - person examinations and attempted technique adjustments to achieve optimal visualization of those examination components . these limitations and optimization techniques
ten subjects with dysmorphic features were evaluated by geneticists and ten subjects with encephalopathy were evaluated by neurologists .
abnormal examination findings and accuracy of identification via telemedicine are indicated in table 2 for the dysmorphology examination and table 3 for the neurologic examination .
most abnormal examination components were identified with an accuracy of greater than 90% . for the dysmorphology examination
, 81 of 87 ( 93% ) dysmorphic features were correctly identified using telemedicine on the initial examination .
optimization of lighting and positioning resulted in visualization of two additional abnormalities ( 83 of 87 , 95% ) .
for the neurologic examination , 36 of 39 ( 92% ) abnormal features were identified using telemedicine on the initial examination .
optimization of lighting and positioning resulted in visualization of one additional abnormality ( 37 or 39 , 95% ) .
all calculations were made comparing the assessments of the remote consultant physician s initial telemedicine examination and their own subsequent in - person examination .
inter - rater agreement with the bedside clinician was not ascertained . following the standardized telemedicine examination
, the bedside clinician and remote consultant physician worked together to optimize visualization of the examination of features that could not be assessed well remotely . in many cases the viewing angle , lighting , or examination technique could be adjusted to optimize the evaluation .
bedside clinician role : the bedside clinician plays a critical and active role in the evaluation , and must report their observations of the subject and their impressions regarding the camera s view
. the bedside clinician must evaluate and adjust the equipment s angle and distance from the subject , camera s position and zoom , lighting conditions , and patient positioning .
for effective evaluation , the camera and/or subject must be repositioned during the evaluation , and often these adjustments must occur in a fluid manner that is responsive to the examination needs and activity of the subject . for example , identifying dysmorphologic abnormalities which are often small and subtle requires optimal positioning , and movement , tone , and reflex examination must occur when the subject is in the relaxed state.image display : although the telemedicine equipment permits different displays for the bedside clinician and remote consultant physician , having the same image displayed allowed the bedside clinician to more readily make changes to enhance the view of the remote consultant .
suboptimal lighting , camera angles or camera focal points sometimes resulted in an inability of the remote consultant to appreciate abnormal skull shape , pigmentary differences ( especially eye color ) , respiratory effort , cleft palate evaluation , eye movement abnormalities , muscle tone abnormalities , and deep tendon reflexes . in all cases ,
the bedside clinician knew the camera images were suboptimal and did not allow them to fully appreciate the examination finding they could see in-person.focal point : prior to the examination , it was useful to identify the minimal focal distance for the equipment s cameras by zooming in on an object in the environment . subsequently , placing the equipment so the camera was at or minimally further away allowed maximal zoom while retaining the ability to focus on features .
for the equipment tested , this was approximately one meter from the patient.lighting : lighting conditions varied based on overhead lighting of different rooms and with different camera positions .
overhead lighting adjustments were often needed to provide lighting that was sufficiently bright enough for the examination without creating bright spots on the subject that exceeded the camera s ability to achieve good contrast balance .
in addition , low angle lighting was at times useful to distinguish skin surface characteristics .
having identical images displayed on the bedside and telemedicine physicians screens allowed the bedside physician to best optimize the lighting.viewing angle : features perpendicular to the plane of view were seen most accurately .
thus , for most components of the examination the optimal camera angle was directly above the isolette and angled down at the prone or supine subject since this approach maximized the amount of the subject perpendicular to the camera .
when examining particular body parts , positioning them perpendicular to the camera through repositioning of the equipment and the patient was essential .
for dysmorphology examination , this was particularly important when evaluating hand / finger characteristics or limb proportions . for neurologic examination ,
this was particularly important when evaluating reflexes or spontaneous movement.multiple views : while it may be difficult to maneuver large equipment around an isolette , the bedside clinician must be an active participant and can turn the subject as needed to optimize examination . for the dysmorphology examination ,
viewing the subject from angles rotated 180 degrees , so that the feet extend away from the camera permits better visualization of skull shape , hand shape , and dorsum of foot evaluation ( syndactyly , overfolded toes ) . turning the subject to the side permitted a lateral view which also aided in assessment of head shape . for the neurologic examination , repositioning of the subject s face perpendicular to the camera aided in evaluation of facial movement , suck , rooting , and eye movement evaluation . placing the subject
so that the angle of the joints was perpendicular to the camera aided in assessment of spontaneous movements and reflexes.special considerations for the hands : because neonates often have their hands clenched at rest , the hand must be held open and flat by the bedside clinician for proper evaluation .
the dorsum of the hand is best evaluated with the subject s head pointed towards the camera .
hand proportions can be distorted when the hand is not placed perpendicular to the camera viewing angle .
however , clinodactyly might not be identified since holding the fingers open made the fingers appear to curve inwards .
bedside clinician role : the bedside clinician plays a critical and active role in the evaluation , and must report their observations of the subject and their impressions regarding the camera s view .
the bedside clinician must evaluate and adjust the equipment s angle and distance from the subject , camera s position and zoom , lighting conditions , and patient positioning . for effective evaluation ,
the camera and/or subject must be repositioned during the evaluation , and often these adjustments must occur in a fluid manner that is responsive to the examination needs and activity of the subject .
for example , identifying dysmorphologic abnormalities which are often small and subtle requires optimal positioning , and movement , tone , and reflex examination must occur when the subject is in the relaxed state .
image display : although the telemedicine equipment permits different displays for the bedside clinician and remote consultant physician , having the same image displayed allowed the bedside clinician to more readily make changes to enhance the view of the remote consultant .
suboptimal lighting , camera angles or camera focal points sometimes resulted in an inability of the remote consultant to appreciate abnormal skull shape , pigmentary differences ( especially eye color ) , respiratory effort , cleft palate evaluation , eye movement abnormalities , muscle tone abnormalities , and deep tendon reflexes . in all cases , the bedside clinician knew the camera images were suboptimal and did not allow them to fully appreciate the examination finding they could see in - person .
focal point : prior to the examination , it was useful to identify the minimal focal distance for the equipment s cameras by zooming in on an object in the environment . subsequently , placing the equipment so the camera was at or minimally further away allowed maximal zoom while retaining the ability to focus on features . for the equipment tested , this was approximately one meter from the patient .
lighting : lighting conditions varied based on overhead lighting of different rooms and with different camera positions .
overhead lighting adjustments were often needed to provide lighting that was sufficiently bright enough for the examination without creating bright spots on the subject that exceeded the camera s ability to achieve good contrast balance .
in addition , low angle lighting was at times useful to distinguish skin surface characteristics . having identical images displayed on the bedside and telemedicine physicians screens allowed the bedside physician to best optimize the lighting .
thus , for most components of the examination the optimal camera angle was directly above the isolette and angled down at the prone or supine subject since this approach maximized the amount of the subject perpendicular to the camera . when examining particular body parts , positioning them perpendicular to the camera through repositioning of the equipment and the patient was essential .
for dysmorphology examination , this was particularly important when evaluating hand / finger characteristics or limb proportions . for neurologic examination ,
multiple views : while it may be difficult to maneuver large equipment around an isolette , the bedside clinician must be an active participant and can turn the subject as needed to optimize examination . for the dysmorphology examination ,
viewing the subject from angles rotated 180 degrees , so that the feet extend away from the camera permits better visualization of skull shape , hand shape , and dorsum of foot evaluation ( syndactyly , overfolded toes ) . turning the subject to the side permitted a lateral view which also aided in assessment of head shape .
for the neurologic examination , repositioning of the subject s face perpendicular to the camera aided in evaluation of facial movement , suck , rooting , and eye movement evaluation . placing the subject so that the angle of the joints was perpendicular to the camera aided in assessment of spontaneous movements and reflexes .
special considerations for the hands : because neonates often have their hands clenched at rest , the hand must be held open and flat by the bedside clinician for proper evaluation .
the dorsum of the hand is best evaluated with the subject s head pointed towards the camera .
hand proportions can be distorted when the hand is not placed perpendicular to the camera viewing angle .
however , clinodactyly might not be identified since holding the fingers open made the fingers appear to curve inwards .
ten subjects with dysmorphic features were evaluated by geneticists and ten subjects with encephalopathy were evaluated by neurologists .
abnormal examination findings and accuracy of identification via telemedicine are indicated in table 2 for the dysmorphology examination and table 3 for the neurologic examination .
most abnormal examination components were identified with an accuracy of greater than 90% . for the dysmorphology examination , 81 of 87 ( 93% ) dysmorphic features were correctly identified using telemedicine on the initial examination .
optimization of lighting and positioning resulted in visualization of two additional abnormalities ( 83 of 87 , 95% ) .
for the neurologic examination , 36 of 39 ( 92% ) abnormal features were identified using telemedicine on the initial examination .
optimization of lighting and positioning resulted in visualization of one additional abnormality ( 37 or 39 , 95% ) .
all calculations were made comparing the assessments of the remote consultant physician s initial telemedicine examination and their own subsequent in - person examination .
following the standardized telemedicine examination , the bedside clinician and remote consultant physician worked together to optimize visualization of the examination of features that could not be assessed well remotely . in many cases the viewing angle , lighting , or examination technique could be adjusted to optimize the evaluation .
bedside clinician role : the bedside clinician plays a critical and active role in the evaluation , and must report their observations of the subject and their impressions regarding the camera s view .
the bedside clinician must evaluate and adjust the equipment s angle and distance from the subject , camera s position and zoom , lighting conditions , and patient positioning .
for effective evaluation , the camera and/or subject must be repositioned during the evaluation , and often these adjustments must occur in a fluid manner that is responsive to the examination needs and activity of the subject .
for example , identifying dysmorphologic abnormalities which are often small and subtle requires optimal positioning , and movement , tone , and reflex examination must occur when the subject is in the relaxed state.image display : although the telemedicine equipment permits different displays for the bedside clinician and remote consultant physician , having the same image displayed allowed the bedside clinician to more readily make changes to enhance the view of the remote consultant .
suboptimal lighting , camera angles or camera focal points sometimes resulted in an inability of the remote consultant to appreciate abnormal skull shape , pigmentary differences ( especially eye color ) , respiratory effort , cleft palate evaluation , eye movement abnormalities , muscle tone abnormalities , and deep tendon reflexes . in all cases ,
the bedside clinician knew the camera images were suboptimal and did not allow them to fully appreciate the examination finding they could see in-person.focal point : prior to the examination , it was useful to identify the minimal focal distance for the equipment s cameras by zooming in on an object in the environment . subsequently , placing the equipment so the camera was at or minimally further away allowed maximal zoom while retaining the ability to focus on features . for the equipment tested ,
this was approximately one meter from the patient.lighting : lighting conditions varied based on overhead lighting of different rooms and with different camera positions .
overhead lighting adjustments were often needed to provide lighting that was sufficiently bright enough for the examination without creating bright spots on the subject that exceeded the camera s ability to achieve good contrast balance .
in addition , low angle lighting was at times useful to distinguish skin surface characteristics .
having identical images displayed on the bedside and telemedicine physicians screens allowed the bedside physician to best optimize the lighting.viewing angle : features perpendicular to the plane of view were seen most accurately .
thus , for most components of the examination the optimal camera angle was directly above the isolette and angled down at the prone or supine subject since this approach maximized the amount of the subject perpendicular to the camera . when examining particular body parts , positioning them perpendicular to the camera through repositioning of the equipment and the patient was essential .
for dysmorphology examination , this was particularly important when evaluating hand / finger characteristics or limb proportions . for neurologic examination ,
this was particularly important when evaluating reflexes or spontaneous movement.multiple views : while it may be difficult to maneuver large equipment around an isolette , the bedside clinician must be an active participant and can turn the subject as needed to optimize examination . for the dysmorphology examination ,
viewing the subject from angles rotated 180 degrees , so that the feet extend away from the camera permits better visualization of skull shape , hand shape , and dorsum of foot evaluation ( syndactyly , overfolded toes ) . turning the subject to the side permitted a lateral view which also aided in assessment of head shape . for the neurologic examination , repositioning of the subject s face perpendicular to the camera aided in evaluation of facial movement , suck , rooting , and eye movement evaluation . placing the subject so that the angle of the joints was perpendicular to the camera aided in assessment of spontaneous movements and reflexes.special considerations for the hands : because neonates often have their hands clenched at rest , the hand must be held open and flat by the bedside clinician for proper evaluation .
the dorsum of the hand is best evaluated with the subject s head pointed towards the camera .
hand proportions can be distorted when the hand is not placed perpendicular to the camera viewing angle .
however , clinodactyly might not be identified since holding the fingers open made the fingers appear to curve inwards .
bedside clinician role : the bedside clinician plays a critical and active role in the evaluation , and must report their observations of the subject and their impressions regarding the camera s view .
the bedside clinician must evaluate and adjust the equipment s angle and distance from the subject , camera s position and zoom , lighting conditions , and patient positioning .
for effective evaluation , the camera and/or subject must be repositioned during the evaluation , and often these adjustments must occur in a fluid manner that is responsive to the examination needs and activity of the subject .
for example , identifying dysmorphologic abnormalities which are often small and subtle requires optimal positioning , and movement , tone , and reflex examination must occur when the subject is in the relaxed state . image display : although the telemedicine equipment permits different displays for the bedside clinician and remote consultant physician , having the same image displayed allowed the bedside clinician to more readily make changes to enhance the view of the remote consultant .
suboptimal lighting , camera angles or camera focal points sometimes resulted in an inability of the remote consultant to appreciate abnormal skull shape , pigmentary differences ( especially eye color ) , respiratory effort , cleft palate evaluation , eye movement abnormalities , muscle tone abnormalities , and deep tendon reflexes . in all cases ,
the bedside clinician knew the camera images were suboptimal and did not allow them to fully appreciate the examination finding they could see in - person .
focal point : prior to the examination , it was useful to identify the minimal focal distance for the equipment s cameras by zooming in on an object in the environment . subsequently , placing the equipment so the camera was at or minimally further away allowed maximal zoom while retaining the ability to focus on features . for the equipment tested , this was approximately one meter from the patient . lighting : lighting conditions varied based on overhead lighting of different rooms and with different camera positions .
overhead lighting adjustments were often needed to provide lighting that was sufficiently bright enough for the examination without creating bright spots on the subject that exceeded the camera s ability to achieve good contrast balance .
in addition , low angle lighting was at times useful to distinguish skin surface characteristics .
having identical images displayed on the bedside and telemedicine physicians screens allowed the bedside physician to best optimize the lighting .
thus , for most components of the examination the optimal camera angle was directly above the isolette and angled down at the prone or supine subject since this approach maximized the amount of the subject perpendicular to the camera . when examining particular body parts , positioning them perpendicular to the camera through repositioning of the equipment and the patient was essential . for dysmorphology examination ,
this was particularly important when evaluating hand / finger characteristics or limb proportions . for neurologic examination ,
multiple views : while it may be difficult to maneuver large equipment around an isolette , the bedside clinician must be an active participant and can turn the subject as needed to optimize examination . for the dysmorphology examination ,
viewing the subject from angles rotated 180 degrees , so that the feet extend away from the camera permits better visualization of skull shape , hand shape , and dorsum of foot evaluation ( syndactyly , overfolded toes ) . turning the subject to the side permitted a lateral view which also aided in assessment of head shape . for the neurologic examination , repositioning of the subject s face perpendicular to the camera aided in evaluation of facial movement , suck , rooting , and eye movement evaluation . placing the subject so that the angle of the joints was perpendicular to the camera aided in assessment of spontaneous movements and reflexes .
special considerations for the hands : because neonates often have their hands clenched at rest , the hand must be held open and flat by the bedside clinician for proper evaluation .
the dorsum of the hand is best evaluated with the subject s head pointed towards the camera .
hand proportions can be distorted when the hand is not placed perpendicular to the camera viewing angle .
however , clinodactyly might not be identified since holding the fingers open made the fingers appear to curve inwards .
we describe a prospective study to determine the accuracy of dysmorphology and neurologic examinations in subjects in the nicu , and to identify optimal approaches and limitations of telemedicine examinations in this setting . for both dysmorphology and neurologic examinations
, telemedicine could be used to perform generally accurate examinations , but certain limitations were identified .
importantly , we found that the bedside clinician played an essential role in optimizing the examination .
the bedside clinician was able to indicate if the displayed image did not accurately reflect the in - person view , which could prompt adjustments to the camera , viewing angle or patient positioning .
these results suggest telemedicine has the ability to improve access to pediatric subspecialty expertise for patients in locations without in - person subspecialty access and may reduce the need to transfer of critically ill patients .
currently , patients may be transferred to a tertiary care center for evaluation by subspecialists who are not available at the referring hospital . while some transfers are necessary so that the subspecialty service can provide ongoing management , some infants are transferred for a single evaluation by a geneticist or neurologist .
significant health care costs , medical risks to the critically ill patient , and stress to the patient and family could be prevented if a subspecialist could reliably assess the infant from a remote setting using telemedicine . by providing for greater subspecialty access
, telemedicine could play a role in making feasible recent calls for enhanced neonatal neurocritical care .
however , there are no published studies addressing telemedicine for subspecialty consultations in a nicu setting .
while telemedicine has been used to facilitate access for patients with more chronic conditions such as epilepsy , it s most widespread use has been to manage patients with neurologic conditions requiring rapid intervention such as stroke .
studies have documented the reliability of a remotely administered stroke scale , shown that telemedicine is associated with a high retention rate at the local level and reduced patient costs , and demonstrated that thrombolysis decisions were more often correct using telemedicine than phone consultation .
therefore , guidelines for stroke management have recommended implementing telemedicine to increase access to acute stroke care . in a more general neurocritical care setting
, telemedicine has been shown to improve physician responsiveness to unstable patients and reduce costs .
a recent report from the american academy of neurology telemedicine work group concluded that tele - neurology would be of greatest benefit to patients with restricted access to neurologic care , would improve neurologists ability to meet the demand for specialty care in underserved areas , and overall could promote rapid evaluation of patients in remote locations .
while most prior work has focused on adults requiring stroke or neurocritical care so the guideline could not address pediatric care specifically , occur data indicate that telemedicine may also be of benefit to neonates and infants requiring neurologic care .
this study focused on the physical exam component of dysmorphology exam , as previous literature has documented patient and provider satisfaction with remote genetic counseling and outpatient visits .
however , these studies did not include an in - person evaluation , and were therefore not able to assess the accuracy of physical examination findings .
most relevant to the current data was a study addressing the accuracy of telemedicine for dermatology examination , although this study used transmission of static images as opposed to our study utilizing real - time audio - video assessments .
they found some skin lesions were readily diagnosed ( e.g. eczema , acne , psoriasis , and skin infections ) while others were more difficult to accurately assess ( e.g. pigmented lesions , actinic keratosis and basal cell carcinoma ) .
this study is the first to provide data about the limitations of the equipment for dysmorphology evaluations , and recommend a systematic approach to an examination using telemedicine .
similarly , while components of the neurologic examination have been validated using telemedicine in the adult ( e.g. for stroke evaluation ) , this is the first study to assess the accuracy of thorough neurologic examination of neonates and infants .
several studies have addressed telemedicine accuracy and impact when used by neonatologists . a recent study comparing bedside and telemedicine neonatologist evaluations of 46 neonates found excellent or intermediate - to - good agreements for almost all components of the evaluation . however , poor agreement was identified for several physical examination features including auditory components ( breath , cardiac , bowel sounds ) and capillary refill time . in a second study , 5 of 19 neonates who underwent tele - consultation avoided transport , and as a result use of telemedicine was associated with substantial cost savings .
there have been more limited studies of telemedicine use for subspecialty evaluation of neonates , and they have generally focused on image interpretation and not performance of a full subspecialty physical examination . an early study with a low cost video - teleconferencing system for surgical consultation reported that in six neonates , an accurate diagnosis was established in all , leading to earlier care implementation for some neonates .
a study of 63 neonates with suspected congenital heart disease reported that a diagnosis could be made in 97% using transmitted echocardiographic images of neonates with suspected congenital heart disease .
congenital heart disease was excluded in 19 , diagnosed but did not require transfer in 28 , and required transfer in 14 .
a telemedicine program focused on retinopathy of prematurity screening performed 582 examinations on 137 infants .
thirteen infants were transferred and nine required laser treatment , and there were no poor outcomes . in addition to being useful for image transmission , our data indicate telemedicine may allow performance of a more complex examination .
one similar study has been reported in 15 older children being evaluated in an emergency department as if they required transport by in - person and telemedicine examinations .
the sensitivity of the telemedicine physician to identify abnormal findings was 88% and the specificity of the telemedicine physician to identify normal findings was 93% .
first , since we aimed to define the limitations of the equipment with optimal use , subspecialists were involved in the patient evaluations at bedside and remote positions .
additional steps are needed to generalize these data since with clinically implementation the bedside clinician would not be a subspecialist . although the bedside clinician was instructed to follow directions of the remote consultant and not provide feedback about their own observations during the first phase of the study , it is possible that having a bedside physician with less familiarity with these specialized examinations could negatively affect accurate demonstration of abnormal findings .
second , this study focused on the ability to identify examination abnormalities and did not assess whether management decisions would differ based on in - person or telemedicine examinations .
further study is needed to determine the impact of examination omissions or inaccuracies on clinical diagnosis or management . additionally , it is unclear whether these management changes would exceed the medical risk and financial cost of transferring these sometimes critically ill patients to a center for a specialty consultation .
third , our study did not select patients based on their hypothesized suitability for avoidance of transfer .
fourth , we utilized a single in - person remote consultant examination as the gold standard . however , even with in - person examination there will be inter - examiner variability .
this study was designed to mimic the real - life scenario of a remote physician evaluating a neonate or infant via telemedicine and then repeating their examination if the patient was transferred to their facility . a more robust strategy to determine the sensitivity of the telemedicine evaluation would employ multiple expert evaluators of the same patient .
finally , we used one model of telemedicine equipment used and the sensitivity may vary depending upon technical specifications of the cameras and data transfer . the telemedicine device used in
this study was similar to that employed in prior evaluations of infants in a nicu setting so it is possible that studies employing different technology could have different findings .
using telemedicine consultation , geneticists were able to accurately identify 93% of dysmorphic examination features and neurologists were able to identify 92% of abnormal neurologic examination features in encephalopathic patients .
these data extend previous evaluations of telemedicine in children by indicating that beyond simple image transmittal , telemedicine may allow subspecialists to interpret more extended physical examinations accurately .
however , our data also indicate that physicians must be mindful of specific telemedicine limitations , that the bedside clinician must play an active and educated role in the process , and that collaborative communication between the bedside clinician and remote consultants is essential .
these findings indicate that while equipment improvements are certainly important , optimization of telemedicine consultation also requires attention to implementation .
our data indicate that telemedicine examinations are generally accurate which provides an impetus to study additional implementation issues .
we did not involve parents in the study , so our study can not provide any data regarding acceptance of telemedicine consultation by parents / families . further study is certainly needed focused on implementation issues , such as family and physician opinions regarding speaking and providing consultation over telemedicine .
establishment of telemedicine programs in which physicians understand the limitations of this approach and continually work to improve upon its use will likely be of most benefit to patients . with further development of these types of programs , telemedicine may offer specialized care to patients in the nicu on a larger scale than could be accomplished by conventional in - person consultation models .
| objectivesevaluate whether telemedicine can be used to perform dysmorphology and neurologic examinations in the neonatal intensive care unit ( nicu ) by determining the examination accuracy , limitations , and optimized procedures.study designprospective evaluation of nicu patients referred for subspecialty consultation for dysmorphic features ( n=10 ) or encephalopathy ( n=10 ) .
a physician at bedside ( bedside clinician ) performed an in - person examination which was viewed in real - time by a remote physician ( remote consultant ) .
standardized examinations were recorded and compared .
subsequently , a qualitative approach established technique adjustments and optimization procedures necessary to improve visualization.resultstelemedicine examinations identified 81 of 87 ( 93% ) dysmorphology examination abnormalities and 37 of 39 ( 92% ) neurologic examination abnormalities .
optimization of remote consultant visualization required an active bedside clinician assisting in camera and patient adjustments.conclusionstelemedicine can be used to accurately perform many components of the dysmorphology or neurologic examinations in nicu patients , but physicians must be mindful of specific limitations . |
mucormycosis , an infection caused by fungi of the order mucorales , typically presents as an aggressive angio - invasive disease in immunosuppressed hosts .
patients with hematological malignancies and recipients of hematopoietic stem cell transplants are at highest risk for pulmonary mucormycosis with reported mortality rates of up to 76% .
early diagnosis and aggressive management with surgical debridement and antifungal treatment are critical for optimal outcomes .
previously , the only two systemic antifungals available with reliable activity against mucorales were amphotericin b and posaconazole .
isavuconazole , a new extended - spectrum triazole with activity against yeasts , molds and dimorphic fungi , has recently been approved for treatment of invasive aspergillosis and mucormycosis .
we report a case of successful salvage treatment of pulmonary / splenic mucormycosis with isavuconazole , in a hematology patient intolerant of primary therapy with liposomal amphotericin b and posaconazole .
a 59 year old male gardener was transferred from another hospital with left lower lobe pneumonia unresponsive to nine days of intravenous broad - spectrum antibiotics .
this was on a background of a recent diagnosis of hypoplastic myelodysplastic syndrome ( mds ) .
his hypoplastic mds had been treated , 38 days earlier , with antithymocyte globulin ( atg ) and five days of pulsed intravenous methylprednisolone ( 1 mg / kg / day ) . at presentation
, he was taking cyclosporine 200 mg orally twice daily and a tapering dose of prednisolone , 5 mg daily .
his neutrophil count was 1.1 cells / mm at presentation , however in the six weeks prior , had been < 1.0 cells / mm .
antifungal prophylaxis with posaconazole oral solution , 200 mg three times daily ( tds ) , had been commenced at the onset of atg and methylprednisolone treatment .
this was increased to 200 mg four times daily ( qid ) , following a sub - therapeutic serum concentration of 0.3 g / ml , seven days after commencement .
his past medical history was significant for chronic kidney disease due to primary focal and segmental hyalinosis and sclerosis , presenting as nephrotic syndrome in 2012 , resulting in chronic proteinuria and a baseline creatinine clearance 91 ml / min .
1a ) revealed a cavitating left lower lobe lesion with contiguous splenic involvement , suspicious for invasive fungal disease ( ifd ) .
empirical treatment was commenced with liposomal amphotericin b ( l - amb ) 5 mg / kg and piperacillin - tazobactam 4.5 mg qid .
histopathology on a ct guided biopsy , obtained on day 2 of admission , demonstrated broad ribbon - like hyphae within necrotic tissue ( fig .
2 ) , consistent with mucormycosis . on day 4 of admission he underwent a left lower lobectomy , with latissimus dorsi muscle flap transposition .
culture and pan - fungal pcr were negative , on both ct - guided biopsy and resected lung tissue .
the patient deteriorated clinically 15 days post lobectomy ( day 19 of l - amb therapy ) with hypoxia , delirium , renal failure and raised inflammatory markers .
repeat chest ct demonstrated bilateral pleural effusions with significant peri - bronchial consolidation in the remaining left lung and evidence of a new subcapsular splenic lesion concerning for disease progression ( fig .
posaconazole oral solution , 300 mg tds , was added to l - amb 5 mg / kg and increased to 300 mg qid following a sub - therapeutic level of 0.38 g / ml , six days after commencement .
the patient achieved clinical stability on day 21 , but was intolerant of high - dose posaconazole with severe nausea unresponsive to multiple anti - emetics , requiring a dose reduction to 200 mg tds . worsening renal function , with a reduction in creatinine clearance to 30 ml / min , after 46 days of l - amb and despite ceasing cyclosporine and
isavuconazole was obtained on compassionate access and commenced on day 48 post lobectomy ( day 52 of l - amb ) according to the manufacturer 's instructions , with a loading dose of 200 mg intravenously tds for 2 days .
the patient received a further four days of intravenous therapy 200 mg daily , before changing to oral therapy , also at a dose of 200 mg daily .
serum concentrations of isavuconazole were monitored at regular intervals according to the manufacturer 's protocol ( fig .
3 ) and were considered to be within therapeutic range . the patient was discharged day 50 following lobectomy and six days after commencing isavuconazole . while on isavuconazole 200
mg orally daily , he continued to experience grade 1 nausea , requiring three anti - emetic agents , which was marginally improved relative to posaconazole therapy .
his renal function improved to his baseline creatinine clearance of > 90 ml / min .
to the best of our knowledge , this is the first report of the use of isavuconazole in australia .
isavuconazole is a broad - spectrum triazole pro - drug , which is licensed for primary and salvage treatment of mucormycosis .
a sub - group of 37 patients with proven / probable mucormycosis in a phase 3 , open - label , non - comparator trial ( vital ) achieved response rates of 31.6% for primary therapy and 36.4% for salvage therapy , .
the mainstay of management of mucormycosis has , until recently , been polyene antifungal therapy and surgical debridement .
amphotericin b has reported response rates of 3139% and l - amb is recommended as first line therapy . in our case ,
empirical l - amb was delayed by nine days with delays in treatment of mucormycosis beyond three days associated with an increased mortality , . despite this , our patient recovered with aggressive surgical debridement and multiple antifungal agents including salvage isavuconazole therapy .
posaconazole and l - amb had to be prematurely ceased due to gastrointestinal side - effects and nephrotoxicity , respectively .
until recently , posaconazole and amphotericin b based formulations have been the only antifungal agents with reliable activity against the mucorales species .
posaconazole has shown response rates of up to 60% as salvage therapy for mucormycosis , however erratic bioavailability of the oral solution has limited its use .
posaconazole serum levels in our patient remained sub - therapeutic despite dose escalation up to 1200 mg daily , resulting in severe nausea .
the favorable physico - chemical and toxicity profile of isavuconazole contributed to our patient 's successful outcome .
isavuconazole has excellent oral bioavailability , a predictable pharmacokinetic profile and the absence of a solubilizing agent such as cyclodextrin , obviates concerns of nephrotoxicity in patients with underlying kidney disease .
it has few reported significant adverse effects and , other than nausea , was relatively well tolerated by our patient over prolonged treatment course lasting 102 days .
similar tolerability was evident among the 5 patients from vital study who had complete response with treatment courses lasting 179 , 180 and 509 days , respectively for primary treatment and 86 and 735 days , respectively for disease refractory to other systemic antifungal therapy , .
case reports have documented complete response of disseminated and sino - orbital mucormycosis with prolonged salvage therapy with isavuconazole lasting 29 weeks and 16 months days , respectively , .
our case demonstrates a role for isavuconazole for treatment of mucormycosis when therapeutic options are limited due either to renal impairment or dose - limiting toxicities of other antifungal agents .
com has been on the advisory board for , received investigator - initiated grants from and given lectures for gilead sciences , merck sharp and dohme and pfizer . | mucormycosis carries a high mortality rate with few therapeutic options available .
we describe a man with pulmonary / splenic mucormycosis complicating hypoplastic myelodysplastic syndrome on a background of chronic kidney disease , who achieved a complete response with salvage isavuconazole therapy following intolerance of consecutive courses of liposomal amphotericin and posaconazole therapy . |
teratomas are tumors that typically , but not always contain all three germ cell layers ectoderm , mesoderm , and endoderm .
teratomas are classified as mature , immature , and malignant based on the degree of differentiation .
these lesions can occur throughout the spinal canal , but there is a thoracic and lumbar predominance among reported cases with the conus medullaris region being the most frequent .
a 12-year - old boy was referred to our hospital with a 2 months history of back pain and 1-week history of bilateral leg weakness , urinary incontinence , and constipation . on physical examination ; he had no gross motor deficit but bilateral numbness in his legs .
lumbosacral spine magnetic resonance imaging revealed a well - delineated , intramedullary , 2 cm 4 cm mass at the l1 - 2 levels of the lumbar spine .
the mass was hyperintense in all sequences , and it was containing a hypointense lesion in the central zone .
magnetic resonance imaging of lumbar spine ; ( a ) sagittal t2-weighted and ( b ) axial t2-weighted images showing a well - delineated , intramedullary , 2 cm 4 cm mass at the l1 - 2 levels of the lumbar spine the patient underwent an emergent surgery .
the operation was performed under a surgical microscope with the assistance of intraoperative neurogenic monitoring evoked potentials .
a midline incision was performed at the conus medullaris level , and a creamy , yellow - white , mucoid substance was discharged .
subsequent to the evacuation of the tumor 's content , calcified solid component , and capsule was resected . however , gross total resection could not be performed because of the tenacious adhesions of the tumor to the adjacent parenchyma .
after removal of the tumor , dura mater and l1 and l2 vertebrae were reconstructed .
the patient had an uneventful postoperative course and his severe back pain and bilateral leg weakness improved after the surgery .
now , as 1 month after surgery , the patient has no reported recurrent symptoms .
( a ) photomicrograph ( h and e , 40 ) showing connective tissue containing mucinous epithelium ( b ) photomicrograph ( h and e , 20 ) showing adipose tissue containing mature muscle cells ( c ) photomicrograph ( ihc , 40 ) showing smooth muscle cells reacted positively with actin
associated anomalies such as metameric cutaneous lesions , sinus tracts , neural tube defects or split cord malformations may be present .
the current classification states that a teratoma is a tumor that composed of derivatives from all 3 primitive germ layers .
histologically , these lesions are divided into three categories ; mature , immature , and malignant .
it is important to study the specimen completely because benign and malignant elements can be present simultaneously .
magnetic resonance imaging is the most valuable diagnostic technique but , even so it can not determine with certainty the differential diagnosis between teratoma and other intramedullary lesions .
the primary treatment for teratomas is surgery , and total surgical resection of the tumors should be the aim of these operations . however , as in our case gross total resection may not be possible in every operation . in a literature review made by poeze et al .
, it was found that intramedullary tumors could be completely removed only in 61.8% of the reported cases .
application of intraoperative electrophysiologic monitoring is important in these operations , and it is important to resect tumor as extensive as possible according to intraoperative electrophysiologic monitoring findings .
removal of the tumor should be stopped when the neurological function is at risk due to the tenacious adhesions of the tumor to the adjacent parenchyma .
radiotherapy is an adjuvant treatment modality that should be indicated when the tumors contain malignant components .
however , adjuvant radiotherapy is not recommended for benign teratomas , and there is not any role of adjuvant chemotheraphy in the treatment of spinal teratomas .
| teratoma is a tumor that derivatives from all three primitive germ layers and spinal intramedullary teratomas are very rare lesions .
the primary treatment modality for these tumors is surgical resection , and total resection should be the aim .
however , subtotal resection is a valid alternative to prevent traumatizing adjacent functional neural tissue . in this report
, we presented a case of a 12-year - old male patient with spinal teratoma of the conus medullaris .
we describe the presentation , evaluation , and treatment of this rare disease . |
zirconia has higher flexural strength than the existing ceramic and is widely used in fixed partial dentures.1,2 it is recommended that prepared tooth with compromised taper be bonded with resin cement to improve the chances of crown retention.3 however , as zirconia does not contain silica and has resistance against acid etching , its bond strength with resin cements can be reduced.4,5 as a stable bond strength is critical in long - term prognosis of prosthesis , various mechanical and chemical zirconia surface treatment methods have been introduced to improve bond strength with resin cement .
airborne particle can be applied to enhance the mechanical retention of resin cement by increasing zirconia surface roughness . however , as zirconia has a high surface hardness , only minimal undercuts were observed indicating a failure to significantly increase the surface roughness.6 also , airborne particle abrasion can form microcracks on the ceramic surface , which reduce the fracture resistance of zirconia.7,8 tribochemical silica coating is widely used in order to obtain effective mechanical retention and chemical bonding.9 however , while the initial bond strength of resin cement to zirconia was high , it decreased after long - term storage or thermocycling in several surface treatments.10 various resin cements contain functional monomers to improve chemical bonding with ceramics , such as methacrylated phosphoric ester , 10-methacryloylxydecyl dihydrogen phosphate ( mdp ) monomer , etc.11,12,13 zcan et al.14 reported that bond strength between zirconia and resin cement containing mdp monomer was high in dry conditions but decreased after thermocycling . in this study , nano - structured alumina coating on zirconia surfaces was used to improve the bond strength with resin cement .
this method can increase the bond surface area and improve the micro - mechanical retention through mechanical interlocking of the resin cement .
the purpose of this study was to evaluate whether nano - structured alumina coating on zirconia surfaces can enhance the bond strength between zirconia and various dual - cured resin cements , compared to the existing surface treatment methods .
tetragonal zirconia polycrystal stabilized with 3 mol% yttria ( y - tzp ) blocks ( hass co. , gangneung , korea ) were sintered at 1,550 for two hours .
a total of 90 disk - shaped specimens were fabricated with a diameter of 15 mm and a thickness of 3 mm .
the specimens were annealed in the air at a temperature of 1,200 to release the surface stress occurring during tetragonal - to - monoclinic phase transformation .
the specimens were divided into three groups of 30 specimens according to the surface treatment methods as follows :
airborne particle abrasion : the surfaces of specimens were abraded with 50 m aluminum oxide particles from adistance of 10 mm in a vertical direction at a pressure of 3 bar for 10 seconds.tribochemical silica coating : the surfaces of specimens were abraded with 110 m silica - modified aluminum oxide particles ( rocatec plus , 3 m espe , seefeld , germany ) from a distance of 10 mm in a vertical direction at a pressure of 4 bar for 10 seconds . the silane coupling agent was applied and dried according to the manufacturer 's instructions .
espe sil ( 3 m epse ag , seefeld , germany ) for relyx arc and relyx unicem , and clearfil ceramic primer ( kuraray , okayama , japan ) for clearfil sa luting were used.nano-structured alumina coating : ultrasonic cleaning of specimens was performed for 2 minutes in alcohol , acetone , and distilled water , respectively .
7.5 g of aluminum nitrate ( aln ) powder ( grade c , 1.2 m median particle size , surface area of 6 m / g , oxygen contents of 2.5 wt%o2 ; h. c. strack , berlin , germany ) was dispersed in 250 ml of deionized water at 75 to make a soluble suspension diluted with 3 wt% aln powder .
the specimens were immersed in the ain suspension for 15 minutes . when dispersed ain powder is exposed to hot water ,
a nano - structured boehmite coating starts to form on the surface as following reaction .
airborne particle abrasion : the surfaces of specimens were abraded with 50 m aluminum oxide particles from adistance of 10 mm in a vertical direction at a pressure of 3 bar for 10 seconds . tribochemical silica coating : the surfaces of specimens were abraded with 110 m silica - modified aluminum oxide particles ( rocatec plus , 3 m espe , seefeld , germany ) from a distance of 10 mm in a vertical direction at a pressure of 4 bar for 10 seconds .
espe sil ( 3 m epse ag , seefeld , germany ) for relyx arc and relyx unicem , and clearfil ceramic primer ( kuraray , okayama , japan ) for clearfil sa luting were used .
nano - structured alumina coating : ultrasonic cleaning of specimens was performed for 2 minutes in alcohol , acetone , and distilled water , respectively .
7.5 g of aluminum nitrate ( aln ) powder ( grade c , 1.2 m median particle size , surface area of 6 m / g , oxygen contents of 2.5 wt%o2 ; h. c. strack , berlin , germany ) was dispersed in 250 ml of deionized water at 75 to make a soluble suspension diluted with 3 wt% aln powder .
the specimens were immersed in the ain suspension for 15 minutes . when dispersed ain powder is exposed to hot water , a nano - structured boehmite coating starts to form on the surface as following reaction .
aln + 2h2o alooh + nh3 the coated specimens were dried at 110 for 2 hours and treated with heat at 900 for 1 hour using an electric resistance furnace .
boehmite that was formed by heat is thermally decomposed , which will form transitional alumina and subsequently undergo continuous polymorphic transformation.15 each group was divided into three subgroups of ten specimens , each of which was bonded with three different dual - cured resin cements ( table 1 ) .
teflon tube with 3 mm inner diameters and 3 mm heights were filled with a composite resin ( estelite quick , tokuyama , tokyo , japan ) and light - cured for 20 seconds from two opposite sides . after the zirconia specimens were fixed in an adhesion mold ,
composite resin cylinders were bonded with three types of resin cements according to the manufacturer 's instructions .
equal quantities of the base and catalyst of resin cement were mixed for 10 seconds . in the relyx arc , a thin layer of adper single bond 2 ( 3 m epse ag , seefeld , germany )
was applied on the zirconia specimens and air - dried for 5 seconds and photo - polymerized for 10 seconds before bonding .
immediately after bonding of the resin cylinders to the zirconia specimens under a static load of 10 n , photo - polymerization was performed in four directions , each for 40 seconds , for a total of 160 seconds .
after storing the specimens in distilled water at a temperature of 37 for 24 hours , 6,000 cycles of thermocycling were performed by immersion in 2 water tanks with temperatures of 5 and 55 water bath , each for 30 seconds .
the shear bond strengths ( sbss ) of the bonded specimens were measured using a universal testing machine ( model 4201 , instron corp . , canton , ma , usa ) at a crosshead speed of 0.5 mm / min .
the failure mode of the specimens were observed with field - emission scanning electron microscopy ( fesem ) after the sbs test .
the failure modes were classified as ( a ) adhesive failure between the zirconia and resin cement ; ( b ) cohesive failure in the resin cement ; and ( c ) mixed failure when a combination of cohesive and adhesive failures occurred .
two - way anova and tukey hsd post - hoc tests were performed to assess interaction of the surface treatments and type of resin cements , as well as their effects on the shear bond strength .
in addition , the chi - square test was performed to determine a possible correlation between surface treatment methods and failure modes .
all analyses were performed using spss statistics program ( spss version 18 . 0 , spss inc . , chicago , il , usa ) and the level was set at .05 .
2 . according to the two - way anova statistical analysis , significant differences were observed in shear bond strength depending on the surface treatment method and type of cement ( table 3 ) .
< .05 . the nano - structured aluminum oxide coating groups showed significantly higher shear bond strengths than the other groups .
also , clearfil sa luting cement showed significantly higher bond strength than the other resin cements . among the airborne particle abrasion groups ,
the shear bond strengths of relyx arc could not be measured due to spontaneous debonding , while clearfil sa luting demonstrated significantly highest shear bond strengths . among the tribochemical silica coating groups , clearfil sa luting had significantly higher shear bond strengths than the other two types of resin cements . among the nano - structured alumina coating groups , both relyx unicem and clearfil sa luting demonstrated significantly higher shear bond strengths than relyx arc .
the groups of the nano - structured alumina coating showed significantly higher shear bond strengths than the groups of the other , regardless of type of cement .
micro - undulated structures were observed on the surface of the specimens treated with airborne particle abrasion ( fig .
silica particles are distributed on the undulated surface , which was treated with tribochemical silica coating ( fig .
nano - structured alumina layer is evenly coated on the surface of y - tzp , which can increase the adhesion surface area and create micro - mechanical interlocking with the resin cement ( fig .
the failure modes of each group are shown in table 4 . in the ca , cu , and cc groups
, the aluminum oxide coating was covered with the resin cement and the cohesive failure was dominant ( fig .
according to the chi - square test result , there was a statistically significant difference in failure mode depending on surface treatment methods ( p < .05 ) . among the airborne particle abrasion groups and tribochemical silica coating groups , the adhesive failure mode was dominant .
in contrast , among the nano - structured alumina coating groups , cohesive failure was prevalent .
various mechanical or chemical methods have been used to enhance the bond strength between zirconia and resin cement .
several studies have reported that the bond strength could be reduced after thermocycling.16,17 although airborne particle abrasion can improve the roughness of surface , only a minimal undercut can be formed and abrasion does not lead to sufficient surface roughness because of high surface hardness . hence , small increase of bond strength is obtained.6 kern and wegner18 also reported that the bond strength with conventional bis - gma resin cement after airborne particle abrasion did not remain stable for a long time . in this study ,
all specimens that were bonded using resin cement without functional monomer after airborne particle abrasion spontaneously debonded after thermocycling . also , the groups that were bonded with other resin cements after airborne particle abrasion showed significantly lower bond strength than the other surface treatment groups . these results supported that as airborne particle abrasion created only a modest amount of undercut on the surface , it failed to achieve enough micromechanical bonding with the resin cement .
many studies demonstrated that a tribochemical silica coating could achieve a higher bond strength than airborne particle abrasion for chemical bonding of a silica surface layer.19,20,21 the bond strength between the silica coated surface and resin cement was initially high but was reduced after long - term storage or thermocycling.10 in this study , the group bonded with relyx arc after airborne particle abrasion debonded spontaneously , while the bond strength of group coated tribochemical silica remained still after thermocycling .
however , tribochemical silica coating groups showed significantly lower bond strength than the nanostructured alumina coating groups .
this result supports that the siloxane bond created on the surface can increase the bond strength with resin cement in some degree , but it fails to achieve an effective improvement .
a substantial amount of silica , which remains on zirconia surfaces after silica coating , is necessary for durable bonding .
however , matinlinna et al.22 reported that the amount of silica remaining after silica coating was not sufficient to obtain a silanization effect due to the high surface hardness of zirconia .
it remains unclear whether the effect of tribochemical silica coating can endure on a long - term basis even in a clinical practice .
nano - structured alumina coating groups showed remarkably higher bond strength with all types of resin cements .
the surface treated with nano - structured alumina coating showed nano - scale undulated structures in fe - sem images .
such structures can improve bond strength with resin cement by increasing the surface area and creating resin cement tags .
jevnikar et al.15 observed the infiltration of resin matrices into inter - lamellar spaces of alumina coating .
the conventional bis - gma resin cements tend to show early fracture patterns due to low bond strength to zirconia.23 relyx unicem resin cement , which consists of multifunctional phosphoric acid methacrylate and alkaline fillers , tends to show a relatively higher bond strength.21,24 the phosphate ester group of the mdp in clearfil sa luting cement is known to bond directly with oxidized metal surfaces or ceramics , including zirconia , and form a stable bond against hydrolysis .
hydrophilic phosphate groups of mdp monomers demineralize the tooth surface and bond with calcium ions or amino groups .
anumber of studies have demonstrated that resin cements containing mdp monomers had higher bond strength compared to those containing different monomers.25,26 in this study , among the experimental groups with airborne particle abrasion or tribochemical silica coating , clearfil sa luting showed significantly higher shear bond strength than the other resin cements .
however , among the experimental groups with nano - structured alumina coating , clearfil sa luting had significantly higher shear bond strength than relyx arc but did not show a statistically significant difference compared to relyx unicem . in the experimental groups treated with airborne particle abrasion or tribochemical silica coating , the shear bond strength could vary depending on the chemical bonding of resin cement monomers because the zirconia surface directly contacted the resin cement
. however , in the experimental groups treated with nano - structured alumina coating , the importance of chemical bonding was less critical because the zirconia surface was not exposed to the resin cement by alumina coating and micromechanical bond strength was substantially increased .
when comparing failure modes depending on surface treatment methods , adhesive failure was common in the airborne particle abrasion groups and the tribochemical silica coating groups .
in contrast , cohesive failure was prevalent in the nano - structured alumina coating groups .
these results support that the nano - structured alumina coating has a higher shear bond strength than other surface treatment .
some studies suggested that if stress is occurred on a y - tzp ceramic by airborne particle abrasion , the phase transformation , which creates a local compressive stress area , subsequently leads to an increase in fracture toughness.8,27 in contrast , other studies reported that if stress concentrates on a surface flaw , it can reduce the fracture strength.28,29,30,31 however , the advantage of nano - structured alumina coating is that it can improve the bond strength with a resin cement without reducing the flexural strength of y - tzp ceramics because it does not result in any surface flaws . in clinical practice , the alumina coating layer thickness can affect the marginal discrepancy . in this study ,
the thickness of the alumina coating was approximately 290 nm , and the possibility of increasing the marginal gap was negligible .
however , as this study applied nano - structured alumina coating to the disc - shaped zirconia specimens in the insufficient thermocycling cycle , further studies must be carried out to determine whether it is possible to form an even and uniform coating on the internal surface of a prosthesis for use in clinical practice .
within the limitation of this study , nano - structured alumina coating of y - tzp ceramics provides remarkably higher bond strength between zirconia and various dual - cured resin cements , compared to the other surface treatment methods . if a zirconia surface is treated with either airborne particle abrasion or tribochemical silica coating , application of resin cement containing mdp monomers is recommended . in addition , as a zirconia surface treated with nano - structured alumina coating can improve the bond strength between zirconia and resin cement , various resin cements can be used . | purposethe purpose of this study was to evaluate the effect of nano - structured alumina surface coating on shear bond strength between y - tzp ceramic and various dual - cured resin cements.materials and methodsa total of 90 disk - shaped zirconia specimens ( hass co. , gangneung , korea ) were divided into three groups by surface treatment method : ( 1 ) airborne particle abrasion , ( 2 ) tribochemicalsilica coating , and ( 3 ) nano - structured alumina coating .
each group was categorized into three subgroups of ten specimens and bonded with three different types of dual - cured resin cements .
after thermocycling , shear bond strength was measured and failure modes were observed through fe - sem .
two - way anova and the tukey 's hsd test were performed to determine the effects of surface treatment method and type of cement on bond strength ( p<.05 ) . to confirm the correlation of surface treatment and failure mode , the chi - square test was used.resultsgroups treated with
the nanostructured alumina coating showed significantly higher shear bond strength compared to other groups treated with airborne particle abrasion or tribochemical silica coating .
clearfil sa luting showed a significantly higher shear bond strength compared to relyx arc and relyx unicem .
the cohesive failure mode was observed to be dominant in the groups treated with nano - structured alumina coating , while the adhesive failure mode was prevalent in the groups treated with either airborne particle abrasion or tribochemical silica coating.conclusionnano-structured alumina coating is an effective zirconia surface treatment method for enhancing the bond strength between y - tzp ceramic and various dual - cured resin cements . |
it is a controversial and difficult problem for surgeons to manage a simultaneously occurring intraabdominal pathologies and abdominal aortic aneurysm .
it is not apparent that which pathology should be treated first or they both shold be treated at the same time .
it is a controversial and difficult problem for a surgeon to manage the simultaneously occurring diseases divirticular abscess and abdominal aortic aneurysm .
it is not apparent that which pathology should be treated first or they both should be treated at the same time .
many of the physicians have agreed that the symptomatic lesions should be provided priority and they should be treated first .
the treatment of abdominal aortic aneurysms can increase the complications risk and the pathology progression of disease that is cancer .
it is further indicated that concurrent elective cholecystectomy , nephrectomy and oophorectomy can be carried during the repair of abdominal aortic aneurysms and also do not causes am increase in the risk of graft rejection . in these types of cases , it is necessary to prevent the infections of graft and it can be ensured by the operation of aaa .
in addition , it is necessary to consider the endovascular aneurysm repair because it provides benefits to those patients who are suffering from associated intra - abdominal diseases .
this issue becomes more apparent in the situations of emergency because there is a possibility of sepsis .
it is connected with an increased risk of rupture of aaa because of its size . in both of these conditions
, the abnormal presentation of a huge aaa associated with a divirticular abscess requires the surgical treatment and there are not any similar cases in literature .
a 71 years old male was presented with 4 days history of an abdominal pain that was increased by movement and eating .
the pain was found to be relieved by rest and analgesia associated with nausea and vomiting .
he was a heavy smoker who smoked about 25 cigarettes a day . at the time of examination he was fully conscious , oriented , afebrile , and not pale or jaundice vital sign were stable .
the abdomen was soft , lax , with marked tenderness at right iliac fossa with pulsating mass at the epigastric area .
his blood tests showed wbc ( 8.4 ) and normal hb ( 123 ) and other parameters .
after performing the abdominal ultrasound it showed huge infra renal abdominal aortic neurysm ( 10 cm 10 cm ) with mural thrombus and right iliac fossa collection 3 3 , 5 cm most likely a divirticular abscess .
because of the risks of graft infection and the complicated nature of presentation , the decision was taken to conduct a two staged procedure .
ten days after the drainage and few days after minimal pus drainage a follow up ct showed complete resolution of the abscess ( fig .
the drain was removed and the next day an elective abdominal aortic aneurysm repair was performed through standard midline tranperoteneal approach .
the patient was asked to visit the outpatient clinic for the follow up after one , three and six months . at the time of making this report ,
according to the literature , the patients who are suffering from intra - abdominal surgical pathology along with the abdominal aortic aneurysm are increasing and their surgical strategies are still controversial .
there are not any studies that focus on the management of huge abdominal aortic aneurysm that needs urgent repairing and the one which is presented with the associated pathologies with an increase risk of sepsis . in many of the situations ,
the concurrent operations should not be carried because there can be a risk of graft infection and the life threatening condition should be given priority .
the second pathology should also be handled as soon as possible and in the same period of admission .
the condition of an abdominal pathology among with the abdominal aortic aneurysm is found to be contentious and it is not cleared that which condition should be treated first or both should be treated same time .
the problem of abcesses takes place in those 16% of the patients who are suffering from diverticulitis .
moreover , it is also present in 3050% of those patients who are in need of surgery for the condition of diverticulitis . before the development of the radiologic techniques for intervention , there were operative interventions for the treatment of the condition and that process was a two stage process .
the percutaneous drainage has provided a single step of surgery in more than half of the patients .
it has also been revealed from the studies that the drainage by catheter can also provide relief in the disease symptoms .
the process of drainage is carried by the anterior abdominal wall as the abscesses are present in the pelvis or sometimes they are hidden by other organs . up till now ,
almost all of the abscesses are found to have complicated diverticulitis and the surgery is recommended after the occurrence of resolution . in addition
, there has been continuous improvement in the ct technology by the help of which very small abscesses can be identified .
there are some abscesses which are not more than 3 cm but they are not amended in such a way that they can be drained . according to a retrospective study that included 22 patients and they also had 23 diverticular abscesses and their diameter was found to be 3 cm , all of the acute episodes of the disease were treated with the help of antibiotics . with the help of follow up , it was shown that 36% of the patients needed the eventual surgery and 41% indicated the complete resolution of the symptoms .
the 23% of the patients were those who had persistent or mild symptoms of disease that did not require surgery .
those patients were provided the treatment by the help of percutaneous drainage that is guided by ct , and they are required to undergo the surgical procedures if the drainage is not able to control the symptoms up till three or five days .
those patients who are successfully treated by the help of percutaneous drainage undergo the process of clinical evaluation in order to identify the risks . in case of increased risks of a patient
he is recommended to have the operative intervention . in patients with concomitant colorectal cancer and aaa
, the symptomatic lesion should be a treatment priority . although evar results in early recovery and a shorter convalescence compared with open aneurysm orrhaphy , this modality is not suitable for our case because the anatomy of the aneurysm .
our patient has divirticular disease of the sigmoid with abscess which was localized and no contamination of the peritoneal cavity and we did aspiration of it and close observation of the patient and control of blood pressure
. then open repair of the aneurysm was done using a dacron tube graft as there was no contamination of the peritoneal cavity . after the completion of treatment the patient was also further provided with several recommendations .
those patients who suffer from diverticular disease are further advised to not to take the fibre whole pieces that include nuts , corns and seeds .
it is further indicated that there is an inverse relation between the risks of development of diverticulitis , bleeding and the consumption of popcorn .
the management of concurrently occurring abdominal aortic aneurysm and another intra - abdominal pathology is controversial and represents a difficult management problem for the surgeon .
the major dilemma is in the management of patients with large aneurysm which require an urgent repair and presented with concomitant pathologies that carry a high risk of sepsis . in this case report
, we described an unusual presentation of a large aneurysm with concomitant divirticular abscess where both needed an urgent surgical intervention .
a.m. al wahbi is the main author who contributed in study design , data analysis , writing , editing , and submitting of the manuscript .
m. al tamimi is the coauthor who also contributed in data collection , literature search , and writing . | introductionit is a controversial and difficult problem for a surgeon to manage the simultaneously occurring diseases divirticular abscess and abdominal aortic aneurysm .
mostly surgeons are not willing to execute a non vascular procedures during the repair of an aneurysm because there can be a risk of graft infection.presentation of casein this case study , we have explained about the presentation of a huge infrarenal abdominal aortic aneurysm ( aaa ) that is found to be associated with a divirticular abscess and both needed an intervention.discussionit has been suggested by various evidences that a one - stage elective surgical treatment is safer and cost effective for the treatment of patients with an abdominal aortic aneurysm associated with other problems like gastro - intestinal malignancies .
however , the high risk of graft infection made the two staged procedure a popular option.conclusionthe major dilemma is in the management of patients with large aneurysm which require an urgent repair and presented with concomitant pathologies that carry a high risk of sepsis . in this case report , we described an unusual presentation of a large aneurysm with a concomitant divirticular abscess where both needed an urgent intervention . |
creating the ear framework for total auricular reconstruction is technically challenging procedure . for novice surgeons
so , a new technique is introduced to practice creating ear framework with silicon impression material . by this
to start with we will require following materials and armamentarium :
impression compound [ figure 1]silicon impression material5/0 stainless steel sutures ( ear set , steelex , b / braun ) [ figure 2]wood carving instruments [ figure 3]thin plastic sheet - to prepare templaterapid cure acrylic materialmodeling waxblue and red markerglass slab
impression compound [ figure 1 ] silicon impression material 5/0 stainless steel sutures ( ear set , steelex , b / braun ) [ figure 2 ] wood carving instruments [ figure 3 ] thin plastic sheet - to prepare template rapid cure acrylic material impression compound cake 5/0 stainless steel sutures(ear set , steelex , b / braun ) wood carving instruments with the help of impression compound , model of costal cartilage harvested for actual surgery is prepared .
this model is positive replica of sixth , seventh and eighth costal cartilage [ figure 4 ] .
then a mould is prepared with rapid cure acrylic , which is negative replica of this model , on a glass slab [ figure 5 ] .
silicon impression material is used to take the impression using the mould , which yields positive replica of sixth , seventh and eighth costal cartilage [ figure 6 ] .
3-d model of harvested costal cartilage prepared from impression compound cake mould prepared with rapid cure acrylic for making silicon cartilage model silicon model of harvested costal cartilage with the help of thin plastic sheet , surgical template is prepared keeping in mind the design of auricular cartilage . in case of unilateral microtia , a template is made following opposite ear 's anatomy [ figure 7 ] .
this is done with placing a surgical template[15 ] on sixth and seventh rib cartilage in such a way that whole of helical rim is placed on the cartilage except crus helicis [ figure 8 ] .
then scaphoid fossa , fossa triangularis and concha are carved in the base frame with the help of wood carving instruments [ figure 9 ] .
helical rim and antihelix portion are sculpted from eighth costal cartilage [ figure 10 ] .
helical rim and antihelix are now fixed to base frame with the help of stainless steel sutures in a way that tags end in the posterior portion of the framework .
now the tragus portion is carved from the remaining pieces of sixth and seventh cartilage and fixed to base frame .
after this all the edges of framework should be rounded so that final framework should not look
boxy type and all suture tags submerged in framework to prevent extrusion . with this a 3d ear cartilage framework is ready for placement [ figure 11 ] .
placement of surgical template on sixth and seventh costal cartilage in such a way that whole helical rim is located on base frame marked area 's for scaphoid fossa , fossa triangularis and concha- to be carved base frame with carved scaphoid fossa , fossa triangularis and concha and helical rim and antihelix completed 3-dimensional model of ear cartilage
with the help of impression compound , model of costal cartilage harvested for actual surgery is prepared .
this model is positive replica of sixth , seventh and eighth costal cartilage [ figure 4 ] .
then a mould is prepared with rapid cure acrylic , which is negative replica of this model , on a glass slab [ figure 5 ] .
silicon impression material is used to take the impression using the mould , which yields positive replica of sixth , seventh and eighth costal cartilage [ figure 6 ] .
3-d model of harvested costal cartilage prepared from impression compound cake mould prepared with rapid cure acrylic for making silicon cartilage model silicon model of harvested costal cartilage
with the help of thin plastic sheet , surgical template is prepared keeping in mind the design of auricular cartilage . in case of unilateral microtia ,
this is done with placing a surgical template[15 ] on sixth and seventh rib cartilage in such a way that whole of helical rim is placed on the cartilage except crus helicis [ figure 8 ] .
then scaphoid fossa , fossa triangularis and concha are carved in the base frame with the help of wood carving instruments [ figure 9 ] .
helical rim and antihelix portion are sculpted from eighth costal cartilage [ figure 10 ] .
helical rim and antihelix are now fixed to base frame with the help of stainless steel sutures in a way that tags end in the posterior portion of the framework .
now the tragus portion is carved from the remaining pieces of sixth and seventh cartilage and fixed to base frame .
after this all the edges of framework should be rounded so that final framework should not look
placement of surgical template on sixth and seventh costal cartilage in such a way that whole helical rim is located on base frame marked area 's for scaphoid fossa , fossa triangularis and concha- to be carved base frame with carved scaphoid fossa , fossa triangularis and concha and helical rim and antihelix completed 3-dimensional model of ear cartilage
the technique was successful in simulating the ear framework , and the consistency of the silicon material with relatively blunt carving instruments was almost similar to real costal cartilage with sharp instruments . hence with this technique one can access the difficulties that may be encountered in real surgery and may reduce surgical time .
a total ear reconstruction with autogenous tissues is one of the greatest technical challenges to a reconstructive surgeon .
major part of difficulty in surgery is creating a 3d cartilage framework out of costal cartilage .
this difficulty lies in the fact that costal cartilage structure is different in each individual .
so , novice surgeons might find it difficult and time consuming to locate future parts of 3d ear in costal cartilage .
but due to less availability of cadavers , it is practically difficult for surgeon to practice on it .
but the technique had disadvantage that consistency and configuration of potatoes and carrots was far different from costal cartilage . to sculpt the framework in one piece from resin block
akira yamada et al suggested technique for preparing silicone rib model by taking impression of harvested costal cartilage and pouring with silicon impression material and fabricating framework with exactly same instruments that were used for real surgery . but
considering these challenges , we herewith present a technique which will be helpful for teaching novice surgeons , and large number of silicone models can be made from a single mould which can be helpful in teaching in large number like in training workshops .
practicing with less sharp instruments on silicon model will simulate working with surgical instruments on costal cartilage .
we believe that our method will help novice surgeons to create framework that will simulate real surgery in a cost - effective way .
by using this novel technique , novice surgeons can practice creating ear framework and improvise their results in the actual surgery . | this article presents the novel method of training of creating cartilage framework for total ear reconstruction in microtia .
replica of costal cartilage harvested for real surgery was simulated by silicon dental impression material .
carving of framework was done with wood carving instruments .
silicon dental impression material gives the consistency and texture almost comparable to real costal cartilage .
sequential steps similar to actual surgery were simulated to create the three - dimensional framework.by using this novel technique , novice surgeons can practice creating ear framework and improvise their results in the actual surgery . |
recently , there has been a hot debate over whether current salt intake is too high from a health perspective .
it is estimated that globally 62% of cerebrovascular disease and 49% of ischaemic heart disease were attributable to elevated blood pressure . a technical report produced by who and the food and agriculture organization of the united nations recommended the consumption of less than 5 g of salt per day as a population nutrient intake goal .
given the adverse impact of excessive salt consumption on health and particularly on blood pressure levels and cardiovascular diseases , the world health organiztion has urged member nations to take action to reduce population wide dietary salt intake to decrease the number of deaths from hypertension , cardiovascular disease and stroke ( who report 2006)1 ) .
salt is an ionic compound composed of sodium chloride , which is 40% sodium and 60% chloride .
the molar mass of salt is 58.443 g / mol , its melting point is 801(1,474 ) and its boiling point 1,465(2,669 ) .
salt is readily soluble in water and dissolved in water it separates into na and cl ions . over the past century , sodium chloride has been the subject of intense scientific research related to blood pressure ( bp ) elevation and cardiovascular mortalities .
however , recently some in the academic society and lay media dispute the benefits of salt restriction , pointing to inconsistent outcomes noted in some observational studies2,3 ) .
for millions of years , human beings ate very small amounts of salt ( 0.1 - 0.5 g / day ) . among the food that humans ingest , meat has the highest salt content ( about 0.6 g of salt per pound ) . during the hunter - gathering period , the main food for human being was meat .
so the intake of salt in paleolithic times was less than 1 g / day . in the mean times ,
population growth led to the introduction of agriculture , and during the first few thousand years after the advent of agriculture , the intake of meat declined and the intake of vegetables increased up to 90% . in the agricultural period ,
human beings consumed about the same amount of salt as did their hunter - gathering ancestors4 ) .
people living in romania were boiling the salt - laden spring water to extract the salts .
the harvest of salt from the surface of xiechi lake near yuncheng in shanxi , china , dates back to at least 6000 bc , making it one of the oldest verifiable saltworks5 ) .
about 5,000 years ago , the chinese discovered that salt could be used to preserve food .
salt then became of great economic importance as it was possible to preserve food during the winter and allowed the development of settled communities . with the increased use of salt ,
about 1,000 years ago , salt intake in the western world had risen to about 5 g / day6 ) .
herodotus , an ancient greek historian , described salt trading routes that united the salt oases of the libyan desert back in the 5 century bc . in the early years of the roman empire
, roads such as the via salaria were built for the transportation of salt from the salt pans of ostia to the capital7 ) . with
the spread of civilization , salt became one of the world 's main trading commodities .
the oppressive salt tax in france was one of the causes of the french revolution . in india
, this tax gave rise to a civil disobedience campaign under mahatma ghandi against the british8 ) .
salt is an essential electrolyte to life in human beings and is used universally in cooking , seasoning , and preserving manufactured food stuffs around the world . for several million years , human ancestors ate a diet that contained less than 1 g of salt per day .
salt was the most taxed and traded commodity in the world , with intake reaching a peak around the 19 century9 ) .
however , salt was no longer required as a food preservative with the invention of the refrigerator .
salt intake had been declining , but with the recently large increase in the consumption of highly salted processed food , salt intake is increasing towards levels similar to those of the 19 century , and is approximately 9 - 12 g / day in most countries around the world10 ) .
iodine is an important micronutrient for humans and iodized table salt has significantly reduced disorders of iodine deficiency .
however , in naturally occurring foods such as meats , vegetables , and fruits , salt is present in small quantities .
there is more salt in animal tissues such as meat , blood and milk , than there is in plant tissues11 ) .
they do not eat salt with their food , but agriculturalists , feeding mainly on cereals and vegetables , need to supplement their diet with salt12 ) .
recently many processed foods use large amounts of salt and over 75% of daily sodium intake comes from salt found in processed foods .
sodium serves as an important nutrient in the body and helps nerves and muscles to function correctly .
it is also involved in the auto - regulation of the water and fluid balance of the body .
high dietary salt intake presents a major challenge to the kidneys to excrete large amounts of salt administered .
one of the main organ systems vulnerable to the adverse effects of excessive sodium in the diet is the cardiovascular system .
the earliest comment relating dietary salt intake to bp was by the chinese medical doctor in 2698 - 2598 bc(huang ti nei ching su wen ; the yellow emperor 's classic of internal medicine ) : " therefore if large amounts of salt are taken , the pulse will stiffen or harden"15 ) .
the association between salt intake in the diet and bp was first suggested by ambard and beaujard in 190416 ) .
salt intake was measured by estimating the salt content of the food and the urine each day .
when the diet contained little salt , the patients went into negative sodium balance and blood pressure fell , even though the intake of protein was raised .
conversely , when the diet was high in salt , less salt was excreted in the urine so that the patient was in positive sodium balance and the bp rose even when the protein intake was low .
they had some success in reducing bp by restricting salt intake . in 1948 , kempner introduced rice diets for the treatment of hypertension .
he treated 500 hypertensive patients with a diet containing 20 g of protein , little fat , and < 0.5 g salt .
he showed that rice diets containing little salt improved bp , decreased heart size , reversed inverted t waves in the ekg , and ameliorated hypertensive retinopathy .
however , a rice diet did not have widespread acceptability because of insipid and unpalatable features of food contents .
the connection between high salt intake and elevated bp became widely recognized after kempner 's rice diet in 1948 and thus , high bp was lowered by a low salt diet and a renewed salt restriction strategy utilized in the treatment of hypertension17 ) . since then
, many animal studies showed a causal relationship between dietary sodium and hypertension and in human studies , established that the average bp in the population is related to habitual salt intake13 ) .
the available data in animal studies confirm the existing relationship between habitual salt intake and bp levels .
a well - documented connection between hypertension and dietary salt intake has been demonstrated in various animals , especially in rats and chimpanzees .
ball and meneely18 ) reported that in groups of young adult rats that were fed different amounts of salt in their diet ( 0.15 , 2.8 , 5.6 , 7.8 , or 9.8% ) with free access to distilled water , the average bp after 9 month increased proportionally to the amount of salt in their diet .
a similar study had been performed in chimpanzees , which are genetically very close to humans , by denton et al.19 ) .
chimpanzees normally consume a diet low in salt , but when their salt intake increased to ~15 g / day for 20 months , they developed hypertension .
the rise in bp was gradual and it was still rising 18 months after they started the high salt diet .
moreover , when the salt was reduced from the diet containing ~15 g salt / day to 0.5 g salt / day , blood pressure levels fell back to the original level after 6 months . in this study
, we can see a progressive increase in bp with added salt and this was completely reversible when the added salt was stopped .
studies on the effect of salt intake on bp in humans have been carried out extensively for the past several decades .
after kempner 's rice diet in 1948 , other investigators showed the bp lowering effect of a low salt diet20 ) .
however , it was a very difficult thing to reduce the salt intake below 1 g / day in the everyday food intakes in humans .
regional differences in habitual salt intake and bp within a population have been studied in various areas of the world . around the newfoundland area ,
a survey of salt intake revealed that a county in the center of the island had a typical salt intake varying between 6.7 and 7.3 g / day .
in contrast , the salt intake varied between 8.4 and 8.8 g / day in a relatively isolated coastal community21 ) .
this difference in salt intake was accompanied by parallel changes in the incidence of hypertension . in individuals aged between 55 and 75 years ,
the incidence of hypertension in the inland community was 15% while it was 27% in the coastal community .
similar evidence had been obtained among the solomon islanders22 ) . in those tribes , which lived away from the coast and had a salt intake below 2 g / day , only 1% of the population had a raised bp . in two tribes with salt intakes between 3 and 8
in one tribe , which lived on the coast and had a salt intake between 9 and 15 g / day , 8% of the population had a raised bp .
studies of population groups that migrated from areas with lower salt intake to areas with higher salt intake have reported increases in bp .
a carefully controlled study from kenya showed that the migrants who had higher mean urinary sodium / potassium ratio compared to the non - migrants had higher systolic bp23 ) .
blood pressure in these migrants rose after a few months ( 6.9/6.2mmhg for systolic and diastolic ) , whereas it did not increase in a control group who did not migrate24 ) .
another example of the effect of life - style changes including dietary sodium intake on bp is that of the yi people , an ethnic minority living in southwestern china .
blood pressure rose very little with increasing age ( 0.13 and 0.23mmhg / year for systolic and diastolic , respectively ) in the yi farmers who lived in a remote mountainous environment and consumed a sodium - poor diet .
in contrast , yi migrants and han people who lived in urban areas consumed a sodium - rich diet and experienced a much greater increase in bp with progressive aging ( 0.33 and 0.33mmhg / year for systolic and diastolic , respectively)25 ) .
these findings suggest that changes in life - style , including higher intake of dietary sodium , contributed to higher bp among yi migrants .
a large number of epidemiologic , evolutionary , and clinical studies have confirmed that salt intake is an important factor in elevating the bp in humans .
the first double - blind controlled study of moderate salt restriction was performed in the early 1980 's by macgregor et al.26 ) .
they recruited nineteen unselected patients with mild to moderate essential hypertension , whose average supine bp after two months ' observation and no treatment was 156/98mmhg .
after 2 weeks of sodium restriction , patients were entered into an 8-week double - blind randomized crossover study of ' slow sodium ' ( ciba , 10mmol of sodium per tablet ) versus slow sodium placebo .
the mean supine bp was 7.1mmhg ( 6.1% ) lower in the fourth week of slow sodium placebo than that in the fourth week of slow sodium ciba ( p<0.001 ) .
urinary sodium excretion in the fourth week of slow sodium ciba was 1629mmol/24 hours and that in the fourth week of slow sodium placebo was 869mmol/24 hours ( p<0.001 ) .
they suggested that moderate sodium restriction should become part of the management of essential hypertension .
followed by this study , many large observational and epidemiological investigations conducted worldwide link between high salt intake and hypertension .
intersalt study was one of the first large international epidemiologic studies on sodium intake and hypertension using a standardized method for measuring 24-hour urinary sodium .
this study , which was a crosssectional assessment of 10,079 subjects aged 20 - 59 sampled from 52 centers around the world , attempted to relate sodium intake to bp from an epidemiological perspective .
sodium excretion ranged from 0.2mmol/24 hours ( yanomamo indians , brazil ) to 242mmol/24 hour ( north china ) . in individual subjects ( within centers ) , salt intake was significantly related to bp .
four centers found very low sodium excretion , low bp , and little or no upward slope of bp with age . across the other 48 centers sodium was significantly related to the slope of bp with age but not to median bp .
the study demonstrated a significant positive relationship between salt intake and bp in individual subjects within centers . in an initial analysis of 48 of the 52 centers ,
however , after inclusion of the remaining 4 centers , in which the average sodium consumption was 0.2 - 50mmol / day , they found a significant association between salt intake and the increase in bp with age .
furthermore , it also found that populations with low average daily salt intakes had low bp and very little or no increase in bp with age27 ) .
intersalt study indicates that bp increases with age only if accompanied by increased salt intake . as part of the intersalt study , bp ,
urinary electrolytes , age , and body mass index were assessed in a sample of 195 yanomamo indians from the brazilian amazon28 ) . mean sodium excretion was 0.9mmol / day .
systolic and diastolic bp were not higher at older than at younger ages in men . in women ,
in this tribe , there was a low average population bp , no hypertension and no positive slope of bp with age in a population with very low salt intake .
salt institute criticized that in an initial analysis of 48 of the 52 centers , no significant association was noted between sodium intake and median bp .
however , the intersalt 's investigators re - analyzed their data and showed that the highly significant within - population association between salt intake and bp across all 52 centers was virtually unchanged . lowering sodium intake by 100mmol was associated with a 3mmhg decrease in systolic bp29 ) .
there are several studies on the effect of reducing the salt intake on bp on a community levels .
forte et al.30 ) studied reducing the dietary salt intake on bp on a community in portugal , which is notorious for its high consumption of salt .
the trial was performed in two communities within the same district , each with 800 inhabitants who had salt intakes of ~21 g / day and a 30% incidence of hypertension . in the intervention community
the reduction in salt intake in one of the communities to ~12 g / day was associated with a highly significant difference in bp . by the end of the second year
, there was a small rise in systolic bp in the control community and a significant fall in both systolic and diastolic bp in the low salt intake community , the difference between the two villages reached 13/6mmhg .
the fall in bp involved the whole community , normotensives and hypertensive individuals alike , and the response did not differ between the young and the old or between men and women .
those with the greatest fall in salt excretion tended significantly to be also those who showed the greatest fall in bp .
the other long - term trial was carried out in tianjin in china as part of a community - based intervention program to reduce non - communicable diseases31 ) .
this intervention was based on examinations of independent cross - sectional population samples in 1989 ( 1,719 persons ) and 1992 ( 2,304 persons ) in the intervention and matched reference areas .
the mean reduction in salt intake was 1.3 g / day in men and 0.7 g / day in women in the intervention area from 1989 to 1992 . during the same period ,
the reduction was statistically significant in men ( p<0.001 ) and near significant in women ( p<0.05 ) . in the intervention area , the mean systolic bp decreased by 3mmhg for the total population and by 2mmhg for normotensive people .
another long - term trial was performed in two belgian towns of 12,000 and 8,000 inhabitants , situated within 50 km of each other32 ) . the low - sodium intervention in one town
was mainly directed at women and implemented through mass media techniques , while the control town was merely observed .
cross - sectional random sampling at baseline and 5 years later was employed , the participation rate being similar ( 67% ) in the two towns . during the study a total of 2,211 subjects were examined . in adult women ( greater than or equal to 20 years ) in the intervention town the 24-hour urinary excretion of sodium(uvna )
decreased by 25mmol/24 h ( p<0.001 ) and this reduction differed ( p=0.01 ) from the concurrent trend in uvna in the control town ( + 8mmol/24 h ) .
however , both systolic bp ( -7.5 versus -7.9mmhg ) and diastolic bp ( -2.3 versus -3.0mmhg ) declined to a similar extent in the women from the two towns . in adult men in the intervention town
, decreases were observed in uvna ( -12mmol/24 h ) and in systolic bp ( -5.6mmhg ) and diastolic bp ( -2.4 mmhg ) , but these trends were not significantly different from the concurrent changes in the control town ( -14 mmol/24 h , -4.9 and + 0.2mmhg , respectively ) .
no significant difference was observed in the evolution of mean systolic and diastolic pressures that declined to the same extent in the two towns during the trial . in women of the intervention town ,
24-hour urinary salt excretion decreased by 1.5 g , whereas in the control town it rose by 0.5 g. this negative result may be explained by the small reduction in salt consumption that would be insufficient to observe a net effect on bp in the belgian environment .
these results suggest that a reduction in salt consumption is difficult to achieve with mass media techniques and in women and in subjects aged 50 years or more , the intervention did achieve some success , but bp was not affected .
there were many randomized clinical trials performed to test the effects of reducing salt intake on bp .
cutler et al.33 ) analyzed previously published randomized clinical trials testing the effects of reducing sodium intake on bp .
thirty - two trials with outcome data for 2,635 subjects were included . pooled bp differences between treated and control groups were highly significant for all trials combined .
the effects on blood pressure by lowering sodium in hypertensive and normotensive subjects were -4.8/-2.5 and -1.9/-1.1mmhg ( systolic / diastolic ) , respectively .
median differences in sodium excretion between sodium - reduction and control groups in these subgroups were -77 and -76mmol/24 h , respectively . weighted linear - regression analyses across the trials showed dose responses , which were more consistent for trials in normotensive subjects .
these analyses yielded estimates , per 100mmol of sodium reduction , of -5.8/-2.5mmhg in hypertensive subjects and -2.3/-1.4mmhg in normotensive subjects , respectively .
there is no evidence that sodium reduction as achieved in these trials presents any safety hazards .
they concluded that the bp reduction with a substantial lowering of dietary sodium in the us population could reduce cardiovascular morbidity and mortality .
however , in two other meta - analyses34,35 ) , it was claimed that salt reduction had very little effect on bp in individuals with normal bp and a reduction in population salt intake was not warranted .
the meta - analysis by midgley et al.34 ) included 56 trials that had randomized allocation to control and dietary sodium intervention groups with outcome measures of both systolic and diastolic bp .
the mean reduction in daily urinary sodium excretion was 95mmol / day ( 71 - 119mmol / day ) in 28 trials with 1,131 hypertensive subjects and 125mmol / day ( 95 - 156mmol / day ) in 28 trials with 2,374 normotensive subjects .
the decrease in bp for a 100mmol / day reduction in daily sodium excretion was 3.7mmhg ( 2.35 - 5.05mmhg ) for systolic ( p<0.001 ) and 0.9mmhg ( -0.13 to 1.85mmhg ) for diastolic ( p=0.09 ) in the hypertensive subjects , and 1.0mmhg ( 0.51 -1.56mmhg ) for systolic ( p<0.001 ) and 0.1mmhg(-0.32 to 0.51mmhg ) for diastolic ( p=0.64 ) in the normotensive subjects .
decreases in bp were larger in trials of older hypertensive individuals and small and non - significant in trials of normotensive individuals .
they concluded that dietary sodium restriction for older hypertensive individuals might be considered , but the evidence in the normotensive population does not support current recommendations for universal dietary sodium restriction .
another meta - analysis by graudal et al.35 ) included 58 trials of hypertensive and 56 trials of normotensive person to evaluate the effect of sodium restriction to blood pressure lowering . in 58 trials of hypertensive subjects ,
the effect of reduced sodium intake as measured by urinary sodium excretion ( mean , 118mmol/24 h ) on systolic bp was 3.9 mmhg ( p<0.001 ) and on diastolic bp was 1.9mmhg ( p<0.001 ) . in 56 trials of normotensive subjects ,
the effect of reduced sodium intake ( mean , 160mmol/24 h ) on systolic bp was 1.2mmhg ( p<0.001 ) and on diastolic bp was 0.26mmhg ( p=0.12 ) .
they concluded that these results do not support a general recommendation to reduce sodium intake .
however , these two meta - analyses were criticized by some authors because the data included was flawed .
both meta - analysis included trials of very short duration with comparing the effects of acute salt loading to abrupt and severe salt restriction for only a few days .
it is inappropriate to include the acute salt restriction trials in a meta - analysis where the implications of the findings are to apply them to public health recommendations for a long - term .
it is possible that acute and large reduction in salt intake increases sympathetic activity , stimulates the renin - angiotensin system which would counteract the effects on bp .
subsequently , several large - scale intervention studies showing significant antihypertensive effects of salt reduction in diet were performed by several groups .
trials of hypertension prevention ( tohp ) phases i and ii were two large randomized controlled trials enrolling 2,182 and 2,382 patients , respectively .
in tohp i36 ) , the patients were randomized to three life - style change groups ( weight reduction , sodium reduction , and stress management ) , one of which was a low sodium diet . at 18 months follow - up ,
weight reduction intervention produced weight loss of 3.9 kg ( p<0.01 ) , diastolic bp change of -2.3mmhg ( p<0.01 ) , and systolic bp change of -2.9mmhg ( p<0.01 ) .
sodium reduction interventions also lowered urinary sodium excretion by 44mmol/24 hours ( p<0.01 ) , diastolic bp by 0.9mmhg ( p<0.05 ) , and systolic bp by 1.7mmhg ( p<0.01 ) .
they concluded that weight reduction was the most effective strategy tested for reducing bp in normotensive persons .
in tohp ii37 ) , from baseline , participants ' sodium excretion decreased 50 and 40mmol / day at 6 and 36 months in the sodium reduction group , respectively .
compared with the usual care group , bp decreased 2.9/1.6mmhg in the sodium reduction group , and 4.0/2.8 mmhg in the combined group ( weight and sodium reduction ) at 6 months ( all groups , p<0.001 ) . at 36 months
, bp decreases remained greater in the active intervention groups than in the usual care group ( weight loss group , 1.3/0.9mmhg ; sodium reduction group , 1.2/0.7mmhg ; combined group , 1.1/0.6mmhg ) .
the long - term , 10- to 15-year trials of hypertension prevention ( tohp ) i and ii show that , apart from lowering bp , net sodium reduction by 44mmol / day ( tohp i ) and 33mmol / day ( toph ii ) leads to significant reduction in cardiovascular events by 25% , even after adjustment for several confounding factors .
toph i and ii will presumably remain the best evidence supporting the beneficial effect of a moderate reduction of salt intake in the general population38 ) .
the intervention studies of salt intake reduction are often conducted with other life - style modifications .
tone study39 ) was performed to determine whether weight loss or reduced sodium intake is effective in the treatment of older persons ( aged 60 to 80 years ) with hypertension .
the authors randomized 585 obese participants to reduced sodium intake , weight loss , both , or usual care , and the 390 non - obese participants to reduced sodium intake or usual care .
the primary endpoint included high blood pressure after discontinuation of the bp lowering medication at entry ( 190/110 , 170/100 , and 150/90mmhg at 1 , 2 , 3 visits - not adjudicated ) , resuming drug treatment , inability to withdraw treatment , and cardiovascular complications ( angina , myocardial infarction , stroke , heart failure , and coronary revascularization ) .
after a median follow - up of 29 months ( range 15 - 36 months ) , the composite outcome occurred less frequently among those assigned vs. not allocated to reduced sodium intake ( hazard ratio 0.69 ; 95% ci , 0.59 - 0.81 ; p<0.001 ) .
the mean change in blood pressure for participants assigned to sodium reduction alone was -3.4mmhg systolic and -1.9 mmhg diastolic , and for participants assigned to combined intervention of sodium reduction and weight loss -5.3mmhg systolic and -3.4mmhg diastolic , respectively ( p<0.001 for comparisons to usual care group ) .
this study , however , has to be interpreted with caution including selection of adherent and well educated patients ( only 11.1% of pre - screened patients were finally randomized ) and uncertain blinding .
there was no difference between sodium - restricted and control patients in the incidence of cardiovascular events ( 44 [ 12.9% ] versus 57 [ 16.7% ] ; p=0.24 ) .
the level of salt restriction effective for maintaining a normal bp after the discontinuation of an antihypertensive drug was -5.6 g per day or less .
tone study demonstrated that a reduced sodium intake and weight loss , alone or combined , could effectively control hypertension39 ) .
another well - conducted landmark study was the dash ( dietary approaches to stop hypertension)-sodium trial40 ) , a 12-week well controlled feeding trial provided the most robust evidence about the effect of salt intake on human bp .
it was a multi - centered clinical trial that studied three levels of sodium intake ( 150 , 100 and 50 mmol / day ) on 412 individuals with and without hyper - tension .
two different diets that is the control diet and the dash diet , which is rich in fruits , vegetables and low - fat dairy products , were tested . in both groups , the participants were assigned randomly to a high sodium diet ( 150mmol / d ) , normal sodium diet ( 100mmol / d ) , or low sodium diet ( 50mmol / d ) for 30 days and were then crossed over within their assigned groups . when the participants were shifted from a high sodium diet to a normal sodium diet ,
the systolic bp decreased by 2.1mmhg ( p<0.001 ) in the control group and by 1.3mmhg ( p=0.03 ) in the dash group . when they were shifted from a normal sodium diet to a low sodium diet
, there was a further reduction in systolic bp of 4.6mmhg in the control group ( p<0.001 ) and 1.7mmhg in the dash group ( p<0.01 ) .
when compared with the controls , the dash diet led to a lower systolic bp of 7.1mmhg in participants without hypertension and 11.5mmhg in participants with hypertension .
the adherence to the diet of participants was monitored , not only by measuring 24-hour urine sodium at the end of each period but also their daily food diaries .
the study demonstrated that a reduction in salt intake lowered bp both on the control diet and on the dash diet .
the combination of a low salt and dash diet had a greater effect on bp than either intervention alone .
there was a very significant difference in systolic ( -6.7mmhg ) and diastolic pressure ( -3.5mmhg ) between participants on the 150mmol sodium / day diet and those on the 50mmol sodium / day .
there was a greater reduction in systolic pressure when blood pressure was initially high and in women , but most importantly the blood pressure - lowering effect of reducing the salt intake was observed in all categories of the population , in particular in normotensive as well as in hypertensive people .
the dash - sodium trial supports that a low sodium diet leads to lower blood pressure .
most acknowledge that this study reliably confirmed the benefit of dietary sodium restriction in bp management . however , the dash diet was significantly different from the control diet in terms of more fruits , vegetables , low - fat dairy foods , fish , nuts , potassium , calcium , magnesium , and dietary fiber .
although the group on the dash diet had a lower urinary sodium excretion , this does not necessarily imply that the benefit was being solely caused by a dietary sodium reduction .
in addition , this study did not evaluate the long - term effects of the intervention and the clinically relevant variables , such as mortality or morbidity .
after the dash - sodium trial , many meta - analyses of intervention studies were investigated . in 2002
, he and macgregor41 ) demonstrated that a modest salt intake reduction caused significant falls in bp in both hypertensive and normotensive individuals . in this study ,
a total of 17 trials in hypertensives ( n=734 ) and 11 trials in normotensives ( n=2,220 ) were included .
the median reduction in 24-hour urinary sodium excretion was 78 mmol in hypertensives and 74mmol in normotensives .
the pooled estimates of bp fall were 4.96/2.730.40/0.24mmhg in hypertensives ( p<0.001 ) and 2.03/0.970.27/0.21mmhg in normotensives ( p<0.001 ) .
weighted linear regression analyses showed a dose response relationship between the change in urinary sodium and bp .
a reduction of 100mmol / day in salt intake predicted a fall in bp of 7.11/3.88mmhg ( p<0.001 ) in hypertensives and 3.57/1.66mmhg in normotensive individuals ( p<0.05 ) .
they demonstrated that a modest reduction in salt intake for a duration of 4 or more weeks does have a significant and important effect on bp in both hypertensive and normotensive individuals .
these findings in conjunction with other previous evidence relating salt intake to bp make a strong case for a reduction in population salt intake , which will lower population bp and therefore reduce cardiovascular mortality .
many meta - analyses , so far , on the effect of salt reduction on bp have shown consistent reductions in bp in those with high blood pressure , but there has been some controversy about the magnitude of the fall in bp in normotensive individuals34,36 ) . in these two meta - analyses ,
it was claimed that salt reduction had no or very little effect on blood pressure in normotensive individuals .
however , detailed examination of these two meta - analyses showed that their data collection and analysis were flawed .
recently , there has been a hot debate whether current salt intake is too high from a health perspective .
there were studies reporting the influence of salt intake on overall cardiovascular diseases such as he et al .
he et al.42 ) estimated that reduction of salt intake by 3 g per day lowered systolic blood pressure by 5.6 mmhg in hypertensive individuals .
they also found that it was the obese and not the non - obese who benefited .
tuomilehto et al.43 ) prospectively followed 1,173 finnish men and 1,263 women aged 25 - 64 years with complete data on 24 hour urinary sodium excretion and cardiovascular risk factors .
the hazards ratios for coronary heart disease , cardiovascular disease , and all - cause mortality , associated with a 100mmol increase in 24 hour urinary sodium excretion , were 1.51 ( 95% ci 1.14 - 2.00 ) , 1.45 ( 1.14 - 1.84 ) , and 1.26 ( 1.06 - 1.50 ) , respectively , in both men and women .
they concluded that high sodium intake predicted mortality and risk of coronary heart disease , independent of other cardiovascular risk factors , including blood pressure . in a study by o'donnell et al.44 ) , the tendency of an excessive salt intake - induced increase in the incidence of cardiovascular diseases
strazzullo et al.45 ) also performed a meta - analysis of prospective cohort studies and reported that excessive salt consumption tended to increase the incidence of cardiovascular diseases .
therefore , a salt reduction strategy may be a useful tool for preventing cardiovascular diseases .
however , to the contrary , alderman et al.46 ) and cohen et al.47 ) repeatedly suggested that excessive salt reduction increased the incidence of cardiovascular diseases . in the presidential address of the 21 international society of hypertension meeting in 2007 , alderman48 ) advocated that the relationship between salt intake and the risk of cardiovascular diseases is j - shaped and that salt intake at 5 to 6 g per day might be characterized by the lowest risk of cardiovascular diseases . in 2011 , stolarz - skrzypeket al.49 ) reported prospective longitudinal population - based study including 3,681 people without cardiovascular disease at baseline . during a median follow up of 7.9 years , cardiovascular deaths decreased across increasing tertiles of 24-hour sodium excretion .
this significant inverse association between cardiovascular mortality and tertile of sodium excretion was retained in multivariable analyses ( p=0.02 ) , with a hr(harzard ratio ) of 1.56 in the low tertile versus the overall risk in the whole study population ( p=0.04 ) .
the 24-hour sodium excretion at baseline did not predict either total mortality or fatal combined with nonfatal cardiovascular events . in a subgroup of 1,499 participants who had both bp and sodium excretion measured at baseline and at last follow - up were followed up for a median of 6.1 years .
the annual increases in bp averaged 0.37 mmhg systolic and 0.47mmhg diastolic ( p<0.001 ) , but on average the 24-hour urinary sodium excretion did not change over time .
however , in multivariable - adjusted analyses of individual participants , a 100-mmol increment in 24-hour sodium excretion was associated with a significant 1.71mmhg increase in systolic bp ( p<0.001 ) , without concomitant change in diastolic bp .
they concluded that lower urinary sodium excretion was associated with higher cardiovascular disease ( cvd ) mortality .
these findings contradict a large body of evidence that established elevated salt consumption as a risk factor for cvd .
however , many researchers including he et al.50 ) criticize the results of this study . in the same year , taylor et al.51 ) published the results of a meta - analysis of intervention studies regarding salt reduction .
this meta - analysis involved randomized , control studies of salt reduction in which follow - up was continued for > 6 months ; three of these studies involved normotensive individuals , two hypertensive patients and one normotensive and hypertensive combined subjects . of these , hypertensive and normotensive individuals were separately analyzed .
salt reduction was associated with reductions in urinary salt excretion of between 27 and 39mmol per 24 hour and reductions in systolic blood pressure between 1 and 4mmhg .
pooled relative risks ( rrs ) comparing the intervention with the reference groups for all - cause mortality were 0.90 ( ci , 0.58 - 1.40 ; 79 deaths ) and 0.96 ( ci , 0.83 - 1.11 ; 565 deaths ) in normotensive and hypertensive participants , respectively .
salt restriction increased the risk of all - cause mortality in patients with heart failure ( rr , 2.59 ; ci , 1.04 - 6.44 ; 21 deaths ) . in both hypertensive and normotensive individuals ,
salt reduction slightly decreased the incidence of cardiovascular diseases , although there was no significant difference .
these authors found no strong evidence that salt reduction reduced all - cause mortality or cvd morbidity in normotensives or hypertensives .
a single rct showed an increase in the risk of all - cause death in those with congestive heart failure receiving a low - sodium diet .
he et al.52 ) simultaneously analyzed normotensive individuals and hypertensive patients from the same studies including taylor 's meta - analysis .
they reported that salt reduction at 2.0 to 2.3 g per day significantly decreased the risk of cardiovascular diseases ( 20% of decrease ) .
however , in most studies supporting the fact that salt reduction increases the risk of cardiovascular diseases , methodological problems have been indicated47,49 ) , or study subjects were high - risk patients46,51 ) . in conclusion , a moderate reduction of dietary salt intake is generally an effective measure to reduce blood pressure .
a reduction in dietary salt from the current intake of 9 - 12 g / day to the recommended level of less than 5 - 6 g / day will have major beneficial effects on cardiovascular health along with major healthcare cost savings around the world . the who strongly recommended to reduce dietary salt intake as one of the top priority actions to tackle the global non - communicable disease crisis and has urged member nations to take action to reduce population wide dietary salt intake to decrease the number of deaths from hypertension , cardiovascular disease and stroke .
however , some scientists still advocate the possibility of increased risk of cvd morbidity and mortality at extremes of low salt intake .
future research may inform aspects of optimal sodium reduction strategies and the intake targets for populations . until then , we have to continue to build consensus around the greatest benefits of salt reduction for cvd prevention , and dietary salt intake reduction strategies must remain at the top of the public health agenda . | over the past century , salt has been the subject of intense scientific research related to blood pressure elevation and cardiovascular mortalities . moderate reduction of dietary salt intake is generally an effective measure to reduce blood pressure .
however , recently some in the academic society and lay media dispute the benefits of salt restriction , pointing to inconsistent outcomes noted in some observational studies . a reduction in dietary salt from the current intake of 9 - 12 g / day to the recommended level of less than 5 - 6
g / day will have major beneficial effects on cardiovascular health along with major healthcare cost savings around the world .
the world health organization ( who ) strongly recommended to reduce dietary salt intake as one of the top priority actions to tackle the global non - communicable disease crisis and has urged member nations to take action to reduce population wide dietary salt intake to decrease the number of deaths from hypertension , cardiovascular disease and stroke
. however , some scientists still advocate the possibility of increased risk of cvd morbidity and mortality at extremes of low salt intake .
future research may inform the optimal sodium reduction strategies and intake targets for general populations . until then
, we have to continue to build consensus around the greatest benefits of salt reduction for cvd prevention , and dietary salt intake reduction strategies must remain at the top of the public health agenda . |
over the past decade , many immune tolerance agents have shown promise in the non - obese diabetic mouse model for prevention and reversal of type 1 diabetes but have not been successful in clinical trials among recently diagnosed type 1 patients .
the trials from decades ago using cyclosporine a in significantly lower dosages than used for organ transplantation and in similar dosages that have increased t regulatory cell populations in conditions such as atopic dermatitis , demonstrated very high initial insulin - free remission rates when administered immediately after diagnosis . over time , all newly diagnosed type 1 patients given cyclosporine a required insulin .
human trials with immune tolerance agents suggest that in addition to an immune tolerance agent , a beta cell regeneration agent may also be necessary to induce long - lasting remission among patients with recent onset type 1 diabetes .
a randomized , double - blind prospective trial among recent onset type 1 diabetes patients has been designed using cyclosporine a and a proton - pump inhibitor , which increases gastrin levels and has been shown to work through the reg receptor to transform pancreatic duct cells into islets .
over the past three decades , there have been several trials using immune tolerance agents demonstrating both prevention and reversal of type 1 diabetes in non - obese diabetic mice , yet these results have not been translated into man . as we look at recent clinical trials among patients using immunotolerance agents , including anti - cd3 antibodies , anti - cd20 monoclonal antibody , interleukin 1-receptor antagonists , anti - thymoglobulin and others that have reversed diabetes in type 1 mouse models , the insulin - free remission rates in humans have not been seen .
it is now clear that unlike the diabetes remissions seen in rodents , making peace with the immune system is not enough to generate new beta cells in humans .
as we begin to understand that autoimmunity is only one aspect of type 1 diabetes in humans and the other being beta cell deficiency , we can now look at the efficacy of the immune tolerance agents in a new light .
lasting insulin - free remissions in humans will only occur when beta - cell mass is sustained and regenerated over time in the milieu of immune protection .
if we now examine the efficacy of immune tolerance agents based on insulin - free remissions at 1 year following initiation , some of the most promising data actually dates back 25 years .
cyclosporine a had a 67.5% insulin - free remission among recent onset type 1 patients , with 50% of patients sustaining the insulin - free state after 12 months 1 .
renal side effects were not seen in the many trials , including a published cohort of 285 patients with recent type 1 diabetes followed for up to 13 years after 20 months of therapy on cyclosporine 3 .
the usage of cyclosporine a among patients with dermatological conditions has been associated with significantly increased t regulatory cells , both in absolute numbers and frequencies and in the reduced ability of t cells to be activated 4 . despite immune intervention , the inability to generate new beta cells has contributed to the necessity for exogenous insulin .
distinct complexities of human islets , including the slower beta - cell turnover rates in humans compared with mice , may account for the need for the use of a beta cell regeneration agent in addition to an immunosuppressive or immunoregulatory agent when intervening in the disease 5 .
novel therapies have emerged with a mechanism of transforming ducts into islets . for example , the reg3 gene proteins , which have been found to be upregulated in acute pancreatic ligation and in new onset human type 1 diabetes , and have been shown to generate new islets from progenitor cells found within the pancreatic ductal population . in vitro studies have shown reg peptides transform human pancreatic ducts to islets 6,7 .
a recent brdu study found that two reg peptides resulted in a twofold increase in the volume of small new islets after 5 days of treatment ( p < 0.05 ) 8 .
five days of reg peptide treatment also demonstrated upregulation of pancreatic transcription factors , including ngn3 , nkx6.1 , sox9 and ins .
the reg3 gamma peptide , islet neogenesis associated protein , demonstrated a 27% rise in arginine stimulated c - peptide area under the curve among 63 type 1 patients , with an average of 20 years of type 1 diabetes with no prior detectable fasting c - peptide levels ( p = 0.0057 ) 9 .
three reg peptides are currently in development . by similar mechanisms of action to the reg peptides , gastrin has been shown to stimulate islet cell hyperplasia 10 .
preliminary studies combining gastrin with epidermal growth factor resulted in up to a 75% reduction in insulin requirements among existing type 1 diabetes patients 11 .
proton pump inhibitors have been shown to increase gastrin levels ninefold in man . a recent study among type 2 diabetes patients demonstrated that the proton pump inhibitor , pantoprazole , given at dosage of 40 mg twice daily for 12 weeks resulted in a decrease in a1c of 7.66.8% ( p < 0.001 ) .
the decrease in a1c significantly correlated with an increase in gastrin and insulin levels and beta - cell function assessed by homeostatic model assessment 12 .
thus , in type 1 diabetes , therapeutic agents that may generate new islets from ductal tissue may hold great promise when used in conjunction with an immune tolerance agent .
such an approach may potentially increase beta - cell mass among type 1 patients in a muted immune environment , in order to protect and sustain both remaining beta cells and newly generated beta cells .
in mice , type 1 diabetes is primarily an autoimmune disease , and rendering mice insulin independent is different than it is for humans .
contributing factors may include their continuous patterns of eating , and the fact that their islets have a higher percentage of beta cells and lower percentage of alpha , delta , pancreatic polypeptide and ghrelin - secreting epsilon cells .
rodents also have less complex vascular , neural and paracrine systems . perhaps , these differences and complexities help explain why diabetes can be cured in a mouse with immunosuppression alone but not in humans , in which the reversal of diabetes requires islet regeneration in addition to immune protection .
these studies posit that , in humans , type 1 diabetes is both an autoimmune disease and a disease of beta - cell deficiency , which can not be corrected with immune agents alone , as illustrated by the diagram below ( figure 1 ) . a novel proposal to tackle type 1 diabetes at diagnosis
because type 1 diabetes is both an autoimmune disease and a beta - cell deficiency disease , it can not be corrected with immune dampening agents alone .
our analysis of the shortcomings of the earlier therapeutic approaches to type 1 diabetes have led us to design a new proposal , utilizing the immune muting agent cyclosporine with the proton pump inhibitor , lansoprazole , a drug that markedly enhances plasma levels of gastrin .
gastrin has been associated with the in vivo expansion of islet neogenesis from ductal populations acting through the reg receptor 10,13,14 . with this in mind
we are now starting a novel trial http://www.clinicaltrials.gov/ct2/show/nct01762644?term=iit&rank=1 utilizing the immune agent cyclosporine a with the proton pump inhibitor , lansoprazole .
each of the agents has a long enough track record so that the anticipated risks of the study are modest . | backgroundover the past decade , many immune tolerance agents have shown promise in the non - obese diabetic mouse model for prevention and reversal of type 1 diabetes but have not been successful in clinical trials among recently diagnosed type 1 patients .
the trials from decades ago using cyclosporine a in significantly lower dosages than used for organ transplantation and in similar dosages that have increased t regulatory cell populations in conditions such as atopic dermatitis , demonstrated very high initial insulin - free remission rates when administered immediately after diagnosis . over time , all newly diagnosed type 1 patients given cyclosporine a required insulin .
human trials with immune tolerance agents suggest that in addition to an immune tolerance agent , a beta cell regeneration agent may also be necessary to induce long - lasting remission among patients with recent onset type 1 diabetes.methodsa randomized , double - blind prospective trial among recent onset type 1 diabetes patients has been designed using cyclosporine a and a proton - pump inhibitor , which increases gastrin levels and has been shown to work through the reg receptor to transform pancreatic duct cells into islets .
2013 the authors .
diabetes / metabolism research and reviews published by john wiley & sons , ltd . |
it represents the fifth most prevalent cancer worldwide and accounts for 500 000 deaths each year [ 1 , 2 ] .
hcc is associated with a poor prognosis due to a lack of effective treatment options .
although new therapeutic strategies have been continuously developed and applied to clinical treatment of hcc , the prognosis is still very poor .
it has a survival rate of less than 5% and an average survival of less than one year after diagnosis .
the sensitivity and specificity of serum alpha - fetoprotein ( afp ) in hcc diagnosis are not optimal . in recent years tumor - associated antigens ( taas ) were studied by researchers in order to find better early stage biomarker of hcc .
the mdm2 oncogene , biochemically as e3 ubiquitin protein ligase , was originally identified by virtue of its amplification in a spontaneously transformed derivative of mouse balb / c cells and the mdm2 protein subsequently was shown to bind to p53 in rat cells transfected with p53 genes . in humans ,
mdm2 protein is encoded by the mdm2 gene and localized in chromosome 12q13 - 14 .
mdm2 is a nuclear phosphoprotein that binds and inhibits transactivation by tumor protein p53 , as part of an autoregulatory negative feedback loop .
it binds to p53 via an n - terminal hydrophobic pocket , and this domain contains the highest identity at the amino acid level .
this transcriptional antagonism can take place within the nucleus , as mdm2 has been detected at p53-responsive promoter elements in chromatin .
however , mdm2 is most abundant in the cytosol in many cell lines , suggesting that cytoplasmic localization is important for their function [ 8 , 9 ] .
mdm2 both functions as an e3 ubiquitin ligase that recognizes the n - terminal transactivation domain ( tad ) of the p53 and can inhibit p53 transcriptional activation .
this protein also affects the cell cycle , apoptosis , and tumorigenesis through interactions with other proteins , including retinoblastoma 1 and ribosomal protein l5 .
overexpression of this gene can result in excessive inactivation of tumor protein p53 , diminishing its tumor suppressor function .
the mdm2 proteins are deregulated in many human cancers and exert their oncogenic activity predominantly by inhibiting the p53 tumor suppressor .
several human tumor types have been shown to have increased levels of mdm2 , including soft tissue sarcomas , bladder cancers , and osteosarcomas as well as breast tumors [ 1215 ] .
diagnosis of hcc was considered as a terminal situation and the leading cause of death in cirrhotic patients . however , when diagnosis is achieved at an early stage , effective therapies that improve long term survival are more likely to be achieved . in such sense , the early diagnosis of hcc is crucial for the treatment of patients . in this study ,
mdm2 was evaluated by immunoassay as a potential taa in hcc , and autoantibody to this protein was also validated to be an early stage biomarker in immunodiagnosis of hcc .
all sera used in this study , including 244 sera from patients with hcc , 30 sera from patients with liver cirrhosis ( lc ) , 30 sera from patients with chronic hepatitis ( ch ) , and 89 normal human sera ( nhs ) , as well as some sera from four hcc patients with serial bleeding samples , were obtained from the serum bank of cancer autoimmunity and epidemiology research laboratory at utep ( university of texas at el paso ) .
all hcc patients were diagnosed according to the criteria described in a previous study and had not received treatment with any chemotherapy or radiotherapy .
patients with ch and lc were followed up at least 18 months after collecting blood to exclude individuals with primary biliary cirrhosis and asymptomatic or clinically undetectable hcc .
normal human sera were collected from individuals at the same locality during annual health examinations , who had no obvious evidence of malignancy . mdm2
construct pgex-4 t mdm2 wt ( plasmid i d : 16237 ) purchased from addgene.com was subcloned into pet28a vector producing a fusion protein with nh - terminal 6x histidine and t7 epitope tags .
the recombinant protein expressed in escherichia coli bl21 ( de3 ) was purified using nickel column chromatography .
polyclonal anti - mdm2 rabbit antibody and monoclonal anti--actin mouse antibody were purchased ( cell signaling technology , inc . , danvers , ma ) .
horseradish peroxidase- ( hrp- ) conjugated goat anti - human igg , hrp - conjugated goat anti - rabbit igg , hrp - conjugated goat anti - mouse igg , and fitc - conjugated goat anti - human igg were purchased ( santa cruz biotechnology , inc . ,
anti - rabbit igg fab2 ( alexa fluor 488 ) was purchased ( cell signaling technology , inc . , danvers , ma ) .
standard protocol for elisa was used as described in our previous study [ 19 , 20 ] . in brief , a 96-well microtiter plate ( immunochemistry technologies , llc , bloomington , mn ) was coated overnight at 4c with recombinant mdm2 protein at a final concentration of 0.5 g / ml in phosphate - buffered saline ( pbs ) .
the antigen - coated wells were blocked with gelatin postcoating solution at room temperature for 2 h. human sera diluted at 1 : 100 with serum diluent were incubated for 2 h at room temperature in the antigen - coated wells , followed by hrp - conjugated goat anti - human igg ( caltag laboratories , san francisco , ca ) at 1 : 4000 dilution .
the average optical density ( od ) value at a wavelength of 405 nm was used for data analysis . the cutoff value designating positive reaction was the mean optical density ( od ) of 89 normal human sera plus 3 standard deviations ( sd ) .
denatured recombinant mdm2 protein and cancer cell lysates were electrophoresed on 10% sds - page and transferred to nitrocellulose papers , respectively . after blocking in pbs with 5% nonfat milk and 0.05% tween-20 for 1 h at room temperature
, the nitrocellulose papers were incubated overnight at 4c with 1 : 200 dilution of human sera , 1 : 500 dilution of polyclonal anti - mdm2 antibody , and 1 : 500 dilution of monoclonal anti--actin mouse antibody , separately .
hrp - conjugated goat anti - human igg , hrp - conjugated goat anti - rabbit igg , and hrp - conjugated goat anti - mouse igg were , respectively , applied as secondary antibody at a 1 : 10,000 dilution .
the ecl - kit was used to detect immunoreactive bands according to the manufacturer 's instructions ( thermo scientific , waltham , ma ) .
indirect immunofluorescence assay was performed on hep2 antinuclear antigen tissue slides ( bion enterprises , des plaines , il ) .
the sera were diluted at 1 : 80 in pbs and ph 7.4 and incubated with the slides for 30 min at room temperature .
after extensive washing , the slides were incubated with fluorescein isothiocyanate- ( fitc- ) conjugated goat anti - human igg secondary antibody ( santa cruz biotechnology , ca ) or anti - rabbit igg fab2 ( alexa fluor 488 ) as secondary antibody diluted 1 : 100 in pbs for 1 h at room temperature .
the slides were washed two times with pbs before adding a drop of mounting media containing 1.5 g / ml 4,6-diamidino-2-phenylindole ( dapi ) ( vector laboratories inc . ,
the slides were then examined under fluorescence microscopy , lsm 700 confocal microscopy ( zeiss ) , at 400x magnification .
the diluted human sera ( 1 : 80 ) were incubated with recombinant protein ( final concentration of recombinant protein in the diluted human sera was 0.03 g/l ) overnight at 4c and then centrifuged at 10,000 g for 10 min .
liver cancer tissue array slide with normal tissue controls ( 38 cases/80 cores , including pathological diagnosis and pathological grades , two normal liver tissues as controls ) was purchased ( us biomax , inc . ,
antigen retrieval was performed by microwave - heating methods in trilogy pretreatment solution for 20 min .
the sections were incubated with polyclonal anti - mdm2 antibody ( 1 : 50 dilution ) for overnight at 4c . hrp detection system ( hrp streptavidin label and polyvalent biotinylated link ) and dab substrate kit were used as detecting reagents .
the mean od value of each group of patients ' sera was compared using the mann - whitney u test ; the frequency of autoantibody to taas in each group of patients ' sera and the expression profile of mdm2 in liver cancer and normal tissue groups were compared using the chi - square ( ) test with fisher 's exact test , and two significant levels ( 0.05 and 0.01 ) were used .
the full - length recombinant mdm2 protein was used as coating antigen in elisa to screen autoantibodies against mdm2 in sera from patients with hcc , lc , ch , and nhs . in total , 244 sera from patients with hcc , 30 from lc , 30 from ch , and 89 sera from normal human individuals were used in this study . as shown in table 1 ,
the prevalence of autoantibody against mdm2 was 19.3% ( 47/244 ) in hcc , which was significantly higher than that in lc ( p = 0.000 ) , ch ( p = 0.002 ) , and nhs ( p = 0.000 ) .
the average titer of autoantibody against mdm2 in hcc sera was higher than that in lc , ch , and nhs ( p < 0.01 ) .
figure 2 shows that representative hcc sera with positive reaction to mdm2 in elisa also have strong reactivity in western blotting compared to the normal sera .
autoantibody to mdm2 in serial serum samples from four hcc patients ( case 1 to case 4 ) was also tested .
anti - mdm2 autoantibody titer was high in nine months before hcc was diagnosed . in hcc case 2 ,
case 3 , and case 4 , anti - mdm2 autoantibody titer became higher at six months before hcc was detected . to further confirm the reactivity of autoantibody in hcc sera to mdm2 and the intracellular location of mdm2 ,
commercially purchased hep2 cell slides were used in indirect immunofluorescence assay to detect hcc sera with anti - mdm2 positive in elisa . as shown in figure 4 ,
a representative anti - mdm2 positive hcc serum had an intense nuclear staining pattern , which was similar in fluorescent staining pattern and cellular location to that shown by polyclonal anti - mdm2 antibody .
the fluorescent staining was significantly reduced when the same hcc serum was preabsorbed with recombinant mdm2 protein . in the current study ,
the expression profile of mdm2 in liver cancer tissues and normal liver tissues was examined by immunohistochemistry with tissue array slides .
tissue array slides were commercially available for this study , including 30 hcc tissue specimens and 10 normal hepatic tissue specimens .
the polyclonal anti - mdm2 antibody was used as primary antibody to detect the expression of mdm2 in liver cancer and normal hepatic tissues . as a result ,
the characteristics of patients and mdm2 expression in liver cancer are shown in table 2 .
the expression of mdm2 in liver cancer and normal hepatic tissues is shown in figure 5 .
many studies demonstrated that sera from patients with cancer contain antibodies , which react with a unique group of autologous cellular antigens generally known as tumor - associated antigens ( taas ) . during the progression from chronic liver disease to hcc
, the novel autoantibodies appearing with malignant transformation will be more likely to be related to events associated with tumorigenesis , and therefore these autoantibodies can be used as reporters identifying aberrant cellular mechanisms in tumorigenesis and also serve as immunodiagnostic markers for hcc detection [ 2123 ] . as we know
, afp is the most common and traditional biomarker being used for diagnosis of hcc in current clinical screening .
there are still about 40% of hcc patients that can not be identified by this approach .
hcc patients with small tumors or with well - to - moderately differentiated tumors may not have high level of serum afp .
the ultrasonic and computer tomography are useful when the tumors are big enough to be detected .
usually it is too late for patients to get effective therapy when the hcc is detected by these methods .
therefore , identification of a better biomarker for the early stage hcc is crucial for reducing the hcc mortality rate in the population .
reporters from the immune system , can be used as biomarkers in immunodiagnosis of early stage hcc . in our previous study , over ten oncoproteins have been evaluated and validated as taas in hcc , and autoantibodies against these taas have been detected in sera from patients with hcc .
but the sensitivity and specificity of autoantibodies to single taa as a diagnostic marker in hcc are not high enough for the diagnosis of hcc .
the method to solve this problem is to combine the known taas making a taa array with multiple taas , by which a higher sensitivity and specificity can be acquired , but it is still not good enough till now .
it is necessary to work on the validation of taas which are good at early stage diagnosis in hcc patients .
mdm2 can lead to nuclear export , ubiquitination , and nuclear and cytoplasmic proteasomal degradation of p53 through directly binding to the p53 transactivation domain [ 27 , 28 ] .
p53-mediated tumor suppressing activity can be inhibited by high levels of mdm2 through p53 inactivation .
the mdm2 protein also interacts with several cell cycle regulatory proteins that may contribute to its tumorigenic ability .
the role of mdm2 in hcc and its overexpression in hcc patients are studied by many researchers .
a study on japanese patients with hcv infection showed that mdm2 gene was significantly associated with development and recurrence of hcc .
mdm2 snp309 plays a major role in the carcinogenesis of hcc , especially among caucasian populations .
the findings from different areas support that mdm2 is significantly associated with increased risk of hepatocellular carcinoma [ 3235 ] .
mdm2 snp309 g allele is a susceptibility gene for the development of viral hepatitis - related hepatocellular carcinoma [ 3638 ] .
the combination of mdm2 snp 309 and tp53 arg72pro genotypes confers higher risk to develop hcc [ 3941 ] .
the mdm2 gene silenced by shrna effectively inhibits hcc tumorigenesis of subcutaneously xenografted hepg2 cells in nude mice .
mdm2 antagonist can inhibit tumor growth in hcc with different types of p53 in vitro .
hcc patients with a low expression of mdm2 survived significantly longer as compared with patients with high expression .
the expressions of mdm2 protein and gene were also related to the high invasiveness of hcc through inactivating the tumor - suppressor function of the p53 gene [ 45 , 46 ] .
mdm2 was considered to be a valuable target for cancer therapy and mdm2 blockade with suitable antagonists was shown to block tumor growth in a number of models .
mdm2 overexpression was also considered to be a useful predictor of poor prognosis in patients with hcc following hepatic resection .
however , there is nothing reported about the possibility of mdm2 as a biomarker of cancer .
mdm2 is predominantly distributed in the cell nucleus , and some of mdm2 can be detected in the cytoplasm .
on the situation of cell injury or the high penetration of the cell membrane , the immune system can find the exposed mdm2 protein and the autoantibodies will appear in the serum . since mdm2 is overexpressed in hcc , we try to evaluate whether the mdm2 protein is a potential taa in hcc and validate if autoantibody to this protein can be used as an early stage biomarker in immunodiagnosis of hcc . in our present study
, the results indicated that almost twenty percent of hcc sera showed immune response to mdm2 recombinant protein .
the mean titer of autoantibodies against mdm2 in sera from patients with hcc was significantly higher than that in lc , ch , and normal individuals .
the results were also confirmed by western blot analysis , which showed that representative hcc sera with positive reaction to mdm2 in elisa also have strong reactivity compared to normal sera . in the further study ,
anti - mdm2 autoantibodies were detected in sera from several hcc patients with serial bleeding samples .
a high titer of autoantibodies against mdm2 in elisa can be seen in sera in 6 months to 9 months before the clinical diagnosis of hcc .
hcc develops when there is a gene mutation to the cellular machinery which causes the cell to replicate at a high rate , which results in the cell avoiding of apoptosis .
constant cycle of damage followed by repair can lead to mistakes during dna repair which in turn lead to carcinogenesis . during the course of hcc formation ,
some new presenting autoantibodies maybe have the value for diagnosis of hcc patients with an early stage of tumorigenesis .
anti - mdm2 autoantibodies are potential biomarkers for immunodiagnosis of hcc patients due to the low positive rate in lc , ch , and normal individuals and higher positive rate in hcc patients .
it has been reported that mdm2 protein is overexpressed in hcc tissue . in a previous report by mf zhang 's group ,
mdm2 expression was examined in 181 pairs of hcc tissues and the adjacent hepatic tissues by using immunohistochemistry , through which the group found that mdm2 was overexpressed in all the hcc cases .
but in another study , the result of immunohistochemistry showed that mdm2 was expressed in 26% of hcc , and its expression correlated positively with p53 mutations . in our present study , eighty percent of hcc liver tissues were positively stained with anti - mdm2 antibody and there were no differences between hcc tissues with grade iii and grade ii in using immunohistochemistry approach with hcc tissue array slide .
ihc study with hcc tissue array has verified that the cellular localization of mdm2 is found in the nucleus of cell .
we also verified the cellular localization of mdm2 by indirect immunofluorescence assay , and anti - mdm2 autoantibody positive hcc serum demonstrated an intense nuclear staining pattern .
the cellular localization of mdm2 is associated with its oncoprotein function as negative regulator of tumor suppressor protein p53 .
data from a chinese population shows that the mdm2 indel polymorphism may be a genetic modifier for developing hcc .
so mdm2 protein maybe appeared in early stage of hcc generation and its antibodies have the possibility of early biomarker in hcc patients .
our current study focused on the possibility of its role as a biomarker in early stage hcc diagnosis .
the data from our study has demonstrated that mdm2 is a potential taa in hcc , and the anti - mdm2 autoantibodies may be used as a biomarker in early stage hcc diagnosis because a higher titer of autoantibodies against mdm2 in elisa can be seen in sera at 6 months to 9 months before the clinical diagnosis of hcc .
the limitation of our study is that no information of these hcc patients with serial bleeding samples was acquired after the hcc diagnosis .
we did not know the reason why the titer of autoantibodies against mdm2 decreased after diagnosis .
the affecting factors may range from the surgery procedures , the drug used by the patients , the chemotherapy , or the radiotherapy .
therefore , in our future study more cases with complete clinical data should be included in order to further investigate the correlation of the role of hepatic tissue mdm2 protein expression and the presence of elevated mdm2 autoantibodies in serum of patients with hcc , to be evaluated as early biomarkers in immunodiagnostic of hcc . | hepatocellular carcinoma ( hcc ) is the fifth most common malignancy worldwide .
although new therapeutic strategies have been continuously developed and applied to clinical treatment for hcc , the prognosis is still very poor .
thus , early detection of hcc may enhance effective and curative management . in this study ,
autoantibody responses to mdm2 protein in hcc patient 's serum were evaluated by enzyme - linked immunosorbent assay ( elisa ) and part sera were evaluated by western blotting and indirect immunofluorescence assay .
immunohistochemistry ( ihc ) over tissue array slides was also performed to analyze protein expression of mdm2 in hcc and control tissues .
the prevalence of autoantibodies against mdm2 was significantly higher than that in liver cirrhosis ( lc ) , chronic hepatitis ( ch ) , and normal human sera ( nhs ) .
the average titer of autoantibodies against mdm2 in hcc serum was higher compared to that in lc , ch , and nhs .
a high titer of autoantibodies against mdm2 in elisa could be observed in the serum in 6 to 9 months before the clinical diagnosis of hcc in the serum of several hcc patients with serial bleeding samples .
our preliminary data indicate that mdm2 and anti - mdm2 system may be a potential biomarker for early stage hcc screening and immunodiagnosis . |
major complications of hysterectomy are rare . however , because hysterectomy is one of the most commonly performed surgical procedures in women , efforts to decrease risks associated with this procedure have the potential for public health impact .
one such effort may include optimizing referral to surgeons with appropriate experience and volumes of major gynecologic cases .
in many surgical fields the relationship between surgical volume and operative outcome has been investigated .
for example , one study of greater than 470 000 medicare patients undergoing either cardiovascular procedures or cancer resections found that the operative mortality rate was strongly and inversely related to surgeon volume for each procedure . in the field of gynecology , this relationship has been examined with regard to procedures including abdominal myomectomy , hysteroscopic myomectomy , cancer surgery , pubovaginal sling placement , and pelvic reconstruction . specifically regarding hysterectomy performed for benign indications , however ,
most past studies on this subject have used relatively low cutoff values to define high - volume surgical status , equating high volume to approximately one hysterectomy per month .
although the number of cases required to achieve initial skill mastery for hysterectomy has been estimated at 30 cases , it is not evident how this translates to annual case volume recommendations because maintenance of performance may require frequency and repetition .
we aim to stratify surgical volume categories beyond what has previously been done to determine whether there is a greater difference observed between very high - volume surgeons and low- or intermediate - volume surgeons .
second , much of the available data are limited by long periods of data collection and isolated examination of only one route of hysterectomy .
this may cause particular difficulty for analysis of laparoscopic hysterectomy because it is characterized by continually evolving technology and more variation in practice patterns . by using the database of all hysterectomies ( including abdominal , vaginal , laparoscopic , and robotic ) performed at brigham and women 's hospital in the years 2006 , 2009 , and 2010 ,
all hysterectomies performed in 2006 , 2009 , and 2010 at brigham and women 's hospital were identified by use of data from the billing department .
all abdominal , vaginal , laparoscopic , and robotic - assisted laparoscopic hysterectomies were included . to limit confounding variables ,
cases performed for oncologic or obstetric indications were excluded . of note , most cases are completed with resident or fellow assistance because the hospital is an academic institution .
the creation of this hysterectomy database has been described in detail elsewhere and was used initially to evaluate evolving hysterectomy practice between the years 2006 and 2009 .
the existing hysterectomy database was expanded to include additional data from 2010 for the purposes of this study .
the number of hysterectomies performed annually was determined for each surgeon by review of case statistics from our database .
consideration was given to the fact that surgeons may also practice at outside institutions ; therefore the report of hysterectomy volume at all other institutions was also taken into account .
information about operative experience at outside institutions was obtained from the department administration and confirmed with medical records at those institutions .
the case load of hysterectomies performed annually over the study period was then averaged to generate the average annual hysterectomy volume for each surgeon .
after review of a histogram of the distribution of hysterectomy volumes ( figure 1 ) , surgeons were categorized as low - volume ( < 11 hysterectomies per year ) , intermediate - volume ( 1150 hysterectomies per year ) , or high - volume ( > 50 procedures per year ) surgeons
information was abstracted from the medical record regarding age , length of hospital stay , and operative time ( reported as time in and time out of the operating room ) .
patient charts were reviewed for body mass index , parity , prior abdominal surgeries , indication , procedure , estimated blood loss during surgery , pathology report , uterine weight , intraoperative complications , and postoperative complications .
intraoperative complications included estimated blood loss 1000 ml , vessel or visceral organ injury , and major cardiac / pulmonary events .
by use of a previously reported complication grading scheme , major postoperative complications included readmission , reoperation , ileus , and pulmonary embolism ; minor postoperative complications included infections ( urinary tract , lung , wound , or fever ) , urinary retention , and deep vein thrombosis .
baseline characteristics and perioperative outcomes were compared across hysterectomies performed by three distinct categories of operators , classified based on their average annual surgical volume .
we used the one - way analysis of variance test to compare continuous variables and the test or fisher exact test to compare categorical variables .
all comparisons were considered to have reached statistical significance at a value of .05 .
all hysterectomies performed in 2006 , 2009 , and 2010 at brigham and women 's hospital were identified by use of data from the billing department .
all abdominal , vaginal , laparoscopic , and robotic - assisted laparoscopic hysterectomies were included . to limit confounding variables ,
cases performed for oncologic or obstetric indications were excluded . of note , most cases are completed with resident or fellow assistance because the hospital is an academic institution .
the creation of this hysterectomy database has been described in detail elsewhere and was used initially to evaluate evolving hysterectomy practice between the years 2006 and 2009 .
the existing hysterectomy database was expanded to include additional data from 2010 for the purposes of this study .
the number of hysterectomies performed annually was determined for each surgeon by review of case statistics from our database .
consideration was given to the fact that surgeons may also practice at outside institutions ; therefore the report of hysterectomy volume at all other institutions was also taken into account .
information about operative experience at outside institutions was obtained from the department administration and confirmed with medical records at those institutions .
the case load of hysterectomies performed annually over the study period was then averaged to generate the average annual hysterectomy volume for each surgeon . after review of a histogram of the distribution of hysterectomy volumes ( figure 1 )
, surgeons were categorized as low - volume ( < 11 hysterectomies per year ) , intermediate - volume ( 1150 hysterectomies per year ) , or high - volume ( > 50 procedures per year ) surgeons
information was abstracted from the medical record regarding age , length of hospital stay , and operative time ( reported as time in and time out of the operating room ) .
patient charts were reviewed for body mass index , parity , prior abdominal surgeries , indication , procedure , estimated blood loss during surgery , pathology report , uterine weight , intraoperative complications , and postoperative complications .
intraoperative complications included estimated blood loss 1000 ml , vessel or visceral organ injury , and major cardiac / pulmonary events .
by use of a previously reported complication grading scheme , major postoperative complications included readmission , reoperation , ileus , and pulmonary embolism ; minor postoperative complications included infections ( urinary tract , lung , wound , or fever ) , urinary retention , and deep vein thrombosis .
baseline characteristics and perioperative outcomes were compared across hysterectomies performed by three distinct categories of operators , classified based on their average annual surgical volume .
we used the one - way analysis of variance test to compare continuous variables and the test or fisher exact test to compare categorical variables .
all comparisons were considered to have reached statistical significance at a value of .05 .
a total of 1914 hysterectomies were performed in 2006 , 2009 , and 2010 for benign non - obstetric indications . of these cases ,
33.7% ( n = 644 ) were abdominal , 20.5% ( n = 393 ) vaginal , and 45.8% ( 877 ) laparoscopic or robotic .
the number of hysterectomies per year remained stable , with 635 procedures in 2006 , 643 procedures in 2009 , and 636 procedures in 2010 .
figure 1 shows the breakdown of surgical volume among the 85 gynecologic surgeons who contributed cases to this study . there were 67 surgeons ( 78.8% ) included in the low - volume group ( < 11 hysterectomies per year ) , 10 ( 11.8% ) in the intermediate - volume group ( 1150 hysterectomies per year ) , and 8 ( 9.4% ) in the high - volume group ( > 50 hysterectomies per year ) .
overall , 43.8% ( 838 ) of the hysterectomies were performed by high - volume surgeons , 35.6% ( 682 ) by intermediate - volume surgeons , and 20.6% ( 394 ) by low - volume surgeons .
the patients who underwent hysterectomy by a low- , intermediate- , or high - volume surgeon did not differ from each other significantly in terms of parity or surgical history before undergoing a hysterectomy ( table 1 ) .
the participants in the cohort of hysterectomies performed by high - volume surgeons were older and were noted to have a slightly higher body mass index . although it did not reach statistical significance , the patients on whom high - volume surgeons operated were noted to have a more frequent history of laparotomy ( 31.7% in both low- and intermediate - volume groups vs 36.75% in high - volume group , p = .061 ) .
baseline characteristics of hysterectomy cohort by surgeon volume category data are given as mean sd or number ( percent ) .
the total does not add up to 100% because cases may have had > 1 finding .
the route of hysterectomy differed significantly by surgical volume category , with abdominal hysterectomy being the most common approach for both the low- and high - volume surgeons ( 42.64% of low - volume cases , 19.65% of intermediate - volume cases , and 40.81% of high - volume cases ; p < .001 ) .
the primary mode of hysterectomy for intermediate - volume surgeons was laparoscopic ( 57.92% of cases ) , whereas robotic - assisted laparoscopic hysterectomies were performed more frequently by the high - volume surgeons ( 2.86% of cases ) .
regarding case mix , low- and intermediate - volume surgeons were predominantly operating on cases of uterine fibroids whereas the high - volume group had more varied indications ( fibroids in 64.47% and 69.65% of cases in the low - and intermediate - volume groups vs 49.4% in the high - volume group , p < .001 ) .
the operative findings of endometriosis , adhesions , or urogynecologic issues ( mainly prolapse ) did not differ significantly in the three groups under study .
perioperative outcomes for all hysterectomies performed by low- , intermediate- , and high - volume surgeons are shown in table 2 . a significant decline in operative time ( 203.35 minutes in low - volume group , 199.19 minutes in intermediate - volume group , and 155.11 minutes in high - volume group
; p < .001 ) and estimated blood loss ( 237.96 ml in low - volume group , 205.58 ml in intermediate - volume group , and 161.09 ml in high - volume group ; p <
.001 ) was seen as the surgical volume increased . on average , surgeries performed by high - volume operators required 44 to 48 fewer minutes and resulted in 44 to 76 ml less blood loss compared with intermediate- and low - volume surgeons .
interestingly , the mean length of hospital stay was found to be slightly higher in the surgeries performed by high - volume surgeons ( 1.8 days in low - volume group , 1.35 days in intermediate - volume group , and 2.06 days in high - volume group ; p < .001 ) .
the three surgical volume groups did not differ from each other significantly with regard to conversion to laparotomy , readmission rate , or incidence of intraoperative or postoperative complications .
perioperative outcomes of hysterectomy cohort by surgeon volume category data are given as mean sd or number ( percent ) .
to better appreciate the potential differences among surgical volume groups , the perioperative characteristics were analyzed separately for each mode of hysterectomy ( tables 35 ) .
when we looked at abdominal hysterectomies alone ( table 3 ) , the relationship of shorter operative time ( 202.54 minutes in low - volume group , 184.39 minutes in intermediate - volume group , and 164.27 minutes in high - volume group ; p < .001 ) and lower estimated blood loss ( 321.96 ml in low - volume group , 379.54 ml in intermediate - volume group , and 253.89 ml in high - volume group ; p <
the length of stay is also longer in the high - volume abdominal hysterectomy group ; however , no differences are seen regarding readmission or intraoperative or postoperative complications .
perioperative outcomes by surgeon volume category for abdominal hysterectomy data are given as mean sd or number ( percent ) .
perioperative outcomes by surgeon volume category for vaginal hysterectomy data are given as mean sd or number ( percent ) .
perioperative outcomes by surgeon volume category for laparoscopic and robotic hysterectomy data are given as mean sd or number ( percent ) .
equals 1270 ( excluding abdominal ) . for the subgroup of vaginal hysterectomy ( table 4 ) ,
the high - volume surgeons had a faster operative time ( 178.64 minutes in low - volume group , 194.12 minutes in intermediate - volume group , and 114.09 minutes in high - volume group ; p < .001 ) , lower estimated blood loss ( 218.37 ml in low - volume group , 215.36 ml in intermediate - volume group , and 106.83 ml in high - volume group ; p < .001 ) , and shorter hospital stay . of note ,
postoperative complications from vaginal hysterectomy were highest in the intermediate - volume group ( 6.25% in low - volume group , 19.44% in intermediate - volume group , and 9.28% in high - volume group ; p = .002 ) .
conversion to laparotomy , readmission rate , and intraoperative complications did not differ by surgical volume cohort .
analysis of solely laparoscopic and robot - assisted laparoscopic hysterectomies also showed a faster operative time ( 217.8 minutes in low - volume group , 205.9 minutes in intermediate - volume group , and 166.83 minutes in high - volume group ; p < .001 ) and decreased blood loss ( 152.56 ml in low - volume group , 144.06 ml in intermediate - volume group , and 92.05 ml in high - volume group ; p < .001 ) in the high - volume group .
in addition , the length of hospital stay was highest in the high - volume group , although no difference was seen in readmissions .
the difference in total complications neared statistical significance , with fewer intraoperative complications seen in the intermediate- and high - volume groups ( 15.07% in low - volume group , 8.42% in intermediate - volume group , and 9.48% in high - volume group ; p = .066 ) .
concerning hospital - level data for abdominal hysterectomy , it has been suggested that academic status or hospital volume may not be as important a modifier of surgical outcomes for this common procedure of relatively lower risk as compared with more complex operations .
similar conclusions were reached when surgeon - level data were analyzed in a retrospective cohort study of greater than 8000 women who underwent abdominal hysterectomy at one institution between 1995 and 2005 . in that study
the authors asserted that although high - volume surgeons ( defined by completion of greater than 10 hysterectomies per year ) have fewer complications and shorter operative times , the overall rate of complications is low enough to deem hysterectomy safely performed by both groups .
the relationship between surgeon volume and perioperative outcomes from vaginal hysterectomy has been assessed through an analysis of greater than 77 000 cases taken from a national database for the years 20032007 .
findings included both lower perioperative complications and resource use in the high - volume vaginal surgeon group ( defined by completion of greater than 13 vaginal hysterectomies per year ) .
volume - related outcomes from laparoscopic hysterectomy have also been examined in a retrospective analysis of 1000 procedures from a single institution during the years 19992008 .
this study did not find any differences between the high- and low - volume surgeon groups aside from a shorter operative time for high - volume surgeons ( loosely defined as completion of greater than 30 cases , not specific to laparoscopic hysterectomy or annual case volume ) .
however , a large database analysis of 124 615 benign laparoscopic hysterectomies performed during the years 20002010 found significantly decreased risk of overall morbidity , complications , prolonged hospitalization , and resource utilization in high - volume surgeons ( defined as greater than 14.1 operations per year ) . finally , a more recent prospective cohort study of 1534 laparoscopic hysterectomies performed over a 1-year study period showed no effect of annual hysterectomy surgical volume on blood loss , operative time , or adverse events .
interestingly , the authors did identify an intangible surgical skills factor that was associated with procedure success and noted that increasing cumulative surgical experience ( up to 125 procedures ) was also associated with improved outcomes . with the benefit of a large hysterectomy database and expanded categorization of surgeon volume , we confirm a strong relationship between operator surgical volume and both shorter operative time and lower estimated blood loss .
these outcomes were strikingly maintained for all modes of hysterectomy , building on prior findings .
interestingly , the incidence of conversion to laparotomy and readmission rate did not differ among surgical volume cohorts for any of the modes of hysterectomy .
few differences were seen in the occurrence of complications , with the highest occurrence of postoperative complications from vaginal hysterectomy for the intermediate - volume cohort and highest occurrence of total complications from laparoscopic / robotic - assisted laparoscopic hysterectomy in the low - volume cohort .
the findings regarding length of stay are inconsistent among modes of hysterectomy , with decreased hospital stay after vaginal hysterectomy by high - volume surgeons but increased hospital stay after abdominal or laparoscopic / robotic hysterectomy by high - volume surgeons .
one possible explanation for the increased length of stay in selected high - volume cohorts is a greater complexity of cases undertaken by the more referral - based high - volume surgeons , although this was not reflected in terms of higher uterine weights for this group .
arguably , uterine weight is an imperfect predictor of case complexity , and other unmitigated clinical factors may be at play , such as surgeon specialization .
for example , many of the surgeons in the high - volume group are gynecologic oncologists who perform predominantly abdominal or laparoscopic / robotic cases ; even when one analyzes only their benign hysterectomies , these surgeons are likely dealing with a more challenging case mix . in addition , hospital stay may reflect physician preference or practice patterns .
strengths of this study include the variety of gynecologic surgeons included , with a selection of subspecialists and generalists . by limiting analysis to a 5-year time frame ( 2006 , 2009 , and 2010 ) at one institution ,
we have also stratified surgical volume categories more finely than previous work on the subject in an attempt to observe the breakdown of operator experience
. limitations of this study include its retrospective nature and inherent potential for errors in data collection .
in addition , one of our main outcomes measures , estimated blood loss , is a subjectively reported value ( surgeon 's estimated blood loss as listed in the operative note ) as opposed to being objectively verified .
although we attempted to accurately code each physician 's average annual hysterectomy case volume , we did not account for cumulative years of experience or other factors such as learning curve or fellowship training .
this variety of providers and types of cases is a benefit but also may introduce confounding factors that are not entirely accounted for in our study design .
in addition , we may have an incomplete sample size to detect rare outcomes such as major complications and conversion to laparotomy .
we have established that high - volume surgeons have shorter operative times and less blood loss for all modes of hysterectomy .
prior work from our institution has shown that charges based on operating room time account for the vast majority of total operative charges for hysterectomy ( unpublished data , presented in abstract form at 2010 american association of gynecologic laparoscopists aagl meeting by k.n .
wright ) . given the current focus on containing health care costs , the importance of operative time savings can not be overlooked .
it is uncertain , however , whether the benefits of operator experience extend to other perioperative outcomes .
this may in part reflect practices already in place at our institution that allow for accurate referral of cases to the appropriate surgical provider , thereby mitigating gradients in perioperative complications . at our hospital
, we have seen a trend of low- and intermediate - volume surgeons transitioning to minimally invasive modes of access , which highlights the need for continued training to maintain optimal patient outcomes , in addition to appropriate surgical referral networks as needed . | background and objectives : to estimate the effect of surgeon volume on key perioperative outcomes after all modes of hysterectomy.methods:we performed a review of 1914 hysterectomies performed at a large , academic tertiary - care hospital .
women who underwent abdominal , laparoscopic , vaginal , or robotic hysterectomy for benign non - obstetric indications in 2006 , 2009 , and 2010 were included.results:gynecologic surgeons were categorized according their average annual hysterectomy case volume : low volume ( < 11 cases per year ) , intermediate volume ( 1150 cases per year ) , and high - volume ( > 51 cases per year ) . taking all modes of hysterectomy together , surgeries performed by high - volume surgeons required a shorter operative time ( 155.11 minutes vs 199.19203.35 minutes , p < .001 ) and resulted in less estimated blood loss compared with low- and intermediate - volume surgeons ( 161.09 ml vs 205.58237.96 ml , p < .001 ) .
the 3 surgical volume groups did not differ from each other significantly in the conversion to laparotomy , readmission rate , or incidence of intraoperative or postoperative complications .
these findings were maintained when subgroup analyses were performed by type of hysterectomy , with few exceptions . in the subgroup of vaginal hysterectomies by intermediate - volume surgeons ,
there were slightly more postoperative complications .
there were fewer intraoperative complications in laparoscopic / robotic hysterectomies performed by high - volume surgeons , though not statistically significant.conclusions:hysterectomies performed by high - volume surgeons at our institution during the 3-year study period were associated with shorter operative times and less estimated blood loss . |
rituximab is a human - mouse chimeric monoclonal antibody that targets the b - cell cd20 cell surface protein and has become indispensible for the treatment of b - cell non - hodgkin 's lymphoma [ 13 ] .
however , complications in the form of severe viral infections have been observed , and methods to counter these infections are presently being evaluated [ 47 ] .
prior to the introduction of rituximab , reactivation of hbv was a major complication of chemotherapy - induced immunosuppression [ 820 ] .
the events leading to hbv reactivation during rituximab / chemotherapy combination treatment have been reported by various groups , as have the effects of preventive administration of the nucleoside analogue lamivudine on the suppression of hbv reactivation [ 2133 ] .
however , a number of issues remain to be determined , such as the level of hbv resistance to lamivudine , the optimal lamivudine administration period , and even whether or not lamivudine is the best preventive drug to use .
hbv reactivation may result in a number of serious outcomes , including death from hepatitis [ 2337 ] .
additionally , even in the event that hepatitis is prevented , possible problems in subsequent lymphoma treatment or in anticipated outcomes may arise , such as lymphoma recurrence or shortened survival prognosis . in this paper based on studies from various groups , we explored issues in preventive nucleoside analog administration , including optimal administration period and the utility of concurrent use of these drugs .
the discussion includes a compilation of our results on preventive administration of nucleoside analogs in hbs antigen- ( hbsag- ) positive cases , which were accrued from the debut of rituximab in japan in 2002 to december 2008 .
acute hepatitis caused by hbv is initially suppressed by cytokines secreted from nk and other types of cells .
since hepatitis is triggered by ctls , a time lag exists from initial infection to hepatitis onset [ 38 , 39 ] .
however , hepatitis that arises from hbv reactivation , unlike that which occurs during conventional hbv infection , is under a state of immunosuppression of the normal immunological responses to hbv , leading to increased viral replication and widespread infection of hepatocytes .
when immunosuppression is removed by discontinuation of chemotherapy , immune competence is restored and infected hepatocytes are rapidly destroyed , leading to hepatitis .
it is therefore likely that reactivated hbv results in a shorter time period to hepatitis progression than seen in conventional hbv infection .
we believe that this mechanism of hepatitis aggravation can explain those case patients who die despite administration of lamivudine in response to hbv reactivation during chemotherapy or use of immunosuppressive drugs . in contrast , hcv tends to become chronic through suppression of the induction and amplification of immune responses ( such as interferons ) from the outset , and this may be why hepatitis caused by hcv reactivation during the use of anticancer or immunosuppressive drugs is less likely to become severe [ 4042 ] .
we have reported on reactivation of hbv and hepatitis during rituximab treatment alone or in combination with chemotherapy .
other groups have reported a rate of hbv reactivation of 20%~55% [ 15 , 1820 ] .
one report claims an hbv reactivation rate of 3% , even in hbsag - negative cases .
reported a 4% rate of hbv reactivation in hbsag - positive patients based on a questionnaire conducted in japan , which is lower than previous studies , and 20% of these patients developed fatal hepatitis .
frequent hbv reactivation is also thought to occur as a complication of chemotherapy during treatment of lymphomas and may be influenced by steroids [ 18 , 45 , 46 ] . since the initial use of rituximab in the clinic , debate has centered on whether rituximab alone can induce hbv reactivation or whether it does so only when used in combination with chemotherapy .
however , subsequent reports by us and by yang et al . have shown that hbv reactivation can occur with rituximab alone , and it is therefore likely that rituximab itself can induce hbv reactivation [ 22 , 37 ] . although reactivation of hbv is deemed more likely when rituximab is combined with chemotherapy or steroid therapy , clearly the use of rituximab alone can not be deemed completely safe .
future prospective analyses are needed to definitively answer whether or not reactivation of hbv is more prevalent in patients given rituximab alone or given chemotherapy without rituximab .
nonetheless , in a questionnaire survey at a hokkaido facility examining and treating blood diseases , hbv reactivation cases were observed only in patients given rituximab alone .
these results support the study by yeo et al . , showing that reactivation of hbv is more prevalent in patients given rituximab alone compared with chemotherapy alone [ 22 , 47 ] .
to date , various investigators have identified a number of risk factors for hbv reactivation which include being male , hbs antigen positivity , hbv - dna level , presence of lymphoma , use of anthracyclines or steroids , second- and third - line anticancer drug treatment , and youth . a review of these factors by yeo et al .
showed that youth , being male , and liver function prior to chemotherapy were potential risk factors .
furthermore , yeo et al . conducted an analysis on hbv reactivation in patients using rituximab , as there is a potential for risk factors to change in such cases . including risk factors which had previously been reported , they list being male , lacking hbs antibodies , and using rituximab as additional risk factors .
further analyses on such risk factors are needed in order to enable the stratification of hbv - infected patients receiving rituximab in order to identify those that require preventive administration with nucleoside analogs [ 20 , 47 , 48 ] .
in hbsag - positive patients , there have been reports of hbv reactivation during chemotherapy [ 820 ] . increased liver function and a tendency for increased jaundice and lowered albumin
have also been reported in hepatitis cases caused by hbv reactivation , compared to primary hbv infection . similarly , hbv reactivation has been reported in patients receiving chemotherapy in addition to rituximab [ 2137 ] . for these patients ,
attempts to prevent hbv reactivation are needed regardless of whether or not rituximab is given .
preventive administration of nucleoside analogs is currently recommended for such patients , and chemotherapy under preventive administration is desirable [ 4963 ] .
although there are few complete reports of cases in which rituximab was used in combination with preventive treatments , he et al . among others
loomba et al . reported that preventive lamivudine therapy reduced hbv - reactivation - related hepatitis by 79% or more , based on an analysis of previous studies .
however , it has been reported that with lamivudine administration , drug resistance increases yearly by 15%20% [ 66 , 67 ] .
thus , with preventative administration of lamivudine , the emergence of resistant hbv strains is highly problematic . a report by pelizzari et al .
suggested that lamivudine administration may result in a lower likelihood of emergent drug resistance compared to treatments for conventional hepatitis b .
however , picardi et al . reported numerous instances of hbv genome mutations in patients receiving chemotherapy with fludarabine .
in addition , they have shown that there is a high risk of hbv resistance to lamivudine in patients undergoing treatments that have powerful immunosuppressive effects . a similar report exists for chemotherapy combined with rituximab , and it is possible that similar occurrences may be observed when steroids or fludarabine are used together with rituximab . from these observations , as well as considering issues of emergent drug resistance , administration of entecavir is preferred over lamivudine for the prevention of hbv reactivation .
additionally , even among hbsag - positive cases , preventive treatment may change in relation to the level of hbv - dna present .
various guidelines recommend that when more than 1 year of long - term nucleoside analog administration against hbv - dna is necessary , the analog should be changed to entecavir . in patients who have a high level of hbv - dna ,
it is desirable to use drugs such as entecavir , based on its effectiveness against the ymdd mutation [ 70 , 71 ] .
moreover , for hbsag chronic hepatitis , the guidelines recommend the use of entecavir when hbv - dna is more than 2000 iu / ml , while lamivudine is adequate at less than 2000 iu / ml .
in addition , in hbv - dna - positive cases , it may be necessary to screen for ymdd mutations in advance as there is a possibility in these cases that other treatments will be necessary , such as the use of tenofovir or the combined use of 2 nucleoside analogs [ 7073 ] .
anti - hbc - positive cases indicate a prior history of hbv infection . these include window period cases as well as cases which are hbv - dna positive and anti - hbs negative or hbsag negative ( occult hbv infection ) [ 43 , 7476 ] .
caution is required in these cases when using rituximab , chemotherapy with anticancer drugs , or immunosuppressive drug preparations [ 70 , 71 ] .
although rare , hbv reactivation during chemotherapy has been reported in anti - hbs - positive , anti - hbc - positive cases or in cases positive only for anti - hbc [ 15 , 26 , 43 , 45 ] .
moreover , in a study by hui et al . , hbv reactivation - induced hepatitis was reported in anti - hbc - positive , anti - hbs - negative cases , and even in those that were hbv - dna negative .
this shows that hepatitis caused by hbv reactivation can occur in anti - hbc - positive cases regardless of hbv - dna status . for these cases ,
the guidelines recommend careful monitoring of hbv - dna , since hepatitis caused by hbv reactivation is uncommon and treatment is expensive [ 7073 , 77 ] .
although reports to date show that hbv reactivation in patients undergoing chemotherapy is rare , when it does occur , the chemotherapy treatment period is extended .
in addition , the treatment intensity of the chemotherapy against lymphoma declines , and fatalities are not uncommon in hbv reactivation - induced hepatitis .
this is because the mortality of hepatitis caused by hbv reactivation is about 30%100% , and once the disease is present , some patients are not able to be saved [ 4345 ] .
based on these facts , administration of nucleoside analogs may be desired in these cases .
it has been reported , however , that hbv reactivation - related hepatitis can be preventable by monitoring hbv - dna level on a monthly basis .
hbv reactivation - induced hepatitis has been reported in anti - hbs - positive , anti - hbc - positive , hbsag - negative cases [ 15 , 26 , 43 , 45 ] .
in addition , while rare , there have been sporadic reports of cases in which hbv reactivation was observed after administration of rituximab in patients that were positive for anti - hbs alone [ 29 , 43 , 78 ] although it is generally thought that such occurrences are primarily due to vaccine administration .
such cases may arise when , for instance , the production of antibodies has declined with age and only anti - hbs remains ; however , details from these types of cases are unavailable .
caution is required , as even in cases that are positive for anti - hbs alone , the chance exists that symptoms are due to hbv reactivation . in our experience
studying the changes in anti - hbs after rituximab administration , we have found that in the large majority of cases , anti - hbs antibody titers decreased with increased number of rituximab administrations [ 21 , 32 , 33 , 79 ] .
furthermore , we have experienced cases which presented with hbv reactivation following an increase in hbv - dna that accompanied a decline in anti - hbs and anti - hbc antibody titers [ 32 , 33 ] .
these results suggest an association between anti - hbs / anti - hbc antibodies and hbv reactivation , and that monitoring these antibodies may provide an index for hbv reactivation .
in addition , regarding hbv reactivation during bone marrow transplantation , onozawa et al . reported that a decrease in anti - hbs titer results in hbv reactivation - induced hepatitis .
as anti - hbs is a part of the humoral immune component which monitors hbv , we surmise that transitions in anti - hbs have the potential to become indices for predicting hepatitis caused by hbv reactivation .
, in a patient with anti - hbs titers of 868 miu / ml , indicating that hbv reactivation may occur even in cases with high anti - hbs titers . this may indicate that predicting reactivation only by monitoring anti - hbs titers would be insufficient .
from each group , treatment guidelines for prevention of hbv reactivation are shown in table 1 [ 7073 , 77 ] . from the information detailed above ,
hbe - ag- and hbsag - positive cases qualify as targets for preventive administration of nucleoside analogs . in hbsag - negative cases ,
it is likely that the frequency of hbv reactivation in anti - hbc only positive cases is not necessarily high .
however , since hbv reactivation may lead to patient death , we believe that these cases would qualify as targets for preventive administration of nucleoside analogs . in contrast , in hbsag - negative , anti - hbs - positive cases , it is desirable while observing changes in antibody titers to additionally monitor hbv - dna in those cases with titers less than 500 . additionally , it is possible that periodic monitoring of hbv - dna may predict hbv reactivation , and it is therefore advantageous , keeping cost in mind , to combine these indices [ 81 , 82 ] .
monitoring of hbv - dna is also likely to be essential in cases having hbv - dna mutations and in whom antibody expression is weak .
it is crucial , however , to stratify groups into those in whom preventive administration can be recommended , since with hbv - dna monitoring alone , the frequency of hbv reactivation has been reported to be higher in those given preventive administration of lamivudine compared to controls . when monitoring for hbv reactivation , it is important to identify hbv reactivation at an early stage using , in addition to hbv - dna monitoring , a variety of additional information including changes in anti - hb titers .
summarized the risks of hbv reactivation during chemotherapy in reference to hbsag , anti - hb , and level of treatment .
these data may be useful for preventive treatment , however , more clinical studies are needed .
preventive administration against hbv is recommended for a period of 6 months following completion of treatment [ 67 , 70 ] .
this may not be sufficient , however , as a number of cases in which hbv reactivation occurred more than 46 months after treatment have been reported [ 8488 ] .
the 6-month timeframe is possibly associated with changes in b - cell number [ 13 , 82 , 88 ] .
the guidelines proposed by lok and mcmahon in 2007 have been more specific compared to past guidelines and have incorporated recommendations such as extending the period of preventive administration depending on the hbv - dna level .
we observed a case in which hbv reactivation occurred 4 years after termination of preventive lamivudine administration following completion of treatment . from this case , we surmise that for hbeag- or hbsag - positive cases in which anti - hbs can not be produced , it is necessary to combine the use of nucleoside analogs from the initial time of treatment and to continue their use .
in addition , for cases which are anti - hbs positive from the outset , we believe that preventive administration of nucleoside analogs is necessary at least until anti - hbs titers return to pretreatment levels , and that the period of nucleoside analog administration needs to be determined using immune recovery , for example , of anti - hbs , as an index .
however , with long - term administration of drugs such as lamivudine or entecavir , issues such as cost arise ; therefore , it is hoped that cases requiring long - term preventive administration will become clarified in the future with followup studies . as this report shows
, preventive nucleoside analog administration involves issues of lamivudine resistance , therefore we believe it is desirable to administer entecavir at an early stage .
although we commonly set lamivudine dosing at 100 mg and entecavir at 0.5 mg , it is recommended that entecavir be increased to 1 mg when dealing with lamivudine - resistant cases [ 7073 ] .
compared to lamivudine , resistance to entecavir is less likely to develop , and administration of entecavir is recommended in cases where preventive administration against hbv will exceed 12 months [ 70 , 71 , 73 ] . in lamivudine - resistant cases ,
some clinicians recommend the combined use of entecavir , adefovir , or tenofovir with lamivudine while others recommend switching over completely .
we believe that combined use with lamivudine is desirable , as it has been reported that adefovir - resistant strains immediately develop when patients with lamivudine - resistant strains are switched over to adefovir alone . in seronegative hbv cases that are treated with immunosuppressive or anticancer drugs ,
however , as mentioned previously , when using rituximab , anti - hbs level decreases and then disappears , and it is therefore possible that no antibodies will be produced after prior administration of vaccine ; therefore , it may be desirable to administer vaccine after completion of treatment . in some cases ,
however , hepatitis caused by hbv reactivation can not be controlled with a vaccine , and it is therefore possible that hbv reactivation will not be prevented with vaccination alone .
future studies on vaccine efficacy in patients receiving rituximab or chemotherapy are needed ; however , it will take considerable time before practical applications of these studies can be realized .
hbv reactivation during chemotherapy treatment is thought to occur from hbv amplification in hepatocytes as a result of immunosuppression caused by anticancer drugs , and subsequently from hbv - infected hepatocytes that are targeted by an immune response that becomes activated after removal of this immunosuppression .
it is likely that one of the causes of hbv amplification during the combined use of chemotherapy and rituximab is the reduction in antibody titers that accompanies a decrease in b cells [ 79 , 91 ] .
in addition , rituximab is able to alter the t lymphocyte population , and it is possible that aggressiveness toward hbv intensifies as a result , further promoting hbv amplification and subsequent recovery of the distribution of immunocompetent cells such as lymphocytes .
uemura et al . found that chemotherapy - induced reactivation of hbv is more lethal compared to acute hbv hepatitis , and the likelihood of survival also decreases ; therefore , it is crucial to prevent this method of disease onset [ 36 , 44 ] .
it is hoped that future studies will further clarify the changes that occur in immune functions of the organism during rituximab use along with the mechanisms of hbv reactivation , which will lead to safer and more effective treatments for malignant lymphomas . | rituximab is a drug used for the treatment of b - cell non - hodgkin 's lymphoma , and its range of use has expanded to the treatment of collagen diseases such as idiopathic thrombocytopenic purpura and rheumatoid arthritis .
one serious complication of rituximab use is the reactivation of dormant hepatitis b virus , and prevention of this phenomenon has become an urgent issue .
this paper provides a general outline of the problem through an analysis of patient cases that we and other groups have experienced to date . |
syphilis , the great imitator , is among the most fascinating of skin disease .
it may present to the dermatologist as a sexually acquired , contagious disease or as a congenitally acquired infection .
syphilis can be transmitted by blood transfusion , but it is very rare now and in this condition , no primary lesion appears .
a 28-year - old unmarried male , office worker , presented to the skin , v.d . and leprosy department with a grayish white painless plaque on the left side of the lower lip for last 8 months and multiple papulosquamous non - pruritic lesions all over the body including palm and sole .
there was history of transfusion of 3 u of stored blood in to the patient for severe anemia 1 year back , in a local nursing home .
on examination , lip lesion was grayish white in color , oval in shape and size was about 4 cm in diameter , soft and non - tender [ figure 1 ] .
there was generalized lymphadenopathy and lymph nodes were palpable , mobile , non - tender and rubbery .
there were also multiple papulosquamous non - pruritic lesions all over the body including trunk [ figure 2 ] , upper and lower limbs , palms and soles but sparing oral mucosa and the genital .
lesion of secondary syphilis - flat papule ( condyloma lata ) on lip lesions of secondary syphilis - papulosquamous lesions on trunk hematological examination showed moderate anemia ( 8.2 gm% ) and all biochemical parameters were within normal range .
serological tests for syphilis were positive ( vdrl in 1:32 dilution and reactive tpha ) and elisa for hiv-1 and 2 were negative .
incisional biopsy of the lip lesion was sent for histopathological study , which showed dense infiltration of plasma cells and few lymphocytes in the dermis , in and around blood vessels in the form of perivasculitis and intimal proliferation in few of the arteries and veins ( endarteritis obliterans ) [ figure 3 ] .
then patient was put on oral azithromycin 1 gm stat and doxycycline 100 mg twice daily and partial regression of lesion was observed after nearly 3 weeks of treatment .
histopathology of lip lesion(condyloma lata ) showing plasma cell infiltration in dermis and endarteritis obliterance ( h and e stain 400 )
syphilis is a chronic disease with a waxing and waning course , the manifestations of which have been described for centuries .
the rate of primary and secondary syphilis , the most infectious stages of the disease decreased throughout the 1990s and in 2000 reached an all time low .
the primary mode of transmission is by sexual contact , and the next most common is transfer across the placenta .
kissing , blood transfusion and accidental inoculation have also been reported as routes of transmission , but are of minor importance today .
the risk of transmission through blood is negligible due to improved donor selection , uniform serological testing of all blood donor , and a shift from transfusion of fresh blood to transfusion of refrigerated blood components .
transmission via blood products is nonetheless theoretically possible since organism may survive for up to 5 days in refrigerated blood .
needle sharing probably does not play a significant role in syphilis , but remains unclear .
the secondary stage is characterized by a polymorphic rash , lymphadenopathy and other systemic manifestations .
the tertiary stage is the most destructive and is marked by cardiovascular and neurologic sequelae and gummatous involvement of any organ system .
our patient presented with lesions of secondary syphilis including soft , non - tender plaque on the left side of lower lip , papulosqumaous lesions on the trunk , palms and soles and lymhadenopathy .
the confirmed diagnosis of primary , secondary or early congenital syphilis are made by demonstration of organism by dark ground microscopy .
serological test for syphilis include vdrl , t. pallidum immobilization test , fluorescent treponemal antibody absorption(fta - abs ) test , t. pallidum hemagglutination assay ( tpha ) test and eia ( treponemal enzyme immunoassay ) test.[811 ] in this case vdrl and tpha tests were reactive .
the characteristic histopathological findings of syphilis are perivascular infiltration of plasma cells and lymphocytes and intimal proliferation of both arteries and veins ( endarteritis obliterans ) , which was seen in the biopsy of lip lesion of our case .
the differential diagnosis of our case were chancre , squamous cell carcinoma lip , lichen planus , actinic granuloma , leukoplakia , psoriasis , drug eruption and graft versus host disease .
theoretically , the possibility of chancre of lip could not be ruled out but strongly denied of any type of sexual contact by the patient and history of blood transfusion indicate syphilis d emblee .
many antibiotics , with notable exceptions of the aminoglycosides and sulphonamides , have some treponemicidal activity .
benzathine penicillin is the recommended first - line therapy for syphilis . in patients who are hypersensitive to penicillin ,
regimens based on tetracycline , doxycycline , erythromycin , azithromycin , ceftriaxone and chloramphenical have all been used .
the patient in this case showed hypersensitivity to penicillin so he was put on azithromycin 1 gm single dose and doxycyclin 100 mg twice daily and he had been responding to it . | a 28-year - old male patient presented to skin , v.d . and leprosy outpatient with a single gray white plaque on the left side of the lower lip for last 8 months and multiple papulosquamous lesions all over the body for last 6 months .
there was history of blood transfusion for anemia 1 year back .
histopathology of lip lesion and reactive vdrl and tpha tests confirmed the diagnosis as syphilis .
we report this rare case of syphilis d emblee . |
the anaerobic threshold ( at ) is a hallmark of the aerobic fitness of athletes1 , physically active individuals3 and on a minor scale for special
populations , such as individuals with type 2 diabetes ( t2d)2 , 4 , 5 . at
has been considered the gold standard along with maximum oxygen
consumption ( vo2max ) for the evaluation of aerobic fitness and the subsequent
prescription of aerobic exercise6,7,8,9 .
moreover , monitoring of aerobic training
adaptations through at determination may be especially interesting because no maximal effort
is needed , and could be safer for individuals with pathologies such as hypertension and
diabetes10 , 11 .
for instance , the at has been determined through the analysis of heart rate
variability ( hrv)2 , blood
catecholamines12 , blood glucose and
lactate3 , ventilation and respiratory
equivalents13 , 14 , and the double product ( dp)15,16,17,18,19,20,21,22 . identification of at through different indices , such as the double product ( dp ) , has been
widely utilized for healthy individuals16 , 17 , 19 , athletes20 and
individuals with cardiovascular diseases15 , 16 , 18 .
however , few studies have identified the at of individuals with t2d
through different methods2 , 23 , while the exercise recommendations for t2d24 indicate that these patients should be
encouraged to undertake an exercise test before being submitted to physical training .
the at of t2d individuals is often observed to be between 6070% of
vo2max6 , 8 , 25 , which is an exercise
intensity that has been widely recommended for this population for better blood glucose and
cardiovascular control24 .
further , previous studies have shown that aerobic exercises performed around at intensities
significantly reduced the blood pressure ( bp ) of individuals with t2d in the post - exercise
period6,7,8 , 25 , 26 , and none of these
previous studies reported complications during the experimental sessions , suggesting the
desirability of at identification for this population , since inadequate physical exertion
can be a powerful triggering factor of myocardial infarction27 .
individuals with t2d often present autonomic dysfunction28,29,30 . compared to healthy individuals , it is therefore likely that they
would show a different hemodynamic reaction [ heart rate ( hr ) and bp , and in turn dp ] during
exercise , such as an exaggerated exercise blood pressure response31 .
in addition , hypertension and t2d very frequently
coexist , since 20% of hypertensive patients have t2d , and 80% of people with t2d are
hypertensive32 .
hypertensiveness , could
also affect the hemodynamic responses during exercise , which may in turn complicate at
detection through cardiovascular responses .
have presented the feasibility of
at identification for t2d individuals through hrv analyses2 and kumagai et al . identified the double product breakpoint ( dpbp )
in t2d individuals , but did not compared with any other anaerobic threshold identification
method23 .
thus , we hypothesized that at
could be predicted through the dpbp during exercise performed by t2d individuals , similar to
healthy individuals16 .
moreover ,
identification of at through the dpbp would be relevant since the dp can provide information
that correlates with myocardial oxygen consumption , which is recognized as a non - invasive
indicator of cardiac overload33 , 34 as well as cardiac ischemic events
including acute myocardial infarction35 .
further , the determination of at through the dpbp is a simple method that does not depend on
the use of complex equipment such as gas analyzers , which are expensive and require the use
of specialized technicians to operate them .
the validity of the dpbp in estimating the at using a sphygmomanometer and stethoscope has
not been investigated yet .
experiments have used either automated blood pressure
monitor15,16,17 , 19 , 20 , 23 or a cannula inserted into the brachial artery18 .
these procedures are not accessible to most practioners
because they require costly equipment as well as highly invasive procedures . in addition , to
the best of our knowledge , no study has examined the dpbp as an estimate of the at in
individuals with t2d .
thus , the purpose of this study was to verify whether or not the at of t2d individuals can
be predicted through the dp method , and to analyze the relationships between the workload
( watts ) , oxygen consumption ( vo2 ) and hr obtained by the dpbp and the ventilatory
threshold ( vt ) methods .
after approval from the local human research ethics committee ( universidade
catlica de braslia : 011/2011 ) , and obtaining the written informed consent of
the volunteers , 11 t2d individuals ( 8 females ) , and 10 controls ( 7 females ) were initially
selected to participate in this study .
exclusion criteria for participation in this study
included previous diagnosis of peripheral neuropathy or retinopathy , as well as diabetic
foot ulcerations osteomioarticular complications , the endogenous use of insulin , and/or use
of medicines that could have directly affected hr or bp .
all the individuals were recruited
at a public hospital , and all had been previously assessed using standard tests conducted by
endocrinologists and cardiologists .
two participants with t2d were excluded from the
analysis because it was not possible to detect their vt and/or dpbp ( table 1table 1.the number of cases in which the ventilatory threshold ( vt ) and the double
product breakpoint ( dpbp ) were detected by all observersparametert2d ( n)nd ( n)totalparticipants initially screened111021vt detected101020dpbp detected91019hr primarily responsible for dpbp112sbp responsible for dpbp549participants included in the analysis91019t2d : individuals with type 2 diabetes ; nd : individuals without t2d ; vt : ventilatory
threshold ; dpbp : double product breakpoint ; hr : heart rate ; sbp : systolic blood
pressure ) .
therefore , the statistical power a priori conferred on this sample ( n=19 )
was 82% ( power=0.82 ) .
the t2d individuals were receiving medical treatments of oral
hypoglycemiants ( metformin , metformin + glibenclamide ) .
the general
characteristics of the volunteers are presented in table 2table 2.characteristics of the individuals with type 2 diabetes ( t2d ) and non - diabetic
( nd ) controlst2d ( n=9)nd ( n=10)age ( yrs)63.2 8.958.3 7.8body mass ( kg)70.2 14.172.5 19.2height ( m)1.59 0.11.61 0.11bmi ( kgm)26.3 5.027.1 4.1body fat ( % ) 37.4 5.634.5 7.0hba1c ( % ) 6.8 1.35.2 0.2*vo2peak ( mlkgmin)21.4 3.522.6 5.1maximal heart rate
t2d : individuals with type 2 diabetes ;
nd : individuals without t2d ; bmi : body mass index ; hba1c : glycated
hemoglobin ; * significant difference ( p<0.05 ) .
t2d : individuals with type 2 diabetes ; nd : individuals without t2d ; vt : ventilatory
threshold ; dpbp : double product breakpoint ; hr : heart rate ; sbp : systolic blood
pressure data expressed as mean standard deviation .
t2d : individuals with type 2 diabetes ;
nd : individuals without t2d ; bmi : body mass index ; hba1c : glycated
hemoglobin ; * significant difference ( p<0.05 ) all experimental sessions were carried out at the laboratory of physical evaluation and
training ( lafit ) of the universidade catlica de braslia 2 h after
volunteers eaten a standardized breakfast of a moderate glycemic index meal without any
substances that could have directly affected the hr and/or the bp .
the participants were
given a physical evaluation including anthropometric measurements ( body mass , height and
body fat percentage ) and then performed an incremental exercise test ( it ) . during the it ,
measurements of hr , bp and ventilatory variables were continuously monitored .
to carry out
the measurement of body fat percentage by bipolar bioimpedance , the individuals were told
to : avoid caffeine and alcohol for 24 hours before the test ; not perform physical activity
or eat a heavy meal for at least 4 hours before the test ; and suspend diuretic medication 24
hours before the test36 .
the it was performed on a cycle ergometer ( monark , 828 e , valburg / sweden ) at 60 rpm , and
began one minute at zero watt ( w ) load , followed by increments of 15 w every 3 minutes until
voluntary exhaustion .
the cardiologist of the laboratory monitored the electrocardiogram
( ecg ) of the volunteers throughout the test in order to identify any abnormality . during the period before the exercise , as well as during the last 30 seconds of each stage ,
blood pressure ( bp )
was measured non - invasively by the auscultatory method using a mercury
column sphygmomanometer ( tycos instrumentos hospitalares , so paulo , brazil ) . expired gases
were measured during the it through a gas analyzer ( metalyzer 3b , cortex biophysik , leipzig ,
germany ) that was previously calibrated with a 3-l syringe ( flow calibration ) and a standard
mixed gas containing 4.9% co2 and 17% o2 ( gas calibration ) .
the values
of ventilation ( ve ) and oxygen uptake ( vo2 ) were recorded breath - by - breath , but ,
they only were analyzed every 20 s in each 3-min stage .
the ventilation ( ve ) kinetics during the it stages were used to identify the occurrence of
vt by two experienced researchers .
this was considered to be the exercise intensity above
which an over proportional increase in ve was observed in relation to increasing
workload37 .
systolic blood pressure ( sbp ) and hr were collected in the final minute of each stage . the
dpbp was considered to be the exercise intensity at which an over proportional increase in
dp was observed in relation to increasing workload .
two experienced researchers determined
dpbp , and when a discrepancy between them occurred , a third researcher was consulted .
all
the plots were blinded , therefore the researchers did not know the identity of the subjects
or the conditions of the test .
it is noteworthy that the same evaluator , which did not
participated in the determination of dpbp and vt , carried out all collections of bp and hr .
blinded to the
analysis . after assessing the normality and homogeneity of data through the shapiro - wilk and levene s
test , respectively ,
the data were presented as means ( ) standard deviation . in order to
compare the characteristics of the groups ,
the student s t - test for independent samples was
conducted . between and within groups comparisons
when any of the dependent variables did not show sphericity in mauchly s
test , the epsilon of greenhouse - geisser was used to analyze the f
statistic .
since there were only two groups , the post hoc is unable to locate the
differences , therefore , it was necessary to apply parallel test , named : pairwise
comparisons . thus , the paired student s t - test was used to compare the anaerobic threshold
identification methods within groups ( vt and dpbp ) . the effect size within ( vt and dpbp ) and
between groups ( t2d and
pearson s correlation coefficients were
used to verify the relationships between the studied methods used to determine anaerobic
threshold . for the sample size calculation , the statistical power ( 1- ) was used a priori ,
using comparison analysis applied ( anova for repeated measures ) , an effect size of
f=0.35 and an alpha of 5% .
furthermore , the bland and altman technique
was used to verify the level of agreement between the different methods39 . a significance level of 5% ( p<0.05 )
all the
procedures were carried out using the statistical package for the social sciences ( spss
20.0 ) and g*power ( version 3.1.9.2 ) .
the dpbp was identified in all 11 nd individuals and in 9 from 11 t2d individuals ( 81.8% ) ,
with sbp being primarily responsible for the dpbp in almost half of all cases ( table 1 ) .
the results presented in table 3table 3.workload ( watts ) , heart rate ( bpm ) and oxygen uptake
( mlkgmin ) measurements at vt and dbpb of the t2d and nd
groupsparametert2d ( n=9)nd ( n=10)workload ( watts)dpbp45 21.260 29.2vt40 24.963 30.6heart rate(bpm)dpbp124.7 13.6119.1 17.4vt122.1 14.6121.4 19.1vo2
( mlkgmin)dpbp14.6 2.415.1 2.9vt14.1 3.415.3 3.6data expressed as mean standard deviation .
t2d : individuals with type 2 diabetes ;
nd : individuals without t2d ; dpbp : double product breakpoint ; vt : ventilatory
threshold show no there were no significant differences between the workloads ( watts ) ,
hr ( bpm ) and vo2 corresponding to the at identified by the vt and dpbp methods .
further , for the t2d group , strong and significant correlations ( p<0.01 ) between
workloads at vt and dbpb ( r=0.853 ) , hr ( r=0.714 ) , and
vo2 ( r=0.863 ) were found .
strong and significant correlations
( p<0.001 ) were also found in the nd group for all parameters investigated ; they were
r=0.923 for the workload at vt and dpbp , r=0.881 for hr ,
and r=0.863 for vo2 .
t2d : individuals with type 2 diabetes ;
nd : individuals without t2d ; dpbp : double product breakpoint ; vt : ventilatory
threshold the bland and altman analyses indicate there was good agreement for the vo2
( lmin ) corresponding to vt and dpbp based on the low bias and narrows limits
of agreement [ bias ( 95% of confidence interval ) ] for vt and dpbp [ 0.03 ( 0.11 )
lo2min ] for the t2d group , and vt and dpbp [ 0.02 ( 0.13 )
lo2min ] for the nd group .
the main finding of this research is that the dpbp can be used to estimate the at of t2d
and nd individuals , since there were no significant ( p<0.05 ) differences between the
workloads ( watts ) , hr and vo2 of the vt and dpbp methods .
furthermore , the effect
size analyses also demonstrated that the visual differences of the variables mentioned
above , had little ( small ) or no effect , according to cohen s d38 . from a physiological point of view , it is presumed that the dp could be a marker of at
determination , to the extent that , when performing an incremental exercise test , there is a
gradual reduction in the vagal activity and consequent increase in sympathetic nervous
activity .
however , at a certain intensity , a complete vagal withdrawal is expected and this
presents as a persistent depression of the autonomic nervous system until the end of the
tests , featuring the hrv threshold2 , 40 , 41 .
accordingly , there is a marked increase in sympathetic nerve
activity , which results in a significant elevation of catecholamines , which is known as the
catecholamines threshold12 .
the increased
levels of catecholamines , in turn , result in a marked increase in bp and hr , and thus on dp ,
and as a result the dpbp15,16,17,18,19 . furthermore ,
increased levels of catecholamines also produce a significant increase in glycolytic
activity42 , enabling for the
identification of the glycemic and lactate thresholds3 .
this increased glycolysis also contributes to a sharp increase in
co2 production which causes hyperventilation and results in the appearance of
the vt43 .
consequently , it is reasonable to infer that , similar to other markers ( hrv ,
catecholamines , glucose , lactate and ventilation ) , the dp can also be used to determine the
at , with the advantage of it being a non - invasive and low cost method .
exercise tests
involving blood collection and biochemical measures , and the use of expensive apparatus for
expired gases analysis , as well as trained professionals who know protocols are high - priced ,
and thus inaccessible to a major portion of the t2d population .
interestingly , although there was no significant difference of the workloads ( watts )
between the groups ( t2d and nd ) , the effect size of the difference was considered medium
( d=0.588 ) for dpbp and large ( d=0.824 ) for vt . from a
practical point of view , it is noteworthy that the effect size is considered the main
finding of a quantitative study , with the statistical significance ( p ) being the least
important44 , 45 , since the relevant point is the magnitude of differences45 .
with regard to the internal load variables ( hr and vo2 ) , these did not present
significant differences ( p>0.05 ) between the groups ( t2d and nd ) , while the effect sizes
of the differences were small or even null . additionally , intra - group differences were
always null .
moreover , the fact that significant differences ( p>0.05 ) were not observed
between the internal load variables ( hr and vo2 ) of t2d and nd groups , may be
partly explained by the higher cardiorespiratory capacity of the healthy individuals , as
they reached an absolute external load ( watts ) , but with a similar cardiorespiratory
responses ( hr and vo2 ) .
additionally , another aspect that should be considered
regarding the absence of significant differences between the t2d and nd groups , is that they
presented quite similar characteristics ( table
2 ) , since the only variable that was different between the groups was glycated
hemoglobin ( hba1c ) .
it is noteworthy that the statistics reveal there were strong and significant correlations
between workloads ( watts ) , hr and vo2 , and also a good agreement in both the t2d
and nd groups .
, who reported a strong
and significant correlation between vo2 at the lactate threshold ( lt ) and dpbp
( r=0.86 ; p<0.01)16 .
likewise tanaka et al . also observed a strong and significant correlation between the dpbp
and the lt ( r=0.90 ; p<0.01 ) in a sample of 90 patients17 .
additionally , kumagai et al . identified
the dpbp in 63 t2d individuals in a submaximal ramp test ( rating of perceived exertion of
15 ) performed on a cycle ergometer23 .
however , it is noteworthy that in that study the dpbp was not compared with any other at
identification method . regarding the comparison of dpbp and vt , brubaker et al
. previously demonstrated a strong
and significant correlation between the methods ( r=0.81 , p<0.01 ) in a
progressive test on a treadmill , and suggested that dpbp could be a valid alternative to vt
for individuals with coronary artery disease15 .
also omiya et al . reported a high and significant association
between workloads ( watts ) ( r=0.95 ; p=0.01 ) of both the dpbp and vt of 15
patients with cardiovascular diseases18 . similarly , hargens et al .
conducted an elegant longitudinal study in order to evaluate the
effect of eight weeks of aerobic training at vigorous intensity on dpbp and vt .
they
evaluated 7 men and 11 sedentary women , using a ramp protocol on a cycle ergometer , and
observed that hr and vo2 at baseline and at the end of the training period were
similar ( p>0.05 ) and showed good agreement19 .
the fact that this previous study reported an increase of dpbp
after training , confirms it is a reliable and sensitive method of identifing at adaptations
to training .
one limitation of the present study was that the intensity of the maximum lactate steady
state ( mlss ) was not determined for comparison with the investigated protocols of dpbp and
vt .
the determination of mlss is considered the gold standard among the exercise assessment
methods1 .
van schuylenbergh indicate that this parameter is not different from the lactate
threshold and vt46,47,48 .
thus , it is reasonable
to infer that the methods investigated in this study , dpbp and vt , which did not differ
between themselves , may also represent the mlss intensity .
the present study demonstrated that the dpbp can be easily determined during incremental
exercise in both t2d and nd controls .
additionally , the dpbp happened at exercise
intensities related to the vt in both the populations studied , suggesting that dpbp can be a
useful marker of exercise intensity associated with at . | [ purpose ] to verify the identification of the anaerobic threshold through the double product breakpoint ( dpbp ) method for individuals with type 2 diabetes . [ subjects and methods ] nine individuals with t2d ( 7 females ; age=63.2 8.9 y ) and 10 non - diabetic ( nd ) ( 7 females ; age=58.3 7.8 y ) performed an incremental exercise test on a cycle ergometer
. heart rate ( hr ) , blood pressure ( bp ) and expired gas were measured at the end of each stage .
the ventilatory threshold ( vt ) and dpbp were considered as the exercise intensities above which an over proportional increases in ve and dp were observed in relation to increasing workload .
[ results ] no differences were observed between the workloads , hr and vo2 corresponding to the at identified respectively by vt and dpbp . for the t2d , strong correlations between vt and dbpb workloads ( r=0.853 ) , hr ( r=0.714 ) , and vo2 ( r=0.863 ) were found .
these relationships were similar to those found for the control group ( r=0.923 ; r=0.881 ; and r=0.863 , respectively ) .
[ conclusion ] these results demonstrate that the dpbp enables for the prediction of at and correlated well the vt in both the t2d and nd participants . |
talon cusp is an uncommon dental anomaly showing morphologically well delineated , accessory cusp - like structure projecting from cingulum to the cutting edge .
it was named as talon cusp due to its appearance as eagle 's talon when viewed from incisal edge .
outfolding of the enamel organ or hyperactivity of the dental lamina during morphodifferentiation stage of tooth development or hyperproductivity of the anterior ends of dental lamina could be the cause for morphological variation in the crown and root , whereas talon cusp on labial surface of the tooth is attributed to the hyperplasia of labial central developmental lobe .
however , the etiology of talon cusp is not well understood , but is proposed to be a combination of genetic and environmental factors .
disturbances during morphodifferentiation , such as altered endocrine function , might affect the shape and size of tooth without impairing the function of ameloblasts and odontoblasts .
talon cusp may be associated with genetic factors and it has been reported in twins , siblings and also children of consanguineous marriages . it has also been reported in a family with father and child exhibiting in permanent and primary dentitions , respectively .
the talon cusp can be considered as one end of a range of hyperactivity of the dental lamina , with macrodontia and double tooth in the middle while supernumerary tooth on the other end .
it was also reported in association with syndromes , such as mohr syndrome ( orofacial digital ii ) , incontinenta pigmentii achromians , ellis van creveld syndrome , struge weber syndrome ( encephalo - trigeminal angiomatosis ) , rubinstein taybi syndrome , and alagille 's syndrome .
the other proposed names for talon cusp are accessory cusp , addition cusp , anterior dens evaginatus , cusp - like hyperplasia , exaggerated cingulum , prominent accessory cusp like structure , supernumerary cusp , interstitial cusp , tuberculated premolar , odontoma of the axial core type , evaginated odontoma , occlusal enamel pearl , occlusal anomalous tubercle .
this anomaly has been reported to be unusual in the mandibular dentition and rare on the facial aspect . to the best of our knowledge , only four cases table 1
have been reported in the literature and a rare case of fusion of the mandibular permanent incisors with labial and lingual talon cusps was also reported .
benefits and risks in a clinical trial the presence of this anomaly is not always an indication for dental treatment unless it is associated with clinical problem .
the complications of talon cusp are diagnostic ( if unerupted , resembles supernumerary , or compound odontomes ) , functional ( occlusal interferences , trauma to lip and tongue , speech problems , and displacement of teeth ) , pathological ( caries , abrasion , accidental cusp fracture , apical periodontitis , and periodontal diseases due to plaque accumulation in the grooves ) , and esthetics .
the purpose of the present report is to emphasize the rarity of mandibular facial talon and treatment options for good prognosis .
a 7-year - old south - indian boy reported to the department of pedodontics and preventive dentistry with a chief complaint of extra tooth in the lower jaw .
clinical examination , a cusp - like projection on the permanent right mandibular lateral incisor was evident [ figure 1a and b ] , which was confirmed as talon cusp on obtaining mandibular anterior occlusal radiograph that revealed an inverted v-shaped radio opaque projection extending from the cemento - enamel junction , a pulp horn overlapping pulp chamber and , an open tooth apex [ figure 1c ] .
the gingiva around the tooth was healthy and the talon was 7 mm in length cervico - incisally , 4.5 mm mesiodistally , and 4 mm anterio - posteriorly at its prominence .
talon cusp was conical in shape , curved towards the incisal edge when viewed laterally with deep groove between the talon and the tooth surface [ figure 1b ] .
as the talon cusp was larger , esthetically unpleasant and occlusally interfering with upper right lateral incisor , periodic gradual reduction ( lateral grinding ) of talon , and subsequent topical fluoride application was planned at every 45 days ( between june 2012-december 2012 ) .
talon on right mandibular lateral incisor ( initial visit ) ( a ) labial view ( b ) lateral view ( c ) anterior occlusal radiograph after obtaining the informed consent from the child 's parent , oral prophylaxis was performed and the bulk of talon cusp was reduced in two planes by merging the borders with the tooth surface using a tapered fissure bur ( mani dia - burs , prime dental products pvt .
half the thickness of enamel was reduced in the first visit and the surface was polished with composite finishing discs ( super - snap , rainbow technique kit , shofu , usa ) and fluoride varnish ( bifluorid 12 , voco , germany ) was coated over the surface to reduce sensitivity and stimulate reparative dentine formation for pulp protection . during 2 visit ,
entire enamel thickness was removed and similar procedure was followed in the subsequent two visits to reduce the dentin thickness [ figure 2a and b ] and composite resin restoration ( tetric n - ceram , ivoclar vivadent , usa ) was placed over the labial surface during the fifth visit .
the patient was reviewed for a period of 12 months , after treatment [ figure 3a and b ] .
talon on right mandibular lateral incisor ( fifth visit ) ( a ) labial view ( b ) lateral view ( c ) anterior occlusal radiograph ( d ) intraoral periapical radiograph talon on right mandibular lateral incisor ( 1-year follow - up visit ) ( a ) labial view ( b ) in occlusion ( c ) anterior occlusal radiograph pulpal status and root development were evaluated by obtaining periodical mandibular anterior occlusal radiograph during 2 , 5 , and 12-month follow - up visits [ figures 2c and 3c ] . at the end of 1-year follow - up ,
talon cusps occur most commonly on the permanent incisors , with more than 90% of them in maxilla and predominantly on permanent maxillary lateral ( 55% ) or central incisors ( 33% ) and less frequently on mandibular incisors ( 6% ) and maxillary canine ( 4% ) .
hattab classified this anomaly as type 1 ( talon ) , type 2 ( semi talon ) , and type 3 ( trace talon ) .
hsu chin - ying et al . , modified this as major , minor , and trace talon . when viewed from incisal aspect , the morphology appear as either
radiographically , major and minor talon appear typically as v-shaped radio - opaque structure superimposing over the normal image of the tooth , whereas tubercle - like and trace talon may not be detected in radiograph .
this appearance varies with location , size , and shape of the cusp and the angle at which radiograph is taken .
the point of v is inverted in mandibular teeth as it is composed of enamel , dentin , and varying amount of pulp tissue as in the present case [ figure 1c ] . due to the superimposition over the main pulp chamber , it is difficult to determine the extent of pulpal extension and is debatable over the extension of pulp in the cusp or not .
however , it has been proposed that major talon cusps , especially that deviate from the tooth crown are more likely to contain pulp as in the present case .
due to large pulp chamber in the young permanent teeth , the periodic lateral grinding should be performed with utmost care and also complete reduction of talon cusp ( major ) should not be done .
the formation of reparative dentin varies with the operative procedure and was reported around 2.8 m and 1.5 m per day for primary and permanent teeth , respectively .
reparative dentin is usually formed when teeth are mechanically prepared to within 1.5 mm of the pulp and it takes around 15 days for the formation of new odontoblasts from pulpal undifferentiated mesenchymal cells and 30 days for microscopic appearance of reparative dentin . in the present case , periodiclateral grinding was done at every 45 days , over a period of 7 months to preserve pulp vitality and minimize sensitivity .
the root was bifid , which is much similar to that of crown and probing the point that morphodifferentiation stage would have been affected during the formation of crown as well as root .
shey and eytel were the first to report periodic grinding method by reducing the entire bulk along the surface rather than only involving the tip for enhanced formation of reparative dentin .
similar method was reported , on permanent maxillary central incisor without any root abnormalities , every 45 days , over a period of 9 months .
early diagnosis and definitive treatment is necessary to prevent complications and periodic lateral grinding of mandibular facial talon was done in the present case , so that the erupting maxillary right permanent lateral incisor would not have any occlusal interference [ figure 3b ] .
the other treatment options were simple prophylactic measures such as fissure sealants or composite restorations , total cusp reduction followed by calcium hydroxide / mineral trioxide aggregate pulpotomy root canal treatment , extraction followed by orthodontic correction and prosthetic rehabilitation .
though , mandibular facial talon is rare to encounter in dental practice , proper diagnosis is mandatory before appropriate treatment is planned .
however , the management and treatment depend upon size , complications and patient 's compliance . role of pediatric dentist is utmost important in early diagnosis to minimize or prevent complications associated with it . | talon cusp is an uncommon dental anomaly showing morphologically well delineated , accessory cusp - like structure projecting from cingulum to the incisal edge of anterior teeth .
this anomaly is rare in the mandibular dentition and rarer on the facial aspect .
a case of this infrequent entity of mandibular facial talon cusp and its management is reported here . |
hiv risk perception studies among men - who - have - sex - with - men ( msm ) have often reported similar findings , namely : inaccurate or misperception of hiv risk being common among msm [ 1 , 2 ] , perception of low risk being associated with no previous hiv testing and considerable high risk behavior [ 35 ] , and moderate / high risk being associated with unrecognized hiv infection . compared to the general adult population , msm in asia have 18.7 times the odds of being hiv infected . across asia , hiv prevention expenditures targeting msm
the lack of funding for these programs is lamentable as prevention and harm reduction strategies targeting msm are successful in reducing high - risk behaviors [ 8 , 9 ] . in malaysia , homosexuality is illegal and stigmatization is a problem .
msm in malaysia are part of the marginalized most - at - risk populations ( marp ) that include commercial sex workers , migrant workers , and transgender people .
hiv prevention programs including hiv testing among the marp in malaysia are mainly spearheaded by several nongovernmental organizations ( ngo ) with very limited resources .
we present the findings of a study where we explored the relationship between hiv risk perception in a cohort of msm who sought vct services at a ngo - run center in kuala lumpur , malaysia , and their sexual behavior and risk factors .
our study shows that there exists a significant association between high hiv risk perception with high risk sexual behavior and hiv positivity .
targeting msm with high risk perception may indeed be the strategy to consider in a resource - limited setting in order to gain maximum benefits among the msm in malaysia .
data were collated from the pre - hiv testing questionnaires filled by clients seeking vct services at a community - based vct centre from january 2008 to december 2008 .
completion of the questionnaire was voluntary and no data identifiable to the client were required .
a total of 740 clients sought vct services at the centre between january 1 , 2008 , and december 31 , 2008 .
of these , 433 clients disclosed their sexuality as either homosexual or bisexual and were collectively categorized as msm in this study .
the profiles , sexual practices , and hiv prevalence of these 433 clients have been reported in earlier studies [ 10 , 11 ] .
information regarding the clients ' sexual behaviors , preferred role during anal sex , and other risk factors such as multiple sexual partners , types of sexual partners , alcohol use before sex , history of sexually transmitted infections ( stis ) , last unprotected sex , and condom use during anal sex , all in the preceding six months , was collated .
rapid test for hiv infection was performed using either the sd bioline hiv test ( standard diagnostics inc . ) or the acon hiv test kits according to the manufacturer 's specification .
descriptive analysis was used for description of the demographic characteristics , sexual behaviors , and risk factors for acquisition of hiv infection .
chi - square analysis was used to determine the odds ratio of hiv acquisition in each of the risk groups .
regressions analysis was used to determine which of the risk factors were predictors of hiv acquisition in the low , medium , high , and unsure risk perceptions groups .
all statistical analyses were performed using spss for windows v21 . a p value of < 0.05 with 95% confidence interval was deemed significant .
four hundred twenty - three clients rated their own hiv risk perception in this study of which 350 ( 82.7% ) identified themselves as homosexuals while 73 ( 17.3% ) as bisexuals .
most of the clients were chinese ( n = 250 ) followed by malays ( n = 115 ) .
most had received tertiary education ( n = 263 ) and earned a monthly income between myr 2001 and 5000 ( n = 152 ) .
two hundred fifty - six clients ( 60.5% ) reported receiving vct services at the same center previously prior to the survey .
the breakdown according to risk categories was : low risk64.4% , medium risk67.8% , high risk77.8% , and unsure risk49.4% , respectively . from a total of 423 clients , 101 ( 23.9% ) clients perceived themselves to be at low risk ( < 25% ) of being infected with hiv at the time of survey , 118 ( 27.9% ) perceived themselves to be at medium risk ( 2575% ) , 36 ( 8.5% ) clients perceived themselves to be at high risk ( > 75% ) , while the remaining 168 ( 39.7% ) clients were unsure regarding their risk .
most had engaged in anal and oral sex ( n = 328 , 79.0% ) , 25 ( 6.0% ) had vaginal , anal , and oral sex , 23 ( 5.5% ) had anal sex only , 22 ( 5.3% ) had oral sex only , 9 ( 2.2% ) had vaginal and oral sex only , 6 ( 1.4% ) had vaginal sex only , and 2 ( 0.5% ) had vaginal and anal sex only ( table 1 ) . out of 287 clients who disclosed their preferred sexual role during anal sex , 140 ( 48.8% ) were versatile , 72 ( 25.1% ) were top , and 75 ( 26.1% ) were bottom ( table 1 ) .
table 2 shows the number of clients in each risk perception group who had exposure to risk factors for hiv transmission in the preceding 6 months .
the risk factors were multiple sexual partners ( 10 or less versus > 10 ) , promiscuity ( regular versus regular and casual / transactional sex partners ) , alcohol use before sex , history of stis , last unprotected sex , and condom use during anal sex .
an item in the questionnaire explored drug use before or during sex in the preceding 6 months . out of 411 clients who responded to this question , only 42 ( 10.2% ) clients reported drug use before or during sex .
as the number of respondents was small , meaningful conclusions using statistical methods could not be made . assigning clients who rated themselves to be at low risk to acquire hiv infection as the reference group
, we determined the odds ratio for testing hiv positive in each of the other risk groups ( i.e. , medium risk , high risk , and unsure risk ) for each of the risk factors for hiv transmission .
the odds of testing positive for hiv infection in clients who rated themselves to be at high risk were 17x higher compared to clients in the low risk group ( or 17.14 , 95% ci : 3.2889.72 , p < 0.001 ) while clients who rated themselves to be at medium risk had 2.6x the odds of testing positive for hiv compared to low risk
clients ( or 2.61 , 95% ci : 0.5113.41 , p = 0.251 ) . clients who were unsure of their risk had 1.9x the odds of testing hiv positive compared to low risk clients ( or 1.96 , 95% ci : 0.409.75 , p = 0.410 ) . the increased odds in the medium risk and unsure risk groups were not statistically significant compared to the low risk group .
risk factors which were significant predictors for hiv acquisition in the high risk clients include having more than 10 sexual partners in the preceding 6 months , having casual and/or transactional sex partners in the preceding 6 months , alcohol use before sex in the preceding 6 months , unprotected sex of more than 6 months ago , and inconsistent condom use during anal sex in the preceding 6 months ( all p < 0.05 ) .
the same factors were not statistically significant predictors in the medium risk and unsure risk groups ( p > 0.05 ) .
history of sexually transmitted infections was not a predictor for hiv acquisition in any of the risk groups in this study ( table 3 ) .
msm who rated themselves to be at high risk for being infected with hiv in this study were significantly associated with high risk factors and were 17x more likely to be infected with hiv compared to those who rated themselves to be at low risk .
the significant predictors of hiv transmission in the high hiv risk perception group were multiple sex partners , promiscuity , alcohol use before sex , unprotected sexual intercourse , and inconsistent condom use during anal sex ( tables 2 and 3 ) .
in contrast , clients who have rated themselves to be at medium risk or were unsure of their risk were not significantly associated with these risk factors and had lower odds of being infected with hiv compared to clients who rated themselves as low risk .
these risk factors have been well documented to be independent risk factors for hiv transmission among msm [ 1214 ] .
the findings suggest that the clients were aware of the degree of their own high risk behaviors which enabled them to properly rate themselves into each of the hiv risk perception categories .
this is in contrast to other studies among msm that have shown that misperception of risk is the usual finding [ 1 , 35 ] .
about 60% of the clients in our study had previously used the vct services at the same center prior to the survey .
these clients would most likely have received the prerequisite pre- and posttest counseling previously which may have contributed to greater awareness of their own risk factors .
this may account for the clients ' ability to rate their perceived risk fairly accurately .
nevertheless , clients who did not rate themselves to be at high risk of hiv infection also reported considerable risk behaviors with similar proportions .
the only exception was that of inconsistent condom use during anal sex with 91.4% of clients in the high hiv risk group compared to 67.9% , 75.2% , and 79% in the low , medium , and unsure risk groups , respectively ( table 2 ) .
surprisingly , history of sti was not a significant predictor for hiv transmission in any of the risk perception groups in our study .
we believe this may be due to the structure and the anonymous nature of the questionnaire used in our study where detailed information regarding the types of sti and therapy received could not be readily obtained nor verified .
most clients were disinclined to reveal more information regarding the sti which they have had .
similarly , drug use is a legal offense in malaysia and is punishable by law with either prolonged incarceration or capital punishment .
this may have led to the reluctance of the clients to honestly disclose history of drug use before or during sexual intercourse making it difficult to interpret the available data meaningfully .
we recognized several major themes that have emerged from the findings in our study , namely , ( 1 ) msm in this study were able to accurately rate their perceived risk of hiv infection which suggests they were aware of their individual risk factors ; ( 2 ) the heightened awareness of the risk factors may have been contributed in part by previous counseling sessions these clients have received ; ( 3 ) msm in the high hiv risk perception group were associated with significant risk predictors of hiv transmission and were at much higher odds testing hiv positive compared to clients in the low risk category ; and ( 4 ) despite rating themselves into lower risk groups , the msm in these groups reported considerable risk behaviors suggesting the problem of misperceptions does exist to a certain degree in this study population .
this study has limited generalizability because it was confined to msm who utilized the vct services in one center and , therefore , may not be applicable to msm who do not use vct services .
nevertheless , in the setting of limited funding and resources , the findings in this study suggest that focused strategies to educate msm regarding hiv and sti risks , especially in the high hiv risk perception group and efforts to raise personal risk perceptions , are critical because self - perception often guides the individual 's testing and preventive behaviors .
because some of the clients who reported lower risk perceptions in this study were found to be hiv - infected ; consistent condom use during sexual intercourse regardless of the self - perceived risk with or without other risk factors should be the primary message to msm during every vct encounter . in conclusion , msm in this study
perceived high hiv risk was associated with many of the established risk factors for transmission of hiv and sti .
strategies designed to change the sexual practices among those who perceived themselves to be at high risk of being infected with hiv should be put in place in a limited - resource setting . | we describe the hiv risk perception , sexual behavior , and hiv prevalence among 423 men - who - have - sex - with - men ( msm ) clients who received voluntary counseling and testing ( vct ) services at a community - based center in kuala lumpur , malaysia .
the mean age was 29 years old .
one hundred one ( 23.9% ) clients rated themselves as low risk , 118 ( 27.9% ) as medium risk , 36 ( 8.5% ) as high risk , and 168 ( 39.7% ) were unsure of their risk .
twenty - four ( 9.4% ) clients tested hiv positive ( 4 ( 4% ) low risk , 9 ( 7.6% ) medium risk , 11 ( 30.6% ) high risk , and 13 ( 7.7% ) unsure risk ) .
we found a positive correlation between risk perception and hiv infection in this study .
clients with high hiv risk perception have 17x the odds of testing hiv positive compared to low risk clients .
high hiv risk perception was significantly associated with multiple sex partners , multiple types of sex partners , alcohol use before intercourse , unprotected sex beyond 6 months , and inconsistent condom use during anal sex compared to low risk clients .
there were no statistically significant differences between medium risk and unsure risk clients compared to low risk clients .
strategies should be targeted towards change in sexual practices among those who are perceived to be at high risk . |
reflecting in 2008 on aids in south africa during the final phase of the anti - apartheid
struggle and the transition to democracy , edwin cameron , aids activist and justice of the
constitutional court , observed that the transition period brought to the fore the intricate
relationships that existed between illnesses , ideologies and ( intimate ) identities ,
resulting , in the unavoidable politicisation of homosexuality.
indeed , as the oral testimonies in this article reveal , this politicisation of
homosexuality often affected both public and private spheres , and , for two health care
professionals on whose narratives this article focuses , their own sexual identity and
personal and professional relationships with ( gay ) men dying of aids mediated the urgency of
responding to aids .
in july 1983 an article by five medical professionals reporting on the first officially
documented aids cases in south africa appeared in the south african medical
journal .
while the two cases were not named in the journal article
the initial responses by the general public
to the first documented cases were primarily characterised by fear and stigma and reflected
prejudice , homophobia and moral panic that were deeply rooted in the dominant conservative
morality that pervaded south africa in the 1980s .
white , flight stewards , not
as two people who had died and would be mourned .
ralph kretzen died on 26 august 1982 and pieter danil ( charles ) steyn died on 1
january 1983 , and we know very little about their personal histories .
we do however know that the association of the new syndrome with an undefined idea of
homosexuality replicated the initial link between homosexuality and aids that had been
made by the centers for disease control in atlanta , usa ( cdc ) in june 1981 .
white
homosexual men by elites both medical professionals guiding and directing early aids
research and advising government departments and ministers ; and people or organisations in
positions of political power including government health departments and ministers , mps and
political parties shaped and influenced responses , research , and narratives about aids in
these and other spheres .
the resultant moral outrage expressed in parliament , from pulpits and in
conservative media provoked by this initial association did not allow for reflective
discussion about sex and sexuality , but rather positioned homosexuality and
homosexuals
negatively in broader public spheres and , in so doing , politicised homosexuality .
moreover , authors of the hegemonic scientific narratives whether in medical journals ,
health care organisations or parliament often used the term
homosexuality only to refer to
undefined sexual contact between white men in a narrative which was preoccupied with an
imagined middle - class ,
white , male , sexuality that was the primary referent against which
normative and non - normative behaviours were judged .
drawing on narratives about aids primarily from the uk and the usa , influential
medical practitioners and scientists , and politicians responsible for addressing aids in
south africa focused attention on this understanding of homosexuality and in doing so
rendered invisible heterosexuals , lesbians , bisexuals , transgendered and intersexed people ,
and linked male homosexuality only to whiteness .
in the 1980s , being gay in south africa was a process mediated by apartheid legislation and
questions of morality ,
race , sex , gender and class amplified by the socio - political and
economic turmoil of the time .
the oppressive , conservative , moral climate of south africa did not allow sexuality
organisations or lgbti
communities to flourish , especially not in a context where ( male )
homosexuality or more specifically , sodomy was illegal .
while gay organisations and communities in the usa and uk had fought for , and built
on , social changes in the 1960s and 1970s , in south africa apartheid s effect on sexuality
politics meant that corresponding socio - political gains had not been made . despite this
,
some gay activists maintained international contacts with gay organisations and aids
education organisations , resulting in a flow of information and exchange of ideas between
south africa and countries with more established and politically active gay communities .
gay groups in south africa were not necessarily concerned with addressing sexism , racism ,
homophobia or sexuality politics .
agendas ranged from providing safe social spaces to gather
and talk , directing legal challenges to homophobic legislation and laws that discriminated
against people because of their hiv status , to aligning the struggle for sexuality rights
within broader anti - apartheid and international human rights campaigns . as mark gevisser and
edwin cameron noted in one of the earliest and most influential works on the histories of
sexualities in south africa the two factors that exemplify homosexual experiences in south
africa are the history of division and resistance and the demographic divergence our
country reflects.
they note that from the developed world , we inherit notions of sexual freedom and
gay subculture ; from the developing world we gain the imperatives of struggle , resistance ,
and social transformation.
importantly , they add , there is no single , essential
what
has passed for the gay experience has often been that of white , middle - class urban
men.
at the outset it was predominantly communities of urban , middle - class ,
white men
with multiple gay identities who came together to organise around aids and it is this
this article is drawn from a larger thesis project which explored how narratives about aids
were constructed by elites in medico - scientific and political communities , and examined
practical responses to aids by governments , the african national congress ( anc ) liberation
movement in exile and progressive health movements .
the thesis revealed the complex relationships between these communities , tracked the
changing hegemonic aids narratives , and revealed the constructions of morality ,
it also showed how the relationships and narratives
shaped and influenced practical responses to aids .
most of the sources used for this project
were written sources which occasionally provided glimpses of the effects of narratives and
practice on personal experiences , but none provided insights into the specific experiences
of gay health care professionals . the relationships ( nationally and internationally ) between
medico - scientific and political elites could be drawn out by combining written and oral
sources , but the personal accounts of people at the nexus of the polemic and politics that
combined to influence practical responses were hidden . while accessible published work and archived sources provide insights into the experiences
of progressive figures and sexuality activists , the stories of gay health care professionals
living and working in a predominantly homophobic society and medical community at the start
of an epidemic linked explicitly to white gay men while important , were not readily
available .
of the twenty oral interviews undertaken with medical professionals , political
figures , activists and academics for the thesis , only two were with health care
professionals who had openly declared their sexual orientation during the time period under
discussion .
their stories offered unique perspectives and a way to record the effects of
politics and polemics on people s lived experiences .
the article uses oral interviews to examine the experiences of two gay health care
professionals addressing aids during the early years of the epidemic .
it follows the example
set by gerald oppenheimer and ronald bayer of recording the experiences of physicians and
nurses in their book in shattered dreams : an oral history of the south african aids
epidemic .
their book incorporates interviews with gay doctors , including dennis
sifris and steve miller , and , in recording their oral histories , begins to address how some
gay health care professionals experienced the epidemic .
this article expands on the oral testimony of sifris that appears in
shattered dreams and contributes the oral testimony of pierre
brouard .
the face - to - face interviews with sifris and brouard were carried out in single sessions
lasting from one and a half to two hours .
they were recorded in 2008 using a digital voice
recorder and verbal permission was sought and obtained from interviewees to record the
interviews . while specific questions relevant to the professional experiences of each
interviewee were devised , all interviews were predominantly open - ended and unstructured
.
during the interview notes were taken by the author , and the interviews were later
transcribed .
the purpose of the interviews and the nature of the research were also
explained to each interviewee and they confirmed the accuracy and representation of their
statements in the original chapter from which this article is drawn .
sifris is a medical doctor who worked with one of the first south african medical
researchers investigating aids , ruben sher , and helped to establish the aids clinic at the
large public and academic johannesburg general hospital ( also widely known as
joburg gen )
and has been involved in aids education campaigns since the early 1980s .
a clinical psychologist
, brouard was involved in some of the earliest counselling
and support initiatives for people who were hiv - positive at a variety of venues , including
the clinic at joburg gen , at atic ( the first aids training and information centre ) that
opened at the south african institute for medical research ( saimr ) in 1987 , and at the city
of johannesburg s esselen street sexually transmitted infection ( sti ) clinic in
hillbrow .
both sifris and brouard moved between and through the spheres of medical science ,
activism and sexual identity while attempting to address aids . while sifris and brouard are
both professional ,
white , middle - class , urban , gay men , whose paths overlapped and who
shared some similarities in their experiences of the early days of the aids epidemic , their
professions , personalities and individual contexts resulted in different experiences .
these early experiences laid the foundations for two men who , now both in their
fifties , have spent almost three decades of their professional lives involved in aids health
care and education .
their personal reflections provide vignettes that contribute to understanding how
narratives about , and responses to aids ( and sexual orientation ) , affected individuals .
their stories provide insight into personal experiences of addressing aids in the early
years of the epidemic when researchers and decision - makers were constructing a hegemonic
aids narrative that was fundamentally conservative and homophobic .
their stories are
micro - narratives that act as a counterpoint to the meta - narratives about aids featured
elsewhere and act as a reminder of the emotional and psychological realities of an epidemic
that continues to take lives , thus contrasting the unemotional writing about aids in
medical journals , the politically charged language in parliament or the official
correspondence of political organisations .
the article presents first sifris and then brouard s recollections of the early years of
the epidemic and some of their reflections on sexuality , health care provision and aids
after almost three decades as health care practitioners .
sifris and sher began working together in the late 1970s after sher had heard that sifris
had a large gay practice and asked if he would collect serum samples for a hepatitis b
study .
after the first official aids death in south africa in 1982 they met to discuss
further collaboration .
sher was based at the saimr and sifris had his own practice in the
city centre . while these collaborations would result in a long - term professional
relationship , sifris s experience as a physician involved in aids research
was mediated by
how colleagues and government officials responded to his sexual orientation as a gay man .
soon after the first recognised aids deaths sifris sought to address aids among the gay
community and contacted other gay doctors ( an anaesthetist , a psychiatrist , and a
neurosurgeon ) to discuss what could be done .
he recalled that , as a starting point in 1983 , they put the word out in the gay
community that there was this disease which nobody really knew about , while calling for
volunteers to give blood samples to be sent to sher at the saimr .
sher and sifris compiled a questionnaire that followed research in the usa and uk
and included questions about the number of sexual partners volunteers had , their medical
history and current state of health , and their use of recreational drugs .
to facilitate the work sifris extended his practice hours to include saturday
mornings : word got out and we actually got about 700800 people coming to my office over a period of
about four or five months and we pulled the bloods and we did nt quite know what we were
looking for .
we did a full blood count , we did an std [ sexually transmitted disease ] profile
and we stored bloods to see if there was anything we could find .
sifris kept a list of names that linked volunteers the majority of whom came from south
africa but also from countries such as lesotho , swaziland , malawi , zambia and zaire to
their samples , but there was little that could be done medically to assist the increasing
number of people who in the mid-1980s started to become sick and die .
the lack of an
effective medical response did not necessarily make the medical community in johannesburg
more interested in learning about other , non - medical , engagements and responses , however , as
is evident from sifris s account of a visit to johannesburg by glen margo in 1985 .
margo , who was originally from south africa , was involved in aids education in san
francisco .
he contacted the gay association of south africa ( gasa ) as he wanted to deliver
talks about aids in johannesburg .
sifris , a gasa member , agreed to arrange the lectures for
margo and organised one at the department of medicine at joburg gen .
sifris recalls : there
were all these cardiologists and respiratory physicians and this hippy san francisco guy
came along with handmade sandals and ringlets in his hair and told them about this funny
[ sic ] disease that was affecting gay men that they should all be aware of
and he was almost laughed off as a joke .
it was a wee bit embarrassing but that was the
context of the medical community .
it is unsurprising that medical elites in johannesburg did not pay serious attention to a
hippy social worker talking about something that apparently only affected
gays. however ,
as sifris was to discover , even being a medical doctor defined as
white and
male , did not guarantee an attentive audience if one did not also
despite the apparent lack of interest , sifris and sher persuaded hospital authorities to
allow them to establish an aids clinic at joburg gen in 1983 . by 1985
sifris was managing
the daily running of the clinic and sher moved between the clinic and the saimr .
they
decided to bring specialists from various fields , including respiratory illnesses and
dermatology , into the clinic . despite the dominant medical and popular association of aids
with gay men
, sifris recalls that the first person they saw as an outpatient was an
hiv - positive ( then htlv - iii positive ) womyn : she came in and we sat there , eight of us
doctors this poor woman came in , i still remember her sitting there
and i think she was
intimidated by all these doctors sitting there in their white coats .
during the mid-1980s
there was an increase in the number of patients attending the clinic
and being admitted to the hospital with aids - related complex .
the
guys we had seen two or three years ago , who were friends of mine .
a lot of them were saa
( south african airways ) people who were now getting sick and dying with this disease , and
this was the time in the hospital where they were doing barrier nursing.
in 1985 the advisory group on aids ( aga ) was established by the department of health to
advise the conservative and ostensibly christian government led by the nationalist party on
aids policy and sher was invited to become a member .
however , a medical background , an
interest in aids , and experience researching and monitoring the epidemic did not guarantee
membership of the group , as sifris discovered .
high - risk group and if we have a gay man on the ( aga ) we
have to have a prostitute , we have to have a haitian , and we have to have a black .
this decision made sifris determined to just go ahead and do my own thing so he called on
medical professionals such as steve miller , des martin and clive evian ( all of whom would
become key aids researchers ) to work with him at joburg gen ; and , in conjunction with gasa ,
established an alternative aids group .
sifris and other concerned professionals and
volunteers formed the aids action group in 1985 , with the intention of providing
psychological , social and concrete support services for people with aids and the
aids - related complex.
the group shared information about aids with interested parties and undertook
education campaigns amongst sections of johannesburg s gay community . on trips to the usa and uk , sifris gathered literature and came home with bundles of
it.
the aids action group decided to distribute information leaflets and safe - sex cards
in gay clubs in johannesburg and approached club owners for support . in one club
the group
set up a little ( information ) thing outside and people walked past and we said , hey , we
are giving out safe sex cards , and everyone kept away from us!.
on one occasion sifris asked if the music in the club could be stopped at midnight
so that he could make an announcement : so they stopped the music and i came out onto the
dance floor with a microphone and i said ,
listen this is not a police raid or anything ,
just relax , nothing is happening here , i just want to make everybody aware that there is a
problem called htlv - iii which is affecting gays in america and all over the world and
everyone just moved away from me .
i stood there in the middle of the dance floor trying to
say , now listen people it is up to you to volunteer , to create awareness , to get the people
aware , to give out pamphlets and please we have a desk in front , please come and sign .
there was no interest , just apathy like we were ruining the
party .
the pre - emptive declaration in sifris s statement that his announcement was not part of a
police raid speaks to the constant stress , suspicion and fear of arrest that was associated
with being gay in south africa .
the majority of people in south africa faced the constant
threat of police and military violence by virtue of being declared
black in the apartheid
state , and whiteness usually afforded privilege and protection to people declared
white gay men maintained significant privilege in most socio - economic and
political spheres , they did not maintain sufficient privilege to remain exempt from raids
because of their sexual orientation .
black gay men maintained some privileges
by virtue of being male in a patriarchal system , but faced persecution for being both
black and gay .
the unwillingness of people to listen or respond to a potential health concern mirrors
responses in most communities when faced with frightening news .
it is not possible to determine how effective these interventions ultimately were ,
nor how much impact the distributed literature may have had on awareness raising .
nonetheless , it is significant that at a time when official responses to aids failed to
cater for a group of people identified and constructed as
high - risk , alternative ,
non - official efforts were made to raise awareness and provide support .
the group decided to focus their energy on talks , engaging with the press and fundraising
for aids - related work .
sifris and sher were already giving talks for audiences ranging from
medical professionals to staff and students at schools , and to a group of jewish single
people over the age of 40. sifris gave talks to gay groups around the country and spoke at
the official opening of anti - apartheid activist simon nkoli s township aids project ( tap ) in
soweto in 1990 .
sifris both received calls from journalists seeking his expert medical opinion , and
challenged journalists and editors about homophobic or inaccurate content in articles .
he
felt frustrated that many journalists were more concerned with writing sensationalist
articles than with conveying educational information .
these challenges extended to his
engagements with the editor of exit the longest running gay magazine in
south africa .
exit ( originally known as link / skakel )
started in the early 1980s as gasa s newsletter .
it was renamed exit in
1985 and published its 200th edition in 2006 when it cast a reflective eye on some aspects
of its reporting on aids .
sifris criticised moolman for running a sensationalist article about a person
apparently getting aids from their dentist , rather than featuring comprehensive aids
education information .
botha reflected on the early years of exit : during the 1980s a new
gay disease was discovered in america and eventually found its way here . although we
started publishing safe sex guides and lists of symptoms , very little
was known about this
mysterious killer affecting at that stage mostly gay men .
i am still haunted by the front - page headline that said something to
the effect aids [ sic ] scare overrated .
an exclusive black - tie event and art auction launched the aids action group s fundraising
initiative .
the event
raised close to r10,000 for what was referred to as the aids action fund. another
shaft 8 included information and education stalls and provided an
opportunity to remember those who had died .
i
bought those jewish yahrzeit candles , and i laid out a table with one
candle for each person who had died of aids .
the shaft 8 event raised a significant amount of money and brought all the gay communities
together a gay christian community , a support group called outreach , a gay motorcycle
group , and gay lesbians. while aids may have connected these communities , it hardly united
them . sifris noted that there was all sorts of internal politics and stress in the
organisation .
black gays or lesbians and often a lack of
gender sensitivity or awareness of racial prejudice .
aids may have created spaces for
collaboration and co - operation , but these spaces were not necessarily all - inclusive or
self - reflexive ; and addressing aids amongst a certain group of gay men who experienced
discrimination did not amount to challenging the economic , social or racial position of that
group of men , nor other forms of discrimination . in order to increase fundraising opportunities and more effectively manage funds , the group
needed an official fundraising number .
it prepared a proposal outlining its plans to print pamphlets and undertake a
countrywide outreach programme and had it endorsed by medical professionals , a professional
in the finance sector , and people in the marketing and advertising sectors .
the group
presented the proposal to a representative from the department of health and asked for a
grant of r20,000 .
however , because the organisations associated with the project were
sexuality organisations , the fundraising number and the grant request were denied .
lombard apparently listened carefully to the
presentation and then said , you know , aids is not a problem in this country , tb is a
problem in the country .
besides which homosexuality is illegal , and we do nt have
homosexuals , so we do nt have aids.
the department of health s rejection of the funding request meant the group was unable to
initiate extensive aids education programmes based on those of the san francisco aids
foundation , the gay men s health crisis , london lighthouse , and the terence higgins trust ,
whose pamphlets and programmes were the template for their own local campaigns .
thus , the content , politics and practice of aids education programmes developed by ,
and for , male gay communities in the usa and uk , shaped and influenced responses amongst
some sectors of the male gay community in south africa . for sifris
the frustration at not
being able to implement ready - made education programmes that could save lives was
compounded by the denial of the very existence of homosexual men .
invisible in official apartheid discourse . had the grant had been successful , there is no guarantee that imported education campaigns
would have been appropriate or effective in the south african context .
however , in the
absence of official responses or help for gay communities , they would at least have
bolstered existing education efforts by gay organisations to prevent new infections .
access to medical scientists and research organisations in the uk and usa shaped
both sifris s and sher s professional engagement with aids , and contact with gay activists
in these countries deeply influenced sifris s personal and political outlook .
sifris remembered going with sher to a research laboratory in bethesda , maryland , and being
drag[ged ] into the halls of the walter reed hospital in washington or to some top - level
research conference.
in 1985 sifris went on ward rounds at the san francisco general hospital and at the
aids unit in london , experiences that really opened ( his ) eyes to the realities of the
epidemic , the strategies being employed to address aids , and the remarkable solidarity and
politicisation occurring around aids .
sifris had his most profound experiences at the points of intersection between personal ,
professional and political spheres .
in the mid-1980s he attended an aids conference in
washington where members of the aids coalition to unleash power ( act - up ) protested over the
lack of action in addressing aids .
the conference changed sifris s understanding of how
people could organise politically to challenge medical and discursive responses to aids :
larry kramer was there from act - up and he was wearing a concentration camp uniform . at the
time , and i thought this was very strange , he would shout and scream and carry on .
they had
a scientific session , and they had one or two sessions of people with aids and it was very
funny because people stood up and said ,
i am so and so , and i have got hiv and everybody
applauded , and everybody clapped for support
i remember
that to this day and i really learnt a hell of a lot [ particularly ] that there was a lot of
mobilisation .
during trips to london , new york and san francisco , sifris took part in aids memorials that
made a lasting impression on him : i was in castro street and they had it at castro s station
there , a candlelight memorial for rock hudson [ it was ] very moving .
they unfurled the quilt and there was nt a
dry eye in the house , everybody was crying .
contacts with gay organisations and ( gay ) doctors working on aids in the usa and uk were
important in providing models of engagement , education , commemoration and grieving . for
sifris and others like miller , who participated in conferences and marches overseas ,
these
were spaces that demonstrated the potential for public protest , and subsequently influenced
education and mobilisation efforts among sectors of the gay community in south africa .
on a trip to san francisco a group called
inspired by the experience of seeing doctors organise around
political issues relating to the realisation of rights , sifris decided to start a gay
doctors group in johannesburg that numbered about twenty clinicians and met monthly at the
hiv clinic .
brought together by an interest in hiv , the primary aim was just to keep
everybody up to date with hiv news , but eventually it just sort of fell to pieces because
nobody was interested in coming anymore because there was not much more we could do.
it was only in the late 1990s that sifris , des martin and others decided to
establish a new association , the southern african hiv clinicians association .
this was open
to all clinicians involved in hiv work and included a number of gay doctors .
martin was
appointed the first chair of the organisation and the first meetings were held at the
national institute of virology . from small beginnings
the organisation continued to grow and
recently claimed to be the largest professional hiv interest group in the world , with over
12,500 members.
the mid-1980s were particularly turbulent times in south africa , both for anti - apartheid
and sexuality activists . in 1986
gasa collapsed , but its demise , gevisser notes , resulted in
two new forms of gay political activity. one found the notion of single - issue gay
politics to be untenable in south africa and sought to align sexuality struggles with
broader human rights and anti - apartheid struggles ; the second insisted on fighting
specifically for the reform of laws that discriminated against homosexuals.
in 1985 president p.w .
interracial ( hetero)sex which had been made
illegal by the immorality act , 1927 ( amended in 1950 ) .
the immorality act also covered other
sexual offences such as prostitution and sodomy between men and botha tasked an ad hoc
committee with investigating these sexual offences , including homosexuality .
this committee
proposed : a strengthening of legal prohibitions against homosexuals ; that greater effort
should be made to determine how to appropriately rehabilitate or punish homosexuals ; and
that a strategy should be formulated to express society s
the collapse of gasa and the suggested law reforms had a direct impact on the aids action
group and on organisations that had come together to raise money for aids programmes .
key
gay activists established a national law reform fund ( nlrf ) to fund challenges to the
proposed legislation .
the organisers of shaft 8 were asked to consider reallocating the
money they had raised to support the nlrf .
numerous meetings ensued and sifris recalls that
it was decided among the committee that the law reform fund was now more of an emergency
than aids , so all the money was given over to the law reform committee .
we thought we could
always just raise more money for aids.
determining which threat aids or the further criminalisation of homosexuality
was the more pressing could not have been an easy task , but the historical coincidence of
the two issues coming into the spotlight at the same time detracted from plans to address
aids .
as it turned out , the investigations into homosexuality and law reform never got
underway and the nlrf was transformed into a charitable trust .
the late 1980s were also difficult times for sifris and others working at the hiv clinic at
joburg gen , primarily because of the inherent homophobia and stigma associated with aids .
although sifris and sher had convinced hospital authorities to provide space for the hiv
clinic it was not easily accessible to clients or visible , and both the name of the clinic ,
and who attended it , became contentious points .
immune disorders clinic but sifris was adamant
that after the identification and naming of the hi virus it was important for the clinic to
be known as the hiv clinic to reduce the secrecy and fear associated with hiv .
after
several years of running the clinic on an honorary basis , sifris was formally employed as
its director in 1985 .
despite vocal opposition , one of the first things sifris did as director was to
rename the clinic the hiv clinic. it retained that name until sifris s resignation in
1990 , when it was renamed the immunity clinic.
the hegemonic aids narrative of the time constructed gay men with aids as
guilty
offenders who got aids because of their sexual orientation and deviant lifestyles.
conversely , haemophiliacs infected through contaminated blood products were
constructed as
innocent victims. so strong was the stigma and homophobia that a separate
clinic was established for haemophiliacs and others .
sifris recalled the haemophiliacs had
their own clinic because they did nt want to come to the gay clinic
they did nt want to
come to the hiv clinic , so they had their own clinic .
we never had anything to do with
them they did nt want anything to do with us because we were gays .
things came to a head for sifris and miller because of a devastating interplay between
discrimination , drug pricing and public health care provision . in 1986 one of the first
antiretroviral ( arv ) drugs , azido - thymidine ( azt )
also known as zidovudine , came onto the
market in the usa . as the first significant medical intervention for aids it was a
breakthrough , but it was staggeringly expensive , costing approximately $ 10,000 a year in the
us at the time .
the number of south africans who could afford it when it was first distributed to
the country in 1987 was minimal .
while paying for the drug was completely impossible for
most patients attending the hiv clinic , it did at least provide hope because it prolonged
the life of aids patients .
the hospital eventually purchased azt but only wanted to
prescribe it to people with haemophilia .
the hospital said we will give azt
to the haemophiliacs but we wo nt give it to the gay people because it is their own fault
and haemophiliacs are innocent victims.
livid at this blatant discrimination , sifris contacted reporters and informed them of the
hospital s policy , resulting in the story being featured in newspapers .
shortly afterwards the superintendent of the hospital informed sifris that they had
been called to a meeting with the secretary of health in pretoria . at this meeting sifris
was reminded that as a member of staff of a public hospital he was not allowed to make
statements to the media about hospital policy , and was duly admonished by the health
secretary .
i do nt
want anything more to do with the hospital.
after sifris s resignation miller took over as head of the clinic , but was not in
that position for long as he was told to leave by hospital authorities after supporting a
patient s legal claim against the hospital .
a final example of the ongoing frustrations experienced by miller and sifris related again
to the aga .
miller , like sifris , had been excluded from joining the group because of his
sexual orientation and his designation as a member of a
high - risk group. nonetheless
sifris made a second attempt to join the group when it decided to expand its membership .
frustrated that the government had sewed together a bunch of old fogies who did nt do
anything , sifris thought that membership of the group might afford him the chance to
facilitate change or action . in his application sifris argued that as head of the largest
hiv clinic in the country he had considerable experience in addressing aids , which was
augmented by the lectures he had given , the travelling he had done both regionally and
internationally in connection with aids work , and the conferences he had attended .
own coffee cup in case you are worried that a gay man is
going to infect you.
sifris s application was rejected , and the group appointed someone who had never
seen an hiv patient in his life.
while sifris has never explicitly commented in interviews on the racial dynamics within
gasa or any of the aids - related organisations that he was involved in , he did discuss the
problems he and sher faced at the clinic in the late 1980s getting black doctors
involved.
they organised a world aids day event and wanted to invite
black doctors to attend
but were unsuccessful : we searched and searched and searched and searched and could nt find
any black doctors to come out. they approached a prominent black doctor in soweto with known links to the anc but were
turned down .
black doctors were nt interested because it was not a
black problem , it was a gay disease .
it was a gay disease and nobody worried about the
gays.
because aids was not being identified among
black patients , and places like the
hiv clinic were either not legally integrated or welcoming of black patients , it was not
seen as important amongst
black doctors may not have wanted to participate in the events , not
least of which were the other health concerns exacerbated by apartheid which at the time
were regarded as being more pressing . in the early days of the epidemic ,
sifris recounts , it was difficult coping with the
deaths , government and community inaction , and general homophobia .
burnout. added to the stresses and strains of battling
government and hospital officials , he experienced a personal loss that caused him to
withdraw from professional and public spheres .
sifris met an hiv - positive man at an aids
conference in stockholm and the two fell in love and lived together for five years .
as his
partner s health began deteriorating , the couple travelled to sweden for treatment twice a
year .
after his partner suffered a mini - stroke that left him paralysed , the couple decided
that he needed to return to sweden to see his doctors and be with his family . as he recalled
some twenty years later , while they were in transit at brussels airport , sifris s partner
suffered a major stroke and a massive brain haemorrhage : as we were getting onto the plane
he collapsed and died
so , i sort of withdrew at that time a little bit , but ( since then )
i ve slowly gotten back.
in 2013 , sifris has semi - retired but continues to deliver lectures on aids , acts as a
medical consultant to a disease management company , and contributes health information to
exit and other media fora .
having seen so many people die of aids over the course of three decades , sifris
expressed disappointment in the interview that successive post-1994 democratically - elected
south african governments have put up so many barriers to procuring and distributing
life - saving drugs .
his recollections reveal the inherent and blatant homophobia that gay men experienced ,
regardless of their race or class .
frustration at official inaction around aids and the
reality of friends , colleagues and lovers dying , provided the impetus for sifris and others
to mobilise in the early 1980s , soon after the seriousness of the epidemic was becoming
apparent to them .
the importance of international connections and experiences is evident in
sifris s personal politicisation around aids . as a doctor involved in the first hiv clinic
in south africa , his professional status and resources allowed him to travel to
international conferences and connect with other doctors and with gay organisations
addressing aids , and to bring important information and ideas back to sectors of the gay
community in south africa .
while sifris and brouard were both involved in addressing aids as health care
practitioners , they occupied different positions in the health care facilities they worked
in , and their experiences correspond and contrast each other , as will be shown below .
pierre brouard is a clinical psychologist and currently the deputy director of the centre
for the study of aids at the university of pretoria .
brouard s recollections on the early
days of the aids epidemic are noticeable for their reflexive and contemplative nature .
his
discussions about the epidemic , and his involvement in various counselling and support
positions , are imbued with a critical awareness of the broader socio - political landscape of
south africa during the 1980s and early 1990s .
brouard came out in the late 1970s and believes that coming out before aids was on the
scene shaped his sexual identity in a different way to people who were coming out during
the epidemic and helped form his perspective on the fears and considerations that framed
peoples sexual lives . reflecting on the late 1970s , brouard view was that one of the key issues that concerned
many ( white ) gay men at the time was that homosexuality was illegal . under the immorality
act and subsequent amendments , there was the continued threat of being arrested , harassed ,
hounded , shamed or imprisoned .
legal concerns aside , from a personal physical health
perspective the most immediate health concerns were stis such as herpes , syphilis or
gonorrhoea , all of which were treatable .
while fear of the law remained , in the early 1980s
physical health priorities were to be radically altered as the
first murmurings that
something was amiss started to surface with news often coming from middle class , white ,
gay men who travelled internationally , and from people working in the airline
industry .
new disease that was framed entirely as a
gay disease , complete with the acronym grid ( gay - related immune deficiency ) .
central to both brouard s and sifris s narratives , is the importance of international
travel and networks for transmitting information about aids .
brouard remembers that people
who travelled internationally brought back newspapers , pamphlets and magazines or knew or
corresponded with people overseas.
in the early 1980s brouard and his partner at the time noticed that one of their
friends , who had moved to the uk but who regularly visited south africa , was getting
increasingly ill .
they speculated as to whether their friend had aids , but he never told
them and they never asked .
the friend subsequently died , ensuring that from early on in the
epidemic , brouard was acquainted with the reality of aids - related deaths .
brouard saw that aids - related illness and death mobilised people both as individuals and as
members of organisations : those early responses were really located in the gay community .
people mobilised around friends , tried to get support groups going tried to understand the
treatment options available , and set up informal networks .
the latter was
the director of gasa-6010 , was hiv - positive and died of aids - related illnesses .
brouard commented on different political outlooks between organisations : gasa was
the main
organisation for more middle - class , mainstream , men and women and was
apolitical.
he recalls that gay organisations that were more left - orientated appeared to be
more active in cape town , than in johannesburg .
regardless of geographic location however , brouard s retrospective sense of the situation
was that
middle of the road , white , gay men were really just trying to hold it all together
in the absence of really much available [ sic].
reflecting on the arrival of a serious new health concern brouard suggested that
aside from the emotional impact of the deaths of friends , family members , lovers and
partners , the association of aids with homosexuality required people to address their
sexuality without internalising aids as something that was inherently related to being gay .
at the same time as challenging ideas that being gay was wrong , evil or sick ,
gay men were
faced with a health crisis that was portrayed as being intrinsically linked to what was
apparently
gay sexuality. gay men thus had to address both internalised homophobia and
societal perceptions of homosexuality being evil or sick , in the context of a new fatal
disease that many medical professionals , politicians , media and religious groups directly
linked to sexuality .
the ramifications of aids affected both individual and community identity formation ,
as brouard recalls : just to try and retain a sense of healthy sexuality and healthy
identities when aids spoils your identity and spoils your sexuality that was a real
challenge of those times .
i remember having feelings like the community is
self - destructing , there s just so much stuff going on , and people just are nt caring for
themselves or each other .
it was an incredibly distressing and painful time to live through and people were just
getting sick and aids struck the old and the young , and the beautiful and the ugly , the
fat and the thin it was a great equaliser .
my sense of the time , perhaps it was a
psychological sense , was that some people who were the beautiful glittering stars of the gay
firmament , were deeply shamed and humiliated and distressed about being hiv positive because
it reduced them to their physicality and was ultimately a reminder that we are physical
creatures and we get ill and we die and nobody is immune from that no matter how beautiful
or glamorous you or your lifestyle are .
it was really an incredibly fraught time to be gay and i do nt know that , historically , if
we look back , how that whole time affected the psyche of the gay community .
to what extent
it damaged it , or created a particular response or a feeling of what it means to be gay in
the twentieth century .
research into how aids , the illegality of homosexuality , socially - sanctioned homophobia ,
apartheid ideologies and race shaped gay male identity in south africa historically is
another area of research that requires further exploration .
the hegemonic narratives around men and aids created by medico - scientific and
political elites in south africa and elsewhere in the early 1980s and not challenged
significantly until the mid-1990s strongly suggested that it was not
types of sexual
practices such as unprotected anal or oral sex , or higher - risk behaviours that needed to be
examined , but rather something
the focus in
medical journals and parliamentary discussions , throughout the 1980s and mid - way into the
1990s , was thus not on sexual acts and practices that could be performed by sexually active
people regardless of their sexual orientation , but rather on constructed aids avatars of
deviant gays , dirty prostitutes , infected foreigners , and sexually rapacious
this prevented serious attention from political and medico - scientific elites falling
on male sexuality more broadly and assumed a normative , healthy ,
while these avatars were used to construct a hegemonic aids narrative , this does not
mean that the narratives were not contested , challenged and reconfigured or rejected by some
health care professionals such as brouard .
conflated identity with risk
and assumed that something about the gay condition ( or ) the gay being
presupposed
vulnerability to aids to hiv.
this fitted in with broader public health paradigms of the time that were modelled
on popular understandings of sexuality , that did not sufficiently interrogate the
relationships between sexual practice , sexual identity and basic biology . in this paradigm
it was not the fluid that was the problem , it was who you were that posed the risk.
this understanding of sexuality and confusion over sexual practice and sexual identity
resulted in a lot of mythologizing even in the medical sphere.
brouard recalled counselling a heterosexual couple in the esselen street clinic
where the man was concerned about their risk of hiv infection because they practised anal
sex .
further questioning revealed that they were both hiv - negative and in a committed
monogamous relationship .
brouard explained that , in light of this , they were not likely to
be at risk of hiv as it was not the practice of anal sex per se , that was
the primary risk , but rather bodily fluids containing the hi virus .
these and other
experiences left brouard with a sense that , for many people , understandings of aids and hiv
transmission , and sexuality more broadly , were very mystifying and mystified.
brouard started counselling people in the early to mid-1980s through his involvement with
gab ( gay advice bureau ) a counselling service providing support for gay people or people
exploring their sexuality . at that time gab was not dealing with many hiv - positive people
but one of brouard s colleagues was diagnosed with aids so gab established an informal
support system for him and his partner .
after his death , gab launched an unsuccessful
support group for people living with hiv . while completing his master s degree brouard volunteered at the aids centre at the saimr ,
which opened to the public in january 1988 .
it offered an evening counselling and testing clinic where brouard did pre- and
post - test counselling with the predominantly white , gay clients .
he and a friend also
established an hiv support group attended by a small number of gay men , which allowed
brouard and his friend to explore what it means to be positive , and what support one
needed , and what was out there.
during his internship year as a master s student in 1989 , brouard volunteered at the hiv
clinic at joburg gen doing informal counselling and support work , and acting as a resource
person. he recalls that people like ruben sher , dennis sifris and steve miller ran the
clinic and it was a fascinating period because it was very
other.
while the hospital was still racially segregated and primarily served the needs of
other because of their sexual
orientation or because of the demands that they made on the hospital on behalf of gay
people :
suddenly this group of gay men who were feeling more entitled in some ways , or more
informed , or more empowered , were coming in and it created some envy and resentment among
the medical fraternity because why was there a need for a special clinic ?
aids was still
very much a disease of gay men , it was still stigmatised , and dennis , ruben and steve had to
really fight to get staff , to get funds .
they had constant battles with hospital
management .
in the context of apartheid ideology that sought to categorise people in unambiguous ways
and thereby create specific , often binary , identities ,
white gay men were problems as
they not only transgressed accepted heteronormative identities , but were also criminalised .
they were men and so were expected to occupy a particular place in patriarchal structures ,
and they were
race. they were ,
however , gay men and therefore their privileged status in terms of masculinities could be
called into question .
instead of being shamed into silence by their sexual orientation
, these men took
advantage of the social capital they had as white elite medical professionals , and
challenged hospital authorities .
unsurprisingly they antagonised others in positions of
privilege both within , and beyond , the health sector .
brouard recalled how the accessibility of arvs at joburg gen highlighted the class dynamics
inherent in a health care system divided into public and private sectors .
he felt
ineffectual at the clinic because it seemed to be an overwhelming problem and you just saw
so much death and dying , and just sadness and grief. sifris and miller saw patients who
could afford arvs at their private practices , but there was no hope of this at the joburg
gen . by the early 1990s
both sifris and miller had left the clinic and worked predominantly
in their private practices .
brouard in the meantime , continued to volunteer at joburg gen
and got a job in hillbrow at the esselen street clinic with mary crewe and clive evian , who
were starting up the johannesburg city council aids programme . despite being state - funded , and having to negotiate the complexities of national and local
budgetary funding processes
, brouard maintains that he , evian , crewe and others pioneered
several innovative programmes relating to sexual health and stis at the esselen street
clinic .
brouard , for instance , started a closed support group along strict psychotherapeutic
lines for hiv - positive gay men which ran for three years .
with so little access to azt the primary focus of the group was on how to adapt to
living with hiv and preparing to die of aids .
brouard
remembers those years as his most significant immersion into counselling people who were
dying or confronting mortality.
brouard recounts that the clinic was not a specific aids or hiv clinic but rather provided
comprehensive sexual health services with an aids training and support component .
it had
family planning services , a tb clinic , an sti clinic that incorporated hiv antibody testing ,
and an outreach programme focused on sex workers and gay men .
brouard remembers the clinic
being used by all sectors of the hillbrow community and being told by clients that it
developed a reputation for meeting the needs of all people in the community and for being
gay friendly.
brouard knew many of the people who came to the clinic for help , and , despite minimal
resources , recalls that they never turned people away .
the clinic became a space where
community members or groups could collaborate and develop new aids intervention strategies
such as drama productions , comic strips and other innovative education materials .
aids weeks , and in 1991 piloted aids
education adverts on public buses .
brouard maintains that they also developed interesting training models and
counselling courses hailed as innovative by other health care centres , and provided
non - judgemental testing services and sti treatments .
in addition , brouard suggested the background and politics of the staff and a guiding
assumption that responses to stis required a multi - disciplinary rather than a solely
medico - scientific approach influenced the integrative outlook of the clinic .
brouard
referred to the interesting mix of people and the variety of skills that coalesced at the
clinic , with clive evian bringing a community medicine perspective while mary crewe applied
her academic knowledge to understanding the sociological roots of aids to provide a
foundation for implementing programmes at the clinic .
while sexuality organisations or gay professionals could exert pressure on policy makers in
the uk and usa , or influence the aids policy - making process itself , there was less
opportunity for this in south africa .
in the 1980s
the criminalisation of homosexuality and the government s homophobic
stance made engagements between sexuality activists and government officials complicated .
the political transition in the early 1990s created more opportunities for sexuality
activists to engage with decision - makers but the focus for sexuality activists was no longer
only on fighting legislation , or even raising awareness about aids , but rather on securing
citizenship rights and equality for people of all sexual orientations .
in the late 1980s and early 1990s the clinic built more alliances , both nationally and
locally with progressive organisations in the gay communities like the gay and lesbian
organisation of the witwatersrand ( glow ) and the tap .
alliances were also built between
progressive organisations involved in aids work ; and between gay organisations , activists ,
and the clinic .
simon nkoli was involved in glow and tap , and with brouard ran hiv prevention
workshops in hillbrow for
black , gay men based on sex - positive , risk reduction
approaches .
in addition to providing information and distributing condoms , they used the
workshops to explore relationships , sexuality and identities .
brouard and nkoli criticised
the abstain , be faithful , use condoms ( abc ) programmes and messages that were most
prominent at the time , concluding that gay communities would not be receptive to the
moralistic , conservative messages .
nkoli had an office in esselen street and started an
organisation for hiv - positive black , gay men .
brouard recalls how you would walk into his
office and there were all these explicit posters of naked black men
so he was
fantastically subversive.
nkoli , brouard and sifris , as well as organisations like the gay peoples health forum , and
gasa-6010/aset , were responsible for subverting the conservative , homophobic , moralistic
narratives about sex and sexuality by virtue not only of their sexual orientation and
politics , but also because of the safer sex material that they distributed or produced . in
stark contrast to the government s
abc messages , the aids education material developed by
gay organisations or aids support groups in the mid-1980s , was radically different in both
messaging and in assumptions about the intended audience .
as the examples pictured below
reveal , images and messages from gay organisations treated the audience as sexually active
and did not assume that abstinence was realistic .
instead , the images and words encouraged
safer sex practices as a normalised part of enjoyable , erotic , healthy , sexual activity , and
depicted sex across
colour lines. brouard was on an editorial board that produced a comic pamphlet called keep it hot , safe
and gay which featured two characters , hot shot and safe sex a lycra - clad super hero and
a condom who delivered unabashed information about anal sex , oral sex , thigh - sex and
mutual masturbation ( figure 1 ) .
information about hiv
infection and transmission was underscored by a central message that playing safe with the
one you love puts the fun back in and takes the danger out.
the comic provided contact information for the esselen street clinic and gay
organisations .
figure 1:panels from keep it hot safe and gay.
panels from keep it hot safe and gay.
safer sex cards from the johannesburg - based gay peoples health forum ( gphf ) were designed
as actual postcards with writing space , and included the organisation s contact information .
some of the cards showed a
white man and a black man in intimate poses and reminded
recipients to protect their partners and declared that healthy sex was not boring
( figure 2 ) .
the gay peoples health forum s safer sex postcards .
the significance of these media needs to be considered in light of homosexuality being
illegal , and
interracial hetero(sex ) only having been decriminalised in 1985 . producing
and distributing these education materials
was socially ( and legally ) subversive and the
content of their sexual health messages was radical by the standards of the day .
much like the keep it hot pamphlet and the gphf postcards , safer sex cards distributed by
aset in cape town combined basic facts about hiv transmission with contact information for
the organisation .
the cards also used frank language and ( for the time ) explicit images ,
such as a
the photographic credits
indicate that some images were reproduced from the terence higgins trust in london , while
others were created for the organisation .
in 1991 john pegge ,
the director of the organisation , confirmed the importance of
information exchanges between international organisations : we run a large safer sex campaign
amongst the gay [ sic ] minority and for this purpose we use safer sex
posters donated to us by gay aids service organisations in other countries .
we have a wide
selection from australia , new zealand , the united kingdom , the netherlands , canada and the
united states of america .
these images depicting a sexuality that was still criminalised flew in the face of south
africa s conservative pornography laws which organisations managed to circumvent .
the state
of transition in the country was such that aset was able to receive such material , despite
it running foul of pornography legislation , because authorities no longer appeared certain
of quite which legislation was applicable . when apartheid laws were being more rigorously
implemented ,
pornography received by mail did not result in criminal prosecution if the
recipient could prove that they had not solicited the material .
nonetheless , pegge urged
caution : i would suggest that you send any materials that might be deemed pornographic in a
separate envelope without a covering letter.
despite the caution urged by pegge , the socio - political milieu of the country seemed
to be changing . in the early to mid-1990s
, brouard witnessed a change in clinic clients . rather than the
initial client base of predominantly
black south africans returnees who had either lived in exile communities or
travelled to other countries .
one womyn that he counselled frequently had been involved with
a senior operative of the armed wing of the anc umkhonto wesizwe ( mk ) .
the
mk soldier attended counselling only once and later died of aids . yet , this time also saw a renewed sense of hope derived both from the country s political
transition to a constitutional democracy and from developments in arv treatments . at the
same time , progressive health organisations became increasingly involved in aids work and
approached the clinic to obtain training materials and discuss mobilising health workers .
othered in south africa it was the build
up to 94 and the whole nacosa ( national aids convention of south africa ) process had
generated an incredible amount of coming together and talking and debating a first national
( aids ) plan .
people were pulling together more it was a fascinating time to be living
through .
the space for gay health care professionals to influence national aids programmes and
policy throughout the 1980s and into the early 1990s was , brouard suggests , hampered by the
level of homophobia in public health structures . while gay men , like brouard himself , were
involved in the government - funded aids training and information centres
( atics ) , they worked
within a conventional public health paradigm ( doing ) testing , counselling training ,
community prevention , ( and ) fairly dull aids in the workplace stuff. within this
conventional paradigm , it was difficult for gay activists to bring about radical changes in
aids education messaging or campaigns , so their influence would have been primarily at the
individual level , with people they counselled , or , possibly , within the atic structures .
brouard maintains that even people such as ruben sher , as an aga member , would have had
limited opportunities to make substantive changes to national aids policies or programmes .
with aids ring - fenced as something
other and outside the public health care system , it was
seen as a challenge to existing health care systems and structures .
brouard recalls that
even at the clinic , which championed an integrated approach to sexual health , nursing and
other staff who were less involved in aids work saw the aids people as different.
brouard noted that the perception of aids as
when interviewed for this project , brouard remarked that he was struck by the irony that
state resources for aids programmes amongst lgbti communities were not available either
during or after apartheid .
brouard had met rina venter ( the last np national minister of
health ) in the early 1990s and he recalled that she had made it clear that because aids was
a
minority issue the gay community needed to look after itself. this attitude , combined
with the criminalisation of homosexuality , meant that state resources were not made
available to gay people .
similarly , in the post-1994 period , when the focus of government
interventions shifted to heterosexual people and pregnant womyn and people started claiming
their constitutionally - guaranteed rights to health care and treatment ( in this instance arv
therapy ) and demanding responses from the anc - led government , gay communities were again
sidelined as minority groups .
this continued marginalisation has resulted in a lack of basic
information about hiv transmission and infection rates amongst gay communities in
post - apartheid south africa .
of the early years of the epidemic brouard
primarily recollects death : there was a lot of
death that s what i remember of those years
the number of funerals one went to , the
number of people who disappeared , it was just horrible.
while in 2013 the funerals continue , the option for a longer life is now available
to people if regular and sustained access to arvs can be maintained by the state , and in
2013 south africa has what is possibly one of the largest arv rollout programmes in the
world .
brouard continued working at the clinic until 1997 and then joined the centre for the
study of aids ( csa ) in 1999 .
as the deputy director of the csa , brouard continues to
conceptualise and develop training methods and materials , and contributes to academic
publications and other fora on topics such as psychosocial issues , gender , sexuality , human
rights , testing and tertiary responses to hiv and aids .
brouard s work still involves the realities of aids - related deaths , but now also
includes components that address prolonging and living life fully , regardless of hiv
status .
personal and professional relationships and networks organised around sexual identity were
important to the first responses to aids in south africa in both medical and social spheres .
sexuality organisations had to take responsibility for addressing the aids epidemic in the
face of government inaction , but much of this response was limited to specific sections of
the gay community , individuals , and to specific geographic areas . while aids provided a point of mobilisation and support in south africa ,
as this article has
explored , sifris and brouard were active in both sexuality organisations and in two key
public health sites involved in addressing aids .
their accounts of the time provide insights
into the experiences of two gay men at the start of an epidemic written on and around gay
men s bodies . their stories ,
as reflected here show how even within hostile
government - aligned health care spaces , they were able to provide support and treatment to
some people , and to engage in , what were for the time , socially subversive activities .
the
different contexts and professional fields in which they found themselves mediated their
individual experiences , but in both men s case , the hegemonic aids narratives about
homosexuality influenced and shaped their responses even as they challenged them .
homophobic beliefs , conservatism , or any other invisible or unquestioned ideologies that
influence understandings of epidemics and practical responses to them , have tangible
repercussions .
these repercussions may be evident in the lack of legal protection or in the
emotional costs of prejudice and intolerance .
brouard , like miller and sifris , challenged
hospitals to provide care for people with aids .
joburg gen was often reluctant to admit
people with aids , as providing palliative care was seen as a waste of resources and of
limited value for instructional purposes .
even when places could be found for people it was difficult to get staff members to
treat them with dignity ; and many staff members did not
deal well with gay men , young men ,
coming in infected and dying of this strange disease ( where ) dementia complicated the death
process further.
for the partners of the men who died the trauma of loss was aggravated by their lack of
legal status or recognition as partners of the deceased .
brouard , for instance , recalls
counselling loving partners who were not able to claim the bodies of the deceased men .
he
witnessed both the deaths and the grief of survivors during this time . in the early phases of the epidemic sifris
challenged homophobic responses to gay men with
aids , but did not necessarily challenge the then dominant narrative that initially linked
aids to being gay . given that the professional and personal spaces he occupied bore out the
narrative that gay men were the ones dying of aids , this is perhaps not surprising .
brouard
worked within gay communities he most identified with , but his professional and personal
spaces seemed to have presented a more complex narrative about aids and its association with
sexual identity and broader identity politics .
the socio - political evocation of stereotypes , or constructions of gender , race , and
sexual orientation have long shaped experiences of ( ill)health and epidemics .
this article
has focused on the micro - narratives of individuals and their responses to the epidemic as
individuals and as part of communities .
it serves as a reminder of the intricate and
intimate relationships between ideologies , illness and identities , but also that behind each
aids avatar evoked in hegemonic narratives are complex , embodied people . | this article focuses on the micro - narratives of two individuals whose responses to aids
were mediated by their sexual identity , aids activism and the political context of south
africa during a time of transition .
their experiences were also mediated by
well - established metanarratives about aids and homosexuality created in the usa and the
uk which were transplanted and reinforced ( with local variations ) into south africa by
medico - scientific and political leaders.the nascent process of writing south african aids
histories provides the opportunity to record responses to aids at institutional level ,
reveal the connections between narratives about aids and those responses , and draw on the
personal stories of those who were at the nexus of impersonal official responses and the
personal politics of aids .
this article records the experiences of dennis sifris , a
physician who helped establish one of the first aids clinics in south africa and emptied
the dance floors , and pierre brouard , a clinical psychologist who was involved in early
counselling , support and education initiatives for hiv - positive people , and counselled
people about dying , and then about living .
their stories show how , even within
government - aligned health care spaces hostile to gay men , they were able to provide
support and treatment to people ; benefited from international connections with other gay
communities ; and engaged in socially subversive activities .
these oral histories thus
provide otherwise hidden insights into the experiences of some gay men at the start of an
epidemic that was initially almost exclusively constructed on , and about , gay men s
bodies . |
diabetes mellitus experiences a rampant growth and is currently affecting more than 150 million people worldwide . the onset of diabetes heralds the beginning of the macrovascular complications of our body .
the correlation between diabetes and cardiovascular diseases is still nebulous and is presumed to be related to hyperglycemia , hyperinsulinemia , altered lipid metabolism , hypercoagulability , and inflammation [ 2 , 3 ] .
the malefactor behind this is more diffused , deep - rooted , and advanced nature of coronary artery disease in such diabetic individuals .
moreover , the anatomy of coronary arteries involves small vessels and long lesions [ 5 , 6 ] .
the chances of platelet aggregation and thrombotic events are more in diabetics than in nondiabetic individuals .
these pose a challenge against treatment with percutaneous coronary intervention ( pci ) due to more repetitive restenosis , late luminal loss , and stent thrombosis . but
much improvements have been seen in medical management by pci , if we compare 1-year mortality rates of bypass angioplasty revascularization investigation ( bari ) with arterial revascularization therapy study ( arts ) [ 2 , 8 , 9 ] .
but still there are multiple opinions and dilemmas regarding favorable outcomes of biodegradable polymer coated sirolimus - eluting stents in diabetic population .
thus , the main aim of our study is to demonstrate safety and efficacy of biodegradable polymer coated sirolimus - eluting stents in diabetic population .
this was a retrospective , single - arm , non - randomized , multicentre registry involving diabetic patients treated with indolimus sirolimus - eluting stents ( sahajanand medical technologies pvt . ltd . ) from june 2012 to may 2014 .
written informed consent was obtained from all the patients enrolled in the study or from their legally authorized representative .
the study was conducted in accordance with the principle of good clinical practice and declaration of helsinki .
patients were included if they were at least 18 years of age , had diabetes mellitus according to world health organization report , and presented with stable or unstable angina or myocardial ischemia or acute or recent myocardial infarction .
patients were excluded ( 1 ) if they had known allergy to aspirin , clopidogrel , cobalt - chromium , heparin , ticlopidine , sirolimus , and polymers or ( 2 ) if the patient had impaired glucose tolerance without pharmacologic treatment , transient hyperglycemia , or gestational diabetes .
the indolimus biodegradable polymer coated sirolimus - eluting coronary stent involves l605 cobalt chromium ( co - cr ) alloy as its stent platform .
the biodegradable polymer gives it a strut thickness of 60 m and drug load of 1.4 g / mm .
about 70% of drug is released within 7 days and remaining drug is released over a period of 48 days ( figure 1 ) .
the drug is released within 7 weeks after the stent implantation from the polymeric layers coated onto the surface of the stent .
the biodegradable polymeric film is a blend of different biodegradable polymers , poly l - lactide , 50/50 poly dl lactide - co - glycolide , and polyvinyl pyrrolidone , which undergoes hydrolysis .
this process takes approximately 9 to 12 months after which all the polymers degrade naturally and excrete from body in the form of their metabolites .
the average coating thickness of indolimus stent is between 5 and 6 m . the indolimus stent is available in lengths of 8 , 12 , 16 , 20 , 24 , 28 , 32 , 36 , and 40 mm and available diameters were 2.5 , 2.75 , 3.0 , and 3.5 mm .
all patients received a loading dose of 300 mg of aspirin and clopidogrel ( 300 mg ) or prasugrel ( 60 mg ) or ticagrelor ( 90 gm ) .
the procedural anticoagulation was achieved with either heparin or bivalirudin . however , the intraprocedural administration of glycoprotein iib / iiia - inhibitor was at the investigator 's discretion .
all the patients received dual antiplatelet therapy ( aspirin 75300 mg / daily indefinitely and clopidogrel 75 mg / daily or prasugrel 10 mg / daily or ticagrelor 90 mg twice daily for at least 6 months ) after the procedure .
the primary endpoint of the study was a conglomeration of cardiac death , myocardial infarction ( mi ) ( q - wave and non - q - wave ) , target lesion revascularization ( tlr ) , target vessel revascularization ( tvr ) , and stent thrombosis ( st ) .
the secondary endpoints will be measured at 12 and 24 months and yearly thereafter for five years .
q - wave mi was considered , when there was development of new q - wave of more than 0.04 seconds in two or more adjoining leads along with increase in cardiac markers like troponin i or t , creatine kinase , or mb isoform .
non - q - wave mi was considered when there was more than three - time elevation in creatinine kinase levels along with elevation in mb isoform and troponin marker t or i without development of new q - waves .
target lesion revascularization was considered when there was stenosis in treated segment ( 5 mm proximal and 5 mm distal edges ) .
target vessel revascularization was considered when there was stenosis in any segment of the treated vessel .
stent thrombosis ( st ) was considered acute when it occurred within 24 hours , subacute when it occurred between 1 and 30 days , and late when it occurred after 30 days .
clinical follow - up , by hospital appointment or telephonic conversation , was scheduled at 30 days ( 7-day window period ) , 6 months ( 15-day window period ) , and 9 months ( 30-day window period ) .
follow - up data were collected pertaining to current anginal status , intake of antithrombotic regimen , and occurrence of any cardiovascular events or any invasive or noninvasive procedure that the patient had undergone .
the average stent length and diameter were 27.1 8.7 mm and 3.1 0.4 mm , respectively .
the baseline demographics of all the treated patients are described in table 1 . out of all the diabetics ( mean age = 53.4 10.2 years ) , majority of them were male ( 68.8% ) .
lesions type b , type c and totally occluded lesions were found in 317 ( 72.5% ) , 51 ( 11.7% ) , and 45 ( 10.3% ) patients , respectively .
the clinical follow - up at 9 months was obtained for 370 ( 99.4% ) patients . at 9-month follow - up
, mace was found to be 2.2% which is a composite of 2 ( 0.5% ) cases of myocardial infarction , 4 ( 1.1% ) cases of target lesion revascularization , and 2 ( 0.5% ) cases of stent thrombosis .
the clinical outcomes of patients at 30-day , 6-month , and 9-month follow - up are shown in table 3 .
the cumulative event - free survival by kaplan meier method was found to be 97.8% at 9-month follow - up ( figure 2 ) .
does diabetes mellitus worsens the prognosis and long - term outcomes of patients with coronary artery disease ?
this long held question still poses a dilemma and the answer quite fluctuates between fact and fiction .
it is long established that diabetes increases the rates of restenosis and repeat revascularization after coronary angioplasty [ 12 , 13 ] .
this belief was so severe that in the germinating era of bare metal stents surgery was considered as the primary option for patients with diabetes and multiple - vessel disease .
the bypass angioplasty revascularization investigation ( bari ) trial demonstrated the equivalence of angioplasty and bypass surgery but the five - year outcomes of bari trial demonstrated crystal clear advantage of surgery in subset of patients with diabetes .
the introduction of drug eluting stents revolutionized the concept of safety and efficacy in diabetic population .
the sirius trial demonstrated that at 9 months tlr rate was reduced in diabetic population from 22.3% in bms group to 6.9% in ses group .
consistent with this , our study also demonstrated a low tlr rate of only 1.1% at 9-month follow - up .
the lower rates of restenosis associated with our stent can be because of lower strut thickness , as stents with lower stent thickness elicit less angiographic and clinical restenosis than stents with thicker struts .
theoretically , due to aggressive atherosclerosis , platelet hyperactivity , impaired fibrinolysis , and endothelial function after arterial injury , diabetes mellitus is associated with antiplatelet resistance and consequent stent thrombosis after angioplasty .
but , paradoxically , our study presented only two cases of stent thrombosis at 9-month follow - up .
this is also supported by the results from isar - diabetes study and diabetes trial [ 18 , 19 ] .
reported that one - year event - free survival after stenting is lower in diabetics ( 73.1% ) versus ( 78.8% ) in nondiabetics .
the event - free survival at 9 months in our study was found to be 97.8% , which is quite appreciable .
contrastingly , there are studies which demonstrate that cumulative event - free survival is not affected by diabetic status . the results of our study are quite promising . however the long - term follow - up of the study would prove maintained safety and efficacy .
the use of indolimus in high risk diabetic population is associated with lower incidence of tlr , st , and consequent mace .
thus , the long held dilemma about the favorable outcomes after implantation of biodegradable polymer coated sirolimus - eluting coronary stent system in diabetic population turns out to be a fact and not fiction . | objective .
the main aim is to evaluate safety , efficacy , and clinical performance of the indolimus ( sahajanand medical technologies pvt .
ltd . , surat , india ) sirolimus - eluting stent in high - risk diabetic population with complex lesions . methods . it was a multicentre , retrospective , non - randomized , single - arm study , which enrolled 372 diabetic patients treated with indolimus .
the primary endpoint of the study was major adverse cardiac events ( mace ) , which is a composite of cardiac death , target lesion revascularization ( tlr ) , target vessel revascularization ( tvr ) , myocardial infarction ( mi ) , and stent thrombosis ( st ) . the clinical follow - ups were scheduled at 30 days , 6 months , and 9 months . results .
the mean age of the enrolled patients was 53.4 10.2 years .
a total of 437 lesions were intervened successfully with 483 stents ( 1.1 0.3 per lesion ) .
there were 256 ( 68.8% ) male patients .
hypertension and totally occluded lesions were found in 202 ( 54.3% ) and 45 ( 10.3% ) patients , respectively .
the incidence of mace at 30 days , 6 months and 9 months was 0 ( 0% ) , 6 ( 1.6% ) , and 8 ( 2.2% ) , respectively .
the event - free survival at 9-month follow - up by kaplan meier method was found to be 97.8% .
conclusion .
the use of biodegradable polymer coated sirolimus - eluting stent is associated with favorable outcomes .
the results demonstrated in our study depict its safety and efficacy in diabetic population . |
malignant tumors of the nasal cavity and paranasal sinuses account for 0.2% of all human primary malignant neoplasms , with an incidence of 0.11.4 new cases / year/100,000 inhabitants [ 13 ] .
adenocarcinomas account for 1020% of all primary malignant neoplasms of the sinonasal tract [ 4 , 5 ] .
many of these have salivary gland origin , while others have histologic patterns resembling those of colon adenocarcinoma .
this second type of sinonasal adenocarcinoma has been named intestinal - type adenocarcinoma ( itac ) and is responsible for less than 4% of the total malignancies of this region .
itacs of the nasal cavity and paranasal sinuses can occur sporadically or are associated with occupational exposure to hardwood and leather dusts .
exposure to wood and leather dusts increases the risk of adenocarcinoma by 500-fold [ 8 , 9 ] .
findings from several studies have suggested clinical differences between itac arising in individuals with occupational dust exposure and itac arising sporadically .
in fact tumors related to occupational exposure affect men in 8590% of cases , showing a strong tendency to arise in the ethmoid sinuses [ 1012 ] .
itacs are aggressive tumors characterized by frequent local recurrences , low incidence of distant metastases , and an overall mortality of approximately 53% .
itac seems to be preceded by intestinal metaplasia of the respiratory mucosa , induced by hardwood dust , leather dust , and other unknown agents , which is accompanied by a switch to an intestinal phenotype .
the molecular mechanisms involved in metaplastic transformation of terminally differentiated epithelium to a phenotypically different epithelium are largely unknown .
the morphological appearance of these tumors is variable , and they may resemble conventional colorectal adenocarcinoma .
the similarities between itac and intestinal adenocarcinoma involve their ultrastructural and immunohistochemical aspects [ 1417 ] .
numerous studies have shown that mutations of the k - ras and tp53 genes are common in colorectal adenocarcinomas [ 18 , 19 ] . in comparison
, there is very little information about the molecular events involved in the pathogenesis of itac [ 16 , 1926 ] , in contrast to even more increasing information about the molecular mechanisms involved in the pathogenesis of head and neck squamous cell carcinomas ( hnscc ) [ 2732 ] .
tumor necrosis factor ( tnf)-related apoptosis - inducing ligand ( trail ) is a tnf - family member , found in a variety of tissues and with a conditional expression in several immune effector cells [ 34 , 35 ] . to date , five different receptors have been identified to interact with trail : trailr1 ( dr4 ) , trail - r2 ( dr5 ) , trail - r3 ( dcr1 ) , trail - r4 ( dcr2 ) , and osteoprotegerin .
dr4 and dr5 are two different death domain - containing membrane receptors , whereas dcr1 and dcr2 are two decoy receptors that compete for trail binding with dr4 and dr5 [ 3639 ] .
the final effect of the trail signaling is the induction of apoptosis by the intrinsic death pathway , recruiting the inactive form of caspase-8 .
expression of trail and its receptors has been detected in various human tumors , suggesting that trail signaling pathway is involved in endogenous tumor surveillance , but the mechanism of how trail and its receptors contribute to carcinogenesis remains unknown .
to gain further insight into the phenotype and possible mechanisms of ethmoidal itac , we investigated the expression of trail , correlating with clinicopathological data .
the samples of 23 primary ethmoidal intestinal - type adenocarcinomas were retrospectively retrieved from the archives of the institute of pathology , marche polytechnic university , ancona , italy .
all the tumors in which the diagnosis of itac was confirmed were subsequently subtyped , as papillary , colonic , solid , mixed , or mucinous types , as described by barnes .
inclusion criteria were complete clinical data , uniformity of histological differentiation throughout the tumor sample , and the availability of sufficient material from the primary tumor for investigations .
patients with previous or synchronous second malignancies or with previous radiation therapy or chemotherapy were excluded from the study .
the follow - up time ranged from 2 to 10 years ( mean 4.8 years ) .
four - micrometer serial sections from formalin - fixed , paraffin - embedded blocks of tumour representative areas were cut for each case .
sections were deparaffinized in two changes of xylene for 10 minutes each , then , were rehydrated through graded alcohols , and immersed in 0,3% hydrogen peroxide in methanol for 30 minutes to block endogenous peroxidase activity .
the tissue sections were placed in a microwave oven ( philips , cooktyronic m720 , 700 w ) in a plastic coplin jar filled with 10 mm sodium citrate buffer ( ph 6,0 ) at 5-minute interval , the fluid level in the coplin jar was removed from the microwave oven and allowed to cool .
slides were incubated overnight with a 1 : 50 dilution of the primary mouse antihuman trail monoclonal antibody ( dako do-7 , glostrup , denmark ) .
slides were washed twice with pbs and incubated with the linking reagent ( biotinylated anti - immunoglobulins ) for 15 minutes , at room temperature . after rinsing in pbs
, the slides were incubated with the peroxidase - conjugated streptavidin label for 15 minutes at room temperature .
the sections were again rinsed in pbs and incubated with diaminobenzidine for 10 minutes , in the dark .
after chromogen development , slides were washed in two changes of water and counterstained with a 1 : 10 dilution of hematoxylin .
trail expression and location were evaluated on histological section using a leitz orthoplan microscope equipped with a x 400 objective .
the percentage of trail positive cells was evaluated from a minimum of 1,000 cells in each case . only nuclear staining of epithelial cells
was observed , and the nuclei with a clear brown color , regardless of staining intensity , were regarded as trail positive .
a negative control for trail immunostaining was performed in all cases by omitting the primary antibody , which , in all instances , resulted in negative immunoreactivity .
both the histological diagnoses and evaluation of the positivity for trail were carried out indipendently by two of the authors ( c.r . and a.z . ) .
immunohistochemical labeling for trail was classified as positive , when more than 5% of nuclei or cells were stained .
the following pathological and clinical parameters were analyzed : tumour grade of differentiation ( g1,g2,g3 ) , tumour extension ( t1,t2,t3,t4 ) , tumor stage ( i , ii , iii , iv ) , and trail immunoreactivity .
differences in trail immunoreactivity between the different groups were compared by the nonparametric kruskal - wallis test . statistical significance was set at p < 0.05 .
all statistical analyses were performed using the spss statistical package ( spps inc . , chicago , il ) .
there were 21 males and 2 females , with a mean age of 66.3 years ( range 5477 ) .
there were 8 grade i , 8 grade ii , and 7 grade iii adenocarcinomas .
3 patients were in i stage , 7 patients were in ii stage , 11 patients were in iii stage , and 2 patients were in iv stage .
all the patients had a known history of occupational exposure to hardwood dust , and intestinal - type adenocarcinoma was localized in all cases in the ethmoid region as confirmed by endoscopic and imaging ( tc and/or mr ) evaluation .
the main clinicopathologic features of the patients and the oncoprotein expression are summarized in table 1 .
the cases classified as itac showed a variable cellular appearance and were composed of a mixture of tall columnar absorptive cells , atypical stratified cylindrical cells similar to the cells seen in conventional colorectal adenocarcinoma , goblet cells , and large round to polygonal nondescriptive epithelial cells .
the percentage of neoplastic cells immunoreactive for trail ranged from 2% to 90% . in all cases ,
there was a relationship between trail overexpression and the histological grading . indeed , poorly differentiated cases ( g2 and g3 ) showed a higher percentage of trail expressing cells in comparison to well differentiated ( g1 ) cases .
no correlation between trail expression and other clinicopathological parameters , including t , stage and relapses was found ( figures 1 and 2 ) .
despite their histological similarity to colorectal carcinomas , there is very little information about the molecular events involved in the pathogenesis of itac .
several crucial pathways of tumorigenesis have been identified in colorectal adenocarcinomas [ 18 , 19 ] .
these pathways involve the mutation and inactivation of multiple oncogenes , tumor suppressor genes , and dna mismatch repair genes including k - ras , apc , p53 , mlh1 , and msh2 [ 1820 ] .
working on the hypothesis that morphological similarities to colorectal adenocarcinomas might reflect equivalent genetic alterations , several authors have investigated the presence of activating mutations of ras oncogenes and tp53 mutations in itac [ 2125 ] .
tp53 mutations were found in 1844% of mostly occupational itacs , whereas , k - ras mutations were found in 1015% of itacs [ 2125 ] .
the results of these studies suggest that mutations of k - ras and other ras genes are relatively uncommon in itac , and similarly , tp53 mutations in itacs have not been widely demonstrated .
other studies have shown that k - ras mutation and c - erb-2 expression could be associated with more aggressive itacs [ 16 , 26 ] .
found the existence of two genetic itacs subgroups , defined by differences in tp53 mutational status or protein functionality , that strongly influence pathologic response to primary chemotherapy and , ultimately , prognosis .
perez - ordonez et al . evaluated the possible role of dna mismatch repair ( mmr ) gene defects or disruptions of e - cadherin/-catenin complex in itac by investigating the immunohistochemical expression of the mmr gene products , e - cadherin , and -catenin in a group of sporadic itacs .
the preserved nuclear expression of mlh1 , msh2 , msh3 , and msh6 suggested that mutations or promoter methylation of mmr genes do not play a role in the pathogenesis of itac .
kennedy et al . found that sinonasal itacs have a distinctive phenotype , with all cases expressing ck20 , cdx-2 , and villin and most itacs also expressing ck7 , so that the expression pattern of ck7 , ck20 , cdx-2 , and villin positive may be useful in separating these tumors from other non - itac adenocarcinomas of the sinonasal tract .
given these findings , to explore other pathways involved in the molecular pathogenesis of itacs , we immunohistochemically investigated the expression of the apoptosis - regulating protein trail in a group of 23 ethmoidal itac associated with occupational exposure .
this immunohistochemical expression was also retrospectively correlated with the patient outcome to evaluate their independent prognostic relevance . to the best of our knowledge , there are no previous reports on the expression of trail protein in itacs .
trail is an apoptosis - inducing protein and a molecule important in inhibiting cellular immunity [ 4446 ] .
similarly , cancer cells may use trail to evade the antitumor immune response . in literature , there are many works that have detected the expression of trail and its receptors in several tumors , correlating this expression with clinicopathological analysis .
trail - r1 was identified as an independent prognostic factor for disease - free survival in 128 patients with colon cancer .
mccarthy et al . showed that high trail - r2 expression was significantly associated with decreased survival and lymph node involvement in patients with primary breast cancer .
revealed that trail and dr4 expression were correlated with the pathological grading in patients with laryngeal squamous cell carcinoma , while the alteration in dr5 expression was correlated with the clinical staging . despite the several works , the specific mechanism of how trail and its receptors contribute to carcinogenesis remains unknown .
the results of our immunohistochemical examination showed a relationship between trail upregulation and the increase of the histological tumor grading .
no correlation was found with other clinicopathological parameters , including t , stage and relapses .
our results suggest that mutations of trail expression , in combination with additional genetic events , could play a role in the pathogenesis of itac .
however , this study showed that trail expression can not be considered as an independent prognostic factor in patients with itacs , because there was not sufficient statistical power to detect significant associations between immunohistochemical expression of this protein and the clinicopathologic parameters of the tumors , therefore , further investigations with a larger sample are needed . | introduction . despite their histological resemblance to colorectal adenocarcinoma
, there is some information about the molecular events involved in the pathogenesis of intestinal - type sinonasal adenocarcinomas ( itacs ) . to evaluate the possible role of tnf - related apoptosis - inducing ligand ( trail ) gene defects in itac , by investigating the immunohistochemical expression of trail gene product in a group of ethmoidal itacs associated with occupational exposure .
material and methods .
retrospective study on 23 patients with pathological diagnosis of primary ethmoidal itac .
representative formalin - fixed , paraffin - embedded block from each case was selected for immunohistochemical studies using the antibody against trail .
clinicopathological data were also correlated with the staining results .
results .
the immunohistochemical examination demonstrated that poorly differentiated cases showed a higher percentage of trail expressing cells compared to well - differentiated cases .
no correlation was found with other clinicopathological parameters , including t , stage and relapses .
conclusion .
the relationship between upregulation of trail and poorly differentiated ethmoidal adenocarcinomas suggests that the mutation of this gene , in combination with additional genetic events , could play a role in the pathogenesis of itac . |
chronic hepatitis b virus ( hbv ) infection is one of the most common and serious infections worldwide . according to the world health organization
, approximately two billion people have been infected with hbv and more than 240 million of them suffer from chronic hbv infection .
chronic hbv infection may be complicated by cirrhosis , hepatocellular carcinoma ( hcc ) , and end - stage liver disease [ 13 ] .
all children with hbv infection should be monitored by physicians due to the risk of progression to chronicity , cirrhosis , and hcc .
progression to chronicity and development of cirrhosis or hepatocellular carcinoma can be detected by invasive methods such as liver biopsy .
galectin-3 ( gal-3 ) is a 30 kda protein that is important in many activities of malignant behaviors , including cancer cells and development of fibrosis .
gal-3 may be important in the pathogenesis of liver fibrosis , as the previous literature suggest that the lack of gal-3 may be associated with resistance to liver fibrosis development .
recently , some experimental disease models suggested that gal-3 was associated with the development of chronic liver disease secondary to inflammatory or toxic insults .
gal-3 can stimulate the production of some cytokines and chemokines via cd98 through interactions with macrophages .
gal-3 may play an important role in sustaining of hbv replication and may contribute to pathologic processes that resulted in chronic status in hbv infection .
based on these data gal-3 was found to be crucial for promoting inflammation that may contribute in the progression of chronic infection and development of liver fibrosis .
thus , gal-3 may be useful for monitoring the chronicity in different phases of hbv infection . to our knowledge
, there is no study regarding gal-3 levels in children with chb . in this study
, we investigated serum gal-3 levels in children with chb compared to inactive hbv carrier patients to help us understand whether gal-3 levels may be beneficial for discrimination and monitoring of hbv chronicity .
thirty - two patients with chronic hepatitis b ( chb ) with no antiviral treatment and 30 inactive hbv carrier children with hepatitis b were followed up between july 2013 and december 2013 .
patients with chronic hepatitis b were defined by the following criteria : 1 ) positive serum hepatitis b surface antigen ( hbsag ) lasting at least 6 months prior to obtaining of blood samples ; and 2 ) for hepatitis b e antigen ( hbeag ) positive patients [ serum hbv dna level 20 000 iu / ml and serum alt level 1.5upper limit of normal values ( uln ) ; for hbeag negative patients , serum hbv dna 2000 iu / ml and serum alt 1.5uln ] .
inactive hbv carrier patients were defined by the following criteria : 1 ) positive serum hbsag from at least 6 months prior to obtaining of blood samples ; 2 ) serum hbv dna level < 2000 iu / ml and serum alt levels < uln ; and 3 ) negative serum hbeag .
control subjects were recruited from children who applied for routine check - up or elective surgery such as hernia repair or circumcision .
control subjects had no abnormal liver function or hepatitis b tests , and no history or findings of any systemic disease .
the basic laboratory tests of all subjects including serum glucose , albumin , alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , gamma - glutamyl transferase ( ggt ) , total bilirubin ( tbil ) , prothrombin time ( pt ) and activated partial thromboplastin time ( aptt ) were recorded .
patients that were under 2 years or older than 18 years of age were excluded from the study .
patients that received hepatotoxic drugs or had other causes of acute or chronic hepatitis ( wilson disease , autoimmune hepatitis , 1-antitrypsin deficiency , metabolic disease and co - infection or superinfection of hepatitis d virus infection and hepatitis c infection ) were also excluded .
informed consent was obtained from parents or legal guardians of the subjects and the study protocol was approved by the local ethics committee .
serum glucose , albumin , alt , ast , ggt , tbil , pt , and aptt were measured by enzymatic colorimetric method using an abbot architect c16000 ( illinois , united states ) .
serum gal-3 levels were measured by using a commercial quantitative enzyme - linked immune sorbent assay ( elisa ) ( eastbiopharm company , hangzhou , china ) according to the manufacturer s instructions .
serum gal-3 levels were shown as ng / ml . the lowest mean detection limit for gal-3 level was 0.015 ng / ml . all data were evaluated by using spss ( statistical package for social sciences ) 16.0 program for windows .
analyses of variance test followed by post hoc scheffe test was used to compare three independent groups .
thirty - two patients with chronic hepatitis b ( chb ) with no antiviral treatment and 30 inactive hbv carrier children with hepatitis b were followed up between july 2013 and december 2013 .
patients with chronic hepatitis b were defined by the following criteria : 1 ) positive serum hepatitis b surface antigen ( hbsag ) lasting at least 6 months prior to obtaining of blood samples ; and 2 ) for hepatitis b e antigen ( hbeag ) positive patients [ serum hbv dna level 20 000 iu / ml and serum alt level 1.5upper limit of normal values ( uln ) ; for hbeag negative patients , serum hbv dna 2000 iu / ml and serum alt 1.5uln ] .
inactive hbv carrier patients were defined by the following criteria : 1 ) positive serum hbsag from at least 6 months prior to obtaining of blood samples ; 2 ) serum hbv dna level < 2000 iu / ml and serum alt levels < uln ; and 3 ) negative serum hbeag .
control subjects were recruited from children who applied for routine check - up or elective surgery such as hernia repair or circumcision .
control subjects had no abnormal liver function or hepatitis b tests , and no history or findings of any systemic disease .
the basic laboratory tests of all subjects including serum glucose , albumin , alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , gamma - glutamyl transferase ( ggt ) , total bilirubin ( tbil ) , prothrombin time ( pt ) and activated partial thromboplastin time ( aptt ) were recorded .
patients that were under 2 years or older than 18 years of age were excluded from the study .
patients that received hepatotoxic drugs or had other causes of acute or chronic hepatitis ( wilson disease , autoimmune hepatitis , 1-antitrypsin deficiency , metabolic disease and co - infection or superinfection of hepatitis d virus infection and hepatitis c infection ) were also excluded .
informed consent was obtained from parents or legal guardians of the subjects and the study protocol was approved by the local ethics committee .
serum glucose , albumin , alt , ast , ggt , tbil , pt , and aptt were measured by enzymatic colorimetric method using an abbot architect c16000 ( illinois , united states ) .
serum gal-3 levels were measured by using a commercial quantitative enzyme - linked immune sorbent assay ( elisa ) ( eastbiopharm company , hangzhou , china ) according to the manufacturer s instructions .
serum gal-3 levels were shown as ng / ml . the lowest mean detection limit for gal-3 level was 0.015 ng / ml .
all data were evaluated by using spss ( statistical package for social sciences ) 16.0 program for windows .
analyses of variance test followed by post hoc scheffe test was used to compare three independent groups .
the study group included 32 chb patients ( 19 male /13 female ) , 30 inactive hbv carrier patients ( 16 male / 14 female ) and 30 healthy children ( 16 male /14 female ) .
the age was 11.73.0 years in the chb group , 12.32.5 years in the inactive hbv carrier group , and 11.72.7 years in the control group .
demographic characteristics including age , gender , body mass index , and laboratory test results of chb , inactive hbv carrier , and the control groups are summarized in table 1 .
significantly higher serum gal-3 levels were found in the chb group compared with hbv carrier and the control groups ( 16.53.6 ng / ml , 1.10.3 ng / ml , 0.70.5 ng / ml , respectively , p<0.001 ) ( figure 1 ) and no significant difference was found in the gal-3 levels of inactive hbv carrier and the control groups ( p>0.05 ) . similarly , serum alt levels ( 80.230.6 , 26.812.6 , 28.14.4
iu / l , respectively , p<0.001 ) were found to be significantly higher in the chb group than other groups ( p<0.001 ) and no significant difference was found in the alt levels of inactive hbv carrier and the control groups ( p>0.05 ) .
chb group had significantly higher ggt levels ( 51.327.5 iu / l ) compared with the inactive hbv carriers ( 35.710.1
no significant differences were found in aptt , inr , and serum albumin and glucose levels between the three groups ( p>0.05 ) ( table 1 ) . there was a positive correlation between gal-3 and serum alt levels in the chb group ( r=0.82 , p<0.001 ) .
however , no correlations of gal-3 were found with serum albumin , glucose , ggt , aptt , and inr values in the chb , inactive hbv carrier , or the control groups ( p>0.05 for each ) .
in this study , we analyzed gal-3 levels in different phases of hbv infections and healthy subjects by elisa to investigate the possible benefit of gal-3 as a diagnostic marker for chronicity .
the results of the present study indicated that , in patients with serologically - proven chb , serum levels of gal-3 levels were significantly higher than those of inactive hbv carrier and healthy controls .
chb infection is a common infection worldwide and has some important clinical complications such as cirrhosis and hcc .
treatment decision is based on alt levels , hbeag positivity , hbv dna level , and liver histology
. family history of hcc , co - existing liver diseases , and patient s treatment history were also important points for treatment decision .
inteleukin-10 ( il-10 ) is a cytokine which is produced by t helper type 2 cells .
il-10 has effect on inhibition of pro - inflammation cytokines , regulation of humoral immunity , and termination of inflammation .
an experimental autoimmune encephalomyelitis model demonstrated that gal-3 plays an important role in decreasing il-10 production .
decreasing il-10 may contribute to the sustaining of hbv replication and may initiate chronic hbv infection , which may direct fibrogenesis via inhibition of ifn- secretion .
although we did not measure il-10 in our patients , gal-3 may contribute to chronicity of hbv infection by decreasing il-10 .
measurement of il-10 together with gal-3 in children with hepatitis b may be a subject of further studies .
histologic changes of liver can be shown by liver biopsy , which is an invasive technique .
in addition to being an invasive method , liver biopsy is a painful process and accuracy of biopsy depends on suitability of biopsy specimen and experience of pathologist .
although , some non - invasive techniques such as fibroscan have been suggested in order to predict to chronicity and fibrosis status of chronic chb infection , these methods are still not validated . in a multicenter study
, fibroscan was shown to be useful for determining diagnostic accuracy for cirrhosis , but not significant for fibrosis .
therefore , further non - invasive methods are needed to predict entering chronic phase or development of complications with avoidance of liver biopsy .
because of possible role of gal-3 in the promoting of inflammation and leading to chronic hepatitis , it can be accepted as a useful biomarker indicating liver inflammation and chronic phase of chb as a non - invasive technique .
to the best of our knowledge , the presented study is the first that investigates association between chb infection and serum gal-3 levels in childhood .
gal-3 has a carbohydrate recognition domain which is activated by the ligand interaction and has powerful affinity for -galactosides .
gal-3 may contribute some cellular processing which may result in fibrosis development , cancer progression , and tissue remodeling .
moreover , galectins are expressed in immune cells such as activated macrophages , mast cells , and eosinophils .
that s why they may have acute and chronic immunologic effects such as production of some cytokines and mediators as well as apoptosis and chemoattraction . in a previous experimental study ,
the severity of the liver damage during the chb infections was assessed by some biochemical markers including alt , ast , ggt , alkaline phosphatase , bilirubin , serum albumin , and globulin levels . in present study , alt levels , which reflect ongoing liver damage , were found to associate with gal-3 levels in chb group .
higher ggt activities have also been reported in cholestatic liver disease , and other hepatic disorders related to the biliary tract .
in addition , previous studies have suggested that increased levels of serum ggt indicated advanced liver fibrosis .
the results of our study showed no significant positive correlation between serum ggt and gal-3 level . probably , our finding related to lack of correlation between ggt and gal-3 levels may be a reflection of hepatocyte injury due to chb infection rather than cholestatic hepatitis in our patient group .
the pathogenesis of chronic hbv infection and how hbv infection maintains its chronic persistent infection is not fully understood .
we believe that our results may be evidence for the role of gal-3 in both the inflammatory process and the development of chronicity in chb infections .
serum il-10 levels of our study population were unavailable which might increase the value of the results .
the present study demonstrated that gal-3 may be beneficial for predicting chronicity in childhood chronic hbv infection .
understanding the role of gal-3 in hbv infections may contribute to the explanation of the pathogenesis of chb infections .
further investigates are needed to explain the precise mechanism(s ) of gal-3 in chb infection . | backgroundchronic hepatitis b virus ( hbv ) infection is common worldwide .
follow - up of patients by the use of non - invasive techniques may be valuable in clinical practice .
the aim of this study was to investigate serum galectin-3 ( gal-3 ) levels for monitoring disease status in children with chronic hbv infection.material/methodsthirty-two patients with chronic hepatitis b ( chb ) , 30 inactive hbv carrier patients , and 30 matched healthy controls were enrolled in the study .
we performed basic laboratory tests : serum glucose , albumin , alanine aminotransferase ( alt ) , aspartate aminotransferase , gamma - glutamyl transferase ( ggt ) , total bilirubin , prothrombin time , and activated partial thromboplastin time .
in addition , serum gal-3 levels were measured by elisa technique.resultssignificantly higher serum gal-3 levels ( 16.53.6 , 1.10.3 , 0.70.5 ng / ml , respectively , p<0.001 ) and alt levels ( 80.230.6 , 26.812.6 , 28.14.4
iu / l , respectively , p<0.001 ) were found in the chb group compared with the inactive carriers and the control groups . there were no significant differences in alt levels and gal-3 levels or between inactive hbv carriers and the control groups ( p>0.05 , for each ) .
significantly higher ggt levels were found in the chb group ( 51.327.5
iu / l ) compared with the inactive hbv carriers ( 35.710.1 iu / l ) and the control group ( 31.39.5
iu / l ) ( p<0.001 , and p=0.004 , respectively ) .
a significant correlation was found between gal-3 and alt levels in the chb group ( r=0.82 , p<0.001).conclusionsour results suggest that serum gal-3 level may be a beneficial indicator of chronicity in hepatitis b infection in children . |
atrial fibrillation ( af ) is a global healthcare problem , currently affecting 2.5% of the population worldwide , its prevalence steeply increases with age ( 1 ) , ranging from 9% between 76 - 85 years to > 10% over 85 years ( 2 ) and it is expected to increase in the future ( 1 ) . although not directly life threatening , af affects quality of life as a direct cause of left ventricular dysfunction ( lvd ) , heart failure ( hf ) , hospitalizations , disability , cognitive impairment ( ci ) , and stroke .
the annual incidence of stroke in people with af is approximately 5% , which is 2 to 7 times higher than the average rate of stroke in the general population , depending on the presence of other risk factors and age , ranging from 1.5% in patients aged 50 to 59 years to 23.5% in patients aged 80 to 89 years ( 3 ) .
strokes associated with af are mostly cardioembolic , tend to be more severe , and result in longer hospital stays and greater disability than atherothromobotic strokes not associated with af , with an average mortality risk that is two - fold higher ( 4 ) . similar to af , hf is a significant and growing epidemic and its prevalence increases with age . af and hf share several common risk factors and pathophysiologic processes such as hypertension , diabetes mellitus , ischemic heart disease , and valvular heart disease . beyond sharing predisposing factors , af and hf are closely intertwined , with each disease predisposing to the other .
when present in combination , af and hf portend a worse prognosis than either condition alone , with a four - fold increased risk of systemic thromboembolism events per year , while asymptomatic lvd is predictive of developing af ( 5 ) . in framingham heart study participants with new - onset af ,
37% had hf and conversely , 57% individuals with new hf had af ( 6 ) .
several comorbidities are associated with af , such as hypertension , diabetes , chronic kidney disease , chronic obstructive pulmonary disease , ci , cerebrovascular disease .
the symptoms of af vary widely between patients and range from none to severe and functionally disabling .
the most common symptoms are palpitations ( 42%-55% ) , fatigue ( 15%-49% ) , dyspnea ( 24%-49% ) , and angina ( 10%-20% ) ( 7 ) .
only 12%-25% of patients are asymptomatic ( 7 ) , more commonly elderly patients ; asymptomatic or minimally symptomatic af patients are not prompted to seek medical care and can initially be seen with a thromboembolic complication such as stroke or hf .
clinical features and pathophysiologic pathways include the loss of atrial contraction , which can decrease cardiac output by up to 25% ( 8) , atrioventricular dyssynchrony , and rapid and irregular ventricular rates , which result in negative consequences on diastolic and systolic function , as well as increased myocardial oxygen demands and an increased risk of tachicardiomyopathy ( fig .
common pathophysiological mechanisms and interactions between atrial fibrillation ( af ) and heart failure ( hf ) .
la = left atrium ; mr = mitral regurgitation ; tr = tricuspid regurgitation . on the other hand , activation of the renin - angiotensin - aldosterone system ( raas ) and maladaptive physiological changes lead to increased left ventricular filling pressure ( lvfp ) ; increased lvfp is transmitted to the left atrium , causing fibrosis and increasing atrial stretch , which will finally lead to conduction abnormalities and proarrhythmic remodeling of the atrial chamber ( 9 ) .
elevated atrial pressure is further increased when secondary mitral regurgitation develops along with lv remodeling ( fig .
despite evidence from registries and study subsets suggesting adverse outcomes with hf and prevalent af , the benefit of a rhythm control strategy versus rate control has never been established .
the atrial fibrillation follow - up investigation of rhythm management ( affirm ) ( 10 ) and the atrial fibrillation in congestive heart failure ( af - chf ) ( 11 ) trials demonstrated similar all - cause hf incidence , hospitalization , and overall mortality in both rhythm control and rate control groups , although only 23% of patients in affirm had clinical hf , so generalization of the results to the hf population should be made with caution .
there are several reasons to explain the lack of improvement of survival with rhythm control , including imperfect effectiveness of normal sinus rhythm maintenance and adverse effects of current pharmacological therapy .
moreover , the rate control efficacy in permanent atrial fibrillation ( race ii ) trial showed no differences in functional outcomes , hospital admissions , or symptoms in patients with af treated to a more lenient heart rate regime ( < 110 beats / min resting heart rate ) versus a stricter rate control ( < 80 beats / min resting heart rate ) ( 12 ) .
catheter ablation has been shown to significantly improve freedom from af in patients who have failed antiarrhythmic drugs , but there is a lack of evidence of its feasibility in older hf patients , as most patients included in trials were relatively young , with little co - morbidity , and normal to mildly reduced left ventricular ejection fraction ( lvef ) ( 13 ) .
different studies have shown that oral anticoagulants ( oacs ) are much more effective than antiplatelet therapy in reducing the risk of stroke , particularly in elderly patients ( 14 , 15 ) .
an analysis of the atrial fibrillation investigators database , including 8932 patients from 12 trials , showed that with increasing age the relative efficacy of antiplatelet therapy to prevent ischemic stroke appears to decrease , whereas it does not change for oacs . because stroke risk increases with age , the absolute benefit of oacs increases as patients get older , despite a significantly increased risk of serious bleeding ( 16 ) .
however , despite benefits , studies have shown that oacs are underused in af , especially in older patients ( 17 ) , due to uncertainty concerning the risk of stroke and the risk of bleeding .
there are several reasons for physicians to withhold therapy with oacs in older patients , including frailty , a high prevalence of chronic diseases and comorbidities , polypharmacy , adverse drug reactions , and changes in pharmacokinetics and pharmacodynamics .
risk of falls is also perceived as a reasonable factor for withholding oacs as it may increase the risk of intracranial bleeding , but it has been demonstrated that it should not prevent treatment in elderly patients with af .
nevertheless , despite patients with many comorbidities and polypharmacotherapy being less commonly represented in clinical trials , these frail elderly patients for whom decisions regarding anticoagulation are a matter of concern are a significant proportion of the patients seen by physicians in their everyday practice .
novel direct oacs ( noacs ) have been extensively investigated across multiple randomized trials in af .
all studies have demonstrated at least non - inferiority when compared with traditional vitamin k antagonists ( vkas ) with no increase in stroke risk or bleeding ( tab .
all of these trials enrolled relatively large proportions of elderly ( 31.2 - 43.7% ) and hf patients ( 32%-63% ) , with only small interstudy discrepancies in the criteria for diagnosis of hf .
characteristics of the populations enrolled in noac studies detailed subgroup analysis in the major noac trials showed similar benefit in the subgroups with hf with reduced ( hfref ) and preserved lvef ( hfpef ) compared to the total study population ( 18 - 19 - 20 - 21 ) .
for example , an analysis from the apixaban for reduction in stroke and other thromboembolic events in atrial fibrillation ( aristotle ) study compared patients with lvef < 40% to those with lvef > 40% , and found no difference in the risk of embolic events in warfarin - treated patients , nor in subsequent reduction of risk with apixaban ( 22 ) .
the randomized evaluation of long - term anticoagulation therapy ( re - ly ) trial ( 23 , 24 ) included patients with a mean chads2 ( congestive heart failure , hypertension , age 75 years , diabetes , stroke or transient ischemic attack ) score of 2.2 and a mean age of 71.5 years .
there was a significant treatment - by - age interaction for major bleeding . in patients aged 75 years ,
dabigatran 150 mg twice daily ( bid ) resulted in a similar reduction in stroke and systemic thromboembolism compared with warfarin ( 1.4%/year vs. 2.1%/year , hazard ratio [ hr ] , 0.88 ; 95% confidence interval [ ci ] , 0.66 - 1.17 ; p = 0.81 ) .
however , when compared with warfarin , dabigatran 150 mg bid was associated with a trend toward more major bleeding in patients 75 years ( 5.1%/year vs. 4.3%/year , p = 0.07 ) ; in the same subgroup of patients , dabigatran 110 mg bid resulted in a similar major bleeding rate compared with warfarin ( 4.43%/year vs. 4.37%/year , p = 0.89 ) .
the rivaroxaban once daily oral direct factor xa inhibition compared with vitamin k antagonism for prevention of stroke and embolism trial in atrial fibrillation ( rocket - af ) included patients with a mean chads2 score of 3.5 and a median age of 73 years with 62.1% prevalence of hf ( 19 ) . in patients aged 75 years , rivaroxaban resulted in a similar reduction in stroke and systemic thromboembolism compared with warfarin ( 2.3%/year vs. 2.8%/year , hr 0.80 ; 95% ci , 0.63 - 1.02 ; p = 0.3 ) .
there was no treatment - by - age interaction for major bleeding , with similar rates of bleeding with rivaroxaban and warfarin in each age stratum , despite a not significant trend toward more major bleeding in patients 75 years ( 4.8%/year vs. 4.4%/year , hr 1.1 ; 95% ci , 0.92 - 1.34 ; p = 0.3 ) .
the aristotle trial included patients with mean chads2 score of 2.1 and a median age of 70 years .
the dose of apixaban , 5 mg bid , was reduced to 2.5 mg bid in patients with two of the following characteristics : age 80 years , weight 60 kg , and creatinine 1.5 mg / dl ( 20 ) . in patients aged 75 years
, apixaban resulted in a higher reduction in stroke and systemic thromboembolism compared with warfarin ( 1.56%/year vs. 2.19%/year , hr 0.71 ; 95% ci , 0.53 - 0.95 ) .
apixaban was associated with a lower risk of major bleeding in both patients < 75 years ( 1.99%/year vs. 2.82%/year , hr 0.71 ; 95% ci , 0.56 - 0.80 ) and in patients aged 75 years ( 3.33%/year vs. 5.19%/year , hr 0.64 ; 95% ci , 0.52 - 0.79 ) when compared with warfarin ( 25 ) .
apixaban was also associated with greater efficacy and safety with increasing age in all major end - points of the study suggesting a significant net clinical benefit in the elderly population .
moreover , no significant interaction with apixaban dose ( i.e. 2.5 vs. 5.0 mg ) was found with respect to treatment effect on major outcomes ( 25 ) ( fig .
the effect of apixaban versus warfarin on major study outcomes according to age in the aristotle study .
modified from ( 25 ) : halvorsen s , atar d , yang h , et al .
efficacy and safety of apixaban compared with warfarin according to age for stroke prevention in atrial fibrillation : observations from the aristotle trial .
the effective anticoagulation with factor xa next generation in atrial fibrillation - thrombolysis in myocardial infarction ( engage - timi ) 48 trial included patients with a mean chads2 score of 2.8 and a median age of 72 years ( 21 ) .
the edoxaban dose was reduced by half in patients with reduced renal function ( 30 - 50 ml / min ) , weight 60 kg , or with concomitant use of verapamil , quinidine , or dronedarone .
edoxaban 60 mg daily was associated with a lower risk of major bleeding among patients aged < 75 years compared with warfarin , and similar rates among those aged 75 years ( edoxaban 60 mg ; 4%/year vs. 4.8%/year , absolute risk reduction 0.8% and edoxaban 30 mg ; 2.3%/year vs. 4.8%/year , absolute risk reduction 2.6% ) ( 26 ) .
barco et al ( 27 ) reviewed the risks and benefits of noacs compared with vkas in elderly subgroups of patients enrolled in phase 3 randomized trials .
the results confirmed that the favorable balance between risks and benefits of noacs is preserved in the elderly population .
the absolute risk reductions are higher in elderly than in younger patients due to the higher absolute risks .
although interpretations of subgroup analyses should always be made with caution , the proportion of patients aged 75 years was consistently over 40% , which provides considerable power to explore age subgroups ( 27 ) ( fig .
primary efficacy outcome of stroke or systemic embolism , major bleeding and intracranial hemorrhage in phase 3 randomized controlled trials comparing a noac with vitamin k antagonists in patients with atrial fibrillation according to age .
bid = twice daily ; hr = hazard ratio ; noac = novel direct oral anticoagulant ; qd = once daily .
2 , barco s , cheung yw , eikelboom jw , coppens m. new oral anticoagulants in elderly patients , pp .
215 - 224 , copyright ( 2013 ) , with permission from elsevier . as reduced lvef
is independently associated with stroke , the combination of hf with af increases significantly the risk of stroke compared with af alone .
although no trials have investigated this specific population , indirect sub - group data from the noacs randomized trial suggest that the effect of anticoagulation for af is similar in patients with concomitant hf ( 15 , 18 - 19 - 20 - 21 ) , and noacs are particularly attractive in these patients due to more favorable net clinical benefit compared to vka therapy . in conclusion ,
in patients aged 75 years with hf , noacs have a favorable risk - benefit profile compared with warfarin for prevention of stroke and systemic thromboembolism .
in advanced age , global health status results from a complex and dynamic interaction between different areas : the changes related to normal ageing , disease severity , comorbid conditions , and social and environmental factors ( 28 ) .
the three main geriatric conditions , frailty , comorbidity , and disability , are frequent in older hf and af patients ( 28 ) .
disability is defined as difficulty or dependency in carrying out activities essential to independent living , including essential roles , tasks needed for self - care and living independently at home .
frailty is common in older people and is clinically recognized as a syndrome of loss of reserves that enhances vulnerability to stressors ( e.g. acute illnesses , hospitalizations , medical procedures ) , thus increasing the risk for major events and disability . because it reflects biological rather than chronological age , frailty may explain substantial heterogeneity in clinical outcomes within older patients ( 29 , 30 ) .
multiple physiological factors are thought to be involved in the development of frailty , including dysregulation of the immune , hormonal , and endocrine systems .
notably , cardiovascular diseases and frailty share several commonalities , particularly a consistent correlation with the upregulation of inflammatory cytokines .
frailty can be evaluated in many ways and several multi - item indices have been proposed for diagnosis of frailty ( 31 , 32 ) .
in contrast to multi - item frailty scales , 4-meter gait speed , and to a lesser extent handgrip strength , has been advocated as a single - item measure of frailty that often outperforms more elaborate and time - consuming scales .
chaudhry et al ( 33 ) showed that in hf patients slow gait speed and weak grip strength were powerful predictors of hospitalizations , and in a recent study , we found that slow gait speed is independently associated with death , hospitalization for hf , and all - cause hospitalization in older hf patients ( 34 ) .
fumagalli et al ( 35 ) have suggested that af may be a marker of frailty in the elderly , and marzona et al ( 36 ) have reported a loss of independence in performing activities of daily living in a follow - up study of af elderly patients .
cognitive impairment is also frequent in older hf and af patients ; both hf and af represent risk factors for significant cognitive decline , through a multitude of pathways including a hypercoagulable and proinflammatory state , thromboembolic events , cerebral microinfarcts and microbleeds , cerebral hypoperfusion with consequent chronic hypoxic injury secondary to impaired cerebrovascular reactivity , reduced cardiac output combined with hypotension , and cycle length beat - to - beat variability ( 37 ) . the mini mental status examination ( mmse )
scores < 24 are suggestive of dementia , but the mmse has a low sensitivity for mild ci . the montreal cognitive assessment ( moca )
was developed as a screening tool for early cognitive decline , and was found to have a sensitivity of 90% in identifying mild ci compared with a sensitivity of only 18% with the mmse ( 38 ) .
vascular ci , as seen in af patients with stroke and transient ischemic attack , is associated with deficits in executive function , attention , and speed of information processing more than other domains , and moca has been shown to be superior to the mmse in identifying ci . in older patients , previous stroke is associated with a two - fold increase in the risk of developing dementia , but af is thought to play a role in cognitive decline beyond stroke ; it is less clear whether this association is directly related to af itself or to an aging population with multiple comorbidities ( 39 ) . in a subgroup of patients of the cardiovascular health study ( mean age > 65 years ) , thacker et al ( 40 )
found that mmse scores declined faster after incident af compared with no prior af ; the 5-year decline in mean mmse score from age 80 to age 85 was -6.4 points for participants without a history of af , but was -10.3 points for participants experiencing incident af at age 80 . a similar link among af , ci , and
recently , alosco et al ( 41 ) examined the associations among af , cognitive function , and cerebral perfusion in 187 patients with hf and found that hf patients with af exhibited worse global cognition , memory , and cerebral blood flow velocity and that decreased cerebral blood flow velocity predicted worse cognition in multiple domains in these patients , but not in those with heart failure and no af . in a cohort of patients with newly diagnosed hf from the cardiovascular health study ( mean age 78.7 years ) , 23% subsequently developed disability .
factors independently associated with disability included impaired gait speed ( hr 2.29 , 95% ci 1.34 - 3.90 ) ; impaired cognition ( hr 1.87 , 95% ci 1.14 - 3.05 ) ; and depressive symptoms ( hr 1.72 , 95% ci 1.04 - 2.83 ) , suggesting that geriatric variables affect prognosis and quality of life in older hf patients ( 42 ) . in a recent observational survey
, we studied the relation between af , ci , frailty and disability in 331 elderly hf patients aged > 70 years ( mean 78 6 ; range 70 - 93 ; 43% women ) ( 43 ) .
gait speed was used as a marker of frailty and measured on a 4-meter distance at usual pace .
ninety - eight patients ( 30% ) had af at enrollment and 20 ( 6% ) had a history of paroxysmal / persistent af .
af patients were more frequently women with severe valvular disease , preserved lvef , and less frequently on beta - blockers .
a cmmse < 24 was present in 19.6% of patients and a geriatric depression scale ( gds)-15 score > 6 in 51.4% .
patients who had af performed significantly worse on the cmmse than those who had not .
af was significantly associated with disability in either basic or instrumental activities of daily living ( fig .
3 ) . on multivariable analyses , af emerged as independently related to ci ( odds ratio [ or ] 1.909 , [ 1.072 - 3.397 ] ; p = 0.028 ) , and to reduced gait speed ( or 4.366 [ 2.104 - 9.060 ] ; p<0.001 ) .
comprehensive geriatric assessment variables according to the presence of atrial fibrillation the effects of oac on cognitive decline , excluding stroke reduction , has not been well established ( 37 ) .
available data on the benefits of vkas are controversial ( 44 ) , and there is no consensus on the effects of this therapy on cognitive function among patients with af .
in some studies , the use of oacs did not affect cognitive decline while others found a trend toward an association .
data from the atrial fibrillation clopidogrel trial with irbesartan for prevention of vascular events ( active - w ) found that among af patients with a mean chads2 score of 2 on warfarin , cognitive dysfunction was associated with lower time in therapeutic range ( ttr ) of anticoagulation , suggesting that maintaining therapeutic anticoagulation may reduce cognitive decline .
there are no studies examining the cognitive effects of the noacs ( dabigatran , rivaroxaban , and apixaban ) ; however , because they mitigate the challenges of ttr , there has been speculation that they may be able to slow or reverse cognitive decline among af patients .
polymedication is common in patients with af , particularly in the elderly population . in a danish
observational study , 53% of af patients took more than five drugs / day ( 45 ) , while an analysis of the rocket af study showed that 36% of patients were on 0 to 4 medications , 51% were on 5 to 9 , 13% were on 10 ; patients taking 10 medications daily were older and showed a trend toward a higher bleeding risk ( hr 1.46 , 95% ci [ 1.29 - 1.64 ] , p = 0.81 ) ( 46 ) . hf also requires complex medication regimens due to the multiple therapeutic targets that exist and the need for routine symptomatic management ; most studies have found medication non - adherence rates between 40% and 60% in patients with hf ( 47 ) .
having multiple conditions also decreases self - efficacy in performing specific self - care tasks such as medication taking ( 48 ) .
polymedication and complexity of treatment are also associated with poorer medication adherence and poor compliance .
it is well known that many medications interfere with dose response to vkas ; amiodarone , antibiotics such as quinolones and macrolides , antifungal agents , nonsteroidal anti - inflammatory drugs , selective serotonin reuptake inhibitors , omeprazole , and lipid - lowering agents are just some of the drugs that should be used with caution . although noacs are less prone to drug interactions , p - glycoprotein and cytochrome p450 3a4 inhibitors like verapamil , amiodarone , rifampicin , antiretrovirals , and azole antimycotics should be prohibited or used with great caution .
kidney function declines with age and chronic kidney disease ( ckd ) is common in patients with af , and increases both the risk of thromboembolic and bleeding events ( 49 ) .
the efficacy of oac therapy for stroke prevention has been demonstrated in patients with moderate ckd , but a prospective cohort study reported that patients with severe ckd ( estimated glomerular filtration rate [ egfr ] < 30 ml / min per 1.73 kg / m ) had less ttr ( international normalized ratio [ inr ] 2 - 3 ) , had a higher risk of over - anticoagulation ( inr > 4 ) , and required lower warfarin dosing when compared with patients with egfr > 30 ml / min per 1.73 kg / m ( 50 ) .
vkas are associated with increased calcification of renal and other arteries ; vascular calcification , arterial damage , and decline in renal function may be triggered by the inhibition of the vitamin k - dependent protein matrix gamma - carboxyglutamic acid ( gla / mgp ) by vk ( 51 ) .
bhm et al ( 52 ) analyzed changes in gfr during long - term treatment with warfarin or dabigatran in patients enrolled in the re - ly trial .
after an average of 30 months , the mean decline in gfr was significantly greater with warfarin compared with dabigatran 110 and 150 mg bid .
it is important to consider that different noacs are eliminated via the kidneys to different degrees : 80% for dabigatran , 50% for edoxaban , 33% for rivaroxaban , and 27% for apixaban ; this results in different plasma concentrations across the spectrum of creatinine clearance and underlies the advice to reduce the doses of each of the noacs in patients with moderate ckd ( 53 - 54 - 55 - 56 - 57 ) , and should be considered in the selection of the appropriate noac in the individual elderly patient . in a post - hoc analysis of the aristotle trial , where most patients aged 75 years ( 89% ) had impaired renal function , halvorsen et al ( 25 ) evaluated the occurrence of stroke or systemic embolism and major bleeding in relation to renal function and showed that the benefits of apixaban compared with warfarin were consistent across the range of egfr , including in the elderly . when considering dialysis patients , vkas represent the most common approach for reducing the risk of stroke in this population .
however , current evidence based on observational studies have provided conflicting results and clinicians are wary of their potential to increase the high risk of bleeding during dialysis . at the moment
, none of noacs can be safely prescribed in dialysis patients because of their potentially dangerous accumulation and the lack of sufficient experience ( 58 ) .
adherence is crucial to achieve the optimal safety and efficacy of oac therapy in af patients .
in fact , up to 40% of patients taking vkas withdraw treatment after one year of therapy and up to 30% have suboptimal adherence ( 59 ) .
reasons for poor adherence in the elderly include lack of support ( e.g. caregiver ) , lack of disease knowledge , and confusion or physical difficulties associated with taking medicines and polypharmacy .
in addition , a perceived high risk of falls or bleeding reduces the prescription rate of oral anticoagulation by general practitioners .
oacs affect quality of life , requiring frequent laboratory testing and attention to food and interactions , thus contributing to poor adherence . a prospective study in 220 inpatients aged
> 70 years with af reported that frail patients were less likely to receive warfarin than non - frail patients on admission and discharge , with a higher stroke risk over 6 months of follow - up ( risk ratio 3.5 , 95% ci 1.0 - 12.0 , p<0.05 ) ( 60 ) .
as most patients with af are frail elderly with comorbid conditions , disability , and ci , treatment with oacs may present special challenges .
af is frequently associated with disability in either basic or instrumental activities of daily living , particularly in dependence in taking medications and use of transportation ( 43 ) .
these dependencies , combined with frailty and cognitive deficits , may increase the risk of falls with subsequent major injuries , need for surgery , and bleeding , and may seriously affect the patient s skills to safely manage oac therapy .
moreover , all of these conditions represent a risk factor for impaired self - care and low adherence , and , as a consequence , these patients may not take medications as prescribed and may be unaware of drug or food interactions , especially in the absence of a caregiver . in an analysis of the active - w study ,
patients with scores < 26 had more vascular events ( 6.7% vs. 3.6%/year ) and more bleeding ( 9.6% vs. 7%/year ) . after controlling for ttr , mmse
no longer conferred increased risk , suggesting that if improved anticoagulation was provided , vascular events and bleeding would be reduced ( 61 ) .
it is not clear if , in cases of suboptimal adherence , vkas may be considered to be safer than noacs because of their longer half - life and because of the planned frequent inr monitoring , helping to improve adherence through systematic laboratory control .
since recently introduced noacs have a more favorable risk - benefit profile and a wide therapeutic window , a predictable anticoagulant effect , and few interactions with food and other medications , these drugs may be preferable to vkas in many frail elderly patients , especially in those at higher risk of falls ( 23 , 25 ) .
furthermore , noacs are very simple to administer and monitor and may be associated with better adherence and safety in patients with ci and mobility impairments .
the clinical picture of the older hf patient with af is complex and heterogeneous with a higher prevalence of comorbidities , frailty , ci , and disability .
however , because of the association of mental and physical impairment with non - administration of oacs , screening for simple geriatric variables in clinical practice may allow better strategies for intervention in this high - risk population .
there is also a need for large multicenter longitudinal studies to examine the effects of vkas and noacs on long - term cognitive function and frailty .
an individualized approach matching the particular noac to the individual patient , taking into consideration the risk of bleeding and other comorbidities , i.e. renal dysfunction , should be taken rather than a generalized one drug fits all approach in elderly adults . | backgroundatrial fibrillation ( af ) and heart failure ( hf ) , two problems of growing prevalence as a consequence of the ageing population , are associated with high morbidity , mortality , and healthcare costs .
af and hf also share common risk factors and pathophysiologic processes such as hypertension , diabetes mellitus , ischemic heart disease , and valvular heart disease often occur together . although elderly patients with both hf and af are affected by worse symptoms and poorer prognosis ,
there is a paucity of data on appropriate management of these patients.methodspubmed was searched for studies on af and older patients using the terms atrial fibrillation , elderly , heart failure , cognitive impairment , frailty , stroke , and anticoagulants.resultsthe clinical picture of hf patients with af is complex and heterogeneous with a higher prevalence of frailty , cognitive impairment , and disability . because of the association of mental and physical impairment to non - administration of oral anticoagulants ( oacs ) , screening for these simple variables in clinical practice may allow better strategies for intervention in this high - risk population .
since novel direct oacs ( noacs ) have a more favorable risk - benefit profile , they may be preferable to vitamin k antagonists ( vkas ) in many frail elderly patients , especially those at higher risk of falls .
moreover , noacs are simple to administer and monitor and may be associated with better adherence and safety in patients with cognitive deficits and mobility impairments.conclusionslarge multicenter longitudinal studies are needed to examine the effects of vkas and noacs on long - term cognitive function and frailty ; future studies should include geriatric conditions . |
melanoma most commonly arises from epidermal skin melanocytes ( cutaneous melanoma ) , but primary tumors can also be found lining the choroidal layer of the eye ( uveal melanoma ) or the mucosal surfaces of the respiratory , genitourinary , and gastrointestinal surfaces .
similar to other tumors , the progression stage of melanoma is predictive for therapeutic success .
early stage melanomas ( thin tumors ) result in a 97% 5-year survival rate of the patients , after surgical removal .
conversely , advanced melanoma patients , comprising metastasis in regional lymph nodes or other organs , face 5-year survival rates of less than 10% .
due to the intrinsic tendency of melanoma to early metastasis , even small primary tumors have already led to metastasis and a substantial portion of diagnosed melanoma cases are of late progression stages .
treatment of advanced or metastatic melanoma has proven a challenge , as the conventional therapeutic approaches failed to translate into improved or significant survival rate in phase iii clinical trials .
newer treatments were established in the last years that elicit unprecedented response rates in late stage melanoma , for example , up to 80% in the case of braf inhibitors .
however , almost all tumors become resistant within months , and the treatment is available only for a subset of melanomas . altogether , despite substantial improvements in therapeutic options during the last years , there is still an urgent need for alternative approaches .
based on clinical and histopathological features melanoma cancer cells undergo four sequential phases before reaching metastasis .
these phases ensue from several genetic , epigenetic , and microenvironmental , modifications . in the last decade
, a number of reports have brought significant insight into melanoma genetics and molecular markers , which are essential for the development of therapies , and in particular targeted regimens .
this paper will focus on melanoma targeted gene delivery ; we aim at providing a general view on melanoma - targeting ligands , and other forms of specifically driving gene expression , reported in the literature , as well as review the most recent and/or relevant nucleic acid therapeutics employed in this field .
the current paper will not dwell upon melanoma mutations or cancer transcriptional regulators ( for reviews , see [ 4 , 5 ] ) .
instead , the following melanoma section serves rather as a comprehensive overview on the key players of the neoplasia , which is essential for the understanding of targeted therapies .
presently , it is generally believed that melanomagenesis instigates from alterations in multiple molecules or pathways rather than a single high - risk melanoma loci .
moreover , melanoma progression is a dynamic process involving several steps , each requiring the activation of different genes .
first , normal melanocytes undergo genetic alterations that lead to their transformation into benign nevi .
benign nevi differ from normal melanocytes in that they have initially proliferated in the basal layer of the epidermis ; however , they entered a long - term dormant status due to the lack of additional oncogenic alterations .
for example , the most frequent activating mutation in the braf gene occurs in the same frequency in nevi , where it causes a dormant status called oncogene - induced senescence .
this progression stage is characterized by noninvasive horizontal growth and spread through the epidermis and has been termed as radial growth phase ( rgp ) .
this phase has been termed as vertical growth phase ( vgp ) . for invasion ,
alterations like loss of adhesive molecules together with an increase in extracellular matrix degrading enzymes are characteristic .
for metastasis , cell populations have to migrate to distant locations . for this , cells have to acquire more alterations that enable the complex processes underlying metastasis .
these processes involve tissue invasion , entering , and evasion of blood or lymphatic vessels to reach distant location but also survival and proliferation at distinct locations .
whole genome sequencing revealed that melanoma is the tumor type with the most dna mutations many being typical for uv - induced mutations . despite the plethora of dna alterations ,
two gene mutations were found to be rather common in melanoma . a general overview on these mutations and their key players are schematically represented in figure 1 . with respect to mutation frequency , the mitogen - activated protein kinase ( mapk ) pathway plays a central role in melanoma .
activation of growth factor receptors leads to activation of ras molecules which activate in a downstream phosphorylation cascade raf , mek , and erk kinases .
ras molecules , comprising hras , kras , and nras , are small gtpases or g proteins , and activating mutations in nras are found in 10%20% of melanomas .
a single nucleotide mutation in braf at amino acid 600whereupon a valine ( v ) aminoacid is replaced by glutamic acid ( e)represents the most common mutation in braf .
this mutant braf leads to an alternative protein structure and to a constitutive active protein .
the outstanding importance of the ras / raf signaling pathway is documented by the observation that braf and nras mutations exclusively nras or braf
is mutated in a tumor together are found in over 80% of melanomas and by inhibitors of mutated braf that are clearly effective in melanoma therapy .
interestingly , braf has also been reported in melanocytic nevi [ 1012 ] , which rarely develop into melanoma .
nevi are described to be senescent , and , similarly , expression of braf in melanocytes induces oncogene - induced senescence .
these findings imply that braf mutations are involved in the first transition state of melanoma progression .
hence , this mutation per se is insufficient to drive tumorigenesis , rather additional alterations are required to avoid dormancy .
several pathways have been shown to cooperate with ras / raf signaling and to reduce ras / raf - mediated senescence .
dna damage due to oncogene - induced dna replication stress has been proposed as an important mechanism of senescence .
accordingly , molecules involved in dna damage signaling have been shown to promote oncogenesis together with braf , for example , the loss of p53 .
pten is a tumor suppressor gene that negatively modulates signal transduction via phosphatidylinositol phosphatase ( pip3 , a cytosolic second messenger ) .
allelic loss or altered expression of pten can be observed in tumors . in melanoma , this lost / modified expression is present in 20%/40% of melanoma tumors , respectively [ 15 , 16 ] . in a mouse model , it was shown that expression of braf in melanocytes leads to benign lesions that do not progress to melanoma . however , when pten was silenced , these mice developed metastatic tumors with high penetrance .
regarding the family history of melanoma , a two - fold risk increase has been reported , and it was associated to the 9p12 chromosome . in 1994 ,
the cyclin - dependent kinase n2a ( cdkn2a ) gene was identified , and it is now hold as a high - risk melanoma locus .
the cdkn2a gene encodes for two tumor suppressor proteins , p16 and p14 , involved in cell cycle and apoptosis , respectively .
explicitly , p14 directly promotes the degradation of human double minute 2 ( mdm2 ) .
accordingly , inactivation of p14 leads to increased mdm2 levels leading to increased degradation of p53 .
the other product of the cdkn2a locus , p16 , prevents cell cycle progression by binding to cdk4/6 and through a series of events prevents the release of e2f1 ( a transcriptional inducer of s - phase genes )
. mutations of p16 , and similarly of cdk4 gene [ 23 , 24 ] , can therefore lead to increased cell cycle progression .
however , despite the contribution of cdkn2a mutations for oncogenesis , the absolute risk of melanoma in mutation carriers is still highly shaped by environmental and pedigree factors . in close relation to pedigree structure
is skin pigmentation ; the positive connection between light skin color and melanoma risks is well known .
melanocortin-1 receptor ( mc1-r ) is responsible for the cutaneous pigmentation , and , interestingly , it has been reported as being overexpressed in both melanotic and amelanotic melanomas .
there are two forms of epidermal melanin : eumelanin ( with a black - brown color ) and pheomelanin ( red - yellow color ) .
the synthesis of eumelanin in charge of uv attenuation is stimulated by the activation of the mc1-r , through the binding of the tridecapeptide -msh , or -melanocortin stimulating hormone [ 2729 ] .
the binding of -msh results in an increment of camp , which in turn upregulates the microphthalmia - associated transcription factor ( mitf ) inducing the transcription of pigment synthetic genes and the production of eumelanin .
mitf , on the other hand , is also involved in the regulation of the cell cycle and proliferation , and few variants of the gene have been found in melanoma patients [ 31 , 32 ] . in particular ,
mitf(e318 k ) was reported to represent a gain - of - function allele for the gene , supporting mitfs role as an oncogene .
however , mitfs expression in melanoma metastasis is yet to be clarified , as there are also studies showing that downregulation and ablation of this gene create a more invasive phenotype in vitro and increase tumor growth in vivo , respectively .
the transcription factor activator protein-2 ( ap2 ) has been suggested as a major key player in the transition from rgp to vgp .
similar to several other mediators , ap2 also modulates a variety of cellular processes , including cell growth and apoptosis . in tumors , ap2 acts as a tumor suppressor , and high cytoplasmatic to nuclear expression ratio was shown to correlate with poor patients ' prognosis [ 35 , 36 ] . in particular
, the promoters for the adhesion molecule mcam / muc18 , which is overexpressed in tumors , and tyrosinase kinase receptor , c - kit ( silenced in 70% of metastatic tumors ) , have ap2 binding sites .
ap2 has been described to directly bind to mcam / muc18 promoter and to inhibit its transcription , whereas it promotes c - kit expression .
therefore , the loss of this transcription factor during melanoma results in high mcam / muc18 levels and c - kit downregulation .
in addition , the loss of ap2 was also appointed as a probable cause for the upregulation of the g - protein - coupled receptor protease activated receptor-1 , par-1 [ 10 , 39 ] . in par-1 promoter region
, there are two binding complexes for ap2 and sp1 . in normal melanocytes , ap2 binds to par-1 inhibiting its transcription . however , upon melanoma progression , the levels of ap2 decrease , and sp1 binds to the par-1 promoter instead , driving its expression .
ras , phosphoinositide-3 kinase ( pi3 k ) , and mapk pathways are all signaling events downstream par-1 , and hence closely related to tumor progression . during the metastatic process ,
following evasion into the blood circulation , tumor cells adhere to the endothelium at distant sites , and herein adhesion molecules are necessary .
together with selectins , integrins have been found to play crucial roles in these steps .
integrins are a family of transmembrane glycoproteins that mediate cell - cell and cell - matrix adhesion .
it is therefore expected that their expression pattern changes during tumor growth , metastasis , and angiogenesis .
in particular , v3 and 41 ( very late activation antigen-4 , vla-4 ) have been reported as overexpressed in numerous cancer types [ 41 , 42 ] and have served as therapeutic targets .
vla-4 has been shown to be used by malignant melanoma cells to adhere to the endothelium ( binding to the ligand vcam-1 ) [ 43 , 44 ] , and to promote transmigration [ 42 , 45 ] and metastasis [ 46 , 47 ] .
overall , melanoma incidence has been increasing over the years , reaching an annually increase of 3.1% during the past two decades .
, unresectable advanced melanoma is characterized by an aggressive behaviour , fast spread and metastasis , and a strong resistance to chemotherapy . therefore , and in spite of the extensive research , the current prognosis for patients with advanced melanoma is limited . the earlier conventional chemotherapeutic treatment approved by us food and drug administration ( fda ) , dacarbazine ,
alternative chemotherapeutic agents include fotemustine , temozolomide , paclitaxel ( often in combination with carboplatin ) , and docetaxel all not yielding larger progression - free survival ( pfs ) or overall survival ( os ) than dacarbazine [ 50 , 51 ] .
generally , chemotherapeutics suffer from a lack of targeting specificity ; their low molecular mass results in easy and fast body secretion , and thus the need of increased doses , which leads to inevitable toxicity .
similarly , immunotherapy based on interleukine 2 ( il-2)also fda approved has comparable response rates , and it is further restricted by the ensuing multiorgan toxicity , requiring management in specialized cancer centers .
although combined therapies resulted in higher response rates , they still failed to translate into improved survival , with no impact on pfs or os compared to dacarbazine alone [ 1 , 52 ] .
another alternative is the combined treatment with the cytokine tnf in combination with the alkylating drug melphalan .
although highly successful , this treatment is limited to local treatment of melanoma in - transit metastases in limbs by isolated limb perfusion due to live threatening systemic toxicity of therapeutically active tnf doses . in the last decade , much progress was achieved due to the discovery of mutations in the braf gene .
this led to the development of therapies interfering with ras / raf signaling and to specific braf inhibitors . in august 2011 ,
an alternative melanoma regimen , for patients positive for braf mutations , was brought into the market with the fda approval of vemurafenib ( zelboraf , plexxikon / roche ) . in phase ii and iii studies ,
vemurafenib showed a response rate up to 50% , yet the response duration varied between the phase studies [ 5456 ] . in addition , vemurafenib induces acanthopapillomas , keratoacanthomas , and cutaneous squamous cell carcinomas in the early treatment [ 57 , 58 ] .
unfortunately , these unprecedented response rates are limited by the fact that almost all tumors become resistant to this therapy and the overall survival of patients was 6.7 months .
in addition , the treatment is only available for 50%60% of patients with mutated tumors because it is not effective in tumors with wildtype braf .
nevertheless , this success has led to the development of other ras / raf pathway inhibitors , for example , for mutated braf or downstream kinases like mek .
alternative activation of ras / raf pathway has been proposed as a resistance mechanism . in line with this ,
the combination of braf inhibition with mek inhibition led to an improved survival of 9.4 months .
other new therapies that add to the therapeutic options for melanoma patients are immunotherapies . an anti - ctla-4 antibody ( ipilimumab ) improved survival of stage ii and iv melanoma patients ( 10.1 versus 6.4 months ) .
cytotoxic t - lymphocyte antigen 4 ( ctla-4 ) inhibits t - cell responses and respectively , ctla-4 blockade promotes immune responses and antitumor activity . in an early analysis of anti - pd - l1 antibody ,
similar to ctla4 , pd-1 reduces immune activation , and its inhibition can lead to reactivation of immune responses . altogether , even with respect to the recent advances in melanoma therapy , the high resistance rates and the restriction to certain patient subgroups demonstrate that there is still an urgent need to develop alternative therapies .
as also observed for other tumor entities , melanoma treatment with low molecular weight chemotherapeutic drugs often results in the rise of resistant cancers cells , especially in case of relapsed disease .
a well - known mechanism of resistance is the elevated expression of multidrug transporter proteins , like p - glycoprotein , which actively pump chemotherapeutics out of the cell . here
as an example , the attachment of chemotherapeutics to polymers via reversible covalent bonds helps to overcome this type of resistance ( for a recent review see ) .
also , biotherapeutics , such as antibodies , have been successfully applied in melanoma therapy ( see above ) , but also here resistance can occur , for example , when blocking of one cellular pathway responsible for cancer cell proliferation can be replaced by another . in this case , the application of therapeutically active nucleic acids comes into play .
firstly , they exhibit a relatively high molecular weight , which prevents resistance mediated by p - glycoprotein upregulation .
secondly , nucleic acids can be designed to affect only malignant cells , for example , by using promoter elements being only activated in tumors , or as rna oligonucleotides ( like sirna ) , which will enable the knockdown of a specific protein overexpressed in tumor tissue .
furthermore , the delivery of more than one sirna targeting different pathways can prevent tumor resistance by blocking different resistance or escape strands .
last but not least , nucleic acid delivery permits systemic delivery of toxic agents , such as diphtheria toxin a or tumor necrosis factor ( tnf ) , as they only become toxic after transcription in the target cell .
solid tumors exceeding a certain size rely on a functional blood supply for access to nutrients and oxygen .
in contrast to nonmalignant tissues , tumor vasculature often exhibits a leaky appearance , which in principle also allows nanosized particles to reach tumor cells .
being packed into nanoparticles or polyplexes , nucleic acids can be protected from nucleases which are present in the bloodstream .
nevertheless , systemic delivery of nanopharmaceutics offers several pitfalls and obstacles , such as aggregation with blood cells , undesired adherence to the vessel wall , or opsonization with plasma proteins followed by clearance through tissue macrophages ( a key component of the reticulo - endothelial system ) .
blood proteins interact both with negatively and positively charged nanosystems , whereas a neutral surface charge enables , in principle , blood circulation , as it has been shown for small nanocrystals , so called quantum dots .
alternatively , nanosystems can be decorated with hydrophilic polymers , which , owing to their excessive hydration , shield the particles ' surface charge , hereby preventing the aggregation with protein components . from the group of hydrophilic polymers , like
n-(2-hydroxypropyl)methacrylamide ( hpma ) , hydroxyethyl starch ( he s ) , or polyethyleneglycol ( peg ) , peg is the most commonly used one .
in addition , targeting entities can be used to direct the nanocarrier to specific cells .
commonly , these are ligands that bind to receptors , or other cell surface molecules , that are overexpressed in tumor cells .
macromolecular drugs , which exceed the renal excretion limit and are able to circulate in the blood stream , can benefit from the so - called enhanced permeability and retention ( epr ) effect : nanopharmaceutics accumulate in tumor tissue as they can penetrate the leaky vasculature but are retained within the tumor tissue due to incomplete lymphatic drainage .
this tumor deposition is a prerequisite for all steps that follow : binding to and internalization of the particles into target cells .
the latter can be promoted by the incorporation of the earlier mentioned cell - binding ligands into the carrier system .
figure 2 summarizes the limitations in nucleic acids delivery , the solutions for such limitations , and the therapeutic advantages of nucleic acid nanosystems .
directed approaches are of special interest as they have the potential to specifically distress malignant cells causing increased local concentrations of the active agent and avoiding undesired side effects .
tracking down melanoma - associated molecular targets involves identifying signaling pathways ' key players , earlier described , as much as cancer cell surface markers . in particular , for gene therapy , cell surface markers are important , and these abide with the conception of a treatment addressing multiple melanoma subgroups as cells with different mutations can still exhibit common surface markers .
ergo , it is crucial to identify critical and idiosyncratic targets for these cells .
already reported in the early seventies , one of the largely explored targets is the melanocortin-1 receptor ( mc1-r ) , which is also overexpressed in numerous melanoma cases .
mc1-r belongs to a class of g - coupled protein receptors ( mc1-r mc5-r ) , where the different receptors allocate in different tissues , reflecting their functions .
while mc1-r is found in hair and skin , mc2-r is localized in adrenal glands , whereas mc3-r and mc4-r are in hypothalamus and mc5-r in kidneys .
however , owing to their similarity their binding domains may share common affinities , and certain peptide motifs can bind to several receptors . for targeting purposes , the most well - known and used mcr-1
the substitution of methionine in position four by norleucine ( nle ) and of phenylalanine for its d - counterpart in position seven ( d - phe ) renders this peptide with higher affinity and resistance to enzyme degradation than its native form .
however , ndp--msh was shown to have a strong nanomolar binding affinity towards mc3-r , mc4-r , and mc5-r , and , for gene delivery , it is crucial to decrease off - target effects .
aiming at the design of ligands suitable for micelle conjugation , and with an adequate selectivity to mc1-r , barkey et al .
this paper allowed the following conclusions : ( 1 ) free rotation of carbons that compose the peptide 's biding motif seems to be required for mc1-r avidity ; ( 2 ) alkyl modifications , for the attachment of triblock polymer micelle , at the n - terminal of the peptide , did not affect binding affinity in the short four amino acid peptide ; ( 3 ) for peptides twice as long , c - terminal modifications for micelles ' attachment did not altered binding affinities . in addition
, the authors have synthesized micelles conjugated to the short peptide version [ 4-phenylbutyril - hist - dphe - arg - trp - gly - lys(hex-5ynoyl)-nh2 ] , through a peg linker . and importantly , in vitro cell - uptake studies showed the ability of conjugated micelles to selectively bind to mc1-r receptor , and , whether due to multivalent interactions or other factors , the micelles had higher avidity for the receptor than the ligand alone . nevertheless , further studies ( i.e. , by flow cytometry or confocal laser microscopy ) to quantify the uptake of these conjugated micelles are needed to better evaluate the delivery efficiency of this platform .
more recently , -msh peptide has been conjugated to a nanoplatform based on the heavy chain of the human protein ferritin ( hft ) .
ferritin can be used to build a hollow nanocage that can transport materials such as fe3o4 , co3o4 , mn3o4 , pt , and au and hence be used for imaging and therapeutic purposes .
unfortunately , the authors have not analyzed the in vivo distribution of their nanoparticles , and the targeting efficiency was evaluated by immunohistochemistry in the tumor tissue in relation to normal skin . in a similar approach to that of hft nanocages , lu and
collaborators have used hollow gold nanospheres , conjugated to ndp--msh , aiming at cancer photothermal ablation . in this study ,
nude mice were subcutaneously inoculated with b16/f10 murine melanoma cells , and the nanoparticles were administered intravenously .
the authors have collected different organs and were able to show the targeting effect by the ndp--msh - gold nanospheres . interestingly , targeting of mc1-r by -msh peptide has been mostly used in radionuclide therapy studies and for diagnostic purposes . currently , 2-[f]fluoro-2-deoxy - d - glucose ( f - fdg ) is the only radioactive probe used in the clinic to detect melanoma .
be that as it may , f - fdg is an unspecific positron emission tomography ( pet ) imaging agent with poor sensitivity towards micrometastatic sites [ 78 , 79 ] , a fact that underlines the general insufficiency in melanoma targeting .
regarding mc1-r targeting , yubin miao and thomas p. quinn 's extensive work is of particular interest , reporting on two generations of an ndp--msh - based peptide used for melanoma imaging by single - photon emission - computed tomography ( spect ) and more recently by pet .
what distinguishes the two -msh peptide generations is mostly the peptide 's length , being twelve aminoacid - long in the first generation ( cycmsh ) [ 8082 ] and six in the second ( cycmshhex ) [ 83 , 84 ] . in both generations , the peptide is cyclized ( cyc ) , and the mc1-r binding motif ( his - dphe - arg - trp ) is conserved .
the peptides have also undergone structural modifications concerning the aminoacid linkers , which are used to support the peptide cyclization and bridge the targeting ligand and the radiometal chelator .
interestingly , the authors have observed that the exchange of single aminoacids in these linkers , and the introduction of glygly linker between the chelator and the peptide resulted in improved melanoma targeting , with decreased renal excretion and liver uptake of the radiolabelled peptide in b16/f1 melanoma - bearing c57 mice .
these studies underscore the structural role of the targeting moiety but also of the integral component being delivered . in other words ,
the addition of a targeting entity to a carrier does not necessarily suffice for efficient deliver ; the number of peptides conjugated to the delivery platform , the site of conjugation and the size and type of the linker play an important role .
after the discovery of adhesion molecules as mediators of tumor metastasis , the identification of their binding motifs opened the possibilities for targeted therapies .
several peptide fragments have been employed to target these mediators , either as antagonists or as ligands for drug delivery purposes .
the formation of new vessels and , by serving as receptor for extracellular matrix proteins , it mediates migration of endothelial cells into the basement membrane , and regulates their growth , survival , and differentiation .
it is therefore no surprise that such integrin is found upregulated in different tumor cells , where it is involved in processes that govern metastasis .
the integrin 's binding peptide motif has been identified in 1990 arginine - glutamine - aspartate or rgd but studies that followed have shown that the cyclic version of rgd ( crgd ) has higher binding affinities towards the integrin [ 86 , 87 ] . either alone or in combination with other ligands ,
crgd has been conjugated to several nanocarriers for both diagnostic and therapeutic purposes [ 8890 ] .
another integrin reported to have a dominant function in the metastatic spread is 41 or vla-4 .
okumura , and more recently schlesinger , have shown , in different settings , that inhibition of vla-4 by natalizumab ( an antibody against 4 integrin ) significantly decreased melanoma lung metastases in murine models [ 42 , 44 , 122 ] . in 1991 ,
makarem and humphries have identified the leucine - aspartate - valine ( ldv ) sequence as the integrin 's motif , and a few years later , vanderslice et al .
have reported on a series of cyclized peptides based on ldv that were assayed for the inhibition of the integrin . however , and despite the numerous reports relating this agent to tumor metastasis , and to melanoma in particular , most of the literature relies on the ldv sequence as an antagonist , rather than for deliver purposes , where , to our knowledge , there is only one paper reporting on in vitro studies .
indeed , vla-4 is found in multiple leukocyte populations ; vla-4 is a vital receptor of leukocytes , and it is involved in the immune response .
hence , a systemic application of vla-4 inhibitors , or binding peptides , could induce undesired partially immunosuppressive effects . in this context , the application of transcriptional - targeting strategies could potentially prevent off - target effects and prove this ligand a promising tool .
in fact , tissue - specific elements as components of the dna vector can provide a tight control over gene expression and complement and strengthen targeted - delivery .
commonly , tumor cells ' surface markers entail receptors that are also present in nontumor cells but are rather overexpressed in their malignant form .
this is the case for both the integrins here described , but also the transferrin receptor all used as melanoma targets .
therefore , off - target effects can occur , and for gene delivery purposes , tissue - specific control elements are an elegant way to bypass undesired side effects .
these control elements consist of nucleic acid sequences that are recognized by proteins or other nucleic acids , which hereby regulate gene expression . for the case of melanoma , tissue specific promoters
have been described , and these include tyrosinase [ 9395 ] and melanoma inhibitory activity ( mia ) [ 96 , 97 ] .
micrornas are a class of short ( 2022 nucleotides long ) regulatory rnas , which are believed to regulate as many as 30% of all genes
. several micrornas are tissue - specific and fine - tune genetic circuits , some of which are critical for normal development , cellular differentiation , and normal cellular homeostasis .
if the target sequence and microrna have perfect complementarity , the mrna is eliminated by a rna degradation pathway . in the context of transcriptional control
, this means that a dna vector that contains specific mir - binding sites is only translated in cells where the mir in question is absent [ 125 , 126 ] . in tumor cells ,
several micrornas are deregulated , while mirs enrolled in cell homeostasis are downregulated those involved in cell proliferation and differentiation are upregulated . for the case of melanoma , mir let-7b , mir-193b , mir-34a , mir-155 , mir-205 , mir148 , mir-137 , and mir-152
have been found downregulated ( for a review on melanoma micrornas , see ) and can therefore be suitable targets for transcriptional regulation when expressed in normal tissue .
as opposed to conventional therapy , traditionally , that is , in the case of loss of function , gene therapy aims at permanent correction of a defected or missing gene by replacing with or providing , respectively , the corrected version for example , by the introduction of plasmid dna ( pdna ) .
ideally , this approach translates into a single treatment , or few initial treatments , rather than several ( or life long ) required to provide the patients with the functional form of the protein .
however , this permanent correction treatment has proven very challenging . in the last twenty years , new nucleic acids with attractive therapeutic properties were discovered , notably , sirna and micrornas .
small interference rna ( sirna ) has the ability to specifically silence protein expression an asset particularly valuable for antiviral and cancer regimens . in general ,
also mirna negatively regulates gene expression , although via two different mechanism depending on the degree of complementarity towards its mrna target .
nucleic acid - based approaches offer several advantages when compared to treatment with small molecules or proteins .
they can be seen as mostly inactive prodrugs , which are activated at the tumor site producing a therapeutically active protein or knocking down a specific target gene .
importantly , nucleic acid targeted delivery systems , preferably also relying in transcriptional targeting , decreasing off - target effects and toxicity , and permitting a systemic administration otherwise not feasible with a therapeutic agent with toxic properties . in parallel with new therapeutic nucleic acid tools , the last two decades brought insight into tumorgenesis in general and unveiled a plethora of therapeutic concepts against cancer ( figure 3 ) .
the following paragraphs will deal with different antimelanoma approaches based on nucleic acids . despite the apparent tumor tolerance , humoral and cellular immune responses
hence , whether the tumor grows as a result of stealth and nonrecognition or as the result of escape and immunological shaping , its recognition by the immune system can still be prompted .
indeed , at a later stage , during the progressive growth phase , tumors may become more immune - activating for varies reasons : damage or disruption of surrounding tissue , generation of reactive oxygen species , upregulation of stress protective factors , or death by necrosis or apoptosis .
however , at this stage , it is not known whether the tumor still needs to escape immune recognition , as it is unclear that these immune responses can cause tumor destruction .
therefore , a number of studies have focused in eliciting earlier and suitable tumor recognition by the immune system . in a nucleic acid therapy context , this transliterates into genetic immunization or dna vaccination : the delivery and transcription of a gene encoding antigens or immunestimulatory molecules that elicit an immune response . as an example , interleukine-12 ( il-12 ) has been used and studied in different animal models [ 104 , 105 ] .
il-12 is originally produced by mononuclear phagocytes and dendritic cells and is responsible for activating nk and cd4 t cells and inducing the production of high levels of interferon gamma ( inf- ) .
interestingly , il-12 has been described to increase antitumor immune responses [ 129 , 130 ] , and later studies investigated its suitability for a dna vaccine approach against melanoma .
il-12 effects appeared to be long lasting and efficient against tumor metastases , although not mainly mediated by inf- .
the murine studies also revealed moderate toxicity caused by il-12 , and while lower il-12-encoding pdna doses can be administered , ideally the gene expression should be controlled , regarding the tissue and the durability of the expression .
although dna vaccination against a strong melanoma tumor antigen should be possible , the authors have not seen an effect on lung metastases when using melanoma - associated glycoprotein 100 ( gp 100)/pmel17 pdna alone .
adjuvants appear to be necessary for a successful dna vaccination : the authors have seen an effect when the gp 100-pdna was administered together with il-12 , similar to other murine study where granulocyte - macrophage colony - stimulating factor was used .
alternatively , in a canine study , the developed vaccine was based on the human ( rather than canine ) gp 100 protein , where the human form of the antigen acted as adjuvant .
together with gp 100 , and for the case of melanoma , two more tumor genes have been described for dna vaccination : mart-1 and tyrosinase [ 108 , 109 ] . also , the expression of chemokines , such as monocyte chemoattractant protein-1 ( mcp-1 ) and interferon - inducible protein-10 ( ip-10 ) , can mediate an immune response . in particular , ip-10 as been described by sgadari et al . as an antitumor agent and found to promote damage in established tumor vasculature as well as tissue necrosis in a murine model for the human burkitt lymphomas .
based on this , and after their studies with il-12 , keyser and collaborators have investigated the efficiency of ip-10-encoding pdna therapy in murine melanoma models .
the authors have used two murine tumor models , whereupon cells have been injected subcutaneously ( originating a solid tumor ) or intravenously , inducing lung metastases .
when administered alone , and intramuscularly ( resulting in systemic circulation ) , ip-10-encoding pdna showed an antimetastatic effect , reducing the number of lung metastases as compared to the control - pdna treated group . when administered with il-12-encoding pdna , ip-10 pdna enhanced the il-12 effect , and decreased its earlier observed toxicity .
this anti - neoplastic effect of ip-10 has been attributed to the engagement of nk cells and the inhibition of angiogenesis and cell proliferation .
alternative antitumor strategies aim at a direct destruction of cancer cells , through the delivery of pdna encoding for a toxic protein dna - based strategies .
this is referred to as a suicide gene therapy or gene - directed enzyme prodrug therapy ( gdept ) , when the nucleic acid sequence encodes for an enzyme , which is not directly toxic but instead converts a nontoxic prodrug into a cytotoxic metabolite .
the first proof of principle of gdept was presented in the mid - eighties and involved the herpes simplex thymidine kinase ( hsv - tk ) and the prodrug ganciclovir ( gcv ) .
presently , hsv - tk as well as other approaches , such as diptheria toxin a chain ( dta ) , have been employed in the clinics , the most successful cases being reported in ovarian and prostate cancers [ 67 , 133 ] . as for melanoma treatments , hsv - tk has been the most commonly used [ 111113 ] , although there is no human clinical trial yet .
suicide gene therapy has also been proven effective when used in combined approaches , such as with cytokine - enhanced vaccine in a clinical trial involving canine melanoma patients . despite promising ,
this strategy is currently restrained by a poor delivery ; most nanocarriers are not as target - specific and efficient as required , and the toxic gene does not reach the tumor cells in efficacious concentrations .
a number of studies have instead focused on mediators of cell proliferation and differentiation , which are upregulated during tumorgenesis , aiming at their downregulation by means of sirna delivery [ 114 , 135137]these are rna - based approaches . as an example , based on the fact that in epithelial cells , n - cadherin induces changes in morphology of a fibroblastic phenotype ( rendering the cells more motile and invasive ) , the laboratory of laidler has investigated the outcome of n - cadherin silencing in human melanoma cell lines .
although the results suggest that n - cadherin positively affects the regulation of the cell cycle and proliferation through activation of the akt kinase pathway , further investigations are needed to describe the mechanism .
, upon the observation that thrombin receptor ( or protease - activated receptor-1 , par-1 ) is overexpressed in highly metastatic melanoma cell lines , has evaluated the therapeutic potential of sirna against par-1 .
the authors have observed a significant reduction of in vivo tumor growth as well as in the number of metastatic lung colonies .
this report showed that downregulation of par-1 decreased the expression of matrix metallopeptidase-2 ( mmp-2 ) , interleukin 8 ( il-8 ) , and vascular endothelial growth factor ( vegf ) , resulting in an overall decrease in angiogenesis and blood vessels . in 2010 , davis et al .
reported on the first human clinical trial ( including three melanoma patients ) on sirna therapy against melanoma .
the sirna targeted the m2 subunit of ribonucleotide reductase ( rrm2 ) , and the protein knock down was confirmed at the mrna level but not corroborated to the same extend by the protein analysis .
nevertheless , the fact that the authors used a delivery vector targeting the transferrin receptor without showing analysis of such receptor expression in melanoma cells was left to be explained .
of special interests are combinatorial strategies involving sirna delivery as these , similar to other combinatorial therapies , cause the most significant outcomes .
the authors targeted bcl2 ( an apoptosis regulator protein ) , which was reported to play a central role in the resistance of melanoma cells to chemotherapy [ 7 , 116 , 139 , 140 ] . by adding 5-triphosphate ends to their sirna
, the authors also activated innate immune cells , induced the expression of interferons , and caused specific cell tumor apoptosis .
these actions are a consequence of the recognition of 5-triphosphate ends by the cytosolic retinoic acid - induced protein-1 ( rig-1 ) and synergized with the silencing effects originated from sirna resulting in massive tumor destruction in the murine lung metastases .
first reported on a promising double stranded rna ( dsrna ) mimic polyinisine - polycytidylic acid ( pic ) .
importantly , the therapeutic effect of the dsrna was significantly increased when delivered in the form of a complex , together with polyethyleneimine ( pei)-[pic ] .
initially , the dsrna mimic was thought to engage toll - like receptors ( tlr ) , hereby mediating cellular tumor immunity . in turn
, further investigation studies showed that it mobilizes the endo / lysosomal machinery of melanoma cells , and through melanoma differentiation associated gene-5 ( mda-5 ) induces self - degradation by ( macro ) autophagy and apoptosis , following the mda-5-mediated activation of proapoptotic factor noxa .
interestingly , at the exact same time , mda-5 and noxa were also reported to play a role in interferon - independent apoptosis in human melanoma cells by besch and collaborators .
not only were these findings meaningful , opening new windows for cancer therapy , but also , in particular in the dama tormo studies , was the murine model used very suited , whereupon mice overexpressing hepatocyte growth factor ( hgf ) and carrying an oncogenic mutation in the cyclin - dependent kinase-4 [ ( cdk4 ) ] developed invasive melanomas in the skin following neonatal exposure to carcinogenics .
while a number of microrna has been described to play relevant roles in melanoma progression , only few in vitro studies have reported on the mirna potential for antimelanoma therapy [ 119 , 120 ] . however
, pertinent therapeutic approaches targeting mirnas described for other tumor types [ 142 , 143 ] foretell the potential and the therapeutic window opportunities entailing these nucleic acids in metastatic melanoma .
as an overview of this section , table 2 presents the therapeutic nucleic acids herein described , and figure 3 schematically summarizes the different strategies in nucleic acid therapies .
it is of general consensus that the last decade of cancer research significantly expanded our knowledge in tumor development and progression .
unfortunately similar to the tumor escape shaped by the immune surveillance in an early growth phase as new therapeutic strategies are applied , tumor cells undergo another round of selection , giving rise to therapy - resistant cells .
it is therefore necessary to combine several approaches to attack different paths of tumor escape a fact that is confirmed by the most significant results reported in studies where such strategies have been used . on this note ,
nucleic acids deliveries are truly advantageous tools as they allow the systemic delivery of potentially toxic molecules that can be combined with chemotherapy aiming at terminating possible resistant - tumor cells . as an example , recently , su and collaborators have reported on an antitumor strategy combining tnf - encoding pdna and chemotherapy . while systemically administered tnf is extremely toxic , in its genetic form , and when reaching specific target cells , tnf revealed to be a powerful antitumor agent . specific and efficient are indeed key words in this type of targeted approaches , as in suicide gene delivery .
it is thus of extreme importance to thoroughly evaluate the target options and to verify the levels of the target molecule in the cells of interest .
the activation of possible target - receptors may be desired , such as in the case reported by poeck et al . , but only when not hampering the therapeutic effect by activation of pathways that can lead to cell proliferation / differentiation , enhanced cell migration , or inhibition of apoptosis .
, this can be the case when targeting the epidermal growth factor receptor ( egfr ) , and it is then desirable to design a ligand that targets the receptor circumventing its activation .
on the other hand , the relevance of analyzing the targeted receptor has been well exposed in the short letter of perris in response to the work published by davis et al . .
to avoid other pitfalls in nanovector development , also the in vivo distribution needs to be assessed , preferably by several approaches ( e.g. , bioluminescence imaging , positron emission tomography ( pet ) , and magnetic resonance imaging ( mri ) ) . to this end
, immunohistochemistry studies may be suitable and very convenient to corroborate and support data collected by different means , but also microscopy ( mostly in vitro but also histochemistry analysis ) has had its traps . in summary , already a number of promising nucleic acid strategies exist , and these certainly present less hurdles for delivery than their protein counterpart , as they are smaller , less antigenic , and can bypass certain resistance mechanisms . nevertheless , further improvements in nonviral targeted delivery appear required to increase the efficacy of such therapies . a small final note regarding the potential of mirna approaches : microrna therapies can aim at ( 1 ) mirna upregulation , when the target nucleic acid is enrolled in cell homeostasis and
is found silenced in tumor cells ; ( 2 ) mirna downregulation by antimirs , when it is upregulated in tumor cells due to its play in cell proliferation ; ( 3 ) alternatively , mirna can also have a role in cell - specific transcription in pdna vectors containing mirna binding - sites , allowing the expression of the gene of interest in cells , where the mirna is silenced . | despite recent advances , the treatment of malignant melanoma still results in the relapse of the disease , and second line treatment mostly fails due to the occurrence of resistance . a wide range of mutations
are known to prevent effective treatment with chemotherapeutic drugs .
hence , approaches with biopharmaceuticals including proteins , like antibodies or cytokines , are applied . as an alternative ,
regimens with therapeutically active nucleic acids offer the possibility for highly selective cancer treatment whilst avoiding unwanted and toxic side effects .
this paper gives a brief introduction into the mechanism of this devastating disease , discusses the shortcoming of current therapy approaches , and pinpoints anchor points which could be harnessed for therapeutic intervention with nucleic acids .
we bring the delivery of nucleic acid nanopharmaceutics into perspective as a novel antimelanoma therapeutic approach and discuss the possibilities for melanoma specific targeting .
the latest reports on preclinical and already clinical application of nucleic acids in melanoma are discussed . |
resource - poor countries , like india , have additional challenges of low awareness about mental disorders , stigma and scarcity of mental health professionals.(1 ) there are few studies on the prevalence and patterns of emotional and behavioral disturbances among indian adolescents,(2345678 ) with the largest knowledge gaps for middle and late adolescence .
although this developmental stage is marked by a range of potential stressors , help seeking rates in traditional mental health services tend to be low .
parents , teachers , school or college counsellors and other supportive adults need to be empowered to recognise and respond to emotional and behavioral difficulties among youth .
brief , low - cost screening instruments , like the strengths and difficulties questionnaire , may help build a national database of youth mental health needs , beyond diagnosable psychiatric conditions.(9 ) however , there are some concerns about inaccurate estimates , particularly concerning cross - cultural variations in rating and in caseness indicators of the sdq.(10 ) while some studies have used the original cut - off scores , others(111213 ) have reported discrepancies in rates while using alternate cut - off points derived from their sample percentile distributions .
the present study was planned in the initial phase of a two - year adolescent mental health and suicide prevention teacher training programme in an educational institution in bangalore , india .
it assessed the rates of vulnerable adolescents at risk for a range of emotional and behavioral difficulties in order to plan targeted training objectives and processes .
additionally , it examined the utility of a brief screening tool and compared the rates of adolescent problems using varied cut - off points .
informed assent and consent for participation was sought from all 1336 students enrolled in a pre - university college , and their parents .
the sample included 1087 male and female students enrolled in the first and second year of a pre - university college in bangalore ( 81.4% participation rate ) .
the sample was almost equally divided between the first and second year of pre - university college , with a slightly higher proportion of students from the commerce stream ( 53.6% vs 46.4% ) .
about 3/4 lived in nuclear families and the majority ( 63.2% ) lived in an urban area .
perceptions of difficulties in the family domain ; e.g. physical punishment , parental marital discord , death of a parent , excessive alcohol / drug use by a family member , financial difficulties in the family ; were assessed in a yes / no format .
strengths and difficulties questionnaire ( sdq)(14 ) this 25 item behavioral screening questionnaire has a three point rating ( not true , somewhat true , certainly true ) .
four problem scales ; emotional symptoms , conduct problems , hyperactivity / inattention , peer relationship problems add to generate a total difficulties score ( 0 - 40 ) .
the original british cut - off points were derived by classifying approximately 10% of the normative sample with the most extreme scores in the abnormal banding , the next 10% in the borderline banding and the remaining 80% in the normal banding categories . some studies in europe ,
asia and africa have used scores above the 90 percentile of their samples to define the
abnormal group.(11121516 ) in the present study , it was planned to contrast rates calculated from using both the original cut - offs and the percentile scores derived from the study sample .
the sdq has good psychometric properties in varied cultures and languages and demonstrated use in indian studies.(7171819 ) institutional ethical clearance was obtained and classroom administration was done after obtaining written parental consent and student assent .
individuals were considered to be in the abnormal range if the sdq total difficulties score was above the 90th percentile for this data .
individuals scoring over the 92.5 percentile , on each of the sdq subscales , were placed in the abnormal range for that domain.(12 ) number ( % ) of individuals falling in the abnormal range using the uk cutoff(20 ) and the cut offs derived in this study are presented .
analyses using t - tests and one - way anovas identified independent variables to be considered in the multiple regression analyses ( at p < 0.1 ) , to identify predictors of total difficulties , from among twelve variables ; gender , education and stream , type of family , residence of family , paternal and maternal education levels , presence of physical punishment by parent / s , parental marital discord , parental death , excessive alcohol / drug use by a family member , family financial difficulties .
informed assent and consent for participation was sought from all 1336 students enrolled in a pre - university college , and their parents .
the sample included 1087 male and female students enrolled in the first and second year of a pre - university college in bangalore ( 81.4% participation rate ) .
the sample was almost equally divided between the first and second year of pre - university college , with a slightly higher proportion of students from the commerce stream ( 53.6% vs 46.4% ) .
about 3/4 lived in nuclear families and the majority ( 63.2% ) lived in an urban area .
perceptions of difficulties in the family domain ; e.g. physical punishment , parental marital discord , death of a parent , excessive alcohol / drug use by a family member , financial difficulties in the family ; were assessed in a yes / no format .
strengths and difficulties questionnaire ( sdq)(14 ) this 25 item behavioral screening questionnaire has a three point rating ( not true , somewhat true , certainly true ) .
four problem scales ; emotional symptoms , conduct problems , hyperactivity / inattention , peer relationship problems add to generate a total difficulties score ( 0 - 40 ) .
the original british cut - off points were derived by classifying approximately 10% of the normative sample with the most extreme scores in the abnormal banding , the next 10% in the borderline banding and the remaining 80% in the normal banding categories . some studies in europe ,
asia and africa have used scores above the 90 percentile of their samples to define the
abnormal group.(11121516 ) in the present study , it was planned to contrast rates calculated from using both the original cut - offs and the percentile scores derived from the study sample .
the sdq has good psychometric properties in varied cultures and languages and demonstrated use in indian studies.(7171819 )
perceptions of difficulties in the family domain ; e.g. physical punishment , parental marital discord , death of a parent , excessive alcohol / drug use by a family member , financial difficulties in the family ; were assessed in a yes / no format .
strengths and difficulties questionnaire ( sdq)(14 ) this 25 item behavioral screening questionnaire has a three point rating ( not true , somewhat true , certainly true ) .
four problem scales ; emotional symptoms , conduct problems , hyperactivity / inattention , peer relationship problems add to generate a total difficulties score ( 0 - 40 ) .
the original british cut - off points were derived by classifying approximately 10% of the normative sample with the most extreme scores in the abnormal banding , the next 10% in the borderline banding and the remaining 80% in the normal banding categories . some studies in europe ,
asia and africa have used scores above the 90 percentile of their samples to define the
abnormal group.(11121516 ) in the present study , it was planned to contrast rates calculated from using both the original cut - offs and the percentile scores derived from the study sample .
the sdq has good psychometric properties in varied cultures and languages and demonstrated use in indian studies.(7171819 )
institutional ethical clearance was obtained and classroom administration was done after obtaining written parental consent and student assent .
individuals were considered to be in the abnormal range if the sdq total difficulties score was above the 90th percentile for this data .
individuals scoring over the 92.5 percentile , on each of the sdq subscales , were placed in the abnormal range for that domain.(12 ) number ( % ) of individuals falling in the abnormal range using the uk cutoff(20 ) and the cut offs derived in this study are presented .
analyses using t - tests and one - way anovas identified independent variables to be considered in the multiple regression analyses ( at p < 0.1 ) , to identify predictors of total difficulties , from among twelve variables ; gender , education and stream , type of family , residence of family , paternal and maternal education levels , presence of physical punishment by parent / s , parental marital discord , parental death , excessive alcohol / drug use by a family member , family financial difficulties .
the cut - off scores derived from the percentile distribution of this study differed from that of the original uk cut - offs in select sdq domains [ table 1 ] .
the difference in the overall difficulties score cutoff was mainly due to the differences observed in domains of emotional symptoms ( rate lowered from 22.4% to 9% ) and conduct problems ( rate lowered from 18.8% to 13% ) .
the rates of hyperactivity / inattention , peer problems and prosocial behavior in the abnormal range were the same using the original cut - offs and the cut - off points derived from the study sample .
percentage of abnormal scores on sdq based on sample percentile cut - offs and uk percentile cut - offs findings related to gender differences indicated significantly higher levels of emotional symptoms and total problems among females [ table 2 ] .
there were no significant gender differences in the levels of self - reported conduct problems and hyperactivity / inattention .
gender differences in sdq total and subscale mean scores multiple regression analysis examined if the following nine variables significantly predicted the level of total problems on sdq [ table 3 ] ; gender , education level , education stream , maternal education level , presence of physical punishment by parent / s , parental marital discord , death of a parent , excessive alcohol / drug use by a family member , financial difficulties in the family . predictors of total difficulty levels on the sdq the results indicated that five predictors explained 14.5% of the variance ( f = 20.61 , p < .001 ) in total difficulty levels : education stream ( commerce ; = 0.09 ; t = 3.08 ; p < .01 ) ; education level ( 2 year pre - university ; = .08 , t = 2.86 , p < .01 ) ; presence of parental marital discord ( = 0.18 , t = 5.76 , p < .001 ) , financial difficulties ( = 0.22 ; t = 7.39 , p < .001 ) and parental physical punishment ( = 0.11 ; t = 3.83 , p < .001 ) .
the study provides a snapshot of vulnerability among students from a pre - university college in bangalore , with 10% of the sample at risk , with both internalizing ( emotional ) and externalizing ( conduct ) manifestations as prominent .
the rates tended to be lower than those in other indian studies using the sdq.(4717 ) at least partially , this could be due to the use of the original cut - offs in this group of studies .
al.(10 ) cautioned that sdq tended to overestimate prevalence in low and middle - income countries , including india . in the present study , cut - offs derived from the percentile distribution of the sample were higher than the original cut - offs for total , emotional and conduct problems , resulting in lower rates in these domains .
the findings support the call for local and contextual knowledge and population specific norms when using screening tools like sdq as an indicator for further exploration and follow - up.(1421 ) the present study identified gender differences in patterns of psychopathology among adolescents .
consonant with theoretical models(22 ) and other studies using the sdq,(7232425 ) emotional symptoms were predominant among girls and peer problems among boys .
interestingly , in contrast to previous research , the present findings did not support the tilt towards higher conduct problems and hyperactivity among males .
the reasons could include the subjective interpretations of the sdq items in these domains and may also reflect the narrowing of the gender gap pertaining to conduct disorder in adolescence.(26 ) clearly , college mental health programs must have a gender - informed perspective in early identification and intervention . within the sample ,
students in the commerce stream and those in the second year , closer to their final examinations , were more at risk .
disturbed home environments can be a potent influence on youth mental health and both parental marital discord and corporal punishment have been linked with adolescent mental health trajectories.(222728 ) the present study identified the triad of parental marital discord , financial difficulties in the family and physical punishment by parents as predictors of total difficulty levels .
overall , the findings suggest that comprehensive community - based youth mental health interventions must encompass family related vulnerabilities and find ways to engage and involve parents .
the study has inherent limitations in terms of a non - representative sample and the purposive sampling method in a single educational institution .
the use of a single stage screening method and the absence of the impact supplement of the sdq could result in inflated estimates of youth at risk .
caseness and planning service delivery could benefit from inclusion of the sdq impact supplement , a second stage using standardised diagnostic interviews and the analysis of sensitivity and specificity .
the importance of specificity would vary depending on the aim ; is the focus on identifying definite psychiatric
caseness or on exploring the magnitude of potential vulnerability among adolescents to plan a range of promotive and preventive inputs along intervention .
youth mental health initiatives in educational settings need to cover a spectrum of vulnerability and sensitive instruments , like the sdq , tapping difficulties in the borderline and abnormal range , could help identify varied youth mental health needs .
the present study highlights the need to examine the psychometric properties of the sdq in a larger , representative community sample of adolescents in india .
roc analysis could clarify the predictive validity of the sdq , while confirmatory factor analysis could assess the proposed five factor structure of the tool .
the study focused on identifying potential mental health needs in an age band where there is scarce indian literature and highlighted issues related to assessment methodologies .
the findings have implications for understanding gender - specific vulnerabilities , family contextual variables and planning targeted youth mental health services .
| background : the understanding of youth mental health needs and development of service delivery models is a national public health challenge.objectives:the rates and predictors of emotional and behavioral problems among 1087 youth were assessed in a pre - university college in bangalore , india .
variations in rates of disturbance , identified by using different cut - off points , were also examined.materials and methods : the strengths and difficulties questionnaire was used as a self - report screening tool.results:results indicated that 10.1% of adolescents had total difficulty levels in the abnormal range , with 9% at risk for emotional symptoms , 13% for conduct problems , 12.6% for hyperactivity / inattention and 9.4% for peer problems . select gender differences were present . cut - off scores derived from the sample yielded lower estimates of disturbance than the published cut - offs .
regression analysis identified predictors of total difficulty levels.conclusions:implications for assessment of youth mental health and planning targeted services in educational institutions are discussed . |
stability of the trunk , often referred to as core stability , has gained considerable attention in the recent years .
it can be operationally defined as : the body s ability to control the trunk in response to internal and external disturbances , including the forces generated from distal body segments as well as from expected or unexpected perturbations
core stability training has become a major element of training programs in sports as well as rehabilitation ( borghuis et al .
it seems plausible that core stability is important for injury prevention and athletic performance , especially in sports with large demands for balance control , such as gymnastics .
the importance of core stability for injury prevention in athletes has received some support from epidemiological studies relating low core stability to incidence of injuries of the back ( cholewicki et al . 2005 ) and lower extremities ( zazulak et al .
in addition , there is limited evidence to support its role as a determinant of performance .
nesser et al . found a moderate correlation between core stability and performance in football players ( nesser et al .
it should be noted that , in several of the studies mentioned ( nesser et al .
2004 ) , the measurements used to determine core stability reflect muscle strength and endurance , possible determinants of stability , rather than stability itself .
the stability of the spine and trunk depends on contributions of the passive , active and control sub - systems ( panjabi 1992 ) .
the passive joint structures such as ligaments and intervertebral discs contribute to stability by providing joint stiffness ( cholewicki et al .
the active sub - system , the trunk musculature contributes by further increasing stiffness through cocontraction ( cholewicki et al .
1997 ; gardner - morse and stokes 1998 ; dien et al . 2003 ; stokes and gardner - morse 2003 ) .
in addition , the control sub - system , i.e. , the nervous system , controls reflexive and triggered muscle activity contributing to stability ( moorhouse and granata 2007 ) , based on sensory feedback from muscle and joint receptors , and feedback from the visual and vestibular systems ( goodworth and peterka 2009 ) .
a model study predicted that the decrease in muscle stiffness that is associated with fatigue ( kirsch and rymer 1987 ; zhang and rymer 2001 ) , may impair trunk stability ( granata et al . 2004 ) . given the importance of reflexive muscle activity for core stability , the reductions in maximum force and rate of force rise that develop with muscle fatigue can also be expected to impair core stability , which may be aggravated by the adverse effects of fatigue on proprioception ( taimela et al .
2004 , 2009 ) , which was attributed to a reduced control over the trunk .
granata and gottipati ( 2008 ) showed that fatigue of the extensor muscles had a negative effect on the trunk s local dynamic stability , i.e. , the responses to the small perturbations that are always present due to neuromuscular noise ( dien et al . 2008 ) .
herrmann et al . found decreased contact forces during an external perturbation of the trunk induced with a swinging pendulum , which indicates reduced trunk stiffness ( herrmann et al .
( 2010 ) , however , found no change in contact forces . in both of these studies , increased electromyographic amplitudes of the reflex responses indicated compensatory mechanisms to counteract the fatigue effects .
moreover , several studies found increased cocontraction in unperturbed standing after inducing trunk muscle fatigue , as a potential compensatory mechanism ( grondin and potvin 2009 ; granata et al .
two of these studies also investigated the response to an external perturbation and found no effects of fatigue ( grondin and potvin 2009 ; granata et al .
the evidence for the effects of fatigue on trunk stability from the studies reviewed above is not consistent . furthermore , all of these studies involved high fatigue levels ( typically a reduction in force producing capacity by 40% ) induced using isolated , non - functional activity of trunk muscles .
it thus remains unclear whether fatigue of the trunk muscles that even well - trained athletes may develop during training or competitive events can induce impairments of trunk stability .
the aim of the present study therefore was to investigate the effects of fatigue induced by a set of exercises as performed regularly in training on trunk stability in elite gymnasts .
we hypothesized that gymnasts show increased sway amplitudes in a seated balancing task and that they are less able to correct an external perturbation of seated balance after a set of gymnastic exercises .
nine gymnasts , all girls , with a mean age of 12.4 ( 2.3 ) years old participated in the study .
their mean ( standard deviation ) height and body mass were 1.47 0.12 m and 39.0 12.92 kg , respectively .
none of the participants reported any recent history of injuries that did not allow training participation .
the experimental protocol , which had been approved by the ethical committee of the faculty of human movement sciences of the vu university amsterdam ( number 2009 - 039 ) , comprised a 10-min fatigue protocol in between the pre- and posttests of trunk stability .
the measurements were performed at the beginning of regular training sessions in the afternoon , after school hours .
prior activities , either during the day before the training or during warming - up were not controlled .
the fatigue protocol contained four series of five dump handstand exercises on the uneven bar . based on subjective report of trainers and gymnasts , this bout of exercises
series of dump handstands are a regular part of normal training and the intensity of this bout of exercises was comparable to the more intensive elements of the participant s normal training activities for the measurements of trunk stability , subjects were seated with arms in their lap on an unstable seat , which required them to dynamically balance by trunk movement only ( fig . 1 ) .
the seat was mounted over a hemisphere ( radius of hemisphere : 25 cm , height of the seat relative to the lowest point on the hemisphere : 17 cm ) , creating instability in all directions .
to trace the center of pressure ( cop ) , the seat was placed on a custom - made strain gauge force plate that was sampled at 200 samples / s .
the force plate was calibrated prior to each measurement session , by placing known weights on the plate .
the footplate was adjusted to support the feet with the knees and hips at 90 angles .
the first task required the subject to sit as still as possible and lasted 30 s. three repetitions of this task were performed before and after the fatiguing exercise , as reliability of single measurements was previously shown to be poor ( dien et al . 2010b ) . in the second task , the subject leaned back on the seat , supported by a strap around the thorax that was attached to two electromagnets on the safety rail in front of the subject .
the length of the strap was adjusted to obtain a constant inclination angle for each subject . after a random interval of 37
s after the start of the measurement , the electromagnets were released and the subject had to regain balance as quickly as possible .
recording of data was continued for a total of 20 s. nine repetitions were performed within a few minutes before and after the fatigue exercise.fig .
1schematic of the unperturbed seated balancing task ( a ) and the sudden release task ( b ) schematic of the unperturbed seated balancing task ( a ) and the sudden release task ( b ) force plate data were low - pass filtered at 10 hz ( fourth order butterworth ) , as the signals contained only very limited power above 3 hz , while sensitivity analysis showed only minor effects of filtering at either a higher ( 20 hz ) or lower ( 2.5 hz ) cutoff .
four independent parameters were derived from the cop time series of the 5th till 30th second of the trial : the rms of the cop in x and y direction ( rmsx , left
right ; rmsy , antero - posterior ) and the mean power frequency of the cop time series in both directions ( mpfx and mpfy ) . for the unperturbed trials ( see fig . 2 for an example ) , the mean cop position determined over the first 2 s was first subtracted from the time series .
next , the maximum cop position in the y direction was determined , which reflects the maximum backward cop displacement after the sudden release ( maxy ) .
subsequently , an exponential decay function was fitted to the cop time series in the y direction from the instant of maxy and the subsequent 7 s ( fig .
2typical examples of : the cop time series in the unperturbed trials with anterior posterior displacement in black and left right displacement in gray ( a ) , the spatial distribution of the cop position in the same trial ( b ) , the rmsx ( black ) and rmsy ( gray ) values for all trials of the same subject ( c ) , the mpfx ( black ) and mpfy ( gray ) values for all trials of the same subject ( d ) .
3typical examples of the anterior posterior cop times series in a sudden release trial with the fitted exponential curve as a thick gray line ( a ) , the spatial distribution of the cop position in the same trial ( b ) , the maxy values for all trials of the same subject ( c ) , the values for all trials of the same subject ( d ) .
the fatiguing exercise was performed between trials nine and ten1\documentclass[12pt]{minimal }
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\begin{document}$$ y(t ) = y^{\prime } + ( { \text{max}}_{y } - y^{\prime } ) \times e^ { - \lambda t } $ $ \end{document}with time t defined as zero at the instant of maxy , y referring to the y coordinate of cop , y referring to the steady state y position of the cop to which the subject converged , estimated as the median position over the fifth till seventh second after the maximum backward displacement , and maxy referring to the maximum displacement .
the parameter was used as an indicator of the rate of recovery after the perturbation , with higher values indicating faster recovery .
typical examples of : the cop time series in the unperturbed trials with anterior posterior displacement in black and left right displacement in gray ( a ) , the spatial distribution of the cop position in the same trial ( b ) , the rmsx ( black ) and rmsy ( gray ) values for all trials of the same subject ( c ) , the mpfx ( black ) and mpfy ( gray ) values for all trials of the same subject ( d ) .
the fatiguing exercise was performed between trials three and four typical examples of the anterior posterior cop times series in a sudden release trial with the fitted exponential curve as a thick gray line ( a ) , the spatial distribution of the cop position in the same trial ( b ) , the maxy values for all trials of the same subject ( c ) , the values for all trials of the same subject ( d ) .
the fatiguing exercise was performed between trials nine and ten for all six dependent variables , median values over the repeated trials per subject were used for statistical analysis to avoid effects of trials with outlying results .
comparisons between the fatigued and unfatigued conditions were made based on averages over subjects , which were tested with paired t tests , with = 0.05 .
the effect of trial order was tested over the trials performed before the fatiguing exercise using repeated measures anova .
figure 2 illustrates a typical example of the data obtained in the unperturbed trials .
however , the rmsy values were on average higher and mpfy values were on average lower after than before the fatiguing exercise , whereas no difference was discernible in the x direction .
the effects in the y direction ( anterior posterior ) were in line with our hypothesis and were found to be statistically significant for the group ( table 1 ) .
no effects were found in the x direction.table 1means and standard deviations of all dependent variables before and after the bout of fatiguing exercise and p values for differences between the fatigued and unfatigued conditions based on paired t testsindependent variablemean sdprmsx ( cm ) unfatigued0.19 0.080.549 fatigued0.21 0.09rmsy ( cm ) unfatigued0.18 0.080.030 fatigued0.31 0.17mpfx ( hz ) unfatigued0.40 0.150.810 fatigued0.41 0.24mpfy ( hz ) unfatigued0.52 0.130.005 fatigued0.35 0.17maxy ( cm ) unfatigued3.81 0.900.003 fatigued5.10 1.00 ( s ) unfatigued1.73 0.580.012 fatigued0.80 0.27 means and standard deviations of all dependent variables before and after the bout of fatiguing exercise and p values for differences between the fatigued and unfatigued conditions based on paired t tests figure 3 illustrates a typical example of the cop data in the perturbed trials . after the sudden release approximately 4 s after the start of the trial , the subject moved further backward to recover and attain a fairly steady position within 3 s after the release .
however , the maxy values were on average higher and values were on average lower after than before the fatiguing exercise .
these effects were in line with our hypothesis and were found to be statistically significant for the group ( table 1 ) . to test whether the differences between the trials before and after the fatiguing exercise could be explained by order effects , we tested for the effect of measurement order over the trials collected before the fatiguing exercise .
none of the variables showed a significant order effect ( p > 0.383 ) .
this study was designed to test the hypotheses that gymnasts show increased sway amplitudes in a seated balancing task and that they are less able to correct an external perturbation of seated balance after a set of fatiguing gymnastic exercises . as hypothesized ,
unperturbed seated balancing performance was decreased in the anterior posterior direction as evidenced by a larger amplitude and lower frequency .
perturbations were performed in the posterior direction only . in line with the hypothesis , the maximum displacement after the perturbation was larger and the recovery of balance was slower after the exercise than before .
it is at present unclear why effects were found in the sagittal plane ( anterior posterior direction ) only .
an explanation could be that the dump hand stands fatigued the abdominal muscles selectively given the mechanics of these exercises . while abdominal muscles can contribute to control of lateral bending of the trunk ( seroussi and pope 1987 ) , the extensor muscles have a higher moment producing capacity in the frontal plane ( dien and kingma 1999 ) and probably allow more precise control in view of their abundant insertions on the multi - segmented lumbar spine .
thus , while control in frontal plane could be done mainly by the extensor musculature , the abdominal muscles are obviously indispensible for control in the sagittal plane .
hence , selective fatigue of these muscles might explain why effects were restricted to the sagittal plane .
as reported in the introduction , the previous studies on the effects of fatigue on trunk stability showed inconsistent effects .
most of these tested trunk stability by applying perturbations to the trunk in either standing or sitting positions .
it was suggested that compensatory mechanisms such as increased cocontraction and increased reflex gains could explain the absence of fatigue effects in some of these studies . in the present study
, we used a dynamic balancing task to test trunk stability . in this task ,
the center of mass unavoidably sways beyond the surface of support , which is the point of contact of the hemisphere on the force plate .
trunk moments , accelerating the upper body relative to the pelvis and seat , must then be used to bring the center of mass back over the surface of support , while stiffening the trunk would not be effective ( cf otten 1999 ) .
it has previously been shown that cocontraction , which would stiffen the trunk is not effective in this task ( reeves et al .
. the present task may therefore be more sensitive to trunk muscle fatigue than tasks in which the support surface is larger .
activity of the trunk muscles was not measured and it is therefore unknown whether subjects increased cocontraction , but this would be consistent with the increase in rmsy ( reeves et al . 2006 ) and decrease in mpfy ( dien et al .
with fatigue of an agonistic muscle , the activation levels need to be increased to maintain force output .
2009 ) , which would cause increased sway and thus could explain the increase in rmsy .
variability of muscle force would increase even more when antagonistic cocontraction would be increased ( selen et al .
2005 ) which , given the fact that stiffening the trunk does not limit kinematic variability in the present task , could further increase sway amplitudes .
fatigue will likely slow down muscular responses due to increased proprioceptive thresholds ( taimela et al .
1999 ) and due to the slower force development in the fatigued muscles ( de ruiter et al . 1999 ; perrey et al .
slower responses to balance perturbation would increase sway amplitude ( radebold et al . 2001 ) and likely decrease sway frequency in the unperturbed task and would also increase the amplitude and reduce the recovery in the perturbed task ( reeves et al .
. increased proprioceptive thresholds might be reflected specifically in an increase in the maximum displacement after the perturbation ( increased maxy ) , while a decreased rate of force development might be more obvious in a decreased rate of recovery ( decreased ) .
finally , fatigue could be associated with an increased respiratory challenge , which might affect trunk stability ( janssens et al .
it is to be expected that the respiratory effect would be most obvious in the sagittal plane , which would be in line with the fact that only effects in the y direction were found .
unavoidably , trials in the fatigued condition were performed after those in the unfatigued condition and this could lead to order effects , for example due to learning , which might dilute the fatigue effect .
the absence of an order effect over the trials performed before the fatiguing exercise suggests that such effects did not occur , possibly due to the fact that the participants were all highly trained athletes .
it is unclear to what extent the loss of trunk stability as found in the present study would affect the performance of gymnastic activities .
furthermore , impaired trunk stability may limit performance and increase injury risk . therefore , the fact that trunk stability was negatively affected by a series of fatiguing exercises that reflect intensity of typical training activities and competitive events suggests several practical implications .
the results suggest that trainers should take fatigue effects into account when planning the order of training activities , e.g. , avoid balance beam exercises after uneven bar or horizontal bar exercises .
furthermore , the results may have implications for the intensity of warming up exercises for the competitive events .
finally , the results indicate that endurance training of trunk muscles and perhaps balance training may need to be performed not only in rested , but also in fatigued condition .
in conclusion , the present study showed that trunk stability in elite gymnasts was negatively affected by a bout of exercises , which reflected normal training activities . both sagittal plane sway in unperturbed balancing and recovery after a backward balance perturbation were affected .
these results suggest that fatigue effects on trunk stability should be taken into account in the planning and design of gymnastics training . | the aim of the present study was to test the hypothesis that fatigue due to exercises performed in training leads to a decrement of trunk stability in elite , female gymnasts .
nine female gymnasts participated in the study . to fatigue
trunk muscles , four series of five dump handstands on the uneven bar were performed . before and after the fatigue protocol
, participants performed three trials of a balancing task while sitting on a seat fixed over a hemisphere to create an unstable surface .
a force plate tracked the location of the center of pressure ( cop ) .
in addition , nine trials were performed in which the seat was backward inclined over a set angle and suddenly released after which the subject had to regain balance . sway amplitude and frequency in unperturbed sitting
were determined from the cop time series and averaged over trials . the maximum displacement and rate of recovery of the cop location after the sudden release were determined and averaged over trials .
after the fatigue protocol , sway amplitude in the fore - aft direction was significantly increased ( p = 0.03 ) , while sway frequency was decreased ( p = 0.005 ) .
in addition , the maximum displacement after the sudden release was increased ( p = 0.009 ) , while the rate of recovery after the perturbation was decreased ( p = 0.05 ) .
fatigue induced by series of exercises representing a realistic training load caused a measurable decrement in dynamic stability of the trunk in elite gymnasts . |
when microvascular option is not available or contraindicated the pedicle options are usually pectoralis major myocutaneous ( pmmc ) flap for lining and deltopectoral ( dp ) flap for cover .
if this procedure can be done in a single stage it becomes advantageous both for the patient in terms of reducing the morbidity , total cost , hospital stay and to the surgeon with no microvascular expertise .
the idea is to transpose a medially based platysma myocutaneous neck flap , to cover the cheek defect after it has been lined by pmmc flap .
the resultant neck defect after elevation of neck flap is covered by transposing a dp flap to the neck .
we are describing the procedure in a 43-year - old female patient with squamous cell carcinoma of right side buccal mucosa with skin involvement and ulcerating neck node [ figure 1 ] .
a medially based platysma myocutaneous flap was planned so as to cover the skin defect of cheek comfortably .
the onco surgeon then proceeded with the neck dissection , which was already bare after elevation of the platysma myocutaneous flap followed by excision of the tumour .
the residual neck skin over which the dp flap is going to sit was marked and elevated as a laterally based platysma myocutaneous flap and transposed to the dp area .
preoperative photograph showing ulcerated carcinoma cheek with ulcerating neck node intraoperative photograph showing planning of the flaps - medially based platysmal myocutaneous flap ( a ) , laterally based platysmal myocutaneous flap ( b ) , deltopectoral flap ( c ) and pectoralis major myocutaneous flap ( d ) showing planning of flaps ( a ) medially based platysmal myocutaneous flap ( b ) laterally based platysmal myocutaneous flap ( c ) deltopectoral flap and pectoralis major ( d ) myocutaneous flap intraoperative photograph showing the inset of the flaps into their respective positions with skin - grafted chest wall ( flap d is used for lining and hence not seen in the picture ) eighth day postoperative photograph of the same patient , showing well settled medially based platysmal myocutaneous flap ( a ) , laterally based platysmal myocutaneous flap ( b ) and deltopectoral flap ( c )
the patients with conventional dp flap usually do not leave the hospital till flap detachment and final inset .
hence the hospital stay is significantly reduced with this procedure . during the second stage ,
some surgeons prefer to excise the bridge segment , which leaves a large skin grafted area on chest which is not aesthetic .
skin graft requirement is less ( approximately 20% ) than a conventional dp since part of the pectoral area is covered by the laterally based cervical flap . by the same logic
overall cost associated with a double procedure and longer stay is also avoided . for the onco surgeon ,
the neck dissection is easier with an elevated platysma myocutaneous flap leaving a bare neck .
since the length of the dp flap to reach the neck is shorter than a cheek defect , it is more reliable .
there is no doubting the fact that free tissue transfer is superior to this technique . but
this flap comes in the armamentarium when the logistics do not favour a free flap .
this is aesthetically superior to a double paddle pmmc and more so in a female patient where the flap is very bulky .
forehead flap is also an option where lining and cover can be provided but it is aesthetically not appealing for obvious donor site deformity .
providing cover with a cervicothoracic flap is another option but it is cumbersome to raise such a huge flap as a single unit .
our technique splits this single cervicothoracic unit into three units which allows a better positioning of regional tissue units .
the plastic surgeon has to precisely plan and raise the neck flap prior to the onco procedure so as allow the onco surgeon access to the neck . in patients with normal neck skin ,
a part of it is sacrificed near its base to avoid dog ear , which was not the case with this patient since she had an ulcerating neck node .
this technique offers an alternative to single - staged microvascular procedure and is superior to double - paddle pmmc or conventional dp with pmmc for classical lining and skin defects . | even though free tissue transfers are a routine in many centres , pedicle flaps still have a huge roll to play in our country .
there are many centres in the country where pedicle flaps are in use because of logistic problems .
deltopectoral and pectoralis muscle flaps are usually preferred for composite cheek defects . when both these flaps are used in combination
it is a two - staged procedure .
we describe a single - stage procedure to reconstruct a composite cheek defect with pectoralis major myocutaneous flap for lining and single - stage deltopectoral flap for cover . in the available literature search
, single - stage dp and pmmc have not been described for management of composite cheek defect . |
by definition , health literacy is defined as the degree to which an individual has the capacity to obtain , communicate , process , and understand basic health information and services to make appropriate health decisions.(http://www.cdc.gov / healthliteracy / learn / index.html ) results from prior studies provide evidence that patients health literacy has a significant impact on the extent of a patients medication adherence and related health care utilization .
adherence can be broadly defined as a patient s ability to accurately follow a prescribed treatment regimen . in research , this is often operationalized as the patient s ability to successfully take the prescribed number of doses of a medication .
a common metric of defining adherence is taking at least 90% of the prescribed doses .
the annual excess of health care costs as a result of medication nonadherence is roughly us$290 billion in the united states . by understanding barriers to adherence , clinicians can help patients become more adherent in managing chronic disease that can alleviate these costs . between 30 and 34 million
american adults fall into the lowest of the 4 levels of health literacy ( below basic ,
basic , intermediate , and proficient ) , according to results from the 2003 national assessment of adult literacy .
even adults with proficient levels of general literacy may have low health literacy due to the stressful nature of health care visits and unfamiliar medical terminology . in a systematic review of literature examining health literacy prevalence rates , up to 50% of us adults were determined to have low health literacy , while approximately 33% of medicare enrollees have low or inadequate health literacy .
however , it is difficult to accurately determine the national prevalence of low health literacy due to different definitions and measurements used across the studies .
it has been suggested that patients with low health literacy experience a higher rate of medication errors .
standard medication guides approved by the food and drug administration have been estimated to require a 10th- to 11th - grade reading level , leaving many patients with a poor understanding of the content .
one study by davis et al found that patients in primary care settings who had low literacy were 3 times less likely to correctly interpret medication label warnings .
another study by the same authors found that low literacy was associated with misunderstanding medication labels , and only 35% of patients with low literacy could correctly identify how many pills needed to be taken daily .
often , these patients do not fully comprehend the benefits and risks of medications or how adherence affects health outcomes .
therefore , medical providers can make use of a patient s health literacy to assist patients in understanding their treatment plans .
when only 12% of adults in the united states are considered health literate , clinicians must acknowledge an opportunity to improve patient outcomes .
therefore , the objective of this study is to evaluate the health literacy levels and medication adherence of patients treated by pharmacists in both the general medicine and the chronic care clinics at the kansas city free health clinic ( kcfhc ) .
the study was conducted by pharmacists at the kcfhc , an urban free health clinic in kansas city , missouri .
the study estimated health literacy levels and medication adherence of patients in both the general medicine and the chronic care clinics .
rapid estimate of adult literacy in medicine ( realm ) was chosen for its ease of use and short administration and scoring time . in order to be eligible for this study , patients had to be 18 to 65 years old , english speaking , and currently receiving medical care at the kcfhc in 2011 .
patients were excluded if their current health condition was so poor that it inhibited normal patient provider communication . level of health literacy was assessed using the realm .
the entire realm can be administered and scored in less than 5 minutes , which is convenient for many health care settings .
the realm can also help practitioners identify those patients who have compensated for their literacy deficiencies by requesting patients to pronounce 66 words that may be used during a medical visit with a health care provider .
as this is a word recognition test , it is intended to screen patients rapidly and provide an estimate of their literacy levels , rather than testing comprehension or diagnosing specific reading deficits or learning disabilities .
the results of realm are broken down into the following reading level categories : below third grade , fourth to fifth grade , seventh to eighth grade , and high school .
patients were asked by the pharmacists on how many medication doses they had missed in the last 3 , 7 , and 14 days prior to their clinic visit .
if any doses were missed , patients were asked to volunteer reasons why this had occurred .
medication adherence rate ( mar ) was calculated by dividing the number of doses taken in 14 days by the total number of doses available .
this calculation was adjusted for each patient s dosing schedule ; for instance , if a medication was prescribed to be taken twice daily , then the total number of doses available in 14 days was 28 .
adherent , and patients with an mar less than 90% were categorized as nonadherent .
medication adherence barriers were collected by the pharmacist using open - ended questions and emphasized with the probe what else to gather all possible medication adherence barriers experienced by these underserved patients .
patients who came to the general medicine clinic were recruited after the patient had been checked into their room and before their examination with their provider .
if the patient agreed , the investigator read the institutional review board - approved consent script describing the nature of the research and the realm process .
participation in this study was voluntary and did not affect the quality of medical care that the patient would receive .
adherence was entered as a dichotomous variable , whereby 1 = mar 90% and 0 = mar < 90% .
multivariate logistic models determined associations between adherence , health literacy level , and patient characteristics .
results were reported in estimated proportion ( % ) and odds ratio ( or ) with 95% confidence intervals ( ci ) .
the study was conducted by pharmacists at the kcfhc , an urban free health clinic in kansas city , missouri .
the study estimated health literacy levels and medication adherence of patients in both the general medicine and the chronic care clinics .
rapid estimate of adult literacy in medicine ( realm ) was chosen for its ease of use and short administration and scoring time . in order to be eligible for this study , patients had to be 18 to 65 years old , english speaking , and currently receiving medical care at the kcfhc in 2011 .
patients were excluded if their current health condition was so poor that it inhibited normal patient provider communication .
the entire realm can be administered and scored in less than 5 minutes , which is convenient for many health care settings .
the realm can also help practitioners identify those patients who have compensated for their literacy deficiencies by requesting patients to pronounce 66 words that may be used during a medical visit with a health care provider .
as this is a word recognition test , it is intended to screen patients rapidly and provide an estimate of their literacy levels , rather than testing comprehension or diagnosing specific reading deficits or learning disabilities .
the results of realm are broken down into the following reading level categories : below third grade , fourth to fifth grade , seventh to eighth grade , and high school .
patients were asked by the pharmacists on how many medication doses they had missed in the last 3 , 7 , and 14 days prior to their clinic visit .
if any doses were missed , patients were asked to volunteer reasons why this had occurred .
medication adherence rate ( mar ) was calculated by dividing the number of doses taken in 14 days by the total number of doses available .
this calculation was adjusted for each patient s dosing schedule ; for instance , if a medication was prescribed to be taken twice daily , then the total number of doses available in 14 days was 28 .
adherent , and patients with an mar less than 90% were categorized as nonadherent .
medication adherence barriers were collected by the pharmacist using open - ended questions and emphasized with the probe what else to gather all possible medication adherence barriers experienced by these underserved patients .
patients who came to the general medicine clinic were recruited after the patient had been checked into their room and before their examination with their provider .
if the patient agreed , the investigator read the institutional review board - approved consent script describing the nature of the research and the realm process .
participation in this study was voluntary and did not affect the quality of medical care that the patient would receive .
adherence was entered as a dichotomous variable , whereby 1 = mar 90% and 0 = mar < 90% .
multivariate logistic models determined associations between adherence , health literacy level , and patient characteristics .
results were reported in estimated proportion ( % ) and odds ratio ( or ) with 95% confidence intervals ( ci ) .
the average patients age was 48 ( range 20 - 66 ) , and majority was female ( 56% ) and white ( 55% ) .
there was a significant association between ethnicity and reading level ( p = .002 ) .
white patients displayed higher literacy levels compared to black patients in the bivariate analysis ( table 1 ) .
while 33% of patients reported completion of their high school degree , 64% scored as much according to the realm .
conversely , 36% of patients could not read at a high school level . in this study , only 21% of patients read at a seventh- to eighth - grade level , which is currently the average american adult literacy level .
it is unknown at this time why this discrepancy exists , but with a larger sample size , results may prove representative of the us population .
p value for chi - square tests of associations between literacy skills and patient characteristics and between adherence and patient characteristics , respectively .
as shown in table 2 , patients with hiv / aids had the highest levels of adherence ( 88% ) , whereas patients with hypertension reported the lowest level of adherence ( 64% ) . when asked to provide a reason for missing a dose , 26% of the patients reported that they simply forgot .
chi - square or fisher exact if < 5 in a cell .
as shown in table 1 , some patients reported multiple disease states .
as such , there may be some overlap in the analysis of adherence versus nonadherence .
significance determined at p < .05 .
health literacy level as determined by realm scores .
we discovered that health literacy was not an indicator of a patient s ability to comply with prescription therapy in treating chronic disease conditions .
general trends for decreased adherence included lower dose frequency ( ie , once - daily dosing ) and lower daily pill burden .
when patients are separated by disease state and evaluated for adherence , those patients who were hiv - positive showed the greatest trends toward adherence , followed closely by those with diabetes and then hyperlipidemia .
this is consistent with the most common reasons given for nonadherence , as the hypertensive population had the lowest daily pill burden and dose frequency .
patients with high health literacy level were 21% more likely to be medication adherent ( or : 1.21 , ci : 0.68 - 2.16 ) ; however , it is not statistically significant .
we also adjusted this relationship by patients age , gender , education , and ethnicity , but the or and significance level remained the same .
male patients were 2.87 times more likely than females to adhere to their medication regimen ( table 2 ) .
moreover , ethnicity , age , and education level were not significantly associated with adherence in the multiple logistic regression model .
this study examined the varying levels of health literacy and medication adherence in a group of patients with a broad range of disease states and medication regimens .
disease state and adherence were only significantly related in the hiv / aids and hypertension subsamples .
similar to our study , a recent trend report showed that diabetes and hypertension medication adherence was below the national average , particularly in the midwest .
notably , in this population , white patients displayed higher literacy level compared to black patients ( table 1 : p = .002 ) .
additionally , patients with high health literacy levels were 21% more likely to be medication adherent ( or : 1.21 , ci : 0.68 - 2.16 ) ; however , it is not statistically significant ( p > 0.05 ) .
therefore , the patients literacy levels according to realm were not significantly associated with self - reported medication adherence in this small study sample .
we relied only on self - reported medication adherence in this study , which could be imprecise .
more accurate and automated medication adherence measures of tracking adherence , such as electronic drug monitoring ( edm ) or partner / proxy reports could have provided more objective data . moreover
, medication self - management approach a new conceptual model including patient s knowledge , skills , and behaviors necessary for patients to correctly take medications may be the accurate way to measure adherence .
however , the medication self - management approach does not take into consideration patient s initial decision to acquire the medication as their primary treatment management option . in this sample , 21% of patients
read at a seventh- to eighth - grade level , which is the average reading level of american adults . therefore , in this underserved population , 64% of the patients read at above average reading level of american adults . similar to general population , this underserved population reports forgetfulness as the most common reason for medication nonadherence .
interestingly , in this underserved population , 9% of the participants reported that they ran out of their medication .
it is not clear whether this is happening because these patients did not have enough money to refill their medication or not .
it is important to mention that many of these underserved patients are on free supply of medication provided by the free health clinic .
although from these study findings we can not truly establish the actual reason for their running out of medication , this finding highlights an issue that warrants further investigation .
it is possible that low - literacy patients did not fully understand their dosing regimen and thus could not accurately report their adherence to said regimen .
if patients with low levels of health literacy were more likely to underestimate nonadherence , this could introduce systematic bias to our results .
other methods of tracking adherence , such as edm or partner / proxy reports would provide more objective data .
this may explain the discrepancy between our results and those of murray et al , which utilized edm to measure adherence .
however , it should be noted that the relationship between health literacy and self - reported adherence has been successfully demonstrated in other studies , such as the sample of hiv - positive patients using triple - drug antiretroviral therapy .
indeed , hiv - positive patients may be more motivated to adhere to their drug regimen , given that nonadherence to antiretroviral therapy can have significant consequences , such as reduced viral suppression , poorer immunologic benefit , and overall treatment failure . in comparison ,
a patient who is nonadherent to their hypertension medication is not susceptible to such risks and therefore may perceive fewer consequences of missing a dose . in light of this ,
an additional limitation of the current study is the relatively small sample size involving a variety of disease states , which prevented the analysis of the relationship between literacy levels and adherence within each disease category .
it is possible that low - literacy patients did not fully understand their dosing regimen and thus could not accurately report their adherence to said regimen .
if patients with low levels of health literacy were more likely to underestimate nonadherence , this could introduce systematic bias to our results .
other methods of tracking adherence , such as edm or partner / proxy reports would provide more objective data .
this may explain the discrepancy between our results and those of murray et al , which utilized edm to measure adherence .
however , it should be noted that the relationship between health literacy and self - reported adherence has been successfully demonstrated in other studies , such as the sample of hiv - positive patients using triple - drug antiretroviral therapy .
indeed , hiv - positive patients may be more motivated to adhere to their drug regimen , given that nonadherence to antiretroviral therapy can have significant consequences , such as reduced viral suppression , poorer immunologic benefit , and overall treatment failure . in comparison ,
a patient who is nonadherent to their hypertension medication is not susceptible to such risks and therefore may perceive fewer consequences of missing a dose . in light of this ,
an additional limitation of the current study is the relatively small sample size involving a variety of disease states , which prevented the analysis of the relationship between literacy levels and adherence within each disease category .
in this underserved population , 64% of the patients read at above average reading level of american adults .
overall mar was 73% . forgetting to take medication was the most popular reason provided for nonadherence .
disease state and adherence were significantly related in patients with hiv / aids and hypertension .
although patients literacy levels were not significantly associated with self - reported adherence in this population , availability of a patient s baseline health literacy level as a part of the medical record may help clinicians to individualize their interaction in order to achieve better health outcomes , including improved medication adherence , especially for underserved populations . | background : a patient s health literacy is not routinely assessed during visits with a health care provider . since low health literacy is a risk factor for poor health outcomes , assessing health literacy should be considered as part of the standard medical workup.objectives:to evaluate the health literacy levels and medication adherence of patients treated by pharmacists in both the general medicine and the chronic care clinics at an urban free health clinic.methods:eligible patients from the free health clinic completed the rapid estimate of adult literacy in medicine ( realm ) , a health literacy measurement tool , during their clinic visit in 2011 .
medication adherence was self - reported by the patients.results:a total of 100 patients participated ( mean age = 48 ) .
the majority of participants were female ( 56% ) and white ( 55% ) .
most ( 64% ) of the patients scored at a high school reading level according to realm .
only 21% of participants read at a seventh- to eighth - grade level . overall medication adherence rate was 73% . forgetting to take medication was the most popular reason given for nonadherence.conclusion:disease state and adherence
were significantly related in patients with hiv / aids and hypertension .
patient s ethnicity was significantly associated with literacy levels ( p < .05 ) .
although patients literacy levels were not significantly associated with self - reported adherence in this population , availability of a patient s baseline health literacy level as a part of the medical record may help clinicians to individualize their interaction based on the patient s health literacy level in order to achieve better health outcomes , including improved medication adherence , especially for underserved populations . |
in the western world , the commonest indications for liver transplantation are end - stage cirrhosis due to hepatitis c virus ( hcv ) infection and , secondly , cirrhosis from alcohol ( ald ) .
the medium and longer term outcome of patients undergoing transplantation for hcv - related cirrhosis is reduced because of the impact of recurrence of hepatitis c : this occurs almost invariably for those who have hcv rna prior to transplant .
early reports suggested that hcv recurrence was a relatively benign condition after liver transplantation [ 24 ] but in 1999 , forman et al .
, using the united network for organ sharing ( unos ) database of patients grafted between 1992 and 1998 , showed that hcv - infected patients had worse patient and graft survival than those with chronic cholestatic diseases , a similar outcome to those transplanted for hepatitis b infection , autoimmune hepatitis , cryptogenic cirrhosis , and ald and better than that in patients undergoing transplantation for cancer .
an analysis of the unos database of patients grafted between 1990 and 1996 by roberts et al . reached similar conclusion . in a relatively large proportion of cases ,
hcv recurrence has an aggressive course , with progression to cirrhosis within 5 years , resulting in survival rates of less than 65% [ 68 ] .
treatment of the hcv is , with current treatments , relatively ineffective and appears to be more toxic than in the native liver .
alcoholic liver disease ( ald ) is , for selected patients , an excellent indication for liver transplantation , with outcomes at least as good as for other indications and with a rate of alcohol relapse acceptably low [ 911 ] .
nonetheless , it remains a controversial indication for liver transplantation , due to concerns that in some cases , the recipient will return to a pattern of alcohol consumption , leading to graft failure , noncompliance , or other potentially fatal complications .
many patients with alcoholic liver disease have associated hepatitis c viral ( hcv ) infection , and , conversely , many people , infected with hcv drink above the recommended limits for alcohol .
there is mounting evidence that alcohol abuse may accelerate the course of chronic hepatitis c [ 1318 ] .
however , there are few data available about the possible interaction between alcohol and hcv on the posttransplant setting .
up to one third of hcv - infected transplant candidates have a history of significant alcohol intake prior to transplantation , but the drinking habits in hcv cirrhotic patients are often not always well explored .
this probably has the effect of significantly underestimating the impact of alcohol in causing end - stage liver disease and overemphasizing the serious consequences of hcv infection .
furthermore , after transplantation , patients with hcv infection ( whether or not associated with alcohol ) tend not to be monitored as closely for alcohol consumption as those with hcv infection .
the effect of other agents , such as use of illegal drugs ( such as cannabis ) , is also not well studied even though such agents are associated with accelerated progression of hcv . in this paper
, we review the current evidence on the effect of hcv and alcohol use on the listing and outcome after liver transplantation , compared to those with hcv or alcohol alone , as a cause of the liver failure .
current evidence supports hcv + ald as an appropriate indication for liver transplantation , as highlighted by several studies suggesting that patients with hcv + ald have similar graft and patient survival outcomes as patients who undergo transplantation for hcv or ald alone [ 2025 ] ( table 1 ) . in an early study ,
dhar et al . in 1999 showed little difference in patients and graft survival between patients undergoing transplantation for hcv alone or for hcv infection plus alcohol .
subsequently , several other early studies [ 2123 ] have analyzed the consequences of hcv infection in patients with ald undergoing olt , and they found that pretransplant hcv infection in ald patients does not affect the survival after olt but these series included only small numbers of patients and followup was relatively short .
analyzed retrospectively the clinical and histological outcome of 60 patients undergoing liver transplantation for cirrhosis of mixed etiology ( hcv and alcohol ) and compared it with that of patients undergoing liver transplantation for hcv - related cirrhosis ( n = 170 ) or alcohol - related cirrhosis ( n = 107 ) .
they found that patients transplanted for hcv + ald had a better survival compared with patients with hcv alone , and similar survival compared with patients with alcohol alone .
patient survival at 1 , 5 , and 7 years was 86% , 73% , and 63% , respectively , in the mixed group , 72% , 49% , and 43% , respectively , in the hcv group , and 90% , 76% , and 67% , respectively , in the alcohol group ( hcv group versus mixed group , p = .0001 ; mixed group versus alcohol group , p = .74 ) .
graft survival was significantly lower in patients with hcv - related cirrhosis compared to the other two groups .
the rate of retransplantation was higher in the mixed group , and the main cause of retransplantation in this group was viral recurrence .
the main causes of death in the mixed group were recurrence of hepatitis c and complications possibly related to excessive immunosuppression such as sepsis .
no difference were found in the incidence of severe recurrent hcv disease or fibrosis stage > 1 at 1 year between mixed group and hcv group .
the authors suggest that the better survival of the mixed group might be explained by two factors : the greater use of antiviral treatment in the mixed group versus the hcv group , because patients belonging to the former group were younger and so more likely to receive an antiviral treatment , and the young age is itself consistently associated with better survival . in the mixed group ,
alcohol was assumed to contribute mainly to the progression of liver disease prior to transplantation .
after transplantation , when alcohol factor disappeared in the majority of patients , the liver damage could be related only to recurrent hepatitis c , similar to that occurring in patients without a history of alcohol intake prior to transplantation ; this would explain the similar histologic hcv - progression in both groups in this study .
the cumulative experience of multiple european transplant centers have confirmed that the survival rates following liver transplantation in the european liver registries are similar for patients with ald and ald plus viral etiology ( hcv and hbv ) , but patients with ald plus hcv had significant lower survival compared to ald plus hbv infection .
results show that concomitant infection with hcv eliminates the progressive improvement of survival over time in ald patients .
an adverse role of hcv coinfection in patients transplanted for ald liver cirrhosis is in keeping with the finding of more aggressive liver damage was in ald plus hcv infected patients . in the same study
, the authors found that de novo tumors were a major cause of death in the ald group , but not in the hcv + ald group and hcv group ( 13.7% , 8% , and 5% , resp . ) .
patients transplanted for ald with and without associated hcv infection had a significantly higher incidence of death due to cardiovascular events compared to patients with hcv alone ( 7.4% , 8% , and 5.3% , resp . ) . also , patient with ald and ald + hcv had a greater incidence of deaths caused by social problems , including suicide , compared to patients with hcv alone ( 1.3% , 1.2% , and 0.6% , resp . ) .
interpretation of findings from registries will inevitably have limitations : the data may be incomplete and definitions and protocols for selection , transplantation , and followup vary between participating centers . furthermore , case mix ( as discussed below ) may differ between the groups .
nonetheless , that the two large registries provide similar outcomes implies that the conclusions are valid .
thus , for those transplanted for hcv , it is important that the recipient and the clinician are aware of the importance of limiting alcohol consumption in order to prolong patient and graft survival .
hcv + ald patients undergoing liver transplantation are usually younger than those transplanted for hepatitis c or alcoholic liver disease alone [ 20 , 24 , 25 ] , in keeping with other evidence that hepatitis c and alcohol act synergistically to cause more aggressive liver disease .
suggested a difference in pre - olt duration of liver disease between those grafted for ald alone compared with those grafted for hcv + ald ( median 25 and 15 years , resp . , but the strength of the conclusions is limited by the numbers of cases in each groups ) .
found that patients with alcoholic cirrhosis were sicker at the time of transplantation than those of the hcv and hcv + ald groups ( percentage of patients with child - pugh - turcotte c : 47% , 30% , and 43% , resp . ;
a history of tobacco consumption was more frequently reported in patients undergoing transplantation for hcv + ald and ald alone than those with hcv alone ( 72% , 68% , and 24% , resp . ; hcv versus mixed , p = .001 ) .
the incidence of hcc pre - olt was greater in hcv and hcv + ald patients than in ald patients ( 44% , 35% , and 18% , resp . ; mixed versus ald , p = .01 ) .
reported that the tumors in the mixed group were larger than those in the ald group ( mean diameter 4.25 versus 0.85 cm ) .
post - olt recurrence of hcc was noted in 25% in ald compared with 50% in those with hcv + ald , suggesting the recurrence rate is related to their size of the tumour at lt rather than any effect of the coexistent hcv ( table 2 ) .
while there may be some element of selection bias and different patterns of referral for those in the three groups , those transplanted for ald and hcv are more likely to have more aggressive disease pretransplant and have more advanced disease at the time of transplant .
several studies pointed to a link between alcohol and progression of liver disease due to viral hepatitis .
alcohol will transiently increase circulating levels of hcv rna , suggesting that alcohol may have a direct effect on viral replication ( 26 ) .
patients with hcv infection who have high alcohol consumption have higher hepatic iron concentrations , which may affect hcv replication and also perhaps enhance hepatic fibrogenesis ( 17 ) .
moreover , the immunologic effects of alcohol may affect the balance between host and virus , resulting in more severe liver disease . however , there is , as yet , no convincing evidence that hcv recurrence in patients transplanted for hcv + ald cirrhosis is more aggressive than that in patients with hcv cirrhosis alone [ 23 , 24 ] .
no differences were found in the incidence of severe recurrent hcv disease ( 45% in the hcv group versus 45% in the mixed group , p < .660 ) , acute hepatitis ( 26% in the hcv group versus 28% in the mixed group , p = .854 ) , or fibrosis stage > 1 at 1 year ( 34% in the hcv group versus 35% in the mixed group , p = .88 ) between patients undergoing transplantation for hcv - related cirrhosis alone compared to those with cirrhosis of mixed etiology .
however , these conclusions must be treated with caution : numbers are relatively small and many factors ( host , viral , graft , and immunosuppression ) may affect rates of rate recurrence .
the risk of recidivism is a major problem in alcoholic cirrhotic patients although the actual incidence is difficult to establish as centers use different evaluation methods [ 2629 ] , definitions of alcohol recurrence , and different followup protocols .
the effect of alcohol recidivism in terms of histological liver damage has been reported in several studies [ 3035 ] , with conflicting results in terms of severity [ 30 , 32 , 33 , 35 , 36 ] .
determining the contribution of alcohol and hcv infection to the extent of graft damage can be difficult [ 22 , 37 , 38 ] .
there is no clear definition of relapse , and this lack of definition may explain the widely different relapse rates reported in the literature , ranging from 7% to 95% [ 35 , 39 , 40 ] .
. showed that heavy drinking reduces the long - term survival ( over 5 years ) of patients transplanted for ald alone .
however , the significance of slips remains uncertain : it has been hypothesized that a slip , which should always be considered an unwelcome event but , in itself is unlikely to cause harm but , for many patients will progress to a more damaging pattern of alcohol intake .
thus , most centers advocate total abstinence in those with a history of alcohol dependence .
reported a similar rate of relapse to alcoholic behaviour in ald and hcv + ald groups ( 8,9% versus 9,4% , resp . ) , but overall return to heavy drinking seemed to be uncommon .
found that the incidence of alcohol relapse was higher in patients undergoing transplantation for ald than in the hcv and hcv + ald groups ( 18% , 3% , and 8% , resp . ) .
studied the effect of alcohol recidivism in terms of liver histopathological damage in patients following liver transplantation for alcoholic cirrhosis , with or without concomitant hcv infection .
they classified the patients as heavy drinkers if they drank more than 200 g of alcohol a week and occasional drinkers if they drank less than 200 g of alcohol a week .
no statistically significant difference emerged between hcv positive heavy drinkers , occasional drinkers , and abstainers in terms of fatty changes in the hepatocytes , pericellular fibrosis , and perivenular fibrosis .
portal tract fibrosis ( p = .0005 ) , portal tract monocyte ( p = .003 ) and lymphocyte ( p = .001 ) inflammation , parenchymal lymphocyte inflammation ( p = .006 ) , and perivenular fibrosis ( p = .025 ) were more common in anti - hcv positive compared with hcv - negative patients , suggesting graft damage due to the virus damage rather than with alcohol consumption .
however , because of the relative low rate and short duration of alcohol use following liver transplantation , these conclusions must be treated with caution .
no significant differences in liver tests were seen at any time after liver transplantation among the three groups of drinkers with or without hcv .
it seems that the majority of patients who resume drinking habit after liver transplant consume moderate amounts of alcohol ( fewer than 12% of patients drink > 200 g of alcohol / week ) , and therefore such liver damage is slow to develop . on the present ,
limited information , alcohol recidivism after liver transplantation in patients with hcv + ald cirrhosis does not appear adversely to influence liver morphology or function .
the role of recurrent hcv in inducing the alcohol behaviour or potentiating the injury is unclear .
hcv patients may be at increased risk for waiting list mortality when compared with patients without hcv , and this effect is greater in subjects with both hcv and ald .
one study from the us showed that for those with hcv infection , there was a significantly greater risk of death on the waiting list ( hr 1.19 , 95% ci 1.091030 , p = .001 ) whereas for those with ald there was no significant increased risk of death .
however , of those with hcv infection , those with ald had a greater risk of waiting list death than those with hcv alone ( hr 1.14 , 95% ci 1.041.25 , p = .006 ) and conversely for those with a primary diagnosis of ald , those with hcv had a greater risk of waiting list mortality than those with ald alone ( hr 1.36 , 95% ci 1.211.53 , p < .0001 ) .
thus , while it may be difficult to distinguish whether ald or hcv is the main cause of end - stage liver disease , it is clear that those with both hcv and ald are more likely to die awaiting transplant .
unadjusted and adjusted posttransplant mortality is greater in hcv+ subjects than hcv recipients ( hr 1.26 , 95% ci 1.101.45 ; p = .0009 ) whereas ald did not influence posttransplant mortality ( hr 0.95 ) .
in contrast , hcv infection increase posttransplant mortality in both ald + hcv recipients ( hr 1.30 , 95% ci 1.071.59 ; p < .01 ) and ald alone recipients ( hr 1.25 , 95% ci 1.081.45 ; p = .004 ) , with the interaction between hcv and ald on posttransplantation mortality being nonsignificant ( table 3 ) . thus ,
while alcohol consumption does lead to graft loss or patient death in some recipients , the effect is too small to show statistical significance so the conclusion is likely to represent a type 2 error rather than support the view that a return to alcohol consumption has no impact on patient or graft survival .
the apparent lack of impact of alcohol may be due to low rate of posttransplantation heavy drinking habit relapse and to the fact that it takes up to 10 years to have an impact on graft [ 4547 ] .
the survival benefit of transplanted patient for mixed etiology has not been well evaluated although it is likely to be significant .
quality of life ( qol ) is an important factor in the evaluation of olt .
the role of hcv infection on qol and specifically the effect of recurrence of the infection after lt has been the focus of several studies [ 4855 ] . in comparison with other olt indications , hepatitis c report much lower health - related quality of life ( hrqol ) scores after transplantation despite similar survival rates [ 49 , 51 , 53 , 56 , 57 ] ; moreover , patient knowledge of the diagnosis of recurrent hcv alone can negatively impact hrqol [ 48 , 5761 ] .
singh et al . evaluated prior alcohol use and the hrqol scores of hcv transplanted patients .
they found that karnofsky scores at 6 months ( mean , 82 versus 97 ) and 12 months ( mean , 84 vsersus 85.7 ) , the beck depression scores at 6 months ( mean , 13.4 vsrsus 14.9 ) and 12 months ( mean , 19.5 versus 21.5 ) , and quality of life scores at 6 months ( mean , 5.0 versus 4.6 ) and 12 months ( mean , 3.5 versus 2.6 ) did not differ significantly between patients with hcv recurrence and alcohol use and patients with hcv recurrence without alcohol use .
multiple studies confirm these findings and show no significant difference in hrqol with alcoholic liver disease as olt indication compared with other olt indications , including hcv [ 6266 ] .
recently , ruppert et al . reported that recipients who had both ald + hvc , but not either diagnosis alone , had consistently worse qol in all domains evaluated ( physical distress , psychological distress , social / role function , personal function , and general health perception ) at the 1-year time point , and they significantly worsen in both physical functioning and physical symptoms over time .
these differences remain after controlling for other medical factors ( age , meld scores , etc . ) before lt .
current evidence suggests that hepatitis c / alcohol - related cirrhosis represents an excellent indication for liver transplantation , and that graft and patient survival are comparable to those of patients with hcv cirrhosis or ald cirrhosis alone .
hcv recurrence in patients with hcv + ald cirrhosis undergoing liver transplantation does not seem more aggressive than that in patients with hcv cirrhosis alone . a return to alcohol consumption , in the short term at least in patients with hcv + ald cirrhosis does not have an important impact on graft survival in patients with alcohol and hcv infection .
however , outcomes are relatively short and , given the known interaction of hcv and alcohol on exacerbation of liver damage in the native liver , it would seem prudent to advise those grafted for hcv infection to have a minimal alcohol consumption , and where there is a history of alcohol dependence , complete abstinence should be advised . | end - stage liver disease due to hepatitis c ( hcv ) and cirrhosis from alcohol ( ald ) are the commonest indications for
liver transplantation in the western countries
. up to one third of hcv - infected transplant candidates have a history of
significant alcohol intake prior to transplantation . however , there are few data available about the possible interaction between alcohol
and hcv in the post - transplant setting .
patients with both hcv and alcohol are more likely to die on the waiting list than those with
ald and hcv alone . however , after transplantation , non - risk adjusted graft and patient survival of patients with hcv + ald are comparable to
those of patients with hcv cirrhosis or ald cirrhosis alone
. in the short and medium term hcv recurrence after transplant in patients with
hcv + ald cirrhosis does not seem more aggressive than that in patients with hcv cirrhosis alone . a relapse in alcohol consumption in
patients with hcv + ald cirrhosis does not have a major impact on graft survival .
the evidence shows that , as is currently practiced ,
hcv + ald as an appropriate indication for liver transplantation .
however , these data are based on retrospective analyses with relatively
short follow - up so the conclusions must be treated with caution . |
primary intraosseous cavernous hemangiomas ( pichs ) of the cranium are rare benign vascular tumors that account for about 0.2% of all bone tumors and 10% of benign skull tumors .
these typically present as osteolytic lesions in the calvarium.[24 ] even though calcification in hemangioma is relatively common , completely ossified cavernous hemangioma of the skull in an adult has hitherto been unreported in literature .
total surgical excision is the treatment of choice and the prognosis after complete excision is excellent and recurrence is usually rare .
we present a rare case of a giant cavernous hemangioma of the skull which was completely ossified .
the clinical presentation , pathology , differential diagnosis and treatment of this rare disorder are discussed .
a 28-year - old female presented to us with history of huge swelling over the right parietal region progressively increasing in size over the past 15 years .
the lesion measured approximately 6 cm in diameter in the right posterior high parietal region .
clinical impression on local examination was that of a hard bony mass arising from the calvarium .
a plain radiograph of the skull was performed which showed a completely radio opaque bony mass in the right parietal region .
plain computed tomography ( ct ) of brain with bone window showed a uniform hyperdense mass in the right high parietal region measuring 6.3 5.3 5.6 cm .
[ figure 1 ] postcontrast ct showed enhancement of extradural soft tissue component with buckling of under lying gray matter .
[ figure 2 ] plain sagittal ct scan showing a completely ossified mass in the high parietal region involving both the inner and outer table of skull postcontrast axial ct scan showing a uniformly hyperdense mass in the right high parietal region with contrast enhancing extradural tissue .
there is evidence of buckling of underlying gray matter patient was operated in left lateral position .
three burr hole right parieto - occipital craniectomy with total resection of lesion was performed .
intraoperatively lesion was extremely vascular and looked like white coral - filled with vascular soft tissue .
patient was in regular follow - up and was subjected to cranioplasty 6 months later . at 3-years follow - up
the lamellar bone showed concentrically arranged lamellae ( collagen ) around vascular canal ( haversian canal ) .
medullary spaces between bony trabeculae showed ectatic thin - walled blood vessels with single layer of flat endothelial cells .
[ figure 3 ] the lesion was consistent with the diagnosis of cavernous hemangioma with ossification .
h and e - stained slide showing bone bits with mature lamellar bone and bony spicules .
medullary spaces between bony trabeculae showing ectatic thin - walled blood vessels with single layer of flat endothelial cells
pichs are rare , benign skeletal tumors most commonly found in the spinal vertebral column .
overall , they represent 0.7% of all osseous neoplasms . the earliest description in the english literature was in 1845 by toynbee , who reported a vascular tumor arising in the confines of the parietal bone .
although the origin of pichs remains obscure , some authors believe that they represent congenital lesions that manifest in adulthood . in a review by wyke
, 70% of cranial pichs were localized to the calvarium , particularly the parietal and frontal bones .
they are predominantly seen in patients in their fourth and fifth decades.[28 ] however , there are a couple of reports of cavernous hemangiomas in neonates or infants which have been misdiagnosed as ossified cephalhematoma .
unlike the age predominance , our patient was a young adult ( 28-year old ) .
hemangiomas are classified histologically by the predominant type of vascular channel in the lesion : capillary , cavernous , venous and arteriovenous . in the cavernous variety
this variety is the commonest in the calvarium with a preference for the frontal and parietal bone .
calcification within the hemangioma is common and may be of three types . the first variant , nonspecific type is either amorphous or , at times , curvilinear .
the second type , which is more specific and is the most frequent type of calcification , is the phlebolith .
occasionally , metaplastic ossification may be found in hemangiomas , and this is the third type of calcification . when bone is involved resulting in reactive osteoclastic and osteoblastic remodeling ,
the characteristic trabeculated , honeycomb appearance is visible on ct . in our case , however , the whole lesion was ossified .
calvarial hemangiomas are slow - growing tumors that are only symptomatic when they become large or compress adjacent neurological structures .
they develop in the diploic space constituted by dilated blood vessels with fibrous septa and their vascular supply is frequently from the middle meningeal artery and branches of the external carotid artery .
they are usually solitary but multiple hemangiomas of the skull have been reported with a frequency of 15% of all identified calvarial hemangiomas .
ct is the investigation of choice and calvarial hemangiomas typically appear as a lytic expansile and bubbly lesion with a sclerotic rim or a spiculated sunburst skull tumor .
ct is also helpful in delineating the osseous extension to adjacent skull structures and possible complications such as inner or outer table bone fracture .
nonenhanced ct usually shows a mass isodense with adjacent muscles with intense enhancement after intravenous contrast administration . in this case
other radiological investigations include magnetic resonance imaging ( mri ) and scintigraphy , which was not done in our case .
a table mentioning various reports and series of solitary intraosseous cavernous hemangiomas of the skull vault in literature show the rarity of ossification in these lesions [ table 1].[34911131419 ] previously reported cases of solitary primary intraosseous cavernomas of the skull vault in various age groups and their radiological presentation the differential diagnosis of a solitary circumscribed expansile intradiploic cranial lesion includes an osteoma , aneurysmal bone cyst , giant cell tumor , fibrous dysplasia , langerhans cell histiocytosis , sarcoma , meningioma , metastatic disease , pagets disease and dermoid tumor .
rare intraosseous meningiomas , also known as primary extradural meningiomas , represent only 12% of all lesions of this group and may be difficult to distinguish from calvarial hemangiomas solely on the basis of location .
radiation has been attempted to treat hemangiomas , and it has been shown to stop tumor growth ; however , it has not been shown to reduce the size of the tumor .
nasrallah et al have done embolization followed by craniotomy , preserving the outer table with no recurrence at 3-year follow - up .
cavernous hemangioma of the calvarium is a benign condition of the skull bone which can be completely resected with good results .
these lesions usually have typical radiological imaging features , which may , however , show some variations .
cavernous hemangiomas should be considered as a differential diagnosis even in completely ossified calvarial masses and adequate preparation to minimize blood loss such as embolization can be considered if they are adequately investigated considering this possibility . | primary intraosseous cavernous hemangiomas ( pichs ) of the cranium are rare benign vascular tumors that account for about 0.2 % of all bone tumors and 10 % of benign skull tumors .
they generally present as osteolytic lesions with honeycomb pattern of calcification .
completely ossified cavernous hemangioma of the calvarium in an adult has not been reported previously .
a 28-year - old female presented to us with a large right parietal skull mass that had been present since the last 15 years .
total resection of the lesion was performed .
pathological examination was suggestive of cavernous hemangioma of the skull bone .
cavernous hemangioma should be considered as one of the differential diagnosis in any case of bony swelling of the calvarium so that adequate preoperative planning can be made to minimize blood loss and subsequent morbidity . |
in women previously treated for breast cancer , surveillance mammography is useful for early detection of tumour recurrence , or for confirming the absence of recurrent cancer , and for the early detection of contralateral cancers .
although published figures vary , it has been estimated that approximately 50% of local recurrences in the breast following breast conservation surgery will be detected by mammography , with the remainder being detected by clinical examination or reported by the patient [ 14 ] .
recurrent tumours detected by mammography are generally smaller and less invasive than those found on clinical examination [ 2 , 4 ] .
lu and colleagues recently conducted a systematic review to determine the impact of early detection of isolated loco - regional and contralateral recurrence on survival .
the authors reported better overall survival for recurrences detected by mammography or in asymptomatic patients , with an absolute reduction in mortality of 1728% if all breast cancer recurrences are detected early . while tumour recurrence displays similar mammographic features to the primary lesion , interpretation of the surveillance mammogram is made more difficult by surgical scarring and changes to breast density caused by primary treatment .
for example , following surgery and/or radiotherapy , detectable abnormalities on mammography may include the presence of old haematoma , scar formation , fat necrosis , skin thickening , increased soft tissue density in the breast and microcalcifications .
approximately 10% of palpable tumours are not clearly visible on mammography and require additional imaging techniques for their demonstration .
surveillance mammography is therefore also associated with the possibility of false - positive results , which then require further investigations that are unnecessary and have a negative impact on a woman s quality of life .
we conducted a national institute for health research health technology assessment programme funded project ( nihr hta project 07/47/01 ) to examine the clinical effectiveness and cost - effectiveness of different surveillance mammography regimens after the treatment of primary breast cancer in the uk in primary and secondary care settings .
the work comprised : a survey of uk breast surgeons and radiologists , a series of systematic reviews ( test performance review presented here ) , and statistical and economic modelling to determine the effectiveness , cost consequences and cost utility of differing surveillance regimens .
the primary objective of this systematic review was to determine the test performance of surveillance mammography , alone or in combination with other tests , in detecting ipsilateral breast tumour recurrence and/or metachronous contralateral breast cancer in women undergoing routine surveillance .
our secondary objective was to compare surveillance mammography performance with alternative tests , alone or in combination , in women with a previous diagnostic test result indicating suspected ipsilateral breast tumour recurrence and/or metachronous contralateral breast cancer ( referred to subsequently as non - routine surveillance ) .
we considered randomised controlled trials of surveillance mammography and diagnostic consecutive cohort studies of surveillance mammography or other comparator tests , involving women previously treated for primary breast cancer without detectable metastatic disease at the time of presentation for their initial treatment .
we also considered indirect ( between - study ) comparisons by comparing cohort studies analysing results of at least 100 women who received surveillance mammography , or a comparator test , or a combination of tests , with the reference standard test in the same population .
comparator tests included ultrasound , magnetic resonance imaging ( mri ) , specialist - led clinical examination and unstructured primary care follow - up ( defined as absence of formal routine secondary care follow - up , which may or may not involve mammography ) .
the reference standard was histopathological assessment for test positives and a period of follow - up for test negatives .
we chose to include studies assessing test performance for routine and non - routine surveillance patients .
adjunct tests are part of breast cancer surveillance management and the performance of diagnostic tests used for this purpose is relevant to our population of interest .
the accuracy of non - routine adjunct imaging tests may differ from the accuracy of first - line surveillance tests as the test operator is primed to evaluate a suspicious finding in the non - routine surveillance patient .
it is unclear what effect this has on test accuracy but it is likely to focus attention on a particular area of the breast and may conceivably increase the diagnostic test sensitivity .
consequently , we have not attempted to mix or compare the performance of tests used for these different purposes .
similarly , because of anatomical differences between a treated and an untreated breast ( due to treatment effects ) it was inappropriate to combine data on test performance for the detection of ipsilateral breast tumour recurrence and metachronous contralateral breast cancer . the following types of outcome were considered : test performance in diagnosing ipsilateral breast tumour recurrence in women undergoing routine surveillancetest performance in diagnosing ipsilateral breast tumour recurrence in women undergoing non - routine surveillancetest performance in diagnosing metachronous contralateral breast cancer in women undergoing routine surveillancetest performance in diagnosing metachronous contralateral breast cancer in women undergoing non - routine surveillance test performance in diagnosing ipsilateral breast tumour recurrence in women undergoing routine surveillance test performance in diagnosing ipsilateral breast tumour recurrence in women undergoing non - routine surveillance test performance in diagnosing metachronous contralateral breast cancer in women undergoing routine surveillance test performance in diagnosing metachronous contralateral breast cancer in women undergoing non - routine surveillance to be considered for inclusion , the studies had to report the absolute numbers of true - positives , false - positives , false - negatives and true - negatives , or provide information allowing their calculation , and report a per - patient analysis . in studies reporting the above outcomes , we planned to record the following additional outcomes , if stated : adverse effects ( defined as physical harms ) of mammography and other testsacceptability of the testsreliability of the testsradiological / operator expertise ( who conducts the test and previous experience)interpretability / readability of the tests adverse effects ( defined as physical harms ) of mammography and other tests acceptability of the tests reliability of the tests radiological / operator expertise ( who conducts the test and previous experience ) interpretability / readability of the tests major electronic databases were searched using sensitive search strategies to identify diagnostic studies of surveillance mammography , mri , ultrasound or clinical follow - up .
searches were conducted from 1990 to march 2009 and were restricted to the english language .
the following databases were searched for primary studies : medline , medline in process , embase , biosis , science citation index , cancerlit , while medion , the cochrane database of systematic reviews ( cdsr ) , database of reviews of effects ( dare ) and the hta database were searched for reports of evidence syntheses .
reports of ongoing and recently completed trials were sought from current controlled trials , clinical trials , who international clinical trials registry platform , nci clinical trials database , nrr archive and nihr portfolio database . in addition , relevant websites were searched and the reference lists of all included studies were scanned for additional reports .
full details of the search strategies used are available from the authors or the full study report , currently in press ( the clinical effectiveness and cost - effectiveness of different surveillance mammography regimens after the treatment of primary breast cancer . by robertson et al . accepted for publication in health technol assess 2011 ) . from an initial first screening round of titles and abstracts we were able to exclude reports that were clearly irrelevant to the review ( e.g. did not include any of our considered diagnostic tests ) .
we then assessed the full text versions of the remaining reports against our eligibility criteria using a screening tool comprising a checklist of our inclusion eligibility criteria , which we developed specifically for this review .
we calculated sensitivity , specificity , positive and negative likelihood ratios and diagnostic odds ratio for each included study .
we evaluated the quality of studies using an adapted version of the quality assessment of diagnostic accuracy studies quadas tool .
higher quality studies were defined as those considering a representative patient spectrum and judged to have successfully avoided partial verification bias ( whether the whole or random sample of the population received reference standard verification ) , differential verification bias ( whether patients received the same reference standard ) and test review bias ( whether index and reference standard test results were interpreted independently ) .
disagreement or uncertainty regarding data extraction or quality assessment was resolved by discussion or arbitration by a third reviewer .
1flow of studies through the review process flow of studies through the review process nine studies met our inclusion criteria .
overall , the nine studies enrolled 4002 participants . after exclusions , due to eligibility or participant drop - out , the studies included 3724 participants in their analyses .
the earliest study took place in 1995 and the latest in 2009 . the earliest participant enrolment date given was 1992 and the latest was 2003 .
four studies did not give any indication of the enrolment time period [ 912 ] .
one study took place in sweden , two in the uk [ 10 , 11 ] , two in germany [ 12 , 13 ] , two in south korea [ 8 , 14 ] , one in italy and one in france . across studies the ages of the participants ranged from 2282 years .
the median age was 53 years ( inter - quartile range 50 to 56 years ) . reported follow - up of test negatives ranged from 5 to 32 months .
table 1 provides details of the characteristics of the included studies.table 1summary of characteristics of the individual diagnostic accuracy studiesstudy idstudy designtype of surveillance and primary surgeryindex testscomparator testfollow - up time for verifying test negative resultsbelli 2002 direct head - to - head cohortnon - routine surveillance breast conservation patientsmri for local recurrencesurveillance mammography , clinical examination , ultrasound for local recurrencemri and clinical examination follow - up performed at 3 months.all mri test negatives underwent cytological examinationbon 1995 direct head - to - head cohortroutine surveillance mastectomy patients , all with breast reconstruction and implantssurveillance mammography for local and contralateral recurrencemri , clinical examination for local and contralateral recurrencemedian 10 months ( range 518 months)drew 1998direct head - to - head cohortroutine surveillance breast conservation patientsmri for local recurrencesurveillance mammography , clinical examination , surveillance mammography + clinical examination for local recurrencemedian 341 days ( range 168451 days)kim 2009direct head - to - head cohortroutine surveillance breast conservation and mastectomy patientsadjunct ultrasound ( surveillance mammography + ultrasound ) for contralateral recurrencenone1 - 2 yearsmumtaz 1997direct head - to - head cohortnon - routine surveillance breast conservation patientssurveillance mammography for local recurrencemri for local recurrencemedian 12 months ( range 615 months)rieber 1997cohortnon - routine surveillance breast conservation patientsmri for local recurrencenoneclinical examination , ultrasound performed at 6 months follow - up . surveillance mammography performed at 12 months follow - up . in 22 patients a control mri was performed at intervals of 216 months ( mean 7.2 months)shin 2005cohortroutine surveillance patients ( primary surgery type not reported)ultrasound for local and contralateral recurrencenone6 monthsternier 2006direct head - to - head cohortnon - routine surveillance breast conservation patientssurveillance mammography for local recurrence clinical examination , ultrasound for local recurrence6 monthsviehweg 2004direct head - to - head cohortroutine surveillance breast conservation patientsmri for contralateral recurrenceconventional methods ( surveillance mammography + clinical examination + ultrasound ) ; mri + conventional methods for contralateral recurrence12 months1 .
study authors considered computed tomography as the index test in this study but this test was not considered as an included comparator in this review summary of characteristics of the individual diagnostic accuracy studies 1 .
study authors considered computed tomography as the index test in this study but this test was not considered as an included comparator in this review table 2 shows test performance in detecting ipsilateral breast tumour recurrence in patients undergoing routine surveillance .
the studies by bon and colleagues and drew and colleagues involved a total of 188 patients and reported the performance of surveillance mammography , mri and clinical examination in routine surveillance patients .
these studies reported sensitivities of 64% and 67% , and specificities of 97% and 85% , respectively , for surveillance mammography . for mri , the studies reported sensitivities of 86% and 100% respectively , and for clinical examination 50% and 89% .
the highest reported sensitivity was for mri , and surveillance mammography combined with clinical examination ( both 100% ) while the highest specificity was for surveillance mammography ( 97% ) .
the lowest reported sensitivity was for clinical examination ( 50% ) and the lowest specificity was for surveillance mammography combined with clinical examination ( 67%).table 2sensitivity , specificity , likelihood and diagnostic odds ratios for detecting ipsilateral breast tumour recurrence in routine surveillance patientsteststudy idprimary surgical treatmentreported sensitivity%reported specificity%lr+lrdor ( 95% confidence interval)surveillance mammographybon 1995mastectomy649722.20.460.3 ( 10.2358.1)drew 1998breast conservation67854.60.411.7 ( 2.652.4)mribon 1995mastectomy86not reporteddrew 1998breast conservation1009314.3icicclinical examinationbon 1995mastectomy50not reporteddrew 1998breast conservation89763.70.225.4 ( 3.0213.9)combined surveillance mammography & clinical examinationdrew 1998breast conservation100673.0icicmri magnetic resonance imaging , lr + likelihood ratio of a positive test result , lr likelihood ratio of a negative test result , dor diagnostic odds ratio , ic incalculable sensitivity , specificity , likelihood and diagnostic odds ratios for detecting ipsilateral breast tumour recurrence in routine surveillance patients mri magnetic resonance imaging , lr + likelihood ratio of a positive test result , lr likelihood ratio of a negative test result , dor diagnostic odds ratio , ic incalculable table 3 shows test performance in detecting ipsilateral breast tumour recurrence in patients undergoing non - routine surveillance , as reported by belli and colleagues , mumtaz and colleagues , rieber and colleagues , and ternier and colleagues .
the studies involved a total of 156 patients . across these studies , for surveillance mammography the median ( range ) sensitivity was 71% ( 50% to 83% ) and specificity was 63% ( 57% to 75% ) . for mri , the studies by belli and colleagues , mumtaz and colleagues and rieber and colleagues , involving a total of 193 patients , reported sensitivity of 93% and 100% ( two studies ) and a median ( range ) specificity of 94% ( 88% to 96% ) .
belli and colleagues and ternier and colleagues reported the test performance of ultrasound , with sensitivities of 43% and 87% , and specificities of 31% and 73% respectively , and for clinical examination , sensitivities of 43% and 62% , and specificities of 56% and 49% respectively . the highest reported sensitivity ( 100% ) and specificity ( 96% )
the lowest reported sensitivities were ( 43% ) for both ultrasound and clinical examination , while the lowest specificity was for ultrasound ( 31%).table 3sensitivity , specificity , likelihood ratio and diagnostic odds ratio for detecting ipsilateral breast tumour recurrence in non - routine surveillance patientsteststudy idprimary surgical treatmentreported sensitivity%reported specificity%lr+lrdor ( 95% confidence interval)surveillance mammographybelli 2002breast conservation71631.90.54.2 ( 2.652.4)mumtaz 1997breast conservation50752.00 .
73 ( 0.614.0)ternier 2006breast conservation83571.90.36.3 ( 2.515.6)ultrasoundbelli 2002breast conservation43310.61.80.3 ( 0.12.1)ternier 2006breast conservation87733.20.217 ( 6.246.5)mribelli 2002breast conservation1009416.0icicmumtaz 1997breast conservation93887.40.191 ( 7.41126 . 9)rieber 1997breast conservation1009624.2icicclinical examinationbelli 2002breast conservation43561.01.01.0 ( 0.25.8)ternier 2006breast conservation62491.20.81.5 ( 0.73.4)1 . mri magnetic resonance imaging , lr + likelihood ratio of a positive test result , lr likelihood ratio of a negative test result , dor diagnostic odds ratio , ic incalculable sensitivity , specificity , likelihood ratio and diagnostic odds ratio for detecting ipsilateral breast tumour recurrence in non - routine surveillance patients 1 .
mri magnetic resonance imaging , lr + likelihood ratio of a positive test result , lr likelihood ratio of a negative test result , dor diagnostic odds ratio , ic incalculable table 4 shows test performance in detecting metachronous contralateral breast cancer in routine surveillance patients .
the studies by bon and colleagues and viehweg and colleagues involving a total of 202 patients , reported 67% and 91% sensitivity and 50% and 90% specificity , respectively , for mri .
only individual studies reported the test performance of surveillance mammography , clinical examination , and combinations of tests involving surveillance mammography . the highest reported sensitivity ( 100% ) was for combined surveillance mammography , clinical examination , ultrasound and mri , while the highest reported specificity ( 99% ) was for combined surveillance mammography and ultrasound . the lowest reported sensitivity ( 0% )
was for clinical examination and the lowest specificity was for surveillance mammography , mri and clinical examination ( all 50% ) .table 4test performance as measured by sensitivity , specificity , likelihood ratio and diagnostic odds ratio for detecting metachronous contralateral breast cancer in routine surveillance patientsteststudy idprimary surgical treatmentreported sensitivity%reported specificity%lr+lrdor ( 95% confidence interval)surveillance mammographybon
1995mastectomy67501.30.72.0 ( 0.178.2)mribon 1995mastectomy67501.30.72.0 ( 0.178.2)viehweg 2004breast conservation91909.40.193.1 ( 11.0786.2)clinical examinationbon 1995mastectomy050combined surveillance mammography & ultrasoundkim 2009mastectomy959961.50.051149.2 ( 148.08937.8)breast conservationcombined surveillance mammography , clinical examination & ultrasoundviehweg 2004breast conservation64843.90.48.9 ( 2.433.0)combined surveillance mammography , clinical examination , ultrasound & mriviehweg 2004breast conservation100898.9icicmri magnetic resonance imaging , lr + likelihood ratio of a positive test result , lr likelihood ratio of a negative test result , dor diagnostic odds ratio , ci confidence interval , ic incalculable test performance as measured by sensitivity , specificity , likelihood ratio and diagnostic odds ratio for detecting metachronous contralateral breast cancer in routine surveillance patients mri magnetic resonance imaging , lr + likelihood ratio of a positive test result , lr likelihood ratio of a negative test result , dor diagnostic odds ratio , ci confidence interval , ic incalculable none of the studies reported diagnostic accuracy of the included tests for diagnosing metachronous contralateral breast cancer in non - routine surveillance patients with a previous suspicious test result .
the study conducted by shin and colleagues was the sole study reporting overall test performance for diagnosing ipsilateral breast tumour recurrence and metachronous contralateral breast cancer .
shin and colleagues evaluated ultrasound in routine surveillance patients , reporting a sensitivity of 71% and a specificity of 98% , lr + 41.4 , lr 0.3 , or 138.25 ( 95% ci 61.26 to 312.04 ) .
none of the studies met all of our criteria specified for higher quality studies , although in five[8 , 9 , 11 , 12 , 15 ] this was due to lack of clarity as to whether reference standard results were interpreted without knowledge of index test results only .
it was unclear in all but one study whether the time interval between a positive test result and the histopathological reference standard was short enough to avoid improvement or progression of the condition occurring in the intervening period ( disease progression bias ) .
we are therefore uncertain of the effects of this type of bias for positive test results in these studies .
all studies were judged to have appropriate follow - up time intervals for confirming negative test results and were therefore considered to be at low risk of disease progression bias for negative test results .
it was unclear in the study conducted by shin and colleagues , however , whether all patients with negative test results received follow - up and so there is a possible risk of partial verification bias for this study . the study by drew and colleages was considered vulnerable to partial verification bias as only those participants testing positively on mri received reference standard verification .
none of the included studies reported data concerning adverse effects , acceptability , reliability , radiological / operator expertise and interpretability / readability of the tests .
we found no discernible pattern for the histology of cancers detected and not detected both within and between diagnostic tests .
table 2 shows test performance in detecting ipsilateral breast tumour recurrence in patients undergoing routine surveillance . the studies by bon and colleagues and drew and colleagues involved a total of 188 patients and reported the performance of surveillance mammography , mri and clinical examination in routine surveillance patients .
these studies reported sensitivities of 64% and 67% , and specificities of 97% and 85% , respectively , for surveillance mammography . for mri , the studies reported sensitivities of 86% and 100% respectively , and for clinical examination 50% and 89% .
bon and colleagues did not report specificity for mri or clinical examination . the highest reported sensitivity was for mri , and surveillance mammography combined with clinical examination ( both 100% ) while the highest specificity was for surveillance mammography ( 97% ) .
the lowest reported sensitivity was for clinical examination ( 50% ) and the lowest specificity was for surveillance mammography combined with clinical examination ( 67%).table 2sensitivity , specificity , likelihood and diagnostic odds ratios for detecting ipsilateral breast tumour recurrence in routine surveillance patientsteststudy idprimary surgical treatmentreported sensitivity%reported specificity%lr+lrdor ( 95% confidence interval)surveillance mammographybon 1995mastectomy649722.20.460.3 ( 10.2358.1)drew 1998breast conservation67854.60.411.7 ( 2.652.4)mribon 1995mastectomy86not reporteddrew 1998breast conservation1009314.3icicclinical examinationbon 1995mastectomy50not reporteddrew 1998breast conservation89763.70.225.4 ( 3.0213.9)combined surveillance mammography & clinical examinationdrew 1998breast conservation100673.0icicmri magnetic resonance imaging , lr + likelihood ratio of a positive test result , lr likelihood ratio of a negative test result , dor diagnostic odds ratio , ic incalculable sensitivity , specificity , likelihood and diagnostic odds ratios for detecting ipsilateral breast tumour recurrence in routine surveillance patients mri magnetic resonance imaging , lr + likelihood ratio of a positive test result , lr likelihood ratio of a negative test result , dor diagnostic odds ratio , ic incalculable table 3 shows test performance in detecting ipsilateral breast tumour recurrence in patients undergoing non - routine surveillance , as reported by belli and colleagues , mumtaz and colleagues , rieber and colleagues , and ternier and colleagues .
the studies involved a total of 156 patients . across these studies , for surveillance mammography the median ( range ) sensitivity was 71% ( 50% to 83% ) and specificity was 63% ( 57% to 75% ) . for mri , the studies by belli and colleagues , mumtaz and colleagues and rieber and colleagues , involving a total of 193 patients , reported sensitivity of 93% and 100% ( two studies ) and a median ( range ) specificity of 94% ( 88% to 96% ) . belli and colleagues and ternier and colleagues reported the test performance of ultrasound , with sensitivities of 43% and 87% , and specificities of 31% and 73% respectively , and for clinical examination , sensitivities of 43% and 62% , and specificities of 56% and 49% respectively . the highest reported sensitivity ( 100% ) and specificity ( 96% )
the lowest reported sensitivities were ( 43% ) for both ultrasound and clinical examination , while the lowest specificity was for ultrasound ( 31%).table 3sensitivity , specificity , likelihood ratio and diagnostic odds ratio for detecting ipsilateral breast tumour recurrence in non - routine surveillance patientsteststudy idprimary surgical treatmentreported sensitivity%reported specificity%lr+lrdor ( 95% confidence interval)surveillance mammographybelli 2002breast conservation71631.90.54.2 ( 2.652.4)mumtaz 1997breast conservation50752.00 .
73 ( 0.614.0)ternier 2006breast conservation83571.90.36.3 ( 2.515.6)ultrasoundbelli 2002breast conservation43310.61.80.3 ( 0.12.1)ternier 2006breast conservation87733.20.217 ( 6.246.5)mribelli 2002breast conservation1009416.0icicmumtaz 1997breast conservation93887.40.191 ( 7.41126 .
9)rieber 1997breast conservation1009624.2icicclinical examinationbelli 2002breast conservation43561.01.01.0 ( 0.25.8)ternier 2006breast conservation62491.20.81.5 ( 0.73.4)1 . mri magnetic resonance imaging , lr + likelihood ratio of a positive test result , lr likelihood ratio of a negative test result , dor diagnostic odds ratio , ic incalculable sensitivity , specificity , likelihood ratio and diagnostic odds ratio for detecting ipsilateral breast tumour recurrence in non - routine surveillance patients 1 .
mri magnetic resonance imaging , lr + likelihood ratio of a positive test result , lr likelihood ratio of a negative test result , dor diagnostic odds ratio , ic incalculable table 4 shows test performance in detecting metachronous contralateral breast cancer in routine surveillance patients .
the studies by bon and colleagues and viehweg and colleagues involving a total of 202 patients , reported 67% and 91% sensitivity and 50% and 90% specificity , respectively , for mri .
only individual studies reported the test performance of surveillance mammography , clinical examination , and combinations of tests involving surveillance mammography . the highest reported sensitivity ( 100% ) was for combined surveillance mammography , clinical examination , ultrasound and mri , while the highest reported specificity ( 99% ) was for combined surveillance mammography and ultrasound . the lowest reported sensitivity ( 0% ) was for clinical examination and the lowest specificity was for surveillance mammography , mri and clinical examination ( all 50% ) .table 4test performance as measured by sensitivity , specificity , likelihood ratio and diagnostic odds ratio for detecting metachronous contralateral breast cancer in routine surveillance patientsteststudy idprimary surgical treatmentreported sensitivity%reported specificity%lr+lrdor ( 95% confidence interval)surveillance mammographybon 1995mastectomy67501.30.72.0 ( 0.178.2)mribon 1995mastectomy67501.30.72.0 ( 0.178.2)viehweg 2004breast conservation91909.40.193.1 ( 11.0786.2)clinical examinationbon 1995mastectomy050combined surveillance mammography & ultrasoundkim 2009mastectomy959961.50.051149.2 ( 148.08937.8)breast conservationcombined surveillance mammography , clinical examination & ultrasoundviehweg 2004breast conservation64843.90.48.9 ( 2.433.0)combined surveillance mammography , clinical examination , ultrasound & mriviehweg 2004breast conservation100898.9icicmri magnetic resonance imaging , lr + likelihood ratio of a positive test result , lr likelihood ratio of a negative test result , dor diagnostic odds ratio , ci confidence interval , ic incalculable test performance as measured by sensitivity , specificity , likelihood ratio and diagnostic odds ratio for detecting metachronous contralateral breast cancer in routine surveillance patients mri magnetic resonance imaging , lr + likelihood ratio of a positive test result , lr likelihood ratio of a negative test result , dor diagnostic odds ratio , ci confidence interval , ic incalculable none of the studies reported diagnostic accuracy of the included tests for diagnosing metachronous contralateral breast cancer in non - routine surveillance patients with a previous suspicious test result . the study conducted by shin and colleagues was the sole study reporting overall test performance for diagnosing ipsilateral breast tumour recurrence and metachronous contralateral breast cancer .
shin and colleagues evaluated ultrasound in routine surveillance patients , reporting a sensitivity of 71% and a specificity of 98% , lr + 41.4 , lr 0.3 , or 138.25 ( 95% ci 61.26 to 312.04 ) .
none of the studies met all of our criteria specified for higher quality studies , although in five[8 , 9 , 11 , 12 , 15 ] this was due to lack of clarity as to whether reference standard results were interpreted without knowledge of index test results only .
it was unclear in all but one study whether the time interval between a positive test result and the histopathological reference standard was short enough to avoid improvement or progression of the condition occurring in the intervening period ( disease progression bias ) .
we are therefore uncertain of the effects of this type of bias for positive test results in these studies .
all studies were judged to have appropriate follow - up time intervals for confirming negative test results and were therefore considered to be at low risk of disease progression bias for negative test results .
it was unclear in the study conducted by shin and colleagues , however , whether all patients with negative test results received follow - up and so there is a possible risk of partial verification bias for this study .
the study by drew and colleages was considered vulnerable to partial verification bias as only those participants testing positively on mri received reference standard verification . none of the included studies reported data concerning adverse effects , acceptability , reliability , radiological / operator expertise and interpretability / readability of the tests .
we found no discernible pattern for the histology of cancers detected and not detected both within and between diagnostic tests .
table 2 shows test performance in detecting ipsilateral breast tumour recurrence in patients undergoing routine surveillance . the studies by bon and colleagues and drew and colleagues involved a total of 188 patients and reported the performance of surveillance mammography , mri and clinical examination in routine surveillance patients .
these studies reported sensitivities of 64% and 67% , and specificities of 97% and 85% , respectively , for surveillance mammography . for mri , the studies reported sensitivities of 86% and 100% respectively , and for clinical examination 50% and 89% .
bon and colleagues did not report specificity for mri or clinical examination . the highest reported sensitivity was for mri , and surveillance mammography combined with clinical examination ( both 100% ) while the highest specificity was for surveillance mammography ( 97% ) .
the lowest reported sensitivity was for clinical examination ( 50% ) and the lowest specificity was for surveillance mammography combined with clinical examination ( 67%).table 2sensitivity , specificity , likelihood and diagnostic odds ratios for detecting ipsilateral breast tumour recurrence in routine surveillance patientsteststudy idprimary surgical treatmentreported sensitivity%reported specificity%lr+lrdor ( 95% confidence interval)surveillance mammographybon 1995mastectomy649722.20.460.3 ( 10.2358.1)drew 1998breast conservation67854.60.411.7 ( 2.652.4)mribon 1995mastectomy86not reporteddrew 1998breast conservation1009314.3icicclinical examinationbon 1995mastectomy50not reporteddrew 1998breast conservation89763.70.225.4 ( 3.0213.9)combined surveillance mammography & clinical examinationdrew 1998breast conservation100673.0icicmri magnetic resonance imaging , lr + likelihood ratio of a positive test result , lr likelihood ratio of a negative test result , dor diagnostic odds ratio , ic incalculable sensitivity , specificity , likelihood and diagnostic odds ratios for detecting ipsilateral breast tumour recurrence in routine surveillance patients mri magnetic resonance imaging , lr + likelihood ratio of a positive test result , lr likelihood ratio of a negative test result , dor diagnostic odds ratio , ic incalculable table 3 shows test performance in detecting ipsilateral breast tumour recurrence in patients undergoing non - routine surveillance , as reported by belli and colleagues , mumtaz and colleagues , rieber and colleagues , and ternier and colleagues .
the studies involved a total of 156 patients . across these studies , for surveillance mammography the median ( range ) sensitivity was 71% ( 50% to 83% ) and specificity was 63% ( 57% to 75% ) . for mri , the studies by belli and colleagues , mumtaz and colleagues and rieber and colleagues , involving a total of 193 patients , reported sensitivity of 93% and 100% ( two studies ) and a median ( range ) specificity of 94% ( 88% to 96% ) . belli and colleagues and ternier and colleagues reported the test performance of ultrasound , with sensitivities of 43% and 87% , and specificities of 31% and 73% respectively , and for clinical examination , sensitivities of 43% and 62% , and specificities of 56% and 49% respectively . the highest reported sensitivity ( 100% ) and specificity ( 96% )
the lowest reported sensitivities were ( 43% ) for both ultrasound and clinical examination , while the lowest specificity was for ultrasound ( 31%).table 3sensitivity , specificity , likelihood ratio and diagnostic odds ratio for detecting ipsilateral breast tumour recurrence in non - routine surveillance patientsteststudy idprimary surgical treatmentreported sensitivity%reported specificity%lr+lrdor ( 95% confidence interval)surveillance mammographybelli 2002breast conservation71631.90.54.2 ( 2.652.4)mumtaz 1997breast conservation50752.00 .
73 ( 0.614.0)ternier 2006breast conservation83571.90.36.3 ( 2.515.6)ultrasoundbelli 2002breast conservation43310.61.80.3 ( 0.12.1)ternier 2006breast conservation87733.20.217 ( 6.246.5)mribelli 2002breast conservation1009416.0icicmumtaz 1997breast conservation93887.40.191 ( 7.41126 .
9)rieber 1997breast conservation1009624.2icicclinical examinationbelli 2002breast conservation43561.01.01.0 ( 0.25.8)ternier 2006breast conservation62491.20.81.5 ( 0.73.4)1 . mri magnetic resonance imaging , lr + likelihood ratio of a positive test result , lr likelihood ratio of a negative test result , dor diagnostic odds ratio , ic incalculable sensitivity , specificity , likelihood ratio and diagnostic odds ratio for detecting ipsilateral breast tumour recurrence in non - routine surveillance patients 1 .
mri magnetic resonance imaging , lr + likelihood ratio of a positive test result , lr likelihood ratio of a negative test result , dor diagnostic odds ratio , ic incalculable
table 4 shows test performance in detecting metachronous contralateral breast cancer in routine surveillance patients .
the studies by bon and colleagues and viehweg and colleagues involving a total of 202 patients , reported 67% and 91% sensitivity and 50% and 90% specificity , respectively , for mri .
only individual studies reported the test performance of surveillance mammography , clinical examination , and combinations of tests involving surveillance mammography . the highest reported sensitivity ( 100% ) was for combined surveillance mammography , clinical examination , ultrasound and mri , while the highest reported specificity ( 99% ) was for combined surveillance mammography and ultrasound . the lowest reported sensitivity ( 0% ) was for clinical examination and the lowest specificity was for surveillance mammography , mri and clinical examination ( all 50% ) .table 4test performance as measured by sensitivity , specificity , likelihood ratio and diagnostic odds ratio for detecting metachronous contralateral breast cancer in routine surveillance patientsteststudy idprimary surgical treatmentreported sensitivity%reported specificity%lr+lrdor ( 95% confidence interval)surveillance mammographybon 1995mastectomy67501.30.72.0 ( 0.178.2)mribon 1995mastectomy67501.30.72.0 ( 0.178.2)viehweg 2004breast conservation91909.40.193.1 ( 11.0786.2)clinical examinationbon 1995mastectomy050combined surveillance mammography & ultrasoundkim 2009mastectomy959961.50.051149.2 ( 148.08937.8)breast conservationcombined surveillance mammography , clinical examination & ultrasoundviehweg 2004breast conservation64843.90.48.9 ( 2.433.0)combined surveillance mammography , clinical examination , ultrasound & mriviehweg 2004breast conservation100898.9icicmri magnetic resonance imaging , lr + likelihood ratio of a positive test result , lr likelihood ratio of a negative test result , dor diagnostic odds ratio , ci confidence interval , ic incalculable test performance as measured by sensitivity , specificity , likelihood ratio and diagnostic odds ratio for detecting metachronous contralateral breast cancer in routine surveillance patients mri magnetic resonance imaging , lr + likelihood ratio of a positive test result , lr likelihood ratio of a negative test result , dor diagnostic odds ratio , ci confidence interval , ic incalculable none of the studies reported diagnostic accuracy of the included tests for diagnosing metachronous contralateral breast cancer in non - routine surveillance patients with a previous suspicious test result .
the study conducted by shin and colleagues was the sole study reporting overall test performance for diagnosing ipsilateral breast tumour recurrence and metachronous contralateral breast cancer .
shin and colleagues evaluated ultrasound in routine surveillance patients , reporting a sensitivity of 71% and a specificity of 98% , lr + 41.4 , lr 0.3 , or 138.25 ( 95% ci 61.26 to 312.04 ) .
none of the studies met all of our criteria specified for higher quality studies , although in five[8 , 9 , 11 , 12 , 15 ] this was due to lack of clarity as to whether reference standard results were interpreted without knowledge of index test results only .
it was unclear in all but one study whether the time interval between a positive test result and the histopathological reference standard was short enough to avoid improvement or progression of the condition occurring in the intervening period ( disease progression bias ) .
we are therefore uncertain of the effects of this type of bias for positive test results in these studies .
all studies were judged to have appropriate follow - up time intervals for confirming negative test results and were therefore considered to be at low risk of disease progression bias for negative test results .
it was unclear in the study conducted by shin and colleagues , however , whether all patients with negative test results received follow - up and so there is a possible risk of partial verification bias for this study . the study by drew and colleages was considered vulnerable to partial verification bias as only those participants testing positively on mri received reference standard verification .
none of the included studies reported data concerning adverse effects , acceptability , reliability , radiological / operator expertise and interpretability / readability of the tests .
we found no discernible pattern for the histology of cancers detected and not detected both within and between diagnostic tests .
our findings should be interpreted with caution , as they are based on only nine studies involving a total of 3724 participants .
furthermore , the study conducted by bon and colleagues included only mastectomy patients who underwent breast reconstruction using implants .
surveillance of the chest wall and/or the reconstructed breast in patients receiving either mastectomy alone , or mastectomy with breast reconstruction and implants , varies according to different health care systems and local protocols [ 1618 ] . these comprise an increasingly relevant sub - group of women , because of increasing rates of breast reconstruction procedures , who might receive routine surveillance mammography in the future . results from this study should be treated as distinct from the others owing to the highly selected patient population who , whilst representing a subset of our considered population , differ greatly from the wider spectrum of women who receive surveillance in practice .
systematic reviews of diagnostic accuracy are highly complex and methodology in this area continues to evolve . in terms of strengths , we believe that the methods adopted for this review are scientifically rigorous and compatible with current guidance in this area .
a limitation was that non - english language studies were excluded , potentially limiting the evidence base .
of the studies included here , few evaluated the performance of the considered tests for similar purposes .
furthermore , even where data were available it was not clinically appropriate to combine them , for example , because of differences between a treated and an untreated breast .
similarly , it was inappropriate to combine data from routine and non - routine surveillance patients
. furthermore , no data were reported by the included studies on other aspects such as adverse effects or acceptability of the tests .
results for the index and comparator tests evaluated in this review were ascertained by subjective operator interpretation , either by visual inspection of an image of the breast ( surveillance mammography , ultrasound and mri ) or by clinical examination of the breast .
data on the level of operator expertise or intra / inter - rater reliability were not reported .
it is therefore unclear whether these factors had any influence on reported test accuracy within , and between , studies and therefore whether any potential test operator bias exists .
none of the studies met all of our criteria specified for higher quality studies , although they were judged to have reasonable internal validity .
all but one study were considered to include a representative sample and therefore have good external validity .
our findings suggest that mri can be considered to have higher diagnostic value than surveillance mammography in women previously treated for primary breast cancer . of the test combinations
reported , surveillance mammography combined with breast ultrasound could be considered the most accurate combination of tests for detecting metachronous contralateral breast cancer
. however these results should be interpreted with caution owing to the paucity of data for all diagnostic tests available for breast cancer surveillance .
further evidence on surveillance mammography and other diagnostic tests in this group of women is required in order to make a robust and informed judgement on their relative performance .
ideally a definitive randomised controlled trial should be undertaken focusing on those women at higher risk of ipsilateral breast tumour recurrence or metachronous contralateral breast cancer .
such a trial might also compare more sophisticated surveillance regimens that vary not only in terms of the frequency of mammography or other diagnostic tests but also in terms of the frequency and setting of clinical follow - up .
alternatively , high - quality , direct head - to - head studies could be undertaken comparing the diagnostic accuracy of tests used in the surveillance population . | objectivesto determine the diagnostic accuracy of surveillance mammography for detecting ipsilateral breast tumour recurrence and metachronous contralateral breast cancer in women previously treated for primary breast cancer.methodsa systematic review of surveillance mammography compared with ultrasound , magnetic resonance imaging ( mri ) , specialist - led clinical examination or unstructured primary care follow - up , using histopathological assessment for test positives and follow - up for test negatives as the reference standard.resultsnine studies met our inclusion criteria .
variations in study comparisons precluded meta - analysis . for routine ipsilateral breast tumour detection , surveillance mammography sensitivity ranged from 6467% and specificity ranged from 8597% . for mri , sensitivity ranged from 86100% and specificity was 93% . for non - routine ipsilateral breast tumour detection , sensitivity and specificity for surveillance mammography ranged from 5083% and 5775% and for mri 93100% and 8896% . for routine metachronous contralateral breast cancer detection ,
one study reported sensitivity of 67% and specificity of 50% for both surveillance mammography and mri.conclusionalthough mammography is associated with high sensitivity and specificity , mri is the most accurate test for detecting ipsilateral breast tumour recurrence and metachronous contralateral breast cancer in women previously treated for primary breast cancer .
results should be interpreted with caution because of the limited evidence base.key points surveillance mammography is associated with high sensitivity and specificity findings suggest that mri is the most accurate test for detecting further breast cancer robust conclusions can not be made due to the limited evidence base further research comparing surveillance mammography and other diagnostic tests is required |
neurofibromatosis type - i ( nf-1 , von recklinghausen 's disease ) is an autosomal - dominant single - gene hereditary disorder of neural crest cells , involving neuroectoderm , mesoderm , and endoderm .
it affects 1:3000 - 4000 individuals and is caused by a defect in the gene responsible for the production of the protein neurofibromin : the nf-1 gene .
the nf-1 gene is a tumor suppressor gene linked to the long arm of chromosome 17 .
the clinical manifestations of nf-1 are as follows : caf - au - lait spots , axillary or inguinal freckling , lisch nodules of the iris , neurofibromas , optic gliomas , and spinal skeletal deformities .
the latter manifest as acquired short , sharp , and angular ( usually thoracic ) scoliotic curves .
dystrophic changes such as rib - penciling , vertebral scalloping , transverse process spindling , apical wedging , foraminal enlargement , and dural ectasia are classic features
various procedures have been reported and entail anterior , posterior , and combined anterior / posterior approaches to establish arthrodesis and prevent curve progression .
we describe a case that highlights the operative management of an nf-1 patient with severe dystrophic thoracic kyphoscoliosis treated by a posterolateral spinal approach .
a 44-year - old patient with nf-1 presented to the department of neurosurgery with myelopathy caused by a 93 thoracic ( t5-t9 ) kyphoscoliosis , thoracic vertebral scalloping leading to a severe thinning of the vertebral bodies , profound stenosis by a bony ridge between two dural ectasias at t4-t5 anteriorly and auto fusion of t4-t7 [ figures 1 and 2 ] . magnetic resonance image of a 93 thoracic kyphoscoliosis and stenosis due to a bony ridge ( red circle ) between two dural ectasias at t4-t5 anteriorly and an autofusion of t4-t7 .
note the dural ctasias ( asterix ) and the vertebral scalloping ( yellow curve ) computed tomography image of the thoracic spine with a 93 kyphoscoliosis .
note the bony anterior autofusion of t4-t7 clinically , she had progressive sensory disturbances and upper motor signs .
operative intervention , performed using intraoperative neuromonitoring , consisted of a posterolateral approach with posterior thoracic cord exposure , anterior decompression of the bony ridge on t4-t5 by a right costotransversectomy [ figure 3 ] at t4 and posterior fusion of c6-t12 with bilateral pedicle screws at the c6 , t11 , and t12 level as well as bilateral bridging rod instrumentation [ figures 4 and 5 ] .
cadaver view of the anterior thoracic spinal cord with the thoracic nerve root lifted upward ( curved arrow ) after a performed right costotransversectomy at t4 .
note the detailed exposure of the anterior spinal cord with its epidural space ( white circle ) , corresponding veins ( black arrow ) , and the posterior longitudinal ligament ( white arrow ) .
remark : the spinous process and the lamina have been removed for demonstrative purposes postoperative x - ray image showing the posterior fusion from c6 to t12 with bilateral pedicle screws at the c6 , t11 , and t12 level and bilateral bridging rods postoperative computed tomography image with posterior fusion of c6-t12 .
note the pedicle screws at the c6 , t11 , and t12 level as well as the resection of the bony ridge at t4-t5 ( red circle )
it occurs usually in the low thoracic region and has a prevalence ranging from 2% to 69% in the literature .
etiological theories regarding nf-1-related scoliosis include erosion or infiltration of bone by neurofibromas , primary mesodermal dysplasia , osteomalacia , and endocrine disturbances .
this classification is based on the absence or presence of skeletal dysplasia , such as vertebral scalloping and associated dural ectasias , rib penciling , vertebral wedging , transverse process spindling , paraspinal or intraspinal soft tissue masses , apical rotation , foraminal enlargement , pedicle dysplasia , and widened interpediculate distance .
the literature states that the likelihood of curve progression and deterioration is proportional to the severity of skeletal dystrophy .
studies have shown that when a combination of three or more dysplastic features are present , the risk of curve deterioration was significantly increased in 85% of patients .
non - dystrophic curves can be managed as idiopathic scoliosis , dystrophic curves , however , need a more aggressive approach . a dystrophic curve pattern , as this was the case in our patient , classically presents as a short segment , sharply angulated , thoracic curve with three or more dystrophic elements .
they require aggressive treatment as there is a strong tendency for curve progression and clinical catastrophic deterioration .
dystrophic scoliosis less than 20 should be closely monitored at 6 month intervals . in scoliosis , measuring 20-40 with less than 50 of kyphosis , a posterior spinal arthrodesis is feasible .
however , when the scoliosis exceeds 40 and/or thoracic hyperkyphosis exceeds 50 , a combined anterior / posterior ( circumferential ) fusion is mandatory .
neurological deficits are usually caused by hyperkyphosis and subsequent attenuation and deformation of the spinal cord . in those cases , an anterior decompression followed by circumferential fusion
other contributory factors include intraspinal tumors , which have to be resected followed by a mechanical stabilization and bony fusion .
one of the difficulties encountered in nf - spinal surgery is the segmental instrumentation and fusion as there can be poor anchorage points for internal fixation due to vertebral destruction .
due to the high incidence of pseudarthrosis , fusion evaluation should be performed at 6 months and at regular intervals until solid arthrodesis is obtained . in case of failure ,
the neurological deficit was caused by a 93 thoracic ( t5-t9 ) kyphoscoliosis and stenosis due to a bony ridge between two dural ectasias at t4-t5 anteriorly [ figure 1 ] .
furthermore , there was an autofusion of t4-t7 [ figures 1 and 2 ] . due to the anterior t4-t7 autofusion , severe kyphosis , and the presence of dural actasias
we choose a posterolateral approach [ figure 3 ] instead with thoracic cord exposure , anterior decompression of the bony ridge on t4-t5 by a right costotransversectomy at t4 and posterior fusion of c6-t12 with bilateral pedicle screws at the c6 , t11 and t12 level as well as bilateral bridging rod instrumentation [ figures 4 and 5 ] .
we state that in selected cases of severe thoracic dystrophic kyphoscoliosis in nf - i , the posterolateral approach [ figure 3 ] is the only possible method to visualize the anterior thoracic spinal cord , perform anterior decompression , and to stabilize the thoracic deformity , because of the anterior dural ectasias and the kyphosis limiting an anterior procedure . | neurofibromatosis type - i ( nf - i ) is an autosomal - dominant hereditary disorder , in which spinal skeletal deformities are one of the manifestations and manifest as acquired abnormalities and present as short , sharp , and angular ( usually thoracic ) curves .
the scoliosis can be severe requiring surgical intervention .
the surgical procedure can be difficult because of scalloping of the vertebral bodies and dural ectasia .
we state that in selected cases of severe thoracic dystrophic kyphoscoliosis in nf - i , the posterolateral approach is the only possible method to visualize the anterior thoracic spinal cord , perform anterior decompression , and to stabilize the thoracic deformity , because of the anterior dural ectasias and the kyphosis limiting an anterior procedure . |
smoking is still the single greatest preventable cause of premature death and disability in modern times.1 it has been estimated that one third of the world 's population , above 15 years of age , are current smokers .
it is much more prevalent among males ( 47% ) than females ( 12%).2 tobacco consumption does not only pose multiple health risks ( respiratory , cardiovascular , gastrointestinal , genito - urinary ) , but also adversely affects the lives of individuals , their families and communities , socially , economically and hence developmentally.34 smoking places are the heaviest morbidity and mortality burden on people , compared to any other risk factor.1 the estimated annual death rate of about 3.5 - 4 million people is expected to rise to 10 millions by the 2020s - 2030s , 7 million of which will occur in developing countries.2 to compensate for the deaths , tobacco companies need to recruit 11,000 new smokers daily , especially young adolescents , to maintain the size of their business.5 most smokers ( 78 - 90% ) take up the habit during adolescence ( 10 - 19 years ) ; less than 2% of them start smoking after their 22 birthday.35 students in the countries of gulf cooperation council ( gcc ) have estimated that the prevalence of smoking among male secondary students ranges from 12 - 50%.611 among gcc male medical students , the rate of prevalence was 13.6 - 33% , compared to only 2.3 - 8.6% among female medical students ( 12 - 16 ) .
very few studies have been done among the students of the allied sciences . however , saeed reported that smoking prevalence among students of the allied medical sciences in riyadh from 12.4% among females to 46.8% among males;17 but was much lower ( 8 - 17.5% ) among students of the secondary health institutes in riyadh.18 while several studies have looked into the knowledge and practice of smoking among secondary school or medical students in the central and south - western saudi arabia,791214 to the best of our knowledge there has been no study of nursing or medical laboratory technology ( mlt ) university students in the eastern province .
this work is aimed at exploring the patterns of smoking among nursing and mlt students and interns of king faisal university college of medicine to estimate the magnitude of the problem ; assess their knowledge on smoking and find out their attitude towards the habit .
this was a cross - sectional study of nursing and mlt students at king faisal university , dammam , saudi arabia in 1998/1999 .
all students and interns enrolled in the nursing and mlt departments during the academic year were included in the study sample ( overall n=266 ; with nursing n1=152 and mlt n2=114 ) .
all students were included in the study to ensure an adequate sample size . a self - administered arabic questionnaire , modified from the world health organization tools was used.1920 the questionnaire was first tested in a pilot study of medical students .
the nursing and mlt students ( 1998/1999 ) were then asked to complete the questionnaire after the objectives of the study and contents of the study tool ( questionnaire ) had been classified by a member of the research team .
the questionnaire , which had been explained to the students , included 3 groups of questions .
the first elicited demographic information , such as college year , age , sex , marital status , nationality , residence , parents education , parents occupation and smoking status .
the second group of questions inquired about pattern of smoking behaviour : e.g. type , duration , frequency , age / source / cause/ where the habit began . who definitions of current and ex - smoking status were adopted.19 the third group included questions about attitude towards smoking and smokers .
after the data collection , all the information was entered into the computer and checked . using the spss / pc programme it was transferred for analysis after it had been rechecked for accuracy.21 univariate and bivariate analyses of the reviewed data were used , as appropriate .
tables 1 to 3 reflect the attributes of the practice of tobacco consumption in the study sample ( n=266 ) .
the mean age of students and interns studied was 19.7 1.8 years ( ranging from 16 - 30 years ) .
as shown in table 1 , only 15 of them were smokers ( 5.6% ) , but the prevalence was higher among nursing students ( 6.6% ) , compared to mlt students ( 4.4% ) . of
the mlt group showed the highest prevalence of smoking was among the interns ( 27.3% ) , while the prevalence was highest among the second year students in the nursing group ( 13.8% ) .
prevalence of smoking comparing nursing with mlt female students and interns by specialty and school year , 1998 - 1999 characteristics of smokers among nursing and mlt students and interns , 1998 - 1999 distribution of smokers according to selected variables 1998 - 1999 as shown on table 2 , the mean age at which smoking started was 17.6 2.5 years ( ranging from 14 - 22 years ) , with a mean duration of 1.7 1.1 years .
as shown on table 3 , most smokers ( 86.7% ) smoked only cigarettes or shisha ( water - pipe ) as well the quantity , ranging from 1 - 6 cigarettes per day .
two thirds of the smokers ( 66.7% ) have been smoking daily for the last 6 months - 5 years .
almost half the smokers ( 46.7% ) indicated that friends or parents had been the source of the first cigarette .
curiosity ( 53.5% ) , and stress ( 33.3% ) were the most important reported motives for initiating the habit .
most of the smokers ( 66.7% ) had started the habit at home . for tables 4 and 5 ,
the mentioned numbers represent the percentage of students who responded with true to these statements ( i.e. , agreed to the statement , whether true or
table 4 deals with a comparison between the views of mlt students and interns and nursing students on tobacco use .
it also deals with those who have never smoked as against those who have . for most statements ( referred to as group a ) , true is the correct answer ; except for the two ( referred to as group b some types of cigarettes are harmless ) to which
false is the correct response and quitting smoking is too difficult ( 74.1 - 97.7% ) . except for the statement : smoking may lead to stomach cancer which was wrongly responded to by more than half the participants of the study ( 54.1% ) group a responded to most statements correctly .
group b , generally got the two questions correct ( only 13.9 - 28.9% ) agreed to these false statements ) .
there was a statistically significant difference between the nursing and mlt with regard to their percent correct responses to some statements such as : smoking increases the heart rate ,
a similar difference was observed when the response of those who had never smoked on the statement .
most of lung cancer patients were regular smokers were compared with those who had .
a comparison of the knowledge on smoking of nursing with mlt female students and interns by specialty and smoking status , 1998 - 1999 smoking attitudes comparing nursing with mlt female students and interns by specialty and smoking status , 1998 - 1999 table 5 compares the attitude of nursing and mlt students / interns ; the ever and never smokers on statements on tobacco use . on the whole ( n=266 )
, most statements were responded to correctly , except for three statements which should have been rejected as false .
i should smoke whenever in the company of smokers , which was wrongly agreed to by 91% of the respondents .
never smokers were compared , a highly significant statistical difference was observed ( p<0.001 ) . the second statement : smoking is a personal matter .
i do it in the time / place of my choice , was wrongly agreed upon by 82.4% of respondents , but similarly when the ever with
never smokers were compared , a highly significant statistical difference was observed ( p<0.001 ) . the third statement :
information gained about smoking in the college is enough for to help people stop smoking , was agreed upon by 47.8% of respondents , but a comparison of the
only three statements showed a statistically significant difference when nursing and mlt students and interns were compared .
these were physicians are not doing enough to help people stop smoking , if you are a smoker , will you be upset when others ask you to quit , and every encounter with a smoking patient should be utilized to convince him / her to quit .
it has been observed that tobacco consumption in this region has escalated with the rising population , increased purchasing power , especially of the youth , campaigns by tobacco companies , and the increase in the number of girls and women who smoke .
another reason to the lack of awareness of the hazards of smoking , insufficient support and inadequate funds for campaigns against the use of tobacco.22 it is unfortunate that this habit which inevitably results in smoking - related diseases and other subsequent social and economic problems is on the increase.23 this study on female university students and interns , from the nursing and mlt departments of king faisal university indicated an overall smoking prevalence of 5.6% . in comparison with other saudi studies on prevalence of smoking among female students , reported by felimban ( 1993 ) as 4.7% among king saud university medical students;13 by saeed et al ( 1993 ) as 8% in secondary health institutes in riyadh,18
as high as 12.4% among allied medical sciences students in 1987 was fair.17 it should be emphasized that these figures are low in comparison with female professionals , such as saudi female physicians ( 15.8% ) as reported in another study by saeed et al in 1989.24 even though these figures , ranging from 4.7 - 15.8% , are much lower than those among male students or health professionals , they are quite alarming .
women / mothers who are to be role models for their children , are in continuous contact with their offspring , should be in good health to function efficiently and fulfil their responsibilities to their families as well as their communities .
the effect of this new epidemic among women on health and socio - economic conditions if allowed to continue would indeed be disastrous . according to our study
this is quite similar to what was reported by saeed in two studies in which he stated that 92% of female smokers among allied medical sciences students formed the habit when they were less than 18 years,17 while all female smokers in secondary health institutes started between 10 - 20 years.18 these figures reaffirm the importance of effective health education as early as possible in life in schools and through the mass media in order to protect children , especially girls , from adopting this dangerous habit . in this
study the most cited motives for the adoption of the habit of smoking were curiosity ( 53% ) followed by relief of stress ( 33% ) .
this is in agreement with the findings of abolfotouh et al in 1998,14 where curiosity/ showing off were each mentioned by 52% of the smokers in king saud university in abha ( currently known as king khalid university ) .
compared to mlt students in our study , the nurses knowledge about smoking and its hazards was generally more satisfactory .
these results are comparable to the results of other studies on medical students done elsewhere in saudi arabia.1214 the knowledge of smokers as compared to non - smokers did not yield any statistically significant difference .
this was not in conformity with the report of abolfotouh et al,14 which showed that non - smokers were more knowledgeable than smokers .
bridging the gap between knowledge and practice is difficult and requires intensive and collaborative effort on the part of the entire community .
our findings were similar to those of abolftouh et al in two studies1425 on the attitude towards public health drives against smoking in that they were less favorable among smokers compared to non - smokers .
a similar pattern was observed for statements of attitude towards time , place and company of smoking , which is again in agreement with the studies mentioned above .
differences in attitude statements between nursing and mlt students strongly reflected the background or training offered to each group .
the nurses attitude was more inclined towards the clinical sciences and patient satisfaction whereas mlts focus more on laboratory technology studies . in conclusion
, an effective sound education on tobacco consumption should be incorporated into our educational system , commencing as early as the primary schools .
in addition , the media engagement in this drive to control tobacco use should be intensified to reach the entire community .
there should be a vigorously pursuit of this objective through curricula designed for future health professionals , including nursing and mlt departments of universities
. these professionals should be able to provide sound advice to the public on the one hand ( as through health promotion and education activities ) , as well as present themselves good role models for their communities . | objective : to study the reported practices of knowledge about and attitude towards smoking among nursing and medical laboratory technology ( mlt ) students , college of medicine , king faisal university at dammam and al-khobar.setting:college of medicine , dammam and king fahd hospital of the university , al - khobar , saudi arabiamethods : a cross - sectional approach involving a sample of 266 students and interns ( 152 nursing and 114 mlt ) , which included all enrolled students in the academic year ( 1998/1999 ) . a self - administered questionnaire was used to collect data covering knowledge , practice and attitude to smoking .
spss was used for statistical analysis.results:the overall smoking prevalence was low ( 5.6% ) , slightly higher among nursing ( 6.6% ) versus mlt ( 4.4% ) students .
knowledge of and attitude towards smoking was generally satisfactory in both groups , although deficient in some key areas , such as the addictive nature of smoking , some of its consequences on health , and difficulty of quitting.conclusion and recommendations : the prevalence of smoking among nursing and mlt students is generally low but their knowledge and attitude need improvement .
health education on facts , dangers and consequences of smoking should start as early as the primary school , and should continue throughout the education of future health professionals ( role models for the community ) . |
periodontitis can be considered as the consequence of broken balances in bacterial components of the dental plaque .
its prevalence drives to its consideration as the most prevalent infectious disease in the community , with 75% of adults affected as reported in published studies .
several studies have identified similarities in the pathogenesis of late periodontitis and peri - implantitis , showing intraoral translocation of periodontal pathogens from teeth showing chronic periodontitis to the peri - implant niche , producing at last the loss of affected teeth or implants .
previous history of periodontitis , poor oral hygiene and smoking are considered risk factors for peri - implantitis , and late dental implant failures are associated with peri - implantitis and/or biomechanical forces .
while peri - implantitis is defined on implant basis ( an inflammatory process leading to deformation of the peri - implant pocket and bone loss around an implant in function ) , periodontitis is defined on subject basis ( individuals with more than one tooth showing alterations not only in the classical measures of bone loss but also in additional parameters as bleeding on probing and probing pocket depth ) . the peri - implant keratinized mucosa is firmly bound to the underlying bone and constitutes a functional barrier between the oral environment and underlying dental implants .
however , after teeth are extracted , the resorption of surrounding bone and keratinized gingiva occurs , which may result in deficiency of keratinized mucosa during subsequent implant placement .
the need for keratinized mucosa around dental implants has been widely discussed . during the early development of endosseous dental implants , the establishment of a dense connective tissue around the implant collar for long - term implant stability
nevertheless , a number of subsequent studies showed that implants had a high survival rate irrespective of the presence or absence of keratinized mucosa . nowadays , in addition to achieving high implant survival following implant therapy , maintenance of functionally loaded implants in an adequate status of health and aesthetics had become a prerequisite for long - term success of implant restoration .
the need for keratinized tissue around the dental implant to maintain health and tissue stability is therefore becoming of increasing concern . because of the vast differences between natural teeth and dental implants , their maintenance is of critical importance for the longevity of successful osseointegrated implants .
a study which purposely banned oral hygiene around dental implants for a short period of time demonstrated a cause - effect relationship between the accumulation of bacterial plaque and the development of peri - implant mucositis .
recent studies have shown that bacterial colonization occurs within 30 minutes following implantation and becomes stable after a 2-week period .
thus , the primary objective of maintenance and recovery of any implant regiment is to remove the bacterial plaque and/or calculus .
of course , the dental provider has a role in guiding implant stability following osseointegration , however , proper maintenance of the peri - implant soft tissue health is largely in the control of the patient s own oral hygiene regimen .
patients self - management includes mechanical methods and chemical ways to control biofilm formation and subsequent plaque / calculus accumulation .
therefore , the aim of the present study is to determine influence of soft tissue condition and plaque accumulation around dental implants on peri - implantitis development .
protocol and registration the methods of the analysis and inclusion criteria were specified in advance and documented in a protocol . the review was registered in prospero , an international prospective register of systematic reviews .
the review included all human prospective and retrospective follow - up studies and clinical trials , cohort studies , case - control studies , and case series studies published between january 2011 and march 2016 , on various soft - tissue conditions and plaque accumulation around dental implant influence on peri - implantitis development .
additionally , a hand search was carried out in dental implant related journals , including
journal of oral and maxillofacial implants , clinical oral implants research ,
, journal of periodontology , international journal of oral and maxillofacial surgery
the references of each relevant study were screened to discover additional relevant publications and to improve the sensitivity of the search .
the keywords and search inquiries that were used during the primary stage were as follows : ( ( attached gingiva or keratinized gingiva or
keratinized mucosa or attached mucosa or soft tissue condition or soft tissue volume or
gingiva volume or soft tissue height or gingiva height or soft tissue width or gingiva width ) ) and ( peri - implantitis or peri - implant pathology or dental implant infections or peri - implant bone loss or peri - implant disease ) or ( ( plaque or plaque accumulation or plaque index or plaque control ) ) and ( peri - implantitis or peri - implant pathology or dental implant infections or peri - implant bone loss or peri - implant disease ) . the choice of keywords was intended to be broad , in order to collect as much relevant data as possible without relying on electronic means alone to refine the search results .
the resulting articles were independently subjected to clear inclusion and exclusion criteria by two reviewers as follows .
reviewers compared decisions and resolved differences through discussion , consulting a third party when consensus could not be reached .
inclusion and exclusion criteria the applied inclusion criteria for studies were as follows : investigated soft - tissue dimensions at implant sites and peri - implantitis occurrence ; followed - up plaque accumulation influence on peri - implantitis development ; all human prospective or retrospective follow - up studies and clinical trials , cohort studies , case - control studies , and case series studies with at least 5 patients ; a follow - up time period of at least 6 months after the placement of definitive prosthesis ; could not be excluded before careful reading .
the following articles were excluded : studies that targeted soft - tissue condition around teeth ; studies where the effect of soft tissue condition and plaque accumulation on peri - implantitis could not be extracted from the data ( e.g. , a combination of other risk factors , including heavy smokers , systemic diseases , personal habits ) ; studies that included unclear data , with authors who could not be contacted for any reason .
sequential search strategy following the initial literature search , all article titles were screened to eliminate irrelevant publications , review articles , case reports , and animal studies .
next , studies were excluded based on data obtained from screening the abstracts . the final stage of screening involved reading the full texts to confirm each study s eligibility , based on the inclusion and exclusion criteria .
the data were independently extracted from studies in the form of variables , according to the aims and themes of the present review , as listed onwards .
data were collected from the included articles and arranged in the following fields : year , follow - up period , patient number , implant number , plaque index , gingiva index , probing depth .
assessment of methodological quality the risk of bias assessment of the included trials was undertaken independently and in duplicate by at least two review authors as part of the data extraction process .
this was conducted using the recommended approach for assessing risk of bias in studies included in cochrane reviews .
relevant data of interest on the previously stated variables were collected and organised into table .
the initial search identified a total of 1071 articles . following the screening of the article titles ,
independent screening of the abstracts resulted in the selection of 46 publications for possible inclusion .
finally , 8 articles that met the predefined criteria were included in the systematic review ( figure 1 ) .
five of them are prospective clinical studies ( patients number : 287 ; implants number : 917 ) , one cross - sectional study ( patients : 109 ; implants : 202 ) , one retrospective study ( patients : 118 ; implants : 320 ) and one present cohort study ( patients : 80 ; implants : 270 ) .
the reasons for excluding studies after full - text assessment were as follows : investigated soft - tissue dimensions at implant sites and peri - implantitis occurrence ( n = 14 ) , followed - up plaque accumulation influence on peri - implantitis development ( n = 8) , all human prospective or retrospective follow - up studies and clinical trials , cohort studies , case - control studies , and case series studies with at least 5 patients ( n = 7 ) , a follow - up time period of at least 6 months ( n = 1 ) , could not be excluded before careful reading ( n = 7 ) .
study characteristics the included studies were further divided into two groups : km 2 mm ; km < 2 mm .
also studies were compared regarding to the follow - up period , number of the patients , implants number and clinical parameters : plaque index ( pi ) , gingival index ( gi ) , probing depth ( pd ) , bleeding on probing / modified bleeding index ( bop / mbi ) .
no meta - analysis could be performed due to the heterogeneity in the study designs and treatment modalities .
influence of keratinized mucosa peri - implant soft - tissue inflammation , marginal tissue recession , pd , and attachment level are the clinical parameters commonly used for monitoring soft - tissue status of dental implants .
the clinical signs of bop , mucosal recession , increasing pd , and loss of attachment level are always present with peri - implant disease .
qualitative change of soft tissue , pi , gi , pd , bleeding index ( bi ) , or bop were used to determine the status of soft tissue inflammation ( table 1 ) .
characteristics of the included studies with report on soft tissue condition around dental implants
pi = plaque index ; gi = gingival index ; pd = pocket depth ; bop / mbi = bleeding on probing / modified bleeding index ; sd = standard deviation .
pi : 6 of 8 studies showed significantly higher difference in the pi of the periimplant soft tissues [ 22 - 27 ] .
gi : four clinical studies reported [ 22 - 25 ] higher scores of gi in implants with narrow keratinized mucosa ( < 2 mm ) .
pd : 2 of 8 studies showed significantly smaller pd at implants with 2 mm width of keratinized mucosa .
bi / bop : using bi / bop as an indicator of the presence of an inflammatory lesion in the peri - implant mucosa , 3 of 8 studies showed significantly higher prevalence of bleeding scores at implants with < 2 mm compared to 2 mm width of keratinized mucosa . however , other studies showed that the width of keratinized mucosa around implants had no impact on pi , gi , pd and bi / bop .
the findings of those studies regarding the effect of the width of keratinized mucosa on soft - tissue inflammation are controversial , and impaired oral hygiene may play a role in the manifestation of mucosal inflammation around implants with minimal keratinized tissue .
several authors reported that significant elevation of gi and bi scores was accompanied by compromised plaque control at sites with narrow keratinized mucosa [ 22 - 25 ] .
these results demonstrated that the amount of keratinized mucosa has little influence on soft - tissue inflammation in the presence of good oral hygiene . however , suboptimal oral hygiene due to difficulty in access for plaque control in the areas of minimal keratinized mucosa may lead to greater tissue damage . for the maintenance of soft - tissue health of dental implants ,
the capability to access oral hygiene at implant sites is more important than the width of keratinized mucosa .
the quality assessment of the included studies revealed an unknown risk of bias ( for one or more key domains ) for the majority of the included studies [ 21 - 25,27,28 ] , one study was classified as low risk ( of bias for all key domains ) ( table 2 ) .
traditionally , the sufficient keratinized gingiva has been recognized to maintain healthy gingival tissues and to prevent gingival recession .
particularly , it has been believed that the success of implants is dependent on the ability of the mucosa endowing the appropriate biologic protective role between the oral environment and the implants . according to several authors [ 30 - 32 ]
reported that , in good oral hygiene conditions , the marginal gingiva around implants were clinically healthy , even when no keratinized mucosa was present .
on the other hand more investigators [ 20 - 27 ] with newer data reported an association between implant survival and width of keratinized gingival . in our findings
, some researches showed that implants with narrow zones of keratinized gingiva had more bop . in the peri - implant sulcus
, the collagen fibers are orientated parallel to the implant surface , in contrast to the collagen fibers adjacent to the natural teeth , which are perpendicularly orientated and anchored in the cementum .
this will lead to a local tissue trauma and some bleeding , even in clinically healthy peri - implant tissue .
our finding is supported by previous studies [ 23 - 26,32 ] that showed that km width was positively correlated to pd .
it was reported that at sites with healthy mucosa or mucositis , the tip of the probe may identify the location of the apical level of the barrier epithelium . at sites with peri - implantitis , however , the probe will penetrate apical to the epithelium and reach the base of the inflammatory lesion at the alveolar bone crest . consequently , an increased probing depth will result .
schou et al . reported that probing depth measurements at implant and teeth yielded different information , and small alterations in probing depth at implants may reflect changes in soft tissue inflammation rather than loss of supporting tissues .
this study showed that implants with narrow zones of keratinized tissue ( < 2 mm ) had significantly more plaque and signs of inflammation than those with wider zones of keratinized gingiva ( 2 mm ) .
these findings are supported by previous studies [ 22,25 - 27 ] that demonstrated that the absence of adequate keratinized mucosa in endosseous dental implants , especially in posterior implants , was associated with higher plaque accumulation and gingival inflammation .
in fact , good oral hygiene is very difficult to achieve around dental restorations without the protection of a band of keratinized gingival tissue .
therefore , in order to achieve long - term stable peri - implant health , it is important to achieve an adequate soft tissue seal around dental implant / restorations .
several studies have shown the use of free soft tissue grafts to augment keratinized gingiva in conjunction with implant placement , around present dental implant or following the restoration of an implant .
the rationale for performing the procedures include making plaque control more effective , facilitating impression taking by the restorative dentist and dissipating muscular and frenal pull , and possibly preventing further recession . in conclusion
, the present study demonstrated that patients with < 2 mm of km exhibited higher levels of peri - implant discomfort during brushing , plaque , and peri - implant inflammation .
further studies are necessary to evaluate whether patients reporting brushing discomfort at implant sites are more likely to develop peri - implantitis .
this systematic review has highlighted a number of studies examining the clinical relevance of keratinized mucosa around dental implants in preventing peri - implant disease .
all studies concluded that the width of keratinized mucosa around dental implants was related with less mucosal inflammation , less plaque accumulation , increased stability of the peri - implant area , and prevention of mucosal recession leading to loss of implant . within the limitations of the current review
, the following conclusions may be drawn : the absence of adequate keratinized mucosa around implants supporting overdentures was associated with higher plaque accumulation , gingival inflammation and bleeding on probing .
only one study reported that in cases with insufficient keratinized gingiva in the vicinity of implants , the insufficiency does not necessarily mediate adverse effects on the hygiene management and soft tissue health condition .
the authors declare that there are no financial or other conflicts of interest related to this publication . | abstractobjectivesto systematically examine influence of soft tissue condition and plaque accumulation around dental implants on peri - implantitis development.material and methodsan electronic literature search was conducted of two databases - medline ( ovid ) and embase from 2011 to 2016 .
sequential screenings at the title , abstract , and full - text levels were performed .
clinical human studies in the english language that had reported soft tissue condition or plaque accumulation influence on peri - implantitis development were included .
the resulting articles were independently subjected to clear inclusion and exclusion criteria by two reviewers as follows.resultsthe search resulted in 8 articles meeting the inclusion criteria .
these studies reported gingival index , plaque index , pocket depth , bleeding on probing / modified bleeding index for sites with adequate
( 2 mm ) and inadequate ( < 2 mm ) width of keratinized mucosa .
results demonstrated that the amount of keratinized mucosa has little influence on soft - tissue inflammation in the presence of good oral hygiene .
however , suboptimal oral hygiene due to difficulty in access for plaque control in the areas of minimal keratinized mucosa may lead to greater tissue damage.conclusionsin cases with insufficient keratinized gingiva in the vicinity of implants , the insufficiency does not necessarily mediate adverse effects on the hygiene management and soft tissue health condition .
nonetheless , the risk of the increase of gingival index , plaque index , pocket depth , bleeding on probing / modified bleeding index is present .
therefore , the presence of an appropriate amount of keratinized gingiva is required . |
eosinophilic protein x is produced by activated eosinophils and can be measured in blood , stools , and urine ( uepx ) [ 1 , 2 ] .
elevated uepx has been reported in asthmatics with or without allergy [ 3 , 4 ] , with great overlap between healthy controls and asthmatics [ 5 , 6 ] .
a relation with asthma symptoms and lung function has been shown , and uepx increases significantly during asthma exacerbations [ 79 ] .
therefore , it has been suggested that measuring urinary epx may be useful for monitoring the effect of ics in asthma [ 3 , 10 , 11 ] .
since measuring uepx is completely noninvasive and independent of patient 's cooperation , it has the potential to be a feasible tool in asthma management in young children .
several studies have demonstrated cross - sectional correlations between uepx and conventional clinical markers of asthma control and airway inflammation [ 3 , 4 , 12 ] .
previously , we reported these cross - sectional data measured at baseline in part of the present study population .
data on longitudinal changes in uepx within subjects compared to changes in markers of asthma control or airway inflammation were examined in only one study in 14 children .
we analysed cross - sectional and longitudinal uepx data from a long - term follow - up study in children with atopic asthma .
we hypothesised that changes in uepx would be related to changes eosinophilic airway inflammation and could provide information that is additional to symptoms and lung function .
asthmatic children took part in a randomised controlled multicentre trial ( cato : children asthma therapy optimal ) reported in detail previously .
briefly , 288 children with a documented clinical history of moderately severe asthma were recruited from 15 paediatric clinics including 7 university hospitals in the netherlands .
al patients had a positive , class 1 , or more radioallergosorbent test result for one or more airborne allergens . during a run - in phase ,
all patients were treated with 100 or 250 g fluticasone bid depending on their equivalent treatment before the study .
children were selected on the basis of symptoms and/or ahr , and 210 children were randomised to a reference strategy ( adjustment of treatment on symptom score ) or an ahr strategy ( treatment adjusted on the basis of ahr and symptom score ) . during the 2-year follow - up study ,
3 monthly visits were performed . at each visit symptoms scores ( diary card over the last 2 weeks ) , forced expiratory volume in one second ( fev1 ) , and methacholine challenge results were obtained , and medication ( five levels : fluticasone 100 or 200 g per day or fluticasone / salmeterol 200/100 , 500/100 , or 1000/100 g per day ( flixotide diskus or seretide diskus , glaxosmithkline , uk ) adjusted according to the algorithms of the reference- and ahr strategy . for the present study , baseline is defined as the moment of randomisation for the cato study .
cough , shortness of breath , and wheezing during night and day were recorded on a 4 point scale ( 0 = no symptoms to 3 = severe symptoms interfering with activity or sleep ) .
the percentage of symptom - free days defined as score 0 for cough , wheeze , and shortness of breath was calculated . were measured during each clinic visit .
the provocation dose causing a 20% fall in fev1 from baseline ( pd20 ) was calculated from a log dose - response plot by linear interpolation .
sputum was induced according to a standardised method by inhaling an aerosol of hypertonic sodium chloride 4.5% w / v [ 18 , 19 ] .
differential cell counts of cytospins were performed and samples containing more than 80% squamous cells were excluded from the analysis .
spot samples of urine were collected at randomisation (= baseline ) , after a treatment period of 1 year , and after 2 years ( the end of the study ) , and immediately stored at 20c .
epx was determined using a commercial enzyme - linked immunosorbent assay ( elisa ) for human epx in 50-fold diluted samples ( medical and biological laboratories , nakaku nagoya , japan ) .
urinary creatinine levels were measured by the alkaline picrate method ( roche , mannheim , germany ) .
urinary epx concentrations were expressed as g per mmol creatinine . repeated measurements anova was used to evaluate associations between the various markers and uepx levels .
sas proc mixed ) the information about the associations at the different study time points can be combined and the method allows for uepx not being available at all three time points for each patient . in these analyses , uepx values , % eosinophils in sputum , and fluticasone doses
were transformed logarithmically in order to get approximate normal distributions and to reduce the effect of outlying observations , and consequently results for these parameters are expressed as geometric means .
percentage eosinophil values equal to 0 were replaced by 0.1% in order to allow the logarithmic transformation . in a substantial part of the study population , especially at time points t1 and t2 , pd20 was higher than the highest dose methacholine used ( 1570 g ) . for that reason
pd20 data were arbitrarily dichotomized in non - ahr ( pd20 > 300 g ) and ahr ( pd20 < 300 g ) .
in total 461 , uepx samples were analysed during the study . during the 2-year treatment period ,
the geometric mean uepx significantly decreased from 159 g / mmol to 104 g / mmol ( p < 0.001 ) ( figure 1 ) .
anova showed a significant treatment effect after 1 year : in the ahr strategy group , uepx had decreased significantly more than in the reference strategy group , with a 29% lower geometric mean epx level at that time point ( p = 0.03 ) .
further analysis showed that at 1 year , there was a significant fluticasone dose effect , and this effect was not found at baseline or at year 2 . at 1 year
a twofold higher dose was associated with a 25% lower uepx level ( 95% ci : 15%34% ; p < 0.001 ) .
children treated according to the ahr strategy had received significantly higher doses of fluticasone at 1 year ( geometric means 580 versus 412 ug / day ; mann - whitney test , p = 0.01 ) .
after adjustment for the higher dose using anova , no significant treatment effect remained .
no correlations were found between uepx and the percentage symptom - free days or changes in uepx and changes in percentage symptom - free days .
cross - sectional analysis showed weak negative correlations of the levels of uepx and fev1 ( baseline : r = 0.18 , p = 0.02 ; at 1 year : r = 0.25 , p = 0.002 ; at 2 years : r = 0.21 , p = 0.016 , resp . ) .
anova showed that the significant associations did not differ between the three time points and adjusting for ics dose did not change this . also the changes from baseline of uepx and fev1 were significantly related with each other ( at 1 year r = 0.24 , p = 0.01 and at 2 years ; r = 0.21 , p = 0.03 ) ( figure 2 ) .
as compared to baseline , a tenfold increase in uepx was associated with a decrease of fev1 ( % pred ) of 2.8 percentage points ( 95% ci : 0.3 to 5.3 ; p = 0.026 ) and this decrease did not significantly differ between the two time points ( p = 0.65 ) .
a similar result was found when changes from the previous assessment were evaluated . at all visits , uepx levels were higher in patients with ahr ( pd20 methacholine < 300 g methacholine ) than in patients without ahr . at 1 year this difference was statistically significant , with 13% higher uepx in patients with ahr ( p = 0.006 ) ( p = 0.08 , p = 0.10 , resp .
this effect remained after adjustment for dose . with regard to percentage eosinophils in sputum a significant correlation with uepx
was found at 2 years ( r = 0.51 , p < 0.001 , n = 33 ) .
a twofold higher value of sputum eosinophils was associated with a 14% increased value of uepx ( 95% ci : 6%22% ; p < 0.001 ) , and this relation did not significantly differ between the 3 time points ( p = 0.11 ) , nor was the relation affected by ics dose . although changes from baseline of uepx and percentage sputum eosinophils did not significantly correlate with each other at the separate time points , a significant relation was found combining both time points using anova .
a within - patient doubling of sputum eosinophils was associated with a mean increase of uepx of 16% ( 95% ci : 0.1%36% ; p = 0.049 ) , and the ics dose did not affect this relation .
we prospectively assessed changes in uepx in relation to changes in asthma control within subjects over an observation period of 2 years .
we found in atopic asthmatic children that changes in uepx measured after 1 and 2 years related significantly with changes in fev1 and % sputum eosinophils , but not with changes in symptoms .
previously we described the results of cross - sectional analysis at baseline in the present study population .
this is in line with the significant correlation between these parameters found by lugosi et al . in 14 asthmatic children .
however the interval of their measurements was much shorter ( 1 - 2 months ) .
it has been shown that correlations between eosinophilic airway inflammation and lung function are weak and may only reach significance in large populations [ 4 , 6 , 20 , 21 ] .
the correlation between uepx and sputum eosinophils was weak , and this is in line with the relations between sputum ecp , sputum eosinophils , and uepx described by mattes et al . .
weak correlations seem to be the rule when different markers of eosinophilic inflammation are compared .
moreover , uepx is secreted by activated eosinophils , and it is therefore possible that uepx provides information about eosinophil activation rather than numbers .
a significant relation between uepx and symptoms has been described during acute asthma exacerbations , spontaneously or provoked by ics withdrawal .
also , significantly higher uepx levels were found in symptomatic asthmatic children than in asymptomatic children [ 1 , 3 , 4 ] . however , a 6-month follow - up study in 14 children with mild asthma found , no significant association was found .
so the severity of symptoms should be taken into account when correlating uepx and symptoms . in our study population ,
the symptom scores were low and this may explain the apparent discrepancy with these previous studies
. it could well be that relations between uepx and parameters of asthma control could have been influenced by other factors as well , including seasonal variations , severity of allergy , and asthma and atopic dermatitis [ 24 , 25 ] .
the use of varying doses of ics is another possibility , for which we have corrected .
what are the clinical implications of our findings ? as uepx does not correlate with symptoms , but does correlate with sputum eosinophils , we could argue that uepx might provide additional information on activity of eosinophilic airway inflammation . however , the correlations were weak and the scatter of individual uepx values wide .
therefore , it seems unlikely that uepx will be useful as a biomarker for monitoring asthma in the individual child . | the aim of this study was to assess cross - sectional and longitudinal correlations between uepx and other markers of asthma control and eosinophilic airway inflammation . methods .
we measured uepx at baseline , after 1 year and after 2 years in 205 atopic asthmatic children using inhaled fluticasone . at the same time points , we assessed symptom scores ( 2 weeks diary card ) , lung function ( forced expiratory volume in one second ( fev1 ) ) , airway hyperresponsiveness ( ahr ) , and percentage eosinophils in induced sputum ( % eos ) . results .
we found negative correlations between uepx and fev1 at baseline ( r = 0.18 , p = 0.01 ) , after 1 year ( r = 0.25 , p < 0.01 ) and after 2 years ( r = 0.21 , p = 0.02 ) .
within - patient changes of uepx showed a negative association with fev1 changes ( at 1 year : r = 0.24 , p = 0.01 ; at 2 years : r = 0.21 , p = 0.03 ) . within - patient changes from baseline of uepx correlated with changes in % eos .
no relations were found between uepx and symptoms . conclusion . in this population of children with atopic asthma , uepx correlated with fev1 and % eos , and within - subjects changes in uepx correlated with changes in fev1 and % eos . as the associations were weak and the scatter of uepx wide , it seems unlikely that uepx will be useful as a biomarker for monitoring asthma control in the individual child . |
adenoid cystic carcinoma ( acc ) accounts for 1% of all head and neck ( hn ) cancers and about 10 - 22% of all malignant tumors of the major and minor salivary glands . minor salivary glands ( 65% ) are more frequently involved than major salivary glands ( submandibular - 19% , parotid - 16% ) . occasionally , they arise from sites other than salivary glands , such as the lacrimal glands , the ceruminal glands of external auditory canal , nose , paranasal sinus , palate , nasophaynx , and larynx .
acc was initially described by bilroth in 1856 and named as cylindroma for its classic histologic appearance .
although these tumors are usually low - grade malignancies as per histologic differentiation , management of these tumors is a distinct therapeutic challenge because of the propensity for local invasion , perineural involvement , distant metastasis and ability to recur over a prolonged period .
studies have demonstrated the utility of adjuvant radiation in terms of superior disease control over either modality alone .
however , the improvement in loco - regional control with combined modality treatment did not translate into a significant improvement in overall survival rate .
the role of chemotherapy is investigational and is often confined to recurrent , metastatic or advanced unresectable tumors .
the response rate ranges from 10% to 25% depending on the choice of drugs . targeted therapy with biological agents has a response rate of < 10% and has failed to improve treatment outcome . hence , no consensus exists on the appropriate modality for the management of these malignancies .
most studies concur that overall survival rate for acc is favorable , with 5-year survival ranging between 64% and 89% and 10-year survival between 37% and 77% .
the current study aimed at reviewing the clinical experience of management of hn acc and analyzing the prognostic factors in this malignancy at a tertiary care institute in north india .
medical records were reviewed and data collected on all hn acc over a 16-year period ( 1995 - 2011 ) from the institutional archives .
fourteen cases of acc of the lacrimal gland and 10 cases with missing data were excluded from analysis .
collected data were analyzed for a demographic profile , clinical presentation , disease site , stage , treatment modalities , and survival outcome .
patients were retrospectively staged as per american joint committee on cancer ( 2010 ) tnm classification .
the philosophy of treatment over the study period has been to assess all the patients in a multi - disciplinary hn cancer clinic comprising of an otolaryngologist , radiation oncologist , and medical oncologist .
baseline staging work - up consisted of contrast enhanced computed tomography or magnetic resonance imaging of hn and x - ray of the chest .
complete blood count , liver function test , and kidney function tests were carried out in all patients .
the type of procedure was dependent on the primary site , the extent of disease , cosmetic considerations , and discretion of the surgeon .
in general , an attempt was made to maximize local control with preservation of cosmetic and functional outcomes .
unresectable tumors were treated with radiation alone , the intent being curative or palliative , depending on the performance status of patients and the loco - regional extent of disease .
the base of the skull was routinely included in the radiation portal for all tumors with named cranial nerve , invasion of the base of the skull or intra - cranial extension ( ice ) .
the dose of adjuvant radiation and addition of concurrent chemotherapy were decided based on the presence of high - risk factors in the postoperative histopathology report e.g. , presence of positive or close margin , perinodal spread or more than one lymph node involvement .
the dose of postoperative radiation ( port ) was 60 - 64 gray at 2 gray per fraction over 6 - 6.5 weeks depending on the presence of the aforementioned high - risk features .
patients treated with reirradiation on recurrence received radiation dose of 45 gray at 1.8 gray per fraction over 5 weeks and for metastatic bone disease , either 8 gray in single fraction or 20 gray in 5 fractions over 5 days were used . extrapolating from the data of use of postoperative concurrent chemoradiation in high - risk squamous cell carcinoma of hn , cisplatin ( 40 mg
/ m body surface area intravenous weekly ) was added to port in patients with positive margin and extracapsular extension in patients considered eligible for this intensified approach , else a higher dose of radiation was used in these cases .
weekly toxicity assessment was done during the course of radiotherapy according to radiation therapy and oncology group acute radiation morbidity scoring criteria .
disease - free survival ( dfs ) was defined as the interval between the date of diagnosis and the date of recurrence and was estimated by kaplan - meier product - limit method .
median age at presentation was 40 years ( range : 17 - 73 years ) .
ten patients had clinical node - positive disease and of 11 necks sampled , 8 patients had pathological node positivity .
patient and tumor characteristics ( n = 66 ) fifty - seven patients underwent surgery . on postoperative
histopathology , positive margin and perineural invasion were noted in 18 and 10 patients , respectively .
three patients underwent surgery alone ( one was early stage oral cavity , and two were salivary gland carcinoma ) .
six patients with advanced disease received palliative radiotherapy ( 20 - 30 gray in 5 - 10 fractions over 1 - 2 weeks ) .
patients were treated with megavoltage photons and/or electron beams after ensuring proper immobilization using customized thermoplastic cast .
treatment planning had evolved with time from two - dimensional fluoroscopy based radiation therapy to three - dimensional conformal radiation therapy ( 3d - crt ) to intensity modulated radiotherapy ( imrt ) and use of image - guidance ( ig ) .
two - dimensional radiation techniques were used in 26 patients , 3d - crt was used in 25 patients , and imrt was used in 4 patients , whereas ig - imrt was used in 6 patients .
mixed beam therapy ( photon : electron combination ratio = 1:4 ) was used in 2 patients .
of the 18 patients with margin positive disease , concomitant chemotherapy ( cisplatin 40 mg / m weekly ) along with adjuvant radiation was used in 8 of these patients , seven patients received higher dose of radiation - 64 gray in 32 fractions over 6.5 weeks and 3 patients had re - surgical excision with negative margins and received conventional radiation doses only .
in addition , 5 patients received 64 gray in 32 fractions over 6.5 weeks due to the presence of other high - risk factors .
the overall rate of grade 2 or higher acute nonhematological toxicity was 8% among those treated with combined modality approach .
median follow - up duration was noted to be 23 months ( range : 12 - 211 months ) .
thirteen patients failed locally , 4 patients had distant metastasis ( 3 had lung metastasis , and 1 had bone metastasis ) , and 2 patients had both local recurrence and distant metastasis ( lung metastasis ) . at failure ,
surgical excision followed by reirradiation ( 3 patients ) , only reirradiation ( 2 patients ) , palliative chemotherapy ( 3 patients ) , palliative radiotherapy alone to involved metastatic sites of bone ( 1 patient ) and rest received only best supportive care .
two years and 4 years dfs rate [ figure 1 ] were 75% and 71% , respectively .
univariate analysis indicated increased predisposition [ table 2 and figure 2 ] toward failure with skull base erosion / ice at presentation ( hr : 3.59 , 95% confidence interval [ ci ] : 1.89 - 14.46 ; p = 0.007 ) , lymph node involvement ( hr : 4.06 , 95% ci : 1.63 - 26.17 ; p = 0.006 ) , use of treatment modality other than surgery and postoperative radiotherapy ( hr : 2.39 , 95% ci : 1.63 - 9.12 ; p = 0.01 ) and higher t stage ( t3/4 ) ( hr : 3.27 , 95% ci : 1.36 - 11.43 ; p = 0.007 ) . other prognostic factors [ table 2 ] viz . age ( p = 0.196 ) , margin positivity ( p = 0.605 ) and extent of surgery ( p = 0.573 ) did not impact dfs significantly . lymph node involvement ( p = 0.038 ) and skull base invasion / ice ( p = 0.038 ) continued to have significant impact on dfs even on multivariate analysis . kaplan - meier survival curve depicting disease free survival of the entire cohort univariate and multivariate analysis of factors predictive of disease free survival kaplan - meier survival curve depicting impact of prognostic factors on disease free survival . ( a ) lymph node positivity ( b ) intra - cranial extension ( ice ) ( c ) treatment modality ( port postoperative radiotherapy ) ( d ) t stage of the disease
in this current single institutional analysis of 66 patients of hn acc , predominantly treated with the multi - modality approach , we observed 2- and 4-year dfs rate of 75% and 71% respectively .
skull base invasion / ice , lymph node involvement and higher t stage were found to be poor prognostic factors with respect to dfs .
finally , lymph node involvement and presence of skull base invasion or ice continued to retain their prognostic significance on multivariate analysis .
the results of this study are in accordance with those of other contemporary series of hn acc [ table 3 ] .
comparative analysis of prognostic factors and survival outcomes in contemporary series one of the largest series on hn acc ( n = 155 ) has been reported by chen et al . from university of california , san - francisco ( ucsf ) .
the 10-year overall survival and distant metastasis - free survival were 64% and 66% , respectively .
the authors concluded that t4 disease ( p = 0.0001 ) , perineural invasion ( p = 0.008 ) , omission of port ( p = 0.007 ) , and major nerve involvement ( p = 0.02 ) were independent predictors of local recurrence .
study by simpson et al . from mallinckrodt institute of radiology also established the superiority of surgery , followed by radiation over surgery alone .
ten years local control was 83% for patients treated with surgery , followed by adjuvant radiation compared with only 25% for those treated with surgery alone . according to single institute data from m.d .
anderson cancer centre , local control rates were 95% and 86% at 5- and 10-year , respectively in patients of hn acc ( n = 198 ) treated with combination of surgery and port . in another series reported from university of california , los angeles by cohen et al .
, local control rates were 82% and 70% for patients treated with or without adjuvant radiation after surgery , respectively .
mendenhall et al . reported 5- and 10-year local control rates of 94% and 91% in a cohort of 56 patients treated at the university of florida with multi - modality approach and identified t stage as an independent predictor of local failure .
failed to show any benefit of port . on the contrary , silverman et al . showed the utility of port in patients of hn acc with t4 tumors and positive margins
however , the benefit of adjuvant radiation was not observed in other subsets of patients of hn acc .
some of the other contemporary series also had comparatively higher rate of positive margins , which might be attributed as a potential reason for the superiority of surgery and port over surgery alone . in the current study , we used port in all patients with positive margin , thus nullifying the negative prognostic impact of margin positivity on local control .
the difference in the results regarding the impact of port and the variation in survival rates across the studies may be attributed to patient selection criteria and different therapeutic approach like the extent of surgery and radiotherapy target volume .
existing literature has heterogeneity regarding elective nodal irradiation of neck due to individualization of treatment decisions .
we have routinely included the base of the skull in the postoperative treatment volume in case of perineural invasion of any major named nerve , the base of skull invasion or ice .
the discrepancies can also be partially due to the difference in the demographic and clinicopathologic factors across these studies done from different parts of the world .
results of our series are in concordance with other reported studies in the literature [ table 3 ] .
lymph node involvement and base of skull invasion or ice were noted to be risk factors for poor dfs on univariate analysis in our series .
study from memorial sloan - kettering cancer centre showed that t4 stage and gross or clinical nerve involvement ( p = 0.002 ) were associated with decreased progression - free survival , whereas t4 stage and lymph node involvement were associated with decreased overall survival .
the association of lymph node involvement with poorer outcome has also been demonstrated by fordice et al .
multiple studies have pointed out the adverse impact of perineural invasion or nerve involvement on local control and survival . however in our study , perineural involvement did not have any significant impact on dfs .
this might be because of our cautious approach in the inclusion of base of the skull in the radiation target volume in all such patients , which nullified the risk of recurrence . in our series , local failure
was observed to be the predominant pattern of failure that is in accordance with the results obtained by khan et al .
however , in the ucsf series reported by chen et al . , distant metastasis was the most common pattern of failure . sites of distant failure in this study were as follows : 25 lung ( 71% ) , 5 bone ( 14% ) , 3 liver ( 9% ) , and 2 brain ( 6% ) metastasis . a longer follow - up in our study
the current study , in accordance with literature from different parts of the world , shows excellent tolerance to port with favorable acute toxicity profile . however , lack of attention to late toxicity , which might be significant in long - term survivors , is a limitation of our study .
although subject to inherent limitations of any retrospective study , the current study highlights the superiority of surgery , followed by adjuvant radiation in managing patients of hn acc .
invasion of skull base or ice was found to be an independent prognostic factor in our study .
however , the adverse prognostic impact of positive margin was negated by the routine use of high dose port .
the median follow - up duration in the present study is comparatively shorter than other contemporary series , which is essentially due to poor compliance to long - term follow - up policy , mandatory for hn acc .
last but not the least ; more attention needs to be focused on late toxicity , functional outcome , and quality of life in long - term survivors .
the current single institutional analysis clearly demonstrates the superiority of multi - modality management in the form of surgery and adjuvant radiation in the management of hn acc .
however , the prognostic impact of positive margin on local control and survival can be nullified by the routine use of high dose radiation therapy in such cases .
special attention needs to be given to late morbidities and quality of life issues in long - term survivors .
| background : adenoid cystic carcinoma ( acc ) accounts for 1% of all head and neck ( hn ) cancers.materials and methods : demographic , clinical , treatment , and survival details of 66 patients were collected ( 1995 - 2011 ) and analyzed . disease - free survival ( dfs ) was estimated by kaplan - meier method.results:primary disease sites were sinonasal ( n = 27 ) , salivary gland ( n = 30 ) , and others ( n = 9 ) .
median follow - up was 23 months ( range : 12 - 211 months ) .
estimated dfs at 2- and 5-year were 75% and 67.2% , respectively .
on univariate analysis , intra - cranial extension ( ice ) ( hazard ratio [ hr ] : 3.59 , p = 0.0071 ) , lymph node involvement ( hr : 4.05 , p = 0.0065 ) , treatment modality ( others vs. surgery plus adjuvant radiotherapy , hr : 2.39 , p = 0.0286 ) and t stage ( t3/4 vs. t1/2 , hr : 3.27 , p = 0.007 ) had significant impact on dfs . lymph node involvement ( p = 0.038 ) and ice ( p = 0.038 ) continued to have significant impact on dfs on multivariate analysis.conclusion:surgery followed by adjuvant radiotherapy remains the treatment of choice for hn acc .
lymph node involvement and ice confer poor prognosis . |
the interest of clinical research in aging males increased in recent years and thereby the interest to measure health - related quality of life and symptoms of aging men .
the aging males ' symptoms ( ams ) scale was originally developed in germany in 1999 based on the assumption that like women during their menopausal transition men also develop similar complaints .
the scale was designed ( a ) to assess symptoms of aging ( independent of those which are disease - related ) between groups of males under different conditions , ( b ) to evaluate the severity of symptoms over time , and ( c ) to measure changes pre- and post androgen replacement therapy .
it was developed in response to the lack of fully standardized scales to measure the severity of aging symptoms and their impact on hrqol in males , specifically .
a review of reliability and validity of the scale was published elsewhere , further details will not be discussed in this paper .
the development of the scale started with the comparison of more than 200 variables in more than 100 medically well characterized males ( aged over 40 years ) .
a factorial analysis was applied to establish the raw scale of complaints or symptoms that are not particularly related to diseases , treatment , social and other variables , but related with aging .
statistical methods were used to identify the dimensions of the scale and to reduce the number of items of the raw scale .
finally , three dimensions of symptoms / complaints were identified in the patients group : a psychological , a somato - vegetative , and a sexual factor that explained 51.6% of the total variance and the number of items of the scale could be reduced to 17 .
this final scale was applied to a large representative population sample of 992 german males in order to establish reference values for the severity of symptoms in males over 40 .
the scoring scheme is simple , i.e. the score increases point by point with increasing severity of subjectively perceived complaints in each of the 17 items ( severity 1 ... 5 points ) . by checking one of 5 possible boxes of " severity " for each of the items
this can be seen in the questionnaires in the additional files linked to this publication .
the composite scores for each of the dimensions ( sub - scales ) is based on adding up the scores of the items of the respective dimensions .
the three dimensions , their corresponding questions and the evaluation are detailed and summarized in an attached file linked to this publication [ see additional file 13 ] .
the german original ams scale was first translated and culturally adapted into english , showing cross - cultural equivalence .
sporadic information about translations into other than the two first languages received by the developer of the scale ( first author lajh ) led to the need to gather more complete information about the translation process from all key persons involved .
it was agreed among the contributors to publish this review as update together with all known language versions and to discuss quality issues .
thus , the aim of this review paper is to present all translations we are aware of and to provide some details about quality - relevant issues of the translation process .
this should help other international research groups to contact key persons , to have easy access to certain language versions , and to prevent double translations .
most but not all of the translations were performed in congruence with international methodological recommendations for linguistic & cultural adaptation of hrqol measures [ 6 - 12 ] using mostly the english version as source language to ensure cross - cultural equivalence among countries .
six steps of the translation process are recommended : forward translations ( at least two independent translators ) , a consensus meeting with the coordinator of the translation , a check by a bilingual expert in the application field of the scale to evaluate the scientific correctness of the wording , a backward translation , a consensus meeting among the translators with the coordinator , and finally a pretest with a few persons the test is designed for ( also called cognitive debriefing ) .
the table shows the results of an inquiry among the authors of different existing language versions of the ams .
it demonstrates that most of the translations followed the recommended process , but a few did not for various reasons such as lack of financial sources , no need of international compatibility of results obtained with the scale or others .
however , potential users of these incompletely adapted scales ( finnish , flemish , russian language ) should be encouraged to complete the translation and assistance of authors of this paper is offered if needed .
review of ams scales available in different languages characteristic of linguistic & cultural translation n.a .
= not applicable * = to be completed in due course as one can see from the table , the translations that followed the recommended process involved mostly 2 forward translators , a consensus meeting between these translators with the coordinator of the translation process .
the objective of all consensus meetings is to find an acceptable solution , i.e. to make corrections , revisions , or confirm the translation .
a bilingual expert of the field , often a physician , compared the translations from the scientific perspective and in a discussion with the coordinator of the translation necessary adaptations or corrections were made , and followed by a backward translation .
this was the first version that was published after the standardization of the original german ams scale in 2000 . in the translation process of the english version we became aware at the preliminary end of the translation process , that from the view of the north american translators further revision was essential .
a consensus meeting finally agreed upon a wording that was compatible in both cultures and also in different social classes .
most of the other existing versions were translated only in the language of the mother - country .
if the scale should be applied in another country with the " same " language ( e.g. the spanish version in mexico , argentine or cuba ) , at least a critical review of the translation has to be made by a group experienced in the field of cultural adaptation . in some cases ,
a new translation could be necessary that may even influence the translation into the original language if compatibility is required .
this is a complex process that needs expert experience , which can be offered from the authors of this publication or from the developer of the scale ( lajh ) .
critical distance is particularly important if results of the ams are intended to be pooled between different populations or regions , and less important if a study is planned as before - after - treatment - comparison in one country with only one language .
the latter applies only if the linguistic / cultural adaptation process considered different social classes and regions with slight language differences , which is standard in experienced groups . for completeness , all ams versions are attached as additional files in pdf - format ( adobe acrobat ) , even the preliminary language versions that obviously did not follow the " state - of - the - art " translation process ( finnish , russian and flemish language ) or are not completed ( korean language ) .
the majority of language versions , however , followed the same , i.e. internationally recommended , complex methodology of language adaptation .
for this reason , we can assume that they have linguistic and cultural equivalence , although different groups did the translation work .
access to the original scales in 12 different languages , please see additional files linked to this publication : additional file 1 : german version of the ams
additional file 7 : spanish version of the ams additional file 8 : portuguese version of the ams
additional file 9 : italian version of the ams additional file 10 : russian version of the ams
additional file 12 : korean version of the ams it should be stressed that persons who are interested in applying the ams scale in their research can download the appropriate language version and use it without any formal permission .
however , it would be important to keep an overview who is using the scale and for what reason .
therefore , an information should be sent to the developer and copyright holder of this scale ( lajh ) . in return , all information on the scale that became known in between will be made available .
the ams scale is in the process of translation in korea ( attached as pdf - file of the forward translation ) , in thailand ( personal communication : somboon leungwattanakij , division of urology , ramathibodi hospital , bangkok , thailand ) , and in indonesia ( personal communication : dr .
in addition , there is a consensus process almost completed to establish a reference version of two existing french ams scales .
the ams scale is obviously a valuable tool for assessing health related quality of life in aging men , because it is used worldwide .
most of the currently available language versions were translated following international standards for linguistic and cultural translation of quality of life scales .
lajh : developer of the scale , involved in the collection of the language versions , and in writing of the paper .
fs : involved in the co - ordination of several translations and collection of information , contributed to writing of the paper .
tz : co - authors of the original german ams scale , contributed to writing of the paper . an : involved in the co - ordination of the translations into dutch , french , and japanese , contributed to writing of the paper .
em : involved in the co - ordination and performance of the translations into french , and spanish , contributed to writing of the paper .
xb : responsible for the translation into spanish , provided the data , and contributed to writing of the paper .
pepo : involved in the co - ordination of the translations into swedish , portuguese , and italian language , provided data from those countries , and contributed to writing of the paper .
gts : responsible for the translation into flemish , provided the data , and contributed to writing of the paper .
pap : responsible for the translation into finnish , provided the data , and contributed to writing of the paper .
sk : responsible for the translation into korean , provided the data , and contributed to writing of the paper .
cg : responsible for the translation into japanese , provided the data , and contributed to writing of the paper .
aging males ' symptoms ( ams ) rating scale : evaluation form german version of the ams english version of the ams swedish version of the ams finnish version of the ams dutch version of the ams flemish version of the ams spanish version of the ams portuguese version of the ams italian version of the ams russian version of the ams japanese version of the ams korean version of the ams | backgroundthe interest of clinical research in aging males increased in recent years and thereby the interest to measure health - related quality of life ( hrqol ) and symptoms of aging men .
the aging males ' symptoms scale ( ams ) became the most commonly used scale to measure hrqol and symptoms in aging males in many countries worldwide .
the aim of this paper is to review the current state of the instrument particularly concerning versions of the scale in different languages in the light of the quality of the translation process.ams versions availablemost of the translations were performed following international methodological recommendations for linguistic & cultural adaptation of hrqol instruments .
mainly the english version was used as source language for the translation into dutch , spanish , portuguese , italian , swedish , and japanese ( attached as additional pdf - files ) .
preliminary versions that were derived only from forward translations are of secondary quality and available in finnish , flemish , and russian .
it is recommended to complete the translation process for the latter languages before using them in international studies.translations in processthe ams scale is in the process of consensus finding of two existing french versions , and the versions in the korean , thai , and indonesian languages have not yet been completed in the translation process.conclusionthe ams scale is obviously a valuable tool for assessing health related quality of life in aging men , because it is used worldwide .
it is a standardized scale according to psychometric norms .
most of the currently available language versions were translated following international standards for linguistic and cultural translation of quality of life scales .
assistance is offered to help interested parties in the translation process . |
reverse transcription of viral rna into double - stranded ( ds ) dna , and the subsequent insertion of the synthesized viral dna ( vdna ) into a host chromosome , are characteristic features of retroviruses including human immunodeficiency virus type 1 ( hiv-1 ) .
the reverse transcription and integration of the viral genome is sequentially catalyzed by the enzymes reverse transcriptase ( rt ) and integrase ( in ) , respectively .
these retroviral enzymes are originally packaged in the viral particle along with the viral genomic rna .
after synthesis of vdna by rt , in acts on the termini of vdna and catalyzes insertion of the vdna into the host chromosome through two sequential enzymatic reactions : 3-end processing and strand - transfer ( katz and skalka , 1994 ) . currently , a clinically approved in inhibitor specifically targets strand - transfer activity but not 3-end processing activity ( summa et al . , 2008 ) .
therefore , the in inhibitor is referred to as a strand - transfer inhibitor ( sti ) .
the sti shows very potent antiviral activity ; however , emergence of sti - resistant variants is inevitable ( metifiot et al . , 2010 ) , as seen in patients treated with a combination of inhibitors against rt and protease activities .
efforts to develop novel drugs with distinct inhibitory mechanisms must continue so that we can effectively treat hiv-1 infections . for development of novel in inhibitors , in addition to its enzymatic action , other non - enzymatic functions of in , as described below , might be the next target(s ) ( luo and muesing , 2010 ) .
human immunodeficiency virus type 1 in is composed of 288 amino acids with three structurally distinct domains ( li et al . , 2011 ) : an n - terminal domain ( ntd ) , a central catalytic core domain ( ccd ) , and a c - terminal domain ( ctd ; figure 1 ) .
the ntd contains a highly conserved ( his - his - cys - cys , hhcc ) motif , which binds to zinc ions ( zn ) and folds a helix - turn - helix ( hth ) structure . through a tetrahedral attachment to the hhcc motif
, zn enhances both multimerization and enzymatic activities of hiv-1 in in vitro ( burke et al . , 1992 ;
the ccd contains the highly conserved asp , asp , and glu ( dde ) residues directly involved in the catalytic activities of in ( engelman and craigie , 1992 ; kulkosky et al . , 1992 ; lafemina et al . , 1992 ; bushman et al . ,
overall topology of the ccd is similar to those of ribonuclease h ( rnaseh ) , the holliday junction resolvase ruvc , and bacteriophage transposase mu . despite lack of sequence similarity between the ccd and rnaseh ,
there is remarkable similarity in the positioning of the two asp catalytic residues ( dyda et al . , 1994 ) .
the ctd , consisting of a structure that closely resembles src homology 3 domains ( sh3-like ) , possesses sequence- and metal ion - independent dna binding activity ( eijkelenboom et al .
, 1995 ; lodi et al . , 1995 ) . each domain has been demonstrated to form a dimer and higher multimerization states ( dyda et al . , 1994 ; eijkelenboom et al . , 1995 ; cai et al . ,
hiv-1 in is encoded by the pol region and composed of 288 amino acids with three structurally distinct domains : an n - terminal domain ( ntd ) ; a central catalytic core domain ( ccd ) ; and a c - terminal domain ( ctd ) .
the ccd contains the highly conserved dde motif , which is directly involved in the catalytic activities of in .
overall topology of the ccd is similar to that of ribonuclease h ( rnaseh ) .
the ntd , contains a highly conserved hhcc motif , which binds to zinc and folds a helix - turn - helix ( hth ) structure .
the ctd , consisting of a structure that closely resembles src homology 3 domains ( sh3 ) , possesses sequence- and metal ion - independent dna binding activity .
recently , the entire prototype foamy virus ( pfv ) in in a complex with its cognate vdna ends , referred to as the intasome , has been successfully crystallized ( hare et al . , 2010 ) .
the crystal structure analysis of the pfv intasome revealed an unprecedented tetramer structure for in ( see cherepanov et al .
the in tetramer structure observed in the pfv intasome demonstrated that two sets of in dimer acts on each vdna end ( figure 2 ) .
the outer subunits of each in dimer might be speculated to have supportive or other functions , such as engagement of target dna or interaction with host factors .
several models for the in tetramer have been proposed from previous structure analysis using partial in fragments possessing the ntd ccd or ccd ctd ( chen et al .
, 2000 ; wang et al . , 2001 ; hare et al . , 2009a ) . however , these in tetramer models are different from those observed in the active pfv intasome ( craigie , 2010 ) . stable interaction of in with 3-end processed vdna in the intasome might be a plausible explanation for the difference .
the stable in tetramer formation observed in the intasome reflects the in - dna complex required for proper concerted integration of both vdna ends into the proximal sites of the target host chromosomal dna .
furthermore , analysis of the pfv intasome interacting with the sti elucidated its inhibitory mechanism . based on the pfv intasome structure as a template , structural modeling of the hiv-1 intasome
structural analysis of this intasome revealed numerous details of retroviral integration and will contribute to the design of the next generation of hiv-1 in catalytic inhibitors .
the functional significance of the dna - independent in tetramer as observed by analysis of partial in fragments ( chen et al . , 2000 ; wang et al . , 2001 ;
after entry into cells , retroviral genomic rna ( vrna ) is reverse transcribed into dna ( vdna ) by rt .
then , vdna is transported into the nucleus ( nuclear import ) and finally integrated into host chromosomal dna ( black lines ) . in forms a functional tetramer with vdna ends ( intasome ) to assist with integration .
subsequent repair processes by host dna repair machinery establishes the proviral dna , in which retrovirus dna is stably integrated into host chromosomal dna . in tetramer structures for its enzymatic ( integration ) and non - enzymatic functions ( reverse transcription and nuclear import ) and possible involvement of host factors
the vrna , vdna , and host dna are shown as blue , red , and black lines , respectively .
originally , we found that introduction of amino acid substitutions at conserved hhcc residues in the ntd of hiv-1 in resulted in almost complete abrogation of proviral dna formation , concomitant with a severe reduction in vdna synthesis .
this suggests the mutations in the in affected the viral life cycle at steps prior to integration ( masuda et al . , 1995 ) .
further genetic analysis of hiv-1 in revealed that the pleiotropic effects of in mutations affected uncoating ( masuda et al . , 1995 ; leavitt et al . , 1996 ; nakamura et al . , 1997 ; briones et al . , 2010
) , reverse transcription ( engelman et al . , 1995 ; masuda et al .
, 1995 ; wu et al . , 1999 ; tsurutani et al . , 2000
, 1997 ; tsurutani et al . , 2000 ; ikeda et al . , 2004 ) , and protein processing during viral particle assembly and maturation ( mohammed et al . , 2011 ) .
importantly , hiv-1 carrying point mutations at the catalytic sites of in ( dde ) affected the integration step , but not vdna synthesis ( masuda et al . , 1995 ) .
thus , the pleiotropic effects of in mutations might not be directly related to the loss of its catalytic function .
these experiments suggest that in may possess non - enzymatic roles throughout the viral replication cycle ( figure 2 ) . among the possible non - enzymatic roles of in
, there has been an accumulation of evidence to suggest involvement of retroviral in during reverse transcription ( engelman et al . , 1995 ;
the contribution of in during reverse transcription has also been noticed in a retrovirus - like element of saccharomyces cerevisiae , ty3 ( nymark - mcmahon and sandmeyer , 1999 ; nymark - mcmahon et al . , 2002 ) .
a previous study from our laboratory showed that reverse transcription of hiv-1 was abrogated by knocking down a host factor , survival motor neuron ( smn)-interacting protein 1 ( sip1/gemin2 ) , which binds to hiv-1 in ( hamamoto et al . , 2006 ) .
gemin 2 is a component of the smn complex that mediates the assembly of spliceosomal small nuclear ribonucleoproteins and nucleolar ribonucleoproteins ( fischer et al . , 1997 ; liu et al . , 1997
; buhler et al . , 1999 ; jablonka et al . , 2001 ; meister et al . , 2001 ) . in a subsequent study
, we demonstrated that hiv-1 in and gemin2 synergistically stimulate rt activity by enhancing the assembly of rt on viral rna in vitro ( nishitsuji et al . , 2009 ) .
chow and colleagues have also reported that hiv-1 in stimulates rt activity through physical interactions with rt ( zhu et al . , 2004 ) .
thus , in might possess a direct function to support efficient reverse transcription by rt .
delineation of the in mutant structure could provide clues for depicting the in conformation required for non - enzymatic functions .
nuclear magnetic resonance ( nmr ) analysis of an isolated ntd has shown that the ntd exists in two conformational states , the e and d forms ( cai et al . , 1997 ) .
a previous study using nmr spectroscopy indicated that the ntd mutant protein , in which the tyr 15 residue was replaced with ala ( y15a ) , folds correctly but only as the e form ( nomura et al . , 2006 ) .
the in tetramer structure was formed through interaction of ntd ccd between the inner subunits in the intasome ( hare et al . , 2010 ) .
the residues 1326 and 4045 in the ntd interact extensively with residues 150196 in the ccd of the other subunit .
ccd interaction to form the in tetramer has been proposed from previous crystal structure analysis using the ntd ccd in fragments ( wang et al . , 2001 ; hare et al . , 2009b ) . as observed in these previous analyses , the hydrogen - bond contacts between the side chains of ccd residues gln164 and arg187 and the backbones of the ntd residues lys14 and tyr15 also persist in the recent structure - based model of the hiv-1 in intasome ( krishnan et al . , 2010 ) .
hiv-1 carrying in mutations at the lys186 , arg187 , and lys188 residues exhibited a reverse transcription - defective phenotype ( tsurutani et al . , 2000 ) as found in the ntd mutants including y15a .
these experimental data suggest that the functional tetramer form , stabilized with the ntd ccd interaction , might be critical for the non - enzymatic function of in during reverse transcription .
numerous host factors that interact with hiv-1 in have been reported ( al - mawsawi and neamati , 2007 ) .
the best characterized factor is lens epithelium - derived growth factor / transcription co - activator p75 ( ledgf / p75 ; cherepanov et al . , 2003 ) for chromosomal targeting of hiv-1 in ( maertens et al . , 2003 , 2004 ;
the crystal structure analysis of the pfv intasome ( hare et al . , 2010 ) together with results from previous studies ( li et al .
, 2006 ; hare et al . , 2009a ) suggest that synapsis formation with dna ends of the virus through tetramerization of in might be critical for proper assembly and/or a stable and functionally active intasome .
importantly , ledgf / p75 stabilizes the functional tetramer of in for its enzymatic function .
meanwhile , a reduction of the tetramer form of in was reproduced when wild - type in was expressed in cells in which endogenous gemin2 was knocked down by rna interference ( nishitsuji et al . , 2009 ) .
we also noticed that the intracellular stability and multimer formation of in , especially the tetramer formation of in , were dramatically reduced by in mutations that led to a reverse transcription - defective phenotype .
thus , in the absence of vdna , host factors might be involved in forming or maintaining highly ordered structures of in , which is critical for non - enzymatic function .
therefore , inhibition of the interaction between in and host factors could be a novel therapeutic approach for the design and development of new classes of in inhibitors targeting non - enzymatic functions .
it is obvious that in must be closely associated with the viral genome complex to form an active intasome upon the completion of reverse transcription . physical interaction of in with rt ( zhu et al . , 2004 ; wilkinson et al . , 2009 )
how is the in structure in the absence of vdna before and during reverse transcription maintained ? what is the contribution of host factors to in conformation that is required for non - enzymatic functions ?
the highly ordered in structure and/or its interface structure for interaction with host factors required for non - enzymatic in function(s ) should be determined .
finally , it should be emphasized that in mutations affecting non - enzymatic function resulted in severe deleterious affects to hiv-1 replication compared with in mutations that specifically affected catalytic activity .
clinical efficacy of the sti that blocks in catalytic activity guarantees that a greater impact on hiv-1 control would be achieved with a novel inhibitor that blocks the non - enzymatic function of in .
the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . | integrase ( in ) is a retroviral enzyme that catalyzes the insertion of viral dna ( vdna ) into host chromosomal dna , which is necessary for efficient viral replication .
the crystal structure of prototype foamy virus in bound to cognate vdna ends , a complex referred to as the intasome , has recently been resolved .
structure analysis of the intasome revealed a tetramer structure of in that was required for its catalytic function , and also showed the inhibitory mechanism of the in inhibitor .
genetic analysis of in has revealed additional non - enzymatic roles during viral replication cycles at several steps other than integration .
however , the higher order structure of in that is required for its non - enzymatic functions remains to be delineated .
this is the next major challenge in the field of in structural biology hoping to be a platform for the development of novel in inhibitors to treat human immunodeficiency virus type 1 infectious disease . |
fundamental molecular
transactions responsible for the cell s
homeostasis ( such as in transcription , translation , and replication )
involve dynamic interactions of multiple components and their regulations
via allostery , conformational changes , or translocation . as an example ,
protein dna interactions play a crucial role in many essential
biological processes in which three - dimensional multicomponent structures
and their dynamic changes are often directly related to their functions .
especially , the dynamic aspects of structural changes that occur upon
protein binding to dna are still difficult to capture with standard
biophysical techniques .
a powerful technique for the study of
dynamic conformational changes
in biomolecules is frster - type resonance energy transfer ( fret ) , which is capable of probing distances comparable
to the size of small - to - midsized proteins , as well as short dsdnas
( 310 nm ) .
two - color fret is a one - dimensional ( one
distance ) ruler that can probe the inter- or intramolecular distance
between donor and acceptor fluorophores attached to a macromolecule
( or to two different components of a macromolecular complex ) .
if the fluorophores are suitably positioned on
the macromolecule / complex , fret can report on the conformational states
and on dynamic fluctuation in their structure .
if three or four different
fluorophores are used simultaneously , up to 6 distances can be probed
simultaneously . at the single molecule level , single molecule fret ( smfret )
can
mostly report on nonequilibrium time trajectories of the distance
reaction coordinate ( when molecules / complexes are immobilized ) and
distinguish subpopulations of different conformational states under
various biochemical conditions ( when molecules diffuse in solution ) .
in protein dna interactions , such as in the case of rna polymerization
and nuclease activity by restriction enzymes , better insights into
the structure and function of these complexes could be gained by simultaneously
measuring multiple distances in the presence or absence of multiple
components within a complex .
however , measuring several distances with the same distance range
is not always sufficient .
in fact , a combination of two molecular
rulers with different distance scales may be beneficial to simultaneously
identify large structural conformational changes ( e.g. ,
of dna ) together with smaller scale structural changes ( e.g. , by changes
in binding mode ) .
we recently demonstrated the combination
of microsecond alternating - laser excitation ( salex)-smfret with a shorter - range single - molecule distance
ruler based on protein - induced fluorescence enhancement ( pife ) . in this technique ( which we dubbed alex - based pife - fret ) , an environmentally
sensitive cyanine fluorophore ( cy3 ) is placed at a position on a double - stranded
dna that is close ( <3 nm ) to a protein binding site . upon binding ,
or upon
small distance translocations of the protein with respect
to the labeled position , the fluorescence intensity of the environmentally
sensitive fluorophore is altered .
the cyanine fluorophore cy3 acted
both as fret donor and as pife proximity probe .
a fret acceptor fluorophore
( atto 647n ) was attached on the same dsdna further away from the cy3
attachment base . utilizing a microsecond alternating laser excitation
( salex ) scheme
, we demonstrated
that the ratiometric observable fret efficiency , e , and the ratiometric observable stoichiometry , s , could report on the interprobe distance and the protein proximity ,
respectively . in this work ,
we
present the theoretical framework of alex - based
pife - fret and demonstrate its ability to quantitate experimental results .
we show that the fold - increase in cy3 emission intensity , fluorescence
quantum yield or fluorescence lifetime ( in single molecule pife experiments ) ,
and changes in stoichiometry ( as utilized in alex - based pife - fret
experiments ) are manifestations of the underlying steric hindrance
of the cy3 isomerization mobility . due to its structure ,
the presence
of a bound protein in close proximity to the dye sterically hinders
its mobility and may also specifically interact with it .
our model
reveals the dependencies of fluorescence emission intensity and quantum
yield on the hindered isomerization rate constants .
we demonstrate
how alex - based pife - fret measurements can be fitted by the proposed
model and provide best fit values for the relevant parameters .
we
then apply alex - based pife - fret to study the restriction enzyme bamhi dsdna interaction and quantify the dynamic
range of the ruler .
in addition , we studied the interaction of the
t7 polymerase gp5/trx with dna .
lastly , we show how to disentangle
fret efficiency values from stoichiometry values in alex - based pife - fret .
the results analyzed
here , were obtained from two different dsdna systems as described
before . in short ,
the distance dependence of pife in te absence of
fret was tested on a 40bp - long dsdna carrying
cy3(b ) and atto 647n at opposite 5-ends .
all dsdnas contained
a palindromic sequence 5-ggatcc-3 for the restriction
enzyme bamhi placed at 1,2,3,5 or 7 bp away from
the donor . for experiments involving unspecific binding of t7 polymerase
gp5/trx and controls for bamhi
, 45 bp - long dsdna
was used as described before labeled
with cy3(b ) at the 5-end of the top strand , and atto 647n
at 8,13,18,23,28 or 33bp of the bottom strand were employed .
alex - experiments were carried out at 2550 pm of dsdna
at room temperature ( 22 c ) .
the imaging buffer contained 50
mm tris - hcl , 100 mm sodium chloride , 10 mm calcium chloride , 0.1 mm
edta and 143 mm bme at ph 7.4 .
the instrumentation ,
data acquisition and data analysis for s - alex have been previously
described in detail .
shortly , the alternation period was set to 50 s , and the
excitation intensity to 60 w at 532 nm and 25 w at 640
nm . a 60 objective with na = 1.35 ( olympus , uplsapo 60xo , germany )
was used .
laser excitation was focused to a diffraction limited spot
20 m into the solution .
fluorescence emission was collected ,
filtered against background ( using a 50-m pinhole and bandpass
filters ) and detected with two avalanche photodiode detectors ( -spad ,
picoquant , germany ) .
all data analysis was performed using a home - built
python - based software .
as described previously , salex spectroscopy records four photon streams : donor - based
donor emission f(dd ) , donor - based acceptor emission f(da ) , acceptor - based acceptor emission f(aa ) and donor - based emission after acceptor excitation f(ad ) ( which can be neglected ) .
bursts in the three channels were
identified applying a dual color burst search with parameters m = 15 , t = 500
s , and l = 25 ; additional filtering removed
slow - varying changes in fluorescence intensity and thresholding selected
for bright single - molecule bursts ( > 100 photons per burst unless
otherwise
mentioned ) .
after burst identification , fluorophore stoichiometries s and apparent fret efficiencies e * were
calculated for each burst .
uncorrected fret
efficiency e * monitors the proximity between the
two fluorophores and is calculated according to1 s is defined as the
ratio between the overall green fluorescence intensity over the total
green and red fluorescence intensity and describes the ratio of donor - to - acceptor
fluorophores in the sample s:2 by binning the detected bursts into a 2d e*/s histogram , subpopulations as free
and protein - bound dna
species can be separated according to their s - values . e*- and s - distributions
were fitted using
a gaussian function , yielding the mean values i of the distribution and an associated standard deviations wi . to obtain accurate e and corrected s values ,
finally , a gamma
factor correction was applied , i.e. , use of cy3b of free cy3b - labeled oligos to all
data sets . applying
the theory in
either simulations respectively for experimental data
fitting was
performed using scripts in matlab r2008b respectively r2013b ( mathworks
matlab , natick , ma , usa ) .
the differential equations ( eqs 5 ) were numerically solved by use
of the ordinary differential equation solver function ode45 . for the numerical solution of systems of coupled ordinary differential
equations ,
the matlab built - in ode package allows
full control over fine - tuning the numerical solution process .
nevertheless ,
the matlab built - in matrix exponentiation function expm allows for a faster and efficient numerical solution and can be
used instead of ode45 .
data fitting was performed
by comparison of experimental s(e ) values to the theoretical computed ones until the square sum of
the fitting residues was minimized .
accessible volume calculations
of cy3 attached to the 5-end of dsdna , in the absence or presence
of bamhi , were performed using the software provided
by sindbert et al . the crystal structure
( pdb code : 2bam )
the
photophysics of cy3 as a molecular rotor used in pife is explained following the jablonski diagram in figure 1 .
although the pi - conjugated system restricts rotations about the one - dimensional
polymethine group , the trans and cis ground - state isomers typically interconvert in the s time
range , probably via vibrational weakening
of the double bond inducing double bond twisting . out of the two ground - state isomers ,
the trans isomer has a larger absorption cross - section at = 532 nm ,
owing to its absorption spectrum peak ( cis isomer
is red - shifted by 20 nm ) , higher
thermodynamic stability , its symmetry ,
and its larger excitation dipole moment ( in comparison to the dipole
of the cis form ) .
we denote the fraction
of molecules excited from the trans ground - state
isomer with , and the fraction of molecules excited from the cis isomer with 1- ( see figure 1c ) .
( b ) excited - state cy3 undergoes
isomerization between a bright trans isomer and a
dim cis isomer via a 90 isomerization intermediate .
in the presence of an acceptor fluorophore ,
the excited trans isomer dtrans * of cy3 is deactivated via one
of three possible pathways : ( i ) by frster - type resonance energy
transfer kfret to the acceptor , ( ii ) direct
deexcitation from excited- to ground - state trans isomer
through either fluorescence or internal conversion kd , and ( iii ) deactivation by excited - state isomerization
( kt90 ) .
the excited intermediate
state d90 * is tightly coupled to the ground state 90-transition
state and hence undergoes fast de - excitation ( kd,90 ) to the ground state of both isomers .
( c ) the steady state
population density between the cis and trans isomers of cy3 is shifted toward the trans isomer
due to changes in the microenvironment of cy3 caused by steric hindrance
from a nearby bound protein .
after excitation , the electronic configuration of the excited
state
allows fast rotation around the central polymethine bond , due to a
reduction in bond order following electronic excitation .
this , in
turn , allows cy3 to isomerize between trans and cis through a 90 isomerization intermediate .
the excited - state isomerization rate constants from trans to 90 intermediate and back are denoted by kd , trans90 , kd,90trans , and from cis to 90 intermediate as kd , cis90 , kd,90cis .
this intermediate has been reported to be tightly coupled
to the ground - state , allowing fast
nonradiative de - excitation with branching into both cis and trans isomers .
therefore , the 90 intermediate serves as an efficient sink
for excited - state deactivation and competes with fluorescence . the
de - excitation rate from the 90 excited - state intermediate is
denoted as kd,90 .
excitation energy can
be depleted radiatively or transferred only from planar forms of cy3 .
altogether , the excited - state cis and trans isomers may yield fluorescence or fret , but the 90
excited - state isomerization intermediate can not .
the de - excitation
rate constants from the trans and cis isomers represent sums of all radiative and nonradiative de - excitation
processes including fluorescence , internal conversion , or frster - type
resonance energy transfer ( fret ) and are denoted as kd , t , and kd , c respectively .
the de - excitation from the cis isomer has been reported
to be mostly nonradiative .
in addition , the transient
absorption in the cis ground - state isomer , hundreds
of ns after excitation , reports on population of the cis ground - state isomer .
the isomer is reached via branching on the
potential energy surface at the 90 transition - state , rather
than directly from the cis isomer after de - excitation .
therefore , out of the three excited - state isomers ,
it is mostly the trans isomer for which de - excitation
yields fluorescence . hence the longer cy3 survives in the trans excited - state isomer after excitation , the higher
the probability it will yield a fluorescence photon upon relaxation
back to ground state .
pife is the results of a modulation in isomerization
rate due to steric hindrance .
this hindrance is responsible for enhancement
in the trans isomer survival ( and reduction in isomerization
into the 90 isomerization intermediate ) . in pife - fret
,
an acceptor fluorophore is placed closely to the
cy3 and acts as yet another nonradiative sink for the excitation energy .
since fret is effective for distances of 310 nm , no
direct steric hindrance is imposed on cy3 by the acceptor fluorophore .
however , the fret process reduces the time cy3 spends in the excited - state
( including in the trans isomer hence reducing the
fluorescence quantum yield of the donor ) , which in turn will lead
to a reduction in the magnitude of a possible pife effect .
if pife probes the binding of a protein proximal to a cy3
placed on dsdna and if the dsdna undergoes a conformational change
upon binding , a pife effect could potentially be masked by an increase
in fret efficiency .
energy transfer from the trans excited - state isomer
is described here as another de - excitation rate constant ( eq 3):3where r0,t is
the frster radius , the distance at which 50% of the excitation
energy is transferred via fret to the acceptor , r is the distance between the dipole moments of cy3 and the acceptor
fluorophore , and e is the fret efficiency from the trans donor .
energy transfer may also occur from the cis cy3 excited - state isomer ( eq 4):4 this treatment
of fret is accurate as long as the dsdna structures
with either a protein bound or unbound can be considered static .
other cases require an extension of the model ( given in the si ) . in the static case ,
since pife modulates
the frster radii r0,t and r0,c , the fret rate constant is a function of
the modulated frster radius r0 and dye distance . since the latter might also be influenced depending
on the chosen protein system , we treat the rates as functions of fret
efficiency in this uniform approach . for further discussion regarding
heterogeneous and dynamic fret systems , see discussion in the si . following the jablonski diagram ( figure 1 ) and the above - mentioned
rationale ,
pife - fret
can be described by the following set of coupled rate equations ( eqs 5):5here , dt * , d90 * and dc * represent
the time - dependent occupancy of cy3 excited - state trans , cy3 excited - state cis , and the 90 isomerization
intermediate , respectively . since the 90 isomerization intermediate
is not expected to be de - excited radiatively , the fluorescence signal
of cy3 and the corresponding acceptor in pife - fret can be calculated
from dt * , dc*. and a * which is the survival
probability of the excited acceptor .
we have recently demonstrated
applications of the pife ruler using
alex - based pife - fret ( i.e. , using fluorescence intensity - based measurements
rather than fluorescence lifetimes measurements ) .
we demonstrated
that our approach could simultaneously provide two dynamic molecular
rulers at different length scales as long as the acceptor is insensitive
to changes in its microenvironment . here , we compare experimental
results obtained before to our derived model ( eqs 5 ) .
this attempt requires knowledge of fret
efficiency e and the stoichiometry s both of which we derive from cy3 ( donor ) intensity and the acceptor
intensity according to67 the intensities
in eqs 6 and 7 are calculated for the alternation period
during which the donor is excited . during the acceptor s excitation
period , only the acceptor is excited , and hence it yields fluorescence
after direct excitation , with a rate and total intensity of8respectively .
a good practice for carrying cy3-based pife - fret
measurements is
the utilization of the cy3b fluorophore in control experiments .
cy3b
is the chemical equivalent of the trans cy3 isomer
without isomerization possibility .
the fret rate equations , for cy3b
acting as a donor , are therefore simplified to9 the fluorescence
intensity of cy3b is then calculated according
to10 the mean values of the fret efficiency e and
stoichiometry s distributions given in an e s histogram are calculated according
to1112 the fret efficiency calculated for
either the cy3-donor or cy3b - donor
assumes both fret pairs have been normalized to the same frster
radius using the r0 of the cy3b - acceptor
pair .
this is achieved following the corrections scheme given
in ploetz and lerner et al . following
this approach
, we treat all data after gamma correction and convert
all fret efficiencies onto the common r0-axis of cy3b by
employing the gamma of cy3b .
the absolute quantum yield changes and
absolute gamma values of complex protein system do not interfere anymore
with the fret efficiency , but are decoupled .
rotational diffusion of
cy3 on dsdna might be hindered upon binding
of a protein close to the attachment site .
the fluorescence quantum
yield ( qy ) of cy3 is directly proportional to the degree of isomerization
inhibition from the excited - state trans isomer . since
isomerization requires available space to accommodate the spatial
change in the structure of cy3 , its fluorescence spectrum is sensitive
to the microenvironment .
the average fluorescence intensity , fluorescence
lifetime , or qy of cy3 are expected to be low when the fluorophore
is free to isomerize .
this occurs partly due to loss of excitation
energy via nonradiative relaxation through the 90 isomerization
intermediate .
an induced change in the microenvironment , such as in
the case of protein binding in close proximity , can sterically restrict
cy3 and hence impede its isomerization .
steric restriction will decrease
the isomerization rate and thus increase radiative recombination from
the trans isomer , leading to increased fluorescence
intensity and an increased fluorescence qy . according to kramers
kinetic theory ,
the isomerization rate constants ( eqs 5 ) depend on the free energy of
the excited - state isomers , on the free energy barrier between isomers
( in the exponential term ) , and on the isomerization mobility / diffusivity
( in the pre - exponential term ) .
steric interactions are the manifestation
of volume excluded by a near - by molecular system ( excluding all other
types of interactions ) .
cy3 isomerization mobility could
be influenced by the viscosity of its microenvironment ( microviscosity ) and/or by steric hindrance .
these will affect the pre - exponential
term of the kramers isomerization rate constant without changing the
activation barrier for isomerization . however , specific interactions
of the fluorophore with a nearby molecule may also induce a delay
in isomerization , which will lead to an increase in activation barrier
for isomerization . here
we provide a model that accounts for pife
as the effect of steric hindrance on cis / trans isomerization .
in diffusion activated reactions , restricted rotational mobility due
to steric hindrance
. the modified isomerization rate of a cy3 in
the presence of a nearby protein could therefore be written as13where kiso represents
all isomerization rates ( kt90,k90t , kc90,k90c ) , kiso represents all modified isomerization rates due to steric hindrance , vt and vf are the
accessible volumes to cy3 ( see figure 2a ) in the absence and presence of steric hindrance ,
respectively , and b is a proportionality constant
that accounts for solvent properties , tethering of the dye to the
dna , other specific interactions , etc .
vf is the excluded volume ,
and = vt/(vt vf ) is cy3s
normalized accessible volume in the presence of steric hindrance ( eq 13 ) . importantly , this
allows to define a universal pife parameter , , which is the
ratio of the isomerization rate constants in the absence and presence
of steric hindrance .
( a ) illustrations of dsdna with donor ( cy3 ) and acceptor ( atto 647n ) ,
separated by 40 bp .
the restriction enzyme bamhi
binds specifically to the dna containing its palindromic dna sequence
( purple ) .
binding of bamhi restricts the available
volume of cy3 at a separation of 1 bp away from the protein - binding
sequence , leading to an increased brightness . at a separation of 7
bp away from the binding site , the available volume of cy3 is maximized ,
and the brightness of cy3 is low .
( b ) alex - based pife - fret experimental
results ( circles ) and a global fit using the pife model ( solid lines )
for dsdna labeled with cy3(b ) at the 5-end and atto 647n as
acceptor on the complementary strand . control experiments with fret
distances of 33 , 23 , 18 , and 13 bp ( absence of bamhi ) are shown in black .
pife - fret results at 33 bp separation in
the presence of bamhi are shown for 1 ( red ) , 2 ( orange ) ,
3 ( green ) , 5 ( blue ) , and 7 ( purple ) bp separation between the donor
and bamhi .
the obtained pife factors for the 5 dna
constructs are shown in the legend and in figure 3 . in order to calculate the pife effect for a particular case ,
one
needs to replace all isomerization rate constants in eq 5 with the modified isomerization
rate constants of eq 13 . for this
, however , the volume explored by the fluorophore in the
absence and presence of the bound protein needs to be evaluated .
if
a protein dna crystal structure exists for a particular interaction
of interest , the volume explored by the mobile fluorophore could be
estimated via published procedures . in alex - based pife - fret ,
a given cy3-acceptor pair will yield a particular s value . in the presence of a nearby bound protein ( or any other steric
hindrance ) , if contact quenching does not occur , this s value will increase ( assuming that there are no changes in e ) .
following directly from the increase in cy3 fluorescence
intensity ( assuming there is no change in the acceptor s fluorescence ) .
such a simple case was reported for the interaction of the restriction
enzyme bamhi with dsdna . since the nonradiative de - excitation of cy3 through isomerization
( modulated by pife ) and through fret to the acceptor
, the molecular
target and its binding position have to be carefully chosen so that
pife and fret are sensitive to binding and conformation , respectively .
to maximize the pife effect , it is advisible to position the acceptor
away from cy3 at a distance which is close - to or larger than the corresponding
frster radius r0 .
in many
cases , protein dna interactions trigger a conformational change
not only to the dna , but both to the protein and the dna . upon protein
binding , dna can exhibit bending ( e.g. , introduced by the restriction
enzyme ecorv ) , bubble - formation ( triggered by rna
polymerases ) and other types of dna conformational changes . such conformational
changes
may alter the fret efficiency , which in turn will introduce
an increase in stoichiometry due to the decreased survival time in
the excited cy3 , regardless of pife effects . e and s values in alex - based pife - fret experiments
are not independent
and require careful calibration . in the absence of pife and
depending on the intrinsic rate constants
and the relevant parameters ( eqs 5 ) , one could plot s values as a function
of e values , i.e. , s(e ) .
this s(e ) curve will dictate
a lower bound on pife related change in s ( see figure 2b , black solid line ) .
if cy3 is replaced with cy3b , and all corrections are applied , the
corresponding s should , by definition , always be
0.5 .
so any result that points to a pife effect
in an alex - based pife - fret measurement should fall above the s(e ) curve for cy3 and below that for cy3b
( see figure 2b , black
dashed line ) .
the experimental ( best fit extracted ; see figure 2 ) pife parameter
( ) and the theoretical normalized ( b = 1 )
accessible volume ( ) are plotted as a function of bp distance
( black and red filled circles ) respectively .
the theoretical ruler
based on the estimation of the accessible volume of cy3 serves as
a lower boundary for the experimental ruler , where specific interactions
of the dye with the bound - protein can delay the isomerization from trans isomer even longer . in
previous published work , we
performed
salex measurement for the restriction enzyme bamhi bound to dsdna , having cy3 or cy3b as a donor , and atto 647n as
an acceptor , at different donor acceptor separations .
the results
of these experiments were fitted to the pife - fret model ( eqs 512 ) and are shown in figure 2 as a family of s(e ) curves .
the extracted best fit values are 1/kd , c = 0.39 ( 0.150.55 ) ns , 1/kd,90 = 0.02 ( 0.010.03 ) ns , 1/kt90 = 1.00 ( 0.901.10 ) ns , 1/k90t = 0.89 ( 0.601.50 ) ns , 1/kc90 = 0.35 ( 0.200.55 ) ns , 1/k90c = 0.09 ( 0.080.30 ) ns , and = 0.95 ( 0.940.99 )
using lifetimes measured by ploetz and lerner et al . for cy3b - atto 647n , we also derive : 1/ka = 4.3 0.2 ns and 1/kd , t = 2.2 0.1 ns .
a striking observation is the fast de - excitation
of cy3 via the 90 intermediate isomer in comparison to the isomerization
rate constants .
this observation is not only consistent with the currently
accepted models for cy3 photoisomerization , but also supports the
hypothesis that this intermediate does indeed serve as a sink to the
excitation energy .
pife is therefore dominated by a decrease in isomerization
yield from the trans isomer .
also note that the fraction
of the excited - trans isomer is very high , as expected . when stepping the bamhi binding site 7 , 5 , 3 ,
2 , and 1 bp toward cy3
, the stoichiometry s is increased
( figure 2b , purple ,
blue , green , orange , and red , in that order ) . this data were globally
fit to the pife - fret model ( eqs 513 ) , while keeping the pife
parameter ( - the fold decrease in isomerization rate ) free ,
and fixing the photophysical rate constants of cy3 .
the best fit results
( figure 2 , colored
lines ) report on the dependence of the pife effect on the distance
of cy3 from the nearby bound bamhi , which is then
explicitly plotted in figure 3 .
figure 3 shows that
the experimentally derived linearly depends on the separation
between the cy3 fluorophore and the interaction interface ( leading
edge ) of the bamhi on dsdna .
the theoretically derived normalized accessible volume ( eq 13 ) is also plotted on
the same graph , showing a linear dependence on distance ( until a pife
separation of 5 bp ) when setting the proportionality factor to be b = 1 .
the experimentally derived pife parameter values
( figure 3 , black ) fit
with the theoretical evaluations of the ratio of cy3 available volumes
for pife distances 1 , 2 , and 7 bp away from the binding sequence ( figure 3 , red )
. however ,
for pife distances 3 and 5 bp , the experimentally derived pife parameter
values are higher than the theoretical estimates of cy3 ratio of available
volumes .
since the theoretical estimation of the pife effect shown
by eq 13 are derived
following the sole effect of steric hindrance and does not provide
information on possible specific interactions , the results for pife
separations 3 and 5 bp may report on specific interactions of cy3
at these positions with the bound bamhi .
the resemblance
between the theoretical estimates driven solely by steric hindrance
effects and some of the experimentally derived pife values supports
the notion that pife of cy3 is caused by steric hindrance from the
nearby bound protein , which leads to a decrease in isomerization rate .
the steric hindrance can be estimated directly from ratios of fluorophore
accessible volume calculations performed on dna in the absence and
presence of a protein , given that the crystal structure of the complex
is available .
this theoretical estimation can act as a lower boundary
for the experimental - derived pife parameters and allows assessing
specific interactions of cy3 with the bound protein or possible deviations
of the binding mode of the protein to the dna that may occur with
the labeled dna .
the best fit of the model to pife data results in
a s(e ) curve with different e and s pair values ( figure 2 , colored lines ) . to provide a control experiment
in which no conformational changes
in the dna duplex are induced by protein binding ( observed in fret )
,
we also reanalyzed published data of unspecific binding of t7 dna
polymerase gp5/trx at different
fret distances .
the unspecific sliding
of the polymerase is so fast that an average pife effect is observed
instead of multiple distinct pife state for different distances of
polymerase and cy3 .
these pife - fret measurements of gp5/trx on dsdna
represent cases in which binding does induce conformational changes
in the dsdna , with data points that move on the s(e ) lines due to
the change in e ( figure 4 ) .
the comparison between the achieved enhancement
factors for gp5/trx with bamhi ( figure 2 , = 2.47 ) clearly points
to the different nature of locally involved amino acids .
this difference
of local environment renders it hence necessary in general , to calibrate
the ruler - characteristic of any protein and applied buffer system
separately in order to link the measured pife enhancement to an actual
spatial separation .
alex - based pife - fret experimental results ( circles ) and
a global fit using the pife model ( solid lines ) . results for dsdna
labeled with cy3 and cy3b as the donor are denoted with filled and
open circles , respectively .
the acceptor in both cases is atto 647n .
control experiments ( absence of gp5/trx ) are shown in black for 33 , 28 , 23 , 18 , 13 , and 8 bp .
pife - fret
results in the presence of gp5/trx for dna constructs with 28 , 23 , and 18 bp separation are shown in
magenta . gp5/trx is known to slide
on dna , hence it does not have a known pife separation from
the bound protein . the best fit value of the pife parameter , ,
to the data , is shown close to the best fit curve ( 4.14 ( 3.944.36 ) ) . in previous works ,
pife was quantified by the fold increase in cy3 fluorescence lifetime
or intensity ( or the related fluorescence qy ) . following the above
description
the fluorescence
quantum yield of cy3 can be described as the ratio of radiative de - excitations
to all de - excitation processes:14 this expression assumes that the time
it takes to reach steady - state for all isomers is much shorter than
the excited - state lifetime .
the probability of occupying the trans excited - state isomer is the highest immediately after
excitation , and it decreases thereafter . as the best fit results to
the model show ( figure 2 , black ) , the isomerization rates are not much different from the
excited - state lifetime .
a temporal definition of the fluorescence qy can be given
by considering the weights on isomers state occupancy probability:15 at each moment after excitation ,
the cy3 qy decreases as the population
of the excited - state trans isomer decreases , until
it reaches a steady - state .
the mean qy should therefore be taken into
account ( figure 5a ) .
the mean qy can be calculated accurately ( figure 5b , black ) , but can also be approximated by
the asymptote value of the qy curve at the long decay times ( figure 5b , gray ) .
when referring
to the pife effect as the fold increase in fluorescence qy , we will
calculate the term in eq 15 in the absence and presence of steric hindrance and compare
the ratio between the two to the pife parameter ( figure 5b ) .
fold - decrease in cy3
mobility and fold - increase in mean fluorescence
quantum yield .
( a ) time dependence of cy3 and cy3b fluorescence quantum
yields ( following eq 15 in the absence ( = 1 ) and presence = { 2,3,4,5,10 } )
of pife .
( b ) time averaged ratio ( black ) and longtime asymptote ( gray )
of cy3 qy in the presence and the absence of pife for different
values , = { 210}. the parameters used for this simulation
are the ones found in the analysis reported above ( figures 2 and 3 ) for a candidate fret efficiency of e = 0.4 .
the fold increase in the fluorescence
qy of cy3 ( either using fluorescence
intensities or lifetimes ) is the measure that was used to quantify
pife in previous works.figure 5 shows that this measure does
not directly report on the fold decrease in isomerization mobility .
the nonlinear dependence between the two measures holds even for large
values of qy ( as in the case of bamhi ) .
however , note that as the pife effect is increasing ,
one pife parameter , the fold - increase in qy , approaches an asymptote ,
while another pife parameter , the fold - decrease in cy3 isomerization
mobility , still increases .
the fold - decrease in cy3 isomerization mobility , ,
however , has a linear dependence on the change in cy3 accessible volume ,
and hence on steric hindrance .
therefore , the parameter is
a more direct measure of the pife effect .
we compared our obtained
values ( figure 5b )
with those reported by hwang et al . through
a direct calculation of the fold - increase in fluorescence intensity
or fluorescence lifetime , performed on the same dna constructs in
the absence and presence of bamhi ( figure s1 ) .
this comparison
revealed that ( 1 ) the intensity - based data found with smpife is higher
than the alex - based pife - fret results using our model with bamhi ; ( 2 ) the lifetime - based data found with smpife is
mostly lower or equal to the alex - based pife - fret results using our
model for bamhi ( note the large error values ) ; however
( 3 ) the intensity- and lifetime - derived smpife data found by hwang
et al
. already differ from each other with statistical significance
( no intersection of error ranges for both ) .
most importantly the results
of our re - evaluation of the bamhi pife experiment
using alex - based pife - fret with the herein proposed model are in the
middle between the intensity- and lifetime - derived smpife results ,
and hence reconcile the two . in alex - based
pife - fret , the mean values of e and s are
determined via 2d gaussian fittings of subpopulations in the e s histogram .
these subpopulations
tend to exhibit intrinsic width which depends on shot - noise and other
photophysical effects .
alex - based pife - fret experiments involve measuring
separately the cy3-acceptor dsdna in order to characterize the system
parameters in the absence of pife . in a next step ,
if the protein binds in close
proximity to the cy3 fluorophore and induces an increase in its fluorescence
due to pife , the fraction of single dna molecules bound to the protein
will show up as a second s(e ) subpopulation .
due to the widths of these subpopulations , it is always better to
have the mean e and s of the two
well separated .
the best fit results shown in figure 2 represent the s(e ) curves for different values of the pife factor .
they allow us to observe what will be the dynamic range of changes
in the stoichiometry for different nominal fret efficiency values :
the higher the fret efficiency value is , the smaller the changes in
stoichiometry will be .
in addition , if a conformational change that
increases e is concurrent with protein binding ( and pife ) , one should
exercise great caution in correctly interpreting the increase in s , since it could be due to increase in e only and vice versa .
the region in figure 2 that is bound by the black cy3 and cy3b
curves ( no protein bound ) is the region in which e and s values will report on pife either with or
without fret changes .
pife is a powerful single
molecule spectroscopy technique that
provides a distance ruler shorter than fret but longer than contact - quenching - based
rulers , such as photoinduced electron transfer .
the combination of
pife with fret allows us not only to probe both short and long distances
simultaneously , but also to study binding - induced conformational changes
in nucleic acids triggered by unlabeled proteins . in this work we
have provided a model that allows correct interpretation of pife experiments
in general and of alex - based pife - fret experiments in particular ,
and elucidated the nonlinear dependence of s on e in a pife experiment .
we identified steric hindrance ( aside
from other possible effects such as a specific interaction of the
fluorophore with the bound protein ) and slowing down of cy3 isomerization
mobility as the main mechanisms responsible for pife .
the resulting
change in the fluorescence qy , the fluorescence lifetime and hence
cy3 s brightness can serve as a signal reporting on pife in
different implementations of confocal smfret experiments .
given that this assay can be
applied for surface immobilized smfret , the presented work represents
a generalized and comprehensive framework for studying protein nucleic
acid interactions in a quantitative manner . | single - molecule ,
protein - induced fluorescence enhancement ( pife )
serves as a molecular ruler at molecular distances inaccessible to
other spectroscopic rulers such as frster - type resonance energy
transfer ( fret ) or photoinduced electron transfer . in order to provide
two simultaneous measurements of two distances on different molecular
length scales for the analysis of macromolecular complexes , we and
others recently combined measurements of pife and fret ( pife - fret )
on the single molecule level .
pife relies on steric hindrance of the
fluorophore cy3 , which is covalently attached to a biomolecule of
interest , to rotate out of an excited - state trans isomer to the cis isomer through a 90 intermediate .
in this work
, we provide a theoretical framework that accounts for
relevant photophysical and kinetic parameters of pife - fret , show how
this framework allows the extraction of the fold - decrease in isomerization
mobility from experimental data , and show how these results provide
information on changes in the accessible volume of cy3 . the utility
of this model
is then demonstrated for experimental results on pife - fret
measurement of different protein dna interactions .
the proposed
model and extracted parameters could serve as a benchmark to allow
quantitative comparison of pife effects in different biological systems . |
these surfactant characteristics result from the combination of the polar hydroxyl groups on the concave -face and the methyl groups on the convex -face .
bile salts in aqueous solution form aggregates mainly due to hydrophobic attractive interactions of the apolar -faces .
it is known that bile salts form micelles at concentrations above the critical micelle concentration ( cmc ) .
the more hydrophobic bile salt is , the lower critical micelle concentration it has , due to more stable hydrophobic aggregations .
bile salts are known as permeability enhancers to increase drug penetration through various biological membranes by interacting with phospholipids in cell membranes .
micelles of bile salts have been investigated in different pharmaceutical formulations where they solubilise poorly soluble drugs [ 10 - 13 ] .
bile salts with more hydrophobic micellar core have greater ability to accept hydrophobic pharmaceutical ingredients .
solubilisation capacity of the micellar systems can be increased by chemical modification of the bile salt steroid skeleton .
another method to improve solubilisation properties of micellar systems is to produce mixed micelles by combining individual surfactants with different aggregation properties ( cmc values ) .
furthermore , intermolecular hydrophilic interactions between polar groups in certain bile salt mixed micelles are known to greatly increase stability of these binary systems . in the pharmaceutical area , various nonionic co - surfactants have been used in bile salt - nonionic binary systems due to ability of these mixtures to improve solubilisation of poorly soluble drugs [ 17 - 23 ] . however , in studies of bile salt - anionic binary mixtures , sodium dodecyl sulfate has been predominantly used as an anionic co - surfactant [ 24 - 27 ] .
triton x-100 ( trx ) is a nonionic surfactant with a hydrophilic polyethylene oxide group ( on average it has 9.5 oxyethylene groups ( oe ) ) and a hydrophobic group .
trx ( p-(1,1,3,3-tetramethylbutyl)phenoxypolyoxyethylene glycol ) differs from conventional nonionic surfactants , having hydrophilic part longer than the hydrophobic region of the molecule .
sodium lauryl ether sulfate ( sles ) is an anionic surfactant with one to four hydrophilic oe groups adjacent to the anionic sulfate group .
its basic structure is c12h25(och2ch2)xoso3na where x represents the number of oe groups in the chain . for this work , sles having two oe groups was used . to the best of our knowledge ,
work on physico - chemical characterisation of binary mixtures consisting of bile salt and polyoxyethylene type of co - surfactant has been done only with individual bile salts . however , there is no comparative data on the physico - chemical properties of mixed micelles of trx or sles with different bile salts .
according to the authors ' knowledge investigations of sles - bile salt binary systems have not been reported previously . in the present work ,
we have tested a set of three bile salts ( sodium cholate ( c ) , sodium deoxycholate ( d ) or sodium 7-oxodeoxycholate ( od ) ) to interact with trx or sles .
the results of the study have been analysed using clint 's , rubingh 's , and motomura 's theories for mixed binary systems [ 30 - 32 ] .
the first aim of this study was to investigate how different combinations of hydroxyl and oxo groups on the concave - face of the bile salt skeleton can influence the interactions with anionic and nonionic co - surfactant having polar oe groups in the hydrophilic region .
the second goal was to evaluate how structural differences in selected anionic and nonionic co - surfactant impact micelization behaviour of developed mixed micelles .
it is envisioned that insight on the formation of the micellar structures obtained in this study could be used to develop drug delivery formulations with optimized physico - chemical properties .
the cmc values of individual bile salts ( d , c , and od ) and sles were acquired through conductivity and surface tension studies .
the experimentally obtained cmc values , with standard deviations of three determinations , for individual surfactants ( cmc ) are presented in table 1 and compared to the literature values ( cmc ) [ 33 - 35 ] .
experimentally obtained critical micelle concentrations of the individual surfactants compared to the literature values a the values obtained from conductivity measurements .
cmcthe mean values standard deviation of three determinations cmcthe cmc values obtained from the literature the critical micelle concentrations of the binary mixtures were studied using conductivity and surface tension measurements , at different mole fractions of trx or sles ( ) .
prepared mixtures consisted of 0.1 , 0.2 , 0.3 , 0.4 or 0.5 mole fractions of trx or sles ( ) .
for the conductivity studies , a break in the conductivity ( specific conductivity , ) versus concentration plot indicated the onset of the micellization process , since this break is characteristic of micelle formation and represents the cmc ( figure 1 ) .
the cmc values obtained in the conductivity measurements were acquired from the intersection of the straight lines created above and below the cmc .
the straight line above the cmc value was determined by linear regression starting at the highest concentration , while the straight line below the cmc value was obtained by linear regression beginning at the lowest concentration of investigated binary mixtures .
the regression diagnostic was constantly performed after addition of each subsequent experimental point . if an experimental point was identified as an outlier , by measuring cook 's distance , the regression would be stopped and the straight line would be formed using previously added experimental points ( figure 1 ) .
dependence of the specific conductivity on concentration of trx - od ( = 0.5 ) binary system .
the discontinuous line represents the gaussian curve obtained by phillips ' method and the arrow denotes the cmc .
however , this method for the determination of cmc values is reliable only if there is an abrupt change in conductivity . as seen in figure 1
, the change in conductivity does not show an abrupt alteration and the intersection point is not clearly detectable . since the uncertainty in determination of cmc values is increased , phillips ' method was applied in order to obtain the cmc values more reliably . in this approach ,
the cmc is considered as the concentration at which the maximum change in a physical property of the solution , , versus concentration curve occurs .
( d3dc3)c = 0 ; c = cmc phillips ' method was employed as proposed by mosquera et al .
the runge - kutta integration was applied and a least squares fitting method , according to levenberg - maquardt , was used .
the usage of phillips ' method for determination of cmc values has been reported in previous studies , as well .
figure 1 shows the application of phillips ' method . in order to validate the results determined by means of conductivity measurements
plots of surface tension ( ) versus concentration of binary system were obtained , where a break point corresponds to the cmc .
dependence of surface tension on concentration of trx - od ( = 0.5 ) binary system .
the cmc determination error did not exceed 3% . extended t - test ( p < 0.05 ) shows that the mean cmc values determined using conductivity and surface tension measurements belong to the same population .
the experimentally obtained cmc values , with standard deviations of three determinations , for the trx - bile salt and sles - bile salt binary mixtures are presented in tables 2 and 3 respectively .
comparision of the experimentally obtained critical micelle concentrations of investigated trx - bile salt binary systems a the values obtained from conductivity measurements .
cmcthe mean values standard deviation of three determinations comparision of the experimentally obtained critical micelle concentrations of investigated sles - bile salt binary systems a values obtained from conductivity measurements .
cmcthe mean values standard deviation of three determinations as seen in tables 2 and 3 the cmc values obtained in conductivity and surface tension studies are in good agreement . since there is no statistically significant difference between the cmc values determined by means of the two methods , the cmc values acquired by conductivity measurements were used in further calculations . in order to study the influence of the structure of bile salts on the formation of mixed micelles with trx or sles , the physico - chemical parameters of the micellar systems
the determined physico - chemical parameters are : the critical micelle concentration of ideal mixtures ( cmc ) , the mole fraction of the more hydrophobic surfactant in the ideal mixed micelle ( x ) , the mole fraction of the more hydrophobic surfactant in the real mixed micelle ( x1 ) and the parameter .
the physico - chemical parameters for the trx - bile salt and sles - bile salt mixed micelles were calculated according to the equations presented in table 4 .
physico - chemical parameters of mixed micelles i the mole fraction in the solution of component i , cmci the experimentally obtained cmc of component i. cmc1 the experimentally obtained cmc values of the more hydrophobic surfactant ( trx or sles ) cmc2 the cmc of d , c or od cmcthe experimentally obtained cmc values of investigated binary systems the mole fraction of the more hydrophobic surfactant in the solution ( trx or sles ) comparison of experimental with ideal parameters and interaction parameter ( ) of the trx - bile salt mixtures cmcthe experimentally obtained cmc values of investigated binary systems , each result is the average of three separate determinations .
cmcthe values for the critical micelle concentrations of ideal mixtures , calculated according to clint 's theory of ideal mixtures .
x1 mole fractions of the more hydrophobic surfactant in the real mixed micelles , according to rubingh .
xmole fractions of the more hydrophobic surfactant in the ideal mixed micelles , according to motomura 's model .
comparison of experimental with ideal parameters and interaction parameter ( ) of the sles - bile salt mixtures cmcthe experimentally obtained values , each result is the average of three separate determinations .
cmcthe values for the critical micelle concentrations of ideal mixtures , calculated according to clint 's theory of ideal mixtures .
x1 mole fractions of the more hydrophobic surfactant in the real mixed micelles , according to rubingh .
xmole fractions of the more hydrophobic surfactant in the ideal mixed micelles , according to motomura 's model .
the results show deviation of the cmcvalues from those calculated according to clint 's theory indicating nonideal behaviour of examined mixtures and existence of mutual interactions of the components in the micelles .
the difference between xand x1 also indicates nonideal behaviour of the mixtures . from tables 5 and 6 ,
it can be observed that values of the parameter come out to be negative at all molar ratios of the mixtures .
the more negative value of the interaction parameter indicates stronger synergistic interactions between the components .
these values correspond to the deviation between the experimentaly obtained and calculated cmc values and indicate synergism in all investigated trx - bile salt and sles - bile salt mixtures . in table 7 , the average values of the interaction parameter ( av ) for analysed binary systems are presented in order to make the results easily comparable .
average interaction parameters ( av ) of analysed mixed binary systems the average interaction parameter values show that trx generates stronger sinergistic interactions with investigated bile salts than sles .
many recent studies have confirmed that synergistic effect generally occurs in anionic - nonionic surfactant mixtures , improving stability of the system [ 41 - 48 ] . in previously investigated sodium deoxycholate -
this value is in good agreement with the average value of the interaction parameter reported in our study ( -0.88 ) .
it is known that in mixed miceles composed of two anionic amphihpiles , repulsive electrostatic interactions between anionic heads are important source of destabilisation .
hence , weak synergistic or antagonistic interactions between individual surfactants are frequently reported in anionic - anionic surfactant mixtures .
therefore , weak synergism that has been noticed for anionic - anionic surfactant mixtures in this article is in agreement with previous studies .
the results presented in table 7 show that trx and sles generate the weakest synergistic effect with d , while od creates the strongest interaction with investigated co - surfactants .
we assume that the hydrophobic regions of trx and sles are incorporated in the micellar core of investigated binary systems .
the polar heads of sles and trx are probably located at the surface of the aggregates , interacting with the polar side of the bile salts , as presented in figure 3 . for trx - od binary system .
consequently , the variation in synergism between investigated bile salts and selected co - surfactants can be attributed to the structural differences in the polar part of the steroid skeleton .
spatial relation of triton x-100 and steroid skeleton of sodium 7-oxodeoxycholate in the mixed micelles . obviously , the larger number of hydrophilic groups on side of the steroid ring generates stronger synergism , since c and od have one polar group more than dc ( 7-hydroxyl and 7-oxo group , respectively ) .
namely , it is possible that -hydroxyl groups on the steroid skeleton create hydrogen bonds with proton acceptor oe groups of the polar head of trx and sles .
it is known that dc lacks 7-hydroxyl group , thus most likely forms fewer hydrogen bonds .
stronger synergistic effect of od in all investigated binary systems compared to the same surfactant systems with c is probably the consequence of the alteration in the spatial position of the oxygen atom at c7 of the steroid skeleton .
it is known that -axial hydroxyl group at c7 position of the steroid skeleton forms a 90 angle with the median plane of the steroid skeleton ( mpsc ) .
oxidation of this group creates -equatorial oxo group which forms a 30 angle with the median plane of the steroid skeleton ( mpsc ) as seen in figure 4 .
therefore , the oxygen atom of the c7 oxo group is moved for 60 towards the mpsc i.e. towards the side of the steroid ring .
spatial position modification of the oxygen atom at c7 ( newman projection of sodium 7-oxodeoxycholate ) .
we assume that this spatial relation of the oxygen atom enables polar head ( oe groups ) of the co - surfactants to be oriented towards the -axial hydroxyl groups of the od steroid skeleton , since hydrophilic region of the co - surfactants is probably located at the micellar surface .
this spatial orientation of trx is probably achieved through the water molecules , as seen in figure 3 .
we assume that similar positioning is attained with sles molecules , through the dipole interactions between the oe group distal to sulfate ion and the 7-oxo group of od .
according to the results , trx shows stronger synergism with investigated bile salts than sles .
apart from difference in hydrophobic region , this variation can be explained due to the smaller number of oe groups in hydrophilic region of sles , forming fewer attractive interactions with the polar part of selected bile salts .
additionally , negatively charged sulfate ion of sles creates repulsive interactions with carboxylate anion of selected bile salts , decreasing stability of mixed micelles .
the cmc values of individual bile salts ( d , c , and od ) and sles were acquired through conductivity and surface tension studies .
the experimentally obtained cmc values , with standard deviations of three determinations , for individual surfactants ( cmc ) are presented in table 1 and compared to the literature values ( cmc ) [ 33 - 35 ] .
experimentally obtained critical micelle concentrations of the individual surfactants compared to the literature values a the values obtained from conductivity measurements .
cmcthe mean values standard deviation of three determinations cmcthe cmc values obtained from the literature
the critical micelle concentrations of the binary mixtures were studied using conductivity and surface tension measurements , at different mole fractions of trx or sles ( ) .
prepared mixtures consisted of 0.1 , 0.2 , 0.3 , 0.4 or 0.5 mole fractions of trx or sles ( ) . for the conductivity studies , a break in the conductivity ( specific conductivity , ) versus concentration plot indicated the onset of the micellization process ,
since this break is characteristic of micelle formation and represents the cmc ( figure 1 ) .
the cmc values obtained in the conductivity measurements were acquired from the intersection of the straight lines created above and below the cmc .
the straight line above the cmc value was determined by linear regression starting at the highest concentration , while the straight line below the cmc value was obtained by linear regression beginning at the lowest concentration of investigated binary mixtures .
the regression diagnostic was constantly performed after addition of each subsequent experimental point . if an experimental point was identified as an outlier , by measuring cook 's distance , the regression would be stopped and the straight line would be formed using previously added experimental points ( figure 1 ) .
dependence of the specific conductivity on concentration of trx - od ( = 0.5 ) binary system .
the discontinuous line represents the gaussian curve obtained by phillips ' method and the arrow denotes the cmc .
however , this method for the determination of cmc values is reliable only if there is an abrupt change in conductivity . as seen in figure 1
, the change in conductivity does not show an abrupt alteration and the intersection point is not clearly detectable . since the uncertainty in determination of cmc values
is increased , phillips ' method was applied in order to obtain the cmc values more reliably . in this approach ,
the cmc is considered as the concentration at which the maximum change in a physical property of the solution , , versus concentration curve occurs .
( d3dc3)c = 0 ; c = cmc phillips ' method was employed as proposed by mosquera et al .
the runge - kutta integration was applied and a least squares fitting method , according to levenberg - maquardt , was used .
the usage of phillips ' method for determination of cmc values has been reported in previous studies , as well .
figure 1 shows the application of phillips ' method . in order to validate the results determined by means of conductivity measurements
plots of surface tension ( ) versus concentration of binary system were obtained , where a break point corresponds to the cmc .
dependence of surface tension on concentration of trx - od ( = 0.5 ) binary system .
. extended t - test ( p < 0.05 ) shows that the mean cmc values determined using conductivity and surface tension measurements belong to the same population .
the experimentally obtained cmc values , with standard deviations of three determinations , for the trx - bile salt and sles - bile salt binary mixtures are presented in tables 2 and 3 respectively .
comparision of the experimentally obtained critical micelle concentrations of investigated trx - bile salt binary systems a the values obtained from conductivity measurements .
cmcthe mean values standard deviation of three determinations comparision of the experimentally obtained critical micelle concentrations of investigated sles - bile salt binary systems a values obtained from conductivity measurements .
cmcthe mean values standard deviation of three determinations as seen in tables 2 and 3 the cmc values obtained in conductivity and surface tension studies are in good agreement . since there is no statistically significant difference between the cmc values determined by means of the two methods , the cmc values acquired by conductivity measurements were used in further calculations .
in order to study the influence of the structure of bile salts on the formation of mixed micelles with trx or sles , the physico - chemical parameters of the micellar systems were calculated using experimentally obtained cmc values .
the determined physico - chemical parameters are : the critical micelle concentration of ideal mixtures ( cmc ) , the mole fraction of the more hydrophobic surfactant in the ideal mixed micelle ( x ) , the mole fraction of the more hydrophobic surfactant in the real mixed micelle ( x1 ) and the parameter .
the physico - chemical parameters for the trx - bile salt and sles - bile salt mixed micelles were calculated according to the equations presented in table 4 .
physico - chemical parameters of mixed micelles i the mole fraction in the solution of component i , cmci the experimentally obtained cmc of component i. cmc1 the experimentally obtained cmc values of the more hydrophobic surfactant ( trx or sles ) cmc2 the cmc of d , c or od cmcthe experimentally obtained cmc values of investigated binary systems the mole fraction of the more hydrophobic surfactant in the solution ( trx or sles ) comparison of experimental with ideal parameters and interaction parameter ( ) of the trx - bile salt mixtures cmcthe experimentally obtained cmc values of investigated binary systems , each result is the average of three separate determinations .
cmcthe values for the critical micelle concentrations of ideal mixtures , calculated according to clint 's theory of ideal mixtures .
x1 mole fractions of the more hydrophobic surfactant in the real mixed micelles , according to rubingh .
xmole fractions of the more hydrophobic surfactant in the ideal mixed micelles , according to motomura 's model .
comparison of experimental with ideal parameters and interaction parameter ( ) of the sles - bile salt mixtures cmcthe experimentally obtained values , each result is the average of three separate determinations .
cmcthe values for the critical micelle concentrations of ideal mixtures , calculated according to clint 's theory of ideal mixtures .
x1 mole fractions of the more hydrophobic surfactant in the real mixed micelles , according to rubingh .
xmole fractions of the more hydrophobic surfactant in the ideal mixed micelles , according to motomura 's model .
the results show deviation of the cmcvalues from those calculated according to clint 's theory indicating nonideal behaviour of examined mixtures and existence of mutual interactions of the components in the micelles .
the difference between xand x1 also indicates nonideal behaviour of the mixtures . from tables 5 and 6 ,
it can be observed that values of the parameter come out to be negative at all molar ratios of the mixtures .
the more negative value of the interaction parameter indicates stronger synergistic interactions between the components .
these values correspond to the deviation between the experimentaly obtained and calculated cmc values and indicate synergism in all investigated trx - bile salt and sles - bile salt mixtures . in table 7 , the average values of the interaction parameter ( av ) for analysed binary systems are presented in order to make the results easily comparable .
average interaction parameters ( av ) of analysed mixed binary systems the average interaction parameter values show that trx generates stronger sinergistic interactions with investigated bile salts than sles .
many recent studies have confirmed that synergistic effect generally occurs in anionic - nonionic surfactant mixtures , improving stability of the system [ 41 - 48 ] . in previously investigated sodium deoxycholate -
this value is in good agreement with the average value of the interaction parameter reported in our study ( -0.88 ) .
it is known that in mixed miceles composed of two anionic amphihpiles , repulsive electrostatic interactions between anionic heads are important source of destabilisation .
hence , weak synergistic or antagonistic interactions between individual surfactants are frequently reported in anionic - anionic surfactant mixtures .
therefore , weak synergism that has been noticed for anionic - anionic surfactant mixtures in this article is in agreement with previous studies .
the results presented in table 7 show that trx and sles generate the weakest synergistic effect with d , while od creates the strongest interaction with investigated co - surfactants .
we assume that the hydrophobic regions of trx and sles are incorporated in the micellar core of investigated binary systems .
the polar heads of sles and trx are probably located at the surface of the aggregates , interacting with the polar side of the bile salts , as presented in figure 3 . for trx - od binary system .
consequently , the variation in synergism between investigated bile salts and selected co - surfactants can be attributed to the structural differences in the polar part of the steroid skeleton .
spatial relation of triton x-100 and steroid skeleton of sodium 7-oxodeoxycholate in the mixed micelles . obviously , the larger number of hydrophilic groups on side of the steroid ring generates stronger synergism , since c and od have one polar group more than dc ( 7-hydroxyl and 7-oxo group , respectively ) .
namely , it is possible that -hydroxyl groups on the steroid skeleton create hydrogen bonds with proton acceptor oe groups of the polar head of trx and sles .
it is known that dc lacks 7-hydroxyl group , thus most likely forms fewer hydrogen bonds .
stronger synergistic effect of od in all investigated binary systems compared to the same surfactant systems with c is probably the consequence of the alteration in the spatial position of the oxygen atom at c7 of the steroid skeleton .
it is known that -axial hydroxyl group at c7 position of the steroid skeleton forms a 90 angle with the median plane of the steroid skeleton ( mpsc ) .
oxidation of this group creates -equatorial oxo group which forms a 30 angle with the median plane of the steroid skeleton ( mpsc ) as seen in figure 4 .
therefore , the oxygen atom of the c7 oxo group is moved for 60 towards the mpsc i.e. towards the side of the steroid ring .
spatial position modification of the oxygen atom at c7 ( newman projection of sodium 7-oxodeoxycholate ) .
we assume that this spatial relation of the oxygen atom enables polar head ( oe groups ) of the co - surfactants to be oriented towards the -axial hydroxyl groups of the od steroid skeleton , since hydrophilic region of the co - surfactants is probably located at the micellar surface .
this spatial orientation of trx is probably achieved through the water molecules , as seen in figure 3 .
we assume that similar positioning is attained with sles molecules , through the dipole interactions between the oe group distal to sulfate ion and the 7-oxo group of od .
according to the results , trx shows stronger synergism with investigated bile salts than sles .
apart from difference in hydrophobic region , this variation can be explained due to the smaller number of oe groups in hydrophilic region of sles , forming fewer attractive interactions with the polar part of selected bile salts .
additionally , negatively charged sulfate ion of sles creates repulsive interactions with carboxylate anion of selected bile salts , decreasing stability of mixed micelles .
we have studied a set of three bile salts ( sodium cholate , sodium deoxycholate or sodium 7-oxodeoxycholate ) to interact with triton x-100 or sodium lauryl ether sulfate , by conductometric and tensiometric method .
the results of the study have been analysed using clint 's , rubingh 's , and motomura 's theories for mixed binary systems .
the critical micelle concentration of the ideal mixed micelle , the mole fraction of the more hydrophobic surfactant in the ideal mixed micelle , the mole fraction of the more hydrophobic surfactant in the real mixed micelle , and the interaction parameter of the mixed micelles were calculated using experimental data .
based on the calculated values of the parameter we have concluded that trx and sles generate the weakest interactions with d , while od creates the strongest interaction with investigated co - surfactants . most probably , hydrogen bonds between -hydroxyl groups of steroid skeleton and proton acceptor oe groups of the polar head of selected co - surfactants are formed and contribute to the synergism .
since d lacks one polar group compared to c and od , it showed the weakest interactions .
we have also noticed that od generates stronger synergism than c in all investigated binary systems .
this is probably due to attractive interactions between 7-oxo group of od and the oxygen atom of the phenoxy ring of trx through the water molecules .
it is assumed that attractive interaction between 7-keto group of od and of the second oe group distal to sulfate group in sles promotes formation of hydrogen bonds and stabilisation of the binary system .
sodium 7-oxodeoxycholate was synthetized by selective oxidation of the 7-hydroxy group of cholic acid , following the tullar procedure .
all bile acids were transformed to the sodium salt by known procedure .cholic and deoxycholic acids were purchased from sigma - aldrich ( germany ) .
sodium lauryl ether sulfate having two oxyethylen groups was provided by stepan company , usa .
surfactant solutions were prepared by dissolving accurately weighed quantities of surfactants in requisite volumes of deionised water .
deionised water ( conductivity < 1 s cm , at 25c ) was used for all purposes .
the cmc values of pure c , d , od and sles were acquired through conductivity and surface tension measurements .
the cmc value of trx was obtained through surface tension studies only , since this surfactant is nonionic amphiphile and does not have influence on electric conductance .
the critical micelle concentrations of all investigated binary mixtures ( d - trx , c - trx , od - trx , d - sles , c - sles , and od - sles ) were determined by means of conductometry and tensiometry .
conductivity measurements were carried out on aqueous solutions of pure c , d , od and sles .
aqueous solutions of mixtures of c , d or od and one of two co - surfactants ( trx or sles ) were also analysed measuring conductivities .
the cmc values of the binary systems were obtained for the different molar ratios of the components .
the cell containing solutions was immersed in a water bath , controlling the temperature variation at 0.1c .
surface tension measurements were carried out on aqueous solution of trx in order to determine its individual cmc .
surface tension measurements were carried out on a sigma 703d tensiometer ( finland ) using a du nouy ring method .
sodium 7-oxodeoxycholate was synthetized by selective oxidation of the 7-hydroxy group of cholic acid , following the tullar procedure .
all bile acids were transformed to the sodium salt by known procedure .cholic and deoxycholic acids were purchased from sigma - aldrich ( germany ) .
sodium lauryl ether sulfate having two oxyethylen groups was provided by stepan company , usa .
surfactant solutions were prepared by dissolving accurately weighed quantities of surfactants in requisite volumes of deionised water .
deionised water ( conductivity < 1 s cm , at 25c ) was used for all purposes .
the cmc values of pure c , d , od and sles were acquired through conductivity and surface tension measurements .
the cmc value of trx was obtained through surface tension studies only , since this surfactant is nonionic amphiphile and does not have influence on electric conductance .
the critical micelle concentrations of all investigated binary mixtures ( d - trx , c - trx , od - trx , d - sles , c - sles , and od - sles ) were determined by means of conductometry and tensiometry .
conductivity measurements were carried out on aqueous solutions of pure c , d , od and sles .
aqueous solutions of mixtures of c , d or od and one of two co - surfactants ( trx or sles ) were also analysed measuring conductivities .
the cmc values of the binary systems were obtained for the different molar ratios of the components .
the cell containing solutions was immersed in a water bath , controlling the temperature variation at 0.1c .
surface tension measurements were carried out on aqueous solution of trx in order to determine its individual cmc .
surface tension measurements were carried out on a sigma 703d tensiometer ( finland ) using a du nouy ring method .
the cmc values of pure c , d , od and sles were acquired through conductivity and surface tension measurements .
the cmc value of trx was obtained through surface tension studies only , since this surfactant is nonionic amphiphile and does not have influence on electric conductance .
the critical micelle concentrations of all investigated binary mixtures ( d - trx , c - trx , od - trx , d - sles , c - sles , and od - sles ) were determined by means of conductometry and tensiometry .
conductivity measurements were carried out on aqueous solutions of pure c , d , od and sles .
aqueous solutions of mixtures of c , d or od and one of two co - surfactants ( trx or sles ) were also analysed measuring conductivities .
the cmc values of the binary systems were obtained for the different molar ratios of the components .
the cell containing solutions was immersed in a water bath , controlling the temperature variation at 0.1c .
surface tension measurements were carried out on aqueous solution of trx in order to determine its individual cmc .
surface tension measurements were carried out on a sigma 703d tensiometer ( finland ) using a du nouy ring method .
this work was financially supported by the provincial secretariat for science and technological development , ap vojvodina , republic of serbia , grant no.114 - 451 - 2113/2011 - 01 | backgroundin order to develop colloidal drug carriers with desired properties , it is important to determine physico - chemical characteristics of these systems .
bile salt mixed micelles are extensively studied as novel drug delivery systems .
the objective of the present investigation is to develop and characterize mixed micelles of nonionic ( triton x-100 ) or anionic ( sodium lauryl ether sulfate ) surfactant having oxyethylene groups in the polar head and following bile salts : cholate , deoxycholate and 7-oxodeoxycholate.resultsthe micellization behaviour of binary anionic - nonionic and anionic - anionic surfactant mixtures was investigated by conductivity and surface tension measurements .
the results of the study have been analyzed using clint 's , rubingh 's , and motomura 's theories for mixed binary systems .
the negative values of the interaction parameter indicate synergism between micelle building units .
it was noticed that triton x-100 and sodium lauryl ether sulfate generate the weakest synergistic interactions with sodium deoxycholate , while 7-oxodeoxycholate creates the strongest attractive interaction with investigated co-surfactants.conclusionit was concluded that increased synergistic interactions can be attributed to the larger number of hydrophilic groups at side of the bile salts . additionally ,
7-oxo group of 7-oxodeoxycholate enhance attractive interactions with selected co - surfactants more than 7-hydroxyl group of sodium cholate . |
since its introduction , clozapine has been associated with haematological abnormalities , with most common abnormality being leucopenia , and other less commonly reported haematological abnormalities include agranulocytosis , neutropenia , thrombocytopenia , leukocytosis , and thrombocytothaemia .
however , there are few reports of eosinophilia associated with the use of clozapine . in this report
, we present two cases of eosinophilia associated with clozapine and discuss the clinical implications of detection of eosinophilia in patients receiving clozapine .
miss x , a 32-year female , diagnosed with schizoaffective disorder , hypothyroidism , drug induced polycystic ovary disease , and migraine presented with a relapse while on quetiapine 800 mg / day , lithium carbonate 900 mg / day , thyroxine 150 mcg / day , amitriptyline 75 mg / day , and metformin 1 g / day .
her treatment history revealed that she had not responded to three antipsychotic trials in the past and resultantly was considered for clozapine .
preclozapine investigations in the form of heamogram , liver function test , renal function test , fasting blood glucose level , electroencephalogram and electrocardiogram did not reveal any abnormality .
however , lipid profile was deranged for which she was advised dietary modifications , regular physical activities ; metformin was continued in consultation with the endocrinologist .
clozapine was started at the dose of 25 mg / day and her heamogram was monitored .
after about 2 weeks ( 13 day of clozapine ) , while she was on clozapine 150 mg / day , her haemogram showed an eosinophil count of 9% with an absolute eosinophil count of 936/cmm . at this time
her physical examination did not reveal any rash or any other abnormality to suggest any infection or allergic reaction . in view of the increased eosinophil count
, she was investigated further in form of complete blood count , urine microscopic examination and culture , peripheral blood smear for malarial parasite and a chest roentgenogram , all of which were found to be within the normal range except for persistence of increased eosinophil count . her investigations for impending myocarditis in form of troponin - t and creatine kinase ( ck- mb ) also did not reveal any abnormality . in view of the response to clozapine and no symptoms suggestive of any infective or allergic pathology , clozapine
was continued and the dose was gradually increased to 200 mg / day along with an empirical trial of mebendazole .
however , on 26 day of clozapine her eosinophil count increased to 42% with absolute eosinophil count of 6090/cmm along with leucocytosis ( total leucocyte count of 14500/cmm ) .
all other investigations were repeated including evaluation for myocarditis , but no abnormality was noted except for t - wave inversion in leads v1 - 5 .
clozapine was stopped and she was started on haloperidol . following stoppage of clozapine her symptoms again worsened , but her eosinophil count reduced to 18% with absolute eosinophil count of 2,880/cmm . in view of the worsening , after a gap of 2 weeks she was restarted on clozapine and dose was increased to 200 mg / day and her eosinophil count was closely monitored . during the initial 2 weeks of re - challenge
later on the eosinophil count settled down to 25% and she was continued on clozapine .
she is on clozapine for more than a year and have been maintaining well with eosinophil count varying between 25% , with no signs of any myocarditis .
miss z , 46 years , diagnosed with paranoid schizophrenia presented to the inpatient unit with a relapse .
her treatment history revealed that she had not responded to adequate trials of trifluperazine , risperidone , olanzapine , and haloperidol . in view of the treatment resistance ,
preclozapine investigations in the form of haemogram , liver function test , renal function test , fasting blood glucose level , electroencephalogram , and electrocardiogram did not reveal any abnormality .
she was started on clozapine at the dose of 12.5 mg / day and on fourth day while on 37.5 mg / day of clozapine her eosinophil count increased to 7% . however , there was no evidence of any rash , fever , or infective pathology .
her investigations in the form of liver function test , urine examination ( microscopic examination and culture ) , chest roentgenogram , echocardiography , and electrocardiogram did not reveal any abnormality .
she was investigated for ova / cysts in stool , serum immunoglobulin e levels , and antinuclear antibodies to rule out allergic etiology or connective tissue disorder .
she tested negative for ova / cysts and serum ana levels were within normal range but the ige levels were raised to 3300 iu / ml ( n = 30300 iu / ml ) . in consultation with the rheumatologist
in view of the clinical response to clozapine , she was continued on clozapine and the dose of clozapine was increased to 150 mg / day . over the next 5 weeks her eosinophil count varied between 58% .
after this her eosinophil count settled down to 12% and she tolerated 150 mg / day of clozapine with no other haematological abnormality .
the studies which have carried out surveillance of side effects of clozapine have reported an incidence of transient eosinophilia to vary widely from 0.261.7% patients .
eosinophilia whenever develops is usually seen during the initial phase of treatment with clozapine , usually within first 4 weeks of therapy . as with other blood dyscrasia with clozapine , various mechanisms have been proposed to be responsible for clozapine associated eosinophilia .
commonly proposed mechanisms include type - i hypersensitivity reaction , which is supported by evaluated ige levels in few reports and stimulation of t - lymphocytes .
clozapine - associated eosinophilia is understood as two different forms : first transient benign eosinophilia and second eosinophilia with end organ damage .
the eosinophilia has been shown to be associated with myocarditis , pancreatitis , colitis , toxic hepatitis , and pleural effusions .
other reports have also linked eosinophilia to predict neutropenia . with regard to the management of clozapine - associated eosinophilia
, literature suggests that decision of continuation of clozapine is determined by the presence or the absence of other organ damage .
the literature emerging from the case reports suggests that invariably clinicians have opted to stop clozapine in the presence of end organ damage .
however , in the absence of end organ damage the eosinophilia is usually benign and transient .
accordingly , whenever eosinophilia is noticed it is important to evaluate the patient for the presence of any specific organ damage and decision making should take the same into account . in occasional cases , authors have also rechallenged patients with clozapine even after an episode of clozapine and have used it successfully with only few cases of recurrence of eosinophilia along with other organ damage
however , clinicians should give due importance to the eosinophil count while reviewing the hemogram .
both our patients developed eosinophilia during the initial phase of the treatment and further evaluation did not reveal any other organ damage and , hence , clozapine was continued in the second case and in the first case rechallenged with clozapine did not lead to the recurrence of eosinophilia .
our cases add to the existing literature that in the absence of other organ damage clozapine can be continued safely . however , close monitoring should be done . | clozapine , an atypical antipsychotic , has been used in the treatment of schizophrenia and other psychotic disorders .
although it has good therapeutic effect but many a time it use is overridden by the associated adverse effects which range from minor to severe life - threatening events . there has been extensive literature for severe side effects like leukocytosis but limited data are available for transient eosinophilia . here , we present two cases of benign transient eosinophilia and discuss the importance of recognizing eosinophilia while using clozapine . |
lung cancer is the leading type of occupational cancer , with 13% to 30% of the cases attributable to occupational exposures [ 1 , 2 ] .
numerous lung carcinogenic hazards were / are present in the work place , in particular asbestos , a lung carcinogen very frequently found in numerous occupational settings such as construction , transport , isolation , and maintenance . in france ,
nurminen and karjalainen showed in a large review dedicated to occupational attributable fractions that 14% of lung cancer cases were due to asbestos , 4.5% to radon , 2.7% to crystalline silica , and 2.5% to diesel motor exhaust ( dme ) ; figures close to those are found recently by de matteis et al . .
in june 2012 , the international agency for research on cancer evaluated the carcinogenicity of diesel engine exhausts and classified these fumes as
this upgrading was achieved with the results of major cohorts which demonstrated dose response relationship between dme exposure and lung cancer in sectors where coexposure was not the main concern ( i.e. , underground mines ) .
this evaluation , however , is discussed by some authors who question the validity of the results of cohort studies among miners [ 6 , 7 ] .
other studies have also demonstrated an increase in lung cancer risk , although no consistent dose - response relationship was observed while considering occupational exposure to specific lung carcinogens such as asbestos or silica [ 814 ] . on the other hand ,
studies that were adjusted for broad groups of carcinogens ( i.e. , list a jobs or high risk jobs for lung cancer ) did find a significant trend between level / duration of dme exposure and lung cancer [ 15 , 16 ] .
from a large population - based case - control study conducted in france in the 2000s , we investigated the relationship between lung cancer and occupational exposure to dme while considering the smoking habits and occupational exposure to asbestos .
we considered first list a as a proxy of asbestos exposure and then used a specific asbestos job exposure matrix to examine the influence of asbestos assessments on the dose - response relationship .
the icare study is a large multicentre population - based case - control study conducted in france between 2001 and 2007 that has previously been described in detail .
briefly , all lung and upper aerodigestive tract ( uadt ) cancer patients who were between 18 and 75 years of age and who were identified during the study period in each cancer registry were eligible for the study .
the industrial activities in the dpartements ( area of residence ) with registry are representative for industrial activities in france .
the cases were all histologically confirmed as primary lung cancer ( c33-c34 icd - o ) , including all histological types . of
the 3865 eligible men cases identified , 403 could not be located , 653 died before any contact could be made , and 197 could not be contacted because of their health status .
accordingly , 2612 patients were asked to participate , and 336 refused , which led to a refusal rate of 12.9% ( comparable in men and women ) .
population controls were randomly selected by a polling institute from the same dpartements as the cases through incidence - density sampling .
the controls were frequency - matched to the cases by age ( < 50 years , 5060 , 6070 , and over 70 ) , gender , and dpartement .
additional stratification was performed to achieve a distribution by socioeconomic status among the controls comparable to that of the general population in each dpartement . among the 3618 eligible men controls , 208 could not be contacted , and 4 died before any contact could be made . accordingly ,
3406 were asked to participate and 626 refused ( refusal rate : 19% ) . overall , 6481 subjects have been included in the study : 2926 cases and 3555 controls .
we restricted the analysis to men ( 78% of our population , n = 2264 cases and 2780 controls ) because only 104 women declared an exposure to dme .
the study was approved by the institutional review board of the french national institute of health and medical research ( irb - inserm , number 01 - 036 ) .
information was collected on demographics , residential history , education , occupation , lifelong cigarette smoking , and alcohol consumption .
if the subject was unable to answer the complete questionnaire , or if a relative answered for the subject ( a spouse or a child ) , a summary version of the questionnaire was used that primarily included information on lifetime cigarette smoking and lifelong occupational history but not education level .
this short version applied to 5% of the respondents ( n = 201/2264 cases and 60/2780 controls ) .
the lifelong occupational history included the beginning and ending date of each job , the industry , a description of the tasks carried out in the job , the type of machines used , and the exposure to dme .
twenty supplementary questionnaires completed the interview for certain tasks or job titles including jobs in mines / quarries or in vehicle repair .
the occupations and branches of industry were coded blindly with respect to case or control status by trained coders according to the international standard classification of occupations ( isco ) of the international labour organisation , 1968 revision , and according to the french nomenclature of activities ( nomenclature d'activits franaises : naf ) of the national institute for statistics and economic studies ( insee ) , 1999 edition , respectively .
lists of occupations or industries associated with lung cancer either conclusively ( list a ) or with suspicion ( list b ) were first established by ahrens and merletti according to the evaluation of carcinogenic risk conducted by iarc .
these lists have been repeatedly updated and extensively used worldwide as a standardized tool to quantify the burden of occupational lung cancer .
we applied list a to our data using the latest update created by mirabelli et al . and detailed by consonni et al . .
assessment of dme exposure was done by job period with the single question of the general questionnaire during this job period
were you exposed to dme ? allowing us to assess a probability of exposure ( pq ) to dme in three classes ( yes / no / do not know ) .
in addition , for those people who answered no or do not know to this question , but who had declared that they were working in mines or quarries close to a diesel motor or in a garage involved in truck repairing , we created a fourth class of probability defined as possibly exposed ( pq became then 0 for no exposure , 1 for possible exposure , 2 for certain exposure , and missing data for do n't known ) .
we created the variable maximal exposure probability for the lifelong occupational history to classify the study population .
if someone did not know if he had been exposed to dme and had no other job with a certain exposure to dme , the maximal exposure probability was considered as a missing data .
however , for the calculation of exposure to dme duration for the lifelong occupational history , each job with an exposure probability to dme was considered , even if the maximal exposure to dme is missing .
asbestos exposure was assessed using an asbestos specific jem [ 23 , 24 ] . for each job
i , according to the combination of isco and naf codes , the asbestos - jem assigned a probability of exposure ( pi ) , a frequency ( fsi ) , and an intensity ( isi ) of exposure related to specific tasks and a frequency ( fai ) and an intensity ( iai ) of exposure related to work environment contamination .
the probability of exposure was expressed with the percentage of workers exposed in the considered combination isco naf in 5 classes : none or less than 1% of workers exposed in the considered job code ; < 5% ; 530% ; 3070% and > 70% .
the frequency of exposure corresponded to the percentage of working time during which a subject of this combination would be exposed in 4 classes ( < 5% , 530% ; 3070% ; 70% ) .
the intensity was given in fibers / ml in 5 classes ( < 0.01 ; 0.010.1 ; 0.11 ; 110 ; > 10 ) .
assessment of silica exposure had been done in a previous work , using a specific silica jem .
similarly to the asbestos jem , for each job i , according to the combination of isco and naf codes , the silica - jem assigned a probability of exposure ( pi ) , a frequency ( fi ) , and an intensity ( ii ) .
the probability of exposure was expressed with the percentage of workers exposed in the considered combination isco naf in 12 classes : none or less than 1% of workers exposed in the considered job code ; < 5% ; ] 515% ] ; ] 1525% ] ; ; 8595% ; > 95% .
the frequency of exposure corresponded to the percentage of working time during which a subject of this combination would be exposed in 12 classes ( 0 ; ] 15% ] , ] 515% ] ; , 95% ) .
the intensity was given in mg / m in 5 classes 0 ; ] 0.020.1 ] ; ] 0.10.5 ] ; ] 0.51 ] ; >
each subject 's exposure to dme was then summarised by the following exposure indices:(i)the maximum probability of exposure based on questionnaires ( pq),(ii)the duration of exposure corresponding to the sum of the duration of exposed job periods , whatever the probability of exposure to dme is,(iii)the cumulative exposure index ( cei ) corresponding to the product of the classes attributed to the exposure estimated by the questionnaire of each job period ( pq ) with the duration of employment of the period , summed over all job periods of a subject .
this calculation excluded the job periods for which the subjects did not know how to answer .
the cei was categorised into 3 classes ( not exposed , low , high ) according to the 50th percentile among controls corresponding to the following cut - off points 0 and 31 for the cei calculated from the questionnaire 's assessment .
the maximum probability of exposure based on questionnaires ( pq ) , the duration of exposure corresponding to the sum of the duration of exposed job periods , whatever the probability of exposure to dme is , the cumulative exposure index ( cei ) corresponding to the product of the classes attributed to the exposure estimated by the questionnaire of each job period ( pq ) with the duration of employment of the period , summed over all job periods of a subject .
this calculation excluded the job periods for which the subjects did not know how to answer .
the cei was categorised into 3 classes ( not exposed , low , high ) according to the 50th percentile among controls corresponding to the following cut - off points 0 and 31 for the cei calculated from the questionnaire 's assessment .
since asbestos exposure was assessed for adjustment purpose only , we solely calculated an asbestos cumulative exposure index as follows : ceiasbestos = di pi [ ( fsi isi)+(fai iai ) ] , where di is the duration of job i. we assessed two asbestos cumulative exposure indices defined as sensitive for one and specific for the other according to the probability of asbestos exposure taken into account to calculate the cumulative index .
the sensitive assessment considered all jobs with a proportion of exposed workers above 1% ( pi from 1% to > 70% ) .
the more specific definition did not consider the jobs with a proportion of exposed workers equal to or less than 30% ( pi > 30% ) . in practice , to compute the cei we assigned a value to the probability corresponding to the middle of the range of the class , that is , for the sensitive definition : nonexposed = 0 ; < 5% = 0.025 ; 530% = 0.175 ; 3070% = 0.5 ; > 70% = 0.85 . the specific definition assigned a value equal to 0 for all jobs with a probability 30% and the same values as in the sensitive assessment for the other classes .
similarly the values assigned to the intensity and the frequency of exposure corresponded to the middle of the range of the class .
in addition , when the exposure was related to an indirect exposure instead of a specific task done by the worker himself , the values were divided by 2 .
the cumulative index for silica exposure was calculated similarly ( i.e. , silica = di pi fi ii ) and then categorized into four classes according to the distribution among controls .
unconditional logistic regression was used to estimate odds ratios ( or ) and 95% confidence intervals ( 95% ci ) for association between lung cancer and different diesel exhaust exposure indices .
lifelong cigarette smoking was captured by the cumulative smoking index ( csi ) ( csi = ( 1 0.5/t models were adjusted for age at interview ( < 50 years , [ 50 - 60 [ , [ 60 - 70 [ , and 70 ) , the dpartement , the csi and the total number of working periods ( <3 jobs , ] 3 - 4 ] , ] 4 - 6 ] and > 6 jobs , according to the quartiles in the controls ) and either employment in a list a job ( yes , if the subject had held at least one job included in this list /no , if not ) , asbestos cei in four classes with cut - off point at 0.27 and 14 or 2.06 and 28 according to the sensitive or specific asbestos assessment , respectively , or silica cei , in 4 classes with cut - off point at 0.048 and 0.495 .
adjustment for list a or for asbestos or silica exposure was never run in the same models .
modification effect between asbestos exposure or smoking status and dme in the risk of lung cancer has been tested with logistic regression by likelihood ratio method .
the analysis was first conducted on the entire male population and was subsequently restricted to men who were never exposed to asbestos . population attributable fraction ( paf ) associated with dme exposure and the corresponding 95% ci were estimated , using the proportion of people definitely exposed to dme among asbestos never exposed subgroup and the or adjusted for age , department , lifelong cigarette smoking , and number of jobs [ 27 , 28 ] .
a total of 5044 men ( 2264 cases and 2780 controls ) who reported their occupational histories were included in this analysis .
the mean age at diagnosis was 60 for the cases and 58 for the controls ( p < 10 ) .
we observed a clear increase in the risk of lung cancer with the cumulative smoking level assessed by the csi ( p trend < 10 ) .
the two main histological types were squamous cell carcinoma ( n = 803 , 35.2% ) and adenocarcinoma ( n = 794 , 34.8% ) .
having held at least one job period with a potential exposure to a carcinogenic agent ( i.e. , list a = yes ) is associated with a higher risk of lung cancer ( or = 1.8 ( 95% ci 1.52.1 ) ) ( table 2 ) .
in addition , results demonstrate a clear dose effect relationship with asbestos exposure , whatever the definition of asbestos assessment , sensitive or specific . in both cases ,
the p value of the trend is highly significant , and the increased risk is manifest from the lowest cumulative exposure class to the highest ( or = 1.5 ( 95% ci 1.21.7 ) or or = 1.2 ( 95% ci 1.01.5 ) for the sensitive or specific asbestos assessment to 2.3 ( 95% ci 1.73.0 ) or 1.7 ( 95% ci 1.22.3 ) , resp . ) . in this study ,
the occupational exposure to dme concerned the period of time between 1944 and 2006 , when data collection ended .
table 3 presents the results of the association between dme exposure and lung cancer assessed with the maximum probability of exposure from questionnaire ( pq ) .
thirty - five percent of the cases and 29% of the controls had declared at least one job period with a dme exposure .
we observed an increased risk of lung cancer for the subjects who declared to be exposed to dme ( or = 1.3 ( 95% ci 1.11.6 ) ) .
the more the cumulative exposure index increases , the more the risk of lung cancer increases ( or = 1.4 ( 95% ci 1.11.6 ) for the highest iec ) .
these results were first adjusted for having held at least one job belonging to list a. no effect modification between asbestos exposure assessed in a sensitive way and dme in the risk of lung cancer could be detected ( p = 0.66 ) . as can be seen in table 3 ,
the adjustment for asbestos cumulative exposure slightly reduced the associations , especially when asbestos was assessed in a sensitive way ( or3 ) .
results were rather similar among nonsmokers ( orpqdefinite exposure = 1.37 ( 95% ci 0.672.7 ) ) , ex - smokers ( orpqdefinite exposure = 1.27 ( 95% ci 0.981.64 ) ) , and among current smokers ( orpqdefinite exposure = 1.46 ( 95% ci 1.11.9 ) ) , and no modification effect could be detected ( p = 0.35 ) ( data not shown ) .
we repeated the analysis among subjects never exposed to asbestos , assessed in a sensitive way ( ceiasbestos equals 0 ) ( table 4 ) .
this subgroup consisted of 638 cases and 1147 controls , corresponding to 511 jobs exposed to dme .
the description of the main tasks of the job ( free text ) reported that it was often jobs with many hours of driving , for example , 16% of transport equipment operators , 11% of farmers , 6% of agriculture and animal husbandry workers , 6% technical salesmen , commercial travellers , and manufacturers ' agents , 5% salesmen , shop assistants , and related workers ) .
interestingly , all associations studied ( maximum of probability pq , duration of exposure ( or > 15 years ) , and cumulative exposure index ) showed significant dose - response relationships . the additional adjustments with list a or silica iec showed an increased significant risk of lung cancer .
we estimated a paf of lung cancer to dme of 7.0% ( 95% ci 1.312.4 ) based on an or of 1.46 ( corresponding to the association obtained among subjects never exposed to asbestos , adjusted for age , department , lifelong cigarette smoking , and number of jobs ) and a prevalence of exposure of 17% among controls and 22% among cases .
considering the whole population , with an or adjusted for asbestos exposure ( specific definition ) of 1.26 ( 95% ci 1.051.51 ) we estimated a paf of 7.2% ( 95% ci 1.812.3 ) .
with this study we were able to find a clear dose - response relationship between cumulative dme exposure and lung cancer risk among subjects who were never exposed to asbestos . because in our population some occupations were exposed to both dme and asbestos , adjusting for asbestos decreased the associations highlighted , predominantly in occupations with a high exposure to dme , particularly when asbestos exposure was assessed in a sensitive way .
icare is a large population - based case - control study that was designed to investigate the role of occupational exposure in the risk of lung cancer .
cases and controls were stratified by sex and age using a single control group for both types of cancer ( lung and upper aerodigestive tract cancers ) .
collaboration with the french network of cancer registries allowed us to recruit lung cancer cases in almost all of the healthcare establishments in the dpartements covered by the registries .
overall , less than 20% of subjects refused to participate in the study , and this percentage was similar in cases and controls .
furthermore , the 10 dpartements included in the study covered a large fraction of the french population ( 13% ) giving a broad view of the different situations of dme exposure .
therefore , our results may be extrapolated to the overall french population and to similar industrialised western countries . in this context
, we found that 7% of lung cancer cases in men could be attributed to dme , thereby accounting for approximately 1975 persons each year in france .
this is almost three times higher than what was observed in the nurminen and karjalainen study , where there was an attributable fraction of 2.5% .
the retrospective assessment of exposure in case - control studies is always a matter of debate .
our questionnaire was specifically designed to assess occupational exposure to a large variety of carcinogens present in the workplace .
diesel exposure is relatively easy to assess , as it comes from classical and typical machines that operate with diesel engine .
in addition , no specific jem had been developed at the department of occupational health of the institut de veille sanitaire
29% of the controls reported at least one job with dme exposure , a number similar to that reported in an italian population and slightly more than the frequency observed in canada ( 14% ) [ 11 , 13 ] .
because this exposure assessment was self - reported by the subjects , it is not possible to exclude completely differential misclassification bias in our results .
nevertheless , in order to minimize it , we presented the study to the subjects as a study aimed at investigating the relation between environmental exposures and health .
in addition , data collection was set up between 2002 and 2007 , before classification of dme in group 1 of carcinogens for lung .
the csi is a smoking index that takes into account the 3 main smoking parameters in the risk of lung cancer ( intensity , duration , and time since cessation ) .
the very low number of cases of never smokers exposed to dme ( n = 14 ) makes unlikely a noncontrolled effect for passive smoking in our results .
our results are in accordance with the two major meta - analyses [ 9 , 29 ] published about a decade ago and more recently with the large pooled analysis from olsson et al . .
our results do show a clear impact of the adjustment for previous asbestos exposure on the crude findings .
asbestos exposure assessment was made in two ways ( i ) using list a as a proxy of occupational exposures including asbestos and ( ii ) using an asbestos specific jem developed at the national institute for health surveillance and already applied in one mesothelioma case - control study .
then , to explore to what extent asbestos could impact the crude relation initially obtained , we considered a sensitive definition to calculate the cumulative index ( taking into consideration all jobs titles with a proportion of exposed workers different from zero ) and a more specific one for which the jobs with a proportion of exposed workers less than 30% were not taken into account in the calculation of cumulative index .
first of all , as can be seen in table 3 , the two definitions of asbestos , sensitive and specific , are both related to an increase risk of lung cancer in a dose dependant way , although the more specific definition is slightly less steep .
we also note that subjects with a low cumulative index were in both cases ( sensitive or specific ) at significant increase risk of lung cancer .
the very minor changes between crude and adjusted results with list a indicate that this list underestimates asbestos exposure and is not a way to accurately take asbestos into account .
although list a is a very useful tool to consider carcinogenic occupational exposures in a whole in investigations not specifically targeted to an occupational agent , it seems that this list is too global to take into account as precisely as possible a particular agent , such as asbestos , very frequently found in different workplaces in particular relevant periods for our cases and controls who were mainly active before the asbestos ban in france .
our results with asbestos exposure adjustment show that workers exposed to diesel fumes may also have been exposed to asbestos .
these jobs belong to the two following isco-68 groups : 8 - 4 ( group including assembler fitters , machine installers , and precision mechanics and a group consisting notably of agricultural , automobile , or truck mechanics , and in particular in that group 8.43 : mechanic of motor vehicles and 8.44 : mechanics of engines of plane ) for 69% of them and 9 - 7 ( group including material - handling and earth - moving machinery drivers , dockers , and freight handlers ) and in particular 9.71 : dockers , 9.72 : riggers , 9.73 : driver of overhead crane , and 9.74.60 : driver of bitumen and tarring machines for the remaining .
adjustment for asbestos exposure decreases the crude findings , more specifically for the subjects who are classified in the highest class of the diesel cei , to an extent that depends on the sensitivity or the specificity of asbestos assessment .
this result is in coherence with the fact that when restricting the analysis to subjects not exposed to asbestos we find almost no subjects with high diesel exposure .
our group of subjects never exposed to asbestos allowed us to bypass the difficulty to adjust for concomitant exposures and confirm that the relation between diesel fumes exposure and the risk of lung cancer is independent of asbestos exposure .
silica exposure is another carcinogen very frequently found in working places and in particular in places where asbestos or dme is also found ( e.g. , construction sites ) . in order to investigate to what extent our results among subjects never exposed to asbestos
were not due to silica exposure , we adjusted for this carcinogen and found similar results .
finally , excluding subjects never exposed to asbestos could also modify the distribution of cases and controls according to socioeconomical status .
we thus further excluded from this group subjects with high school or university degree and found again a consistent ( even if not significant ) relationship between dme exposure and lung cancer that was likely not due to silica exposure or asbestos .
these results suggest that adjustment for asbestos exposure decreases the association between dme exposure and lung cancer that could explain why the studies that have specifically considered asbestos have in most cases failed to find a significant dose / duration respond relationship .
overall , our study highlights dme exposure as a risk factor of lung cancer , a result that is not due to smoking or to asbestos exposure .
efforts should be made to reduce dme emissions to decrease the occurrence of lung cancer among the workers exposed to these fumes , even if the composition of dme has evolved over time .
our results highlight that , because dme and asbestos exposure are closely related in the occupational histories of the subjects , analysis by exposure subgroups must be considered
. this consideration could also be applicable to other occupational exposures and emphasizes the importance of initiating large - scale studies . | background . in a french large population - based case - control study we investigated the dose - response relationship between lung cancer and occupational exposure to diesel motor exhaust ( dme ) , taking into account asbestos exposure .
methods . exposure to dme was assessed by questionnaire .
asbestos was taken into account through a global indicator of exposure to occupational carcinogens or by a specific jem .
results .
we found a crude dose response relationship with most of the indicators of dme exposure , including with the cumulative exposure index .
all results were affected by adjustment for asbestos exposure .
the dose response relationships between dme and lung cancer were observed among subjects never exposed to asbestos .
conclusions .
exposure to dme and to asbestos is frequently found among the same subjects , which may explain why dose - response relationships in previous studies that adjusted for asbestos exposure were inconsistent . |
supplementary figure 1 . regional assembly supplementary table 1 . bacterial data sets used to test polymorphism discovery with vaal supplementary table 2
. nature of called , uncalled polymorphisms of m. tuberculosis f11 vs h37rv supplementary table 3 .
effect of depth of sequence coverage on discovery of polymorphisms in m. tuberculosis f11 vs h37rv supplementary table 4 .
effect of depth of sequence coverage on discovery of polymorphisms in m. tuberculosis f11 vs h37rv , using vaal and velvet + vaal steps 3,4 supplementary table 8 .
vibrio cholerae : observed differences between resistant isolates and their sensitive parents , found using velvet ( + vaal steps 3,4 ) supplementary methods supplementary results supplementary references supplementary resources | our variant ascertainment algorithm vaal uses massively parallel dna sequence data to identify differences between bacterial genomes at high sensitivity and specificity .
vaal found ~98% of differences ( including large indels ) between pairs of strains from three species while calling no false positives .
further , vaal pinpointed a single mutation between vibrio genomes , identifying an antibiotic s site of action by finding the difference(s ) between a drug sensitive strain and a resistant derivative . |
renal cell carcinoma ( rcc ) has a propensity to metastasize along the hematogenous route .
about 20% to 25% of patients with rcc have distant metastases at presentation,1 and another 50% develop distant metastases or local recurrence after nephrectomy.2 metastasis can occur at any time , and the most frequent sites include the lungs , bones , liver , and brain .
however , rccs can also metastasize to unusual sites , including the pancreas , thyroid , adrenal gland , skeletal muscle , and skin.3 clinical courses vary among patients with rcc who initially present with metastatic disease or who develop tumor dissemination during follow - up .
rcc metastases to the stomach , including solitary synchronous gastric metastases from small , localized rccs , are extremely rare.4 we here describe a patient with a solitary synchronous metastatic gastric cancer originating from a small rcc who was treated with endoscopic resection .
we also review the clinical characteristics of , therapeutic modalities for , and clinical outcomes of , patients with this disease .
he had previously undergone surgery for a duodenal ulcer as well as an open cholecystectomy .
, we did not detect abdominal tenderness or a mass , and laboratory tests revealed no abnormal findings .
upper endoscopy showed an erosive lesion , approximately 0.6 cm in length , in the anterior wall of the midbody ( fig .
a biopsy of the lesion showed tumor cells , consistent with metastatic rcc of the clear cell type . computed tomography ( ct ) showed a mass , approximately 5 cm long , in the right kidney , but there was no evidence of lymph node enlargement or metastatic lesions in the abdomen ( fig .
positron emission tomography showed an isometabolic mass ( maximum standardized uptake value [ maxsuv ] , 2.7 ) in the right kidney ( fig .
the resected specimen showed a well - defined lobulated mass , 5.23.83.5 cm in size , in the lower pole of the right kidney .
the tumor cells had clear cytoplasm and round monotonous nuclei , both typical of clear cell carcinoma ( fig .
the tumor was confined to the renal parenchyma , and no lymphovascular or renal vein invasion was apparent .
microscopic examination of the resected specimen showed an ill - defined tumor , about 0.6 cm long , with clear resection margins , a finding consistent with metastatic rcc ( fig .
upper endoscopy and a ct scan performed 6 months later showed no evidence of local recurrence or additional metastasis .
although metastatic gastric cancers are uncommon , those observed are frequently metastases of lung cancer , breast cancer , and malignant melanoma.5 - 8 the clinical symptoms most commonly requiring endoscopy of patients with gastric metastasis include anemia , gastrointestinal bleeding , dyspepsia , and epigastric pain .
metastases are most common in the gastric body , and are more likely to be single rather than multiple .
grossly , such lesions can resemble submucosal tumors , with or without central depressions , or primary gastric cancers.8 the outcome of patients with metastatic gastric cancer is generally poor , because concomitant metastases to other organs are common.5 metastasis to the stomach from rcc is extremely rare,8 although we identified as many as 36 such patients ( including the patient described here , but excluding those identified on autopsy ) by systematic review of articles in english using a computerized search of the pubmed database ( table 1).1,9 - 37 we found that gastric metastases from rcc were more common in males than in females ( 26 males vs 9 females ) .
median age at presentation was 67 years ( range , 48 to 84 years ) . among the most frequent clinical symptoms were upper gastrointestinal bleeding , anemia , and epigastric pain .
the most frequent location of metastatic lesions was the gastric body , followed by the fundus and the antrum .
the average period from rcc surgery to the presentation of gastric metastasis was approximately 6.5 years ( range , 0 to 24 years ) .
twenty - one patients showed disseminated tumor spread to other organs , most frequently to the lungs .
two patients presented with metastatic gastric cancer arising from rcc before detection of a renal mass ; one patient was diagnosed with advanced rcc with metastatic gastric cancer as well as lung and bone metastases,32 and the second patient , described here , had a single gastric metastasis arising from a small rcc without evidence of additional metastases .
fourteen previously reported patients underwent surgical resection ( total / partial gastrectomy or wedge resection ) ; 3 ( including the patient described here ) received endoscopic resection / excision ; 7 were treated with systemic therapy including interferon , tamoxifen , sunitinib malate , or chemotherapy ; 6 received palliative embolization or endoscopic ablation for control of bleeding ; whereas 7 received no specific therapy .
the clinical course in patients with rcc and metastasis to the stomach appears to be unpredictable .
generally , the outcome of patients with rcc and gastric metastases is poor.5 some patients die within a few weeks , whereas six ( including ours ) showed no tumor recurrence at a median of 17 months ( range , 6 to 36 months ) after metastatectomy.20 - 22,28,35 of 10 patients with solitary gastric metastases , 6 underwent surgical metastatectomy , 2 underwent endoscopic resection ( snare polypectomy and endoscopic submucosal dissection ) , 1 was treated with sunitinib malate , and 1 received no therapy .
five patients showed no evidence of tumor recurrence at a median of 12 months ( range , 6 to 18 months ) after metastatectomy , and one patient lived for 7 years after diagnosis .
although the optimal treatment for patients with solitary gastric metastasis arising from rcc remains unclear , patients with potentially surgically resectable primary rcc and a solitary resectable metastatic cancer are candidates for nephrectomy and metastatectomy.38 patients in generally good condition showed prolonged survival after nephrectomy and complete resection of the metastatic lesion.4,38 - 40 similarly , complete metastatectomy may offer survival benefits in patients with solitary gastric metastases arising from rcc.21,28 the optimal treatment modality in a patient with a small , solitary gastric metastasis confined to the mucosa and submucosa may be endoscopic resection . | metastasis to the stomach from renal cell carcinoma ( rcc ) is extremely rare .
usually , gastric metastasis seems to be a late event in patients with rcc and is accompanied by disseminated tumor spread to other organs .
solitary synchronous gastric metastasis from small , localized rcc has rarely been reported .
we report a case of 79-year - old man with synchronous gastric metastasis presenting with a single erosive lesion from pt1 rcc .
the patient underwent radical nephrectomy and endoscopic resection for metastatic gastric cancer .
the resected specimen showed an ill - defined tumor , approximately 0.6 cm long , with a clear resection margin .
the morphologic features of the tumor cells were consistent with those of metastatic rcc of the clear cell type . at 6 months 's follow - up
, the patient did not show local recurrence or additional metastasis on upper endoscopy and computed tomography scan . |
cystic fibrosis ( cf ) is an autosomal recessive disease caused by defect in the cystic fibrosis transmembrane conductance regulator ( cftr ) gene .
the protein product of the gene is a chloride channel and a member of the atp - binding cassette membrane transporter superfamily .
the cftr channel is critical for the normal function of epithelial cells in the lungs , pancreas , intestine , gall bladder , and sweat glands .
excess mucus in the respiratory system of cf patients facilitates chronic bacterial infections , leading to respiratory failure , which is the major cause of mortality .
most of the patients also fail to produce digestive enzymes in the pancreas , resulting in pancreatic insufficiency .
approximately one in 2000 to 3000 newborns in populations of european ancestry are affected , and the average carrier frequency is about 1:25[2 , 3 ] .
the p.f508del ( f508 ) allele of the gene is the most common mutation observed worldwide and is probably very old , dating to pre - neolithic times[2 , 4 ] .
in addition to the f508 mutation , more than 1000 other mutations in the cftr gene have been identified ( cf genetic analysis consortium http://www.genet.sickkids .
these mutations vary greatly in their frequency and distribution , but most are very rare . only four ( p.g542x , p.n1303k , p.g551d and p.w1282x ) have overall frequencies higher than 1% . intriguingly , p.g542x and p.n1303k are found on the same haplotype background as f508 , suggesting that they arose in the same population .
few previous reports of cftr mutations in iran have been published[79 ] . in the present study , 5 of the 27 cftr exons of 30 unrelated northern iranian cf patients
in addition , the clinical presentations and laboratory findings were studied based on hospital and outpatient records .
we selected five mutations , deltaf508 , n1303k , g542x , r347h and w1282x based on previous reports in iran and neighboring countries .
the knowledge of the mutation spectrum and its frequency in this population will improve the diagnosis and medical care of cf patients and could also be used as a basis for carrier screening and prenatal diagnosis programming in iran .
several techniques such as allele specific oligonucleotide ( aso ) dot - blot , reverse dot - blot , amplification refractory mutation ( arms ) , and an oligo - ligation assay , are available to detect the most common mutations .
the reverse dot - blot ( rdb ) technique is one of the most widely used techniques to diagnose cf[1114 ] .
this technique was evaluated as routine first - line analyses of the cftr gene status[12 , 15 ] .
rdb is more advantageous than other techniques since it does not necessitate the use of radiolabled probes as in allele specific oligonucleotide assay and the use of positive controls to show the proper functioning of pcr reaction in each tube as in arms assays , since in arms technique , results are based on the absence of pcr products . in this study
the 30 patients were diagnosed as cystic fibrosis based on elevated sweat chloride values ( > 60 meq / l ) and were 6 weeks to 11 years old .
the patients were recruited from a pediatric hospital ( amirkola children 's hospital ) in mazandaran province , northern iran .
families of the patients were informed of the nature of the research and consented to participate in this study .
mutation detection was performed using reverse dot blot ( rdb ) procedure , as described by lappin et al . in this assay
, oligonucleotide probes were synthesized using a c6-amino - linker on the 5 ' end of the product and were attached onto biodyne c nylon membrane ( pall corporation ) activated by 1-ethyl 3-dimethyl aminopropyl carbodiimide ( edac ) .
the target dna was amplified with 5 ' biotinylated primers and hybridized to immobilized oligonucleotides on the membrane .
hybridization was detected by adding streptavidin - horseradish peroxidase to the membrane where it bound to biotinylated dna that was hybridized to the oligonucleotides . a positive signal
cftr amplifications took place in 50-l reactions containing 1x pcr buffer ( 10 mmol / l kcl , 10 mmol / l tris - hcl , ph 8.3 , 1.5 mmol / l mgcl2 ) and 6 mmol / l mgcl2 , 200 mol / l each of dntps , 10 pmol of each primer , and 1.5 units of taq dna polymerase . all pcr reagents ( except primers which were from mwg , germany ) were from roche company .
thermo - cycling conditions were uniform for all reactions and included an initial denaturation step at 95c for 3 minutes , followed by 38 cycles of denaturation at 95c for 30 seconds , annealing at 54c for 30 seconds and polymerization at 72c for 30 seconds .
pcr products were visualized after electrophoresis on 1.5% agarose le gel under 302 nm uv lamp .
the hybridization reaction took place after adding the pooled pcr products to biodyne membranes pre - wet with hybridization buffer [ 2x saline sodium citrate ( ssc ) , 0.1% sodium dodecyl sulfate ( sds ) ] at 42c , the amplicons and membranes were boiled and permitted to hybridize for 1 hour at 42c .
hybridization solutions were poured off and membranes were washed twice in excess of wash buffer ( 0.75 _ ssc , 0.1% sds ) .
membranes were then agitated in a freshly prepared conjugate solution ( 1:4000 dilution of streptavidin - horseradish peroxidase conjugate , ( boehringer mannheim , part no .
1089153 ) , in 0.5x ssc , 0.1% sds for 30 minutes at room temperature .
at the end of the incubation , membranes were washed three times for 3 minutes each with 0.5x ssc , 0.1% sds ; then twice for three minutes each with 0.1 mol / l sodium citrate , ph 5.0 .
the membranes were then exposed to 0.01% w / v tetramethylbenzidine dihydro - chloride ( tmb ) substrate ( sigma t-8768 ) in a freshly prepared , dilute solution of hydrogen peroxide ( 0.00225% v / v ) in 0.1 m sodium citrate , ph 5.0 with agitation for 10 to 20 minutes .
the details of probes and primers ( mwg biotech , germany ) used in this study are presented in tables 1 and 2 respectively and were described previously by lappin et al .
the 30 patients were diagnosed as cystic fibrosis based on elevated sweat chloride values ( > 60 meq / l ) and were 6 weeks to 11 years old .
the patients were recruited from a pediatric hospital ( amirkola children 's hospital ) in mazandaran province , northern iran .
families of the patients were informed of the nature of the research and consented to participate in this study .
mutation detection was performed using reverse dot blot ( rdb ) procedure , as described by lappin et al . in this assay
, oligonucleotide probes were synthesized using a c6-amino - linker on the 5 ' end of the product and were attached onto biodyne c nylon membrane ( pall corporation ) activated by 1-ethyl 3-dimethyl aminopropyl carbodiimide ( edac ) .
the target dna was amplified with 5 ' biotinylated primers and hybridized to immobilized oligonucleotides on the membrane .
hybridization was detected by adding streptavidin - horseradish peroxidase to the membrane where it bound to biotinylated dna that was hybridized to the oligonucleotides . a positive signal
cftr amplifications took place in 50-l reactions containing 1x pcr buffer ( 10 mmol / l kcl , 10 mmol / l tris - hcl , ph 8.3 , 1.5 mmol / l mgcl2 ) and 6 mmol / l mgcl2 , 200 mol / l each of dntps , 10 pmol of each primer , and 1.5 units of taq dna polymerase . all pcr reagents ( except primers which were from mwg , germany ) were from roche company .
thermo - cycling conditions were uniform for all reactions and included an initial denaturation step at 95c for 3 minutes , followed by 38 cycles of denaturation at 95c for 30 seconds , annealing at 54c for 30 seconds and polymerization at 72c for 30 seconds .
pcr products were visualized after electrophoresis on 1.5% agarose le gel under 302 nm uv lamp .
the hybridization reaction took place after adding the pooled pcr products to biodyne membranes pre - wet with hybridization buffer [ 2x saline sodium citrate ( ssc ) , 0.1% sodium dodecyl sulfate ( sds ) ] at 42c , the amplicons and membranes were boiled and permitted to hybridize for 1 hour at 42c .
hybridization solutions were poured off and membranes were washed twice in excess of wash buffer ( 0.75 _ ssc , 0.1% sds ) .
membranes were then agitated in a freshly prepared conjugate solution ( 1:4000 dilution of streptavidin - horseradish peroxidase conjugate , ( boehringer mannheim , part no .
1089153 ) , in 0.5x ssc , 0.1% sds for 30 minutes at room temperature .
at the end of the incubation , membranes were washed three times for 3 minutes each with 0.5x ssc , 0.1% sds ; then twice for three minutes each with 0.1 mol / l sodium citrate , ph 5.0 .
the membranes were then exposed to 0.01% w / v tetramethylbenzidine dihydro - chloride ( tmb ) substrate ( sigma t-8768 ) in a freshly prepared , dilute solution of hydrogen peroxide ( 0.00225% v / v ) in 0.1 m sodium citrate , ph 5.0 with agitation for 10 to 20 minutes .
the details of probes and primers ( mwg biotech , germany ) used in this study are presented in tables 1 and 2 respectively and were described previously by lappin et al .
30 unrelated patients ( 17 males and 13 females ) aged 6 weeks to 11 years originating from mazandaran province , iran were analyzed in this study .
half of the patients were issued from consanguineous marriage ( mostly between first cousins ) .
other manifestations such as meconium ileus , intestinal obstruction , electrolyte abnormality , rectal prolapse , nasal polyps , sinus disease , diabetes mellitus , liver or gallbladder problems were observed in a small number of patients .
phenotype of subjects carrying cftr mutations * other : acrodermatitis like rash , vitamin deficiency states , hypoproteinemic edema , hypoprothrombinemia with bleeding , meconium plug syndrome , diabetes mellitus mutation screening of the cftr gene in 60 alleles by reverse dot blot hybridization for five common mutations showed that 13 ( 21.6% ) alleles were f508 .
six patients were homozygous for f508 and one patient was compound heterozygous for whom only one allele was specified .
the other four mutations tested ( n1303k , g542x , r 347h and w1282x ) were not encountered in these patients .
table 4 shows phenotypes of 7 patients carrying deltaf508 mutation . all had respiratory difficulties and failure to thrive .
other manifestations such as edema and hypoproteinemia , liver and gallbladder problems , rectal prolapse , or diabetes mellitus were encountered in a lower incidence ( one or two patients ) .
30 unrelated patients ( 17 males and 13 females ) aged 6 weeks to 11 years originating from mazandaran province , iran were analyzed in this study .
half of the patients were issued from consanguineous marriage ( mostly between first cousins ) .
other manifestations such as meconium ileus , intestinal obstruction , electrolyte abnormality , rectal prolapse , nasal polyps , sinus disease , diabetes mellitus , liver or gallbladder problems were observed in a small number of patients .
phenotype of subjects carrying cftr mutations * other : acrodermatitis like rash , vitamin deficiency states , hypoproteinemic edema , hypoprothrombinemia with bleeding , meconium plug syndrome , diabetes mellitus
mutation screening of the cftr gene in 60 alleles by reverse dot blot hybridization for five common mutations showed that 13 ( 21.6% ) alleles were f508 .
six patients were homozygous for f508 and one patient was compound heterozygous for whom only one allele was specified .
the other four mutations tested ( n1303k , g542x , r 347h and w1282x ) were not encountered in these patients .
other manifestations such as edema and hypoproteinemia , liver and gallbladder problems , rectal prolapse , or diabetes mellitus were encountered in a lower incidence ( one or two patients ) .
mazandaran cf patients were underrepresented ( 2 out of 69 patients ) or not included in those studies , and regarding the high genetic heterogeneity observed among different provinces of iran in other disease causing genes[1719 ] and also considerable heterogeneity in the nature of cftr mutations observed among iranian cystic fibrosis patients[79 ] , the molecular characterization of cf in mazandaran province seems a necessity . using reverse dot blot method , 5 common cftr mutations were analyzed in this study in 30 cf patients ( 13 males and 17 females ) aged 6 weeks to 11 years , originating from mazandaran province and presenting acute or persistent respiratory symptoms and failure to thrive .
this method is a simple , rapid and reliable method which allows simultaneous detection of up to 20 different mutations in a single hybridization assay .
delta f508 mutation was present in 13 ( 21.6% ) alleles tested by rdb assay .
six patients were homozygous and one was compound heterozygote for this mutation . the other four mutations tested : n1303k , g542x
these mutations were previously reported in most parts of the world[21 , 22 ] including neighboring countries[2328 ] and also other provinces of iran[79 ] .
only one disease - causing mutation was identified in this study . although the frequency of the f508 among mazandarani patients was lower ( 21.6% ) than its frequency in european countries , it was still the most frequent mutation among those reported to date in iran .
previous studies representing overall iranian cf patients reported a frequency of 16 - 17.8% for this mutation .
this difference could be due to the small number of mazandarani patients in this study and suggests heterogeneity of iranian provinces regarding cf mutations . moreover , its frequency is comparable with those reported in several countries in west asia , north africa and indian subcontinent ( algeria 16.7% , tunisia 18% , turkey 24 - 27% , saudi arabia 13% , pakistan 17% , india 19 - 27%[6 , 20 , 21 , 24 , 2630 ] .
previous studies showed that the mutation spectrum in iran had a significant overlap with that observed in turkey and was less similar to its spectrum observed in europe .
specifically , the four most common turkish mutations were found in iran , including f508 , c.1677delta , p.g542x , and c.2183aa > g . the " mediterranean mutation " , p.g542x ,
table 5 shows the comparison of the frequency of all five mutations considered in this study in iran and some neighboring countries .
comparison of the frequency of common cftr mutations f508 , n1303k , g542x , r347h , w1282x in europe and north africa with iran and some neighboring countries regarding the sample size in this study , underestimation of the other four mutations analyzed may be possible due to their low frequency .
all patients presented acute or persistent respiratory symptoms and more than 80% had failure to thrive , malnutrition and abnormal stools .
only 23.3% of patients ( 7 cases ) had meconium ileus and intestinal obstruction among which 4 had homozygote f508 genotype .
the high prevalence of meconium ileus among f508 patients was also reported in other studies[3233 ] .
liver disorders which are present in 2 - 3% of children and 5% of adults occur in 25% of cf patients . in this study 4
frequency of carriers of a disease - causing cftr mutation in iranians ( 1:40 ) is very similar to that in european populations ( 1:25 ) .
consequently , it is believed that the incidence of cystic fibrosis is also similarly high , and that the low incidence commonly believed to be associated cystic fibrosis is also similarly high , and that the low incidence commonly believed to be associated with this non - european population is likely to be due to under - diagnosis .
moreover , intermediate sweat chloride levels may exist in some cf patients , leading to misdiagnosis of these patients if the selection criteria is based on sweat chloride positive test as is the case in this study .
furthermore , the frequency of cf may be higher in some isolated populations due to consanguinity , as has been previously reported in an isolated population .
therefore , in addition to europe and the united states , cf is likely to be prevalent in many other countries as well , although the mutation spectrums may be different .
thus ascertainment of cftr mutation carrier frequencies and cf incidence among iranians seems to be a necessity .
f508 was the only mutation detected and showed a frequency comparable to that reported in overall iranian population and neighboring countries .
identification of the remaining mutations and their frequency requires to design appropriate tests to improve the clinical diagnosis and establishing cf prevention programs by carrier screening and prenatal diagnosis .
| objectivecystic fibrosis and its distribution vary widely in different countries and/or ethnic groups .
common cystic fibrosis transmembrane conductance regulator ( cftr ) mutations were reported from iran , but the northern population was not or underrepresented in those studies .
the aim of this study was to determine the frequency of common cftr mutations in children from northern iran.methodsthirty unrelated iranian cystic fibrosis patients aged less than 11 years and living in mazandaran province ( in iran ) were screened for 5 common cftr gene mutations .
deltaf508 , n1303k , g542x , r347h and w1282x using reverse dot blot method.findingsonly one mutation , deltaf508 , was found in 7 patients accounting for 21.7% ( 13/60 ) of alleles.conclusionthese findings can be used for planning future screening and appropriate genetic counseling programs in iranian cf families . |
scientometric methods can be used to show potentially fruitful areas for drug discovery , based on the assumption that higher levels of scientific activity , including publication rates , will point to specific targets for novel therapies.1 bordons et al demonstrated the usefulness of bibliometric analyses to detect trends in the research of a therapeutic drug including the evolution of drug studies over time.2 can a scientometric indicator reflecting initial drug coverage in biomedical journals be used to assess a specific drug s potential ?
the first attempt to predict the clinical success of drugs by using bibliometric data was made by windsor in 1976.3 later he wrote , just as rabbits leave rabbit tracks and squirrels leave squirrel tracks successful drugs leave different bibliometric tracks than do unsuccessful drugs . sometimes these track records can be used to make predictions.4 he used bibliometric traits of the journal literature on levodopa over a 14-year period to identify predictors of the drug s success .
he concluded that the bibliometry of single - author papers may have promise in this regard . in 2011 a scientometric indicator , the top journal selectivity index ( tjsi ) , was suggested for use in the assessment of therapeutic drugs.5 it represents the ratio ( as percentage ) of the number of all types of articles on a particular drug in the top 20 journals relative to the number of articles in all ( > 5000 ) biomedical journals covered by medline over the 5 years since the drug s introduction .
five years is a relatively long period of time , but the accurate assessment of a drug s value takes much longer .
the true assessment of a drug usually comes with the confirmation of its effectiveness by meta - analyses ( the process of combining results of many clinical studies to draw conclusions about the therapeutic value of a drug ) .
the conclusion on a drug s value reached via this process can take 1020 years.6 however , even this period may not be sufficient , because the comparison of various drugs in meta - analyses is usually indirect , using a placebo as a common comparator.7 as a result such comparisons are not often reliable ; only direct head - to - head comparisons between drugs can provide dependable information on drugs relative efficacy thus an accurate conclusion on a drug s value via this process can take longer than 20 years .
the relatively rapid ( 35 years ) publication response to a new drug may have an important predictive power related to the whole period of a drug s protracted evaluation . in the assessment of the success of new analgesics over the past 50 years , we observed a difference in the publication response to a new drug between biomedical journals in general and in the top journals : the number of published articles on a drug increased ( or declined ) more rapidly in the top journals.6 this observation prompted the introduction of tjsi as an early indicator of drug success
. this feature of tjsi can probably be explained by the high - caliber experts involved in the assessment of manuscripts evaluating new drugs in the top specialty journals .
one of the tjsi determinants is the number of all types of journal articles with or without abstracts ( including editorials , case reports , reviews , and letters to the editor ) covered by the web site of us national library of medicine pubmed ( see http://www.ncbi.nlm.nih.gov/pubmed ) .
pubmed comprises over 21 million citations for the biomedical literature and covers approximately 5000 journals .
the pubmed web site was used to count the number of articles in english published in all types of journals ( original research and review journals ) .
boolean operations were used in which variables were the selected keywords and years of publications .
another tjsi determinant is the number of articles published in the 20 top journals ( also counted using the pubmed web site ) .
the selection of the top 20 journals was based on two factors : the rank of a journal sorted by ( 1 ) the impact factor and ( 2 ) the journal specialty area related to the drug s specific pharmacological class .
sixteen journals were common for all selected drugs . they represent medicine in general ( 10 journals ) and pharmacology ( six journals ) : ann intern med , annu rev pharmacol toxicol , bmj , clin pharmacol ther , j clin invest , j pharmacol exp ther , jama , lancet , n engl j med , nat med , nat rev drug discov , nature , pharmacol rev , proc natl acad sci u s a , science , and trends pharmacol sci .
for example , in the case of drugs used by neurologists the selected journals were : ann neurology , brain , nat neurosci , and trends neurosci .
journals were sorted by the rank of their impact factor presented by journal citation reports , science edition ( see http://science.thomsonreuters.com ) .
the choice of the specific journals is more important than the total number of top journals .
when we used 100 top journals instead of 20 , the difference in the values of tjsi was not distinct .
it should be noted that the chosen determinants are not mutually exclusive ; one relies on a subject of the information used to create the other .
figure 1 gives an example of tjsi calculated in this manner for drugs in six pharmacological classes when the first - in - class drug ( ficd ) had multiple follow - on drugs ( fod ) .
the assessment period could be decreased to 3 years if the increase in the rate of publications in all journals is sufficiently high from the very beginning .
ten analgesics approved by the us federal drug administration ( fda ) during the period 19862009 were selected for analysis.5 they included new molecular entities ( nme ) developed as analgesics and also drugs developed for nonpain indications with subsequent fda approval for the treatment of pain as an additional indication : sumatriptan , tramadol , remifentanil , gabapentin , zolmitriptan , celecoxib , ziconotide , pregabalin , topiramate , and duloxetine . in counting the numbers of articles published over the 5 years since a drug s introduction , specific keywords were selected according to the name of an analgesic in addition to using pubmed medical subject heading [ mesh ] terms .
terms added to the name of a drug were pain or headache disorder [ mesh ] or migraine .
the analgesic success score was determined based on the following criteria : ( 1 ) novelty of molecular target ( completely novel target , novel modification of existing target , or incremental improvement of existing drug ) ; ( 2 ) analgesic efficacy ( magnitude of pain relief , strength of evidence regarding effectiveness , and universality in use ) ; and ( 3 ) response by the pharmaceutical market ( based on the number of nme similar to the initial drug ) .
the contributions of these three criteria to the total success score were weighted differently : much more weight was given to analgesic efficacy ( up to the maximum of 5 points ) than to the two other criteria ( up to the maximum of 2 points each ) .
the strength of the relationship between tjsi and success score was quite impressive : r = 0.876 , p < 0.001 ( figure 2 ) . even if the
sumatriptan is not taken into account , the correlation coefficient continues to be high at 0.694 ( p < 0.05 ) .
tjsi was compared with two other bibliometric indices : the number of all types of articles presented in pubmed ( aji ) and the number of articles covering only randomized controlled trials ( rct ) .
it was found that although there was some tendency for positive relationships between the all articles index ( or rct articles ) and the success score , these relationships did not reach statistical significance .
there are other approaches trying to find relationships between the success rate of drugs and different factors of drugs research performance .
the most interesting in this report is the study by koenig.9 he defined the composite drug output index , which served as a good predictor for past ( or ) future success .
his finding of a high correlation between the number of all published articles and expert judgment of the quality of pharmaceutical research is of particular interest .
his data also indicated that the number of highly cited clinical articles correlated with drug research success ( assessed by expert judgment ) even better than the number of all clinical articles . because the top biomedical journals selected for our index calculations have a much higher citation rate than the rest of the journals , the previously mentioned better correlation rate for highly cited clinical articles compared to all clinical articles could service as an indication going in the same direction as our conclusion .
two groups of drugs were selected to test the hypothesis that the difference between the most successful ( breakthrough ) and less successful drugs of the same category can be detected by tjsi.8 the fda web site for approved drug products ( see http://www.fda.gov/drugs ) was used for selection of drugs approved between 19801989 .
one group of 10 agents included the most successful ( breakthrough ) drugs belonging to different pharmacological classes .
the other group of 10 drugs was selected using a paired design : the paired drug belonged to the same pharmacological group with a mechanism of action as similar as possible .
the selection of drugs for both groups was based on two principles : the degree of advancement in the new drug development and the level of commercial success .
the drug in the first group had a novel mechanism of action and also a highly competitive market status ( six or more pharmaceutical companies had applied to the fda for a generic alternative ) .
the drug in the second group provided only an incremental improvement on the existing drug and had a lower marketing status as a generic drug ( one to five pharmaceutical companies applied to the fda ) .
the following breakthrough drugs were included : acyclovir , buprenorphine , captopril , diltiazem , enalapril , fluoxetine , gemfibrozil , lovastatin , omeprazole , and zidovudine .
the paired group of drugs included : abacavir , alfentanil , felodipine , fenofibrate , fluvastatin , foscarnet , lansoprazole , maprotiline , moexipril , and perindopril .
the two groups were compared using three publication indices : the tjsi , the number of all types of articles on a drug in journals covered by pubmed ( aji ) , and the number of articles presenting only randomized controlled trials ( rct ) .
it was found that tjsi can detect the difference between the two groups of drugs better than the two other indices .
the results indicated that the mean tjsi of the breakthrough drugs was much higher than that of similar , but less successful , drugs ( 18.6 2.9 vs 7.4 4.2 , difference of 11.2 5.2 ; p < 0.0001 ) . for the aji
the mean difference between the groups was 423 457 ( p < 0.02 ) , and for the rct index the mean difference was statistically insignificant ( 8 42 ; p > 0.5 ) .
table 1 indicates that the higher value of tjsi was always that of the breakthrough member of a pair . with aji ,
the value of a breakthrough member was higher , in only eight of 10 pairs .
with rct , the value of an index was higher with a breakthrough member in only four of 10 pairs .
the percentage of times that an index for the breakthrough drug was higher than that of its corresponding pair , and related 95% confidence interval ( ci ) is presented in table 1 .
the table indicates that the tjsi was distinguishable from random chance was 100% of the time with a 95% ci at 69% and 100% . for the aji ,
the value was 80% of the time with a 95% ci at 44% and 98% .
for the rct index , the distinguishability from random chance was 40% of the time with a 95% ci at 12% and 74% .
thus , tjsi will detect the breakthrough drug at least 69% of the time with 95% confidence .
for the other two indices , their cis contain 50% , and thus they are not distinguishable from random chance ( 50% ) in selecting the breakthrough drug .
thus , tjsi can detect the difference between a breakthrough drug and a less successful drug from the same pharmacological class .
usually the introduction of a ficd is followed by the development of many similar drugs , referred to as fod . while some fod have pharmacological properties that distinguish them from the ficd , others do not
. drugs without distinguishing features are often called me - too drugs , because they offer no significant benefits ( including safety ) over the previous agents.10 the marketing of each new drug without distinguishing features becomes questionable , especially when the sequential number of the market entry is as high as five or more .
it was shown that tjsi can provide help in the assessment of late market entrants without distinguishing features.11 to prove that there is a relationship between the tjsi of me - too drugs and the order ( sequential number ) of their market entry , the 43 fod without distinguishing features were assessed .
the study was based on drug classes approved by the fda for marketing between the 1960s and early 2000s .
the analysis of the remaining 43 fod without such distinguishing properties demonstrated ( figure 3 ) that the relationship between tjsis for fods and the order of the drugs market entry had a negative correlation ( r = 0.372 ; p = 0.014 ) : the higher the order ( sequential number ) , the lower the tjsi .
the negative correlation between fod without distinguishing features and the order of market entry can be used for the identification of me - too drugs .
it was found that if tjsi is less than 0.5 of ficd and market entry order is 5 or higher , the fod is a me - too drug ( with false negative rate of only 9.1% . )
the ficd and the first three fods that followed it were excluded from this analysis to eliminate the possibility of counting as me - too drugs those that might be involved in the process of competition for being the first drug in a new class . to underline the notion that the determinants of the tjsi are not mutually exclusive one can indicate that with each new me - too drug a reasonable increase in the number of publications on the related nme could occur .
this would increase both the tjsi and the aji for the nme . among quantitative studies regarding the effect of fods order - of - entry on the drugs commercial success
there was a publication indicating that despite the average success of pioneers over later entrants , there are examples of opposite phenomenon ; later entrants have gained dominant market share.12 the development and the marketing of me - too drugs has been criticized as duplicative , wasting resources that should be used instead to develop more innovative products.13,14 the view that investment in the development of me - too drugs is not an effective use of community resources dates back to the 1959 kefauer us senate committee review.15 since that time many arguments have been made on both sides of the debate regarding how many me - too drugs is too many.16 identifying which fod offer no significant benefits over previous agents , ie , how to distinguish me - too
drugs among fod is a difficult question , and the tjsi could confirm the presence of a me - too drug among fods .
tjsi is a scientometric index reflecting the potential importance of a new drug and can be an indicator for sustained use of a drug : a higher score increases the probability of continuing success .
studies on drugs with narrow , limited use do not have many publications ; some of such studies are not published . in addition
for example , among the 14 analgesics analyzed previously5 only 10 had enough publications to calculate the tjsi .
four drugs ( ketorolac , pentosan , topical lidocaine , and valproate ) were excluded from the calculations , either because they did not have sufficient number of publications in the all journal category ( more than 10 articles ) or not a single article in the top 20 journals category .
the other tjsi limitation is that this index does not differentiate between publications characterizing a drug in a positive and negative way . sometimes after a drug introduction ( due to the possible discovery of unexpected adverse effects )
a significant number of articles can be devoted to the serious problems associated with its administration .
for example , in 20012008 , publications on the cardiovascular risk related to cycloxygenase-2 ( cox-2 ) selective inhibitors reached up to 12% of all publications on cox-2 inhibitors.6 the evaluation of the tjsi is based on the three presented examples .
the real - life utility of this index for various groups of drugs and different situations should be further explored . | the top journal selectivity index ( tjsi ) is a scientometric index reflecting the potential importance of a new drug .
it represents the ratio of the number of all types of articles on a particular drug in the top 20 journals relative to the number of articles in all ( > 5,000 ) biomedical journals covered by medline over the 5 years since the drug s introduction .
the tjsi can be an indicator of a drug s potential for sustained use : a higher score increases the probability of continuing success . |
the 30-item inventory of depressive symptomatology ( ids30 ) ( rush et al 1996 , 2000 ; trivedi et al 2004b ) has been widely used and evaluated using classical test theory methods .
the standard total score is obtained by summing the ratings of 28 of the 30 items .
either weight loss or weight gain , appetite loss or appetite gain is scored because only one member of each pair is applicable to any given respondent .
each of the 28 items is scored on a 0 to 3 scale ( 0the absence of pathology ; 3severe pathology ) .
standard scoring assumes a traditional model of tests known as classical test theory ( ctt ) in which the trait score ( depression in this case ) represents the scale score total plus random error of measurement .
items are the unit of analysis . totaling individual items is not the only way to score a test . for example , the 16-item quick inventory of depressive symptomatology ( qids16 ) ( rush et al 2000 , 2003b ; trivedi et al 2004b ) uses domain scoring such that when more than one item belongs to the same general domain ( eg , four items assess sleep disturbance ) , the items are grouped and assigned a single score for that domain based upon the highest ( most pathological ) score for the domain - related items .
thus , for the qids , the scores for three domains are based on more than one item ( 4 items for sleep disturbance , 2 items for psychomotor disturbance , and 4 items for the appetite / weight domain ) .
each of the remaining 5 items is individually scored for each domain ( eg , sad mood , concentration , decision making ) .
this method allows the use of ctt analyses with the nine domains rather than items as the units of analysis .
the total score ranges from 0 to 27 rather than the 0 to 48 which would have been the case had each of 16 items been scored individually and totaled .
item response theory ( irt ) methods ( in particular the samejima model ) ( samejima 1997 ) is particularly suited for graded item responses ( eg , 03 ratings on items or domains ) as with the ids and qids .
all irt models scale individual items in terms of their location on an inferred continuum using a complex mathematical procedure .
the underlying continuum , denoted as , refers to depression severity in this report .
one can employ either the ctt or irt approach to evaluate items or domains assessed by scales like the ids or qids .
the level of response ( or severity of pathology ) is the item mean ( x ) .
the relation of the item to overall depression is the item / total correlation ( rit ) .
the larger the value of the individual item or domain , x the more severe the symptom .
the higher the value of rit , the more closely the rated symptom relates to overall depression .
the item ( or domain ) mean ( x ) and the item ( domain ) total correlation ( rit ) may not be strongly related to each other .
for example , sleep disturbance items on both the ids and qids generally have among the highest values of x , but these sleep disturbance items are only modestly related to overall depression severity as judged by the total scale score ( ie , their rit values are not particularly large ) .
conversely , sad mood may have a lower x value , but it is more highly related to overall depression than sleep disturbance , which is expected since sad mood is a core depressive symptom ( apa 2000 ; bernstein et al 2006 ) .
irt allows one to formally equate scores on different scales so that a total score , say x , on one depression scale can be shown equivalent to a score of y on another .
for example , we recently used irt ( carmody et al 2006b ) to equate total scores on the qids and the montgomery - sberg depression rating scale ( madrs ) ( montgomery and sberg 1979 ) .
we did the same ( carmody et al 2006a ) with the madrs and the 17-item hamilton rating scale for depression ( hamilton 1960 , 1967 ) .
secondly , irt allows for a comparison of groups defined , for example , by gender or other baseline demographic or clinical features in terms of both individual item responses and the frequency of different item responses in relation to overall depression severity .
ctt also easily allows tests of differences in x , but with ctt testing difference in rit is somewhat complex ( see rush et al 2006 ) .
when specific items perform differently in different respondent groups , the term differential item functioning ( dif ) is used .
finally , irt ensures a more linear relationship between the construct of depression and individual items than does the ctt approach , which might lead the resulting scores to have more optimal properties . on the other hand
, ctt methods always produce results even if the scale has undesirable properties such as low internal consistency .
the irt analyses may not be feasible in some cases because irt analyses require stronger assumptions .
for example , most irt models assume an s - shaped relation between the magnitude of the trait and the item response .
ctt analyses have been conducted with the ids ( rush et al 1996 ) , but irt analyses have not been reported .
the sample was obtained from the texas medication algorithm project ( tmap ) ( rush et al 2003a ; trivedi et al 2004a ) , which was conducted in accordance with international guidelines for good clinical practice and the declaration of helsinki .
tmap was approved by the institutional review boards at the university of texas southwestern medical center and the university of texas , austin , as well as by each local institutional review board where applicable .
adult outpatients with major depressive disorder ( mdd ) were recruited from the public sector ( bernstein et al 2006 ; trivedi et al 2004a , 2004b ) .
the original sample of 547 out - patients with mdd was reduced to 428 by excluding those with mdd with psychotic features . both the self - report ( ids - sr30 ) and clinician - rated ( ids - c30 ) versions of the 30-item inventory of depressive symptomatology ( rush et al 1996 , 2000 ; trivedi et al 2004b ) were obtained at exit by a research coordinator not involved in patient treatment .
the goal of the analyses was to jointly fit the samejima irt model to the ids - sr30 and the ids - c30 and to evaluate differences between these two scales .
we compared the successive eigenvalues ( scree ) to those obtained by randomly generated correlations using the same number of variables and observations in a procedure known as parallel analysis ( horn 1965 ; humphreys and ilgen 1969 ; humphreys and montanelli 1975 ; montanelli and humphreys 1976 ) .
the number of components ( dimensionality ) is the number of components in the real data for which eigenvalues exceed those that were randomly generated .
since each item on each scale has four response alternatives ( ratings on a 03 scale ) , the samejima model generated 4 parameters per item .
one parameter describes how strongly each of 3 functions relates item ( or domain ) responses ( ie , symptoms ) to overall depression .
these three functions respectively denote : ( a ) the tendency for a symptom to be reported as a 1 , 2 or
3 relative to a 0 , ( b ) the tendency for a symptom to be reported as a 2 or a 3 relative to a 0 or 1 , and ( c ) the tendency for a symptom to be reported as a 3 relative to a 0 , 1 , or 2 . the locations of the respective functions are symbolized b0 , b1 , and b2 ( collectively bi ) .
these locations denote the relative frequency of the dichotomized responses . a scale with mean of 0 and standard deviation 1 is common .
thus , if the estimate of b0 ; to equal 0 , it would imply that a 0 response is made half the time and a 1 , 2 , or
3 is made the remainder of the time . the slope is symbolized a , which corresponds to the item / total correlation of ctt in measuring how strongly a given symptom domain relates to overall depression severity . to illustrate how the irt approach works , consider our previous work with the qids - sr16 and qids - c16 , each of which scores 9 domains ( the criterion symptoms to diagnose a major depressive episode ) ( bernstein et al 2006 ; rush et al 2006 ) .
a base model was constructed pooling the two scales into a single 18-domain scale using exit data from the tmap database ( trivedi et al 2004b ) .
the four parameters ( a , b0 , b1 , b2 ) from a given item on the qids - sr16 were allowed to take on different values from the four parameters of the corresponding item on the qids - c16 , resulting in 72 free parameters ( 2 scales 9 domains 4 parameters / domain ) .
the individual a parameters were then tested individually by constraining each , one at a time , to be the same value in the two scales .
the difference between the two fits is approximately distributed as a form of chi - square known as the likelihood - ratio chi - square ( g ) and was tested for significance with 1 df , representing the one parameter that was constrained .
a significant value implies that the item slope ( a ) differed across the two scales .
next , the a parameters were allowed to vary freely , but the three b parameters / item within each domain were constrained to equality .
values of g were again obtained by comparing the value obtained from the constrained version to the value obtained from the base model .
each of these nine tests was based upon 3 df , representing the three intercepts that were constrained for each domain .
a significant result implies that there are intercept differences between the two scales involving that domain .
that would have meant that symptoms in that domain are reported with different frequencies by the qids - c16 and the qids - sr16 .
in fact , no significant slope differences were found , and only one intercept difference was found ( for agitation / retardation ) .
when slopes or intercept differences are found between groups or measurement methods ( in this case ) , the term differential item functioning ( dif ) is used . in the case of the qids , only the agitation / retardation domain performed differently when one scale as opposed to another was used . even then , the difference was not in the degree of relationship between the symptom and overall depression ( ie , the a parameter was not different ) .
an anomalous result means that a better fit is found with a more constrained than a less constrained model , which leads to a spurious negative
g. such a result can arise from various sources : ( a ) very high correlations between individual items , ( b ) small cell frequencies , or ( c ) long scales .
when we conducted similar analyses with the 30-item ids scales , such anomalies were encountered .
consequently , we made the following modifications to successfully fit the model and to test for dif : ( a ) we replaced scoring of individual items with domain scores for sleep , psychomotor , and appetite / weight domains ; ( b ) we pooled items with few positive responses ; and ( c ) we changed how the base model was tested .
specifically , ids items 14 were combined into a single sleep domain ; items 1114 were combined into a single appetite / weight domain , and items 2324 were combined into a single psychomotor domain ( analogous to the standard scoring of the qids16 ) ( rush et al 2003b ) .
next , items 68 , 16 , and 21 ( diurnal mood variation , distinct quality to mood , distinct mood quality , interest in sex , and gastrointestinal complaints ) were dichotomized into 0 vs 1 or greater because responses of 2 or greater to each of these items were rare .
finally , the tested strategy was reversed by first generating a base model in which all parameters were constrained to equality and then freeing a parameters individually and bi parameters in groups of 3 .
this procedure ( the converse of what was used with the qids16 ) maintains the idea of comparing more vs. less constrained models . as an addendum to this testing
, individual bi parameters were tested specifically by freeing them whenever the entire group of three parameters differed .
for example , if there was a difference in the overall distributions of the sad mood response frequencies between the two rating scales , more specific differences involving the three specific dichotomies ( 0 vs 1 to 3 , 0 or 1 vs 2 or 3 , and 0 to 2 vs 3 ) were examined individually .
finally , the test information functions of the ids and the qids ( obtained by extracting the relevant items from the ids were compared . in the present context
, the test information function ( tif ) describes how well a test can discriminate small differences in depression as a function of the score the higher the value , the more discriminating the test .
tif bears similarities to the internal consistency ( coefficient alpha ) obtained by ctt , but the tif reveals how test information varies over different levels of depression rather than being computed as a constant .
the sample was obtained from the texas medication algorithm project ( tmap ) ( rush et al 2003a ; trivedi et al 2004a ) , which was conducted in accordance with international guidelines for good clinical practice and the declaration of helsinki .
tmap was approved by the institutional review boards at the university of texas southwestern medical center and the university of texas , austin , as well as by each local institutional review board where applicable .
adult outpatients with major depressive disorder ( mdd ) were recruited from the public sector ( bernstein et al 2006 ; trivedi et al 2004a , 2004b ) .
the original sample of 547 out - patients with mdd was reduced to 428 by excluding those with mdd with psychotic features .
both the self - report ( ids - sr30 ) and clinician - rated ( ids - c30 ) versions of the 30-item inventory of depressive symptomatology ( rush et al 1996 , 2000 ; trivedi et al 2004b ) were obtained at exit by a research coordinator not involved in patient treatment .
the goal of the analyses was to jointly fit the samejima irt model to the ids - sr30 and the ids - c30 and to evaluate differences between these two scales .
we compared the successive eigenvalues ( scree ) to those obtained by randomly generated correlations using the same number of variables and observations in a procedure known as parallel analysis ( horn 1965 ; humphreys and ilgen 1969 ; humphreys and montanelli 1975 ; montanelli and humphreys 1976 ) .
the number of components ( dimensionality ) is the number of components in the real data for which eigenvalues exceed those that were randomly generated .
since each item on each scale has four response alternatives ( ratings on a 03 scale ) , the samejima model generated 4 parameters per item .
one parameter describes how strongly each of 3 functions relates item ( or domain ) responses ( ie , symptoms ) to overall depression .
these three functions respectively denote : ( a ) the tendency for a symptom to be reported as a 1 , 2 or
3 relative to a 0 , ( b ) the tendency for a symptom to be reported as a 2 or a 3 relative to a 0 or
1 , and ( c ) the tendency for a symptom to be reported as a 3 relative to a 0 , 1 , or 2 .
the locations of the respective functions are symbolized b0 , b1 , and b2 ( collectively bi ) .
these locations denote the relative frequency of the dichotomized responses . a scale with mean of 0 and standard deviation 1 is common .
thus , if the estimate of b0 ; to equal 0 , it would imply that a 0 response is made half the time and a 1 , 2 , or 3 is made the remainder of the time .
the slope is symbolized a , which corresponds to the item / total correlation of ctt in measuring how strongly a given symptom domain relates to overall depression severity . to illustrate how the irt approach works , consider our previous work with the qids - sr16 and qids - c16 , each of which scores 9 domains ( the criterion symptoms to diagnose a major depressive episode ) ( bernstein et al 2006 ; rush et al 2006 ) .
a base model was constructed pooling the two scales into a single 18-domain scale using exit data from the tmap database ( trivedi et al 2004b ) .
the four parameters ( a , b0 , b1 , b2 ) from a given item on the qids - sr16 were allowed to take on different values from the four parameters of the corresponding item on the qids - c16 , resulting in 72 free parameters ( 2 scales 9 domains 4 parameters / domain ) .
the individual a parameters were then tested individually by constraining each , one at a time , to be the same value in the two scales .
the difference between the two fits is approximately distributed as a form of chi - square known as the likelihood - ratio chi - square ( g ) and was tested for significance with 1 df , representing the one parameter that was constrained .
a significant value implies that the item slope ( a ) differed across the two scales .
next , the a parameters were allowed to vary freely , but the three b parameters / item within each domain were constrained to equality .
values of g were again obtained by comparing the value obtained from the constrained version to the value obtained from the base model .
each of these nine tests was based upon 3 df , representing the three intercepts that were constrained for each domain .
a significant result implies that there are intercept differences between the two scales involving that domain .
that would have meant that symptoms in that domain are reported with different frequencies by the qids - c16 and the qids - sr16 .
in fact , no significant slope differences were found , and only one intercept difference was found ( for agitation / retardation ) . when slopes or intercept differences are found between groups or measurement methods ( in this case ) , the term differential item functioning ( dif ) is used . in the case of the qids , only the agitation / retardation domain performed differently when one scale as opposed to another was used .
even then , the difference was not in the degree of relationship between the symptom and overall depression ( ie , the a parameter was not different ) .
an anomalous result means that a better fit is found with a more constrained than a less constrained model , which leads to a spurious negative
g. such a result can arise from various sources : ( a ) very high correlations between individual items , ( b ) small cell frequencies , or ( c ) long scales .
when we conducted similar analyses with the 30-item ids scales , such anomalies were encountered .
consequently , we made the following modifications to successfully fit the model and to test for dif : ( a ) we replaced scoring of individual items with domain scores for sleep , psychomotor , and appetite / weight domains ; ( b ) we pooled items with few positive responses ; and ( c ) we changed how the base model was tested .
specifically , ids items 14 were combined into a single sleep domain ; items 1114 were combined into a single appetite / weight domain , and items 2324 were combined into a single psychomotor domain ( analogous to the standard scoring of the qids16 ) ( rush et al 2003b ) .
next , items 68 , 16 , and 21 ( diurnal mood variation , distinct quality to mood , distinct mood quality , interest in sex , and gastrointestinal complaints ) were dichotomized into 0 vs 1 or greater because responses of 2 or greater to each of these items were rare .
finally , the tested strategy was reversed by first generating a base model in which all parameters were constrained to equality and then freeing a parameters individually and bi parameters in groups of 3 .
this procedure ( the converse of what was used with the qids16 ) maintains the idea of comparing more vs. less constrained models . as an addendum to this testing
, individual bi parameters were tested specifically by freeing them whenever the entire group of three parameters differed .
for example , if there was a difference in the overall distributions of the sad mood response frequencies between the two rating scales , more specific differences involving the three specific dichotomies ( 0 vs 1 to 3 , 0 or 1 vs 2 or 3 , and 0 to 2 vs 3 ) were examined individually .
finally , the test information functions of the ids and the qids ( obtained by extracting the relevant items from the ids were compared . in the present context
, the test information function ( tif ) describes how well a test can discriminate small differences in depression as a function of the score the higher the value , the more discriminating the test .
tif bears similarities to the internal consistency ( coefficient alpha ) obtained by ctt , but the tif reveals how test information varies over different levels of depression rather than being computed as a constant .
figure 2 contains the successive eigenvalues of the ids - c30 and ids - sr30 ( scree ) and those randomly generated .
note that both scales meet the criteria for two factors since the first two eigenvalues exceed the randomly generated data .
the disparity between the real and randomly generated second eigenvalue , however , was modest .
the presence of two factors imposes a limitation on the irt solution to be provided , since that solution assumes unidimensionality . to the extent that depression is represented by the first principal component ,
the items related to the second principal component define something other than depression that is contributing to the score .
table 1 contains the first and second principal component loadings for the ids - sr30 and ids - c30 , respectively . using an arbitrary cutoff of 0.4 to denote a large weight on a given component , the second principal component of the ids - sr30 is defined by aches and pains ( domain 18 ) , symptoms of sympathetic nervous system arousal ( domain 19 ) , and gastrointestinal complaints ( domain 21 ) .
these same three items plus the presence of the capacity for pleasure ( negative loading ) ( domain 15 ) formed the second principal component for the ids - c30 .
thus , the three items common to both forms deal with somatic symptoms that may not necessarily reflect depression per se , especially in this sample of socially disadvantaged individuals with high rates of general medical conditions ( trivedi et al 2004a ) .
scores on the first and second components were then generated for the ids - sr30 and ids - c30 .
as expected , the correlation between the first component scores for the ids - sr30 and ids - c30 was extremely high ( r=0.92 ) because both represent the dominant depression component of both scales .
the correlation between the two second component scores was also moderately high ( r=0.73 ) .
the remaining correlations ( eg , between the first component of the ids - sr30 and the second component of the ids - c30 ) were 0.11 or less , which establishes the independence between the first and second components of each scale .
these findings indicate that the multidimensionality within the ids - c30 and ids - sr30 is consistent .
table 2 shows the samejima a and bi parameter estimates for the ids - sr30 and ids - c30 when they were each scored to create 23 domains .
the last column ( diff . ) identifies domains for which there is a significant difference between the clinician and self - report ratings in the a or bi parameter estimates ( ie , differential item functioning ) ( dif ) .
note that three of these are also qids items ( concentration / decision making , capacity for pleasure , restlessness / agitation ) vs one that is peculiar to the ids ( diurnal variation ) .
none of the former provided any evidence of a slope difference when scored as part of the qids .
therefore , it is not the item itself but rather the broader definition of depression used by the ids that accounts for these differences .
each version of the 23 domain - scored ids may be scored two ways by ctt simply as the as the sum of the 23 domains or by a fairly complex irt algorithm .
the correlation between the ctt and irt scores on each of the two scales is high ( 0.92 for ctt and 0.91 for irt ) .
the correlation between the two methods of scoring is even higher , 0.97 , for both versions of the scale .
however , the ctt and irt methods of scoring the 23 domain - scored versions of the ids - sr30 and ids - c30 reveal that total scores using the two scoring methods are not linearly related .
figure 3 shows a scatter plot of the two sets of ids - c30 scores , with irt - generated scores along the abscissa and ctt - generated score along the ordinates . according to irt , the irt - generated scores are , by definition , linearly related to depression ( ) .
consequently , the ctt - generated scores are an ogival ( s - shaped ) function of depression . if one accepts the irt scoring of true depression , then scores at the high and low ends of the ctt scale represent less difference in depression than scores in the middle .
scores in the middle of both scales are linearly related to one another . in other words , very low scores as defined by ctt tend to somewhat underestimate depression , as seen in the minimal changes in such scores following changes in irt - generated scores .
similarly , very high scores defined by ctt tend to overestimate depression . however , this effect is modest and may or may not be of clinical significance .
the next step was to compare the results of standard 28-item ctt scoring to the two alternative 23 domain scoring methods ( ctt and irt ) .
thus , the two ctt methods correlated to nearly identical degrees with the irt scoring . of course , correlations between the 28-item ctt scoring with irt scoring were the same as the correlations between the 23-domain ctt scoring and irt scoring ( 0.97 ) .
thus , despite the curvilinearity of ctt scoring , the major properties of both ctt and irt scoring , such as the rank - ordering of individuals , were preserved .
finally , test information functions ( tifs ) were generated for the ids and the qids for the 23 domain scored versions of both the self - report and clinical irt versions .
these functions describe to what degree change in the level of depression is reflected in changes in irt - defined test scores .
the formula for the test information function may be found in nunnally and bernstein ( nunnally and bernstein 1994 , p 408 ) and lord ( lord 1980 , p 68 ) .
the tif serves a role that is similar to the internal consistency ( coefficient alpha ) of ctt , but it is a function of the trait being investigated rather than a constant for the test as a whole .
thus , the ids - c30 and ids - sr30 relate similarly to overall depression when scored using the 23-domain method . as can be seen
, the test information of the ids is approximately twice that of the qids at each level of depression ( generically symbolized in the language of irt ) .
this means that the ids provides a more sensitive measure of depression than the qids .
technically , this applies to the irt - scored ( 23 domain ) version of the ids , but the similarities between this scoring and the 28 item scoring plus the clinical and self - report versions make this a rather general conclusion . this result is expected given the greater length and greater breadth of symptom coverage with the ids . also , note that both tests are maximally sensitive with patients who are of average depression to one standard deviation above average in this sample , which means that it is less useful in discriminating among patients low in depression ( remitted depressives and normals ) and those who are extremely depressed .
these results may reflect , in part , that relatively few depressives were remitted in this sample .
figure 2 contains the successive eigenvalues of the ids - c30 and ids - sr30 ( scree ) and those randomly generated .
note that both scales meet the criteria for two factors since the first two eigenvalues exceed the randomly generated data .
the disparity between the real and randomly generated second eigenvalue , however , was modest .
the presence of two factors imposes a limitation on the irt solution to be provided , since that solution assumes unidimensionality . to the extent that depression is represented by the first principal component ,
the items related to the second principal component define something other than depression that is contributing to the score .
table 1 contains the first and second principal component loadings for the ids - sr30 and ids - c30 , respectively . using an arbitrary cutoff of 0.4 to denote a large weight on a given component , the second principal component of the ids - sr30 is defined by aches and pains ( domain 18 ) , symptoms of sympathetic nervous system arousal ( domain 19 ) , and gastrointestinal complaints ( domain 21 ) .
these same three items plus the presence of the capacity for pleasure ( negative loading ) ( domain 15 ) formed the second principal component for the ids - c30 .
thus , the three items common to both forms deal with somatic symptoms that may not necessarily reflect depression per se , especially in this sample of socially disadvantaged individuals with high rates of general medical conditions ( trivedi et al 2004a ) .
scores on the first and second components were then generated for the ids - sr30 and ids - c30 .
as expected , the correlation between the first component scores for the ids - sr30 and ids - c30 was extremely high ( r=0.92 ) because both represent the dominant depression component of both scales .
the correlation between the two second component scores was also moderately high ( r=0.73 ) .
the remaining correlations ( eg , between the first component of the ids - sr30 and the second component of the ids - c30 ) were 0.11 or less , which establishes the independence between the first and second components of each scale .
these findings indicate that the multidimensionality within the ids - c30 and ids - sr30 is consistent .
table 2 shows the samejima a and bi parameter estimates for the ids - sr30 and ids - c30 when they were each scored to create 23 domains .
the last column ( diff . ) identifies domains for which there is a significant difference between the clinician and self - report ratings in the a or bi parameter estimates ( ie , differential item functioning ) ( dif ) .
note that three of these are also qids items ( concentration / decision making , capacity for pleasure , restlessness / agitation ) vs one that is peculiar to the ids ( diurnal variation ) .
none of the former provided any evidence of a slope difference when scored as part of the qids .
therefore , it is not the item itself but rather the broader definition of depression used by the ids that accounts for these differences .
each version of the 23 domain - scored ids may be scored two ways by ctt simply as the as the sum of the 23 domains or by a fairly complex irt algorithm .
the correlation between the ctt and irt scores on each of the two scales is high ( 0.92 for ctt and 0.91 for irt ) .
the correlation between the two methods of scoring is even higher , 0.97 , for both versions of the scale .
however , the ctt and irt methods of scoring the 23 domain - scored versions of the ids - sr30 and ids - c30 reveal that total scores using the two scoring methods are not linearly related .
figure 3 shows a scatter plot of the two sets of ids - c30 scores , with irt - generated scores along the abscissa and ctt - generated score along the ordinates . according to irt , the irt - generated scores are , by definition , linearly related to depression ( ) .
consequently , the ctt - generated scores are an ogival ( s - shaped ) function of depression . if one accepts the irt scoring of true depression , then scores at the high and low ends of the ctt scale represent less difference in depression than scores in the middle . scores in the middle of both scales
are linearly related to one another . in other words , very low scores as defined by ctt tend to somewhat underestimate depression , as seen in the minimal changes in such scores following changes in irt - generated scores .
similarly , very high scores defined by ctt tend to overestimate depression . however , this effect is modest and may or may not be of clinical significance .
the next step was to compare the results of standard 28-item ctt scoring to the two alternative 23 domain scoring methods ( ctt and irt ) .
thus , the two ctt methods correlated to nearly identical degrees with the irt scoring . of course , correlations between the 28-item ctt scoring with irt scoring were the same as the correlations between the 23-domain ctt scoring and irt scoring ( 0.97 ) . thus , despite the curvilinearity of ctt scoring , the major properties of both ctt and irt scoring , such as the rank - ordering of individuals , were preserved .
finally , test information functions ( tifs ) were generated for the ids and the qids for the 23 domain scored versions of both the self - report and clinical irt versions .
these functions describe to what degree change in the level of depression is reflected in changes in irt - defined test scores .
the formula for the test information function may be found in nunnally and bernstein ( nunnally and bernstein 1994 , p 408 ) and lord ( lord 1980 , p 68 ) .
the tif serves a role that is similar to the internal consistency ( coefficient alpha ) of ctt , but it is a function of the trait being investigated rather than a constant for the test as a whole .
thus , the ids - c30 and ids - sr30 relate similarly to overall depression when scored using the 23-domain method . as can be seen
, the test information of the ids is approximately twice that of the qids at each level of depression ( generically symbolized in the language of irt ) .
this means that the ids provides a more sensitive measure of depression than the qids .
technically , this applies to the irt - scored ( 23 domain ) version of the ids , but the similarities between this scoring and the 28 item scoring plus the clinical and self - report versions make this a rather general conclusion . this result is expected given the greater length and greater breadth of symptom coverage with the ids .
also , note that both tests are maximally sensitive with patients who are of average depression to one standard deviation above average in this sample , which means that it is less useful in discriminating among patients low in depression ( remitted depressives and normals ) and those who are extremely depressed .
these results may reflect , in part , that relatively few depressives were remitted in this sample .
the samejima model was applied to the clinician and self - rated versions of the ids . as noted above
whereas classical methods allow the frequency of symptoms to be compared with ease , classical methods are less suitable to evaluate differences in the relation of symptoms to depression ( or any other trait ) .
however , with the ids there was greater difficulty and more strain on the assumption of unidimensionality than was the case with the qids , which , as our earlier papers have shown , was clearly unidimensional ( bernstein et al 2006 ; rush et al 2006 ) .
these data suggest that scoring the ids conventionally is satisfactory for making judgments about patient care .
conventional scoring ( ie , totaling the 28 items ) allows one to take advantage of the far simpler ctt scoring .
it would appear that creating 23 domains offers little advantage because of the relatively large number of items .
on the other hand , an irt model generated using the 23-domain scoring method , is important for virtually any research involving the ids , since it means that groups and/or conditions can be compared using the relatively straightforward methods based upon testing for fit differences .
this is a major advantage in looking for differences in the relation of domains to depression as a whole .
for example , when comparing patients with postpartum depression and depression outside the postpartum period , the revised ids scoring ( 23 domains ) is preferred . at the same time , the fact that ctt and irt scoring lead one to comparable results means that conventional scoring of the ids is appropriate .
it is reasonable to ask if the revised ( 23 domain ) scoring in either ctt or irt form is sufficient .
the answer is that it is . at the same time , this revised scoring does not seem to be more sensitive to mild depression .
one could also ask about jettisoning those items that seem to induce multidimensionality ( ie , gastrointestinal symptoms , somatic complaints , sympathetic arousal ) .
however , the loss in comparability with previous studies would probably be greater than any largely theoretical gain . finally , there is the question of whether it is good for many of the items that load on the first principal component to reflect anxiety .
that question seems very difficult to answer without considering whether anxiety is or is not an inherent part of depression or whether a large sample of depressed patients could have included some with anxious depression and others with minimally or non - anxious depression .
limitations in the assertions that an irt model can be created for the ids30 and that it is consistent across methods needs be noted .
first , there is a slight , but consistent , degree of multidimensionality in the ids30 that is counter to the assumption of unidimensionality made in standard usage of the samejima model .
the precise nature of the second dimension might be different in a sample with less medical comorbidity .
standard scoring of the ids - c30 and ids - sr30 provides simplicity and comparability to published data .
the present results based on the irt model enhance the validity of comparisons between groups or conditions . while clinical decisions can be made about patients with standard scoring ( ie , totaling all items to obtain a scale score ) , researchers may wish to use this revised irt scoring method to improve the use of the ids when comparing groups .
limitations in the assertions that an irt model can be created for the ids30 and that it is consistent across methods needs be noted .
first , there is a slight , but consistent , degree of multidimensionality in the ids30 that is counter to the assumption of unidimensionality made in standard usage of the samejima model .
the precise nature of the second dimension might be different in a sample with less medical comorbidity .
standard scoring of the ids - c30 and ids - sr30 provides simplicity and comparability to published data .
the present results based on the irt model enhance the validity of comparisons between groups or conditions . while clinical decisions can be made about patients with standard scoring ( ie , totaling all items to obtain a scale score ) , researchers may wish to use this revised irt scoring method to improve the use of the ids when comparing groups . | background : both the clinician ( ids - c30 ) and self - report ( ids - sr30 ) versions of the 30-item inventory of depressive symptomatology have acceptable psychiatric properties and have been used in various clinical studies .
these two scales , however , have not been compared using item response theory ( irt ) methods to determine whether the standard scoring methods are optimal.methods:data were derived from 428 adult public sector outpatients with nonpsychotic major depressive disorder .
the ids - c30 and ids - sr30 were compared using samejima s graded response model.results:a model was constructed jointly fitting the ids - c30 and ids - sr30 .
an improvement in scale performance was obtained by grouping selected items into domains ( specifically sleep , psychomotor , and appetite / weight domains ) analogous to the standard scoring of the 16-item quick inventory of depressive symptomatology.conclusions:for the ids - c30 and ids - sr30 , standard scoring ( ie , computing total score using all individual items ) provides simplicity , comparability to published data , and a basis for clinical decision making .
the revised scoring method , however , improves the utility of both scales when comparing groups as it provides explicit tests of item parameters . |
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