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primary osteosarcoma of the head and neck represents 6 to 13% of osteosarcoma cases,1 and 1.7 to 5% of head and neck cancers.2 males and females are equally affected , and patients usually present in the third and fourth decades of life .
contributing factors include prior chemotherapy , radiation , and underlying pathological conditions such as fibrous dysplasia and paget disease.2
osteosarcoma is subdivided based on histopathologic characteristics .
these subtypes include conventional , telangiectatic , small cell , epithelioid , osteoblastoma - like , chondroblastoma - like , fibrohistiocytic , and giant cell - rich ( gcro ) .
gcro , first described by bathurst et al , is a rare variant accounting for 1 to 3% of all osteosarcoma cases.3 most cases have been reported in the appendicular skeleton with two cases arising in the maxilla and the mandible.4
5 due to overlapping histopathological features , this subtype can be difficult to differentiate from other pathological entities including gcr variety of malignant fibrous histiocytoma , chondrosarcoma , and giant cell tumor .
because of the different prognostic features and management strategies of these pathologies , it is imperative to make the correct diagnosis in a timely fashion . to date , there have been no published cases of primary gcro of the skull base .
we report a case of gcro arising from the clivus and describe its radiologic and histopathologic characteristics , as well as our surgical treatment through an endoscopic endonasal approach .
an 18-year - old female patient presented with a 3-day history of progressive decreased left visual acuity .
imaging revealed a large enhancing and expansile central skull base mass involving the upper two - thirds of the clivus and eroding the dorsum sella ( fig .
there was possible erosion of the medial aspect of the carotid sulci bilaterally at the level of the paraclival internal carotid arteries ( icas ) .
the lesion was also extending to the paraclival , parasellar , and paraclinoid icas bilaterally .
the radiological differential diagnosis included chordoma , chondrosarcoma , interosseous meningioma , and osteosarcoma .
computed tomography angiogram scans with bone window sagittal ( a ) and axial ( b ) showing large central skull base lesion with mass effect over the internal carotid arteries and basilar artery .
there is erosion of the floor of the sella turcica with extension into the sphenoid sinus .
( c ) axial enhanced magnetic resonance imaging disclosing invasive expansile mass in the central skull base with signal intensity most consistent with partial osteoid matrix .
( d f ) magnetic resonance imaging axial ( d ) , sagittal ( e ) , and coronal ( f ) disclosing large central skull base mass with mass effect on internal carotid arteries and encasing the left carotid artery .
the patient underwent a planned two - stage endoscopic endonasal approach to obtain tissue diagnosis and decompress the optic apparatus .
briefly , we raised a right standard nasoseptal flap6 and performed a posterior septotomy , bilateral maxillary antrostomies , ethmoidectomies , and sphenoidotomies for access and to widen the nasal corridor ( fig .
a left - sided transpterygoid approach was undertaken.6 the vidian canal was identified and delineated to the level of the petrous ica ( fig .
this step was essential to allow for proximal control of the vessel on that side .
we elected not to perform the same maneuver on the right side to avoid compromising the vascular supply to the nasoseptal flap .
tumor debulking was initiated centrally and progressed laterally on the left and then right side in a counter - clock wise fashion .
the right medial optic carotid recess , lateral optic carotid recess , and right paraclinoid ica were identified .
following the plane between the tumor and the right paraclinoid ica proximally allowed for complete detachment of the tumor from the dura of the medial compartment of the cavernous sinus and the paraclival ica .
the micro doppler probe was used frequently throughout the process to identify and gauge proximity to the ica .
the tumor was well encapsulated in most areas and elevated well off the lateral recesses of the sphenoid sinus and the planum sphenoidale .
however , it had clearly eroded the middle third of the clivus at the posterior wall of the paraclival recess as well as the floor and anterior wall of the sella turcica . because of the fibrosity of the tumor and its large size , an automated side suction - cutter aspirator ( myriad ) ( nico corp .
, indianapolis , indiana , united states ) was used for a significant part of the debulking process .
although there was dural tumor involvement , the surgeons opted not to resect the dura because of the benign intraoperative pathology report .
d. , pituitary gland ; ppg , pterygopalatine fossa ; t , tumor ; v.n . , left vidian nerve .
immediately , postoperatively , the patient 's vision improved and then normalized by postoperative day 7 .
a postoperative magnetic resonance imaging ( mri ) showed more than 98% tumor resection , with a small remnant at the dura ( fig .
magnetic resonance imaging axial ( a ) , coronal ( b ) , and sagittal ( c ) showing significant reduction in size of lesion .
postoperatively , the initial pathological diagnosis was giant cell lesion , and the differential diagnoses included giant cell tumor and gcro .
the final description found that the tumor was composed of atypical epithelioid and spindle cells within fibrous tissue .
the presence of osteoid matrix with focal mineralization , consistent with osteosarcoma , was identified .
6 ) . eventually , after consultation with several north american pathologists , the final diagnosis of gcro was confirmed .
biopsy of lesion composed of atypical epithelioid and spindle cells within fibrous tissue and osteoid matrix which is focally mineralized .
after 3 months of surgery at the same time the pathological diagnosis was being finalized , there was evidence of tumor recurrence on a follow - up mri ( fig . 7 ) .
further surgical resection was performed to reduce tumor burden in preparation for adjuvant chemoradiation .
enhanced magnetic resonance imaging coronal ( a ) and axial ( c ) disclosing recurrence of the clival mass .
we searched the medline database through pubmed and ovid interface using the terms giant cell rich osteosarcoma and head and neck .
one article presented a case of gcro of maxilla and the other presented a case of gcro of the mandible.4
5 a retrospective review of 27 cases of osteosarcoma arising in the head and neck by ha et al reported one case of gcro of left posterior occiput.7 there were no cases of gcro of skull base .
primary osteosarcoma of the cranial base is a rare and challenging condition with fewer than 150 cases reported in the literature since 1945.8 clinical findings may include proptosis , cranial nerve palsies , visual impairment , and headaches.2 the patient reported in this study presented with a decrease in visual acuity and chronic headaches .
the majority of osteosarcoma subtypes are high - grade conventional , which include osteoblastic , chondroblastic , and fibroblastic subtypes.1 gcro is an extremely rare morphological variant of high - grade conventional osteosarcoma .
to our knowledge , this is the first case of gcro of the skull base .
the differential diagnosis of giant cell lesions can present a diagnostic challenge , especially if they occur in an unusual location . in this case , it was difficult to differentiate between a gcro and giant cell tumor .
this case was sent out to pathologists in other north american centers , and the diagnosis of gcro was eventually confirmed .
an osteosarcoma may contain so many giant cells , and only focal atypia , that the malignant elements in the background are obscured , and the lesion is mistaken for a giant cell tumor.9 also , the presence of osteoid could be quite rare in an obviously malignant lesion .
malignant giant cell tumor is a controversial entity that should be ruled out by a careful examination for osteoids , important for the diagnosis of gcro.1
contributing to the diagnostic challenges , the radiological features of gcro differ from that of conventional osteosarcomas .
bathurst and sanerkin described a typical radiographic pattern in long bones : an ill - defined margin surrounding a predominantly lytic lesion of the diaphysis or metaphysis .
a soft tissue mass is usually not present and the periosteal reaction is weak.10 in the reported case of gcro of the maxilla , a contrast - enhanced ct scan showed an expansile heterogeneous mass with areas of speckled calcification within the tumor and adjacent sclerosis.4 in the reported case of gcro of the mandible , the radiographic appearance showed an osteolytic lesion.5 radiographic changes in conventional osteosarcoma typically show rapid invasion and destruction of metaphyseal area of the long bone with periosteal reaction and soft tissue extension.3
osteosarcoma of skull base is rare and evidence for its treatment is consequently sparse .
although , there is a lack of consensus on the optimal treatment , surgical resection is the mainstay of treatment.11 the most important prognostic factor is the feasibility of complete tumor resection with negative margins.11 our patient presented with an extensive but well - encapsulated tumor of the skull base , almost completely encasing both carotid arteries medially . with advances in endoscopic surgical techniques , image - guidance , doppler ultrasound , and the expertise of the skull base team
the tumor site has a significant impact on survival , and extragnathic tumors are usually associated with worse prognosis due to incomplete resection owing to the complex anatomy of that region .
the anatomy of the head and neck and especially skull base is complex and complete resection without causing morbidity may not be feasible .
in addition , surgeons may be reluctant to undertake complete resections that affect function in younger patients.12 in osteosarcoma of long bones , the addition of adjuvant chemotherapy and radiation has resulted in improved survival .
however , its use in head and neck osteosarcoma has not been established.12 in a study by guadagnolo et al , the role of radiotherapy with respect to outcome in head and neck osteosarcoma was analyzed .
the authors found that radiotherapy in addition to surgery improved the overall and disease - specific survival in cases where resection margins were positive or uncertain.13 in the case of gcro of the maxilla , the patient underwent resection of the lesion followed by 60 gy radiotherapy with five cycles of chemotherapy .
she was disease free at her last follow - up.4 gcro of the mandible was treated with surgical resection followed by chemoradiation and patient was disease free at her 1-year follow - up.5 local recurrence is the main cause of fatality in head and neck osteosarcoma and distant metastasis is only seen in 7 to 17% of cases.2
this report details a patient presenting with a rare tumor , gcro , in a difficult anatomic location , the skull base involving both carotid arteries , treated with surgical resection using expanded endonasal approach followed by chemoradiation .
our experience adds to the very few similar cases in the literature , and should help other surgeons who are presented with similarly challenging cases . | introduction giant cell - rich osteosarcoma ( gcro ) is a rare pathologic diagnosis , and most cases have involved the appendicular skeleton .
we present a challenging diagnosis of gcro of the skull base treated with an endoscopic endonasal approach .
case presentation an 18-year - old female patient presented with acute monocular visual loss .
imaging revealed a large clival mass encasing the internal carotid arteries bilaterally with pituitary and optic nerve compression .
the lesion was resected via a staged endoscopic endonasal approach and the patient 's vision normalized postoperatively .
the final pathological diagnosis was challenging and , after consultation with multiple north american centers , was concluded as gcro .
the tumor recurred and further surgery was performed , followed by adjuvant chemoradiation .
conclusion we highlight diagnostic challenges of gcro of the skull base , and describe , with intraoperative pictures , successful surgical resection via an endoscopic endonasal approach . based on our literature review ,
this is the first published case report of gcro of the skull base . |
the translation of its scientific name , linen most useful [ 1 , 2 ] , aptly describes its versatility and importance to world economy .
linen , the long , strong fibers from flax stems , is considered one of the earliest successes in textiles .
while evidence does not exist on how early people learned to separate fibers from the stems , flax as a major textile in ancient egypt is well documented in depictions of its cultivation and processing .
linen samples have been reported in the remains of swiss lake dwellings dating back some 10,000 years .
production and use expanded beyond the mediterranean countries to central and northern europe , making its way to great britain about 2,000 years ago from the middle east by phoenician traders .
linen , as one of the primary fibers for europe throughout the middle ages and the renaissance period , was used extensively for clothing .
linen was important to russia and its economy through various stages of its political history .
flax became the greatest export item and the basis of economic life in russia in the late 1800s and into the twentieth century .
at one time , russia produced about 80% of the world 's fiber flax crop and before 1936 was the greatest exporter of flax .
production of flax in connecticut was reported as early as 1640 . while flax was grown in several regions of the united states , particular states developed well - organized commercial efforts , particularly michigan and the willamette valley in oregon .
the production of flax fiber in oregon in the early 1900s , led by efforts of the us department of agriculture and the oregon agricultural experiment station , was remarkably well documented and included production yields , processing and mills , and other advancements [ 5 , 8 ] .
as in europe , specially designed equipment to pull , turn , deseed , and scutch flax was developed to increase agricultural efficiency .
oregon 's commercial enterprise for flax fiber , however , ended in the 1950s due to introduction of synthetic fibers and loss of government subsidies .
the linen industry also declined in europe due to the coming of synthetic fibers , such as nylon and polyester for apparel [ 3 , 4 ] .
cotton before then , however , had overtaken the high position of linen and industrial flax fibers , which had existed for millennia , due to large amounts of inexpensive cotton and its improved mechanical processing .
generally , cotton has led as the natural fiber of choice since this time , with only short times of reversals such as blockades during the american civil war ( 18611865 ) and disruptions caused by world war ii ( 19391945 ) .
after the war , the lower production levels returned . in traditional linen - producing areas ,
promotional programs by linen industries in northern ireland and western european countries led to a strategic organization to promote linen in the 1960s . in the 1980s , the fao ( food and agriculture organization of the united nations ) sponsored workshops on flax , and in 1993 ,
european cooperative research network on flax , with coordination through the institute of natural fibres , poznan , poland .
this program continues to compile data on crop production , facilitates interaction of several working groups , and sponsors numerous workshops thus promoting global interests in flax fiber .
recently , however , production of textile flax fiber has decreased in most of the reporting european countries and including the baltic states and the russian federation . in the european union ( eu )
countries , production of flax declined from 122,379 ha in 2005/2006 to 73,029 ha in 2010/2011 . declining eu subsidies since the 1990s have reduced production levels and regions .
production in china , which has varied over the last two decades , has occupied a prominent position for the last several years .
however , the quality of chinese - produced linen is not high enough for textiles , and china imports considerable amounts of long fiber from france and belgium . the belgium market for the prices of long fiber , which varies based on quality , recently was reported to range from 130 to 210/100 kg , with prices for unscutched short fiber of 30 to 50/100 kg . after flax production ended in oregon and effectively ended flax fiber production in the us , efforts to renew a flax fiber industry in the united states have occurred over the years .
most have not been successful . in 1998 , naturally advanced technologies ( nat ) and the alberta research council in canada developed crailar flax .
nat and the hanes clothing company promoted the development of this product for complementary use with cotton in textiles . while the method is proprietary , popular news stories and promotional websites indicated that crailar flax increased performance characteristics in textiles . despite the reduction in production and usage from previous times , linen imparts characteristics of comfort , drape , and a distinctive appearance that have maintained a share of the market , particularly the luxury market for textiles [ 4 , 10 , 11 ] .
blending of cottonized flax , that is , short , refined flax fibers , with cotton and other fibers offers a potential for nontraditional , flax fibers to impart distinctive properties in textiles .
for example , blending cotton with increasing levels of cottonized flax fiber for rotor spinning improved air permeability and wicking rates for moisture and modified the fabric structure .
the fine linen used for painting canvasses also requires strong and clean fibers from linen - type production systems , such as that from belgium . despite the reduced production of traditional linen and
move to other sources , likely markets will exist for the long , strong , and clean linen fiber in specific , high - value applications along with cottonized flax fibers for blends .
the major source for flax fiber in north america , however , is straw from the linseed industry .
linseed provides an industrial oil widely used in paints , varnishes , cosmetics , and linoleum . in the past and even more recently , flax seeds are being recognized as a health food , with the intact seeds providing a laxative effect and linseed products providing lignans and omega-3 fatty acids [ 16 , 17 ] .
it is likely , then , that production of linseed will continue and provide a consistent source of stems for flax fiber production .
in contrast to agronomic systems to optimize fiber production in flax , linseed production seeks to optimize seed yield .
stems are thicker in these plants than in fiber - type flax plants . in north
america , as well as europe and russia , linseed production is in colder climates , where fiber extraction from the stems is very difficult due to reduced microbial activities and poor retting ( see later ) .
most of the north american flax fiber is extracted from linseed stalks by hammer - milling to obtain all the fiber , regardless of length , to be used in specialty papers .
the quality of linseed fibers processed in this manner is considered too low for apparel or other high - value uses under the current commercial production and cleaning operations . even with this hammer milled product for paper and pulp , however , there is currently interest in producing a cleaner fiber product , that is , less shive or nonfiber fractions , in order to reduce the amount of chemicals needed to remove lignin from the flax in pulping .
several research programs are in place to promote the use of flax and other natural fibers from various sources , including linseed stalks , in new products and in particular biocomposites .
efforts are currently under way to improve processing methods and find more uses for value - added fibers in industrial , for example , biocomposite , applications .
in fact , industrial applications with composites and nonwoven materials may provide the greatest potential for expanded use of flax fibers in the future [ 1921 ] .
canada , which is the world 's leading producer of linseed , currently promotes expanding the flax industry , both for fiber and seed , through genetics research programs and organizations such as the saskatchewan flax development commission , the composites innovation centre , and biolin research , inc . in saskatchewan , canada . to this end , canadian research into flax has recently increased , particularly towards improving fiber quality of high - value applications such as biocomposites .
north dakota , the main producer of linseed in the us , follows the trend of the linseed industry in canada .
about 20% of the straw from the linseed industry satisfies the specialty paper ( mostly cigarette ) industry in north america .
the remainder of this straw by - product ( greater than 1 million tons from western canada ) is now burned or chopped to spread on fields . with the closing of the ecusta operation , only one large processor for flax fiber from linseed straw , that is , schweitzer mouduit international , winkler , canada , still produces fiber and mostly for paper and pulp and lower - end uses . a very great opportunity exists with linseed straw to improve farm economy , replace synthetic fibers with natural fibers , and provide a value - added product for myriad applications .
consistency in supply and in fiber characteristics must be addressed when flax fiber is sought for large - scale industrial usage .
the degree of processing for fiber cleanliness will depend upon the end product desired , and for many products , the levels of cleanliness and processing costs are considerably less than for linen in textiles . with the north american textile industry based on short staple fibers ( e.g. , cotton )
, the possible use of new processing methods to provide textile - grade fibers from linseed straw should not be overlooked .
the goal will be to find improved ways of processing the fibers for consistent quality and with properties for applications in high - end uses , even for apparel .
globally , an urgent and increasing need exists for new sources and products for agricultural , manufacturing , and health industries . while linen from fiber - type cultivars is still a valuable commodity for textiles , the desire for natural fibers in biocomposites points to linseed stalks as a largely untapped source of industrial fibers .
production of a higher quality fiber for value - added applications from the linseed stalks , however , requires some changes in fiber production and processing .
biotechnological approaches can supply innovative , efficient , and more directed methods for fiber production .
the chemical composition and the anatomical location of constituents within the flax stem define processing , properties , and applications of flax .
the anatomy of the flax stem and chemical composition in the cell types is well documented in many publications ( e.g. , [ 24 , 37 , 38 ] ) .
figure 1 shows the arrangement from outermost to innermost layers as follows : cuticle connected with a single layer epidermis , bast fibers in cortical region , and woody core tissues .
lipids , including waxes and cutin , and aromatics comprise this layer and provide a protective barrier to water loss and to invading microbial pathogens into the internal stem tissues [ 24 , 25 , 39 ] .
the cuticle can be readily observed with the histochemical stain oil red [ 40 , 41 ] , which stains the wax in the cuticle a bright red color and thereby provides a distinctive , visible marker specific for the cuticle ( figure 2 ) .
flax fibers do not stain with oil red , indicating no or a small amount of wax on fibers per se . by staining processed fibers with oil red ,
a quick , visible assessment of cuticle contamination is possible in cleaned flax fiber [ 25 , 40 ] .
the cuticle and the adjacent single layer of thin - walled , epidermal cells are closely connected , coming off as a unit ( figure 1 ) .
the outer layer of cuticle / epidermis comprises 13%24% ( by weight ) of the bast fraction of selected fiber and linseed cutivars ( table 1 ) .
this layer , and mostly the cuticle , provides a rich assortment of lipids , including waxes , cutin , and sterols , while aromatics are present in small amounts ( table 1 ) . near - infrared fourier
transform raman microscopy indicated , by absorption at specific wavelengths in stem thin sections , the presence of wax and soluble aromatic pigments in the cuticle .
ultraviolet absorption microspectroscopy further indicated an absorption near 280 nm indicative of aromatics in the cuticle but not in the epidermis .
the parenchyma cells , including those below the cuticle / epidermis layer , lack aromatics and are a pectin - rich layer generally amenable to degradation by microbes and enzymes ( discussed later ) , allowing separation of the cuticle / epidermis fragment during proper retting .
the cuticle is generally impervious to microbial attack as observed in field - retting of flax , although some disruption and penetration of the cuticle can occur by field - retting fungi [ 43 , 44 ] . with pectinolytic enzymes , the cuticle can be sloughed off and freed from the fiber .
when this cuticle / epidermis fragment is intact , however , as when flax is underretted ( figure 2 ) , it remains attached to several fiber bundles , resulting in large fragments and reducing the quality of fiber and yarn .
total wax and cutin contents were significantly higher ( p < 0.05 ) in commercial grades of low versus high quality fiber and yarn , likely because of the contamination by cuticle .
cuticle , short fibers broken during processing , and woody core fragments comprise the dust particles that are emitted during fiber processing .
the dust so generated is considerable , and dust bags or other suitable collection containers are used to maintain a clean environment .
potential lipid coproducts from the dust fraction , as well as from a purer cuticle fraction removed during experimental enzyme retting , have been evaluated [ 46 , 47 ] .
a hot alcoholic extraction was effective in removing the wax components , which could then be separated by controlled cooling .
these waxes and other lipids have potential as commercial products from this waste material of flax fiber processing .
the central woody core tissues are the primary xylem and other structural cells , which provide support and water conduction for the plant ( figures 1 and 4 ) .
the main sugars are glucose , representative of cellulose , and xylose , representative of hemicelluloses ( table 2 ) .
almost all of the lignin in flax stems is present in the core cells . the histochemical stain for lignin , acid phloroglucinol , stains the entire core cell walls a bright red color , indicating coniferyl - rich constituents throughout this tissue ( figures 4 and 5 ) .
positive reactions with acid phloroglucinol for coniferyl lignin ( monomethoxylated aromatic rings ) and chlorine - sulfite for syringyl lignin ( dimethoxylated aromatic rings ) indicated that both lignin types are present in core cells .
results , however , vary with the analytical method used , such as klason lignin with 72% sulfuric acid or that derived from permanganate oxidation .
often , klason lignin gives higher values because components other than aromatics are in the residue . by alkaline extraction and subsequent summation of similar structures ,
aromatic levels totaled about 1.5% , with a higher level of guaiacyl to syringyl units ( table 3 ) .
of the low molecular weight aromatics , only small amounts of ferulic acid were found ( table 3 ) [ 24 , 49 ] .
nuclear magnetic resonance ( nmr ) spectrometry confirmed that the aromatics were present as lignin .
further , ultraviolet ( uv ) absorption microspectrophotometry of thin sections also showed a strong absorbance near 280 nm in the core cell walls and no absorbance beyond , but absorbance maxima shifted slightly in cells more towards the stem center .
the presence of a single strong absorption near 280 nm , as well as lack of absorbance near 320 nm , suggests a complex structure of the lignin similar to that in woody plants . during field retting by indigenous fungi ,
the core cell walls remain intact and are virtually impervious to fungal attack , showing a typically high resistance to degradation for this type of lignocellulose .
these lignin - rich core cells make up a large portion of the shive waste fraction generated during fiber processing .
with such a high proportion of stem material , shive waste is a huge by - product of commercial fiber production .
unless there are uses for it , shive becomes an economic deficit in processing , and its disposal is necessary .
shive , however , has economic value in the overall flax processing system , generally now finding application in low - value uses typical of lignocelluloses , such as animal bedding , mulch , particle boards , and thermal energy from burning .
these additional sources of revenue provided by selling the shive , 9.00/100 kg , are essential to guarantee a positive economic position of the flax processing facility .
more recently , the lignocellulosic nature of shive has shown to be exceptional as activated carbon for heavy metal absorption and for removal of chlorinated hydrocarbon , outperforming selected commercial products .
extraction and separation of cellulose , hemicellulose , and lignin from flax shives by various methods , notably pressurized low polarity water extraction and under different conditions of ph and temperature , are reported as effective means of obtaining aromatic feedstocks from this by - product of fiber processing [ 49 , 53 ] .
the fact that the core cells , that is , shive , contain by far the most aromatics in the plant stem while fiber contains very little gave rise to a rapid method to assess fiber cleanliness .
near infrared reflectance ( nir ) spectroscopy coupled with chemometric models for ratios of shive : fiber from 0% to 100% was used to develop a method to predict shive content in cleaned fiber [ 24 , 5457 ] .
this method has been further developed and employed for at - line assessment of quality in which commercial bales were assessed over several weeks . in converse , the cleanliness of shive from residual fiber left from processing can be similarly predicted in materials sought for clean woody lignocellulosic materials .
the industrially important bast fibers are long , slender , and strong specialized cells that develop in bundles in the cortex region , located between the cuticle / epidermis layer and the innermost woody cells ( figure 1 ) .
these cellulose - rich cells are the source of linen and other commercial fibers . based on quality properties such as length , strength , and cleanliness ,
the bast fibers are sought for high - value apparel and other textiles , natural fiber composite reinforcement , and specialty papers such as cigarette , currency , and bible sheets [ 15 , 18 , 19 ] . in nature
, these fibers exist in bundles of individual ( or ultimate ) fibers encircling the lignified core tissues ( figure 1 ) . ten to 40 spindle - shaped ultimate fibers , each 2 to 3 cm long and 15 to 20 m in diameter , form in 20 to 50 discrete bundles [ 3 , 37 ]
dislocations perpendicular to the axis , occur in fibers and entire bundles , as clearly shown by microscopy ( figure 6 ) .
what creates fibernodes is not clear , and kink bands are similar in structure and apparently give similar responses .
possibly , pressure exerted during mechanical handling or even through growth and expansion of tissues may create their presence .
these fibernodes are important , because dyes , enzymes , and other liquids preferentially react there first [ 6163 ]
. cellulases also attack preferentially at the fibernodes , even across bundles ( figure 7 ) .
breaks during stress and compression tests occur preferentially at fibernodes and kink bands [ 64 , 65 ] .
cellulose is the main component in bast fibers , with values of 65%80% of the dry weight reported .
in addition to cellulose , bast fibers contain pectins , hemicellulose , and aromatic compounds in small amounts ( tables 4 and 5 ) .
field - retted fiber showed an expected increase in glucose ( by weight ) indicative of cellulose , while increases also occurred in mannose and galactose .
the fact that hemicelluloses , such as galactoglucomannans and xylans are substantial components in flax fibers has been shown by considerable research [ 63 , 6668 ] . distinguishing characteristics of linen , such as high moisture regain , may be influenced by the presence of these noncellulosic carbohydrates within the cellulosic structure .
proteins and proteoglycans are also associated with secondary walls of flax fiber and possibly provide structure .
data on lipids associated with bast fibers were collected from several studies and presented in table 5 .
fibers that had been manually separated and cleaned of all other visible materials showed the presence of low levels of waxes , cutins , and sterols , with amounts of about 0.2% of fiber dry weight and 1/20th or less of levels in the cuticularized epidermis .
other work , however , on a series of dew - retted and water - retted flax fibers from europe showed that lipids were present on fibers , with higher levels on the better quality ( i.e. , stronger , and finer ) water - retted ones ( table 5 ) . with these latter samples , it was not clear if the lipids represented residual cuticle or if these fibers had more inherent lipids on the surfaces .
chemical analyses of highly cleaned fibers from linseed flax and mature plants ( i.e. , grown for seed ) had higher lipid levels than did bast fibers of the fiber - type plants ( table 5 ) .
these compounds in linseed may reflect a higher level of waxes on the fibers or may reflect more difficulty in removing all the cuticle from fibers during processing .
the use of oil red as a histochemical stain , however , did not indicate wax on the clean fiber surfaces , and so likely it is residual cuticle ( figure 2 ) . while the question of lignin in bast fibers frequently arises
, most data indicate that the amount of aromatics present is small [ 23 , 24 , 28 ] .
localization of aromatics , using histochemical stains [ 24 , 70 ] and ultraviolet ( uv ) absorption microspectrophotometry showed that aromatic compounds were limited to middle lamellae and cell corners in bundles , and by far , the greatest levels were in cell corners ( figures 3 and 5 ) .
the deposition of aromatics , however , as shown by both methods , was sporadic in the bundles .
other work , however , using solid phase c nmr ( nuclear magnetic resonance ) spectrometry indicated that aromatic material in flax fibers was predominately an anthocyanin , rather than lignin .
spectroscopic analysis of water - soaked , manually - separated , and then enzyme - retted fibers , which were free of all nonfiber materials , indicated only trace aromatics in fibers from fiber- and seed - type flax stems .
so , while trace amounts of aromatics are found in fiber bundles , the amount is small and does not appear to impede fiber / core separation in either fiber - type or linseed straw
. often pockets of acid - phloroglucinol staining are observed on fibers , without the presence of obvious shive material , and likely represent a residual from either core or cuticle or the aromatic material from cell corners .
possibly , heavily localized areas of aromatics that remain on retted fiber could influence fiber quality properties or reduce processing efficiency .
extraction of flax bast tissue , which included fibers and cuticle / epidermis , with a series of organic solvents ( i.e. , hexane , propanol , methanol , and water ) and analysis by reverse phase high pressure liquid chromatography ( hplc ) and c nmr indicated a variety of aromatic constituents including flavonoids and hydroxy - methoxy cinnamic acids .
the water extract from these flax samples contained a complex mixture of compounds , including sugars and aromatics .
the phenolic - containing extracts inhibited cellulase and pectinase activities , suggesting a possible influence on retting enzymes if such compounds were released . based on the previous discussion , the most likely source of these aromatic compounds in this study was the cuticle of the bast layer , rather than the fiber . in the cellulose of plant cells , generally , a structure of unbranched linear glucose units allows zones of higher order , that is , crystalline regions , as well as areas of lower order , that is , noncrystalline regions . in flax fibers ,
x - ray diffractometry shows region of crystalline and less - ordered structure , with the linear orientation of both regions higher in flax than the other cellulosic fibers cotton and ramie .
the secondary cell walls of flax fibers at maturity are reportedly locked in an almost axial direction , giving lower elongation and a more brittle nature for flax fibers compared to cotton fibers .
while flax fiber is primarily a cellulosic fiber , its chemistry and characteristics provide specific properties that differ from cotton and many other natural fibers .
parenchyma , cambium , and the middle lamellae , which bind ultimate fibers in the bundles , are particularly rich in pectins , hemicelluloses , and other matrix polysaccharides as shown by response of these tissues to pectinolytic enzymes .
the separation of fibers from the woody core occurs at the outer surface of the cambium and is facilitated when stems have been stored in dry climates for an extended time .
it is the pectin - rich regions that are of prime importance in retting , and considerable work has been done on the pectinases and ways to degrade the pectin in flax .
pectin , while often low in amounts , is strategically located and binds cell walls within plants .
while pectin is , therefore , particularly important in maintaining the structure of flax stems , its degradation is of fundamental importance for retting and the resulting quality of flax fibers [ 37 , 73 ] . chemically , pectin is a heteropolysaccharide consisting mainly of 1,4-linked -d - galacturonic acid , with various degrees of methylesterification at the carboxyl position and with various attached side chains . in some cases ,
pectin in primary plant cell walls may have a high proportion of oligosaccharide chains on the backbone and longer chains than the pectin in the middle lamellae .
nmr spectrometry indicated that a rhamnogalacturonan structure of type i pectin , which is a prominent form in plants , likely forms the backbone of the high molecular weight polysaccharides in flax fiber .
in retting of flax , pectin degradation was reported to be faster in flax harvested during flowering than in mature flax stems , and a residual pectin level of 7 to 10 g / kg remained after retting .
nonmethoxylated carboxyl groups on galacturonic acids are often cross - linked by ca to form stable bridges across pectin molecules .
mid infrared microspectroscopy mapping of mature flax fiber indicated that pectin types varied among plant types and regions , with the potential to influence retting efficiency .
immunocytochemical staining methods , using gold - labeled antibodies against specific pectin structures , provide further indications that the sites of specific pectin types vary within areas and even layers of flax fibers [ 82 , 83 ] .
both nonmethoxylated pectin and calcium levels are higher in the epidermal regions of the flax stem and lower in the fibers .
inductive coupling plasma ( icp ) emission spectrometry showed that calcium levels in the cuticle / epidermis tissue was 5.5-fold greater than in the bast fibers of ariane flax .
high amounts of calcium in the rigid cuticle / epidermis fragment further stabilize an already formidable barrier to retting in flax stalks .
endopolygalacturonase , a pectinolytic enzyme that is present in many enzyme mixtures , was reportedly inhibited by steric hindrance through calcium linkages in pectin .
the reported levels of pectin in flax vary considerably and are influenced by various factors [ 8688 ] . for decorticated ,
that is , processed , flax fiber and cell walls of various cultivars , the pectin content ranged from 20.5% to 34% .
chemical treatment with dilute hydrochloric acid followed by ammonium citrate resulted in a pectin content for flax fibers of 1.6% . while only an approximation , the sum of uronic acids , rhamnose , and arabinose was 1.7% of the total carbohydrates for field - retted ariane flax fiber .
to obtain bast fibers for commercial use , flax stems undergo a process called retting to separate fibers from nonfiber materials , namely , cuticle / epidermis and the woody core .
the method first used for separating and cleaning linen fibers is not known and was possibly an accidental observation of the fact that flax stems turned to fibers under certain conditions of exposure to weather or immersion in water .
the historical value for this information has importance in some contexts , and searches are underway to discover the method early linen producers used to produce their fine strong fibers for textiles .
the main idea is to degrade the pectins and other cementing compounds that bind the bast fibers and fiber bundles to other tissues and thereby separate fibers from nonfiber materials [ 3 , 18 , 37 ] .
the separated fibers are then cleaned of nonfiber materials by mechanical processing . for long fibers used for linen ,
specialized equipment is employed ; the first stage is called scutching , which uses a specialized system to beat and stroke long fibers to remove shive , and the second is hackling , relying on a specialized instrument to comb , straighten , and align fibers .
insufficient retting , or under - retting , results in poor separation of the cuticle / epidermis layer and the woody inner tissues ( i.e. , shive ) from the fibers [ 25 , 31 ] .
subsequent cleaning is then problematic , because the nonfiber materials become entangled in the fibers and reduce fiber yield , processing efficiency , and ultimate fiber quality .
in this situation , the cellulosic fibers are weakened by overly active cellulases ( figure 7 ) , resulting in poor fiber quality .
even though currently most of the linseed straw fiber is of fairly low quality and used for paper and pulp , a desire exists to have less shive in the hammer milled fiber material in order to reduce chemicals used for delignification in making pulp .
the fact that linseed straw is produced in regions unsuitable for dew retting is problematic in having high fiber yields and clean fiber .
two main methods have been historically employed commercially to ret flax for textile - grade fibers , namely , water retting and dew ( or field ) retting [ 3 , 37 ] . in traditional water retting , as practiced in western europe when the linen industry was flourishing , flax stems were pulled and submerged in bodies of water , for example , lakes , rivers , and ponds , for five to seven days .
because the process was understood to some extent , technological methods were adapted to improve the process .
selected microorganisms were chosen to improve water retting . at times , tanks were aerated to modify the bacterial consortium and thereby the bacterial metabolism , reducing the problems of pollution and stench from anaerobic metabolism that caused widespread concern in areas of western europe .
water retting resulted in long , strong , and fine fibers of excellent quality for apparel and other textiles .
the high cost of this method and resulting pollution , with the stench residing in water - retted fibers , however , resulted in water retting being abandoned for the most part in the mid 1950s in western europe .
water retting has been mostly replaced by dew retting , although some water retted flax was commercially available in the early 2000s .
reports indicate that china , the largest producer of flax , may still produce water retted fiber in aerated tanks , but the fiber quality is not reported to be of good quality for textiles .
dew ( or field ) retting is the method used in western europe for obtaining high quality fibers for textiles .
field retting , however , is reported to be the oldest method of retting flax , practiced thousands of years ago by the egyptians .
field retting is carried out by pulling flax stems and laying them in even layers of rows for the moisture to encourage indigenous fungi to colonize and grow on the stem .
the farmers of western europe reportedly produced the best dew - retted fiber because of the climate and their knowledge of when to turn and harvest the flax for uniform retting . in areas of proper climate and expertise ,
commercially dew retting works and has been the method of choice for linen and other flax fiber production .
the quality of flax fiber has declined over the years since dew retting replaced water retting as the main method for getting textile grade fibers in western europe .
dew retting continues to be the main retting method over water retting because production costs are lower , however , and fiber yields are higher and there is no stench .
dew retting , though , has a number of disadvantages other than poor and inconsistent quality compared to water - retted fiber .
certain areas formerly known for their linen production are unable to ret because of the noncompliant climates , such as england , scandinavia , and ireland .
another disadvantage of field retting is that large tracts of land are tied up for weeks until flax is suitably retted . in intensive agricultural areas ( e.g. , for multiple cropping ) ,
too dry weather results in poor fungal growth and lack of proper retting ; wet weather delays field harvest and also interferes with fungal growth , resulting in pockets of anaerobic degradation . over - retting
occurs with excessive growth of cellulolytic fungi in the retting consortium and results in weakened fiber .
so , even in the best regions for field retting , crop losses of about one - third are expectedly for one reason or another . from the early 1900s ,
this subject has been briefly and recently reviewed by this author [ 18 , 38 ] .
methods for improved retting included modifying water retting or dew retting to remove the inherent problems and to select specific retting microorganisms .
stand retting , where standing plants are dried with a herbicide , notably glyphosate ( n - phosphonomethyl glycine ) , and allowed to ret by indigenous microorganisms in a modified form of field retting , has been tried [ 93 , 94 ] and is still being developed .
often the fiber properties were shown to be improved over dew retting , although dry weather interfered with retting and was problematic with this herbicide .
this method is still promising , particularly with new forms of the herbicide , and has been used to produce test plots of flax for cottonized fiber in england .
in addition to modification of the traditional water and field retting methods , much research has focused on chemical retting approaches , sometimes with and sometimes without microorganisms or enzymes [ 80 , 98106 ] .
as mentioned previously , the use of chelators , notably edta , has been pursued , and its value is shown by strong sequestration of ca at various phs to disrupt the pectin linkages .
autoclaving flax straw with the chelators edta and oxalate has been used with breeding programs to effectively extract unretted flax fibers .
a patent exists for a mechanical process to produce fiber strips followed by a chemical / cooking process under pressure .
flash hydrolysis or steam explosion treatment , with or without impregnation before steam treatment , has been used to remove pectins and hemicelluloses from decorticated flax to produce small bundles and ultimate fibers [ 104106 ] .
ultrasonic treatment , following decortication and opening of green flax or hemp stalks , has been used to obtain fibers from diverse sources without the use of chemicals .
the use of low energy , uniform ultrasonic treatment , combined with enzymes , has shown increased activity of various enzymes for cotton fabrics and could be a useful method to improve the efficiency in retting flax .
chemical separation has resulted in successful laboratory results , but at times , fiber properties are less satisfactory than those from other methods .
efforts are reported to be still underway to assess physical and chemical methods to separate fiber .
the use of enzymes , focusing on pectinases , also has been researched for some time [ 37 , 108110 ] .
enzyme retting , however , has proven to offer promise as a biotechnological improvement and is still undergoing research and development for improving fiber quality .
sharma and his colleagues in europe and the united kingdom in the 1980s carried out a major effort on enzyme retting , primarily attempting to mimic water retting with a consortium of plant cell wall - degrading enzymes .
these efforts , as well as other topics related to flax production for textiles , have been documented in numerous papers and in the book the biology and processing of flax .
since the plant cell wall is a complex lignocellulosic material , it was believed that a mixture of cellulases , hemicellulases , and most notably pectinases was required to ret flax as occurred with the plant - associated natural microbial consortia in water or dew retting . in hindsight ,
part of this approach could have been due to the lack or cost of specific enzymes available at the time , as shown by attempts to use highly pectinolytic microorganisms .
fungal cultures contained this mixture of cell wall - degrading enzymes but with some different profiles and activities .
microorganisms with high levels of pectinases were chosen , but most culture filtrates also contained some cellulases .
a product from novo nordisk ( copenhagen , denmark ) called sp 249 was used in a side - by - side test of enzyme versus water retting .
g / l in an 11 : 1 liquid - to - solid ratio at 45c for 24 hours .
oxidizing agents , however , were required to denature the enzymes and stop the continuing action of cellulases in the enzyme mixture .
the study showed the successful application of enzyme retting in pilot plant scale , promising that retting of flax could occur with enzymes and with a reduced time of retting .
the liquid method also ensured a more consistent product and could be carried out in any location with proper equipment . from the work of sharma and colleagues , flaxzyme , which was a patented liquid preparation of balanced cellulases , pectinases , and hemicellulases from aspergillus species ,
this enzyme also resulted in fiber yield and properties equal to or better than fiber from water retting .
later , lyvelin ( lyven , caen , france ) , a pectinase , but not pure , from aspergillus niger was marketed specifically for retting of flax . despite these positive results and developments , an enzyme retting method
did not replace dew retting . while the reasons are complex , likely enzyme costs and lack of industry support prevented further development .
the united states was the largest per capita user of flax - containing textiles , but no linen or flax fiber for textiles was produced domestically .
this statistic prompted the agricultural research service ( ars ) of the us department of agriculture to begin research toward developing a flax fiber program for textiles .
results from the work in europe , particularly with pectinases , were the basis of research .
canada and the us northern plains states had a thriving linseed industry , with tons of waste straw available .
producers wished to remove the straw from their fields soon after harvest , and the straw did not degrade readily .
a small portion of this crop , estimated around 25% , was used for hammer milling and pulp for specialty papers , such as cigarette , currency , and bible sheets .
most of the linseed straw was not used and was ( and still is ) burned to remove it from the fields .
further , the us textile industry was tied to cotton fiber processing and not the long - line linen processing of europe .
in fact , no specialized wet - spinning equipment required for long - line linen yarn existed in the us . the textile spinning technology was based on short staple fibers like cotton and synthetic blends .
a flax tow product , which is short fiber as a by - product of long - line linen , was used in blends with cotton , but that product , too , was imported .
so , quickly , the ars research effort incorporated linseed stems as a source of fibers , with attempts to improve retting and processing for higher , quality in the fibers .
further , since the fibers desired were short - staple like cotton , total fiber production , with collection of all bast fiber regardless of length , was employed with later cottonizing to shorten and refine the fibers .
the ars flax research focused on lowering enzyme amount , finding purer and more active enzymes , and developing a protocol for enzyme retting .
earlier research indicated the value of calcium chelators for disrupting pectins in retting ( see ealier ) .
tests with oxalic acid showed that chelators could greatly reduce the amount of enzyme needed for retting .
so , with following tests , the retting mixture was almost always an enzyme / chelator mix .
excellent field retting of flax had been noted in research experiments at clemson university , south carolina , for winter production of flax . from this material ,
the major fungi colonizing the stems were cultured and isolated in a search for more active retting enzymes .
this fungus , identified eventually as rhizopus oryzae sb nrrl 29086 , produced a potent endopolygalacturonase ( epg ) and few other enzymes in the filtrate [ 30 , 112 ] .
this enzyme was purified and tested for its ability to separate fibers in stems and compared in mixtures with potentially complementary cell wall - degrading enzymes .
in these early tests , fiber separation was judged by light microscopy and the fried 's test , which is an in vitro test to judge fiber separation from stems by comparing visual images .
results indicated that this purified epg with oxalic acid alone was sufficient to separate flax fibers ( table 6 ) .
the other potentially complementary enzymes tested , namely , pectin methyl esterase , xylanase , and cellulases , did not improve fiber separation .
data , therefore , indicated that enzymes other than epg were not required to separate fiber from nonfiber fractions .
based on these results , a search for a potential commercial enzyme , with high pectinase and low cellulase activity , indicated that viscozyme l ( novozymes north america ) had similarities to sp 249 and flaxzyme .
oxalic acid in later tests proved not to be suitable as a chelator , as a precipitate formed and remained on the fibers . while tests continued on a series of chelators of different types and under different conditions [ 113 , 114 ] , edta replaced oxalic acid because of efficiency at ph 5 to 10 and its commercial availability for the textile market .
the retting test method of choice , then , was a combination of the commercial products viscozyme l and edta ( later using mayoquest , a 36%38% edta commercial product ) for other comparisons and modifications . a series of viscozyme / edta formulations , with increases in each of the enzyme and chelator , was used to ret a mature , fiber - type flax . rather than the fried 's test , cotton fiber tests were used for strength by stelometer and fineness using a modified microaire system equilibrated with a series of fineness standards fibers .
an estimate of the percentage of fine fiber , collected by passing retted and mechanically cleaned fiber through the shirley analyzer , was calculated from the starting material .
the shirley analyzer , which is an instrument to separate and collect trash in cotton , provided a percent of fine , cleaned fiber as an additional statistic to judge quality .
experiments with a series of retting combinations indicated that increasing viscozyme levels increased fine fiber yield but reduced strength , regardless of chelator levels ( table 7 ) . increasing levels of chelator , within each enzyme series , increased fine fiber yield and resulted in finer fibers up to 18 mm .
fibers from this study were blended with cotton ( 50 : 50 ) , spun as yarn in a miniature spinning system , and the yarn properties determined .
results indicated that enzyme treatments affected yarn properties , with the highest enzyme level producing finer fibers , easier yarn construction , and better quality yarns .
assessments are difficult to compare , however , because , for example , the highest level of enzyme produced a finer but weaker fiber .
possibly , finer but weaker fiber was also less stiff and brittle and therefore more amenable for blending with cotton .
the clearest result from this work was that the miniature spinning system has value in predicting the optimal retting formulations for yarn quality . in order to develop a new pilot plant method for enzyme retting , a spray enzyme method , or a brief ( 2 min ) soaking , to deliver the enzyme / chelator mixture was used to reduce liquid : solid ratio compared to the former method of retting flax in submerged tests .
since the cuticle / epidermis layer protected the internal stem tissues , methods were explored to facilitate the entry of enzymes into stems .
physically crushing stems with fluted rollers to breach this barrier improved enzyme retting over that by increased or reduced atmospheric pressures .
although inhibitory , aromatic compounds had been shown to be released by chemical treatments , presoaking of fiber with water to remove these compounds showed no clear benefit with enzyme retting and was not included as part of the enzyme retting protocol .
the enzyme retting method , termed ser , was tested on seed- and fiber - type plants , with various levels of enzyme and chelator .
ultimately , enzyme retting would have to be integrated to a commercial cleaning system for fiber production , but such cleaning systems did not exist in the us .
accordingly , arrangements were made to produce pilot scale amounts of enzyme - retted flax for commercial processing in europe .
about 12 kg of retted flax for each formulation was commercially cleaned by ceskomoravsky len ( cml ) in humpolec , czech republic , using a unified line scutching system and la roche cottonizing system .
these fibers were tested for quality parameters and then spun into blended yarns with cotton at 50 : 50 and at 10 : 90 flax : cotton amounts .
fibers were tested with modified cotton testing equipment ( table 8) and the yarn properties by commercial testing equipment and methods ( table 9 ) .
results indicated that the enzyme mixtures retted both fiber- and seed - type plants ( table 8) .
higher levels of enzyme reduced fiber strength but produced finer and higher amounts of fine fiber .
other than strength , fineness and fine fiber yield were better than these characteristics in dew - retted fibers .
chelator levels did not seem to vary in their impact , with 25 mm amounts equal to the 50 mm levels .
the seed flax fibers were of less quality than fiber - flax fibers with similar formulations .
retesting of the fibers 30 months later showed no further loss in strength , indicating the washing step after enzyme retting was sufficient to stop further enzyme activity .
yarn properties were compared favorably between dew retted and enzyme retted at the higher level of enzyme ( table 9 ) .
seed - flax fibers processed into blends much like the fiber - flax fibers , even the dew - retted sample .
since retting , processing , and yarn construction are interrelated , a flax pilot plant was constructed based on the unified line at cml in order to have a commercial method for cleaning retted flax stems .
this system was developed by engineers at cml but reduced in size and with each of four parts separately positioned for research .
the parts were : 9-roller calender for breaking stems , 5-roller calender for further crushing shive , scutching wheel to produce a total fiber from the stems , and an upper pinned shaker to remove loose shive and straighten fibers .
further , a series of test protocols was developed for objective test results of fibers .
fibers were tested using cotton equipment and protocols where applicable , such as strength and elongation by stelometer .
further test standards were developed for flax , including color , fineness , and predicted shive through the flax and linen subcommittee of astm international [ 56 , 57 ] .
the following tests methods adopted were ( 1 ) percent of fine fiber yield produced by passing through a shirley analyzer , ( 2 ) tensile strength and elongation by stelometer , ( 3 ) fineness based first on a modified cotton airflow method and later refined for a new astm test method , and ( 4 ) the percent shive in cleaned fiber using near infrared spectroscopy and chemometric models from fiber : shive combinations [ 54 , 55 , 59 ] .
this latter method was accepted as a new astm test method in 2005 . for certain assessments ,
the fried 's test and light and scanning electron microscopy were used when appropriate . a color test method ( d-6961 - 03 )
enzyme retting results in a lighter fiber color than that by field retting , and various enzyme retting formulations resulted in different color characteristics based on the cielab l , a , and b values [ 123 , 124 ] .
enzyme mixtures that included cellulases , such as viscozyme l , weakened fibers ( figure 7 ) , as shown by previous work ( table 8) .
purer enzymes were becoming more readily available at this time , and research had showed that purified epg alone could separate fibers from core without the other cell wall - degrading enzymes .
further , results indicated these methods worked on both fiber - type and seed - type flax cultivars , indicating that these enzymes should be applicable to linseed straw .
commercial enzyme products , developed for various applications , were tested with the intent of finding purer pectinases ( i.e. , low or no cellulases ) that effectively retted flax without loss of fiber strength .
( franklinton , nc , usa ) released a commercial pectate lyase ( pl ) product for removing the cuticle of cotton fibers as an environmentally friendlier way of scouring cotton , which traditionally used high levels of naoh [ 125 , 126 ] .
bioprep 3000l is a liquid commercial pl produced by multiplying the native gene for alkaline pl in bacillus lichniformis , placing the genes back into the bacterium and allowing expression of these genes for high levels of enzyme production .
we used a product marketed under the trade name dextrol bioscour 3000 ( dexter chemical llc , bronx , ny ) , which was shown to separate the fibers ( figure 8) . at this time
, other enzymes were developed and applied especially for enzyme retting [ 127 , 128 ] .
fibers produced by various enzyme retting formulations were assessed through use of the fiber processing pilot plant and objective test methods .
all the enzymes were tested using suppliers ' recommendations for optimal activity . the fried 's test suggested initial levels and times for effective flax retting of these various enzymes and formulations .
texazym bfe effectively separated fiber from core at 2% , 5% , and 10% levels after 24 h ; only 10% bfe retted flax at 7 h. the addition of edta ( 18 mm concentration ) improved retting , showing effective fiber separation at 7 h for 5% bfe .
edta at 18 mm concentrations improved retting of all enzymes except dlg , which alone was ineffective in fiber separation by this method .
the effect of retting was further evaluated using bfe , dlg , multifect pectinase fe , and bioprep 3000 l in several modifications of formulas and retting conditions . while 1% bfe was effective at 24 h , the addition of edta facilitated enzyme retting with all levels of this enzyme .
the 2% level appeared to be effective enough to warrant further study , and temperatures in the 50 to 60c range were more effective than lower temperatures .
incubation of stems with dlg at 5% , even with edta , did not result in fiber separation by this test .
multifect pectinex fe was effective at 0.2% with edta , but not without the chelator ; lower levels were less effective than other enzymes even with edta .
addition of dlg as high as 0.5% did not improve fiber separation efficiency of 1% bfe plus edta by the fried 's test .
similarly , addition of xylanase , to treat animal feed and reported low in cellulase activity , included up to 0.15% with multifect fe plus edta did not improve fiber separation .
bioprep 0.05% at phs 8 and 9 and with chelators effectively separated fibers from the core .
many of the enzyme mixtures tested contain multiple types of enzymes active against plant cell walls , including cellulases .
a companion study was carried out to test the activities of several commercial polygalacturonases from various sources .
tests of enzyme activities and flax fiber properties , including strength , indicated different cellulase activities within these products that affected fiber properties . microscopic analysis and incubation of commercial flax fibers with these enzymes over several days
bioprep is listed as an alkaline pectate lyase , while the optimal conditions for activity of texazym , which is not identified as to type of pectinase , is similar to that for bioprep .
it should be noted that most of these commercial enzymes are not marketed for flax retting .
their use , for example , texazym dlg , however , could modify fiber and yarn properties as will be discussed later .
stems retted with various enzyme formulations by the ser method and with fiber processed through the pilot plant are shown in table 10 .
fine fiber yield was highest for texazym bfe and bioprep formulations but not significantly different from viscozyme plus mayoquest .
all enzyme - retted and shirley - cleaned samples were cleaner than unretted fiber , and differences were not large among the enzyme treatments .
differences , however , occurred in strength and fineness among retting formulations . further evaluation of the use of these enzymes for retting included the following : the amount of formulation uptake during brief ( i.e. , 2 min ) soaking , fine fiber yield , and cleanliness .
uptake of the amount of various formulations of bioprep was similar and about 300 ml ( ranging from 272 to 408 ml ) for 150 g initial fiber , giving a liquid - to - fiber ratio of 22.7 to 1 .
additional assessments tests were made on linseed varieties grown under commercial - type conditions in north dakota .
results showed that all retting enzymes were more efficient with chelating agents , particularly edta .
edta has substantial ca binding activity even at ph 5 , providing a positive effect of edta at low phs , which is optimal for some enzymes such as epg and viscozyme .
the binding capacity of edta for ca is , however , considerably greater at alkaline ph , and the use of edta at a higher ph should be more efficient in separating fiber from core .
it is well known , however , that pl requires ca for activity . the suggested method for cotton
scouring with bioprep is to first apply the enzyme and later apply the chelator ( s. salmon , novozymes , personal communication ) .
the enzyme retting methods developed earlier indicated that viscozyme could ret linseed varieties of flax but reduced fiber strength .
tests were conducted on the efficiency of bioprep and viscozyme to ret two linseed varieties , and fiber properties were determined .
hermes and omega were grown to full seed maturity under production conditions in north dakota .
stems were enzyme retted using formulations with bioprep or viscozyme in side - by - side tests ( table 11 ) .
the omega sample had rain prior to baling , and substantial weathering had occurred as indicated by darkening of the straw .
hermes , in contrast , was light and showed no effects of weathering prior to enzyme retting .
bioprep effectively retted both cultivars and resulted in higher fiber yield and fiber strength than viscozyme .
hermes was finer after retting with viscozyme plus chelator ( table 11 ) . in this test
, the chelator was used subsequent to the soaking with bioprep but with the viscozyme in a single solution .
tests for incubation times with bioprep , level of bioprep ( without chelator ) , and levels and incubation times of chelator were further assessed .
based on fine fiber yield and percentage shive content , incubation with bioprep for 1 h followed by incubation with 18 mm edta for 24 h was equal or better than other conditions .
retting effectiveness , however , improved with increased amounts of bioprep up to 0.5% , which was the highest level tested in this experiment and suggested that further increases in enzyme level may improve fiber separation .
furthermore , scanning electron microscopy of retted fibers indicated that bioprep levels of 5% appeared to remove more contaminants than 0.1% .
based on earlier results and general recommendations for bioscouring cotton with bioprep ( personal communication , s. salmon , novozymes north america , inc . ) , a series of evaluations was carried out to optimize the use of bioprep and edta for retting flax ( table 12 ) .
linseed variety hermes was selected for these tests . the recommendation for bioscouring cotton was to treat with bioprep about 15 min prior to adding chelators ( s. salmon , personal communication ) .
the use of mayoquest 200 to supply edta as chelator at 18 mm concentration , which had been determined from use with viscozyme , appeared to work adequately with bioprep . to further optimize the formulation and method for enzyme retting
, hermes was retted with a range of bioprep levels from 0.1% to 5% and followed by chelator or combined with chelator in the formulation .
the higher levels of fiber with the lower enzyme levels arise from fiber plus shive in varying amounts , as shown by predicted shive amounts . for shirley - cleaned fine fiber ,
bioprep at 1.0% to 5.0% followed by chelator produced the highest fiber yields and the lowest shive contents , ranging from 1.5% to 2.3% .
bioprep at 5% did not produce higher yields or cleaner fibers than 1% or 2% levels .
shirley - cleaned fibers do not represent all the fibers that could be extracted in commercial , cottonizing systems .
therefore , fiber yields from a single pass through the shirley analyzer were used only to rank enzyme formulations .
fiber strength was maintained at all levels of bioprep ( table 12 ) , showing a significantly greater strength than for fibers retted with viscozyme ( table 11 ) .
therefore , a major objective of enzyme retting with increased fiber strength was reached with pectate lyase followed by edta .
the commercial enzymes used in the present study represented a mixture of polysaccharidases , for example , cellulases and hemicellulases in some , as well as different types of pectinases .
viscozyme , or epg , and pectate lyase were effective in attacking pectin and retting flax , but the two enzymes have different optimal conditions for activity and different modes of action .
polygalacturonase ( pg ) and pectate lyase ( pl ) are both depolymerizing enzymes for pectin but work in different ways and under different conditions .
pg is reported to catalyze random hydrolysis of -1,4 polygalacturonic acid , and pl carries out a nonhydrolytic breakdown of pectates and pectinates by a trans - elimination split of the pectic polymer .
pl is activated by ca and usually is active at higher phs ( e.g. , 810 ) and temperatures ( 5560c ) than pg .
research has been carried out to correlate fiber separation with the degradation of different pectins , that is , various functional groups and linkages , using a series of commercial pgs and pl with low xylanase and cellulase activities .
retting efficiency was highly correlated ( correlation coefficient of 0.99 ) with sparsely esterified pectin , but correlations between retting and activities against other pectins were low . since ca binds acidic groups of pectin molecules and various types of pectin are in different regions of the bast [ 66 , 81 ] , these data further reveal a coordinated mechanism for degradation of nonesterified flax pectins with chelators and pectinases .
further , these and other spectroscopic data suggest that the pectins in the middle lamella and those binding the cuticle / epidermis to fiber bundles in flax stems are targeted by this mechanism . towards a more cost - effective enzyme retting system along these lines , other work indicated that weak acid with enzymes was effective in separating bast fibers with reduced enzyme levels , likely by removing the ca in pectin .
work with commercial pl has indeed shown efficiency in separating bast fiber from stems ( figure 8 , tables 1012 ) .
bioprep levels around 2% with 18 mm edta were optimal with the flax samples used and conditions tested ( table 12 ) .
sequential treatment of bioprep followed by edta was the most effective for retting , but combining both enzyme and edta also retted flax .
the procedure most effective for producing fine , clean fiber was as follows : ( a ) saturate crimped flax stems with bioprep at 2% , ( b ) incubate for 1 h at 55c , ( c ) without washing , resoak with 18 mm edta at ph 12 , ( d ) continue incubation at 55c for about 24 h total time , and ( e ) wash and dry fiber in preparation for mechanical cleaning . other work has shown the potential of alkaline pectinases such as pl to ret flax and the bast plant ramie [ 127 , 133 ] .
bioprep - treated fiber was not tested in the miniature spinning system or for biocomposites to this author 's knowledge .
an engineered pectate lyase from xanthomonas campestris , however , was developed and used at 37c and ph 8.5 to bioscour commercially grown and decorticated linseed flax [ 134136 ] .
pl - treated fibers were cleaner and finer than untreated fibers . in some biocomposites , pl - treated fibers performed better than similar but untreated fibers . other linseed straw samples , which had been left in the field for a few weeks and then decorticated and treated with x. campestris pl for various times up to 46 hr followed by chelator , did not result in improved biocomposites . still further tests of commercially decorticated linseed straw showed that pl - retted fibers , although finer and cleaner than untreated ones , performed better in tensile strength tests but not in interfacial shear strength tests .
further assessment is required to optimize use of bioprep and other pectate lyases as a retting enzyme for flax .
it is clear that many factors influence the successful production and processing of fibers for textiles and biocomposites .
further , while weak cellulases , such as those in viscozyme , reduced fiber strength , the resulting finer fibers often spun better than fibers produced by lower viscozyme levels in blends with cotton .
further assessment is required on fiber characteristics for specific applications ( e.g. , blended textiles or biocomposites ) , as well as the economics of enzyme retting .
it is clear , however , that pectate lyases can separate fiber of nonfiber components and retain fiber strength .
the designed purpose of bioprep as a cotton scouring agent , which acts by removing the cotton fiber cuticle , has shown to be effective in several large tests [ 125 , 126 ] .
the ars research on enzyme retting of flax had ended by 2012 , with the retirement of key individuals and closing of the usda pilot plant .
research and development continues for retting and other fiber applications with enzymes [ 127 , 134 ] .
inotex ( dvur kralove n.l . , czech republic ) has developed enzymes to assist with field retting , particularly towards producing consistent fibers in varying weather conditions and including use of oilseed straw ( j. marek , inotex , czech republic , personal communication ) .
genetics for plant modification of flax to improve fiber properties for linen and biocomposites are active areas of research . related to the idea of improved retting
, some research is focused on genetically modifying flax for improved fiber extraction from linseed stems ( michael deyholos , university of alberta , canada , personal communication ) .
one goal of another program , fibragen , is to identify genetic markers , including those determining anatomy and physical properties , for expanding flax markets in textiles and biocomposites ( jrg mssig , hochschule bremen , university of applied sciences , department for biometrics , personal communication ) .
advances in plant modification coupled with knowledge of specific action of enzyme systems for extracting fiber bode well for new systems to economically extract fibers of high and consistent quality and for directed purposes .
perhaps one of the most effective uses of enzymes may be in postharvest treatments of flax fibers to impart specific properties .
fiber - modifying enzymes are marketed for a variety of purposes , including enrichment of dew retting , repair of poor quality flax ( such as underretted material ) , cottonization of bast fiber tows for textiles or biocomposites , tailored fiber length , and processing of rovings to reduce noncellulosic content .
the use of enzymes pertinent to field retting includes spray applications after stalk pulling to minimize the effect of inclimate weather and to better utilize linseed stalks in europe ( j. marek , inotex , czech republic , personal communication ) .
weak cellulases may have applications where precision in limited attack on cellulose may be beneficial , such as for cottonization or shortening of fiber .
to this purpose , laboratory tests of flax pulp treated with commercial pectinases and cellulases showed improved characteristics of hand sheets compared to those prepared by traditional ( nonenzymatic ) methods .
for pulping , the breakdown of the fiber bundles by pectinases and the shortening of the cellulosic fibers by attack of cellulases at the fibernodes improved some paper properties in laboratory studies .
the nature of flax fibers , that is , the lack of limiting lignin in bundles and presence of susceptible fibernodes , provided opportunities for use of these enzymes not possible in highly lignified , woody sources of pulp .
the authors further suggested that a more precise attack by specific enzymes may provide additional attributes in the pulp .
research with an atomized enzyme delivery system showed that endoglucanases could be effectively delivered in small amounts onto field - retted fibers , likely resulting in attack at the fibernodes to reduce fiber length , strength , and elongation .
application of the atomized method with endoglucanase and extended to other enzymes modified the properties in flax fiber and in flax / cotton blended ( 50/50 ) yarns ( table 13 ) .
these enzymes were used as supplied , and such mixtures usually have multiple enzyme profiles against fibers [ 34 , 109 , 129 ] .
while further work is needed to assess specific activities , data suggest that all enzyme types were active in atomization , and various properties could be modified . for example
, lipase and arabinase improved certain yarn properties , such as increased strength and elongation and reduced neps and thick and thin places .
this paper has focused on flax structure and composition , with potential for enzyme retting .
emphasis has been placed on the nature of the flax bast fiber and bundle , specifically the lack of high levels of lignin , the binding of ca in pectin molecules in the cuticle , and the presence of the more susceptible fibernodes and kink bands within the fibers and bundles .
pectinases , either polygalacturonases or pectate lyases , alone are able to separate fibers from cuticle and core .
flax is just one of many bast plants that are economically important for myriad uses throughout the world .
ramie , which is a nonlignified , cellulose - rich bast fiber - like flax ( unpublished data ) , has been retted with pectin lyase .
enzyme retting of hemp , which is more heavily lignified than flax , showed some success , but different enzymes or protocols from those with flax were needed .
kenaf is highly lignified in the secondary walls and middle lamella of the bast fibers and bundles and has been retted by chemical means [ 140 , 141 ] .
use of a commercial enzyme , having cellulase and xylanase activities , with chelators and a crimping pretreatment separated the bast tissue to fiber bundles .
this process only produced coarse fiber bundles , and a delignifying process seems to be required for effective retting of kenaf . to this end ,
enzymes from noncellulolytic , lignin - degrading white rot fungi to remove aromatics and leave cellulose [ 142 , 143 ] may find applications .
successful , cost - effective , and commercial technologies will have requirements such as the following : selected flax material , enzyme formulations and conditions to tailor fibers with specific properties , integrated cleaning procedures , objective assessment methods to assure high and consistent quality , and directed applications .
the long , fine , and strong bast fibers provide apparel and other textiles , and other varieties of flax provide linseed and its oil .
the textile industry that once flourished in western europe has declined , but the desire for flax and linen is still strong .
quality fibers are still marketed in europe , and china and other regions desire more quality flax fibers for products .
even the paper and pulp industries desire cleaner fibers to reduce the amount of chemicals required for delignification .
biocomposites and nonwoven materials are predominant areas of interest , with the automotive industry continuing to focus on natural fiber composites . in particular , biocomposites are sought for weight and cost savings , improved structural properties , processing benefits , and design flexibility and ease .
compared to glass , flax fibers are lower in cost , lower in density , biodegradable , and similar in elongation at break ; tensile strength is lower for flax . woven flax fibers as insets with resins particularly provide good strength and rigidity in composites .
substantial savings in energy costs are possible with natural fiber mats , which reportedly require about 80% less energy than those made with glass .
the replacement of glass fibers with flax for this application , even with its important advantages , is nonetheless a considerable challenge .
consistency in supply and in fiber characteristics is required for flax fiber to expand further into markets , especially higher value - added products .
reportedly , the best flax fiber is still produced in western europe , where climate and grower experience provide quality fibers for textiles .
the drive for fibers in biocomposites and other industries has focused on getting flax fiber from nontraditional linen plants , namely , linseed straw .
field retting , which is the primary method of flax production , is problematic in that the fibers are often poor and inconsistent in quality .
climate is a major factor in quality , and outside western europe , the major areas of flax production are often in harsh climates for field retting .
it is in regard to all these factors that research has focused on other methods , including enzyme retting , to improve retting and thereby fiber processing and quality .
replacement methods for field retting have been sought for a long time , but currently , there are no such methods used commercially .
there have been positive results , and there is considerable interest in postretting and treatment of roving and yarn to improve their properties .
new developments in enzyme production by commercial companies have provided purer and more active pectinases that have promise in enzyme retting .
our work examined several commercial enzymes for retting and focused on the endopolygalacturonase - rich , mixed product viscozyme plus edta and the purer alkaline pectate lyase product bioprep followed by a commercial edta chelator .
protocols were developed on enzyme concentrations and conditions to separate fibers , which were then cleaned in a fiber processing pilot plant and characterized by objective test methods .
while bioprep - retted fibers had good properties of fineness , strength , and cleanliness , tests in textiles or composites have not been carried out .
the vast amount of research on enzyme retting indicates that pectinases without the need of complementary enzymes are effective in separating fiber from flax straw , even linseed straw .
the inclusion of a chelator , such as edta , greatly reduces the amount of enzyme required and is particularly effective in separating fibers from the cuticle - epidermis layer in linseed straw .
improving the quality and consistency of fiber from the huge biomass resource of linseed straw has great potential in addressing needs of myriad industries , even that of cottonized flax fiber for textiles .
the degree of processing for fiber cleanliness will depend upon the end product desired , and for some products , the requirements of cleanliness and processing costs are considerably less than for linen fabrics . the desire for quality apparel , however , continues to be important in flax and linen products , and improved processing methods should not overlook this important and historical industry . to this end , the crailar process reportedly uses enzymes in a proprietary process to produce soft , fine flax fibers for blending with cotton in an agreement with the hanes clothing industry .
one of the most important uses of enzyme might be in tailoring specific properties in postharvested materials to improve low - quality fibers .
fibers and seeds are two historical products of flax with traditional and continuing economic importance around the world .
it is likely , however , that the usefulness of flax will not be limited to just fiber and seed , as physical , chemical , and biotechnical methods uncover more products .
production of flax fibers by enzymatic or other means of retting , followed by mechanical processing , generates bast fibers for many industrial needs and massive amounts of by - product wastes .
this waste material , consisting of cuticle , shive , and fiber fragments , is already paid for and is localized at the processing plant .
mention has been made of lipids ( sterols , policosanol - type lipids , and waxes ) from the cuticle in dust and of activated carbons and extracted aromatics and sugars from the shive .
currently , there is a burgeoning interest in microcrystalline cellulose from plants and their potential for value - added products ranging from biocomposites for medical devices to solidified liquid crystals .
considerable work is still needed to overcome substantial problems and directed applications towards reaching the huge potential for cellulose nanocomposites . the highly crystalline and oriented nature of cellulose in flax fibers warrants consideration for its properties in nanotechnology .
the source , chemistry , structure , and crystalline nature of the native flax bast fibers , particularly in regard to the response to specific enzymes , may offer a contribution to this growing area of research and technology .
indeed , linen most useful , linum usitatissimum , is poised to continue to expand as a supplier of useful products to mankind throughout the world . | the components of flax ( linum usitatissimum ) stems are described and illustrated , with reference to the anatomy and chemical makeup and to applications in processing and products .
bast fiber , which is a major economic product of flax along with linseed and linseed oil , is described with particular reference to its application in textiles , composites , and specialty papers .
a short history of retting methods , which is the separation of bast fiber from nonfiber components , is presented with emphasis on water retting , field retting ( dew retting ) , and experimental methods .
past research on enzyme retting , particularly by the use of pectinases as a potential replacement for the current commercial practice of field retting , is reviewed .
the importance and mechanism of ca2 + chelators with pectinases in retting are described .
protocols are provided for retting of both fiber - type and linseed - type flax stems with different types of pectinases . current and future applications are listed for use of a wide array of enzymes to improve processed fibers and blended yarns .
finally , potential lipid and aromatic coproducts derived from the dust and shive waste streams of fiber processing are indicated . |
occlusal adjustment can be defined as the removal of occlusal interferences , through selective tooth grinding or through the use of restorative materials .
the aim of such an intervention is to obtain a stable occlusal relationship , with no premature contacts or mandibular excursion .
the adjustments can be performed in different situations , such as 1 ) after performing direct or indirect restorations , in order to establish a better relationship with the antagonist teeth , or 2 ) in the presence of non - uniform tooth wear , which might result in premature contacts and deviations on excursive mandibular movements .
several studies have evaluated the role of occlusal interferences on the etiology of temporomandibular joint disorders.110 nevertheless , there is no consensus on the importance of such a theme for the development of tmj dysfunctions .
the greater limitation on obtaining an appropriate explanation for the role of occlusal adjustment on the etiology of tmds is the number of variables associated with such dysfunction , which renders difficult the studies standardization and the consequent discussion of their results .
also , physiologic factors , traumas , articular disc dislodgment , condylar position , and pathologies can lead to tmds.11,12 given the elevated controversy and extreme importance of such a theme for clinical practice , it might be helpful for correct clinical management to review the information on the influence of occlusal interferences on the etiology of tmds , increasing the possibility of success in the treatment of this highly prevalent dysfunction .
studies have tried to establish the relationship between occlusal interferences and tmds . kopp13 analyzed the effect of professional advisement during occlusal therapy . during the first appointment for this study
, patients had received instructions only about their habits and about the possible causes of tmds . in a second appointment ,
one group of patients was submitted to occlusal therapy , and another group remained untreated , but received different instructions about their habits .
the results showed that both situations ( advisement and occlusal therapy ) resulted in decreased symptoms . in spite of its interesting findings ,
this study presented some limitations ; for example , the occlusal interventions consisted of several modalities of treatment , such as occlusal adjustment , installation of occlusal splints , and even complete dentures . because of these factors , it might be reasonable to assume that such results can not be directly extrapolated to the clinical management of patients with tmds . in 1986
forty - eighty patients were treated with occlusal adjustments , of which 19 used occlusal splints .
an additional 43 patients received a false occlusal adjustment , consisting of a placebo group .
symptoms associated with tmds were investigated , and a neurologic examination of headaches was performed .
the results demonstrated the reduction in clinical symptoms in both groups , although it was significantly greater for treated patients than for the ones from the placebo group .
thus , this study was able to effectively demonstrate the efficiency of occlusal adjustment in the remission of symptoms related to tmds .
the statistical analysis demonstrated that this reduction in symptoms happened irrespective of the use of occlusal splints , reaffirming the effectiveness of the occlusal adjustment .
these results were confirmed in another similar study,15 reinforcing the effectiveness of the occlusal adjustments . the effectiveness of the occlusal adjustment and the use of occlusal splints , whether or not associated with occlusal interventions , were evaluated by wenneberg et al.16 patients were randomly divided into two treatment groups and were examined after two months by means of questionnaires , visual analogical scales ( vas ) , and clinical exams .
the questionnaires indicated clinical improvement in the patients treated with both therapies , but the findings of the clinical exam were statistically significant only for patients from the mixed techniques group .
the authors concluded that the use of occlusal splints in combination with occlusal adjustment is more effective for the treatment of tmds than occlusal adjustments alone .
tmds are not restricted to headaches in the region of the temporal or masseter muscles .
the symptoms can be extended to the neck or to the superior region of the back .
karppinen et al8 investigated the importance of occlusal adjustment for the treatment of chronic neck- and headaches .
forty patients were selected after the observation of some criteria : need for neck and/or headache treatment , absence of clinical signs of tmd , nonexistence of removable prosthesis , and presence of posterior teeth .
patients were randomly allocated into two groups ( n=20 ) ; one group received occlusal adjustment , and the other received a false adjustment .
patients were analyzed after 6 weeks , 12 weeks and 60 months , using a visual analogical scale ( vas ) , a physical exam performed by a physiotherapist , an electromyography , and radiographies ; however , the last two were not performed during the 60-month exam .
the final analysis demonstrated that the occlusal adjustments were efficient in 80% of the cases after 60 months .
the physiotherapists observed a positive response in the occlusal adjustments as compared to the false adjustment , after comparing aches during head and neck movements .
the authors have concluded that occlusal adjustment can be effective for the treatment of head and neck chronic pains .
le bell et al3 performed a study in which patients presenting tmds ( 21 patients ) and those who did not ( 26 patients ) were distributed into four groups that received proper adjustments or simulations . during a two - week period
, patients were instructed to report the intensity of nine different symptoms daily , using the vas .
the symptoms were occlusal discomfort , chewing difficulties , sensitivity of teeth , fatigue of the jaw , headache , facial pain , opening difficulty , bruxism , and ear symptoms .
the results were statistically associated with the presence or absence of tmj disorders and to the implementation of occlusal adjustments .
patients with a history of tmds , as well as the ones that received occlusal interventions , presented greater symptoms than the other groups .
the most evident symptoms were occlusal discomfort and chewing difficulties , and the interaction between them was statistically significant .
patients with no history of tmds who had received occlusal adjustments demonstrated symptoms similar to those of patients with a history of tmds ; nevertheless , these symptoms decreased until the final observation period .
the authors have concluded that the different answers from patients with and without a history of tmds may suggest dissimilar vulnerability to occlusal interferences , indicating the need for a reassessment of the etiology of tmds .
the tmds are a multidisciplinary and multifactorial dysfunction , since factors like occlusal balance , vertical dimension , muscle function , and psychological aspects are involved .
twenty - six healthy women without a history of tmds and 21 women with a history were evaluated .
each group of patients was divided into two groups ( performance of occlusal adjustments or placebo ) , and evaluated before the occlusal adjustments with a questionnaire .
after the intervention , patients were examined for the level of psychological and somatic stress , coping strategies , personality characteristics , and health beliefs .
the authors concluded that health , health hardiness , a positive socialization history , social support - seeking , and inhibition of aggression are associated with better adaptation to occlusal interferences ; and patients with a history of tmds presented more prominent results compared to the other group .
occlusal interferences not only can affect the development and severity of tmds , but also can influence the body posture and equilibrium . this postural instability can lead to damage , especially in patients who need the equilibrium to perform their actions adequately.17 gangloff et al17 evaluated the influence of occlusion on the postural control and gaze stabilization of professional shooters .
thirty - six patients were selected ; nevertheless , in the 6 months before the study , not all patients presented the muscular , articular , and osseous pains that could interfere with postural control .
patients with meniscus subluxation or luxation of the temporo mandibular joint were excluded from the study .
eighteen volunteers with great oral hygiene were included in the control group for postural evaluation .
the test group was completed by the high - level shooters , who were investigated for gaze stabilization and postural control . for the evaluation ,
four mandibular positions were used : first , intercuspal occlusion ( io ) , defined as the interdigitation of all teeth without any type of manipulation ; this is a neutral position , but not a symmetric one .
the other three positions were tested by means of acrylic devices : centric relation ( cr ) and left and right laterality . the postural evaluation through posturographic measurements ( qfp systemes , nice , france )
were recorded in the same place and under the same environmental conditions for all control group subjects , and the results were statistically analyzed .
for each occlusal relation , the test was conducted with opened and blinded eyes . in the professional shooters group ,
four series of five shots ( shooting distance : 10 m ) were performed in each of the four predefinite mandibular positions .
the best results were obtained in rc , followed by io , then lateral occlusion .
the authors have suggested that the mandibular position can be important to a high level sportsman who requires optimum balance control and posture for a better performance .
the effect of different mandibular positions on postural stability was also evaluated by bracco et al.18 ninety - five subjects , with no signs and symptoms of temporomandibular disorder , were evaluated .
the posture of patients was analyzed in three different occlusal positions : maximum intercuspidation ; rest position , which is the habitual postural position of the mandible when at rest , with the condyles in a neutral , unstrained position in the glenoid fossa ; and myocentric position , which is most commonly between 1 and 2 mm of vertical closure from the physiologic rest position that occurs when postural and masticatory muscles are simultaneously at their resting length and in balanced tonus with respect to one another and to a selected terminal contact therapeutic position .
the authors concluded that a good relation between chewing muscles and head and neck seems to be an important factor for postural stability .
cuccia and caradonna19 performed a review in which they searched such databases as medline , pubmed , embase , web of sciences , cochrane library , cinahl , and health star for information on issues like body posture , temporomandibular disorders , and chewing muscles .
the authors analyzed these and other issues , making correlations with the findings and demonstrating the importance of the theme and the need for further studies in this area .
they have suggested that an interdisciplinary approach to tmds , involving diverse specialists like dentists , physiotherapists , orthopedists , and psychologists , is fundamental for a broad , reliable , and successful treatment of the subject .
tmds are pathologic conditions involving the masticatory muscles and head and neck postural muscles or , at the least , a combined pathologic condition of the muscles and temporo - mandibular joints.18 considering this definition , the treatment of tmj disorders by addressing the condylar position in relation to the glenoid fossa or to the chewing muscles like an individual approach , have a great chance of being unsuccessful , but may be necessary in some situations . in accordance with the literature , occlusal interferences can play an important role in the development of tmds.3,4,20,21 these interferences can be formed by uneven tooth wear , but also by restorative procedures performed incorrectly , which can leads to a disharmonic relation between the arches . therefore , after conducting direct or indirect restorations , a careful analysis of occlusal contacts should be performed , in order to avoid the creation of iatrogenic interferences that can produce the signs and symptoms of tmds and postural disorders .
an occlusal interference can cause the alteration of muscular tonus , which can lead to pains in chewing and in the head and neck muscles .
patients with no history of tmds are less vulnerable to occlusal alterations;3 nevertheless , in vulnerable patients , the symptoms can be exacerbated if the occlusal adjustment of rehabilitations is wrongly performed , compromising the harmonic relation between the arches .
if the trigeminal afferences and proprioception are altered , these disorders can generate , in descending action , into an imbalance of postural muscles chains , finally causing postural modifications.18 from the information gathered , it seems to be evident that occlusal interferences can lead to the development of or to an increase in the severity of tmds .
since occlusal therapy could induce a re - equilibrium of masticatory muscles , this re - equilibrium could influence , in descending action , the whole body s postural muscles , resulting in an improved posture.18 also , the relation between the upper and inner arches and the temporomandibular joint can generate alterations in the gaze stabilization and in the body posture , which indicate that the relevance of the occlusal adjustment on the treatment of tmds should be reevaluated .
the authors should clarify that not only occlusal adjustments can be considered in tmd treatment , but also the use of occlusal splints , psychological therapy , and physiotherapist treatment .
each case should be considered singly and analyzed according the clinical situation . as a further consideration ,
adjustments should be conducted only after the influence of interferences on the disorder has been confirmed through evaluating the signs and symptoms , and never in a prophylactic manner.1 finally , a multidisciplinary approach should be employed in order to provide the most correct treatment and to achieve a greater likelihood of success . | this brief review discusses the role of occlusal interferences on the development and progression of temporomandibular dysfunction ( tmd ) and postural disorders .
the eligible literature is described and critically presented to demonstrate that occlusal interferences are an important factor in the risk of tmd .
several studies have demonstrated through their findings that the use of occlusal adjustments , whether or not associated with restorative procedures , might avoid the development of articular problems in vulnerable patients .
the occlusal interferences caused by restorative procedures or orthodontic treatment can cause tmds in susceptible patients , and occlusal adjustments can be an alternative in the treatment of these tmds . |
gene expression levels during adipocyte differentiation were obtained by stimulating the sgbs pre - adipocyte cell line with a mix of differentiation inducing compounds and collecting rna samples at 0 , 4 , 8 and 12 h and on days 1 , 3 and 12 of adipogenesis for hybridization on illumina ht-12 microarrays .
triplicate samples were prepared following the differentiation protocol modified from ( exception is 12 h time point that has only duplicate samples ) as shown in table 1 .
sgbs cells differentiate within 1012 days as determined by microscopic analysis ( oil red o staining ) . at this time point the cells are filled with small sized lipid droplets and are most responsive , whereas at later time points ( 20 days ) the lipid droplets fuse and cells are less active ( personal communication , dr .
specifically , sgbs cells were cultured in dulbecco 's modified eagle 's medium ( dmem)/nutrient mix f12 ( gibco ) containing 8 mg / l biotin , 4 mg / l pantothenate , 0.1 mg / mg streptomycin and 100 u / ml penicillin ( of medium ) supplemented with 10% fbs in a humidified 95% air/5% co2 incubator .
the cells were seeded into 10 cm plates , which were coated with a solution of 10 l / ml fibronectin and 0.05% gelatine in phosphate - buffered saline .
confluent cells were cultured in serum - free of medium for 2 days followed by stimulation to differentiate with of media supplemented with 0.01 mg / ml human transferrin , 200 nm t3 , 100 nm cortisol , 20 nm insulin , 500 m ibmx and 100 nm rosiglitazone ( cayman chemicals ) .
after day 4 , the differentiating cells were kept in of media supplemented with 0.01 mg / ml human transferrin , 100 nm cortisol and 20 nm insulin .
1 ml of trisure was added per a confluent 10 cm dish to lyse the cells .
rna was extracted with 200 l chloroform and precipitated from the aqueous phase with 400 l isopropanol by incubating at 20 c overnight .
the longer isopropanol incubation allowed the precipitation of micrornas and other small rnas from the same samples .
the total rna samples were processed according to the manufacturer instructions to prepare cdna that was hybridized on microarrays ( turku centre for biotechnology , microarray and sequencing facility , turku , finland ) .
total rna integrity was confirmed using an agilent 2100 bioanalyzer ( agilent technologies , santa clara , ca , usa ) . the raw data files were processed and quality controlled using the r / bioconductor lumi package .
control probe data was included and used to background correct the signal values with the lumib bgadjust method .
we provide this data and sample data in a format that is directly compatible with the lumi analysis package through our web resource at http://systemsbiology.uni.lu/idare.html .
the data was then transformed with the vst method and normalized with robust spline normalization ( rsn ) method . the probe intensity value distribution and sample relation
are plotted in fig . 1 , fig . 2 , with sample naming described in table 1 .
no outliers were detected based on data value range at this step and the samples clustered according to the biological sample group .
the code that can be used to download processed data from geo or to process them from the files that we provide through our website is available ( see additional data file 1 ) .
only genes that had a detection p - value < 0.05 within all samples of at least one time point were selected for statistical analysis , resulting in a total of 12 756 detected probes .
the f - test was used to assess significance of overall dynamic response over the differentiation while a two - tailed t - test was performed to compare specific time points to day 0 undifferentiated cells . in both analyses benjamini
hochberg adjusted p - value < 0.01 was considered statistically significant . in total , 1936 refseq transcripts changed their expression more than 2-fold up or down during the differentiation time series .
the code that can be used to filter non - expressed genes and to perform the statistical analysis is available ( see additional data file 1 ) .
several of these genes were metabolic genes , represented by 2-fold more differentially expressed genes compared to other gene categories with similar numbers of genes ( extracted from the go online sql environment , as of 12th of august 2013 : cell projection , envelope , locomotion and receptor activity ) .
the annotation data from recon1 was obtained and checked against the current entrezgene and refseq annotations ( hg19 refseq ; feb 02 2012 ) .
the reaction to gene mappings was updated with current gene ids ( see table s1 ) . withdrawn ids and pseudogenes present a difficulty in the recon1 annotation .
as there were only few such genes ( see table s1 ) , they were left out from visualizations and assigned expression level 0 in modeling .
lpin1 was missing and due to its central role in adipocytes , it was added to the triacylglycerol pathway reaction catalyzed by phosphatidic acid phosphatase ( ppap ) .
the expression profiles of metabolic genes ( from recon 1 ) or tfs ( from ) were clustered for visualization using self - organizing maps ( gedi software ) and autosome as instructed in the tool documentation . the settings to reproduce the results presented in
were the following : gedi grid size was adjusted based on input gene number and settings were tuned in order to minimize data missing grid points ( gene density map ) ( see table s2 ) .
clustering was done for columns ( samples ) on precision mode , with the fuzzy cluster network option and network visualization with cytoscape .
a consistent version of the generic human metabolic model recon1 was used as modeling platform for prediction of network activity distributions .
the recon1 model was downloaded from the bigg database ( 04.11.11 ) and the consistent version was derived using the function reducemodel from the cobra toolbox 2.0 , which resulted in the exclusion of 1273 reactions ( 34% ) of the initial model ( table s3 ) . to include the microarray data as soft - constraints for reaction activity prediction , the probes were mapped to entrez gene ids .
first , continuous log2 normalized expression values for the probes were discretized into three categories : lowly expressed ( 1 ) , moderately expressed ( 0 ) and highly expressed ( 1 ) based on the mean expression 0.5 standard deviation cutoffs across all arrays . then ,
one unique discretized value per gene was selected taking the rounded discretized mean of all probes for a gene .
each gene was then assigned to the recon1 reaction based on gene protein - reaction associations .
this dataset is comprised of whole transcriptome gene expression profiling data derived using the illumina beadarrays .
moreover , discretization of the metabolic gene expression levels allowed using them as soft - constrains for metabolic activity modeling .
further , this dataset is part of a geo superseries ( gse41578 ) and we have used it in combination with next - generation sequencing data and microrna expression profiles to associate putative regulators to the metabolic genes in . to further analyze the data in an integrative manner , we introduced gene metanodes and the web portal idare ( integrated data nodes or regulation ) in for interactive data exploration of various data types within the metabolic network context , available at http://systemsbiology.uni.lu/idare.html , including a detailed user guide .
the following are the supplementary data related to this article.table s1reaction to gene mapping to current gene ids.table s2setting for gedi visualization.table s3blocked reactions in recon1.supplementary material 1the script for transforming and normalizing illumina datasets.supplementary material 2the script to perform statistical analysis of gene expression .
reaction to gene mapping to current gene ids . setting for gedi visualization . blocked reactions in recon1 .
gene expression levels during adipocyte differentiation were obtained by stimulating the sgbs pre - adipocyte cell line with a mix of differentiation inducing compounds and collecting rna samples at 0 , 4 , 8 and 12 h and on days 1 , 3 and 12 of adipogenesis for hybridization on illumina ht-12 microarrays .
triplicate samples were prepared following the differentiation protocol modified from ( exception is 12 h time point that has only duplicate samples ) as shown in table 1 .
sgbs cells differentiate within 1012 days as determined by microscopic analysis ( oil red o staining ) . at this time point the cells are filled with small sized lipid droplets and are most responsive , whereas at later time points ( 20 days ) the lipid droplets fuse and cells are less active ( personal communication , dr .
specifically , sgbs cells were cultured in dulbecco 's modified eagle 's medium ( dmem)/nutrient mix f12 ( gibco ) containing 8 mg / l biotin , 4 mg / l pantothenate , 0.1 mg / mg streptomycin and 100 u / ml penicillin ( of medium ) supplemented with 10% fbs in a humidified 95% air/5% co2 incubator .
the cells were seeded into 10 cm plates , which were coated with a solution of 10 l / ml fibronectin and 0.05% gelatine in phosphate - buffered saline .
confluent cells were cultured in serum - free of medium for 2 days followed by stimulation to differentiate with of media supplemented with 0.01 mg / ml human transferrin , 200 nm t3 , 100 nm cortisol , 20 nm insulin , 500 m ibmx and 100 nm rosiglitazone ( cayman chemicals ) .
after day 4 , the differentiating cells were kept in of media supplemented with 0.01 mg / ml human transferrin , 100 nm cortisol and 20 nm insulin .
total rna was extracted using trisure ( bioline ) . 1 ml of trisure was added per a confluent 10 cm dish to lyse the cells .
rna was extracted with 200 l chloroform and precipitated from the aqueous phase with 400 l isopropanol by incubating at 20 c overnight .
the longer isopropanol incubation allowed the precipitation of micrornas and other small rnas from the same samples .
the total rna samples were processed according to the manufacturer instructions to prepare cdna that was hybridized on microarrays ( turku centre for biotechnology , microarray and sequencing facility , turku , finland ) .
total rna integrity was confirmed using an agilent 2100 bioanalyzer ( agilent technologies , santa clara , ca , usa ) .
the raw data files were processed and quality controlled using the r / bioconductor lumi package .
control probe data was included and used to background correct the signal values with the lumib bgadjust method .
we provide this data and sample data in a format that is directly compatible with the lumi analysis package through our web resource at http://systemsbiology.uni.lu/idare.html .
the data was then transformed with the vst method and normalized with robust spline normalization ( rsn ) method . the probe intensity value distribution and sample relation
are plotted in fig . 1 , fig . 2 , with sample naming described in table 1 .
no outliers were detected based on data value range at this step and the samples clustered according to the biological sample group .
the code that can be used to download processed data from geo or to process them from the files that we provide through our website is available ( see additional data file 1 ) .
only genes that had a detection p - value < 0.05 within all samples of at least one time point were selected for statistical analysis , resulting in a total of 12 756 detected probes .
the f - test was used to assess significance of overall dynamic response over the differentiation while a two - tailed t - test was performed to compare specific time points to day 0 undifferentiated cells . in both analyses benjamini
hochberg adjusted p - value < 0.01 was considered statistically significant . in total , 1936 refseq transcripts changed their expression more than 2-fold up or down during the differentiation time series .
the code that can be used to filter non - expressed genes and to perform the statistical analysis is available ( see additional data file 1 ) .
several of these genes were metabolic genes , represented by 2-fold more differentially expressed genes compared to other gene categories with similar numbers of genes ( extracted from the go online sql environment , as of 12th of august 2013 : cell projection , envelope , locomotion and receptor activity ) .
the annotation data from recon1 was obtained and checked against the current entrezgene and refseq annotations ( hg19 refseq ; feb 02 2012 ) . the reaction to gene mappings
was updated with current gene ids ( see table s1 ) . withdrawn ids and pseudogenes present a difficulty in the recon1 annotation .
as there were only few such genes ( see table s1 ) , they were left out from visualizations and assigned expression level 0 in modeling .
lpin1 was missing and due to its central role in adipocytes , it was added to the triacylglycerol pathway reaction catalyzed by phosphatidic acid phosphatase ( ppap ) .
the expression profiles of metabolic genes ( from recon 1 ) or tfs ( from ) were clustered for visualization using self - organizing maps ( gedi software ) and autosome as instructed in the tool documentation . the settings to reproduce the results presented in
were the following : gedi grid size was adjusted based on input gene number and settings were tuned in order to minimize data missing grid points ( gene density map ) ( see table s2 ) .
clustering was done for columns ( samples ) on precision mode , with the
a consistent version of the generic human metabolic model recon1 was used as modeling platform for prediction of network activity distributions .
the recon1 model was downloaded from the bigg database ( 04.11.11 ) and the consistent version was derived using the function reducemodel from the cobra toolbox 2.0 , which resulted in the exclusion of 1273 reactions ( 34% ) of the initial model ( table s3 ) . to include the microarray data as soft - constraints for reaction activity prediction , the probes were mapped to entrez gene ids .
first , continuous log2 normalized expression values for the probes were discretized into three categories : lowly expressed ( 1 ) , moderately expressed ( 0 ) and highly expressed ( 1 ) based on the mean expression 0.5 standard deviation cutoffs across all arrays . then , one unique discretized value per gene was selected taking the rounded discretized mean of all probes for a gene .
each gene was then assigned to the recon1 reaction based on gene protein - reaction associations .
this dataset is comprised of whole transcriptome gene expression profiling data derived using the illumina beadarrays .
moreover , discretization of the metabolic gene expression levels allowed using them as soft - constrains for metabolic activity modeling .
further , this dataset is part of a geo superseries ( gse41578 ) and we have used it in combination with next - generation sequencing data and microrna expression profiles to associate putative regulators to the metabolic genes in . to further analyze the data in an integrative manner , we introduced gene metanodes and the web portal idare ( integrated data nodes or regulation ) in for interactive data exploration of various data types within the metabolic network context , available at http://systemsbiology.uni.lu/idare.html , including a detailed user guide .
the following are the supplementary data related to this article.table s1reaction to gene mapping to current gene ids.table s2setting for gedi visualization.table s3blocked reactions in recon1.supplementary material 1the script for transforming and normalizing illumina datasets.supplementary material 2the script to perform statistical analysis of gene expression .
reaction to gene mapping to current gene ids . setting for gedi visualization . blocked reactions in recon1 . | obesity is an ever - growing epidemic where tissue homeostasis is influenced by the differentiation of adipocytes that function in lipid metabolism , endocrine and inflammatory processes .
while this differentiation process has been well - characterized in mice , limited data is available from human cells . applying microarray expression profiling in the human sgbs pre - adipocyte cell line
, we identified genes with differential expression during differentiation in combination with constraint - based modeling of metabolic pathway activity . here
we describe the experimental design and quality controls in detail for the gene expression and related results published by galhardo et al . in nucleic acids research 2014 associated with the data uploaded to ncbi gene expression omnibus ( gse41352 ) . |
small - cell lung cancer ( sclc ) , which comprises approximately 1015% of all lung cancers , is a neuroendocrine tumor , previously known as oat cell carcinoma , that is closely linked with tobacco smoking and characterized by an aggressive and rapid pattern of metastases , limited treatment options following progression , and overall poor prognosis .
the disease is traditionally divided into two stages : limited - stage disease ( ld ) , confined to one hemithorax encompassable in a single radiation port , and extensive - stage disease ( ed ) , extending beyond those boundaries , which comprise the vast majority of patients at initial diagnosis [ 5 , 6 ] . for extensive - disease sclc
, the standard first - line regimen is etoposide - platinum ( ep ) doublets followed by topotecan , approved for second - line treatment , since progression is the rule after first - line ep despite the fact that sclc is initially chemosensitive ( with response rates to first - line treatment on the order of 7090% in ls disease and 5060% in ed disease ) . in ed , despite a response rate of 1528% to topotecan , the median os rarely exceeds 6 months . according to the current nomenclature
sensitive relapsed ( pfs <3 months ) , resistant ( pfs <3 months ) and refractory ( progression through first - line treatment ) . in general ,
subsequent readministration of ep doublets is contraindicated in resistant and refractory disease due to the likelihood of undue toxicities ( myelosuppression and nephrotoxicity , for example ) without benefit , since therapeutic resistance in these contexts is , with exceptions , construed as a stable and immutable trait .
pretreatment or priming with epigenetic inhibitors , like the approved dna demethylators , e.g. , 5-azacitidine ( azacitidine , aza ) and 2-deoxy-5-azacitidine ( decitabine , dac ) and the histone deacetylases , e.g. , vorinostat and romidepsin as well as experimental rrx-001 , has been identified as a nascent strategy , still in its infancy , to attenuate or reverse resistance to chemotherapy and immunotherapy [ 12 , 13 , 14 ] , particularly in non - small cell and ovarian cancers .
rrx-001 , in particular , has demonstrated evidence of episensitization to refractory irinotecan in the context of a phase ii clinical trial entitled rocket in metastatic colorectal cancer [ 16 , 17 , 18 ] .
triple threat ( nct02489903 ) is an ongoing open - label phase ii trial in non - small lung cancer , sclc and high - grade neuroendocrine tumors , which investigates a resensitization strategy to previously administered , but now refractory , platinum doublets following treatment with and progression on ( by recist vs. 1.1 criteria ) single agent rrx-001 mixed ex vivo with 100 ml of autologous blood prior to infusion .
the rationale for this unusual method of administration is related to the red blood cell partitioning of rrx-001 , which binds with high affinity to a cysteine residue in the -chain of hb ( cys 93 ) , displacing nitric oxide ( no ) in the process .
when no is displaced in vivo via standard intravenous infusion , it stimulates venous nociceptors , with a mild to moderate sensation of pain or discomfort for patients ; however , in the infusion bag , mixed with blood , the no is reabsorbed by the red blood cells , which appears to completely abrogate the hyperalgesia , resulting in a well - tolerated and rapid ( ~30 min ) infusion .
herein , we report a case of a patient with extensive refractory ed - sclc who was episensitized to cisplatin and etoposide , with a partial response , after treatment with weekly intravenous rrx-001 autologous blood mix .
patient 001001 , a 49-year - old male veteran , former smoker , with a diagnosis of extensive - stage sclc , completed six cycles of carboplatin and etoposide in december 2012 . having progressed approximately 6 months later with
sensitive relapsed ( pfs > 3 months ) disease , he was enrolled on a clinical trial at the nih in july 2013 where he received six cycles of cisplatin and etoposide followed by maintenance with the hdac inhibitor belinostat . approximately 1 year after completion of the nih trial , with symptoms of fatigue and inappetence due to disease progression as well as dyspnea and decreased exercise tolerance from a large unilateral pleural effusion , he enrolled as the first patient ( 001001 ) on the triple threat clinical trial at walter reed in june 2015 .
after the first dose of rrx-001 , and continuing for the next 89 weeks , the patient reported a significantly increased appetite for and consumption of fried foods , specifically pork chops , and desserts , which led to dyspeptic symptoms .
his fatigue , shortness of breath and exercise tolerance also significantly improved to the point that he resumed uphill running and lifting weights . a ct scan at 6 weeks ( fig .
1 ) , correlating with the symptomatic improvement , demonstrated near complete resolution of the unilateral malignant pleural effusion . in addition
unfortunately , nearly 10 weeks after the start of triple threat , patient 001001 developed
travelers thrombosis and pulmonary embolism presumably from a prolonged ( 10 h ) car ride , which prompted the pi to discontinue rrx-001 on suspicion of symptomatic progression since the patient began to complain of fatigue and inappetence as well as dyspnea associated to pulmonary embolism , even though repeat ct scans did not confirm radiologic progression . at this point , per protocol , since the patient still met all inclusion / exclusion criteria , he was rechallenged for a third time with ep doublets and , despite expected toxicities of fatigue and anorexia , he reported improved performance status . at 6 weeks after reintroduction of therapy , repeat ct scans demonstrated a partial response with an approximate 32% decrease in tumor size ( fig .
one month later , a confirmatory scan showed a 50% decrease in tumor size , indicative of continued cytotoxic activity .
to the best of our knowledge , triple threat is the first and only clinical trial to investigate resensitization or episensitization , since rrx-001 is an epigenetic inhibitor in sclc as well as nsclc and neuroendocrine tumors . as a highly aggressive tumor with a uniformly dismal prognosis and an unmet need for effective therapeutic options
, sclc is extremely difficult to treat : the median survival time following recurrence rarely exceeds 6 months , the 2-year survival is < 10% , and there are virtually no 5-year survivors , requiring innovative strategies to address the difficult challenge of salvage treatment of relapsed sclc .
dna methyltransferase and hdac inhibitors have demonstrated the potential to reverse therapeutic resistance in multiple tumor types because epigenetic alterations , an important driver of carcinogenesis and progression , are , in theory , reversible . paradoxically , these alterations are also stably maintained through mitotic cell division , leading to a kind of epigenetic memory , with priming of responses to temporally separate and subsequent , rather than simultaneous , therapy since the induced pattern of gene transcription persists .
the clinical course of this sclc patient ( 001001 ) suggests , with the usual caveats about making generalizations from
n - of-1 results , that reintroduction of etoposide - platinum after initial treatment with rrx-001 , leading to episensitization , is a safe and effective strategy for solid tumor types previously treated with platinum doublet - based chemotherapy .
the phase ii triple threat clinical trial is ongoing , having enrolled 5 patients to date , none of whom , besides patient 001001 , have progressed on rrx-001 , and their data will also be reported , if episensitization , anticipated here as elsewhere , is demonstrated .
any subjects have given their informed consent in the study and the study protocol has been approved by the relevant institute 's institutional review board , also known as an independent ethics committee , ethical review board , or research ethics board .
the authors disclose that the clinical trial in which this case was observed is funded by epicentrx , inc . | rrx-001 is a pan - active , systemically nontoxic epigenetic inhibitor under investigation in advanced non - small cell lung cancer , small - cell lung cancer and high - grade neuroendocrine tumors in a phase ii clinical trial entitled triple threat ( nct02489903 ) , which reexposes patients to previously effective but refractory platinum doublets after treatment with rrx-001 .
the purpose of this case study is first to report a partial response to carboplatin and etoposide in a patient with small - cell lung cancer pretreated with rrx-001 , indicating episensitization or resensitization by epigenetic mechanisms , and second to discuss the literature related to small - cell lung cancer and episensitization . |
the successful isolation of helicobacter pylori ( h. pylori ) infection from the stomachs of patients with chronic gastritis and peptic ulcer disease in 1983 has fundamentally changed our concept of the etiology , pathogenesis and management of upper gastrointestinal ( gi ) diseases .
nowadays h. pylori is thought to be one of the most important factors in the pathogenesis of upper gastroduodenal diseases , particularly in developing countries .
h. pylori is a common bacterium that infects about half of the world s population .
the prevalence of h. pylori varies by geographical location , ethnic background , socioeconomic conditions and age .
h. pylori is the etiology of peptic ulcer disease , gastritis , gastric mucosa - associated lymphoid tissue lymphoma and gastric adenocarcinoma and is present in 95 - 99% of duodenal ulcers and most gastric ulcers .
studies from both the northern and southern regions of iran have demonstrated a high rate of h. pylori infection with the frequent rate of development of duodenal ulcer and gastric cancer .
ghanaei and colleagues found a 40% prevalence of h. pylori among children in northern iran .
the majority of patients infected with h. pylori initially present with dyspeptic symptoms , with or without alarming signs .
in this case , many can and should be treated for h. pylori infection even though , in the absence of endoscopy , the primary care physician ( pcp ) may not have an accurate diagnosis of the underlying disease pathology .
numerous initiatives have been undertaken to educate health care professionals regarding the appropriate diagnosis and management of this infection .
however , results from several recent surveys have suggested that significant confusion and discrepancies are present in the thinking concepts of specialists and gps with respect to the understanding of the pathogenesis , diagnosis and treatment of h. pylori infection .
the major uncertainties surround the management of patients with dyspepsia when the pcp needs to make a decision between testing for h. pylori infection and treating if positive , or the referral of patients to specialists .
because general practitioners ( gps ) are more available than internists to most patients , the present study elucidates the knowledge and practice of gps and internists about diagnosis and treatment of h. pylori infection in order to help future educational strategies about this infection .
this cross - sectional study was performed over an 8 month period from april 2009 until december 2009 in rasht , guilan , a northern iranian province .
the questionnaire was derived by searching textbooks and different articles , and was created and validated by a panel of experts of guilan and tehran universities of medical sciences .
its reliability was documented by a test - retest study , and the validity and reliability was confirmed by cronbach s alpha ( alpha = 0.7 ) .
the demographic part consisted of questions on sex , specialty ( gp or internist ) , work place ( public or private ) , work history , source of information and participation in continuing medical education programs .
the knowledge part consisted of 14 questions about the prevalence of h. pylori infection in the population , its role in peptic ulcer disease , dyspepsia and gastric cancer .
finally , in the practice part , six questions were asked in an interview format . if the physicians answered yes to the first question they were requested to answer the remainder of the questions about its diagnosis and treatment methods .
scores higher than the mean score indicated good knowledge or attitude and lower than the mean score indicated poor knowledge and attitude .
knowledge questions were completed by physicians , themselves , whereas the practice questions were asked by two researchers in interviews .
physicians who were not interested in participating in the study were excluded , and the identity of all participants remained secret .
data were collected and analyzed by spss version 14 by descriptive statistics and compared means .
the mean ( sd ) knowledge and practice scores of participants were 12.13.13 and 2.371.54 , respectively .
two hundred eighty six ( 67.1% ) gps and 44 ( 91.7% ) internists scored above the mean value for knowledge ( p = 0.001 ) .
the number of gps and internists who achieved above the mean value for practice was 305 ( 72.4% ) and 46 ( 95.8% ) , respectively ( p < 0.001 ) .
the best knowledge scores were achieved for the question about the relationship of h. pylori infection and peptic ulcer disease , both among gps ( 90.7% ) and internists ( 90.9% ) . in practice questions ,
67.7% of gps and 60.4% of internists correctly answered the diagnosis - related question and 67.9% of gps and 70.8% of internists correctly answered the question regarding type of medical treatment . the most common suggested diagnostic test ordered was serologic exam by gps ( 66.5% ) and endoscopy by internists ( 91.7% ) .
both gps ( 71.6% ) and internists ( 60% )
gps mostly preferred ( 60% ) a combination of amoxicillin , omeprazole , metronidazole and bismuth .
internists , however , ordered a combination of triple or quadruple therapies that consisted of amoxicillin , metronidazole , omeprazole and bismuth ( 20% ) ; amoxicillin , tetracycline , metronidazole and bismuth ( 2.2% ) ; amoxicillin , omeprazole and clarithromycin ( 6.8% ) ; amoxicillin , furazolidone , omeprazole and bismuth ( 8.8% ) ; or omeprazole , amoxicillin , clarithromycin and bismuth ( 2.2% ) . among both gps and specialists , the most prevalent source of information was a combination of media ( 61% ) and continuing medical education programs ( 54% ) .
mean results for knowledge and practice scores according to sex , specialty , workplace , working history , source of information and participation in continuing medical education programs are reported below .
after statistical analysis and based on lsd post hoc , physicians who used books or educational programs achieved a significantly higher mean knowledge score compared to others .
those physicians with a working history of more than 10 years had a lower knowledge score than those with less working history ( table 2 ) .
ns : not significant
in this study , the mean practice score for internists was significantly higher than gps .
the mean practice score of physicians who worked in public units was higher than those who worked in private units .
those physicians who had a working history of less than 5 years had a lower practice score than physicians who had more working history ( table 3 ) .
those physicians who used books or attended educational programs had a significantly higher mean practice score than those who used media for education ( table 3 ) .
overall , there was significant correlation between physicians mean knowledge and practice scores ( r = 0.2 ) .
over the past 17 years , the status of h. pylori infection and its relation to upper gi diseases has evolved from a medical curiosity to a well - recognized and extensively investigated pathogen of considerable clinical significance .
because of the extremely high prevalence of h. pylori infection in the general population in many countries and the existence of several controversies , a proper and efficient approach to the management of h. pylori infection is crucial .
there are several surveys which have suggested that significant confusion and discrepancies are present in the concepts of specialists and gps regarding their understanding of h. pylori infection and its pathogenesis , diagnosis , and treatment .
this study aimed to gauge the current practices , understanding and approaches of gps and internists regarding the diagnosis and management of h. pylori infection in a city in northern iran that has a high prevalence of h. pylori infection .
in the present study ,
the main source of information was a combination of media and continuing medical education programs .
in ahmed s study , medical journals were the source of information for 32% of gps , while in canbaz s study in turkey the main source of information to gps were pharmaceutical company sponsored symposia .
in the study by behnoud and colleagues ,
26.7% of gps and 25.9% of the internists knew about the role h. pylori in peptic ulcers and in a study by darvish - moghaddam , 41.5% of the gps and 9.4% of internists were knowledgeable about h. pylori .
however , the present data suggested a degree of confusion among physicians about the pathological role of h. pylori infection . in a study by huang ,
61% of respondents recognized h. pylori infection as a gastric carcinogen and over 80% believed that both duodenal ulcer and gastric ulcer were related to the infection , while 63% of physicians knew of the relationship between h. pylori eradication and the treatment for dyspepsia .
the comparatively low proportion of gps who recognize h. pylori as a gastric carcinogen may be associated with the fact that gastric carcinogenesis is a multistep and multifactorial process , and the relative risk of gastric cancer associated with h. pylori infection is only modestly increased . nevertheless , the results suggest an increase in the awareness of the pathological role of h. pylori infection in upper gi diseases when compared to previous surveys
.
there are different diagnostic methods for h. pylori infection , of which the gold standard method is endoscopic exam . in ahmed s study , physicians preferred serological based testing of h. pylori in 43% and only 22% of korean gps in kim s study opted for serology as a first test for the detection of h. pylori , whereas 55% of italian physicians in maconi s study preferred gastroscopy with biopsies for initial diagnostic testing . in a study by canbaz , 92 ( 84.4% ) of the gps reported having used one or more tests and 17 ( 15.6% ) never used any test for the diagnosis of h. pylori infection .
only 9.8% had used the stool antigen test for diagnosis and gps reported that they would prescribe symptomatic treatment without ordering diagnostic tests for 29 ( 26.6% ) . in an israeli study by shirin in 2004 , 94.1% of gastroenterologists and 88.9% of internists chose the ubt ( urea breath test ) for detecting h. pylori . in huang s study in 2003 ,
the results of the present study and similar surveys have shown incomplete knowledge of gps in comparison with internists about the correct diagnostic methods for h. pylori infection which may be due to lessened availability of endoscopy among gps for the detection of h. pylori in symptomatic patients , with more referrals of symptomatic patients to internists .
peptic ulcers due to h. pylori are treated with drugs that eradicate bacteria
( antibiotics ) , lower peptic acid ( ppis and h2 blockers ) and protect peptic epithelium ( bismuth ) .
there has been a dramatic evolution in the history of h. pylori treatment , starting from a relatively single ineffective agent 16 years ago to the current most commonly used triple therapies . in huang s study , 89% of respondents use triple therapies and only 10% treated patients with dual combinations . among all triple therapies , ppi combined with clarithromycin and amoxicillin is the most commonly used treatment regimen , followed by ppi administered with metronidazole and clarithromycin , or with amoxicillin and metronidazole . in ahmed s study , a clarithromycin , amoxicillin
, ppi - based triple therapy has been used as first line treatment by 261 ( 61% ) physicians whereas only 2% of physicians used quadruple therapy . in the present study ,
the main treatment ordered by both gps and specialists was quadruple therapy which may be due to the increased recurrence rate and resistance to therapy in this region that necessitates the use of quadruple therapy .
in the present study , most gps and internists
were informed of drug resistance while in huang s study only 62% of gps showed concern for bacterial resistance .
their study included gps from asia ( 69% ) , south america ( 63% ) and europe ( 70% ) who showed greater concern than gps from oceania ( 35% ) , africa ( 45% ) and north america ( 52% ) .
in ahmed s study , eradication of h. pylori was confirmed by 247 ( 57% ) of physicians only in selected patients .
one of the limitations of the present study is that it only reflects the approach of physicians who reside in guilan province and may not be representative of the entire country , although data from the current study is similar to other iranian studies .
overall , as compared to other studies , current knowledge and attitude of gps is to some extent satisfactory , but more effort should be considered for better education of gps and internists .
in this study , most physicians showed interest in participating in continuing medical education programs .
this indicates the high prevalence of h. pylori infection and its related complications , physicians daily contact with this infection , and adequate interest in obtaining more education in this regard .
the current information on the management of h. pylori infection has been useful for gps in iran .
as a whole
, there was a weak but significant correlation between physicians mean knowledge and practice scores .
internists have higher knowledge because they have performed more studies in this field , are experienced in practice because they have visited more patients with these complaints .
| background
this study aims to elucidate the knowledge and practice of general practitioners ( gp ) and internists regarding diagnosis and treatment of helicobacter pylori ( h. pylori ) infection in a high prevalent area , with the intent to assist with future educational strategies for h. pylori infection .
methods
in this cross - sectional study in guilan , a northern province of iran , all gps and internists in the city of rasht were included .
questionnaires consisted of questions on demographic characteristics in addition to physicians knowledge and practice regarding h. pylori infection .
the questionnaire was verified by a panel of experts , validated by the test - retest method , and distributed among participants .
scores higher than the mean score indicated good knowledge or attitude .
those lower than the mean score indicated poor knowledge and attitude .
data were collected and analyzed by spss version 14 software .
results
the mean ( sd ) knowledge and practice score of physicians was 12.13.13 and 2.371.54 , respectively .
overall , 67.9% of gps and 91.7% of internists exhibited good knowledge scores , while 72.4% of gps and 95.8% of internists showed good practice .
physicians who used books or educational programs and had working histories of less than 10 years scored significantly higher in terms of mean knowledge . the mean practice score of physicians who worked in public units and had working histories of more than 5 years and those who had used books or educational programs was significantly higher .
conclusion
since h. pylori infection is prevalent in iran and gps practices are directly under the influence of knowledge , it is necessary to attempt to increase the level and quality of information among gps by educational and continuing medical education programs and seminars . |
arterial pseudoaneurysms are a well - documented occurrence in the setting of both penetrating and blunt traumatic injuries .
however , traumatic lumbar artery pseudoaneurysms ( lapas ) are less frequent , their documentation in the literature being limited to several case reports [ 25 ] .
traumatic lapas are often accompanied by serious concomitant injuries to the retroperitoneum , peritoneal cavity [ 2 , 4 ] , pelvis and/or spine . here , we report the case of an isolated lapa presenting as a delayed hemorrhage in a patient with a single stab wound to the paraspinal region .
an 18-year - old male presented to our institution with a chief complaint of intermittent , recurrent hemorrhage from the right lower paraspinal region , where he had sustained a knife stab 10 days prior .
he was initially evaluated at an outside hospital , where he underwent contrast - enhanced computed tomography ( ct ) scan of his abdomen and pelvis , which demonstrated only soft tissue changes consistent with a superficial stab injury .
the wound was sutured , and the patient was discharged from emergency department in hemodynamically stable condition . at his follow - up on day 9 , he had his sutures removed and on the next day noticed blood tracking from the wound down to his ankle .
he presented again to the outside institution where no bleeding was visualized , and he was discharged once again . on day 11 ,
his dressing saturated , and contrast - enhanced ct scan at this time showed active hemorrhage in the soft tissue and musculature lateral to the right paraspinal muscles without involvement of the retroperitoneal space . at that time , the patient was transferred to our institution for further care .
examination on admission revealed an actively bleeding 2-cm wide stab wound in the right lumbar paraspinal region .
a contrast - enhanced ct of the pelvis showed an enhancing lesion consistent with a pseudoaneurysm extending from a branch of a right lumbar artery ( figs 1 and 2 ) .
the patient was transferred to the interventional radiology suite where angiography confirmed the diagnosis ( fig .
the pseudoaneurysm was then successfully embolized using platinum microcoils ( fig . 4 ) , which were positioned in the lumbar artery , proximal and distal to the origin of the pseudoaneurysm .
the patient tolerated the procedure well and was discharged the following day without any further complication .
figure 1:contrast - enhanced ct scan of the lower abdomen and pelvis showing a single lobe of a presumed , bilobed pseudoaneurysm ( a ) as well as a 3.5 5.5 6 cm rim - enhancing , lobular collection of the superior right gluteal subcutaneous tissues , just superior to the right iliac crest and lateral to the paraspinal musculature , consistent with a hematoma ( b ) .
figure 2:axial cut from the same contrast - enhanced ct scan of the lower abdomen and pelvis showing a 1.6-cm bilobed , enhancing lesion , consistent with pseudoaneurysm , presumably extending from a branch of a right lumbar artery ( a ) .
figure 3:digital subtraction angiography showing a small pseudoaneurysm originating from the peripheral aspect of the distal right lumbar artery ( a ) .
figure 4:digital subtraction angiography demonstrating successful embolization of the proximal and distal entry points of the pseudoaneurysm using platinum microcoils ( a ) .
contrast - enhanced ct scan of the lower abdomen and pelvis showing a single lobe of a presumed , bilobed pseudoaneurysm ( a ) as well as a 3.5 5.5 6 cm rim - enhancing , lobular collection of the superior right gluteal subcutaneous tissues , just superior to the right iliac crest and lateral to the paraspinal musculature , consistent with a hematoma ( b ) .
axial cut from the same contrast - enhanced ct scan of the lower abdomen and pelvis showing a 1.6-cm bilobed , enhancing lesion , consistent with pseudoaneurysm , presumably extending from a branch of a right lumbar artery ( a ) .
digital subtraction angiography showing a small pseudoaneurysm originating from the peripheral aspect of the distal right lumbar artery ( a ) .
digital subtraction angiography demonstrating successful embolization of the proximal and distal entry points of the pseudoaneurysm using platinum microcoils ( a ) .
traumatic pseudoaneurysm of a lumbar artery is an uncommon occurrence and is most often noted in the setting of high - energy , blunt force trauma or penetrating trauma with significant concomitant injury to intra - abdominal structures [ 2 , 4 ] .
a review of the literature suggests the time from injury to presentation of a traumatic lapa is highly variable [ 6 , 7 ] .
they may present acutely with hemodynamic instability in the setting of an expanding retroperitoneal hematoma [ 6 , 7 ] , or they may present 12 weeks after the inciting injury [ 2 , 4 , 5 ] . some reports document presentations delayed for months or even years after the initial injury . in many reports ,
the diagnosis of lapa in the setting of trauma was initially missed , often because of concomitant overshadowing intra - abdominal [ 24 ] and/or spinal injuries .
this study represents an example from the opposite end of the spectrum , wherein the diagnosis was missed on the patient 's readmission because of the isolated nature and apparent simplicity of the inciting event .
nevertheless , delayed diagnosis of lapas can be life threatening , thus illustrating the necessity of a thorough initial trauma evaluation .
management of a lapa in a stable patient can often be achieved through endovascular embolization [ 1 , 3 ] as supported by our case . to our knowledge , this is the first report of such a complication occurring after an isolated paraspinal stab wound , without evidence of any additional injury .
this case emphasizes the importance of maintaining an appropriate index of suspicion in a patient with penetrating trauma and recurrent bleeding and/or pain several days to weeks after initial evaluation .
pseudoaneurysm rupture has the potential to be acutely life threatening , and a thorough trauma evaluation with diligent follow - up care is paramount even for patients with a seemingly uncomplicated pattern of injury .
| lumbar artery pseudoaneurysms are infrequent complications of penetrating trauma . when present , they are often accompanied by other injuries ; however , we report the case of an isolated traumatic lumbar artery aneurysm resulting from a single knife stab .
the lesion was successfully treated with endovascular microcoil embolization .
while these injuries are uncommon , the surgeon must maintain a high index of suspicion , even with seemingly uncomplicated injuries , as a missed traumatic pseudoaneurysm may be life threatening . |
trade - offs and adaptive phenotypic plasticity are central to our evolutionary understanding of biological variation in metazoan organisms .
fundamental to life history theory is the idea that , whilst natural selection acts to maximize some measure of fitness , the possible combinations of phenotypic traits that can be expressed are limited by trade - offs and constraints ( roff 1992 ) .
trade - offs can take many and diverse forms , but theoreticians have most commonly considered the role of resource allocation trade - offs : organisms have limited resources to invest in the processes ( e.g. growth , maintenance , and reproduction ) required to successfully transmit gene copies to future generations ( roff 1992 ; stearns 1992 ) .
natural selection is expected to optimize the allocation of resources between different processes so as to maximize fitness , assuming that sufficient genetic variation exists to allow the optimum to be reached .
understanding life history decisions and the reasons for phenotypic variability becomes more complicated when one considers that phenotypes do not evolve under constant environment conditions .
the concept of adaptive phenotypic plasticity has been central to our understanding of the evolutionary consequences of environmental variation ( schlichting and pigliucci 1998 ) .
phenotypic plasticity is broadly defined as a change in the phenotype of a given genotype in response to a change in environmental conditions ( schlichting and pigliucci 1998 ) .
if natural selection favours different phenotypes under different environmental conditions , we expect the evolution of plastic phenotypic responses to information or cues that predict environmental change ( gotthard and nylin 1995 ; via et al .
phenotypic plasticity can evolve providing that the environment is predictable and suitable genetic variation exists .
plastic responses may involve facultative changes in a single trait , whole suites of life history , behaviours , or resource allocation strategies ( stearns 1992 ; pigliucci 2001 ) .
phenotypic plasticity is expected to be costly , in that its expression will require the diversion of resources away from other functions such as reproduction or maintenance .
costs are diverse and can include the expenditure of resources on maintaining sensory systems , gathering and processing information , or expressing alternative phenotypes . the ability to respond appropriately to changes in the environment will also be limited by organisms ability to detect reliable cues and process the resulting information ( de witt et al . 1998 ) .
selection will act to maximize the fitness advantages of plasticity whilst minimizing the costs involved .
this trade - off could result in the evolution of a single , fixed plastic response across a population , variation in plastic responses across genotypes within a population ( genotype - by - environment interactions ;
gxe ) , or nonplastic , environmental specialist phenotypes ( pigliucci 2001 ) . in the event that environmental variation is completely unpredictable , selection may instead favour the production of randomly or alternately varying phenotypes ( bet hedging strategies ) .
whilst individual organisms are readily identifiable as targets of natural selection in metazoans , the genotype within an infection represents the comparable target in parasites ( west et al .
when infections consist of a single genotype , all parasites are genetically identical and trade - off resolution will maximize the fitness of all co - infecting parasites ( hamilton 1963 ; foster 2005 ) .
when infections are composed of multiple genotypes , parasites from the same genotype are more closely related to each other than to con - specifics .
each genotype could have different optimal strategies because factors such as variation in competitive ability or immune - evasion may influence the relative importance of investing in different processes .
malaria ( plasmodium ) and related apicomplexan parasites comprise a diverse group of pathogens that are responsible for some of the most serious infectious diseases of humans , wildlife , livestock and companion animals ( garnham 1966 ) . despite more than a century of research
, these parasites have resisted efforts to eradicate and control them and remain responsible for between 1 and 3 million deaths per year .
there are over 170 described plasmodium species , which infect mammals , birds and a wide variety of reptiles ( garnham 1966 ) . despite their diversity , their life - cycle ( fig .
1 ) always includes several rounds of asexual replication in a vertebrate host and sexual reproduction in a dipteran vector . when an infected vector bites a host , infective stages ( sporozoites ) are released and start the pre - erythrocytic phase by invading a specific tissue .
after several rounds of replication , erythrocyte - invading stages are released into the blood .
these blood - stage parasites replicate asexually and disease symptoms develop ( anaemia , weight loss and cerebral malaria in some cases ) . every cell cycle a small proportion of asexually produced parasites develop into nonreplicating , male or female sexual stages , termed gametocytes ( smith et al .
asexual parasites do not survive when taken up by a vector in a blood meal , but the gametocytes rapidly differentiate into gametes and fertilization occurs within an hour ( micks et al .
each male gametocyte can differentiate into up to eight gametes whereas each female gametocyte differentiates into a single gamete . to make their gametes
, males must leave their red blood cells , undergo three rounds of genome replication / mitotic division , and construct eight flagella , each with a genome ( janse et al .
conversely , female gametocytes become gametes once they have condensed and left their red blood cells .
zygotes undergo a series of morphological transformations to establish infections in their vector , leading to the formation of thousands of host - infective sporozoites that migrate to the salivary glands ready for transmission ( baton and ranford - cartwright 2005 ) .
whilst the basic features of their life - cycle are conserved , malaria parasites exhibit considerable variation in traits associated with growth and reproduction .
2 ; proportion of gametocytes that are male ) occurs across species , within species , and during infections ( paul et al .
; paul et al . 2003 ; robert et al . 2003 ; reece et al . 2005 , 2008 ) .
whilst striking species differences in some traits are well documented , most have been largely overlooked . in table 1 , we highlight examples of traits for which variation has been observed between and within species and during infections .
empirical data are scarce but do show that there is interesting and important biological variation to be explained
. sex ratios of malaria parasites vary considerably across species ( a ) , within species ( b ) , and during infections ( a ) and ( b ) .
data shown are mean se for infections of : ( a ) three different rodent malaria species , followed until either the infections were cleared or the experiment was terminated ; and ( b ) six different plasmodium chabaudi genotypes that follow four significantly different patterns throughout infections ( reece et al .
means are calculated from between 6 and 30 independent infections and all were initiated by 10 parasites in the same host genotype ( mf1 ) .
variation in life - history traits associated with growth and reproduction exhibited by malaria ( plasmodium ) parasites .
we highlight traits for which variation is observed across species ( species ) , within species ( genotype ) , and during infections ( infection ) .
this list is by no means exhaustive , but is indicative of the empirical literature to date sequestration : withdrawal of infected rbcs from the peripheral circulation to the microvasculature where they adhere to endothelial cells . rosetting : adherence of uninfected rbcs to infected cells leading to the formation of small clusters of cells
a fundamental question in evolutionary biology is whether or not genetic variation exists in a focal trait .
natural selection can not produce an evolutionary response without appropriate genetic variation underlying phenotypic variation . in malaria parasites , within - species genetic variation
can be tested for when genotypes ( clonal isolates ) can be detected ( e.g. schall and vardo - zalik 2007 ) .
one source of such isolates is the bank of p. chabaudi genotypes in the who registry of standard malaria parasites , held at the university of edinburgh , uk .
experimental work has revealed that whilst these con - specific genotypes follow broadly similar infection dynamics , they vary in the maximum parasite and gametocyte densities achieved , and the degree of harm caused to their hosts in terms of weight loss , anaemia and mortality risk ( mackinnon and read 1999 ; timms et al .
analogous field experiments using wild caught parasites of the lizard malaria ( p. mexicanum ) have also revealed evidence for genetic variation in these traits ( eisen and schall 2000 ) .
the details of infection patterns exhibited by a given parasite genotype can also be influenced by host genotype , as observed in p. chabaudi ( de roode et al .
these studies clearly show there is significant genetic variation underlying both important parasite life history traits and their expression in response to changes in the in - host environment .
another key evolutionary question is how variation in the environment will influence selection on life history trade - offs .
the environment that malaria parasites experience throughout their life - cycle is remarkably complex as it encompasses the internal biotic environment of at least two other living organisms ( the host and vector ) , and the external environment experienced by those organisms .
parasites will encounter different types of host , for which there could be different optimal solutions to resource allocation problems . during infections , the in - host environment shifts radically due to complex interactions between factors driven by both parasites and hosts ( e.g. immune factors , erythropoietic state , presence of competing parasites ) . to thrive in such variable conditions
, parasites should alter their life history decisions and behaviours to best suit each situation they encounter .
the ability of natural selection to shape how parasites respond to environmental variation will depend on : the availability of reliable information on environmental conditions and the ability of parasites process it , the presence of suitable genetic variation in parasite populations ( gxe ) , and the costs of that particular form of plasticity . in the next section ,
we describe how evolutionary theory has been successfully used to understand variation in sex ratio , an important fitness - determining trait .
in the subsequent sections we outline how this approach can be applied to explain other parasite life history traits that are central to survival and transmission .
whilst the concepts we present are applicable to all host and vector stages of the life - cycle , we focus on parasite traits expressed during the erythrocytic phase of infections because the necessary tools and techniques to experimentally test our predictions have recently become available .
sex allocation is one of the most well understood topics in evolutionary biology ( charnov 1982 ; hardy 2002 ; west 2009 ) . in many cases ,
simple theory can successfully predict when , why and by how much organisms should adjust their offspring sex ratio in response to a change in their circumstances . as shown in fig .
2 , sex allocation of malaria parasites varies considerably , but the application of evolutionary theory to explain this variation has been controversial ( shutler et al . 1995 ; ferguson 2002 , 2003 ; west et al . 2003 ) .
sex ratios in malaria parasites are generally female - biased and evolutionary theory predicts that sex ratios reflect the inbreeding rate ( read et al .
gametocytes taken up in a blood meal represent the genetic composition of their host 's infection .
the inbreeding rate is high when mating occurs between gametocytes from one or a small number of genotypes , but the inbreeding rate is low when multiple genotypes are present in a mating group .
female - biased sex ratios are expected when inbreeding occurs because this represents the most efficient allocation of resources to maximize the fertilization success of a mating group .
because each male can fertilize more than one female , a female - biased sex ratio reduces competition between related males and maximizes the number of females available to mate with ( taylor 1981 ) .
in contrast , when outbreeding , the greatest fitness returns come from increasing investment in males .
in this situation , because unrelated females belonging to other genotypes are present , a genotype that produces more males will have the greatest genetic representation in the zygote population . this is
hamilton 's local mate competition theory ( lmc ; hamilton 1967 ) and it explains why female - biased sex ratios are favoured in spatially structured populations for a variety of taxa , from plants to insects to snakes ( west et al . 2005 ) . the application of lmc theory to malaria parasites has proved controversial because it does not explain why sex ratios vary throughout infections or why population sex ratios in some related apicomplexans do not correspond to their inbreeding rate ( shutler and read 1998 ; west et al .
however , cases where the data do not correspond to lmc can be explained by a simple extension of the theory , known as fertility insurance ( west et al .
fertility insurance is important when there is a risk that not all females in a blood meal will be fertilized .
this problem can arise when gametocyte density is low , due to red cell limitation in anaemic hosts or low conversion , or when transmission - blocking immune factors impair the ability of male gametocytes to make viable gametes .
when male gamete viability is low or few gametocytes are able to interact in a blood meal , parasites are expected to alter their sex ratio by increasing their investment in male gametocytes , resulting in a less female - biased sex ratio than their inbreeding rate suggests . by increasing investment in males , parasites
can ensure that they have produced enough males to fertilize their females . the recent development of reverse transcription quantitative pcr assays for malaria parasites that are both genotype- and sex - specific ( drew and reece 2007 ) have enabled the predictions of lmc and fertility insurance theory to be tested
. recently conducted , conceptually simple experiments in which the sex ratios produced by focal p. chabaudi genotypes were followed in single- and double - genotype infections , showed that parasites can evaluate their likely inbreeding rate and faculatively adjust their sex ratio according to the predictions of lmc theory ( reece et al .
furthermore , the double - infection data revealed a negative correlation between sex ratio and the proportion of gametocytes contributed by focal genotypes that quantitatively fits lmc theory .
first , female - biased sex allocation in response to lmc decreases as infections progress and parasite densities decline due to resource competition , anaemia and development of the host 's immune response .
second , within - infection sex ratio patterns , for which there is significant genetic variation , can be explained by variation in anaemia and parasite densities .
third , sex ratios are adjusted in response to erythropoetin ( epo ; a hormone produced by anaemic hosts ) and subsequent changes in the availability of rbc resources ( paul et al .
facultative sex - ratio adjustment in response to changes in the genetic diversity of infections and within - host environment confirm that : ( i ) sex allocation strategies in malaria parasites are as sophisticated as those observed in multi - cellular taxa ; ( ii ) malaria parasites can discriminate kin from non - kin and infer their own relative frequency in infections ; ( iii ) malaria parasites are able to detect and respond to changes in host anaemia and the availability of preferred rbcs ; and ( iv ) crucially , sex ratio variation can be explained by evolutionary theory ( shutler and read 1998 ; knowles and sheldon 2008 ; reece et al .
in this section , we develop this successful application of evolutionary theory to predict how parasites should solve other trade - off problems .
specifically , we consider how plasticity in the allocation of resources to reproduction and growth should be expressed , discuss evidence for plasticity in the underlying traits , and describe how our predictions can be tested . from a life history perspective
, the asexual replication of a single - genotype malaria infection can be viewed as growth and maintenance and gametocyte density as reproduction. as in metazoans , a fundamental trade - off exists resources are invested into growth at the expense of reproduction and vice - versa ( stearns 1992 ) . in metazoans , quantifying how an individual has resolved this trade - off in terms of reproductive effort is rarely possible . in contrast , the reproductive effort of malaria parasites can easily be calculated as the proportion of a cohort of parasites that develop into gametocytes ( conversion rate ) .
though rarely measured , conversion rate frequently varies throughout infections and is increased in response to epo and the presence of antimalarial drugs ( buckling et al .
2005 ) . here , we outline explanations for these observations and predict how parasites are expected to alter conversion in response to changes in their in - host and social circumstances .
these scenarios are not mutually exclusive and in reality we expect that parasites will simultaneously be resolving many of these trade - offs
so the currently held belief is that parasites have nothing to lose , and should increase conversion as a last attempt at transmission before being cleared .
however , theory could also predict the opposite : parasites in stressful circumstances should reduce their conversion to maximize their chances of survival ( schneider et al . 2008 ) . in this scenario , by investing in asexual replication , parasites benefit from the future reproductive opportunities that can be attained from surviving ( mideo and day 2008 ) . if this is the case , why do the data show the opposite pattern ? to adopt the most appropriate strategy , parasites need the ability to determine when continued survival is impossible ( e.g. due to terminally decreasing parasite density or inevitable host death ) . for novel situations like exposure to drugs ,
parasites may not have been under selection for long enough to be able to evaluate their survival chances .
in contrast , parasites will frequently experience severely anaemic hosts , so the ability to infer when host death is inevitable should have evolved .
available data are encouraging : p. gallinaceum parasites can determine whether their infections are lethal or will be resolved by hosts ( paul et al .
1999 ) . in metazoans the analogous hypothesis ( known as terminal investment ; williams 1966 ) is very difficult to test because measurements of relative reproductive effort are required and confounding variables ( e.g. decline due to senescence or infection ) make it hard to measure the intrinsic state of individuals .
these issues can be overcome with malaria parasites because the performance of replicate infections of genetically identical parasites can be followed in progressively deteriorating in - host conditions or drug treatment regimes .
the terminal investment hypothesis was developed from the prediction that the reproductive strategy an organism adopts will depend on the value of the fitness gains made from current reproductive effort , relative to future reproductive effort . in humans ,
relatively few malaria infections result in host death during the acute phase and parasites enter a longer - term ( chronic ) phase in which they can persist for years ( snounou et al .
because these infections have a long life - span , resource allocation strategies will reflect the trade - off between current and future reproductive success .
more data on transmission success from acute and chronic stage infections are needed to test for this trade - off , but parasites at extremely low densities in chronic p. falciparum infections can infect mosquitoes , ( schneider et al .
the idea that current reproductive effort should be traded - off against future reproductive effort applies to two other situations : in - host competition and resource availability .
parasites frequently experience in - host competition with con - specific and con - generic parasites ( paul et al . 2003 ; de roode et al .
because the best competitors are predicted to be genotypes that acquire the greatest share of rbc resources , a decrease in conversion will result in faster replication and increased survival ( mideo and day 2008 ) .
this prediction has been tested but the data are inconclusive : focal p. chabaudi genotypes monitored in single- and double - genotype infections revealed only a small reduction in conversion in the competitively inferior genotype in one of the two host strains used ( wargo et al .
if preferred rbcs are scarce , parasites should invest in survival , but only if their resource limitation situation is temporary . depending on their preference for young or old rbcs , host anaemia can mean either the loss or production of preferred rbcs .
in line with this prediction , p. chabaudi , a species that can infect young and old rbcs , increases investment in gametocytes when hosts receive epo , but p. vinckei , which can only infect mature rbcs , does not alter conversion and maintains transmission by adjusting sex ratio instead ( reece et al .
parasites must maximize survival and transmission in the face of competition and the inevitable attack of the immune response .
theory predicts that success in these endeavors is correlated with parasite virulence ( damage caused to hosts ) because fast growing parasites benefit from a larger pool of mature parasites to produce gametocytes .
a trade - off between the rate and duration of transmission occurs because virulent parasites risk killing their host and losing future transmission opportunities ( frank 1996 ) .
however , as slow growing parasites are poor competitors and at risk from rapid clearance by the host immune response , intermediate levels of virulence are favoured by natural selection ( read 1994 ; frank 1996 ; day 2003 ; day and proulx 2004 ; frank and schmid - hempel 2008 ) . of all parasite traits , virulence is unique in having a large body of theory but , like most traits , the data lag far behind .
the idea that parasites should adjust their virulence by facultatively altering traits that underlie replication rate is both theoretically and empirically unexplored .
in addition to conversion rate , plasticity in components of replication rate would also regulate virulence .
traits likely to exhibit adaptive plasticity include the number of daughter cells ( merozoites ) produced by mature parasites ( schizonts ) , cell cycle duration and synchronicity , and the ability of merozoites to invade different types of rbc . in this section ,
we explore how parasites should adjust these traits in response to the type of host they infect and changes in their in - host environment .
the type of host that malaria parasites encounter will vary substantially in health and immune status .
if parasites face a trade - off between growth / maintenance and reproduction , it follows that changes in conversion rate will influence replication rate and virulence .
could parasite use their conversion rate to regulate virulence ? for example , reducing conversion to prioritize replication will be the best strategy when attempting to establish an infection in an adult host with an already up - regulated immune response , and with competing parasites .
but , if the next host is an immunologically nave infant , parasites will be provided with plentiful resources and no competition or immediate danger of immune attack .
uncontrolled replication in this host type will result in premature host death , and instead , we expect parasites to adopt high conversion rates to prevent host death and prolong the lifespan of their infection , with the added bonus that plenty of gametocytes result ( alizon and van baalen 2008 )
. could this be the evolutionary explanation for why more gametocytes are observed in children than adults ?
the use of conversion rate to regulate virulence may seem intuitively unlikely because gametocytes usually represent a small ( < 1% ) proportion of the parasites present in an infection .
indeed , a frequently posed question is why are there so few gametocytes? ( taylor and read 1997 ; mideo and day 2008 ) .
the answer may lie in the trade - off between investing resources into growth / maintenance and reproduction : the fitness benefits of increasing gametocyte number may rarely outweigh the survival costs incurred in immune hosts and in competition with con - specifics .
alternatively , parasites could also regulate virulence by altering a key component of their cell cycle : the number of merozoites released by each mature schizont .
the pre - requisites for plasticity to evolve in this trait are met : there is genetic variation for the number of merozoites produced by p. falciparum schizonts
( reilly et al . 2007 ) and this trait is under fine - scale control because each nucleus within a developing schizont can divide a different number of times ( leete and rubin 1996 ) .
despite the vast species differences in the number of merozoites produced by schizonts , remarkably little is known about the ecological factors and evolutionary pressures shaping variation in this trait . however , increasing merozoite number would clearly be advantageous under deteriorating environmental conditions ( e.g. immune attack ) , when survival is at risk .
data on variation in the number of merozoites in mature schizonts are extremely scarce , which is partly due to the challenges of collecting them in many species , they sequester in tissues .
new cell and molecular methods can overcome these difficulties because parasites can be collected prior to sequestration and incubated in vitro to maturity ( reece and thompson 2008 ) , or located during sequestration by whole - organism imaging methods ( franke - fayard et al .
also , a variety of methods can be used to identify mature schizonts ( e.g. fluorescent microscopy or cell sorting ) , which can be prevented from rupturing by incubation with cysteine protease inhibitors ( rosenthal et al .
these tools can now be used to investigate when maturing parasites commit to the number of nuclear divisions they will undertake and which factors influence this . for example , does merozoite number vary according to whether schizonts contain parasites committed to asexual or sexual differentiation ? there may also be trade - offs involved between the quantity of merozoites produced and their invasion capabilities , or time required to complete a cell cycle . in addition to variation in the duration of cell cycles , the synchronicity and development time of parasite cohorts varies substantially ( gautret et al .
conventional wisdom assumes that cell cycles are timed to match gametocyte maturation with the diurnal patterns of vectors , and synchronous development enables parasites to withstand immune attack through safety in numbers ( hawking et al .
yet , these explanations are unsatisfactory because it is precisely the synchronous bursting of schizonts that elicits a rapid and short - lived immune response that sterilizes gametocytes for several hours and attacks merozoites ( naotunne et al .
such high costs to transmission suggest that synchronicity yields substantial fitness benefits or is the outcome of a constraint imposed either by host factors or parasite physiology .
the latter is unlikely because parasites increase their synchronicity and developmental rate in response to host melatonin ( p. berghei;hotta et al .
2000 ) and co - culture with other parasites ( p. falciparum;dyer and day 1993 ) .
this could allow parasites to avoid attack from immune factors or be directly caused by host immunity .
experiments to distinguish between parasite and host factors are possible using mice without immune systems or by blocking the actions of specific factors . the fitness costs and benefits of synchronicity can be quantified by assaying the expression of stage specific genes in synchronous ( using purified merozoites ) , or asynchronous ( a mix of stages ) infections ( mons et al .
with transformed parasites in which genes underlying cell cycle regulation have been deleted would also be extremely useful .
replication rate and virulence are also determined by parasites ability to acquire rbcs for replication .
cell and molecular data indicate there is substantial scope for adaptive plasticity in rbc preference , but variation in this trait is yet to be integrated into an evolutionary framework .
the rbc preference of different species ranges from reticulocytes for p. vivax and p. ovale to mature rbcs for p. malariae , p. falciparum has the ability to switch its invasion strategy , and similar variation is found in the rodent malarias ( stubbs et al .
2005 ) . given the variation in the age and density of rbcs available in healthy and anaemic hosts during infections , the ability to facultatively alter rbc preference would be advantageous ( paul et al .
the ability to invade all available rbc types may seem preferable , but different merozoite surface machinery is required for invading rbc of different ages ( cowman and crabb 2006 ) , and because the genes involved in invasion are also a source of antigenic variation , is it important for parasites to avoid expressing them simultaneously ( meunier 2001 ) .
this could explain why each p. yoelii merozoite only expresses one of the 25 genes that determine its rbc preference at a time ( preiser et al .
1999 ) and p. falciparum parasites can switch their invasion strategy ( stubbs et al . 2005 ) . if so , invasion strategies will reflect the trade - off between the benefits of being a generalist that is able to invade a range of rbcs , versus the costs of exposing antigens to the immune system .
an additional trade - off may be operating between the range of rbcs parasites can invade and their ability to grow within them . for example , p. berghei , parasites have a stronger preference for reticulocytes , and produce more merozoites per schizont in reticulocytes ( killick - kendrick and peters 1978 ) . the molecular tools ( e.g. reverse transcription quantitative pcr ) required to measure gene expression and invasion abilities are available and can be applied to experiments in which host haemopoetic state
is manipulated . however , to understand how parasites cope with changes in resource availability , quantitative data on rbc preference throughout infections are required . here
, mathematical models would prove particularly useful because complex erythropeotic processes determine what rbcs are available to parasites and this can not accurately be inferred from snapshot observations of the type of rbcs that parasites have developed within ( cromer et al .
from a life history perspective , the asexual replication of a single - genotype malaria infection can be viewed as growth and maintenance and gametocyte density as reproduction. as in metazoans , a fundamental trade - off exists resources are invested into growth at the expense of reproduction and vice - versa ( stearns 1992 ) . in metazoans , quantifying how an individual has resolved this trade - off in terms of reproductive effort is rarely possible . in contrast , the reproductive effort of malaria parasites can easily be calculated as the proportion of a cohort of parasites that develop into gametocytes ( conversion rate ) . though rarely measured
, conversion rate frequently varies throughout infections and is increased in response to epo and the presence of antimalarial drugs ( buckling et al .
2005 ) . here , we outline explanations for these observations and predict how parasites are expected to alter conversion in response to changes in their in - host and social circumstances .
these scenarios are not mutually exclusive and in reality we expect that parasites will simultaneously be resolving many of these trade - offs
so the currently held belief is that parasites have nothing to lose , and should increase conversion as a last attempt at transmission before being cleared .
however , theory could also predict the opposite : parasites in stressful circumstances should reduce their conversion to maximize their chances of survival ( schneider et al . 2008 ) . in this scenario , by investing in asexual replication , parasites benefit from the future reproductive opportunities that can be attained from surviving ( mideo and day 2008 ) . if this is the case , why do the data show the opposite pattern ? to adopt the most appropriate strategy , parasites need the ability to determine when continued survival is impossible ( e.g. due to terminally decreasing parasite density or inevitable host death ) . for novel situations like exposure to drugs ,
parasites may not have been under selection for long enough to be able to evaluate their survival chances .
in contrast , parasites will frequently experience severely anaemic hosts , so the ability to infer when host death is inevitable should have evolved .
available data are encouraging : p. gallinaceum parasites can determine whether their infections are lethal or will be resolved by hosts ( paul et al .
1999 ) . in metazoans the analogous hypothesis ( known as terminal investment ; williams 1966 ) is very difficult to test because measurements of relative reproductive effort are required and confounding variables ( e.g. decline due to senescence or infection ) make it hard to measure the intrinsic state of individuals .
these issues can be overcome with malaria parasites because the performance of replicate infections of genetically identical parasites can be followed in progressively deteriorating in - host conditions or drug treatment regimes .
the terminal investment hypothesis was developed from the prediction that the reproductive strategy an organism adopts will depend on the value of the fitness gains made from current reproductive effort , relative to future reproductive effort . in humans ,
relatively few malaria infections result in host death during the acute phase and parasites enter a longer - term ( chronic ) phase in which they can persist for years ( snounou et al .
, resource allocation strategies will reflect the trade - off between current and future reproductive success .
more data on transmission success from acute and chronic stage infections are needed to test for this trade - off , but parasites at extremely low densities in chronic p. falciparum infections can infect mosquitoes , ( schneider et al .
the idea that current reproductive effort should be traded - off against future reproductive effort applies to two other situations : in - host competition and resource availability . parasites frequently experience in - host competition with con - specific and con - generic parasites ( paul et al .
because the best competitors are predicted to be genotypes that acquire the greatest share of rbc resources , a decrease in conversion will result in faster replication and increased survival ( mideo and day 2008 ) .
this prediction has been tested but the data are inconclusive : focal p. chabaudi genotypes monitored in single- and double - genotype infections revealed only a small reduction in conversion in the competitively inferior genotype in one of the two host strains used ( wargo et al .
if preferred rbcs are scarce , parasites should invest in survival , but only if their resource limitation situation is temporary . depending on their preference for young or old rbcs , host anaemia can mean either the loss or production of preferred rbcs . in line with this prediction , p. chabaudi , a species that can infect young and old rbcs , increases investment in gametocytes when hosts receive epo , but p. vinckei , which can only infect mature rbcs , does not alter conversion and maintains transmission by adjusting sex ratio instead ( reece et al . 2005 ) .
parasites must maximize survival and transmission in the face of competition and the inevitable attack of the immune response .
theory predicts that success in these endeavors is correlated with parasite virulence ( damage caused to hosts ) because fast growing parasites benefit from a larger pool of mature parasites to produce gametocytes .
a trade - off between the rate and duration of transmission occurs because virulent parasites risk killing their host and losing future transmission opportunities ( frank 1996 ) .
however , as slow growing parasites are poor competitors and at risk from rapid clearance by the host immune response , intermediate levels of virulence are favoured by natural selection ( read 1994 ; frank 1996 ; day 2003 ; day and proulx 2004 ; frank and schmid - hempel 2008 ) . of all parasite traits ,
virulence is unique in having a large body of theory but , like most traits , the data lag far behind . the idea that parasites should adjust their virulence by facultatively altering traits that underlie replication rate is both theoretically and empirically unexplored .
in addition to conversion rate , plasticity in components of replication rate would also regulate virulence .
traits likely to exhibit adaptive plasticity include the number of daughter cells ( merozoites ) produced by mature parasites ( schizonts ) , cell cycle duration and synchronicity , and the ability of merozoites to invade different types of rbc . in this section ,
we explore how parasites should adjust these traits in response to the type of host they infect and changes in their in - host environment .
the type of host that malaria parasites encounter will vary substantially in health and immune status .
if parasites face a trade - off between growth / maintenance and reproduction , it follows that changes in conversion rate will influence replication rate and virulence .
for example , reducing conversion to prioritize replication will be the best strategy when attempting to establish an infection in an adult host with an already up - regulated immune response , and with competing parasites .
but , if the next host is an immunologically nave infant , parasites will be provided with plentiful resources and no competition or immediate danger of immune attack .
uncontrolled replication in this host type will result in premature host death , and instead , we expect parasites to adopt high conversion rates to prevent host death and prolong the lifespan of their infection , with the added bonus that plenty of gametocytes result ( alizon and van baalen 2008 )
. could this be the evolutionary explanation for why more gametocytes are observed in children than adults ?
the use of conversion rate to regulate virulence may seem intuitively unlikely because gametocytes usually represent a small ( < 1% ) proportion of the parasites present in an infection .
indeed , a frequently posed question is why are there so few gametocytes? ( taylor and read 1997 ; mideo and day 2008 ) .
the answer may lie in the trade - off between investing resources into growth / maintenance and reproduction : the fitness benefits of increasing gametocyte number may rarely outweigh the survival costs incurred in immune hosts and in competition with con - specifics .
alternatively , parasites could also regulate virulence by altering a key component of their cell cycle : the number of merozoites released by each mature schizont .
the pre - requisites for plasticity to evolve in this trait are met : there is genetic variation for the number of merozoites produced by p. falciparum schizonts
( reilly et al . 2007 ) and this trait is under fine - scale control because each nucleus within a developing schizont can divide a different number of times ( leete and rubin 1996 ) .
despite the vast species differences in the number of merozoites produced by schizonts , remarkably little is known about the ecological factors and evolutionary pressures shaping variation in this trait .
however , increasing merozoite number would clearly be advantageous under deteriorating environmental conditions ( e.g. immune attack ) , when survival is at risk .
data on variation in the number of merozoites in mature schizonts are extremely scarce , which is partly due to the challenges of collecting them in many species , they sequester in tissues .
new cell and molecular methods can overcome these difficulties because parasites can be collected prior to sequestration and incubated in vitro to maturity ( reece and thompson 2008 ) , or located during sequestration by whole - organism imaging methods ( franke - fayard et al .
also , a variety of methods can be used to identify mature schizonts ( e.g. fluorescent microscopy or cell sorting ) , which can be prevented from rupturing by incubation with cysteine protease inhibitors ( rosenthal et al .
these tools can now be used to investigate when maturing parasites commit to the number of nuclear divisions they will undertake and which factors influence this .
for example , does merozoite number vary according to whether schizonts contain parasites committed to asexual or sexual differentiation ? there may also be trade - offs involved between the quantity of merozoites produced and their invasion capabilities , or time required to complete a cell cycle .
in addition to variation in the duration of cell cycles , the synchronicity and development time of parasite cohorts varies substantially ( gautret et al .
conventional wisdom assumes that cell cycles are timed to match gametocyte maturation with the diurnal patterns of vectors , and synchronous development enables parasites to withstand immune attack through safety in numbers ( hawking et al .
1968 ) . yet , these explanations are unsatisfactory because it is precisely the synchronous bursting of schizonts that elicits a rapid and short - lived immune response that sterilizes gametocytes for several hours and attacks merozoites ( naotunne et al .
such high costs to transmission suggest that synchronicity yields substantial fitness benefits or is the outcome of a constraint imposed either by host factors or parasite physiology .
the latter is unlikely because parasites increase their synchronicity and developmental rate in response to host melatonin ( p. berghei;hotta et al . 2000 ) and co - culture with other parasites ( p. falciparum;dyer and day 1993 ) .
this could allow parasites to avoid attack from immune factors or be directly caused by host immunity .
experiments to distinguish between parasite and host factors are possible using mice without immune systems or by blocking the actions of specific factors . the fitness costs and benefits of synchronicity can be quantified by assaying the expression of stage specific genes in synchronous ( using purified merozoites ) , or asynchronous ( a mix of stages ) infections ( mons et al .
. comparing cohort development in wild type parasites with transformed parasites in which genes underlying cell cycle regulation have been deleted would also be extremely useful .
replication rate and virulence are also determined by parasites ability to acquire rbcs for replication .
cell and molecular data indicate there is substantial scope for adaptive plasticity in rbc preference , but variation in this trait is yet to be integrated into an evolutionary framework .
the rbc preference of different species ranges from reticulocytes for p. vivax and p. ovale to mature rbcs for p. malariae , p. falciparum has the ability to switch its invasion strategy , and similar variation is found in the rodent malarias ( stubbs et al .
2005 ) . given the variation in the age and density of rbcs available in healthy and anaemic hosts during infections , the ability to facultatively alter rbc preference would be advantageous ( paul et al .
the ability to invade all available rbc types may seem preferable , but different merozoite surface machinery is required for invading rbc of different ages ( cowman and crabb 2006 ) , and because the genes involved in invasion are also a source of antigenic variation , is it important for parasites to avoid expressing them simultaneously ( meunier 2001 ) .
this could explain why each p. yoelii merozoite only expresses one of the 25 genes that determine its rbc preference at a time ( preiser et al .
1999 ) and p. falciparum parasites can switch their invasion strategy ( stubbs et al . 2005 ) . if so , invasion strategies will reflect the trade - off between the benefits of being a generalist that is able to invade a range of rbcs , versus the costs of exposing antigens to the immune system .
an additional trade - off may be operating between the range of rbcs parasites can invade and their ability to grow within them .
for example , p. berghei , parasites have a stronger preference for reticulocytes , and produce more merozoites per schizont in reticulocytes ( killick - kendrick and peters 1978 ) . the molecular tools ( e.g. reverse transcription quantitative pcr ) required to measure gene expression and invasion abilities are available and
can be applied to experiments in which host haemopoetic state is manipulated . however , to understand how parasites cope with changes in resource availability
, mathematical models would prove particularly useful because complex erythropeotic processes determine what rbcs are available to parasites and this can not accurately be inferred from snapshot observations of the type of rbcs that parasites have developed within ( cromer et al .
in addition to the specific investigations we have suggested , a substantially broader understanding of how within - infection dynamics are influenced by host factors , parasite traits , and interactions between them is required .
this can be readily achieved for malaria parasites because : ( i ) focal genotypes can be followed during replicate infections in a range of scenarios ; ( ii ) multiple focal genotypes can be investigated to establish whether gxe underlies patterns of trait variation and plasticity ; ( iii ) multiple parasite and host variables can be measured and their potentially confounding influence on traits of interest can be statistically controlled for ; and ( iv ) the use of mathematical models to examine within - host dynamics offers a new tool for hypothesis testing : iterations of theoretical developments and empirical testing promise to be a powerful approach ( mideo et al .
evolutionary theory has been developed as a tool for investigating the ultimate causes of variation in life history traits and can provide new directions in the search for the proximate ( mechanistic ) processes .
for example , evolutionary approaches have revealed adaptive phenotypic plasticity in sex ratio strategies , informing the search for underlying mechanistic processes ( paul et al .
proximate approaches have identified mechanisms likely to be involved in trade - offs and adaptive responses to environmental change .
experimental manipulations can be used to investigate what environmental information parasites use , whether these are direct or indirect cues , and what costs are paid for phenotypic plasticity . by understanding how , when and why parasites respond to different cues could enable interventions to induce parasites to make suboptimal choices that are clinically or epidemiologically beneficial . identifying the physiological and genetic constraints on plasticity
is of central importance in predicting how parasites will respond in the short- and long - term to drug and vaccine interventions .
a synergy between research directed at evolutionary and mechanistic explanations for trait variation is surely the most efficient way to progress both fields . | adaptive phenotypic plasticity in life history traits , behaviours , and strategies is ubiquitous in biological systems .
it is driven by variation in selection pressures across environmental gradients and operates under constraints imposed by trade - offs .
phenotypic plasticity has been thoroughly documented for multicellular taxa , such as insects , birds and mammals , and in many cases the underlying selective pressures are well understood .
whilst unicellular parasites face many of the same selective pressures and trade - offs , plasticity in their phenotypic traits has been largely overlooked and remains poorly understood . here
, we demonstrate that evolutionary theory , developed to explain variation observed in the life - history traits of multicellular organisms , can be applied to parasites .
though our message is general we can expect the life - histories of all parasites to have evolved phenotypic plasticity we focus our discussion on malaria parasites .
we use an evolutionary framework to explain the trade - offs that parasites face and how plasticity in their life history traits will be expressed according to changes in their in - host environment . testing
whether variation in parasites traits is adaptive will provide new and fundamental insights into the basic biology of parasites , their epidemiology and the processes of disease during individual infections . |
head and neck cancer is a major problem that occurs in asia , especially in the indian subcontinent . more than 2 lakh new cases of head and neck cancers are diagnosed each year .
india contributes up to 7.8% of the global cancer burden and 8.33% of global cancer deaths .
lack of improvement in prognosis over the years is due to the fact that a significant proportion of oral squamous cell carcinoma ( oscc ) is not diagnosed or treated until they reach an advanced stage .
although arising de novo in many instances a significant proportion of oscc develop from premalignant lesions and conditions such as leukoplakia , oral submucous fibrosis and lichen planus .
the strong association between cancers of the oral cavity and pharynx with use of cigarette smoking , snuff and chewing tobacco is well established .
alcohol consumption has a synergistic role and dietary factors such as low intake of fruits and vegetables may also be related to an increased cancer risk .
few studies have also indicated that presence of candidal infection increases the risk of malignant transformation in premalignant lesions .
identification of these premalignant lesions is very important in order to prevent malignant transformation . in order to do so
, we must develop criteria which indicate the potential for malignant transformation in individual lesions .
the role of fungal infections has been studied in this respect and holds much promise as such an indicator .
certain strains of candida albicans and other yeasts play a causal role in the development of oral cancer by means of endogenous nitrosamine production .
the association of candida with various precancer and cancer lesions has been reported as a causative agent .
the association of candida with premalignant states has been studied extensively and many authors have shown an increase in candida colonization in these lesions as compared to controls .
further , this persistent infection along with other cofactors may also induce epithelial atypia and dysplasia leading to malignant change .
it appears that the risk of carcinoma developing in these lesions and conditions depends on whether the lesion is homogenous or nonhomogeneous , the presence and degree of epithelial dysplasia and possibly the method of management adopted .
but , literature shows very few studies linking the association of dysplasia and related lesions with candida .
hence , we designed a study to evaluate the prevalence of fungal hyphae in most common oral potentially malignant lesions and conditions such as leukoplakia , lichen planus and submucous fibrosis and to find out a correlation , if any with presence or absence of fungal hyphae with epithelial dysplasia .
the present study was undertaken by retrieving archived records and paraffin - embedded tissue blocks of previously diagnosed cases of 70 potentially malignant lesions and conditions ( 29 leukoplakia , 16 submucous fibrosis and 25 lichen planus ) .
relevant information of age , sex and histopathological grading of dysplasias was obtained from the records of the patients and was tabulated [ tables 1 and 2 ] .
comparison of age and sex distribution between three study groups distribution of grading of epithelial dysplasia between three study groups clinically suspected and histopathologically diagnosed cases having hyperkeratosis and different grades of dysplasia .
three sections of 5 m thickness of each case were stained employing hematoxylin and eosin ( h and e ) , periodic acid
the tissue section of diagnosed case of mucormycosis served as positive control for staining of hyphae .
the entire data was entered in ms excel and analyzed using statistical package for social sciences ( spss ) .
chi - square test of independence of attributes was used to find out the significant differences and linearity trend of proportion was tested using chi - square test to find out association .
three sections of 5 m thickness of each case were stained employing hematoxylin and eosin ( h and e ) , periodic acid schiff 's reagent ( pas ) and grocott 's methenamine silver ( gms ) .
the tissue section of diagnosed case of mucormycosis served as positive control for staining of hyphae .
the entire data was entered in ms excel and analyzed using statistical package for social sciences ( spss ) .
chi - square test of independence of attributes was used to find out the significant differences and linearity trend of proportion was tested using chi - square test to find out association .
the present study was undertaken to determine the prevalence of fungal hyphae in potentially malignant lesions and conditions such as leukoplakia , lichen planus and oral submucous fibrosis ; and to find out association if any , with presence or absence of fungal hyphae with various grades of dysplasia .
fungal hyphae stained with pas appeared magenta red and with gms they appeared brownish black [ figure 1a and b ] .
( a ) dark brown to black fungal hyphae in tissue section ( gms stain , x400 ) .
( b ) magenta red colored fungal hyphae in tissue section ( pas stain , x400 ) .
pas = periodic acid schiff , gms = grocott 's methenamine silver demographic details of the patients in groups i ,
ii and iii are shown in table 1 . in all the study groups , majority of patients were men and above 50 years [ table 1 ] .
statistically significant number of cases of leukoplakia ( n = 27/29 ; p = 0.001 ) showed signs of dysplasia , but not in cases of lichen planus and oral submucous fibrosis [ table 2 ] . in leukoplakia group , 12 out of 29 cases showed presence of fungal hyphae , in lichen planus nine out of 25 were positive for hyphae , but in oral submucous fibrosis group only four were positive for fungal hyphae .
distribution of prevalence of fungal hyphae in each study group with or without dysplasia in total cases distribution of prevalence of fungal hyphae in each study groups in total cases the prevalence of fungal hyphae in cases of leukoplakia with dysplasia was 44.44% , in lichen planus was 66.66% and in oral submucous fibrosis , it was 100% .
out of 27 dysplastic cases of leukoplakia group , 12 cases showed presence of fungal hyphae . in lichen
planus group out of six cases with dysplasia , four cases showed presence of fungal hyphae and in oral submucous fibrosis group , two cases with dysplasia also showed presence of hyphae . on comparison of the prevalence , with respect to presence of fungal hyphae between cases with dysplasia and cases without dysplasia , we found that cases of lichen planus and oral submucous fibrosis showed proportionately higher prevalence of fungal hyphae in cases with dysplasia as compared to leukoplakia .
but the above results were not statistically significant [ table 4 and graph 2 ] .
distribution of prevalence of fungal hyphae in cases with dysplasia between three study groups distribution of prevalence of fungal hyphae in cases with dysplasia between three study groups in cases of leukoplakia , the degree of epithelial dysplasia was significantly associated with presence of fungal hyphae [ table 5 ] .
out of twelve cases of mild dysplasia three were positive for fungal hyphae by pas and gms staining .
[ figure 2a and b ] out of nine cases of moderate dysplasia , five were positive for fungal hyphae by pas and gms staining .
[ figure 3a and b ] in severe dysplasia ; out of four cases , three were positive for fungal hyphae [ figure 4a and b and table 5 ] .
( a ) superficial colonization of fungal hyphae in cornified layer in mild dysplasia ( pas stain , x200 ) .
( b ) superficial colonization of fungal hyphae in cornified layer in mild dysplasia ( gms stain , x400 ) ( a ) fungal hyphae in cornified layer in moderate dysplasia ( pas stain , x400 ) .
( b ) fungal hyphae in cornified layer in moderate dysplasia ( gms stain , x400 ) ( a ) hyperkeratosis and hyperplasia , superficial colonization of fungal hyphae in severe dysplasia ( pas stain , x400 ) .
( b ) fungal hyphae in cornified layer in severe dysplasia ( gms stain , x400 ) association of degree of epithelial dysplasia with presence of fungal hyphae in cases of leukoplakia ( group i ) in lichen planus , the degree of epithelial dysplasia was not significantly associated with presence of fungal hyphae .
out of three cases each of mild and moderate dysplasia , two each were positive for fungal hyphae by pas and gms [ figure 5a and b and table 6 ] .
( a ) invading fungal hyphae in cornified layer in lichen planus a ( pas stain , x100 ) .
( b ) invading fungal hyphae in cornified layer in lichen planus ( gms stain , x100 ) association of degree of epithelial dysplasia with presence of fungal hyphae in cases of lichen planus ( group ii ) in oral submucous fibrosis , the degree of epithelial dysplasia was significantly associated with presence of fungal hyphae .
one case each with mild and moderate dysplasia showed presence of fungal hyphae by pas and gms [ figure 6a and b and table 7 ] .
( a ) fungal hyphae in cornified layer in oral submucous fibrosis ( pas stain , x 400 ) .
( b ) fungal hyphae in cornified layer in oral submucous fibrosis ( gms stain , x400 ) association of degree of epithelial dysplasia with presence of fungal hyphae in cases of oral submucous fibrosis ( group iii ) the association of degree of epithelial dysplasia with presence or absence of fungal hyphae was statistically significant ( p - value 0.003 ) in all three study groups [ table 8 and graph 3 ] .
association of degree of epithelial dysplasia with presence or absence of fungal hyphae in all cases association of degree of epithelial dysplasia with presence or absence of fungal hyphae in all cases
oral cancer is the sixth most common cancer in the world and is largely preventable .
it accounts for approximately 4% of all cancers and 2% of all cancer deaths worldwide . in india ,
oral cancer is the commonest malignant neoplasm , accounting for 2030% of all cancers with a death : registration ratio of 0.45 .
more than 80% of oral cancers are squamous cell carcinomas arising from the oral mucosa with the tongue being the commonest site .
potentially malignant lesions are those in which the oral mucosa locally has undergone sufficient cytological change as to render it more likely than normal tissue to become malignant . the majority , if not all potentially malignant conditions are systemic disorders that result in atrophy of the oral mucosa resulting in increased penetrance of carcinogens . at the present time
it is not possible to differentiate the lesions that will ultimately progress to invasive carcinoma and as such warrant early aggressive treatment from those that will regress and can safely be left alone .
reibel in 2003 reviewed the concept of a two - step process of cancer development in the oral mucosa .
intraepithelial neoplasia and potentially malignant have been used in the international literature to broadly describe clinical presentations that may have a potential to become malignant .
follow - up studies have shown that between < 1 and 18% of oral premalignant lesions will develop into oral cancer and few clinical subtypes of leukoplakia are at a higher risk for malignant transformation than others .
the presence of epithelial dysplasia is more important in predicting malignant development than the clinical characteristics .
sir james paget is credited with the first description of an association between an oral lesion ( which he termed ichthyosis ) and subsequent development of tongue carcinoma . since then , there has been clinical , morphological and experimental evidence of a correlation between certain lesions in the oral cavity and subsequent development of oral cancer .
conflicting evidence exists for the role of various infectious agents in the etiology of precancer and oral cancer that includes human papillomavirus ( hpv ) and herpes simplex virus ( hsv ) . among the fungi , c.
albicans is the most common microorganism to pose a possible risk factor for the malignant transformation in premalignant lesions and conditions .
few studies have also indicated that presence of candidal infection may increase the risk of a premalignant lesion and conditions turning malignant .
oral lesions probably caused by candida have been reported since the time of hippocrates , as early as 377 bce .
langenbeck in 1839 is credited as a pioneer in recognizing a fungus , consistent with c. albicans in an oral aphtha ( ulcer ) of a patient with typhoid fever .
the association of candidal infection with leukoplakia was first reported by cernea et al . , and jepsen and winther in 1965 .
indeed , it is well - known that candida species are keratophilic and they tend to colonize thick layers of keratin .
the role played by various candida species in initiating oral epithelial lesions is ill understood .
cawson and lehner ( 1968 ) first suggested a possible etiological relationship between leukoplakia and candidal infection on finding mycelial elements of c. albicans infiltrating the cornified layer of the epithelium in some cases of human oral leukoplakia .
but other authors have regarded c. albicans as a secondary invader rather than the cause of the leukoplakia in those cases in which it is found .
the concept of carcinogenic potential of candida has received support from a study which demonstrated that certain c. albicans biotypes are capable of producing carcinogenic nitrosamine n - nitrosobenzyl methylamine from its precursors .
they also showed that strains with high nitrosation potential were associated with lesions showing more advanced precancerous changes .
the present study was carried out in patients with potentially malignant lesions and conditions such as leukoplakia , lichen planus and submucous fibrosis with the aim to compare the prevalence of presence of fungal hyphae ; to find out the association if any , with presence or absence of fungal hyphae and various grades of dysplasia ; and to test the prognostic significance of this information .
biopsy - based studies have reported candidal hyphae to constitute 750% in leukoplakia . in our study , we found 41.44% prevalence of presence of fungal hyphae in cases of leukoplakia with or without dysplasia [ table 3 and graph 1 ] .
this finding was in close approximation with the findings of the other studies , such as studies by krogh et al .
in contrast to our findings , few studies showed much lower prevalence of fungal hyphae in tissue sections ; they are , berret et al . , 1998 ( 7.1% ) ; banoczy and csiba 1976 ( 13.5% ) , silverman et al . , 1984 ( 23% ) . in our study among 29 cases of leukoplakia , 12 ( 44.44% )
cases showed presence of fungal hyphae and all the 12 ( 100% ) cases showed dysplasia in various grades [ table 4 ] .
the prevalence of fungal hyphae in cases of lichen planus in our study was 36% with or without dysplasia [ table 3 and graph 1 ] . in biopsy - based studies , the reported candidal colonization rates ranged from 0to 17.7% , without preference for a particular clinical presentation .
our study showed substantially greater prevalence of fungal hyphae in lichen planus than that suggested by previous biopsy - based studies by vuekovic et al . , 2004 ( 22.22% ) ; lundstrom et al . ,
1984 ( 7% ) ; holmstrup and dabelsreen , 1974 ( 2.3% ) ; and hatchuel et al .
, 1984 could have been caused by prior treatment with amphotercin b before biopsy in six of 39 patients . in a study by krogh et al .
, 1987 ; only reticular lichen planus was included and possibly this restriction excluded infected cases that could have had an altered clinical presentation , further possibility might be that in study by holmstrup p and dabelsreen 1974 lichen planus cases with candidal infection could have been classified as candidiasis and hence would not have been accessible in retrospective studies .
comparatively higher prevalence of candidal hyphae in lichen planus biopsies in our study suggests that lichen planus could be predisposing condition to candidal infection . in lichen planus ,
the oral mucosal pathology alters the integrity of the oral epithelium and predisposes the lesion to fungal infection ( lundstrom et al . , 1984 ) .
similarly , we found 25% prevalence of fungal hyphae in cases of submucous fibrosis with or without dysplasia [ table 3 and graph 1 ] .
our results are compatible with results obtained by kumar et al . , 2009 ; where they found positive hyphae in eight ( 33.33% ) out of 24 cases .
lack of published data on the occurrence of fungal hyphae in biopsy - based studies of submucous fibrosis makes comparison of our data with other studies difficult .
the prevalence of fungal hyphae was studied in cases with signs of dysplasia and we found significantly higher proportion of fungal hyphae in dysplasia cases ( leukoplakia 44.44% , lichen planus 66.66% and submucous fibrosis 100% ) [ table 4 and graph 2 ] .
we obtained statistically significant association between histopathologically determined fungal hyphae and various grades of epithelial dysplasia in these individual lesions [ tables 58 and graph 3 ] .
the results of our study were compatible with results obtained by zhang et al . , ( 1994 ) ; berret et al . , ( 1998 ) ; mccullough et al . , ( 2002 )
; spolidorio et al . , ( 2003 ) . the discrepancy in percentage prevalence of fungal hyphae in tissue sections in our study when compared to various studies
it is possible that overall detection rate of 5% represents an underestimate and there is abundant evidence that values of fungal infection as assessed by pas staining are lower than those obtained by culture and using the pas stain there is 13% chance of missing fungal infection , particularly if hyphae are scarce or only one section is analyzed ( roed - petersen et al . , 1970 ) .
on the other hand , histopathology is the quickest means of identifying those cases in which microorganisms have invaded the epithelium where they are more likely to be pathogenic .
non - invasive hyphae and yeasts may be detected in smears or grown in culture , but lost during histological processing ( cawson , 1968 ) , producing a negative result on staining the section with pas .
despite this possible underestimate , our study has observed a significant association of fungal hyphae with various grades of dysplasia and only in few cases without dysplasia .
our study confirms an increased frequency of fungal hyphae in some potentially malignant lesions and conditions and these results are in agreement with field et al . , 1989 .
though the staining pattern of fungal hyphae by pas and gms was similar , we found that the staining intensity was better with gms than pas .
our study suggests that , routine pas or gms staining should be advised whenever signs of candidal infection such as superficial abscess formation , chronic inflammation in lamina propria , epithelial hyperkeratosis and hyperplasia and presence of weak hematoxyphilic hyphae are observed in h and e stained sections of oral mucosal biopsies .
it also emphasizes the important role histopathology plays in the diagnosis of fungal infection . on histopathological confirmation of epithelial dysplasia
a histopathological report of unexpected fungal infection is likely to be acted on clinically and may affect the patient 's management and prognosis ( berret et al . ,
so we suggest future studies involving larger sample size and species isolation in potentially malignant lesions and conditions to bring about a revolution in the early detection , prevention , management and prognosis of these lesions .
epithelial dysplasia and prevalence of fungal hyphae in leukoplakia was significantly higher as compared to lichen planus and submucous fibrosis .
the degree of epithelial dysplasia was significantly associated with presence or absence of fungal hyphae in all cases .
this indicates a significant association between presence of fungal hyphae and various grades of epithelial dysplasia . | background : oral cancer is a major public health problem in india . a key factor that has led to lack of improvement in prognosis of oral cancer over the years , is delay in diagnosis and treatment . in many instances ,
a significant proportion of oral squamous cell carcinomas develop from premalignant lesions and conditions .
identification of such lesions and conditions is very important in order to prevent malignant transformation .
the role of fungal infections has been studied and holds promise as an indicator to predict malignant transformation .
so we designed a study to analyze the prevalence of fungal hyphae in biopsies of patients with clinically diagnosed cases of premalignant lesions and conditions.aims and objectives : to determine and compare the prevalence of fungal hyphae in biopsies of patients with clinically diagnosed cases of potentially malignant lesions and conditions and to assess the possible association between the degree of epithelial dysplasia and presence or absence of fungal hyphae.materials and methods : clinically suspected and histopathologically diagnosed 70 cases of potentially malignant lesions and conditions ( 29 leukoplakia , 16 submucous fibrosis and 25 lichen planus ) made up the study group .
three tissue sections ( 5m ) of each were stained with hematoxylin and eosin ( h and e ) , periodic acid schiff 's reagent ( pas ) and grocott 's methenamine silver ( gms ) and evaluated for fungal hyphae .
the data collected was statistically analyzed by using chi - square test and statistical package for social sciences ( spss ) software.results:the estimated prevalence of fungal hyphae in cases with or without dysplasia in leukoplakia was 41.4% , lichen planus 36% and submucous fibrosis 25% .
there was a significant association between degree of epithelial dysplasia with presence or absence of fungal hyphae in all the study groups.conclusion:presence of fungal hyphae in potentially malignant lesions and conditions may prove to be a useful indicator in predicting malignant transformation . |
to become long - term memory , newly learned information undergoes progressive changes and a stabilization process known as memory consolidation ( mcgaugh , 2000 ; dudai , 2004 ) .
this term was proposed more than 100 years ago by muller and pilzecker ( 1900 ) to indicate that , after learning , memory is initially in a labile state but , over time , becomes stable and resistant to disruption .
since then , many studies in different species and with numerous types of learning have shown that several kinds of interference , including blockade of new rna and protein synthesis , disruption of the expression or function of specific proteins but also new learning , brain cooling , seizure , brain trauma , and brain regional lesions can disrupt the consolidation of the new memory .
although all these treatments disrupt the formation of a new memory , the different types of interference revealed the existence of distinct temporal phases or processes that take place during memory consolidation . molecular interference with protein and rna synthesis inhibitors , blockers of receptors , kinases , neurotrophins , and transcription factors in species ranging from invertebrates to mammals showed that the biosynthetic - dependent phase of memory consolidation is relatively brief and is completed within the first day or two after training ( taubenfeld et al .
, 2001a , b ; bailey et al . , 2004 ; bekinschtein et al . , 2007 ; ou et al . , 2010 )
several reviews provide comprehensive summaries of what is known about the gradients of new protein and rna synthesis requirements , as well as the nature of genes and proteins that are critically recruited during memory consolidation ( kandel , 2001 ; davis and laroche , 2006 ; reissner et al . , 2006 ; wang et al . , 2006 ; bramham et al
proteins whose expression is induced by training and are required for long - term memory consolidation include several immediate early genes , such as the transcription factor ccaat enhancer binding proteins ( c / ebps ) , c - fos , and zif268 , as well as effector genes such as activity - regulated cytoskeletal protein ( arc ) and tissue - plasminogen activator ( tpa ) , to mention just a few ( melchor and strickland , 2005 ; alberini , 2009 ) . on the other hand , animal model and clinical studies based on retrograde and anterograde amnesia caused by brain trauma , stroke , or brain region inactivation or ablation
have shown that , in some types of memories such as the so - called explicit or declarative memories , consolidation lingers for a prolonged period .
these memories engage the medial temporal lobe , and particularly the hippocampus and related structures , including the fornix and entorhinal cortex , for their consolidation .
hippocampal damage impairs human declarative memories that are several years old and animal contextual memories that are up to 30 days old ; older memories remain unaffected ( smith and squire , 2009 ) . thus it has been proposed that the hippocampus initially works with the neocortex to consolidate memory but , over time , gradually becomes less critical .
in contrast , changes in the neocortex over time become more uniquely essential for storing the information by increasing connectivity among distributed cortical areas ( squire , 2009 ) .
given the very different temporal scales of memory fragility in response to different types of interference , one mediated by gene expression during the first 12 days after training and the other mediated by trace rearrangement among brain regions , it is unclear whether a relationship exists between the two temporal mechanisms of consolidation .
it is , in fact , possible that the term consolidation is used in different settings and by different disciplines just to indicate memory maturation processes .
thus , it would be important to determine whether or not different types of interferences and amnesia gradients have mechanistic commonalities , and whether or not the terminology is the only common ground . as mentioned below , it is , however , intriguing to note that the reconsolidation sensitive period seems to overlap with the trace rearrangement consolidation temporal evolution . despite this question
remains to be addressed , for several decades it was believed that memory consolidation is a unitary process : a newly formed memory requires gene expression for several hours , after which it becomes stable or consolidated ( davis and squire , 1984 ) .
however , studies done in the last decade inspired by findings reported earlier , starting from the 1960s , demonstrated that the initial gene expression - dependent phase required for memory consolidation is not the only one .
in fact , if memories that have become resistant to inhibitors of gene expression are retrieved , they again become labile for a limited time . during this time
the process , whereby a retrieved or reactivated labile memory re - stabilizes over time , is known as memory reconsolidation ( nader et al .
( 2000b ) awakened the scientific community to the retrieval - dependent fragility of memory .
these authors reported that an established auditory fear conditioning memory , 24 h or 2 weeks after its encoding , when it generally is not sensitive to inhibition of new protein synthesis , again becomes transiently sensitive if it is retrieved .
since then , many studies in several species , and with respect to different types of learning , have reported similar conclusions proving the generalization of memory reconsolidation ( dudai and eisenberg , 2004 ; alberini , 2005 ; tronson and taylor , 2007 ; nader and einarsson , 2010 ) .
these results also led to many questions : is reconsolidation a recapitulation of the initial consolidation process ?
do all types of memories undergo reconsolidation ? what is the relationship between reconsolidation and extinction ?
what mechanisms underlie reconsolidation ? what is the function of reconsolidation ? is the age of a memory a critical variable for reconsolidation ? can we target the reconsolidation process to weaken memories that contribute to diseases such as post - traumatic stress disorder ( ptsd ) and addiction ? in this article
, i will focus on the last three questions , then propose a model of memory reconsolidation .
it is important to answer this question , not only because it will allow a better understanding of how the memory process works , but also because this knowledge is essential to developing accurate clinical approaches that will use post - retrieval strategies to disrupt or weaken pathogenic memories .
at least two , non - mutually exclusive hypotheses have been proposed to explain the function of reconsolidation . according to one , memory becomes labile because through reconsolidation new information is integrated into the background of the past , thus allowing the memory to be updated ( lewis , 1979 ; sara , 2000a ; dudai , 2004 ) .
the other proposes that memory reconsolidates in order to become stronger and longer lasting ( sara , 2000b ) .
the conclusions reached are contradictory . some studies state that reconsolidation does not mediate memory updating , others that it does .
this controversy seems to be due in part to the fact that the same term was used to indicate different types of behavioral updating , and in part to conflicting interpretations of the results . in general terms
, we can define memory updating as all changes that are incorporated into a reactivated memory due to current perceptual input .
thus , every time a memory is retrieved it undergoes changes because , in fact , it is perceived in a different moment and with different current inputs .
this implies that both a distinct experience that becomes linked to a first , reactivated memory , as well as a second identical learning experience , can be defined as memory updating .
one has to do with how distinct experiences , that is , two distinct traces , become linked or associated ; the other has to do with how the same learning experience adds to a first one by repetition .
the studies that tested whether reconsolidation mediates updating after the reactivation of the same experience ( e.g. , multiple training trials ) need to carefully define the use of the terms consolidation and reconsolidation in those behavioral paradigms .
moreover , an important caveat should be kept in mind when testing the differential role of reactivated or new traces : that both consolidation and reconsolidation are sensitive to many of the same interfering agents , including protein synthesis inhibitors , pharmacological and molecular interferences , and new learning .
thus , a result showing that both the old and updated traces are disrupted by one treatment does not prove that reconsolidation mediates updating . in order to dissect whether reconsolidation differentially contributes to the updating of memory ,
it is necessary to use interfering approaches that selectively disrupt either reconsolidation of a reactivated memory or the consolidation of new traces .
a factor that has been considered critical to the definition of memory reconsolidation is whether amnesic agents applied after memory reactivation disrupt the original memory trace or whether reactivation , by definition , produces a new memory that undergoes consolidation . if reactivation produces a new trace , then disrupting this trace should not affect the old memory ; there should be no decrease in memory retention below the levels reached by the old memory ( debiec et al . , 2002 ) .
reconsolidation experiments in many different tasks and species have shown that this is not the case . to the contrary
, it has been established that , after retrieval or reactivation , the old trace is indeed , in significant part , sensitive to disruption . however , although this argument is clear with reactivations evoked by an experience that is different from the original training , for example a non - reinforced reminder after a reinforced conditioning , it is confusing in the case of the contribution of repeated training trials .
should it be called consolidation or reconsolidation the process induced by a second learning trial that , if challenged with an amnesic agent , results in memory disruption that goes below the retention level produced by the first trial ?
i will review some of the results and then return to these general issues about defining consolidation and reconsolidation . in my laboratory , we focused on testing an updating produced by linking two different experiences ( tronel et al . ,
this study asked whether linking new information to a previously established memory via its reactivation critically depends on the reconsolidation of this memory .
in other words , does reconsolidation mediate the incorporation of new information ? to address this question , we used an inhibitory avoidance task , modified to a second - order conditioning paradigm and the anatomically distinct requirement of the transcription factor c / ebp , specifically in hippocampus but not amygdala during consolidation and in amygdala but not hippocampus during reconsolidation .
second - order conditioning promotes the formation of associations between a new conditioned stimulus ( cs2 ) and a conditioned response elicited by another cs ( cs1 ) that was previously associated with an unconditioned stimulus ( us ; rescorla , 1980 ; gewirtz and davis , 2000 ) .
thus , the stimulus response learning that occurs during second - order conditioning represents the formation of an association between new ( cs2 ) and reactivated information ( memory of cs1us ) , which makes this paradigm proper for investigating the mechanisms involved in linking new to reactivated information .
we found that linking new information to a reactivated memory is not mediated by the reconsolidation of the reactivated memory .
in fact , when we selectively disrupted the reconsolidation of the cs1us , the second - order cs2-(cs1us ) remained intact .
in contrast , when we blocked consolidation selectively , only the new linking ( the update ) was disrupted , whereas the original memory remained intact .
we concluded that when two distinct experiences , one reactivated and one new , become linked , the reconsolidation of the reactivated trace does not mediate this updating ( tronel et al . , 2005 ) .
( 2006 ) , using rat auditory fear conditioning have found that disrupting the reconsolidation of cs1us by inhibiting amygdala protein synthesis after reactivation does not affect the association cs2cs1us .
results similar to ours were found with associative memories in the crab chasmagnatus ( surez et al . , 2010 ) , as well as in humans ( forcato et al . , 2010 ) .
hence , the function of reconsolidation must be different from that of linking a reactivated memory to a novel , distinct experience .
in general terms , we can infer that memory updating via formation of complex networks requires memory retrieval , but not reconsolidation and that the reconsolidation of a reactivated memory does not alter the entire network of updated associative memories .
other studies tested whether reconsolidation mediates memory updating by examining the contribution of the repetition of similar training trials or retrievals as compared to that of new encodings . these studies concluded that with multiple learning trials or reactivations , memory does indeed become labile .
however , the fragility is seen only when learning is in a non - asymptotic mode . when memory has reached an asymptotic level , it is resistant to disruption by post - reactivation amnesic treatments , but then becomes labile again if it is in a new encoding mode ( rodriguez - ortiz et al .
for example , morris et al . ( 2006 ) , using rat learning to locate a platform in a water maze concluded that : the engagement of a memory - encoding mode during the act of spatial memory retrieval may be one requirement for reconsolidation to be observed .
mismatch that triggers exploration and the updating of their cognitive representation of space and proposed that a dual encoding and retrieval state is required for the reconsolidation of spatial memory .
however , an alternative interpretation of the data , is that when new encoding is presented after the reactivation of a memory , two traces exist and are both in a labile state : one that mediates the reconsolidation of the reactivated trace if in a non - asymptotic phase and another that represents the newly encoded trace which may undergo a new consolidation process .
since the results of these studies do not clearly dissect the contribution of reconsolidation of an established memory versus consolidation of a new trace , they do not conclusively demonstrate a role for reconsolidation in the new encoding . nevertheless , it is interesting to note that inhibition of protein synthesis after repeated trials during a non - asymptotic phase returns the memory performance to the pretraining chance level , suggesting that the original trace had remained labile .
similar results have been reported in other studies that used multiple trial learning tasks to investigate the effects of amnesic treatments on memory consolidation ( e.g. , meiri and rosenblum , 1998 ; luft et al . , 2004 ; touzani et al . , 2007 ) .
, it is unclear whether the disruption of the memory ( object recognition , in this case ) occurs because , in the so - called reactivation trial , the rat associates the old information ( the objects ) with a novel environment and the mk-801 disrupts consolidation of this new memory trace , which , in fact , includes the old objects .
thus , my interpretation of these and other studies based on multiple learning trials , including attenuation of neophobia , incentive instrumental learning , and the morris water maze ( hernandez et al . , 2002 ; rodriguez - ortiz et al .
first , in agreement with the authors conclusions , during a learning curve , post - trial applications of amnesic treatments disrupt memory retention only when the memory is not in an asymptotic level .
however , when retention has reached an asymptotic level and no further learning or increased retention is evident , a previously consolidated memory remains stable and resistant to disruption .
second , if then new events are presented and associated with this memory , a new trace is formed which is labile because it undergoes consolidation . if part of the old information is incorporated in a new trace , its retention might be disrupted if the new trace is challenged by amnestic treatments , following the rules of the predominant active trace ( eisenberg et al . , 2003 ) .
should the process of fragility and restabilization resulting from an identical learning trial that is in a non - asymptotic phase of learning be called consolidation or reconsolidation ? is the distinction between consolidation and reconsolidation related to the level of memory disruption ( whether is goes below the incremental level ) ?
if one defines reconsolidation as the process that occurs after each sequential trial during the non - asymptotic phase of a learning curve , then the classic definition of the consolidation process of a multiple trials task coincides with what here would be called a process of consolidation reconsolidation .
interestingly , this stabilization , which would occur through rounds of reconsolidation and result in a memory resilient to disruption , is highly reminiscent of that found after a salient single - trial learning , which over time develops a graded increased resistance to reconsolidation disruption ( see below ) .
this view of reconsolidation as an integral part of the consolidation process was proposed a few years ago by both dudai and eisenberg ( 2004 ) and myself ( alberini , 2005 ) .
in other words , it seems that reconsolidation , as part of a lingering consolidation process , strengthens memory retention by following the rules of trace dominance ( eisenberg et al . , 2003 ) .
notably , what the classical consolidation hypothesis did not account for , nor explored , are the mechanistic contributions of each reactivation of the trace that can be elicited by each learning trial , or the fact that non - reinforced reminders can produce a similar result . supporting the conclusion that reconsolidation increases memory strength , lee ( 2008 )
he then showed that when hippocampal injection of bdnf antisense oligodeoxynucleotide ( odn ) , which had been found to disrupt the initial consolidation but not reconsolidation of this memory ( lee et al . , 2004 ) ,
however , injection of zif268 antisense , which had been shown to disrupt reconsolidation but not consolidation of contextual fear conditioning , when given before the second training trial , completely disrupted memory retention .
his results are in agreement with several lines of evidence that memory reconsolidation , like strengthening ( lee , 2008 ) , uses mechanisms that differ from those used during the initial wave of consolidation ( taubenfeld et al . , 2001a ; lee et al .
, 2004 ; tronel et al . , 2005 ; von hertzen and giese , 2005 ) .
recent results from our laboratory , obtained by using inhibitory avoidance in rats and non - reinforced reminders , lead to similar conclusions ; we found that retrievals of a young memory , which are accompanied by its reconsolidation , result in memory strengthening and contribute to its overall consolidation and concluded that a function of reconsolidation is to mediate memory strengthening and prevent forgetting ( inda et al . , 2011 ) .
in agreement with this conclusion , earlier evidence indicated that , following reactivation , memory can indeed be strengthened .
for example , memory reactivation through the re - experience of a single contingent - reinforcing stimulus given in the same context as previous intermediate - term memory training boosts a residual memory trace into long - term memory ( parvez et al . , 2006 ) .
electrical stimulation of the mesencephalic reticular formation ( mrf ) improves the retention of memories , including fear conditioning and linear maze , when the stimulus is applied after reactivation ( devietti et al .
, memory retention can be increased by pharmacological modulation ( frenkel et al . , 2005 ; tronson et al . , 2006 )
thus , memory enhancement , like memory disruption , can be achieved by targeting a post - retrieval or post - reactivation memory process .
this opportunity to enhance memory strength following retrieval , like that of disrupting it , has important clinical implications . in summary , based on the current knowledge , i suggest that the functional role of reconsolidation , whether induced by a second training trial or a non - reinforced reminder , is to mediate memory strengthening and prevent forgetting .
in contrast , memory updating with new learning and encoding results in a new memory trace that is accompanied by a new consolidation process .
it seems likely that traces that are retrieved or reactivated will reflect which information will undergo reconsolidation and strengthening .
thus , the strengthening evoked by a second training trial will be qualitatively and , perhaps , quantitatively different from that evoked by a non - reinforced reminder . understanding the functions and boundaries of the reconsolidation process is critical for designing treatments that aim at memory disruption or enhancement
a key question is : what happens to an encoded and consolidated memory over time ?
time is an extremely important parameter in memory formation , allowing the selection of what will be stored as long - term memory and dictating what and for how long information will be stored .
clinical studies of retrograde amnesia inspired the concept of memory consolidation ( squire and alvarez , 1995 ; mcgaugh , 2000 ) , according to which medial temporal lobe structures are necessary for the formation and maintenance of a memory for a limited time . during this time
after this reorganization is completed , the medial temporal lobe structures are no longer essential for maintaining the information . indeed ,
clinical and experimental evidence indicate that damage to the medial temporal lobe does not interfere with the storage of remote memories , but does impair recent memories in a temporally graded fashion . as mentioned earlier , the temporal window of this circuitry rearrangement seems to be approximately a few weeks in animal models and a few years in humans ( squire , 2009 ) . in agreement with this temporal evolution of memory consolidation , the expression of activity - related genes such as c
- fos , as well as the uptake of 2-deoxyglucose induced by recall of contextual fear conditioning in rats decrease in the hippocampus and increase in cortical areas as the memory ages over several weeks ( frankland and bontempi , 2005 ) .
neuroimaging studies in humans have shown a similar anatomical distribution of activity when subjects recall memories ranging from 1 to 30 years old .
other support to this temporal frame comes from recordings from cell populations in the medial prefrontal cortex ( mpfc ) in rats during and after eyeblink conditioning .
moreover , firing - rate activity selective for memory associations starts to develop during the late stage of acquisition and continues for up to 6 weeks independently of whether or not the animals are continuously trained .
this slow time course of firing - rate changes agrees with a previously observed time window in which the mpfc becomes important for retrieval . finally , in line with the key principles of the consolidation theory ,
associative learning seems to initiate a temporally graded process that leads to long - lasting changes in cortical areas ( e.g. , the mpfc ) , even without continued training ( takehara - nishiuchi and mcnaughton , 2008 ) .
it is possible , as indicated by animal studies , that the rearrangement of the memory representation from hippocampal to cortical areas , occurs together with a qualitative transformation ; hence , initial memory , which would be highly detailed and context - specific may become one that is more general ( moscovitch et al .
in other words , is the post - retrieval lability of memory a function of time ? understanding the temporal boundaries of reconsolidation is critical for the development of efficacious clinical approaches that target the reconsolidation process .
the initial hypothesis of memory reconsolidation proposed that , when a memory is active ( following training or reactivation ) , it is in a labile state , but that when it is inactive , it is stable ( nader et al . , 2000a ) .
this implies that every time a memory is reactivated ( active ) it becomes labile .
the results have shown that , in most cases , in a variety of species , including rat , mouse , chick , and medaka fish , the passage of time critically influences the stability of a memory , so that the older a memory becomes , the less susceptible it is to disruption following its reactivation .
these studies report increasing resilience to disruption following memory reactivation over a time window of several weeks .
for example , in rat inhibitory avoidance , 2- and 7-day - old memories are disrupted by protein synthesis inhibitors administered after retrieval , while 14- and 28-day - old memories are resistant to the same treatment ( milekic and alberini , 2002 ) .
similarly , in medaka fish , the administration of a na channel blocker at the time of memory retrieval disrupts a 4-day - old memory and has an intermediate effect on a 9-day - old memory , but exerts no effect on a 15-day - old memory ( eisenberg and dudai , 2004 ) .
studies of other species and different types of learning , including both aversive and appetitive , have confirmed the existence of a temporal gradient of progressive resilience to memory disruption after reactivation ( litvin and anokhin , 2000 ; suzuki et al . , 2004 ; boccia et al . , 2006 ; frankland et al . , 2006 ; robinson and franklin , 2010 ) .
additional findings have shown that , in addition to the age of a memory , its strength and the reactivation intensity interact to influence the degree of vulnerability of a reactivated memory ( suzuki et al . , 2004 ,
2004 ) showed that whereas protein synthesis inhibitors can disrupt a reactivated 24-h- to 3-week - old contextual fear memory in mice , an 8-week - old memory remains unaffected by a similar reactivation .
however , if the reactivation session is prolonged , even the older memory can be disrupted . finally , weak ( 1 shock ) versus strong ( 3 shocks ) training protocols correlate , respectively , with more or less susceptibility to disruption after reactivation .
it is important to keep in mind that memory changes over time and that long - lasting reactivation preferentially evokes extinction ( a decrease in the conditioned response resulting from cs presentations ) over reconsolidation ( eisenberg et al . , 2003 , bustos et al . , 2009 ) .
thus , for stronger memories , which are insensitive to reconsolidation disruption but become sensitive when a long reactivation is applied , it is important to exclude the possibility that memory disruption is the result of a facilitated extinction .
the fact that , as noted earlier , memory changes over time is also supported by molecular experimental evidence , including reconsolidation studies .
reconsolidation has distinct requirements than the initial consolidation ( alberini et al . , 2006 ) .
reconsolidation of an associative memory in lymnaea , which is evident at both 6 or 24 h after training by using protein synthesis inhibitors , shows different molecular requirements : only reconsolidation at 6 h after training , but not at 24 h , requires protein kinase a activity .
hence , even seemingly consolidated memories undergo further selective molecular maturation processes ( kemenes et al . , 2006 ) . in summary , over time , memory undergoes many molecular , anatomical , and qualitative changes and these include its increased resilience to reconsolidation disruption .
they view reconsolidation as a general process that occurs following memory retrieval without temporal constraints , and explain temporal boundaries as experimental limitations ( nader and einarsson , 2010 ) .
( 2002 ) showed that 2-day- and 2-week - old cued - fear conditioning memories , as well as a 45-day - old contextual fear conditioning memories in rats , are disrupted , respectively , by post - retrieval bilateral injection of anisomycin into the amygdala or hippocampus . in disagreement with these contextual fear conditioning results ,
other groups have reported that , with time , contextual fear memories in mice become less sensitive to post - retrieval interference ( suzuki et al . , 2004 ; frankland et al . ,
other memories have been reported to have a long - lasting temporal window of post - reactivation fragility .
for example , a 21-day - old appetitive instrumental response ( sucrose self - administration ) is disrupted by systemic injection of the -adrenergic antagonist propranolol , while a 27-day - old cocaine - induced conditioned place preference is disrupted by post - reactivation amygdala injections of zif268 antisense ( diergaarde et al .
in contrast , studies on morphine - induced conditioned place preference show that increased resistance to post - reactivation treatment with propranolol or the gaba ( a ) agonist midazolam develops with stronger training ( robinson and franklin , 2010 ) .
these opposing results can be explained by the findings discussed earlier , which have shown that the nature of the learning task and therefore its underlying circuitry as well as the intensity of training and reactivation are all contributing to produce gradients of post - retrieval protein synthesis requirement that have different temporal evolutions .
it is consistent with a lingering consolidation and the effects of retrograde amnesia on medial temporal lobe - dependent memories that the stabilization process may take a long time .
hypotheses that could be explored are whether the increased resilience to reconsolidation disruption is related to its increased cortical representation or increased distribution over brain areas ( nadel and moscovitch , 1997 ) , or the implicit or explicit nature of the memory and its storage distribution .
it is important that these issues be addressed because it will provide useful information for designing clinical trails .
importantly , the existence of a gradient does not exclude the possibility that reactivation of fully consolidated memories or a memory in an asymptotic phase is accompanied by a phase of de novo protein synthesis .
however , new molecular changes induced by the reactivation of a fully consolidated memory may occur without manifesting fragility of the consolidated information .
the findings on memory reconsolidation have triggered great interest in the medical community , particularly among mental health professionals who treat disorders that are based on pathogenic memories .
two pathologies that could benefit from targeting memory reconsolidation as a potential therapeutic approach are ptsd and drug addiction , both of which are characterized by the presence of strong , repeatedly recalled memories .
post - traumatic stress disorder can develop after exposure to an emotionally or physically traumatic event .
one hallmark of the disorder is that the subject keeps reliving the trauma through repeated , intrusive memories of the initiating experience .
these symptoms can be so severe and persistent that they significantly impair patients ability to function .
although no animal model is yet available to reproduce ptsd fully , various behavioral paradigms have been used to represent some component of the disorder , using a rationale description of how ptsd develops .
according to this description , the traumatic event ( us ) triggers a strong hormonal stress response , which mediates the formation of a robust and enduring memory of the trauma .
subsequent recall of the event in response to cues and reminders ( cs ) releases more stress hormones ( conditioned response ) and even further consolidates the memory , leading to ptsd symptoms such as flashbacks , nightmares , and anxiety ( pitman and delahanty , 2005 ) .
the persistence of ptsd can be explained in terms of trauma - induced strengthening of the memory trace .
for example , it is hypothesized that noradrenergic hyperactivity and stress hormones facilitate the encoding and consolidation of the memory ( pitman , 1989 ; o'donnell et al . , 2004 ;
thus , animal models that evoke strong fear conditioning memories and high - anxiety behavior have been used to study questions that are clinically relevant to ptsd . in principle , the pharmacological and behavioral interferences thus far found to be effective in disrupting fear memory reconsolidation or enhancing extinction ( not discussed in this paper ) could potentially be useful for identifying new treatments that can be tested in clinical trials . some of the compounds found to affect reconsolidation , including antagonists of -adrenergic or glucocorticoid receptors ( grs ) , are already used in clinical pharmacology for treating other diseases .
hence , they are the most readily available potential therapies for targeting reconsolidation in ptsd and addiction .
antagonists of the -adrenergic receptor , such as propranolol , have already been explored at preclinical and clinical levels .
propranolol , which is most commonly used to treat hypertension , has been administered in concert with the retrieval of a fearful or traumatic event in both animals and humans .
( 1999 ) provided one of the first reports on the effect of propranolol on memory reconsolidation . using rat inhibitory avoidance
, these authors found that systemic administration of propranolol after the reactivation of an inhibitory avoidance memory disrupted the memory on subsequent tests .
however , we recently reached the opposite conclusion . because the shock used by przybyslawski et al .
( 1999 ) was very weak ( 0.2 ma ) , we set out to determine whether the reconsolidation of a memory induced by a greater shock , which would more closely approximate a traumatic event , was sensitive to propranolol treatment .
hence , we tested the effect of the same propranolol treatment in rats given either before or after the retrieval of an inhibitory avoidance memory evoked by a 0.6- or 0.9-ma foot shock .
we found that although these memories could be disrupted by several other treatments , like anisomycin or the grs antagonist ru38486 , propranolol had no effect ( muravieva and alberini , 2010 ) .
however , to provide solid preclinical information , it is important that the potency and generalization of the propranolol treatment on fear memories be established . using another paradigm known as auditory fear conditioning , debiec and ledoux ( 2004 )
have found that propranolol injected either systemically or into the lateral nucleus of the amygdala after reactivation of a 1-day- or 2-month - old memory weakened the fear response when tested 48 h later . in agreement , in recent studies based on contextual auditory pavlovian fear conditioning , we found that systemic propranolol injection following a retrieval elicited by cue exposure interferes with the reconsolidation of both cued and contextual fear conditioning . on the other hand , propranolol administered after contextual reactivation only affects contextual fear conditioning and has no effect on the auditory fear .
thus , it seems that the efficacy of systemically administered propranol in disrupting the reconsolidation of fear memories might be limited ( muravieva and alberini , 2010 ) .
other studies reported divergent results on the effect of propranolol on memory reconsolidation : whereas the reconsolidation of eyeblink conditioning potentiation and conditioning to natural or drug - associated reward is disrupted by propranolol ( diergaarde et al .
2009 ) , the reconsolidation of appetitive pavlovian memories in rats ( lee and everitt , 2008 ) and neutral and emotional verbal memories in humans ( tollenaar et al . , 2009 )
interestingly , declarative measures for the acquired contingency between the cs and us ( kindt et al . , 2009 ) are insensitive to propranolol treatment , but the fear response is sensitive . hence , as kindt et al .
( 2009 ) have suggested , propranolol may target the fear response , but not the cognitive or explicit components of that response , and further studies should be able to address this question .
the endogenous stress hormone corticosterone bidirectionally modulates memory retention ( mcgaugh and roozendaal , 2002 ; roozendaal , 2002 ) .
recent studies have shown that glucocorticoids administered after the reactivation of a contextual fear memory have an amnesic effect on the original memory .
these studies also have provided evidence that a possible mechanism for this effect is enhancement of extinction of the expression of the original memory ( cai et al . , 2006 ) .
my laboratory investigated the effect of blocking the grs on inhibitory avoidance memory reconsolidation in rats .
we found that post - retrieval , intra - amygdala blockade of grs by the antagonist ru38486 significantly disrupts inhibitory avoidance in rats ( tronel and alberini , 2007 ) .
further investigations have shown that also systemic administration of ru38486 , either before or after retrieval , consistently weakens inhibitory avoidance retention in a dose - dependent manner .
the efficacy of treatment appears to be a function of the intensity of the initial trauma ; however , highly traumatic memories are disrupted by changing the time and number of interventions .
furthermore , we found that one or two treatments are sufficient to maximally disrupt the memory and that the treatment selectively targets the reactivated memory without interfering with the retention of another unreactivated memory ( taubenfeld et al .
in light of these results , the glucocorticoid pathway appears to be a promising site of pharmacologic intervention for trauma - related pathologies , including ptsd . a novel and very interesting alternative to pharmacological disruption , which uses a behavioral design , has recently been provided by animal and human studies based on sequential retrieval ( reconsolidation ) and extinction .
extinction training after fear conditioning retrieval leads to permanent memory impairment if it is presented within a post - retrieval reconsolidation temporal window ( monfils et al . , 2009 ; schiller et al . ,
substance abuse generally leads to a chronic condition believed to result from an addict 's inability to permanently abstain from drug use .
this pathologic behavior is frequently induced by the recall of memories and environmental stimuli that are intimately connected to the rewarding effects of the drug ( o'brien et al . ,
therefore , disruption of memory reconsolidation provides an unprecedented potential strategy to disrupt memories that facilitate drug addiction .
promising results have recently been achieved in animals dependent on morphine or cocaine by injecting , after memory reactivation , inhibitors of protein synthesis , extracellular signal - regulated kinase ( erk ) , or -adrenergic receptors , or by disrupting the expression of the immediate early gene zif268 either peripherally and within specific brain regions , such as the amygdala , hippocampus , or nucleus accumbens ( lee et al . , 2005 ; miller and marshall , 2005 ; milekic et al . , 2006 ; valjent et al . , 2006 ; fricks - gleason and marshall , 2008 ; robinson and franklin , 2010 ) . in some of these studies ,
inhibitors were injected in animals that had acquired a place preference in response to the drug of abuse , a learning known as conditioned place preference . in this paradigm , animals learned to associate the effects of the drug with a specific location and chose to spend time there .
the administration of several of these inhibitors after reactivation of the drug - related memory interfered with its reconsolidation and abolished the place preference on later tests .
recently , we also found that disrupting the reconsolidation of a conditioned place preference induced in rats by morphine also leads to a loss of motivational withdrawal evoked in the same place .
interestingly , the hippocampus has a critical role in linking the place preference memory to the context conditioned withdrawal , as interfering with hippocampal molecular mechanisms after the reactivation of morphine conditioned place preference significantly weakens the motivational withdrawal .
thus , targeting the reconsolidation of memories induced by drugs of abuse may prove to be an important strategy for attenuating context conditioned withdrawal and , therefore , relapse in opiate addicts ( taubenfeld et al .
other studies have investigated a different type of task in which animals form a cs drug association during drug self - administration training , a model of drug seeking ( lee et al . , 2005 , 2006 ) .
these experiments showed that infusion of zif268 antisense odn into the basolateral amygdala before the reactivation of a cs cocaine association abolishes its impact on the learning of a new cocaine - seeking response or maintenance of cocaine - seeking , as well as relapse to a previously established drug - seeking behavior .
the same group later demonstrated that conditioned withdrawal can be disrupted following the reactivation of a cs - withdrawal association ( hellemans et al . , 2006 ) .
from all these investigations , it emerges that finding agents or strategies that disrupt memory reconsolidation is a potentially important approach for developing novel treatments that aim at weakening pathogenic memories .
it is important that future studies determine precisely what response is affected by the post - reactivation treatments and how the age of the memories changes their sensitivity to treatments .
finally , one other potentially important clinical application of targeting memory reconsolidation , which has not yet been much explored , is memory enhancement .
initial findings indicate that memory can be significantly enhanced by targeting mechanisms activated in the post - retrieval phase of memory ( frenkel et al .
, 2005 ; tronson et al . , 2006 ; chen et al . , 2011 ) .
this intriguing question requires future exploration and the results thus far obtained imply that , in order to identify memory enhancers , we may need to explore the temporally limited post - training or post - retrieval active phases .
memories of a single event that become very long - lasting are evoked by the experience of a very salient or emotional event .
studies of consolidation and reconsolidation , particularly in animal models , have often used models of salient single or few learning trials because they easily allow temporal analysis of the changes that occur after a learning trace is evoked .
these studies show that the temporal evolution of the labile consolidation and reconsolidation phases of memory is a function of the intensity of training , reactivations , and the passage of time .
? learning of a salient event leads to a long - lasting memory through molecular cellular and circuitry changes that evolve over an extended time ( weeks in rats ) . during the first 12 days
, memory is disrupted by treatments that interfere with the synthesis of a number of proteins ; however , following this time , memory becomes resistant to the same amnestic treatments .
however , the memory still lies for some time in a sensitive , critical period , during which the memory can again return to a labile state if reactivated for example by retrievals or retraining , and , while in this fragile state , its retention can be bidirectionally modulated . with the passage of time ,
a gradient of memory stabilization sets in , along with increased resistance to post - retrieval interference . what is the contribution of time to memory strengthening and consolidation ?
first , the memory trace strengthens because it undergoes implicit reactivations , perhaps as a consequence of rythmicities like circadian rhythm or sleep ( stickgold and walker , 2007 ) . a salient , aversive or traumatic event is also frequently recalled over and over , especially during the first days or weeks ( rubin et al . , 2008 ) .
perhaps these reactivations , implicit , and explicit , serve the biologically important function of consolidating an aversive memory without repeating the aversive experience .
similarly , multiple trials of weaker learning events would produce the same outcome through rounds of consolidation and reconsolidation .
reactivation of the memory also has another important function , which is to update our memories and add new information to the past experience .
if the information is a repetition of a previous experience , its reactivations may overlap and perhaps even override the network of the original trace and finally lead to strengthening of the same behavior .
if the new information encountered is novel and distinct from the recalled experience , the new trace , may become linked to the reactivated trace and create a new , parallel memory that coexists with the original one .
importantly , as this new memory is established via a new consolidation process , new associations can be made even when the old reactivated memory is insensitive to post - reactivation interference ( morris et al .
trace activation and reactivation creates a complex network of stored experiences that is highly dynamic , thus allowing us to adapt to the changing environment .
a better understanding of the dynamics of memory consolidation , reconsolidation , and forgetting should help in the design of novel strategies that can either weaken pathogenic memories of memory components or , when advantageous , enhance memory strength .
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 . | it is becoming increasingly clear that the processes of memory formation and storage are exquisitely dynamic . elucidating the nature and temporal evolution of the biological changes that accompany encoding , storage , and retrieval is key to understand memory formation . for explicit or medial temporal lobe - dependent memories that form after a discrete event and
are stored for a long time , the physical changes underlying the encoding and processing of the information ( memory trace or engram ) remain in a fragile state for some time . however , over time , the new memory becomes increasingly resistant to disruption until it is consolidated .
retrieval or reactivation of an apparently consolidated memory can render the memory labile again , and reconsolidation is the process that occurs to mediate its restabilization .
reconsolidation also evolves with the age of the memory : young memories are sensitive to post - reactivation disruption , but older memories are more resistant .
why does a memory become labile again if it is retrieved or reactivated ? here
i suggest that the main function of reconsolidation is to contribute to the lingering consolidation process and mediate memory strengthening .
i also discuss the literature and results regarding the influence of the passage of time on the reconsolidation of memory .
these points have important implications for the use of reconsolidation in therapeutic settings . |
in recent years , several cowpox virus ( cpxv ) infections in humans have been described in europe [ 1 , 2 , 3 , 4 , 5 , 6 ] .
cowpox virus , as well as monkeypox , vaccinia and variola virus , belong to the genus orthopoxvirus , which can infect humans .
wild rodents are considered the reservoir host of cpxv and transmission to humans was shown to occur through infected animals , mostly domestic cats , but also pet rats [ 4 , 5 ] and captive circus animals [ 3 , 6 ] . in immunocompetent humans , cowpox remains localized at the level of skin lesions which heal after several weeks ; however , severe clinical cases have been described and a fatal infection was reported in an immunocompromised patient .
rodent - to - human transmission was reported after physical contact with animals or infection through skin abrasions , resulting in successive lesions for 2 weeks , but not often after a bite .
we report a case of cpxv infection that occurred in a boy without documented contact with animals .
a 10-year - old boy with no significant medical history , living in the southwest of france , presented with an initially small , red , infiltrated patch on the outside of his right arm which evolved quickly into a necrotic ulcer of 3 cm in size , surrounded by an erythematous infiltrated area ( fig .
the lesion was painless and the patient noted a low - grade fever of 38c during the first days . within several days , the erythema surrounding the ulcerated lesion extended to the entire arm , shoulder and axilla , associated with a morbilliform rash on his cheeks ( fig .
the patient was first treated with amoxicilline - clavulinate for 5 days then with azithromycin ( 10 mg / kg / day for 5 days ) with no improvement .
the rash disappeared after 10 days , while lymph node enlargement persisted for 6 weeks and skin ulceration healed within 2 months by using hydrocolloid dressings .
laboratory investigations were unremarkable except for a slight elevation of eosinophils ( 870 g / l , 11% ) .
bacterial cultures were negative ; no leishmania parasite was identified on microscopic examination of skin scrapings .
serology for coxiella burnetii , rickettsia conorii , borrelia burgdorferi , rickettsia typhi , rickettsia felis , and parvovirus b19 were negative .
assuming that the patient could suffer from tick - borne lymphadenopathy , detection of rickettsia slovaca using pcr of skin biopsy was performed ( d. raoult , who collaborative center for rickettsial reference and research , marseille , france ) and was negative .
light microscopic examination of a skin - punch biopsy showed full - thickness necrosis of the skin .
he had no recent contact with animals such as rodents or cats , but he reported that he had played extensively in the hay on a farm near his home 12 days before the first lesions appeared .
his parents reported that he never had direct contact with rodents , but the hay barn where he played regularly houses rodents .
orthopoxvirus was diagnosed , after dna extraction , in both the original swab and after culture in vero cells by a 14-kda fusion protein real - time pcr .
monkeypox virus - specific real - time pcr on both n3r and f3l genes was negative . to identify the isolate ( designated cpxv - f2010 ) ,
the ha sequence ( genbank hq634150 ) was compared to other orthopoxvirus and cpxv ha sequences available in genbank and to cpxv ha sequences of clinical isolates including the viruses involved in the 2009 cpxv outbreaks in france and germany [ 4 , 5 ] .
a blast search performed for species identification of the reported case matched 8 published cpxv strains with an identity from 85 to 97% ( www.ncbi.nlm.nih.gov/blast/blast.cgi . ) .
the cpxv - f2010 strain clustered with 6 other cowpox virus strains available in genbank including the brighton red strain ( cpxv - br ) .
cpxv - f2010 is relatively close to cpxv - br ( 97% identity ) and differs from outbreaks of human cowpox which occurred from december 2008 to january 2009 in france and germany ( cpxv - f2009 and cpxv - ger - rat2009 , respectively ) . to improve the phylogenetic distribution of cpxv strains ,
7 genes of the cpxv - f2010 and the cpxv - f2009 case were amplified ( table 1 ) and sequenced to be concatenated .
this second phylogenetic tree confirmed the cpxv species of the reported cpxv - f2010 case .
the sequences of 7 concatenated genes from cpxv - f2010 and cpxv - br formed a clade , which is clearly distinct from other cowpox virus strains reported .
the cpxv - f2010 strain differs from cpxv - br by 2% at the nucleotide level .
next , we investigated the in vitro host range , plaque phenotype and replication rate of cpxv - f2010 and cpxv - br , the closely related strain identified by phylogenetic analysis ( table 2 ) .
four mammalian cell lines from various species and tissues were infected with the 2 viruses .
cpxv - br has a broad host range and multiplied in all the cell lines .
cpxv - f2010 multiplied only in vero , mrc5 and bhk-21 cells but not in mouse la-4 cells .
the lytic plaques obtained with cpxv - f2010 in vero , veroe6 and mrc5 cells were significantly smaller than those induced by cpxv - br ( p = 0.0009 , p = 0.0078 , and p = 0.0146 , respectively ; unpaired t - test ) .
we could not determine the foci size on bhk-21 because of their imprecise outline morphology .
moreover , compared to cpxv - br , cpxv - f2010 had significantly reduced virus multiplication in all the cell lines ( data not shown ) .
the data of in vitro phenotypic analyses suggest that cpxv - f2010 is different from cpxv - br .
orthopoxvirus was diagnosed , after dna extraction , in both the original swab and after culture in vero cells by a 14-kda fusion protein real - time pcr .
monkeypox virus - specific real - time pcr on both n3r and f3l genes was negative . to identify the isolate ( designated cpxv - f2010 ) ,
the ha sequence ( genbank hq634150 ) was compared to other orthopoxvirus and cpxv ha sequences available in genbank and to cpxv ha sequences of clinical isolates including the viruses involved in the 2009 cpxv outbreaks in france and germany [ 4 , 5 ] .
a blast search performed for species identification of the reported case matched 8 published cpxv strains with an identity from 85 to 97% ( www.ncbi.nlm.nih.gov/blast/blast.cgi . ) .
the cpxv - f2010 strain clustered with 6 other cowpox virus strains available in genbank including the brighton red strain ( cpxv - br ) .
cpxv - f2010 is relatively close to cpxv - br ( 97% identity ) and differs from outbreaks of human cowpox which occurred from december 2008 to january 2009 in france and germany ( cpxv - f2009 and cpxv - ger - rat2009 , respectively ) . to improve the phylogenetic distribution of cpxv strains , 7 genes of the cpxv - f2010 and the cpxv - f2009 case
this second phylogenetic tree confirmed the cpxv species of the reported cpxv - f2010 case .
the sequences of 7 concatenated genes from cpxv - f2010 and cpxv - br formed a clade , which is clearly distinct from other cowpox virus strains reported .
the cpxv - f2010 strain differs from cpxv - br by 2% at the nucleotide level .
next , we investigated the in vitro host range , plaque phenotype and replication rate of cpxv - f2010 and cpxv - br , the closely related strain identified by phylogenetic analysis ( table 2 ) .
four mammalian cell lines from various species and tissues were infected with the 2 viruses .
cpxv - br has a broad host range and multiplied in all the cell lines .
cpxv - f2010 multiplied only in vero , mrc5 and bhk-21 cells but not in mouse la-4 cells .
the lytic plaques obtained with cpxv - f2010 in vero , veroe6 and mrc5 cells were significantly smaller than those induced by cpxv - br ( p = 0.0009 , p = 0.0078 , and p = 0.0146 , respectively ; unpaired t - test ) .
we could not determine the foci size on bhk-21 because of their imprecise outline morphology .
moreover , compared to cpxv - br , cpxv - f2010 had significantly reduced virus multiplication in all the cell lines ( data not shown ) .
the data of in vitro phenotypic analyses suggest that cpxv - f2010 is different from cpxv - br .
we report cpxv infection in a 10-year - old boy , with wild rodents as a possible source of infection .
although wild rodents are considered a natural reservoir of cpxv , only few reports have described wild rodent - to - human transmission [ 1 , 14 ] .
the presence of wild rodents in the hay barn where the boy played 2 weeks before skin lesion appearance , suggests he might have been infected by contact with one of the rodents .
neither bites nor scratches having been felt by the patient , the mode of transmission is uncertain .
the virus was identified as cpxv by em , cell culture isolation , pcr and ha gene sequence analysis .
genotypic analyses on 7 concatenated genes showed that cpxv - f2010 formed a separate clade with cowpox virus strain cpxv - br , isolated originally from a milker in the united kingdom .
however , the phenotypic in vitro study showed that cpxv - f2010 differs from cpxv - br , suggesting a new strain distinct from other cpxv strains reported so far .
this case highlights the risk for cpxv zoonoses in europe where wild rodents are largely infected .
the risk of infection has increased since the cessation of smallpox vaccination and the consequent decrease of orthopoxvirus immunity .
it also underscores the need for physicians and veterinarians to be aware of the risk for cpxv zoonoses and to investigate for cowpox in case of characteristic skin lesions in order to diminish the delay after several diagnostic and therapeutic detours .
although no cases of inter - human cpxv transmission have been reported , the risk of self - contamination has to be taken into account due to the scraping of the primary lesion by fingers .
semipermeable bandage should be used to cover the lesion site in order to provide significant protection from exposure to the virus on the outside of the bandage , and recommendations are necessary to avoid self - contamination with contaminated bandages .
this work was supported by research grants from the service de sant des armes ( ssa ) and the dlgation gnrale pour l'armement ( dga ) .
| the case presented here points towards the fact that skin lesion observed with a cowpox virus is a rare event but should be considered more as the number of cases has increased in the last years .
cowpox virus ( cpxv ) belongs to the poxviridae family .
the transmission of cpxv to humans is caused by wild rodents or mostly by domestic animals and pet rats . in humans , cpxv is responsible for localized skin lesions regularly accompanied by lymphadenopathy .
the lesions remain localized but self - inoculation from the primary lesions could occur . then physicians have to be vigilant concerning bandages . in this case report , a necrotic and ulcerated lesion of a cpxv infection in a young boy is reported .
the cpxv was possibly transmitted by wild rodents .
the importance of performing the diagnosis is also pointed out .
virus information was obtained from phylogenetic analyses showing that the cpxv isolate was distinct from outbreaks of human cowpox which occurred in 2009 in france and germany but was close to the cpxv brighton red strain . for several years
, cases of viral zoonosis caused by cpxv have increased and physicians should be made aware that people could be infected without history of direct contact with animals . |
portal hypertension in liver cirrhosis results from the anatomical changes and the development of contractile element in the liver vascular bed secondary to progressive hepatic fibrosis and formation of regenerative nodules [ 1 , 2 ] .
the increase in portal pressure triggers splanchnic vasodilation , increased cardiac output , and fluid / salt retention leading to a hyperdynamic circulation and increased portal flow .
formation of collaterals between the portal and systemic systems such as those found in the lower esophagus and gastric cardia ( gastroesophageal varices ) may not only relieve some of the pressure , but also pose a risk for rupture and bleeding .
the prevalence of gastroesophageal varices ranges from 040% in compensated cirrhosis to 7080% in decompensated liver disease , while their growth and progression occur at an estimated 7% per year [ 2 , 3 ] .
advanced liver disease ( child b or c ) , large varices , and varices with red wale marks are bad prognostic signs associated with higher incidence of bleeding .
six - week mortality with each episode of bleeding varies between 15 and 20% and is largely dependent on the severity of the liver disease ( 0% for child a and 30% for child c ) [ 57 ] .
the current treatment of gastroesophageal varices has substantially reduced the rate of first and recurrent bleeding while decreasing the mortality of acute variceal hemorrhage [ 9 , 10 ] .
the purpose of this paper is to summarize the management of gastroesophageal varices in terms of primary prophylaxis ( prevention of first episode of bleeding ) , treatment of acute hemorrhage , and secondary prophylaxis ( prevention of recurrent bleeding ) .
nonselective beta - blockers are the current mainstay of therapy in the prevention of first episode variceal hemorrhage [ 911 ] . 1 inhibition reduces cardiac output while 2-blockade induces splanchnic vasoconstriction and together it results in decreased portal flow and pressure .
nonselective beta - blockers used for primary prophylaxis in north america include propranolol and nadolol .
carvedilol has recently been investigated in portal hypertension given its alpha - blocking component and its potential to better diminish portal pressure . however
, more data will be needed on its effectiveness and long - term safety . at this time it is premature to endorse carvedilol as a first - line agent for primary prophylaxis . in terms of dosing and goal of treatment , it is recommended to start at a low dose and to titrate up as tolerated until a heart rate of 55 beats / minute is achieved [ 911 ] . in patients with low - risk small varices ( child a without red wale marks ) ,
there is limited evidence showing that nonselective beta - blockers may slow the growth of varices but they do not reduce mortality and their use can not be universally recommended over regular endoscopic surveillance ( every 2 years and annually with hepatic decompensation ) .
however , nonselective beta - blockers are recommended in patients with small varices and high - risk features such as red wale marks and/or child b - c cirrhosis . a large meta - analysis looking at propranolol / nadolol versus placebo , in patients with cirrhosis and medium to large varices , found a significantly lower incidence of first variceal bleeding in the treatment group : 14% compared to 30% .
also , these nonselective beta - blockers may be equivalent to endoscopic variceal band ligation ( evbl ) in terms of primary prevention and mortality rate [ 15 , 16 ] .
in addition , they are inexpensive and can potentially prevent other complications of cirrhosis such as spontaneous bacterial peritonitis and bleeding from portal hypertensive gastropathy [ 17 , 18 ] . however , 1520% of patients treated with nonselective beta - blockers are noncompliant due to common side effects such as fatigue , dizziness , and shortness of breath .
evbl is associated with fewer side effects and does not rely on patient compliance but requires technical expertise and can lead to serious complications such as bleeding from ligation - induced ulcers [ 12 , 19 ] .
finally , a randomized controlled trial comparing evbl and propranolol to evbl alone in patients with large varices did not show a difference in terms of mortality or incidence of first bleed .
therefore , depending on patient / physician preference and available expertise either nonselective beta - blocker or evbl alone should be used for primary prophylaxis in cirrhosis with medium to large varices .
the management of acute variceal bleeding with the combination of vasoconstrictors , endoscopic therapy , and antibiotics has decreased mortality substantially [ 9 , 10 ] . initial assessment of a patient with acute variceal hemorrhage begins with the evaluation of airway , breathing , and circulation .
many of these patients are at risk for aspiration and intubation is often performed for airway protection , although there are limited data to justify this practice [ 21 , 22 ] .
volume resuscitation with blood and fluid is essential in the initial stabilization ; however , experimental studies suggest that overly aggressive volume repletion can worsen bleeding and increase the rate of rebleeding and mortality . therefore ,
meticulous resuscitation with a target hemoglobin level of 8 g / dl is recommended [ 23 , 24 ] .
in addition , animal studies suggest that blood transfusion may be superior to fluid administration given that fluid resuscitation may decrease blood viscosity , which can exacerbate portal pressure and potentially worsen acute variceal hemorrhage .
correction of significant coagulopathy and thrombocytopenia with fresh frozen plasma and platelet transfusions should also be considered .
studies on factor viia have failed to show benefit in terms of mortality and control of bleeding and its use is currently not recommended .
acute variceal hemorrhage has been shown to increase the risk for severe bacteremia , which is associated with higher mortality rates and greater incidence of rebleeding [ 2628 ] .
meta - analyses have revealed that antibiotic prophylaxis can improve short - term survival while decreasing bacterial infections and rebleeding rates [ 26 , 29 , 30 ] .
oral norfloxacin or intravenous ciprofloxacin for 7-days , administered at the time of acute bleeding , works by decreasing the amount of gram - negative bacteria in the gut believed to be the most common source for infection in cirrhosis [ 27 , 28 ] .
however , in patients with advanced cirrhosis ( child b / c ) ceftriaxone may be superior to norfloxacin in preventing infections .
this is likely due to ceftriaxone 's extended coverage against nonenterococcal streptococci and quinolone - resistant gram - negative bacteria .
therefore , cirrhotic patients without advanced liver insufficiency and acute variceal hemorrhage should receive either oral norfloxacin or iv ciprofloxacin for 7 days while ceftriaxone is preferred in patients with child b / c cirrhosis or previous quinolone use .
in addition to antibiotics , vasoactive agents such as vasopressin , terlipressin , somatostatin , and octreotide play a major role in controlling acute esophageal variceal hemorrhage through their ability to induce splanchnic vasoconstriction thereby reducing portal flow and pressure [ 9 , 10 ] .
in fact , they may be equally as effective as endoscopic sclerotherapy in controlling initial bleeding and in preventing rebleeding with less adverse effects [ 31 , 32 ] .
these agents , when administered at the time when variceal bleeding is suspected , can achieve initial hemostasis in 6080% of the cases .
vasopressin is a potent vasoconstrictor that reduces blood flow to all splanchnic organs leading to substantial decreases in portal pressure .
however , its use has been limited by its side effects such as hypertension , cardiac , and peripheral ischemia , and ischemic bowel .
terlipressin is a synthetic analogue of vasopressin with longer pharmacological activity and fewer side effects [ 4 , 10 , 34 ] .
the intact molecule has immediate vasoconstrictive activity , which is followed by a delayed effect secondary to a slow enzymatic breakdown of terlipressin into vasopressin .
it is the only agent that has been demonstrated to decrease mortality in acute variceal hemorrhage ; however , it is not yet available in north america . in terms of dosing ,
terlipressin is given intravenously and should be started at 2 mg every 4 hours for 48 hours , followed by 1 mg every 4 hours .
the optimal duration of treatment is unknown but current recommendations suggest a total of 25 days .
somatostatin is a naturally occurring tetradecapeptide that has inhibitory effects on exocrine / endocrine hormones , gastrointestinal motility , and systemic blood flow leading to a decreased circulation and pressure in the portal and porto - collateral system .
octreotide , a synthetic analogue of somatostatin with greater potency and longer half - life , is the only substance available in north america mainly due to its safety profile and its apparent effectiveness when used in combination with evbl [ 9 , 34 , 35 ] .
however , its effectiveness in controlling acute variceal hemorrhage has not been firmly established [ 11 , 34 ] . in terms of dosing
, it is administered intravenously and should be initiated with a 50 mcg bolus followed by an infusion at 50 mcg / hr .
a bolus can be repeated in the first hour if variceal hemorrhage is uncontrolled . as with terlipressin therapy should be continued for 25 days ( table 2 ) . following initiation of vasoactive agents
the combination of evbl with pharmacological therapy is the current standard of care and when compared to evbl alone it improves initial control of bleeding and 5-day hemostasis without a mortality benefit .
the use of transjugular intrahepatic portosystemic shunt ( tips ) in acute variceal hemorrhage has been historically reserved for salvage therapy in patients who have failed endoscopic and pharmacological treatment . however , a recent randomized controlled trial looking at early tips , defined as within 72 hours of standard therapy ( evbl + antibiotic + vasoactive agent ) , versus standard therapy alone showed that in patients with child b / c cirrhosis the early use of tips was associated with a reduction in the failure to control bleeding , lower incidence of rebleeding , and decreased mortality rate .
in addition , the tips group did not have an increased incidence of hepatic encephalopathy .
although more studies will be needed to confirm these findings , the early use of tips should be considered in patients with severe liver disease who present with acute variceal bleeding following initial standard therapy .
patients who survive an episode of acute variceal hemorrhage are at high risk of recurrence .
overall , 60% of these individuals will rebleed within 2 years with a mortality rate of 33% [ 4 , 8 ] .
therapy aimed at reducing this risk is essential and should be implemented as soon as the initial hemorrhage is controlled [ 9 , 10 ] .
multiple modes of treatment have been investigated including monotherapy with nonselective beta - blockers , combination medical therapy , evbl with or without pharmacological adjunct , and tips .
nonselective beta - blockers have been shown to decrease rebleeding rates from 60% to 42 - 43% likely secondary to the decrease in portal pressure [ 4 , 8 , 24 ] .
further portal pressure reduction can be achieved when they are combined with oral nitrates ( ismn ) .
nitrate - induced venodilation decreases cardiac output and blood pressure , which can lead to a baroreceptor - mediated splanchnic vasoconstriction and fall in portal pressure .
it may also have a direct vasodilatory effect on the portosystemic circulation ; however , a randomized trial and a recent meta - analysis did not show any benefit in adding a nitrate .
in addition , combined therapy is associated with more adverse effects leading to discontinuation of treatment [ 40 , 41 ] . in terms of endoscopic therapy ,
evbl is superior to sclerotherapy and is the method of choice [ 42 , 43 ] .
meta - analysis of several randomized controlled trials ( 719 patients ) comparing evbl versus combination medical therapy , with nonselective beta - blockers and nitrates , showed no difference in rebleeding rate and increased survival in the medically treated group [ 4447 ] . also , two prospective trials suggest that the combination of evbl with medical therapy ( nadolol ) may be superior to evbl alone [ 48 , 49 ] .
the use of evbl and a nonselective beta - blocker is the current recommendation for secondary prophylaxis and should be instituted without delay following initial bleed .
however , a recent randomized controlled trial looking at combination therapy ( evbl + nadolol + nitrate ) versus medical therapy alone ( nadolol + nitrate ) found no difference in rebleeding rates , need for rescue therapy , or mortality while the combination therapy was associated with more adverse events .
more studies will be needed to confirm these findings but future guidelines may move towards medical therapy alone . finally , tips in secondary prophylaxis has been shown to lower rebleeding rates when compared to the aforementioned medical / endoscopic therapy [ 5153 ] .
however , no mortality benefit has been demonstrated with tips and its use is associated with higher costs and incidence of hepatic encephalopathy .
therefore , the use of tips in secondary prophylaxis is not recommended ; however , its use may be considered following failure with conventional medical therapy .
this may change with the advent of polytetrafluoroethylene- ( ptfe- ) covered prostheses , which substantially improves tips patency . in summary ,
evl in combination with nonselective beta - blockers is the method of choice in preventing recurrent variceal bleeding .
the addition of nitrates can theoretically potentiate the portal pressure drop ; however , it has not been shown to decrease mortality or rebleeding rates and is associated with greater side effects .
tips is not recommended for secondary prophylaxis and should only be considered following failure with usual medical therapy .
it decreases rebleeding rates without a mortality benefit while being associated with higher costs and incidence of hepatic encephalopathy .
whether the new ptfe covered stent will improve tips efficacy in secondary prophylaxis remains to be seen , but for the moment its use is restricted to those cases where other therapies have failed .
the management of gastroesophageal varices has evolved over the last decade resulting in improved mortality and morbidity rates . primary prevention with nonselective beta - blockers or evbl should be initiated in all patients with medium to large varices and in patients with small varices associated with high risk features such as red wale marks and/or advanced cirrhosis . while prophylaxis in patients with small varices without high risk features is considered optional . in acute bleeding , vasoactive agents such as octreotide or terlipressin should be initiated along with antibiotics followed by evbl within 12 hours of presentation .
these patients are at increase risk for rebleeding and secondary prevention should be initiated immediately following control of initial hemorrhage with serial evbl and nonselective beta - blocker .
currently , tips ' role in secondary prophylaxis is limited except for failure with conventional therapy ; however , this may change with the advent of ptfe covered stents .
although therapy for patients with varices has made significant progress , it will continue to improve with better endoscopic technique , novel pharmacological agents , greater efficiency of liver transplant , and more effective rescue therapy . | variceal hemorrhage is one of the major complications of liver cirrhosis associated with significant mortality and morbidity .
its management has evolved over the past decade and has substantially reduced the rate of first and recurrent bleeding while decreasing mortality . in general , treatment of esophageal varices can be divided into three categories : primary prophylaxis ( prevention of first episode of bleeding ) , management of acute bleeding , and secondary prophylaxis ( prevention of recurrent hemorrhage ) .
the goal of this paper is to describe the current evidence behind the management of esophageal varices .
we will discuss indications for primary prophylaxis and the different modes of therapy , pharmacological and interventional treatment in acute bleeding , and therapeutic options in preventing recurrent bleeding .
the indications for tips will also be reviewed including its possible benefits in acute variceal hemorrhage . |
takotsubo cardiomyopathy ( tc ) is an increasingly reported syndrome characterized by transient apical left ventricular dysfunction in absence of low limiting coronary artery disease .
prolongation of qtc interval associated with tc has been previously reported in published case series .
bradycardia induced qt interval prolongation seemed to be amplified by the occurrence of tc , resulting in torsade de pointes .
temporary ventricular pacing at a high rate decreased the qt interval and prevented the recurrence of torsade de pointes .
we report a case of torsade de pointes associated with qt prolongation and tc . because qt prolongation and bradycardia persisted even after the resolution of tc , the patient received a permanent pacemaker .
a 78-yr - old female with a medical history of hypertension was admitted to our hospital due to syncope on 20 july , 2010 .
her antihypertensive medications were nifedipine ( 30 mg ) and valsartan ( 80 mg ) . upon arrival to hospital , the patient was fully conscious and alert with a pulse rate of 40 beats per minute and a blood pressure of 140/80 mm hg .
electrocardiography ( ecg ) on admission showed an escape junctional rhythm of 35 beats per minute combined with t wave inversion in precordial leads and qt prolongation measuring 580 ms in the qtc interval ( fig .
the serum levels of potassium , magnesium , and ionized calcium were within normal limits .
the serum troponin t level was slightly elevated to 0.081 ng / ml ( 0 - 0.1 ng / ml ) .
echocardiography showed akinesia of mid and apical left ventricular ( lv ) walls with the systolic ballooning of the ventricular apex ; lv ejection fraction was estimated to be 35% by a modified simpson 's method ( fig .
she was diagnosed with takotsubo cardiomyopathy based on echocardiographic and coronary angiographic findings . on the second day ,
ecg showed severe bradycardia ( heart rate 20 beats per minute ) with marked prolongation of qt ( qtc = 720 ms ) followed by torsade de pointes ( tdp ) ( fig .
after temporary ventricular pacing , which was performed at a rate of 80 beats / min , the qtc interval decreased to 480 ms . on the fourth day , the temporary pacemaker was removed . at that time
, ecg showed a marked bradycardia ( heart rate of 40 beats per minute ) with qt prolongation and r on t phenomenon ( fig .
1c ) . on the 10th day , we checked follow - up echocardiography , which showed normal lv systolic function without previous wall motion abnormalities ( fig . 2c ,
ecg still showed a severe bradycardia with qt prolongation while we checked daily serial ecg up to the 20th day ( fig .
therefore , we planned to insert a permanent pacemaker to prevent recurrent tdp , although lv systolic dysfunction of the patient was recovered . on the 21st day
one and 3 months after the index procedure of permanent pacemaker insertion , ecg still showed qt prolongation ( qtc = 552 ms ) with t - wave inversion at precordial leads ( fig .
tdp denotes a polymorphic ventricular tachycardia associated with long qt syndrome , and is a life - threatening arrhythmia .
qt prolongation is the surface ecg manifestation of abnormal repolarization of myocardial cells due to problems with cellular ion channels .
acquired qt prolongation may be associated with the following conditions : 1.electrolyte depletion ( especially potassium or magnesium ) 2 .
also , the association of tc with tdp has rarely been reported ( 1 , 2 ) .
patients with tc generally show st elevation , t - wave inversion , q - wave formation and qtc interval prolongation in ecg .
the prevalence of qtc interval prolongation among tc patients is high , ranging from 50% to 100% according to different case series .
this is probably because systolic dysfunction is associated with both tc and qt - interval prolongation ( 3 ) .
although qt - interval prolongation is prevalent among tc patients and might precede tdp , the later has rarely been reported in tc patients .
danney et al . ( 5 ) first reported tdp occurring in patients with tc .
since then , numerous reports of tdp related to tc have been published ( 4 , 5 ) .
also , bradycardia with or without av block and suspicious of congenital long qt syndrome are the risk factors of tc - associated tdt ( 4 ) .
however , permanent pacemaker insertion has been performed on a few patients ( 6 , 9 ) .
in general , qt interval prolongation and st - t changes occur during the acute or subacute phase of illness , and these ecg changes typically normalize within several weeks of improvement in lv wall motion ( 3 ) . in our case , qt prolongation and st - t change persisted for 3 months .
inoue et al . ( 6 ) reported a patient who underwent permanent pacemaker insertion due to complete av block associated with tc .
in their case , the ecg of the patient showed complete av block even after recovery from illness .
( 8) also reported 2 cases of suspicious congenital long qt syndrome associated with tc . during the previous regular medical check up of the present case ,
after development of her illness , qtc interval did n't improved , even after normalization of lv wall motion and cardiac enzyme level . based on her previous medical check up ecg and history , a diagnosis of acquired long qt syndrome associated with tc
, we inserted a permanent pacemaker because sinus bradycardia ( 40 beats per minute ) and qt prolongation ( qtc = 550 ms ) persisted for 2 weeks even after recovery from lv dysfunction . to date , the association between tc , bradycardia and tdp is not exactly known .
( 9 ) stated that prolongation of bradycardia - induced qt interval may be augmented by the occurrence of takotsubo cardiomyopathy , resulting in tdp . in their cases ,
permanent pacemaker insertion was also performed because bradycardia and complete av block persisted after the recovery from lv dysfunction .
although tdp has rarely been reported in patients with tc , the clear relationship between the 2 conditions has yet to be determined .
however , qt prolongation and bradycardia could be persistent even after full recovery of tc , and permanent pacemaker insertion may be a treatment option of long qt syndrome related with tc . | prolongation of qtc interval associated with takotsubo cardiomyopathy ( tc ) has previously been reported in published case series .
we report an unusual case of a patient who presented with tc associated with long - qt syndrome and developed cardiac arrest secondary to torsade de pointes . since qt prolongation and bradycardia persisted after the resolution of tc , the patient received permanent pacemaker .
since then additional event did not occur .
qt prolongation and bradycardia could be persistent even after recovery of tc , and permanent pacemaker insertion may be a treatment option of long qt syndrome related with tc . |
it is well documented that transplants save lives and improve quality of life for patients suffering from kidney , liver , and heart failure .
but in many countries , moral courage and public health policies have not kept pace with need . in a recent issue of critical care , fieux and
colleagues demonstrate remarkable results obtained through a coordinated effort to obtain viable kidneys from people who suffer out - of - hospital cardiac arrest in paris . in the us ,
however , lack of organs results in 8,000 patients who die or become too sick to receive a transplant every year .
furthermore , recent trends suggest the number of patients awaiting organs is increasing by several thousand yearly .
the us institute of medicine ( iom ) suggested that the transplantation community pursue donations from non - heart beating donors in the out - of - hospital setting ( that is , uncontrolled donations after cardiac death ( udcd ) ) to meet the demand for solid organs . at the time , such conclusions were based on case series experience from the us and spain demonstrating that this pool of potential kidney donors yielded transplantation outcomes similar to that of donation after neurological determination of death ( dndd ) and controlled donations after cardiac death ( cdcd ) .
although fieux and colleagues report high rates of delayed graft function compared to the madrid udcd program , it is encouraging that , even using cold perfusion techniques , they achieved similar excellent rates for graft survival . the current strategies of cdcd and living donation have practical and ethical limitations . using donors where care is withdrawn in a hospital setting ( that is , cdcd ) has raised issues about how the time and manner of death will be determined and whether the patient is actually ' dead ' .
living donation raises its own set of concerns : the closer the donor is to the recipient , the more concerns emerge about coercion ; the more distant the donor , the more worries about commodification . as the recent scandal about
illegally purchased organs in the us illustrates , need dictates action ; illegal , mercenary , or altruistic .
current cdcd strategies still yield less than 1,000 organs annually in the us , and the number of living donor kidneys has been steadily declining since 2004 .
udcd programs avoid the dangers of live donation and evade the controversy that surrounds cdcd .
udcd respects the ' dead - donor rule ' and complies with the deep medical traditions of respecting life at all costs and respecting the body after death . during resuscitation
, emergency medical service rescuers must focus completely on their task of restarting the heart to achieve the return of spontaneous circulation in a patient in cardiac arrest .
fortunately , techniques for resuscitation have advanced and survival after out - of - hospital cardiac arrest has improved greatly in the past years ( new york city fire department , unpublished data ) .
when survival is no longer a possibility , and the decision to terminate resuscitative efforts is made independently of organ donation considerations , the ethics clearly allow for the pursuit of udcd .
if it is evident that udcd is an effective and ethical alternative to existing efforts ( that is , living donations , cdcd , and dndd ) , why has udcd not been more widely accepted ? in order for organs to remain viable , interventions for organ preservation must be initiated within minutes after pronouncement of death .
it may be difficult to obtain necessary consent for these interventions from grieving family members . to address this challenge ,
countries such as france and spain have passed legislation allowing ' presumed consent ' for preservation .
therefore , preservation measures may be initiated unless the patient has specifically ' opted - out ' .
other societies should consider first - person consent for organ donation ( as may be indicated through organ donor consent registries or on donor cards such as drivers ' licenses ) .
the latter approach is that advocated for the us in the recent work by dubois and the new york city udcd study group .
currently , people who die from an out - of - hospital cardiac arrest are denied the opportunity to be organ donors except in those few locations where out - of - hospital udcd programs are active .
the results reported by fieux and colleagues in this journal , and the continuing success of the madrid and barcelona out - of - hospital udcd programs , demonstrate the viability and reproducibility of such protocols .
the iom conservatively estimated that in the us about 22,000 decedents could become udcd donors .
it is conceivable that widespread dissemination of udcd could obviate the waiting list for kidney transplants .
cdcd : controlled donations after cardiac death ; dndd : donation after neurological determination of death ; iom : us institute of medicine ; udcd : uncontrolled donations after cardiac death .
the nyc udcd study group is composed of the following : new york city health and hospitals corporation , new york organ donor network , new york university school of medicine , and the fire department of the city of new york . it is supported by the us health resources and services administration ( hrsa ) grant # r380t08761 .
the opinions expressed in this publication are solely those of the authors with lewis goldfrank , md , responsible for its content . | it is well documented that transplants save lives and improve quality of life for patients suffering from kidney , liver , and heart failure . uncontrolled donation after cardiac death ( udcd ) is an effective and ethical alternative to existing efforts towards increasing the available pool of organs .
however , people who die from an out - of - hospital cardiac arrest are currently being denied the opportunity to be organ donors except in those few locations where out - of - hospital udcd programs are active , such as in paris , madrid , and barcelona .
societies have the medical and moral obligation to develop udcd programs . |
cerebral small vessel disease ( svd ) manifests on conventional mr images ( i.e. t1 and fluid attenuated inversion recovery ( flair ) ) as white matter hyperintensities ( wmh ) and lacunes of presumed vascular origin ( wardlaw et al . , 2013 ) .
svd is related to cognitive impairment and may , in some , ultimately lead to dementia ( de groot et al . , 2000 ;
this is supposedly due to the disruption of important white matter ( wm ) tracts . despite the high prevalence of svd ( de leeuw et al . , 2001 ;
, relatively few develop evident cognitive decline or dementia ( vermeer et al . , 2003 ) .
other factors , apart from wmh and lacunes , presumably play a role in the transition from relative intact cognitive performance to severe cognitive decline in these few individuals .
one of these factors could be the ( loss of ) microstructural integrity of the largest part of the wm ; the on flair imaging normal - appearing white matter ( nawm ) surrounding the svd .
pathological studies have demonstrated loss of microstructural integrity in the nawm ( grafton et al . , 1991 ) that can not be visualized with conventional imaging , but can be investigated with diffusion tensor imaging ( dti ) .
dti measures the local water diffusion profiles by : fractional anisotropy ( fa ) , which represents a normalized ratio of diffusion directionality ; mean diffusivity ( md ) , which reflects the overall magnitude of water diffusion ; axial diffusivity ( ad ) , which reflects the diffusivity parallel to the wm tracts and radial diffusivity ( rd ) , which is the diffusivity perpendicular to these tracts ( pierpaoli et al . , 1996 ) .
loss of microstructural integrity is typically reflected by a reduction in fa and/or an increase in md ( sen and basser , 2005 ) ; the latter can result from different combinations of changes in ad and rd .
few studies in patients with cerebral svd demonstrated a relation between higher md and lower fa and loss of cognitive function ( della nave et al .
these studies had , however , small sample sizes and were not able to properly adjust for possible confounders
. one large population - based cohort study demonstrated relation between microstructural integrity of both wmh and nawm and cognitive function .
however , the regional differences of microstructural integrity were not taken into account ( vernooij et al . , 2009 ) .
we hypothesized that cognitive performance in subjects with svd would not only be related to loss of wm microstructural integrity within the wmh , but also to specific areas within the nawm .
we conducted dti using tract - based spatial statistics ( tbss ) analyses to investigate the location of microstructural wm loss related to cognitive disturbances .
also , additional adjustments for the wmh and lacunes were made to examine whether the associations in the wm were primarily explained by the typical manifestations of svd on conventional mri .
the radboud university nijmegen diffusion tensor and magnetic resonance imaging cohort ( run dmc ) study prospectively investigates the risk factors and clinical consequences of brain changes among 503 non - demented elderly with cerebral svd .
the selection procedure of the participants and study protocol were described previously in detail ( van norden et al . , 2011 ) .
in short , on the basis of established research criteria svd was defined as the presence of lacunes and/or wmh on neuroimaging ( erkinjuntti , 2002 ) .
symptoms of svd include acute symptoms , such as tias or lacunar syndromes , or subacute manifestations , such as cognitive and motor ( gait ) disturbances and/or depressive symptoms ( roman et al . , 2002 ) .
inclusion criteria were : ( a ) age between 50 and 85 years ; and ( b ) cerebral svd on neuroimaging .
the main exclusion criteria were dementia ( american psychiatric association , 2000 ) , ( psychiatric ) disease interfering with cognitive testing or follow - up , wmh or svd mimics and mri contraindications or known claustrophobia .
consecutive patients referred to the department of neurology between october 2002 and november 2006 were selected for participation .
participants were selected for participation in the study by a three - step approach . after reviewing the medical history ,
1004 individuals were invited by letter . of those 1004 , 7these numbers seem odd the way they are displayed .
is this correct??27 were eligible after contact by telephone and 525 agreed to participate . in 22
individuals exclusion criteria were found during their visit to our research center , yielding a response of 71.3% ( 503/705 ) .
for the present study , 59 subjects were additionally excluded because of territorial infarcts ( n = 55 ) and inadequate quality of the mri image ( n = 4 ) , resulting in a final population of 444 participants .
cognitive function was assessed by a standardized neuropsychological test battery and has been described in detail elsewhere ( van norden et al . , 2011 ) .
performance across tests was made comparable by transforming raw test results in z - scores .
global cognitive function was evaluated by the mini mental state examination ( mmse ) and the cognitive index .
the cognitive index is a compound score that was calculated as the mean of the z - scores of the 1-letter subtask of the paper pencil memory scanning task , the mean of the reading subtask of the stroop test , the mean of the symbol digit substitution task and the mean of the added score on the three learning trials of the rey auditory verbal learning test and the delayed recall of this last test ( vermeer et al . , 2003 ) .
verbal memory is a compound score of the mean of two z - scores from the rey auditory verbal learning test ; one for the added scores of the three learning trials of this test , and one for the delayed recall of this test .
visuospatial memory is a compound score of the mean of the z - scores of the immediate recall trial and the delayed recall trial of the rey 's complex figure test .
psychomotor speed was calculated as the mean of the z - scores of the 1-letter subtask of the paper pencil memory scanning task , the reading subtask of the stroop test and the symbol digit substitution task .
fluency was calculated from the mean of the z - scores of both verbal fluency tasks .
concept shifting was calculated as the z - score of the third subtask of the stroop .
attention is a compound score of the z - score of the total time of the verbal series attention test ( de groot et al .
the protocol included , among other sequences , the following whole brain scans : 3d t1 magnetization - prepared rapid gradient echo ( mprage ) sequence ( tr / te / ti 2250/3.68/850 ms ; flip angle15 ; voxel size 1.0 1.0 1.0 mm ) , a fluid - attenuated inversion recovery ( flair ) sequence ( tr / te / ti 9000/84/2200 ms ; voxel size 1.0 1.2 5.0 mm , interslice gap 1 mm ) and dti sequences ( tr / te 10,100/93 ms ; voxel size 2.5 2.5 2.5 mm ; 4 unweighted scans , 30 diffusion weighted scans with b - value 900 s / mm ) .
wmh were manually segmented on flair images and the number of lacunes was rated according to a standardized protocol ( van norden et al . , 2011 ) .
in addition , the visual fazekas scale was used on the flair images to rate the severity of changes in the white matter ( fazekas et al . , 1987 ) .
all imaging analyses were performed by two trained raters blinded to clinical information . in a random sample of 10% , interrater variability for total wmh volume yielded an intra - class correlation coefficient of 0.99 .
the probability map of the white matter hyperintensities were created using a method previously described by de laat and colleagues ( de laat et al . , 2011 ) .
in short , we registered the wmh maps to the t1 images using the transformation matrix from the registration parameters of skull - stripped flair images to the t1-images that were obtained using functional mri of the brain linear image registration tool ( http://www.fmrib.ox.ac.uk/fsl/fnirt ) .
next , we normalized the wmh maps non - linearly to the group - specific template using the transformation parameters of t1 images to the group - specific template obtained from functional mri of the brain non - linear registration tool ( http://www.fmrib.ox.ac.uk/fsl/flirt ) . finally , we averaged the normalized wmh maps to create a probability map of the wmh of the study population ( fig .
we computed gray ( gm ) and wm tissue and cerebrospinal fluid ( csf ) probability maps using spm 5 unified segmentation routines on the t1 mprage images ( ashburner and friston , 2005 ) .
total gm , wm and csf volumes were calculated by summing all voxel volumes that had a p > 0.5 for belonging to that tissue class .
total brain volume ( tbv ) was taken as the sum of total gm and wm . intracranial volume ( icv )
tract - based spatial statistics ( tbss ) is a relatively new method that mitigates the limitations of vbm analysis ( smith et al . , 2006 ) .
this analysis is restricted to those wm voxels that constitute the skeleton ( core ) of the brains connectional architecture .
this skeleton can be matched more accurately ( compared to whole - brain normalization ) across subjects , enabling robust voxel - wise analysis of the microstructural wm integrity across subjects .
diffusion data were first preprocessed to detect and correct head and cardiac motion artifacts , using an in - house developed iteratively re - weighted - least - squares algorithm named
fa , md , ad and rd images were then calculated using dtifit within the functional mri of the brain diffusion toolbox , which were fed into the tbss pipeline ( smith et al . , 2006 ) .
the thinning procedure was conducted on the mean fa image to create a common skeleton , which represents the core - structure of the wm tract .
this skeleton was thresholded at the fa - value 0.3 to include the major wm tracts and to account for the inter - subject variability .
these skeleton projection factors were then applied to the md , ad and rd images .
in addition , we obtained fa , md , ad and rd for three parts of the corpus callosum by performing region - of - interest analyses .
the corpus callosum provides interhemispheric connections between cortical and subcortical regions and might play an important role in cognitive function ( bloom and hynd , 2005 ) .
we created masks for genu , body and splenium of the corpus callosum by applying the wm atlas ( johns hopkins university wm labels , provided by functional mri of the brain software library ( fsl ) ) on the mean fa skeleton .
the masks were visually inspected and miscellaneous voxels that belonged to other regions , such as the cingulum bundle , were excluded .
age , sex and level of education , depressive symptoms and normalized tbv were considered possible confounders .
depressive symptoms were assessed using the center of epidemiologic studies on depression scale ( ces - d ) ( radloff , 1977 ) .
functional independence was assessed using the barthel index ( range 020 ) ( mahoney and barthel , 1965 ) . for assessment of vascular risk factors , structured questionnaires were used together with measurements of blood pressure taken on separate occasions .
the risk factors included presence of hypertension ( mean blood pressure 140/90 mm hg and/or use of anti - hypertensive medications ) ( rosendorff , 2007 ) , diabetes ( treatment with antidiabetic drugs ) , hypercholesterolemia ( treatment with lipid - lowering drugs ) and smoking status .
baseline characteristics were presented as mean standard deviation ( sd ) and for the skewed parameters the median and interquartile ranges were calculated . for the tbss analyses , we assessed voxel - wise correlations between the skeletal dti parameters ( fa and md ) and cognitive performance on several cognitive domains , while adjusting for age , sex , education , depressive symptoms and normalized tbv . to test whether these associations were independent of wmh and lacunes , we adjusted for wmh volume and number of lacunes in a second model . for the voxel - wise statistical analyses , we applied permutation - based statistical interference tool for non - parametric approach , with number of permutation tests set to 5000 ( nichols and holmes , 2002 ) .
significant clusters were identified using the threshold free cluster enhancement with a p - value < 0.05 , corrected for multiple comparisons ( smith and nichols , 2009 ) . for the roi analyses , we computed regression coefficients of the mean fa , md , ad and rd of the three roi in the corpus callosum with cognitive performance , while adjusting for age , sex , education , depressive symptoms , normalized tbv , white matter hyperintensities and number of lacunes .
demographic and neuroimaging characteristics are shown in table 1 . mean age of the population ( n = 444 ) was 65.3 years ( sd 8.9 ) and 54.7% were male . mean wm volume was 467.4 ml ( sd 65.4 ) .
the largest part of the wm consisted of nawm , with a median percentage of 98.6 ( iqr 96.499.3 ) . according to fazekas visual rating scale ,
10 participants ( 2.3% ) had fazekas score 0 , 296 participants ( 66.7% ) had fazekas - score 1 , 88 participants ( 19.8% ) had fazekas score 2 and 50 participants ( 11.3% ) had fazekas score 3 .
1 ) . 13 participants ( 2.9% of the study population ) were diagnosed with mild cognitive impairment .
2 shows the relation between fa and the cognitive domains tested ( p < 0.05 , fwe - corrected for multiple comparisons ) .
fa in the frontal , parietal , occipital , temporal but also in the infratentorial voxels of the skeleton was positively related to the cognitive index , attention and verbal memory performance .
a lower fa in almost all regions was associated with higher scores on psychomotor speed and concept shifting ( indicating lower performance ) .
we found a similar distribution for the inverse association with md and the cognitive domains . with regard to mmse , visuospatial memory and fluency
a significant ( p - corrected < 0.01 ) association was identified in almost all voxels of the skeleton in the relation between fa and cognitive index and concept shifting . the strongest significant ( p - corrected < 0.01 )
relation between md and psychomotor speed and cognitive index was located in the frontal lobe and the corpus callosum .
the relation between fa and md and verbal memory was most outspoken along the whole course of the corpus callosum and the cingulum bundle ( p - corrected < 0.01 ) .
2b ) weakened the relations between the fa and cognitive index , psychomotor speed , verbal memory and concept shifting , but remained present ( p - corrected < 0.05 ) .
there were no associations between for fa and attention after additional adjustment for wmh and lacunes .
there were no associations between md and attention after additional adjustment for wmh and lacunes , while the associations between md and psychomotor speed and concept shifting weakened but remained significant .
voxels with the highest significance for fa in relation to cognitive index , psychomotor speed , verbal memory and concept shifting were located in the corpus callosum ( p - corrected < 0.01 ) .
in addition , fa in the cingulum bundle and corpus callosum remained highly associated with verbal memory performance ( p - corrected < 0.01 ) . as post - hoc analysis we analyzed the corpus callosum in more anatomical detail by segmenting it in three regions ( table 2 ) .
we found that microstructural integrity of genu and splenium of the corpus callosum related to cognitive index , but also with executive domains , psychomotor speed and concept shifting ( p < 0.05 after bonferroni corrections ) .
we found no relations between microstructural integrity in corpus callosum and attention , visuospatial memory , mmse and fluency .
the associations with md and cognitive performance were mainly driven by changes in rd , and not by changes in ad .
in this study , we examined the relation between the microstructural integrity of the wm and cognitive performance in subjects with cerebral svd .
we demonstrated that low fa and high md in multiple regions of the wm were associated with lower scores on cognitive performance .
the corpus callosum showed the highest significant relation with cognitive function , especially in the genu and splenium .
the microstructural integrity of the cingulum bundle showed the highest significant relation with verbal memory performance and the frontal wm with psychomotor speed .
a limitation is the cross - sectional nature of our study , which limits causal inference .
the run dmc study has a longitudinal design and follow - up is currently being executed to evaluate the effect of progression of svd on ( changes in ) cognitive performance ( van norden et al . , 2011 ) .
in addition , we did not intentionally adjust for vascular risk factors , such as hypertension or diabetes , as they were considered a part of the causal chain between svd and cognitive performance .
major strengths of the study included the large sample size , the single center design , the use of a single scanner and the high response rate .
extensive neuropsychological assessment was performed by two investigators and all analyses were adjusted for potential confounders .
structural abnormality of the white matter tracts has been found in alzheimer 's disease and mild cognitive impairment and has been demonstrated in a population - based cohort and a cohort of healthy adults ( acosta - cabronero et al . , 2010 ; barrick et al . , 2010 ; damoiseaux et al . , 2009 ; vernooij et al .
, 2008 ; zhuang et al . , 2010 ) . in patients with svd , clinical manifestations of cognitive impairment are usually of a characteristic and fairly homogeneous
subcortical pattern and include psychomotor slowing due to impaired executive function , deficits of attention , planning and set - shifting and forgetfulness ( roman et al . , 2002 ) . in this study
, we demonstrated that in patients with svd the white matter 's microstructural integrity was associated with these measures of cognition .
the strongest significant relations between microstructural integrity and verbal memory performance were located in the cingulum bundle .
this bundle , which connects the medial temporal lobe and the posterior cingulate cortex , is an important structure in verbal memory performance ( sepulcre et al . , 2008 ; van der holst et al . , 2013 ) .
we also found verbal memory to be associated with the anterior parts of the corpus callosum .
episodic memory is partially dependent on interhemispheric interaction ( christman and propper , 2001 ) .
loss of microstructural integrity of the corpus callosum can lead to impaired interhemispheric interaction , resulting in impaired ( verbal ) memory performance . with regard to psychomotor speed
, we found that the strongest significant relation with microstructural integrity was located in the corpus callosum and frontal lobe . the pre - frontal
subcortical circuits are known to be involved in executive function , which affects the psychomotor speed ( roman et al . , 2002 ) .
corpus callosum has been related to global cognitive status and , based on a topographical organization , genu has been related to frontal - lobe - mediated executive function and attention ( jokinen et al . , 2007 ) , whereas splenium is associated with visuospatial construction ( fryer et al . , 2008 ) , which corresponds with its connection with the parietal , temporal and occipital regions ( huang et al . , 2005
this might be explained by the fact that mmse is a too crude measure not designed for subcortical damage related function loss and hence not sensitive to detect subtle cognitive changes that correlate with macro- and microstructural svd changes ( pasi et al . ,
the whole spectrum of cognitive disturbances in patients with svd can not solely be explained by the degree of wmh and lacunes .
our results show that the majority of the relations between microstructural integrity and cognitive performance were located in the nawm .
this suggests that the microstructural integrity of the nawm has an important role in the cognitive disturbances in our subjects with svd and might explain a part of the cognitive impairment beyond the svd - visible on conventional mri .
findings from functional and pathological studies in combination with our results suggest a disruption of cortical cortical and cortical subcortical connections , and a subsequent disconnection - syndrome accounting for cognitive disturbances in patients with svd ( o'sullivan et al .
in concordance with the findings from previous studies ( van norden et al . , 2012 ; vernooij et al . ,
2009 ) , this suggests that microstructural integrity of the nawm should also be taken into account when investigating the relation between svd and cognitive function and that dti should be considered part of the imaging protocol in future studies on cognitive performance .
the abnormalities in microstructural integrity of the nawm are partially svd - related , as our results showed that the presence of wmh and lacunes weakened the relation between the wm integrity and cognitive performance .
first , it may be that these associations were , at least in part , explained by the presence of the coexisting wmh and not so much by the loss of integrity of the nawm .
second , the structural changes of the nawm could be caused by the same risk factors that are involved in the development of svd , such as hypertension ( gons et al .
brain barrier permeability in the nawm in subjects with svd ( topakian et al . ,
finally , the observed association between the microstructural integrity of the nawm and cognitive performance could reflect indirect damage to the nawm by distant effects of wmh by means of antero-(wallarian ) or retrograde degeneration ( dziedzic et al .
in conclusion , our study showed that cognitive disturbances in elderly with svd are related to the microstructural integrity of the cerebral wm areas with wmh and lacunes , but also in nawm , the largest part of the wm .
our results suggest that loss of microstructural integrity in the nawm is part of the svd - spectrum , which is not visible on conventional mri .
dti could therefore serve as an additional tool to conventional mri in order to investigate the cognitive consequences of cerebral svd .
if the predictive value is proven , dti could possibly serve a surrogate marker for development of cognitive decline and dementia and could be a starting point for therapeutic trials aiming to prevent disease progression . | cerebral small vessel disease , including white matter hyperintensities ( wmh ) and lacunes of presumed vascular origin , is common in elderly people and is related to cognitive impairment and dementia .
one possible mechanism could be the disruption of white matter tracts ( both within wmh and normal - appearing white matter ) that connect distributed brain regions involved in cognitive functions . here , we investigated the relation between microstructural integrity of the white matter and cognitive functions in patients with small vessel disease . the radboud university nijmegen diffusion tensor and magnetic resonance cohort study is a prospective cohort study among 444 independently living , non - demented elderly with cerebral small vessel disease , aged between 5500 and 85 years .
all subjects underwent magnetic resonance imaging and diffusion tensor imaging scanning and an extensive neuropsychological assessment .
we showed that loss of microstructural integrity of the white matter at specific locations was related to specific cognitive disturbances , which was mainly located in the normal - appearing white matter ( p < 0.05 , fwe - corrected for multiple comparisons ) . the microstructural integrity in the genu and splenium showed the highest significant relation with global cognitive function and executive functions , in the cingulum bundle with verbal memory performance .
associations between diffusion tensor imaging parameters and most cognitive domains remained present after adjustment for wmh and lacunes . in conclusion ,
cognitive disturbances in subjects with cerebral small vessel disease are related to microstructural integrity of multiple white matter fibers ( within wmh and normal - appearing white matter ) connecting different cortical and subcortical regions . |
nuclear remodeling relies on a timely translation of stabilized mrna transcribed at the round spermatid stage ( brock et al . , 1980 ) .
the first transcripts are testis - specific core and linker histone variants ( gaucher et al . , 2010 ,
testis - specific histones have a lower affinity for dna than somatic ones ( gaucher et al . , 2010 ) , and their subsequent post - translational modification ( goudarzi et al . , 2014 , rousseaux and khochbin , 2015 , morinire et al . , 2009 )
subsequently , acetylated histones are recognized by bromodomain testis - specific proteins ( brdt ) ( pivot - pajot et al .
, 2003 , gaucher et al . , 2012 ) , proteins that prepare the ground for the incorporation of transition proteins ( tps ) .
tps cooperate with topoisomerases to relieve torsional stress ( singh and rao , 1988 ) and with dna repairing enzymes ( akama et al . , 1999 ) .
the substitution of tps with protamine ( prm ) completes nuclear remodeling , conferring a toroid structure to dna and the unique shape of male gametes ( miller et al . , 2010 ) .
paternal chromosomes rapidly lose prm and testis - specific histones ( van der heijden et al . , 2005 , wu et al .
when , instead , a somatic cell is used to fertilize an oocyte , as in somatic cell nuclear transfer ( scnt ; wilmut et al . , 1997 ) , the nucleosome organization of the chromatin is only occasionally reversed by the oocyte , often leading to developmental failure ( loi et al . , 2013 ) .
the current study set out to explore the possibility of conferring a somatic cell nucleus with a prm - based toroid organization .
here , we demonstrate that the nuclei of adult somatic cells undergo a dramatic chromatin reorganization following the induced expression of the prm1 gene , transforming the nuclei into spermatid - like structures .
the prm - induced nuclear remodeling is reversible upon nuclear transfer ( nt ) of protaminized cells into enucleated oocytes , and the resulting embryos develop normally in vitro .
our first approach to remodel somatic cell nuclei was to induce the co - sequential and sequential expression of four main testis - specific genes in primary cultures of sheep fibroblasts , with the aim to repeat the nuclear remodeling occurring in spermatids .
to this end , expression vectors for the bromodomain testis - specific ( brdt - gfp tag ) , tps i and ii ( ha tag ) , and protamine 1 ( prm1-red variant of gfp tag ) were generated and transfected into the fibroblasts .
however , our attempts to induce chromatin remodeling of somatic cells following either a co - stepwise or a stepwise transfection of all four expression vectors failed .
the nuclear reorganization in spermatids relies on a hierarchic translation of synthetized mrna , and each of the protein prepares the ground for the next one ( barckmann et al . , 2013 ) , a scenario we failed to repeat in cultured somatic cells .
there are , however , exceptions to the canonical nucleosome disassembly pathway during spermatogenesis . in the male germline of the cephalopod sepia officinalis , for instance
, histones are replaced directly by prm - like proteins ( martnez - soler et al . , 2007 ) .
hence , we attempted to induce a direct nuclear remodeling in somatic cells through the transient expression of prm .
we selected prm1 because ram spermatozoa contain prm1 only and because prm1 is synthetized as a mature protein with no further processing ( de mateo et al . , 2011 ) .
we transfected sheep adult fibroblasts ( safs ) with pprm1-rfp , wt prm1 , and rfp tag - only vectors using a lipofectamine transfection kit
expression of mrna and protein prm1 was confirmed by rt - pcr , by western blotting , and by tracking the rfp - tag ( figures 1 , s1a , and s1b ) .
prm1-rfp co - localized with nuclei stained by hoechst ( figure 1 ) , whereas in control , i.e. , prfp transfected fibroblasts , the red signal was diffused in the cytoplasm and nucleus ( figure 1 ) .
fibroblasts transfected with wt prm1 showed nuclear reorganization similar to that detected in pprm1-rfp transfected ones ( figures 1 and s2 ) . at 40- to 48-hr post - transfection /
trichostatin a ( tsa ) treatment , 93.3% of prm1-cells did uptake prm in nuclei , and 83.3% of prm1-positive cells completely changed nuclear morphology ( table 1 ) .
prm1 contains numerous cysteine residues that might form intra - prm / inter - prm disulfide cross - links ( balhorn et al . , 1992 ) .
to exclude nuclear prm1 polymerization , chromatin immunoprecipitation ( chip)-seq assay was carried on of sorted prm1-positive cells .
the chip - seq assay confirmed the effective binding of prm1 to dna at 42 dna binding sites on 10 of 27 chromosomes , as early as 16-hr post - pprm1 transfection ( figure 1j ; table s1 ) .
cell - cycle analysis by cytofluorimetry of transfected cells revealed that prm1 was incorporated in all cell - cycle stages ( 72.6% g1 , 4.4% s , 23% g2 stage ) ( table s2 ) .
moreover , fibroblasts forced to enter g0 ( by serum starvation ) were transfected with pprm1-rfp , and even in this case , prm1 incorporation and chromatin compaction took place ( table 1 ) .
. a likely mechanism for prm1 deposition on chromatin might be the conformational changes that nucleosomes undergo as a result of chromatin remodeling enzymes or thermal fluctuations ( chereji and morozov , 2015 ) .
these nucleosomal dynamics make available free stretches of 11 bp between the dyads , enough for prm1 docking on dna ( zhang et al . , 1996 ) .
the low - off rates of prm1 ( brewer et al . , 2003 ) might facilitate the prolonged condensation of dna even after transient prm1 expression . to indirectly test this hypothesis , we transfected gfp - h2b fibroblasts with prm1-rfp .
a histone - to - prm exchange was observed in gfp - h2b nuclei 48 hr after prm1 transfection ( figure 1k ) .
unequivocally , the red fluorescent signal of prm replaced the green h2b , confirming the over competition of prm1 on histones ( figure 1k ) .
furthermore , we observed that the prm replaced the histone h3 trimethylated on lysine 9 ( h3k9me3 ) , a critical epigenetic barrier of scnt reprogramming in 30% ( 36 of 120 ) of prm - positive cells ( figure 1l ) .
prm1 assembly on dna of somatic cells leads to an overlapping of nuclear morphology like the one found in elongating spermatids ( figures 1g1i ) .
prm1 appears in foci scattered throughout the nucleus 20-hr post - transfection ( figure 2b ) and fully coalescences 48 hr later in an elongated structure ( table 1 ) .
the final outcome is an elongated and flattened nucleus , very much similar to the spermatozoa s , but larger , due to the diploid dna content ( figure 2i ) .
the degree of nuclear compaction in protaminized somatic cells approaches that of spermatozoa when observed by transmission electron microscopy ( tem ) ( figures 2f2 g ) .
histone hyperacetylation opens chromatin structure in spermatids and is a pre - requisite for histone / prm replacement .
accordingly , transfected cells treated with tsa incorporated more and faster prm1 than control without tsa ( at 16- to 20-hr post - transfection : tsa 47.8% [ 33 of 69 ] ; without tsa 25.8% [ 38 of 147 ] , p = 0.0018 ; at 4048 hr : tsa 83.3% [ 50 of 60 ] ; without tsa 60.8% [ 116 of 191 ] , p = 0.0010 ; table 1 ) .
physiologically , the massive dna compaction proper of spermatozoa is achieved through the induction of double strand dna breaks and repair ( rathke et al . , 2014 ) .
therefore , we tested whether the same would apply to our forced conversion of somatic chromatin to a protaminized structure .
we analyzed via immunofluorescence the nuclei for the presence of the histone variant h2a.x ( a marker of double - strand dna breaks ) around the prm1 condensed foci , but no positive h2a.x signal was detected ( h2a.x positive : ctr 10.2% [ 6 of 59 ] ; ctr + [ uv irradiated ] 63.2% [ 48 of 76 ] ; prm1-rfp 10% [ 6 of 60 ] ; prm1-rfp / tsa 12.5% [ 8 of 64 ] ; figure 2j ) .
a comet assay also excluded the presence of dna fragmentation in fully protaminized fibroblasts ( figure s3 ) .
protaminized cells showed signs of degeneration on the third day after transfection , likely a consequence of the global transcription shut down resulting from chromatin compaction ( figure 2c ) .
finally , we wanted to verify the reversibility of genome protaminization using nt as a biological assay ( figure 3c , a and a1 ) .
a large pronucleus was observed in the injected oocytes starting 6 hr after activation and swelling in size ( 16.2 2.3 m ) by 8 hr .
meanwhile , prm progressively disappeared in the pronuclei of 77% of injected oocytes ( 28 of 36 ; figure 3a ) and replaced by th2b ( figure 3b ) , an oocyte - specific histone variant that plays a key role in nuclear reprogramming ( shinagawa et al . , 2014 , montellier et al . , 2013 ) .
hence , protaminized somatic nuclei decondense and reacquire a nucleosomal organization following a physiological path after artificial activation of the injected oocyte .
the enucleated oocytes reconstructed with protaminized somatic nuclei were further cultured in vitro in seven separated replicates , indicating their full competence to direct early embryonic cleavages until the blastocyst stage ( figure 3c , a2 ) .
the quality of the embryos ( total cell count and karyotype ) was comparable to that of normal in vitro fertilized embryos ( figure 3d ) .
here , we have found that ectopic expression of prm remodels the interphase chromatin of somatic cells , leading to a nuclear compaction strikingly similar to that of elongated spermatids .
the absence of h2ax immune localization was surprising , for it is hardly conceivable a genome - wide protaminization without torsional stress alleviation through dna double - strand breaks ( laberge and boissonneault , 2005 ) . probably , other dna repairing enzymes not tested here
might be involved ; accordingly , h2ax has not been detected during chromatin remodeling in human spermatids ( de vries et al . , 2012 ) .
another possibility is that the histone variant h2ax might have been evicted by prm binding ; however , a comet assay of fully protaminized cells excluded major dna damage ( figure s3 ) .
the chip - seq dataset demonstrated the effective binding of prm1 to dna on 42 dna binding sites on 10 out of 27 chromosomes ( 2n = 54 in sheep ) ( figure 2j ; table s1 ) .
binding occurred in 28 gene - rich domains14 ( 50% ) genic and 14 ( 50% ) intergenic while the other 14 were classified as scaffold ( table s1 ) .
early nuclear remodeling in round human spermatids is marked by the appearance of a single doughnut - like structure .
the doughnut is the morphological expression of nucleosome disassembly , for nucleosome - destabilizing histones ( h4k8ac and h4k16ac , h3k9me2 ) co - localize with it ( de vries et al . ,
these findings suggest that genome remodeling starts at a defined site in the genome and spreads in a spatially regulated manner , ending up with the nuclear compaction found universally in male gametes .
live imaging of the histone - to - prm transition could be a powerful tool to test this hypothesis , but it is only available in drosophila ( awe and renkawitz - pohl , 2010 ) , not in mice or man .
also , currently unavailable are in vitro models to monitor the whole process of spermatogenesis .
the protaminization of somatic cells might provide unique insights on the early steps of chromatin remodeling , allowing the identification of the very first genome domain(s ) binding with prm . the simplified model for nucleosome - to - prm exchange reported here
might also add to the ongoing debate on nucleosomal retention in male gametes ( samans et al .
. it would be interesting to map on a genome - wide scale , the prm - histones footprints on prm1-expressing somatic cells , to see whether the pattern described in spermatozoa is conserved .
scnt is a promising technology whose full implementation is on hold because of its low efficiency .
recent successes on human embryonic stem cells isolated from somatic cell - derived cloned embryos have boosted interest in scnt ( tachibana et al . , 2013 , chung et al . , 2014 ) .
this renewed interest in scnt goes along with targeted nuclear reprogramming strategies , like rnai - mediated downregulation of xist ( ogura et al . , 2013 ,
2011 ) or the depletion of h3k9 methyltransferases in somatic cells before nt ( matoba et al . , 2014 ) . in vitro protaminization of somatic cells
simplifies the nuclear structure of a somatic cell enormously , formatting it in a way that is easily readable by the oocytes and thus holds potential to improve scnt efficiency .
safs were derived from ear biopsy of three female sarda breed sheep ( 2 years old ) .
safs ( between second and eighth passage ) were cultured in dmem ( gibco ) containing 10% fetal bovine serum ( fbs ) , 2 mm glutamine , 3.7 g / l nahco3 , and 0.5% gentamicin .
sperm prm1 cdna ( genbank : nm_002761.2 ) was amplified from a human testis cdna library with appropriate primes and cloned into a ptagrfp vector ( evrogen ) .
the identity of the cloned cdna and its in - frame cloning c - terminal to rfp was verified by sequencing .
mouse tp1 and tp2 cdnas were amplified by pcr from testis total rna and cloned in a ha - tagged pcdna vector ( derived from a life technologies pcdna his - tagged vector ) .
safs at 80% confluence were transfected with 3 g of pbrdt - gfp , ptp1 , ptp2 , pprm1-rfp , pprm1 , or prfp by lipofectamine 2000 ( invitrogen ) , according to the manufacturer s instructions . at 4-hr post - transfection
, medium was changed for dmem containing 5 nm tsa or control ( no tsa ) , and safs were cultured for additional 16 , 20 , 40 , 48 hr . for cell - cycle analysis , safs before transfection were starved with 0.5% fbs for 5 days . to visualize histone - to - prm exchange , safs were first transfected with construct expressed gfp fused to histone 2b using bacmam 2.0 technology ( c10594 , celllight histone 2b - gfp , bacmam 2.0 , life technologies ) according to the manufacturer s instructions and then with pprm1-rfp , as described above .
histone 2b - gfp and prm1-rfp behavior in live cells using fluorescent imaging were observed .
cells were fixed in glutaraldehyde ( 2.5% in 0.1 m cacodylate buffer [ ph 7.4 ] ) for 24 hr . after washing in ddh2o , cells were post - fixed in 2% oso4 in ddh2o for 2 hr .
next , cells were dehydrated through a graded series of ethanol solutions ( 30% , 10 min ; 50% , 15 min ; 70% , 24
hr ; 80% , 10 min ; 96% , 10 min ; 100% , 10 min ; acetone , twice for 15 min ) and were infiltrated with graded concentrations of epoxy 812 resin ( epon ) resin in 100% acetone ( 1:3 , 20 min ; 1:1 , 24 hr ; 3:1 , 2 hr ) , infused twice for 1 hr in pure epon resin , and polymerized at 65c for 24 hr .
images were captured using a slow scan ccd camera ( proscane ) and esivision pro 3.2 software ( soft imaging systems gmbh ) .
cells were fixed in 4% paraformaldehyde ( pfa ) for 15 min , permeabilized with 0.1% ( v / v ) triton x-100 in pbs for 30 or 20 min , and blocked ( 0.1% bsa/0.05% tween 20 [ v / v ] in pbs ) for 1 hr at room temperature ( rt ) .
then cells were incubated with the anti - phospho - histone h2a.x ( ser139 ) ( h2a.x ) mouse antibody ( 1:100 ) ( 05 - 636 , millipore ) at rt for 2 hr or anti - trimethyl - histoneh3 ( lys9 ) rabbit antibody ( 1:200 ) ( 07 - 523 , millipore ) at 4c overnight .
subsequently , the secondary anti - mouse igg - fitc ( 1:100 ) or anti - rabbit igg - fitc ( 1:200 ) was added for 2 hr or 50 min at rt .
chip assay was performed with the ez - magna chip kit ( millipore ) according to the manufacturer s instructions with minor modification .
protein and dna complexes from pprm1-rfp - transfected cells were cross - linked with 0.5% formaldehyde for 10 min at rt .
sonications were done in nuclear buffer ( six 30-s pulses , power setting 10 and six 30-s pulses , power setting 15 in ice with 50-s rest between pulses ; bandelin sonopuls ) .
soluble chromatin was immunoprecipitated with anti - rfp antibody ( evrogen ) directly conjugated with magnetic protein a beads .
dna and protein immune complexes were eluted and reverse cross - linked , and then dna was extracted using a spin filter column .
indexed libraries were prepared from 10 ng / each chip dna with truseq chip sample prep kit ( illumina ) according to the manufacturer s instructions ( for details see supplemental experimental procedures ) . in vitro embryos production
was adapted from those previously described ( ptak et al . , 2002 ) .
oocytes manipulation was carried out with a piezo - driven enucleation and injection pipette ( piezoxpert ) , as previously described .
enucleate oocytes were injected with a nucleus , either from ctr or a prm1-rfp fibroblasts suspended in pbs with 6% polyvinylpyrrolidone ( sigma - aldrich ) .
activation and culture of reconstructed oocytes were processed as described ( ptak et al .
the zona pellucida of embryos at pronuclear stage ( 10- to 12-hr post - activation ) was removed by incubation in 0.5% ( w / v ) pronase and acid tyrode s solution for 30 s. then embryos were fixed in 4% pfa for 15 min and subjected to immunofluorescence analysis as described in torres - padilla et al .
( 2006 ) with the rabbit anti - th2b antibody ( 1:700 , ab23913 abcam ) .
finally , zygotes were mounted with vectashield mounting medium with 5 g / ml of dapi .
the fisher s exact were used to compare quantitative data on nuclear incorporation of prm1 and in vitro development .
performed all nt and characterization work of prm cells , and m.c . performed the vector expressions and f.z | summaryprotamines confer a compact structure to the genome of male gametes . here , we find that somatic cells can be remodeled by transient expression of protamine 1 ( prm1 ) .
ectopically expressed prm1 forms scattered foci in the nuclei of fibroblasts , which coalescence into spermatid - like structures , concomitant with a loss of histones and a reprogramming barrier , h3 lysine 9 methylation .
protaminized nuclei injected into enucleated oocytes efficiently underwent protamine to maternal histone th2b exchange and developed into normal blastocyst stage embryos in vitro .
altogether , our findings present a model to study male - specific chromatin remodeling , which can be exploited for the improvement of somatic cell nuclear transfer . |
when the intervention occurred , she was 40 years old ( 5.5 years after stroke ) and had severe paralysis in her left hand with no residual movement .
she had received conventional therapy for two years and no significant functional improvement had been observed before her participation to this study .
she participated in 10 recoverix training sessions at the rehabilitation hospital of iasi , romania .
we recorded patients sensorimotor rhythm using 45 active eeg electrodes ( g.ladybird , g.tec medical engineering gmbh , austria ) .
fpz was used as ground electrode , and a reference electrode was placed on the right earlobe .
eeg signals were transmitted to an biosignal amplifier ( g.hiamp , g.tec medical engineering gmbh , austria ) , which then delivered the data to a computer for further processing required in online bci .
after preprocessing , common spatial patterns ( csp ) was applied to transform the data to a new matrix with minimal variance of one class and maximal variance of the other class , which is represented by a transformation matrix , w in equation ( 1 ) .
the transformed matrix reflects the specific activation patterns of the data during motor imagery of left or right hand in this study .
the decomposition of a trial
equation ( 1 )
x is described by z = wx the variance of x was projected onto the rows of z. the variance for one class is largest in the first row of z and decreases in each subsequent row due to the transformation matrix , w. the optimal number of csps is four to classify the left or right trials . only first and last two rows ( p=4 ) of w were used to process new input data x. next , the variance ( varp ) was calculated .
these values were normalized and log transformed according to the formula :
equation ( 2 )
where fp ( p=1.4 ) were the normalized feature vectors and varp was the variance of p - th spatially filtered signal .
the patient was seated in a comfortable chair in front of a monitor that presented cues and feedback ( see figure 2 ) with fes pads placed over the forearm of the affected side .
the patient was instructed to imagine the movement of either left or right hand while an arrow was presented on a monitor indicating its movement site and cue . after a delay of 0.5 seconds
a blue bar moved and updated every 4 ms to the left or right indicating both the direction and magnitude of the motor imagery as visual feedback .
the fes would activate with 50 hz updating rate if the user was imagining hand movement of instructed side .
the muscle contraction by fes was sufficient enough to cause movement in the affected hand .
the feedback period lasted four seconds , and the inter - trial interval was two seconds .
when the intervention occurred , she was 40 years old ( 5.5 years after stroke ) and had severe paralysis in her left hand with no residual movement .
she had received conventional therapy for two years and no significant functional improvement had been observed before her participation to this study .
she participated in 10 recoverix training sessions at the rehabilitation hospital of iasi , romania .
we recorded patients sensorimotor rhythm using 45 active eeg electrodes ( g.ladybird , g.tec medical engineering gmbh , austria ) .
fpz was used as ground electrode , and a reference electrode was placed on the right earlobe .
eeg signals were transmitted to an biosignal amplifier ( g.hiamp , g.tec medical engineering gmbh , austria ) , which then delivered the data to a computer for further processing required in online bci .
after preprocessing , common spatial patterns ( csp ) was applied to transform the data to a new matrix with minimal variance of one class and maximal variance of the other class , which is represented by a transformation matrix , w in equation ( 1 ) .
the transformed matrix reflects the specific activation patterns of the data during motor imagery of left or right hand in this study .
the decomposition of a trial
equation ( 1 )
x is described by z = wx the variance of x was projected onto the rows of z. the variance for one class is largest in the first row of z and decreases in each subsequent row due to the transformation matrix , w. the optimal number of csps is four to classify the left or right trials . only first and last two rows ( p=4 ) of w were used to process new input data x. next , the variance ( varp ) was calculated .
these values were normalized and log transformed according to the formula :
equation ( 2 )
where fp ( p=1.4 ) were the normalized feature vectors and varp was the variance of p - th spatially filtered signal .
the patient was seated in a comfortable chair in front of a monitor that presented cues and feedback ( see figure 2 ) with fes pads placed over the forearm of the affected side .
the patient was instructed to imagine the movement of either left or right hand while an arrow was presented on a monitor indicating its movement site and cue . after a delay of 0.5 seconds
a blue bar moved and updated every 4 ms to the left or right indicating both the direction and magnitude of the motor imagery as visual feedback .
the fes would activate with 50 hz updating rate if the user was imagining hand movement of instructed side .
the muscle contraction by fes was sufficient enough to cause movement in the affected hand .
the feedback period lasted four seconds , and the inter - trial interval was two seconds .
figure 4 presents bci classification accuracy across 10 sessions . the patient reported that she actively participated in motor imagery tasks as instructed .
the accuracy in the first two sessions is slightly over the chance level of 50% , and the accuracy of the remaining eight sessions was substantially higher than that of the first two sessions ( see figure 4 ) .
the accuracy dropped to 82.5% in the session number 8 because of the lack of sleep during the previous night .
this result means that the classifier distinguished two different tasks between left mi and right mi in the most training sessions other than the first two sessions .
the visual and proprioceptive feedback was not properly provided to the patients with incorrect classification ( in the left side of figure 5 ) , and both feedbacks were presented with correct classification during feedback period ( in the right side of figure 5 ) .
event related desynchronization plot also showed that the patients was able to perform the mi tasks and here are two examples of the first and last sessions .
erd is observed in both sessions and the statistical comparison between two erd plots is necessary in the future .
after ten training sessions , the patient was able to voluntarily relax and extend the wrist of her paretic side seen in figure 6 .
we showed that the patient could follow the left or right hand motor imagery instructions .
the higher bci accuracy and lda values of later sessions implies that the patients learned to use the bci .
this chronic stroke victim did not have any residual movement of her paretic hand even after several conventional physiotherapy sessions , but she regained her partial control of the affected hand after recoverix training .
it was not possible to practice the nine - hole peg test ( 9-hpt ) and measure the electromyogram ( emg ) due to complete paralysis of her left hand before the session started , and alternative behavioral measurements are not available in this pilot study .
the recoverix system and current training paradigm will be examined with higher number of patient population in comparison with control group for meaningful statistical outcomes .
figure 7 presents our new g.estim device ( g.tec medical engineering gmbh , austria ) , which will replace the current fes device .
the new g.estim is developed mainly for bci applications , and we are currently developing ce and fda clearance .
in addition , the bar feedback will be replaced with an avatar of patients upper limbs , and eeg will be wirelessly transmitted to a computer by g.nautilus ( g.tec medical engineering gmbh , austria ) , as seen in figure 8 . | conventional therapies do not provide paralyzed patients with closed - loop sensorimotor integration for motor rehabilitation
. paired associative stimulation ( pas ) uses brain - computer interface ( bci ) technology to monitor patients movement imagery in real - time , and utilizes the information to control functional electrical stimulation ( fes ) and bar feedback for complete sensorimotor closed loop .
to realize this approach , we introduce the recoverix system , a hardware and software platform for pas .
after 10 sessions of recoverix training , one stroke patient partially regained control of dorsiflexion in her paretic wrist .
a controlled group study is planned with a new version of the recoverix system , which will use a new fes system and an avatar instead of bar feedback . |
oral squamous cell carcinoma ( oscc ) is the most common type of cancer of the mouth ,
accounting for more than 90% of all malignant oral neoplasms .
however , some patients
develop this neoplasm without exposure to these risk factors , a fact suggesting the
involvement of other etiological factors such as viral agents , particularly human
papillomavirus ( hpv ) .
the role of hpv in the development of cancer of the uterine cervix has been well
established , but the association between this virus and oral cancer is not well
defined .
studies evaluating the role of hpv in oral carcinogenesis have yielded
conflicting results , with the reported prevalence of hpv dna in oral cancer tissue
ranging from 0 to 100% .
the main
cells involved in the elimination of altered cells are t lymphocytes . in the
presence of an antigen ,
t cells need to be activated and this activation is mediated
by antigen - presenting cells , including langerhans cells .
langerhans cells are found in the stratified epithelia of the epidermis and mucosa
and their main function is to capture and transport protein antigens to the lymph
nodes for effective presentation to t lymphocytes .
thus , langerhans cells are able to induce either
a cellular or a humoral immune response .
langerhans cells are believed to play an important role in tumor immunology , exerting
a function against the emergence of new antigens expressing malignant
transformation .
however , t cells need
to be activated upon antigen presentation , which is mediated by the antigen
presenting cells , one of which is the langerhans cell . reduced immune surveillance may contribute to the severity of hpv - associated
lesions .
mota , et al.(1999 ) found a progressive decrease in the number of
langerhans cells with increasing severity of cervical intraepithelial lesions . in
contrast , levi , et al.(2005 )
observed an increase in the number of these cells with the progression of cervical
neoplasms . in cancer of the uterine cervix , which is strongly associated with hpv ,
the onset and progression of this neoplasm
are related to changes in langerhans
cells inside the squamous epithelium of this mucosa .
the aims of the present study were to investigate the presence of hpv dna and to
compare the immunohistochemical staining of langerhans cells between hpv - positive
and hpv - negative cases of oscc .
all specimens were taken from
formalin - fixed , paraffin - embedded tissues from files of the oral pathology
laboratory , school of dentistry , federal university of rio grande do norte and from
archives of dr .
luis antonio hospital , natal , rn , brazil . of the 27 cases of ossc
evaluated , tumor primary sites included lip ( 7 cases ) , tongue
( 4 cases ) , floor of the mouth ( 5 cases ) and other locations ( 9 cases ) .
the mean
patient 's age was 63 years ( range 30 - 93 ) , the sample comprised 19 men an 8
women .
ten 10-m thick histological sections were obtained for dna extraction and two 3-m
sections were stained with hematoxylin / eosin for review by a pathologist .
dna was extracted using the ammonium acetate - isopropanol technique which does not
require the use of phenol - chloroform .
this method consisted of the initial removal
of paraffin by baths in xylene heated to 65c .
next , the tissues were hydrated in a
decreasing ethanol series ( absolute , 95% , 70% and 50% ) .
then , 400 l sterile lysis
buffer ( 50 mm nacl , 5 mm tris - hcl , ph 8 , 12.5 mm edta , ph 8 , and 0.25% sds ) and
proteinase k at a final concentration of 500 g / ml were added to the tissue pellet
of each sample and the samples were incubated at 55c for 3 to 5 days until complete
dissolution of the material .
next , 200 l of a 4 m ammonium acetate solution was
added to each sample for protein precipitation , followed by 600 l 100% isopropanol
for dna precipitation .
finally , the dna pellets obtained were washed with 70%
ethanol , dissolved in 50 l te buffer and stored at -20c . polymerase chain reaction ( pcr )
was carried out in an eppendorf thermocycler in a
reaction mixture containing the following components in a final volume of 50 l : 0.5
m of the pco3 and pco4 primers ( invitrogen , life
technologies , carlsbad , ca , usa ) , 1.0 u taq dna polymerase ( invitrogen , life
technologies ) , 20 mm tris - hcl , ph 8.4 , 50 mm kcl , 1.5 mm mgcl2 , 200 m
dntp ( ge healthcare , little chalfont , bucknghamshire , uk ) , and 0.7 or 7 l sample
dna . the pcr conditions for amplification of the - globin gene were : initial
denaturation at 95c for 4 min , followed by 40 cycles of amplification at 95c for 1
min , 50c for 1 min and 72c for 1 min , and a final extension step at 72c for 10
min .
the pcr products were analyzed by electrophoresis on 2.5% agarose gel ( nusieve ,
cambrex bio science rockland , wokingham , berkshire , united kingdom ) stained with
ethidium bromide .
the samples positive for - globin were analyzed by pcr regarding the presence of hpv
dna using pair of generic primers gp5 + ( 5'tttgttactgtggtagatactac3 ' ) and gp6 +
( 5'gaaaaata aactgtaaatcat attc3 ' ) , which flank a fragment of about 140 bp of the l1
gene , a highly conserved sequence in the genome of mucosal ( genital and oral ) hpvs .
the use of this primer pair permits the detection and amplification of this dna
segment from at least 23 individual mucosal ( genital and oral ) hpv types , including
high - risk hpv types .
the reaction mixture contained 1.0 m gp5+/gp6 + ( invitrogen , life technologies ) , 1.0
u taq dna polymerase ( invitrogen , life technologies ) , 20 mm tris - hcl , ph 8.4 , 50 mm
kcl , 1.5 mm mgcl2 , 200 m dntp ( ge healthcare , little chalfont ,
buckinghamshire , uk ) , and 0.7 to 7.0 l dna , in a final volume of 50 l . the pcr
conditions for the detection of hpv were : initial denaturation at 95c for 5 min ,
followed by 40 cycles of amplification at 95c for 1 min , 45c for 2 min and 72c
for 1.5 min , and a final extension step at 72c for 10 min .
the amplified pcr
products were typed by dot blot hybridization using radioactive probes specific for the 19 hpv types most
common in mucosal ( genital and oral ) infections .
each dot blot membrane included
several negative and positive controls for hpv types , as well as pcr products from
patients and controls .
the membrane was hybridized overnight as 56c in 2x standard
saline citrate ( ssc ) , 0.5% sodium dodecyl sulfate ( sds ) and 200 g / ml dna with 19
hpv types probes ( 6,11,16,18,31,33,34,35,39,40,42,43,44,45,51,52,54,56,58 ) and label
[ p]datp .
after hybridization , the unbound probe was of 2x ssc and
0.5% sds at room temperature and two 10-min washes of the same solution at 56c .
the
membrane was exposed to x - ray film at -20c for 24 h. immunohistochemical staining was performed on 3-m - thick formalin - fixed and
paraffin - embedded sections .
the sections were mounted on glass slides previously
treated with organosilane as adhesive ( 3-aminopropyltrietoxi - silan , sigma chemical
co. , st .
the immunohistochemical technique used was a
streptavidin - biotin method ( streptavidin - biotin complex ) .
the sections were
incubated with antibody against s-100 protein ( clone cow s-100 , dako corporation ,
carpinteria , ca , usa ) , 1:200 dilution for 2 h. the reaction was developed with
diaminobenzidine as chromogen , and the sections were counterstained with mayer 's
hematoxylin .
section from nervous tissue was used protein - positive control . in
negative controls ,
after immunohistochemistry , the immunoreactive cells were analyzed taking into the
presence or absence of immunostaining ( positive , negative ) , following immunostaining
density established by the determination of the number of cells positive in five
fields of all specimens .
after the immunohistochemical
analysis , these data were correlated with a presence of hpv .
this research was
approved by research ethics committee of the federal university of rio grande do
norte ( process number 146/05 ) .
with respect to hpv infection , of the 27 cases analyzed , 9 ( 33.3% ) specimens were
hpv - positive and 18 ( 66.7% ) were hpv - negative .
hpv-18 was detected in all 9
hpv - positive cases ( 100% ) and infection with hpv-16 ( co - infection ) was detected in
only 1 case ( figure 1 ) .
lanes 1 - 7 : samples
from patients ; cp : positive control- hela cell dna infected with hpv18 ; cn :
negative control ( h2o2 ) ; pm : molecular marker ( 100
pb ) the presence of langerhans cells in the tumors was demonstrated by
immunohistochemical staining for the anti - s-100 antibody , which resulted in a brown
staining of the cytoplasm of cells with a dendritic shape ( figures 2 and 3 ) . in the
oscc specimens analyzed ,
a mean number of 49.4830.89 langerhans cells were stained
for the anti - s-100 antibody , ranging from 6 to 139 cells present in five fields
observed at 400x magnification . immunostained langerhans cells exhibiting characteristic dendritic shape .
( sabc 400x ) immunostained langerhans cells in a hpv - positive case of oral squamous cell
carcinoma ( sabc 400x ) the median number of immunostained cells was smaller in hpv - positive cases ( 38 cells )
compared to hpv - negative cases ( 42.5 cells ) , but this difference was not
statistically significant ( p=0.382 ) .
analysis of the presence or absence of hpv
infection in relation to the number of s-100-stained langerhans cells showed a wider
variation in the number of immunostained cells in hpv - positive cases .
the possible association between the number of anti - s-100-positive langerhans cells
and hpv infection was also investigated .
according with the median number of
immunostained cells , cases were categorized into low ( corresponding number of
positive cells 38 ) and high ( > 38 positive cells ) . in the present sample
, no
significant association was observed between the number of s-100-stained langerhans
cells and hpv - positive or hpv - negative cases of oscc ( p=1.000 ) ( table 1 ) .
cases categorized according with the median number of langerhans cells
immunostained , presence or absence of hpv infection and statistical
significance
oral cancer is a multifactorial disease , with smoking and regular alcohol consumption
being the main etiological agents involved in oral carcinogenesis .
however , these habits do not
always explain the development of this type of neoplasm . over the last few years
, hpv has called the attention of researchers as a possible
etiological agent of oral cancer .
this virus is
strongly associated with cancer of the uterine cervix and is detected in almost 100%
of cases of this neoplasm , but its role in oral carcinogenesis is still
inconclusive .
several techniques have been used for the detection of hpv , with pcr being considered
the most sensitive method .
the
most widely used primers are the generic primer pairs gp5+/gp6 + and my09/my11 , which
are able to amplify various virus types . in the present study , the presence or
absence of hpv
was confirmed by pcr using the gp5+/gp6 + primers because it amplifies
a small fragment ( 140 bp ) .
this is important when evaluating archived
paraffin - embedded tissue , as done in the present study .
nine cases of the present
sample ( 33.3% ) were hpv - positive and 18 ( 66.7% ) were hpv - negative .
hpv-18 was detected in all hpv - positive cases , in agreement with previous
studies , in
which hpv-18 was the most frequent type .
however , other investigators detected hpv-16 in 71% , 35% ,
66.6% , 55.6% , 33.3% and 85% of cases , respectively , with this virus type being the
most prevalent .
local cellular immunity plays an important role in the response to infection with hpv
and its progression in terms of neoplastic alterations .
impaired immune function results in an increase of the
frequency of clinically detected hpv infections .
langerhans cells are antigen - presenting and -processing cells present in the
epithelium of skin and mucosa and are the main cell population responsible for the
capture of antigens close to the epithelium , presenting these antigens to t helper
cells ( cd4 + t lymphocytes ) and thus inducing a specific immune response . a possible
method for the evaluation of the patient 's immune system
it has been suggested that hpv infection exerts an immunosuppressive effect , reducing
the number of langerhans cells in the epithelium of lesions infected with this
virus .
this suggestion has encouraged the present investigation , which analyzed the
immunohistochemical staining of langerhans cells in cases of oscc infected with hpv ,
a fact not described in the literature .
all 27 oscc specimens analyzed presented positive staining for langerhans cells
( s-100 + ) , a finding also reported by mota , et al .
( 2003 ) , who investigated cases of
oscc , cervical intraepithelial lesions and laryngeal carcinoma , respectively . in the present study
, a mean number of 49.48 langerhans cells were stained for the
anti - s-100 antibody in each case .
this result differs from those reported by connor ,
et al.(1999 ) , who observed
6.02.3 immunostained langerhans cells in high - grade cervical intraepithelial
lesions , and karakk , et al .
( 2003 ) , who found 5.37.9 s-100 + langerhans cells in squamous cell carcinoma of the
larynx .
( 2005 )
detected about 3 langerhans cells in cervical carcinomas , whereas levi , et
al .
( 2005 ) found a mean
number of 6.1 immunostained langerhans cells in high - grade intraepithelial
neoplasms .
these discrepant results might be attributed to the heterogeneity in the
types of lesions analyzed , as well as to the different sites of the lesions since ,
despite similarities , each mucosa presents its peculiarities .
( 2001 ) detected a
larger number of langerhans cells in tumors infected with hpv ( more than 100 cells
per field ) compared to lesions not infected with this virus
( fewer than 10 cells per field ) in specimens of adenocarcinoma and
squamous cell carcinoma of the lung .
in the present study , we found a wide variation
in the number of immunostained cells , but no significant association was observed
between the number of langerhans cells and the presence of hpv in the oscc cases
analyzed .
( 2004 ) , who did not observe
differences in the density of langerhans cells in cervical lesions between
hpv - positive and hpv - negative groups . however , these authors noted a change in the
morphology of these cells from their characteristic dendritic shape to a more round
shape in hpv - positive cases . this fact was not observed in the present study .
this
divergence between the two studies might reflect peculiarities in the site of the
lesions , in view of the fact that the oral mucosa markedly differs from the cervical
and pulmonary epithelium . although no significant association was observed between the number of langerhans
cells and the presence of hpv , the number of s-100 + cells tended to be smaller in
hpv - positive cases . in agreement with this observation , connor ,
( 2006 ) found that
langerhans cells were significantly reduced in cases of hpv - positive cervical
neoplasms compared to hpv - negative lesions .
we agree with arany and tyring ( 1998 ) who suggested that this
depletion of langerhans cells is apparently associated with replication of hpv since
oncoproteins of this virus may affect antigen presentation .
( 2006 )
suggest that there is not a precise explanation for the decrease of dendritic cells
found in the hpv - infected cervix , although several possibilities exist , including :
reduction of the dendritic cells ( or their immediate precursors ) colonizing this
area ; increased exiting or traffic of the normally resident cells ; or promoted
dendritic cell death .
they believe that the last possibility , although feasible , is
less likely to occur because hpv is known to induce proliferation rather than cell
death , at least in epithelial cells .
when classifying the mean number of langerhans cells into high and low , we found no
association between the number of these cells and hpv - positive or hpv - negative
cases .
though , we believe that hpv infection might create a localized
immunodeficient microenvironment . according to gianini , et al.8 ( 2002 ) and
jimenez - flores , et al .
( 2006 ) , a
consequence of this could be a more permissive microenvironment for the hpv to
initiate and establish infection within the regional tissue , so local dc deficiency
is likely to impede efficient t - cell recruitment into this microenvironment .
so far , no study is available correlating langerhans cells and hpv in oscc , a fact
impairing the comparison of the present data with the literature on this specific
neoplasm .
hpv infection may provoke changes in the immune system by interfering with
langerhans cell - associated antigen presentation , permitting the escape from this
defense mechanism .
however , the present results showed no interference of hpv with
langerhans cells in the oscc cases analyzed .
in conclusion , we observed no association between the immunostaining of langerhans
cells ( s-100 + ) and infection with hpv in the oscc cases analyzed . despite the
reduced sample analyses , mostly due to insufficient amount of tissue found in
paraffin blocks for dna extraction from various cases , which led to the exclusion
these
, our findings suggest that the presence of hpv in such cases of oscc could not
alter the immunological system , particularly the langerhans cells .
further studies
will be necessary to understand the real role between langerhans cells and hpv in
oral carcinogenesis . | the human papillomavirus ( hpv ) has been strongly implicated in development of
some cases of oral squamous cell carcinoma ( oscc ) .
however , the immunological
system somehow reacts against the presence of this virus . among the cells
involved in such mechanism of defense langerhans cells ( lc )
stand out , which are
responsible for processing and presenting antigens.objectivesthe purposes of this study were to investigate the presence of hpv dna and to
evaluate the immunohistochemical reactivity for langerhans cells between
hpv - positive and hpv - negative oscc .
twenty - seven cases of ossc were
evaluated .
material and methodsdna was extracted from paraffin - embedded tissue samples and amplified by
polymerase chain reaction ( pcr ) for the detection of hpv dna .
viral typing
was performed by dot blot hybridization .
immunohistochemistry was performed
by the streptavidin - biotin technique .
resultsfrom the 27 cases , 9 ( 33.3% ) were hpv - positive and 18 ( 66.0% ) hpv - negative .
hpv 18 was the most prevalent viral type ( 100% cases ) and infection with
hpv-16 ( co - infection ) was detected in only 1 case . in the oscc specimens
examined ,
immunoreactivity to s-100 antibody was detected in all cases , with
a mean number of 49.4830.89 langerhans cells positive for immunostaining .
the mean number of immunostained langerhans cells was smaller in the
hpv - positive cases ( 38 cells / case ) than in the hpv - negative cases ( 42.5
cells / case ) , but this difference was not significant ( p=0.38 ) .
conclusionsthe low frequency of detection of hpv dna in oscc indicates a possible
participation of the virus in the development and progression of only a
subgroup of these tumors .
there was no association between the
immunohistochemical labeling for langerhans cells ( s-100 + ) and hpv infection
of in ossc .
these findings suggest that the presence of hpv in such oscc
cases could not alter the immunological system , particularly the langerhans
cells . |
ehlers - danlos syndrome ( eds ) is a heterogeneous group of heritable connective tissue disorders characterized by skin hyperextensibility , joint hypermobility , easy bruising , and tissue fragility . as 1 of 6 major types of eds , vascular eds ( veds )
collagens provide structure and strength to connective tissue , which is mostly found in skin , blood vessels , and internal organs .
veds is the most rare form of eds ( < 5% of all eds patients ) but the most severe because of complications related to the vascular system , which can result in arterial rupture in young adults .
such complications are dramatic and unexpected , often presenting as sudden death , acute abdomen , retroperitoneal hemorrhage , uterine rupture at time of delivery , and/or shock .
therefore , establishing a correct diagnosis of veds is extremely important because the timing of diagnosis can influence prognosis .
hypermobility of the large joints and skin hyperextensibility , characteristic of the more common eds forms , are unusual in veds ; thus , colonic perforation or aneurysm rupture may be the first presentation of the disease .
veds is typified by a number of characteristic facial features ( eg , large eyes , small chin , sunken cheeks , thin nose and lips , lobeless ears ) . here , we describe veds lacking these characteristic facial attributes in a healthy man .
in december 2012 , a 24-year - old healthy japanese man with abdominal pain was admitted to the emergency room .
, we learned his mother had a history of extensive bruising since childhood , idiopathic hematothorax , and aortic dissection in youth . on admission
, he showed stable vital signs but presented with thin translucent skin , extensive bruising , and toe - joint hypermobility . on examination , hypermobility of the small joints and subcutaneous hemorrhage were also detected ( figure 1 ) .
however , we did not observe hypertelorism , bifid uvula , or cleft palate , which are features of loeys dietz syndrome .
enhanced computed tomography revealed free air , diverticula , and perforation in the sigmoid colon .
the lesion of the sigmoid colon perforation was removed , and hartmann procedure was performed because of severe contamination in the emergency operation . during the surgery , the control of bleeding was required because of the vascular fragility of the patient .
the patient had skin necrosis on day 12 but was discharged from the hospital 1 month post - operatively .
he was asked to avoid hard labor since veds was suspected based on the major clinical criteria of thin skin with visible veins , colon rupture , and easy bruising .
the characteristic facial features of this condition ( eg , large eyes , small chin , sunken cheeks , thin nose and lips , lobeless ears ) were not observed ( figure 2 ) .
reversal of the hartmann procedure was performed 4 months later . to confirm the diagnosis of veds
the patient showed none of the characteristic facial features ( eg , large eyes , small chin , sunken cheeks , thin nose and lips , lobeless ears ) of vascular ehlers - danlos syndrome .
dermal fibroblasts were obtained from explants of a skin biopsy specimen taken from the upper arm of the patient and a sex- and age - matched control , after appropriate informed consent had been given .
the cultures were maintained in dulbecco modified eagle medium containing 10% fetal bovine serum in a co2 incubator at 37 c .
the study was approved by the ethical committee of dokkyo medical university , school of medicine ( japan ) .
collagen synthesis in the cultured dermal fibroblasts was assessed to determine whether the production of type iii collagen was reduced in the patient .
after the fibroblasts were cultured with [ h]proline , type iii collagen and type i collagen [ 1(i ) + 2(i ) ] were isolated from the cultured dermal fibroblasts of the patient and sex- and age - matched control .
afterwards , they were separated using sodium dodecyl sulfate - polyacrylamide gel electrophoresis and imaged using fluorography .
the amount of the 1 chain of type iii collagen secreted into the culture medium significantly decreased compared with that secreted from the control ( figure 3 ) .
cultured fibroblasts were pulse - labeled with [ h]proline in fresh medium , and the secreted proteins were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis under normal ( ) or reducing ( + ) conditions .
the synthesis of type i collagen in this patient was the same as in controls .
however , the synthesis of type iii collagen was reduced compared with the normal control values ( inside circle ) .
n , normal control ; p , patient with vascular ehlers - danlos syndrome . based on the reduction in the 1 chain of type iii collagen
, we directly amplified and sequenced the col3a1 gene from genomic dna and detected a single base - pair change ( g > t ) predicted to convert a glycine to a cysteine in the encoded protein .
subsequently , total cellular ribonucleic acid was extracted from cultured dermal fibroblasts , and a complementary dna of the col3a1 gene was synthesized by priming with random hexamers .
after the amplification products from a polymerase chain reaction were directly sequenced , we identified the same heterozygous mutation within exon 32 of the col3a1 gene in proband c.2221 ( g > t , gly cys ) ( figure 4 ) .
a heterozygous mutation was found within exon 32 of the col3a1 gene in proband c.2221 ( g > t , gly cys ) .
subsequently , the patient was diagnosed with veds based on reduced type iii collagen synthesis and identification of a mutation in the col3a1 gene .
after confirmed diagnosis , the patient and mother were prescribed oral celiprolol ( beta blocker ) .
dermal fibroblasts were obtained from explants of a skin biopsy specimen taken from the upper arm of the patient and a sex- and age - matched control , after appropriate informed consent had been given .
the cultures were maintained in dulbecco modified eagle medium containing 10% fetal bovine serum in a co2 incubator at 37 c .
the study was approved by the ethical committee of dokkyo medical university , school of medicine ( japan ) .
collagen synthesis in the cultured dermal fibroblasts was assessed to determine whether the production of type iii collagen was reduced in the patient . after the fibroblasts were cultured with [ h]proline , type iii collagen and type i collagen [ 1(i ) + 2(i ) ] were isolated from the cultured dermal fibroblasts of the patient and sex- and age - matched control .
afterwards , they were separated using sodium dodecyl sulfate - polyacrylamide gel electrophoresis and imaged using fluorography .
the amount of the 1 chain of type iii collagen secreted into the culture medium significantly decreased compared with that secreted from the control ( figure 3 ) .
cultured fibroblasts were pulse - labeled with [ h]proline in fresh medium , and the secreted proteins were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis under normal ( ) or reducing ( + ) conditions .
the synthesis of type i collagen in this patient was the same as in controls . however , the synthesis of type iii collagen was reduced compared with the normal control values ( inside circle ) .
based on the reduction in the 1 chain of type iii collagen , we directly amplified and sequenced the col3a1 gene from genomic dna and detected a single base - pair change ( g > t ) predicted to convert a glycine to a cysteine in the encoded protein .
subsequently , total cellular ribonucleic acid was extracted from cultured dermal fibroblasts , and a complementary dna of the col3a1 gene was synthesized by priming with random hexamers .
after the amplification products from a polymerase chain reaction were directly sequenced , we identified the same heterozygous mutation within exon 32 of the col3a1 gene in proband c.2221 ( g > t , gly cys ) ( figure 4 ) .
a heterozygous mutation was found within exon 32 of the col3a1 gene in proband c.2221 ( g > t , gly cys ) .
subsequently , the patient was diagnosed with veds based on reduced type iii collagen synthesis and identification of a mutation in the col3a1 gene .
after confirmed diagnosis , the patient and mother were prescribed oral celiprolol ( beta blocker ) .
we describe here the treatment of a patient with veds lacking the typical characteristic facial attributes of this condition and identified a previously undocumented mutation in the col3a1 gene .
no standardized procedure for colonic perforation in veds patients has been developed . here , we performed reversal of the hartmann procedure , as in most previous cases .
however , some cases have been managed with subtotal colectomy as the first - line management because colonic reperforation has been reported .
thus , conducting genetic assays to confirm diagnosis could be important for determining the operative technique or in saving relatives through treatment with beta blockers . in the present case ,
the patient and his mother were prescribed the 1-adrenoceptor - antagonist celiprolol . in a recent randomized multicenter trial , treatment with celiprolol
was shown to result in a threefold decrease in the number of arterial events ( rupture or dissection ) during a mean follow - up of 47 months , and therefore might delay the need for invasive treatment . in the present case ,
the overall life expectancy of patients with veds is dramatically shortened , largely as a result of vascular rupture , with a median life span of 48 years ( range , 673 years ) .
in addition , the prognosis after treatment is still poor . in particular , previous reports have observed that the mortality rate ranged from 29 to 50% .
thus , close outpatient follow - up and appropriate guidance to the patient and family ( eg , encouragement to contact the doctor when the patient suddenly notices unexplained pain ) should be provided .
in addition , the patient should carry a medical attention bracelet and papers noting information on the condition and blood group .
our case illustrates an awareness of the clinical characteristics of veds and the family history is important because of high mortality even in young people .
the mutation in our case has not been previously documented and might explain the non - characteristic facial appearance .
thus , genetic assays could help in determining the surgical procedure and offer benefits to relatives , since this condition is inherited in an autosomal dominant manner . | abstractas a type of ehlers - danlos syndrome ( eds ) , vascular eds ( veds ) is typified by a number of characteristic facial features ( eg , large eyes , small chin , sunken cheeks , thin nose and lips , lobeless ears ) .
however , veds does not typically display hypermobility of the large joints and skin hyperextensibility , which are features typical of the more common forms of eds .
thus , colonic perforation or aneurysm rupture may be the first presentation of the disease .
because both complications are associated with a reduced life expectancy for individuals with this condition , an awareness of the clinical features of veds is important.here , we describe the treatment of veds lacking the characteristic facial attributes in a 24-year - old healthy man who presented to the emergency room with abdominal pain .
enhanced computed tomography revealed diverticula and perforation in the sigmoid colon .
the lesion of the sigmoid colon perforation was removed , and hartmann procedure was performed . during the surgery , the control of bleeding was required because of vascular fragility .
subsequent molecular and genetic analysis was performed based on the suspected diagnosis of veds .
these analyses revealed reduced type iii collagen synthesis in cultured skin fibroblasts and identified a previously undocumented mutation in the gene for a1 type iii collagen , confirming the diagnosis of veds . after eliciting a detailed medical profile
, we learned his mother had a history of extensive bruising since childhood and idiopathic hematothorax .
both were prescribed oral celiprolol .
one year after admission , the patient was free of recurrent perforation.this case illustrates an awareness of the clinical characteristics of veds and the family history is important because of the high mortality from this condition even in young people .
importantly , genetic assays could help in determining the surgical procedure and offer benefits to relatives since this condition is inherited in an autosomal dominant manner . |
a male patient , 65 years of age reported to adhiparasakthi dental college and hospital outpatient department with a chief complaint of painful swelling in the lower lip for 1 month .
on intraoral examination , a well - defined sessile swelling of size 1 cm 1.5 cm was noticed in the lower lip .
secondary surface changes such as pseudomembrane was noticed over the swelling [ figure 1a ] , and it was tender on palpation and firm in consistency .
preoperative ( a ) and postoperative ( b ) photographs of the lesional area based on the clinical findings , provisional diagnosis of traumatic fibroma was made .
surgical excision of the lesion was done [ figure 1b ] under 2% lignocaine local anesthetic solution and submitted for histopathologic examination in 10% formalin fixative solution .
on histopathologic examination , a layer of hyperplastic stratified squamous epithelium with bulbous rete ridges was noticed with an underlying inflamed connective tissue [ figure 2 ] .
fibrinopurulent membrane replaced the epithelium at one end of the section [ figure 3 ] .
the underlying connective tissue showed a wide distribution of chronic inflammatory cell infiltrate amidst which foci of acute inflammatory cells mainly neutrophils were evident .
areas of mucin spillage were noticed which were surrounded by a connective tissue wall made of granulation tissue .
large cells with a foamy cytoplasm resembling macrophages were seen in the areas of mucin spillage along with chronic inflammatory cells .
large areas of adipose tissue were seen in the connective tissue [ figures 3 and 4 ] . based on the histopathological report , it was diagnosed as mucous extravasation phenomenon with the adjoining reactive lesion .
hyperplastic epithelium , loss of acinar architecture , largely replaced by adipose tissue , and areas of mucin pooling walled off by granulation tissue wall , ( h and e stained section , 10 ) replacement of epithelium by fibrinopurulent membrane with dense inflammation underneath , ( h and e stained section , 10 ) foci of acute inflammatory cells amidst a sea of chronic inflammation , ( h and e stained section , 40 )
resolution of mucocele can happen by means of enzymatic digestion of acini by neutrophils and macrophages . in this case ,
mucocele was not the provisional diagnosis because it did not present as a soft fluctuant mass with a bluish tinge .
instead , the lesion appears as a firm mass with yellowish surface changes which was more in favor of traumatic fibroma . as such mucocele is a benign lesion .
however , in this case , repeated insult to the lesion has prevented it from autoresolution , and the persistent chronic irritation has prompted the reactionary features to set in .
the repeated trauma can be attributed to the reduction in the occlusal height due to generalized attrition of teeth which makes the swelling in lower lip more vulnerable to irritation .
acute episodes of trauma could be the reason for the foci of acute inflammatory cells seen amidst the sea of chronic inflammatory cells in the long - standing lesion .
the replacement of epithelium by fibrinopurulent membrane with subjacent dense chronic inflammatory cells again shows the effect of body 's response to chronic irritation .
loss of mucous salivary gland architecture could be attributed to the long - standing chronic inflammation .
one interesting aspect of the lesion is the increased adipose tissue found in the hematoxylin and eosin section .
however , the fact that with advancing age , the normal salivary glands are replaced by adipose tissue made us to consider this as a normal response to aging and thus lipoma was ruled out
. the habit of chronic smoking could also be the reason for the change observed .
this shows the importance of detailed case history taking along with the necessary correlation with the histopathological picture before the final diagnosis .
one thing of concern is the hyperplastic epithelium with bulbous rete ridges pushing into the underlying connective tissue
. it could be the reason for the parallel arrangement of fibers observed subepithelially giving the effect of a fibrous capsule .
the basilar hyperplasia and acanthosis clearly show that the epithelium is making an attempt to overcome the effects deleterious associated with persistent irritation and protect the underlying structures .
the basement membrane was intact indicating that there was no risk of malignancy in this patient as the lesion has been excised in toto .
however , the risk of uncontrolled proliferation of epithelial cells leading to invasive carcinoma could have happened due to persistent irritation had it not been intervened at this stage .
the quality of life of patient could be severely compromised if that be the case .
this implies the importance of early surgical intervention and to histopathologically examine any excised tissue which will guide us to plan the postoperative follow - up schedule in the best interest of the patient .
this case report is an effort to emphasize the importance of good clinicopathologic correlation and also to understand the basic concept as how the body 's response to chronic irritation can transform a trivial benign lesion to reactive lesion and possibly to malignancy .
the vigilant role of a dentist in the handling of a lesion is reinforced through our case report .
| oral mucocele is the most common benign lesion of minor salivary gland caused due to any form of mechanical trauma to the excretory duct of the gland .
it is of two types mucous extravasation phenomenon and mucous retention type .
extravasation type is more common . here
, we report a case of a 65-year - old male patient with a complaint of painful swelling in the lower lip for 1 month with a history of trauma in the lower lip .
it was clinically diagnosed as traumatic fibroma , and an excisional biopsy was done . however , the histopathology was basically that of mucous extravasation phenomenon coexisting with the features of a reactive lesion implicating the potential role of chronic irritation in the impairment of normal healing process .
considering the fact that long - standing chronic inflammation in a benign lesion can even lead to malignant transformation of that lesion ; here , we highlight the role of a good clinicopathologic correlation and the significance of prompt intervention and treatment . furthermore ,
emphasizing the potential need for postoperative follow - up by the dentist , how much ever trivial the lesion may appear to be in the best interest of the patient . |
a 41-year - old woman presented to the outpatient ward of our hospital with a two - month history of reddish urine and blood clots on voiding and intermittent left flank pain .
the medical history revealed that the patient had undergone a medical examination three years prior and results were within the normal ranges .
a urinalysis showed 2 + hematuria ( 0.5 mg / dl ) , but no proof of the presence of sugar in the urine or proteinuria .
laboratory findings showed no elevation of the white blood cell count and a normal level of c - reactive protein ( crp ) .
initial abdominal ultrasonography ( iu 22 unit ; philips medical systems , bothell , wa ) demonstrated the presence of a heterogeneous hypoechoic solid mass protruding into the renal pelvis from the upper pole of the left kidney .
1b ) . three - dimensional kidney multidetector ct imaging ( lightspeed ; ge healthcare , milwaukee , wi ) was done .
on pre - contrast scans , a 5.6 5.5 cm hypodense mass with a slightly bulging contour was noted in the left upper polar area . within the hypodense mass
there were also high density lesions in the dilated left proximal ureter . on post - contrast
axial scans , a hypodense mass with hydronephrosis and hydroureter was noted in the left kidney ( fig .
, there was no definite evidence of an enhancing lesion . a multi - locular cystic and solid tumor of the left upper polar area and extending to the renal pelvis and proximal ureter was the provisional diagnosis .
transitional cell carcinoma could not be ruled out from the pre - operative imaging diagnosis .
we did a left radical nephroureterectomy , and the resected specimen measured 14 8 6 cm .
an 8 6 cm myxoid lobulated solid mass was situated in the cystically dilated pelvocalyceal region in the upper pole , which was accompanied by hydroureter ( fig .
the tumor was seen to have a tan , yellow and brown glistening myxoid lobulated cut surface with multiple scattered hemorrhages .
microscopically , the tumor was mostly composed of primitive - looking or poorly differentiated mesenchymal cells within the loose myxoid and vascular stroma ( fig .
numerous epithelial - lined cysts and entrapped tubules were scattered throughout the tumor , which was lined by simple or stratified cuboidal to columnar cells with eosinophilic , granular cytoplasm .
mitotic figures were not found in the epithelial cells , but the cells showed a very focal carcinomatous change ( fig .
based on immunohistochemical staining , the stromal tumor cells were diffusely reactive for vimentin and non - reactive for desmin and myoglobin .
cyst - lining epithelial cells were positive for vimentin , pan - cytokeratin and epithelial membrane antigen ( ema or muc1 ) ( fig .
ultrastructurally , the stromal tumor cells showed round to ovoid nuclei with regular outlines and an electron - dense rim of heterochromatin .
the final diagnosis was cesk , based on findings of several previous studies found in the literature .
cystic embryonal sarcoma of the kidney is an extremely rare renal tumor with a poor prognosis . to the best of our knowledge ,
only a few cases have been reported in the pathological and urological literature , and there have been no reports about radiological imaging of the tumor .
recently , the who classification of renal neoplasms in children and adults has been revised , but various rare and unclassifiable renal tumors remain . a novel type of malignancy , cesk was reported in 1998 by delahunt et al .
( 1 ) , who found 25 similar cases in the files of the national wilms ' tumor study pathology center . this series of 25 cases was designated as embryonal sarcoma of adult and pediatric kidneys at the 1995 meeting of the united states and canadian academy of pathology .
a review of the english literature revealed that only three cases of cystic embryonal sarcoma have been reported ( table 1 ) .
( 1 ) proposed cesk as a new nosologic entity in 1998 for a tumor in a 19-year - old caucasian male who was injured playing rugby ( 1 ) .
ct scans showed a large , well - defined , heterogeneous mass in the right kidney . following a laparotomy ,
( 2 ) reported another case of cesk with localized submucosal invasion of the renal pelvis in a 12-year - old boy in japan in 2000 . in this case , an imaging study was not done .
following a laparotomy , the lesion was found to be very small , ill defined and mainly localized to the submucosal areas and sinuses of the renal pelvis .
matsumoto et al . ( 3 ) in 2002 reported another case of cesk in a 10-year - old boy in japan . in this patient ,
abdominal sonography showed the presence of an isoechoic mass that protruded into the renal pelvis from the lower pole of the right kidney .
cystic embryonal sarcoma of the kidney has been reported to consist predominantly of undifferentiated malignant mesenchyma and numerous epithelial - lined cysts in myxoid stroma .
microscopically , the tubules and cysts of this neoplasm are lined with low cuboidal to flat epithelium composed of cells with spherical nuclei .
vimentin is not expressed by the epithelium in cesk and entrapped glomeruli are not seen .
in addition , there is an absence of the typical architectural pattern of haphazardly arranged cells supported by a duplicate fibrovascular stroma , which is seen at least focally in all cases of clear cell sarcoma ( 1 ) .
cesk appears to be a novel form of a renal malignancy and prognosis is poor if there is early disease recurrence postoperatively or after aggressive chemotherapy . in our patient , we did not administer chemotherapy and the patient was free of disease over an eight - month follow - up period .
the imaging characteristics of cesk have not been well documented because of the rarity of the tumor . in our patient , a poorly enhancing cyst - like mass with hemorrhagic components in the upper polar area that extended to the renal pelvis was found .
these findings are similar to findings for mixed epithelial and stromal tumor of the kidney , synovial sarcoma , collecting duct carcinoma and multilocular cystic renal cell carcinoma .
mixed epithelial and stromal tumor of the kidney is a rare benign neoplasm of unknown histogenesis .
a similar lesion was previously referred to as cystic hamartoma of the renal pelvis , adult type of mesoblastic nephroma , or cystic nephroma ( 4 ) .
cystic hamartoma of the renal pelvis forms as an intrarenal multicystic mass adjacent to the pelvocalyceal system , as originally reported by pawade et al .
histopathologically , it is also characterized by a biphasic proliferation of epithelial and mesenchymal elements .
adsay et al . ( 6 ) recommended that cystic hamartoma of the renal pelvis and multilocular cystic nephroma should be presented under the name of mixed epithelial and stromal tumor of the kidney and stated that the spindle cells of all mixed epithelial and stromal tumor cells were diffusely and strongly positive for muscle markers such as sma ( smooth muscle actin ) .
another recent report , one by jung et al . ( 7 ) , presented two cases of a mixed epithelial and stromal tumor of the kidney with malignant transformation .
primary synovial sarcoma of the kidney is also extremely rare but might present with a multiloculated cystic tumor .
furthermore , a true cystic component with regional lymphadenopathy was a more common finding for collecting duct carcinoma when compared with cortical renal cell carcinoma ( 8) .
multilocular cystic renal cell carcinoma is a distinct subtype of renal cell carcinoma that is distinguished by the presence of a solid mass comprising less than 10% of the volume ( 9 ) , which consists of a well - circumscribed globular cystic mass demarcated from the kidney and surrounding tissues by a fibrous wall . in our case ,
the diagnosis between a cystic embryonal sarcoma and mixed epithelial and stromal tumor of kidney with malignant transformation was debated .
histologically , however , the main differential points are the sarcomatous components . mixed epithelial and stromal tumor of the kidney with sarcomatous transformation does n't show loose myxoid stromal lobules with occasional condensation of round stromal cells as in cesk . also , the former consists of highly cellular malignant spindle cells with heterogeneous chondrosarcomatous or rhabdomyosarcomatous components , with ovarian - type stroma , which is not found in cesk . in conclusion
, cesk should be considered in the differential diagnosis for a multilocular cystic renal tumor . | here we report a case in a 41-year - old woman histologically proven cystic embryonal sarcoma of the kidney , with emphasis on the imaging findings and pathological features .
a large lobulated solid mass in the cystically dilated pelvocalyceal region was accompanied with hydroureter as depicted on both ultrasound and contrast - enhanced ct images . |
we have recently studied two cases of advanced primary duodenal adenocarcinoma ( pda ) . in both cases , we were unable to determine the primary location of the carcinoma by contrast - enhanced computed tomography ( ct ) .
ct images showed a large tumor ( 7.9 6.5 8.5 cm and 6.7 6.4 7.0 cm ) of unknown origin with metastases , ascites , and lymph node invasion .
the aim of the present work is to offer a further contribution to the resolution of therapeutic and diagnostic problems by reporting a new case of adenocarcinoma of the 1st duodenal portion and reviewing the relevant literature .
an 85-year - old woman was admitted to our hospital with melena , hypotension , and a 2-month history of appetite loss .
her medical history included an operation to correct a right inguinal hernia and hypertension , and her vital signs were within normal limits in the emergency room .
routine blood tests showed leukocytosis , hypoalbuminemia , and elevation of c - reactive protein , aspartate aminotransferase and lactate dehydrogenase , along with an elevation of tumor markers , of which carcinoembryonic antigen , ca19 - 9 , du - pan-2 and span-1 antigen were subsequently confirmed .
the presence of a tumor ( 7.9 6.5 8.5 cm ) and irregular thickening of the wall , multiple metastases , multiple lymphadenopathy , and ascites were confirmed by ct scan ( fig .
1 ) . endoscopy and histologic examination revealed a poorly differentiated adenocarcinoma of the duodenum ( in the 1st portion ) , which did not contain hormone and was not differentiated into a neuroendocrine carcinoma ( fig .
a 90-year - old woman was admitted to our hospital with transient loss of consciousness and a 1-year history of palpitation associated with light effort .
routine blood investigations showed leukocytosis , anemia , hypoalbuminemia , and elevation of c - reactive protein and tumor markers , with carcinoembryonic antigen being confirmed subsequently .
a tumor ( 6.7 6.4 7.0 cm ) , irregular thickening of the wall , decreased blood vessel density , bilateral pleural fluid , and ascites were confirmed by ct scan ( fig .
3 ) . endoscopy and histologic examination revealed a well - differentiated adenocarcinoma of the duodenum ( in the 3rd portion ) , which did not contain hormone and was not differentiated into a neuroendocrine carcinoma ( fig .
after having given informed consent , she and her family were offered palliative care . in these two cases ,
the progress of duodenal cancer did not constrict the lumen , and both patients were able to eat until end of life .
written informed consent was obtained from these patients for the publication of this case report and accompanying images .
( a copy of the written consent is available for review from the editor - in - chief of this journal on request .
an 85-year - old woman was admitted to our hospital with melena , hypotension , and a 2-month history of appetite loss .
her medical history included an operation to correct a right inguinal hernia and hypertension , and her vital signs were within normal limits in the emergency room .
routine blood tests showed leukocytosis , hypoalbuminemia , and elevation of c - reactive protein , aspartate aminotransferase and lactate dehydrogenase , along with an elevation of tumor markers , of which carcinoembryonic antigen , ca19 - 9 , du - pan-2 and span-1 antigen were subsequently confirmed .
the presence of a tumor ( 7.9 6.5 8.5 cm ) and irregular thickening of the wall , multiple metastases , multiple lymphadenopathy , and ascites were confirmed by ct scan ( fig .
1 ) . endoscopy and histologic examination revealed a poorly differentiated adenocarcinoma of the duodenum ( in the 1st portion ) , which did not contain hormone and was not differentiated into a neuroendocrine carcinoma ( fig .
a 90-year - old woman was admitted to our hospital with transient loss of consciousness and a 1-year history of palpitation associated with light effort .
routine blood investigations showed leukocytosis , anemia , hypoalbuminemia , and elevation of c - reactive protein and tumor markers , with carcinoembryonic antigen being confirmed subsequently .
a tumor ( 6.7 6.4 7.0 cm ) , irregular thickening of the wall , decreased blood vessel density , bilateral pleural fluid , and ascites were confirmed by ct scan ( fig .
3 ) . endoscopy and histologic examination revealed a well - differentiated adenocarcinoma of the duodenum ( in the 3rd portion ) , which did not contain hormone and was not differentiated into a neuroendocrine carcinoma ( fig .
after having given informed consent , she and her family were offered palliative care . in these two cases ,
the progress of duodenal cancer did not constrict the lumen , and both patients were able to eat until end of life .
written informed consent was obtained from these patients for the publication of this case report and accompanying images .
( a copy of the written consent is available for review from the editor - in - chief of this journal on request .
as there have been no studies so far reporting pda without stenosis and occlusion of the lumen , this may be a major addition to our understanding of pda .
pda is relatively rare [ 1 , 2 , 3 ] , representing 3345% of all small bowel tumors , which themselves represent 45% of all gastrointestinal tumors .
moreover , it is responsible for only 1% of deaths due to gastrointestinal neoplasms [ 2 , 4 ] .
approximately 3050% of small intestinal adenocarcinomas occur in the duodenum . according to a report from china , only 0.02% of all inpatients suffered from pda .
duodenal adenocarcinomas represent approximately 0.30.5% of all malignant gastrointestinal tumors [ 4 , 5 ] , with studies showing that 115% of tumors are located in the 1st portion , 5075% in the 2nd portion , 1433% in the 3rd portion , and 517% in the 4th portion of the duodenum [ 2 , 3 , 4 , 5 , 6 ] .
the causative factors for this disease have not been clearly identified , although patients with familial adenomatous polyposis and gardner syndrome are considered to have a higher likelihood of developing duodenal cancer .
similarly , patients with duodenal polyps but no predisposing family history are also at increased risk .
histologically , adenocarcinoma is the most common type , with lymphoma and leiomyosarcoma being encountered less frequently [ 3 , 4 ] .
symptoms may be absent until the tumor has progressed , thus leading to a delay of several months in presentation .
patients typically present with a long history of variable and vague symptoms , and many present with advanced disease .
the diagnosis of pda is often delayed because its symptoms and signs are nonspecific [ 2 , 3 , 5 , 6 ] . as a result , an average delay of 215 months from the onset of symptoms to the time of diagnosis of pda
clinical manifestations include upper abdominal pain , nausea , vomiting , jaundice , fatigue , weakness , anemia , gastrointestinal obstruction , gastrointestinal bleeding and weight loss , with abdominal pain being the most common symptom ( 56.0% ) .
the majority of these tumors are found to have infiltrated the duodenal wall at presentation , with many being unresectable due to local and distal invasion .
one reason for this is that an examination of the entire duodenum by upper gastrointestinal endoscopy is challenging ; adenocarcinomas of the 3rd and 4th portions of the duodenum are frequently inaccessible using endoscopy , and most cases require multiple investigations .
however , new modalities such as double - balloon enteroscopy or capsule endoscopy can make the diagnosis of small bowel or duodenal adenocarcinoma easier .
esophagogastroduodenoscopy and gastrointestinal barium radiography are key diagnostic tests for pda , detecting 88.6 and 83.3% of tumors , respectively . as such , they are effective in screening this rare tumor
. however , their location is often inaccessible to endoscopic viewing , which may result in a failure to diagnose them endoscopically [ 2 , 4 ] .
consequently , endoscopy with extra - long fiber optic scopes may be of benefit . in some cases , ultrasonography ( us ) or ct findings have prompted repeat endoscopy with deeper than usual advancement into the 3rd and 4th duodenal portion , which allowed diagnosis in our patient .
lesions appear as irregularly marginated hypoechoic masses , although tumors smaller than 2 cm may not be detected .
thus , us can contribute significantly to the choice of the endoscopic procedure and patient management .
contrast - enhanced ct is also useful for the diagnosis and the determination of malignancy and resectability , with a sensitivity of 94% and a specificity of 82% , although , again , tumors smaller than 2 cm may not be detected [ 2 , 4 ] .
features indicative of malignancy are an exophytic or intramural mass , central necrosis , and ulceration , whereas an entirely intraluminal location indicates a benign tumor . however , these features are sensitive but nonspecific
. vascular encasement , invasion of contiguous organs other than the head of the pancreas , distal lymphadenopathy , or metastasis precludes curative resection .
although not commonly used to date , endoscopic us and magnetic resonance imaging are useful for the diagnosis , staging , and determination of resectability of these tumors .
the preoperative correct diagnosis rate by bronchoscopic ultrasound is 41.7 and 69.4% by ct , 75.0% by magnetic resonance imaging , 84.0% by duodenal endoscopy , and 80.9% by air barium double radiography .
furthermore , patients may have at least one negative gastrointestinal contrast study before a positive result is obtained in a subsequent study .
although early - stage duodenal carcinoma should be considered for endoscopic mucosal resection , mucosal duodenal cancer is extremely rare , and endoscopic submucosal dissection of a lesion in the duodenum requires a high level of skill .
in contrast , endoscopic polypectomy using information from endoscopic us would appear to be a safe and effective method for the diagnosis and treatment of pedunculated early duodenal cancers .
the treatment of choice is radical surgical resection [ 4 , 5 ] , although the optimal surgical procedure remains controversial .
worldwide , there is no general agreement as regards the optimal surgical procedure for the treatment of primary nonampullary adenocarcinoma of the duodenum , especially for early - stage disease .
thus , some authors prefer local excision or segmental resection whereas others perform duodenopancreatic resection , even in the case of early - stage duodenal cancer , in order to avoid tumor recurrence , with pancreaticoduodenectomy being considered the procedure that best satisfies the principles of an adequate curative cancer operation . amongst the surgical procedures available , pancreaticoduodenectomy is recommended for tumors located in the 1st and 2nd portion of the duodenum , and segmental resection may be appropriate for selected patients , especially for tumors of the distal duodenum .
in contrast , duodenocephalopancreatectomy remains the standard treatment for adenocarcinomas of the 1st and 2nd portion of the duodenum .
patients with locally advanced ( n1 ) tumors ( stage iii ) can benefit from this aggressive approach with good long - term results .
some studies have reported good survival results following segmental duodenectomy , despite limited lymphadenectomy occurring during the surgical procedure , and considerably lower postoperative morbidity .
segmental duodenectomy is the preferred resection method for patients with adenocarcinoma of the 3rd and 4th portions of the duodenum . in advanced disease , and particularly in duodenal obstruction , palliative resection , gastrojejunal bypass or duodenal stents may be indicated .
the role of adjuvant chemotherapy is still unclear , with some studies showing important statistical benefits as regards medium- and long - term survival . in light of this evidence
some studies have reported that metastases to lymph nodes , positive resection margins , pancreas invasion , duodenum invasion , and tumor size are prognostic predictors for survival in patients with duodenal adenocarcinoma who underwent surgery [ 4 , 5 , 6 , 11 ] .
in contrast , other studies have reported that tumor size , tumor location within the duodenum , depth of partial penetration , local nodal metastases , tumor differentiation , and invasion of the pancreas were not predictive of prognosis .
the location of the duodenal neoplasia seems to be another factor affecting survival , with tumors of the proximal duodenum appearing to have a worse long - term prognosis since such tumors promote retroperitoneal neoformation . in the absence of lymph node involvement , tumors with a proximal location
have a survival rate of between 0 and 25% 5 years after surgery , whereas this rate is 62% for distal locations .
the worst prognosis for neoplasms in the 1st duodenal portion seems to be due to the close topographic relationship with the surrounding organs , which may be affected rapidly .
the same does not occur , however , for neoplasms in the 3rd and 4th duodenal portion , which have an independent embryological development from the surrounding organs . in light of the above , an aggressive surgical approach that achieves complete tumor resection with negative margins
as regards surgery types , pancreaticoduodenectomy is usually required for cancers of the 1st and 2nd portion of the duodenum , whereas segmental resection may be appropriate for selected patients , especially for tumors of the distal duodenum .
the overall 1- , 3- and 5-year survival rates in two studies were 62.678.1 , 43.743.8 and 18.833.1% , respectively [ 3 , 10 ] , with resected and nonresected patients with pda having 5-year survival rates of 2575 and 0% , respectively [ 3 , 5 , 6 , 10 ] .
similarly , 5-year survival rates for t4 stage and non - t4 stage patients with pda were 20 and 40% , respectively [ 3 , 5 , 6 , 10 ] , and a 5-year survival rate of 100 and 52% for stage i and ii , respectively , and of 45 and 0% for stage iii and iv , respectively , has been reported .
life expectancy after resection of the 1st and 2nd portions of the duodenum is reported to be shorter than that after resection of the 3rd and 4th portions .
the median survival times after resection of the 1st and 2nd portions of the duodenum and after resection of the 3rd and 4th portions were 30 and 46 months , respectively .
the 5-year survival rate for the resection of the 3rd and 4th portions of the duodenum was 58% , whereas that for the resection of the 1st and 2nd portions was 32% . in conclusion ,
having examined our cases and the literature data , and taking into account the rarity of adenocarcinoma of the duodenum , an early diagnosis of this neoplasm is crucial .
a higher degree of suspicion and a more aggressive and persistent investigation should lead to earlier treatment , higher curative resectability rates , and , therefore , better long - term results . in these two cases ,
the patients and their families were not offered an aggressive medical treatment because of the patients age . in general , while fit elderly patients with malignancy can receive the same therapeutic treatment as their younger counterparts , a palliative approach should be considered for frail elderly patients and for those with a short life expectancy . in these two cases , the progress of duodenal cancer did not result in constriction of the duodenal lumen , and both patients were able to eat until end of life .
this is a unique finding given the absence of published studies reporting pda without stenosis .
| this article focuses on the symptomatic and diagnostic problems of primary duodenal adenocarcinoma ( pda ) by way of two case reports and a literature review .
an 85-year - old woman with an adenocarcinoma in the 1st duodenal portion was offered palliative care . a 90-year - old woman with an adenocarcinoma in the 3rd duodenal portion
was also offered palliative care .
a unique finding in the two cases reported herein is that pda did not cause stenosis and occlusion of the lumen .
as no reports of pda without stenosis have been published so far , these cases may add to our knowledge of pda .
the diagnosis of pda is often delayed because its symptoms may be absent until the tumor has progressed , thus leading to a delay of several months .
patients typically present with a long history of variable and vague symptoms , and many are diagnosed with advanced disease .
as regards clinical manifestations , abdominal pain is the most frequent symptom .
the majority of these tumors are found to have infiltrated the duodenal wall at presentation , with many being unresectable due to local and distal invasion .
esophagogastroduodenoscopy and gastrointestinal barium radiography are the main diagnostic tests for pda , detecting 88.6 and 83.3% of tumors , respectively . in some cases , ultrasonography or computed tomography are useful for detecting pda and determining vascular invasion . |
the availability of the mouse genome sequence and advances in genetic manipulation has resulted in multiple initiatives , both in the public and private sector , to produce mouse mutants on a large scale .
coordinated efforts are on way to systematically knock out all mouse genes to provide researchers targeting constructs , vectors , live mice , and phenotypic data at a variety of level and detail .
the relationship between the effect of genetic manipulation and the resulting phenotype is a key component to establishing a fundamental understanding of molecular and cellular process .
parallel to the growth in mouse models to study disease patterns , rapid strides have been made in developing imaging instruments specifically targeted to the small animal .
instruments like mri , pet , spect , ct , in vivo fluorescence , in vivo bioluminescence , and intravital imaging allow one to visually , and sometimes quantitatively , evaluate biological processes at the cellular and subcellular level in a living subject . however , the major challenge in successfully characterizing morphological features of transgenic mouse models has been the trade - off between resolution and field of view .
researchers particularly want the ability to detect gross structural change or , in the absence thereof , localize on subtle variations at microscopic level in 3d in tissue and cell structures .
thus the ideal system used in characterizing transgenics should offer multiscale 3d visualization at a very high native resolution . in this paper
we describe the application of cryo - imaging system and the multiscale approach to characterize a transgenic mouse .
our group has developed a cryo - imaging system , which provides contrast rich , bright field anatomical , and fluorescence cellular and molecular imaging of an entire mouse with micron - scale resolution .
the system acquires three - dimensional ( 3d ) , very high - resolution , large field of view ( fov ) , brightfield anatomy and fluorescence molecular image volumes from sequential images of the tissue block face by alternately sectioning and imaging . in an earlier report , we have demonstrated few applications of cryo - imaging ranging from high - resolution anatomical imaging of whole mouse , vascular imaging , and molecular fluorescence imaging of fluorescent mouse models and embryos .
the system has been used for validation of mr studies of human blood vessel lesions , ex vivo characterization of human atherosclerotic iliac plaque , and very recently for single cell detection of fluorescently labeled cancer and stem cells .
true color enhanced volume rendering techniques provide fast 3d visualizations of cryo - imaged samples .
cryo - imaging is unique among all 3d in vivo ( e.g. , micro - mri ) and microscopic techniques , because it allows micron resolution and information - rich color contrast mechanisms over very large 3d fields of view .
mri imaging of whole mouse can produce gray - scale images after administering contrast agents with significantly less resolution .
optical projection tomography is limited to small samples such as embryos and requires treatments to reduce scatter and increase transparency .
diffuse optical tomography while tackling the light scatter problem with advanced algorithms can image a whole mouse albeit at limited resolution providing little anatomical detail .
johnson et al . used very high - resolution ct and osmium tetroxide staining to image tissues and embryos .
serial histology techniques to create a 3d volume involve serially cutting the sections , applying histological processing , mounting on glass slides , digitizing the slides , and then creating a 3d volume from the two - dimensional ( 2d ) images .
such systems have been used for a variety of biomedical applications [ 13 , 14 ] including characterizing phenotypical change in large histological mouse model datasets .
however , such processing suffers from tears , tissue shrinkage , errors in image alignment , and typically time - consuming manual editing of 3d reconstructions .
systems have been mostly applied for imaging excised organs such as brains [ 16 , 17 ] and embryos [ 18 , 19 ] with adult whole mouse block - face imaging being limited to low - resolution photographs of sections taken for autoradiography studies and ct and block - face imaging for creating a mouse atlas .
we have developed the robotic 3d cryo - imaging system and first applied it to high - resolution tiled imaging of a wild - type mouse .
now we report , for the first time , morphological characterization of a transgenic mouse recently generated by researchers at case western reserve university .
this mouse , dubbed supermouse by popular press , contains a chimeric gene in which the cdna for pepck - c was linked to the -skeletal actin gene promoter , expressing pepck - c in skeletal muscle .
the transgenic pepck - c mice were seven times more active than their controls in their cages , ran for up to 5 km at a speed of 20 m / min on a mouse treadmill ( control animals run for 0.2 km at this speed ) .
as reported earlier , the mice eat almost twice as much as controls but are half the body weight ; they also have an extended life span relative to the controls . in their seminal report
, the researchers have detailed the study of biochemical pathways to better understand the repatterning of energy metabolism .
the battery of tests involved assay of metabolites in blood and tissues , home cage activity testing , exercise capacity testing , respiratory quotient ( rq ) measurements during exercise , and histological and electron microscopy analysis of skeletal muscle .
characterization of the pepck - c mouse with a cryo - imaging , multiscale approach involves detailed high - resolution imaging of a whole mouse and a control mouse , processing and manipulations of enormous giga - byte sized data volumes , and enhanced 3d visualizations . in the following sections we describe the cryo - imaging setup , the imaging techniques , and enhanced visualizations in 3d at multiscale and qualitative and quantitative comparison of organs .
briefly , it consists of a cryo - microtome , a microscope imaging system , a robotic xyz positioner , and a computer control system .
the cryo - microtome is a motorized , large section , whole body cryo - microtome with section thickness adjustable between 240 m and a maximum specimen dimension of 250 mm 110 mm 50 mm .
an xyz robotic positioner carries the imaging system comprising of a stereo microscope , coaxial fluorescent attachment with multiple filter cubes , low light digital camera , and brightfield and fluorescent light sources .
the computer control system automatically pans the positioner over the specimen for a high - resolution tiled image acquisition and controls the sectioning and image acquisition sequence through the custom developed programmable sectioning and cryo - imaging ( prosci ) software .
the image processing and visualization system is a quad - core windows 64-bit pc with 32 gb of ram capable of handling large cryo - image volumes .
a suite of matlab custom programs and custom amira scripts are used for image preprocessing tasks and 3d visualization .
in order to acquire cryo - image data , mice covered under iacuc - approved projects , were euthanized in a method approved by case animal resource center which consists of either inhalation of carbon dioxide delivered from tank or anesthetization using an agent such as pentobarbital , at a dose prescribed by the case arc .
a cryo - embedding compound , oct ( optimal cutting temperature , tissue - tek , terrance , ca ) , was used to completely immerse the mouse in an aluminum foil mould .
the mould was then covered in more foil and placed inside a freezing chamber made of styrofoam and filled with liquid nitrogen .
after about 15 minutes , the mould assembly was removed from the liquid nitrogen bath and placed inside the cryo - microtome chamber to equalize the specimen temperature to the cryo - microtome temperature .
the mould was removed from the specimen after two to three hours and the frozen mouse block was mounted on the microtome stage using more oct . an initial facing - off
then the section thickness was set to 40 m , the imaging system was readied for a pixel size of 15.6 m , and the system was started to alternately slice and acquire tiled brightfield images of the block - face .
the system was run completely unattended with the user periodically alerted by the prosci program about imaging progress through email and cell phone text messages .
details of the transgenic mice are available elsewhere . the transgenic animal used in this study weighed 25.8 gm and was 421 days old when sacrificed .
once the mouse specimen was mounted on the stage , the operator selected the boundaries of the acquisition volume and the system calculated the number of tiles required per block - face taking into account the operator - entered minimum overlap zone between adjacent tiles , typically 15% .
the robotic system computed the coordinates of each tile based on the pixel size and automatically sectioned and imaged the entire volume .
brightfield images ( each 1360 by 1036 pixels ) were acquired in 6 4 tiling pattern to cover each block - face .
a total of 586 sections were needed for the transgenic mouse and 616 for the control . a metadata file stored the global coordinates , imaging parameters , and experiment parameters . following acquisition of 2d images on the imaging workstation ,
nonuniform illumination pattern was compensated using a reference image of a white card and compensated images for each tiled block - face were composited together to yield a typical 2d whole mouse section image of 6500 by 3600 pixels .
these stitched images were then automatically aligned to each other to correct for minor misalignments due to the repositioning error of the stage at the return point .
the total stitched 2d image stacks amounted to a data size of > 60 gb . since such a large dataset can not be loaded fully in the memory of the visualization workstation , we converted the stack of images into a large data access ( lda ) volume inside our customized amira visualization software .
lda allows us to subsample at various resolutions an entire mouse or organ , or to sample the data at full resolution within smaller cropped subvolumes . from a relatively low - resolution 3d mouse
the whole mice were segmented from the oct blocks through our fully automated volume visualization . since
cryo - image data is in color , unlike other gray - scale medical imaging data ( mri , ct , etc . )
we have designed color feature detectors which take into account the dominant color of a region to segment it .
examples of red , green , and blue ratio feature detectors ( fr , fg , fb ) are shown below , where r , g , and b refer to the 8-bit data for red , green , and blue channels , respectively ,
( 1)fr = rr+g+b , fg = gr+g+b , fb = br+g+b .
for instance , regions containing a high proportion of red such as liver , lungs , and vasculature can be extracted using the red feature detector fr . for detecting stomach and intestinal regions , which are predominantly brown in color , we exploited the fact that brown is composed of one part of r , two parts of g , and zero ( 0 ) parts of b. a brown feature detector is therefore expressed as a weighted linear combination of red and green feature detectors :
( 2)fbr=(0.33fr+0.67fg ) .
for segmenting out the embedding compound oct , which has a uniform white color
a balanced color detector was used :
( 3)f=[abs(rg)+abs ( gb)+abs ( rb)]3imax , where imax = 255 .
subsequent to feature detection , opacity transfer functions ( otfs ) were used to assign an appropriate opacity value to each voxel .
below , we show an example of color - based step otf that we have employed in our visualizations :
( 4)={255,f > t,0,ft ,
where t is an empirically determined threshold . in other algorithms
, we have optimized opacity by imposing several simultaneous constraints on an opacity function which maps each voxel to a custom opacity value .
enhanced volume rendering techniques for high - resolution color cryo - imaging data have been described elsewhere .
individual organs such as the heart , brain , kidney , spleen , and glands were delineated using a combination of feature detection for initial automatic segmentation and then manually correcting the results . in organs such as the ovaries , where lack of uniform color and lack of high contrast between neighboring tissues prevented automatic approaches , manual segmentation was carried out in selected sections and 2d interpolation
volumes of organs and tissues were computed from pixel count of 2d segmentation , the 2d pixel dimension and section thickness .
the gross animal weights and brain volumes were used as normalizing factors for comparing organ volumes between the transgenic and the control .
for comparing specific bone lengths such as the femur , digital sections through the axis of the bone were created through interactive multiplanar reformatting of the original 3d data volume .
measurement tools available in the software package were used to compute bone length and cross - sectional areas .
the length of the animal ( nose to base of tail ) was used as the normalizing factor for comparison across animals .
standard tools available in amira were used for volume presentation such as snapshot capture , and movie making .
the image acquisition and multiscale volume visualization is illustrated in figure 1 . from the stitched 2d image stack ( figure 1(a ) ) , a 60 gb 3d volume was created and visualized at multiple resolutions . in the down - sampled whole mouse rendering , major organs were clearly seen through transparent skin ( figure 1(b ) ) .
a bounding box was placed in the abdomen area to extract the region in highest resolution and organs such as the kidneys , adrenal glands , ovaries , and vasculature segmented and reconstructed in 3d ( figure 1(c ) ) .
for example , a section through the left ovary shows the inner medulla consisting of loose connective tissue and large blood vessels and a peripheral cortex region containing a number of ovarian follicles in different stages of development as small as 50 m in diameter .
a few fimbriae of the infundibulum of the uterine tube can also be seen . at this full resolution , these and other many small structures like 50 m villi of the small intestine , mesenteric blood vessels 20 m in diameter , thin walls of the vessels 30 m and skeletal muscle and nerve fibers were very clearly seen .
coronal section images composited from 24 (= 4 6 ) individual tiles are shown in figure 2 .
major organs like the heart , lungs , liver , salivary gland , stomach , spleen , pancreas , small intestine , colon and lymph node are visible .
the very visible peritoneum of the control mouse encases a large amount of visceral adipose tissue as compared to the pepck - c transgenic mouse .
this extraordinary difference in adipose tissue depots is a general finding for these animals . using semiautomatic segmentation techniques , we computed fractional vascularization in the visceral adipose tissue encased by the peritoneum in the lower abdomen area around the cervix .
the gross adipose tissue volume , estimated from voxels in 86 sections , was 38% more in the younger control mouse compared to the pepck - c transgenic mouse .
the percentage of tissue classified as vasculature within this limited adipose tissue mass was higher for the pepck mouse as compared to the control ( 2.5% vascularized in the transgenic animal ; 0.4% in control ) .
the highly vascularized pancreas extending from the spleen to the duodenum can be also seen in both the section images . in figure 3 ,
the dermal layers were distinct with an 83% thinner hypodermis for the transgenic as compared to the control ( 502 mm for control and 84 mm thick for the pepck mouse ) .
the epidermis of the control was 16% thinner than that of the transgenic ( 407 mm transgenic , 348 mm control ) .
the dermis was similar in thickness ( 578 mm transgenic , 621 mm control ) .
the parietal peritoneum , visible below the skin , was also measured and found to be same .
cutaway through the 3d reconstructed hearts of the transgenic and control mice reveal the chambers of the heart , pulmonary trunk , and pulmonary veins .
manual segmentation of the heart from the 2d images also enabled computation of the heart volume .
the transgenic heart was 351.52 cubic mm in volume while the control heart was 366.87 cubic mm .
multiplanar reformatting allowed bone length comparison of femur and tibia of the pepck mouse and the control animal ( figure 5 ) .
the femur measured 11.46 mm ( transgenic ) and 12.41 mm ( control ) while the tibia measured 17.26 mm ( transgenic ) and 15.12 mm ( control ) . the body length from tip of nose to base of tail
was estimated to be 87.6 mm for pepck mouse and 83.8 mm for the control mouse .
when normalized to body length , the measures were similar for femur ( 13% for transgenic and 15% for control ) .
however the tibia was longer for transgenic ( 20% for transgenic and 18% for control ) .
the tibia to femur length ratio was calculated to be 24% higher for transgenic as compared to control .
cortical area was measured at right midtibial and midfemoral diaphysis by extracting digital planes transverse to the bone axes .
tibia cortical area was 0.74 mm for transgenic and 0.71 mm for control .
femora cortical area was 1.35 mm for transgenic and 1.38 mm for control .
the pericardial fat around the heart was segmented from the 2d section images and volume visualized in 3d ( figure 6 ) .
the pericardial fat depots were more pronounced in the control mouse as compared to the pepck mouse which is visible readily in the 3d volume visualization . from the 2d segmentations ,
the major organs like brain , heart , kidney and spleen and glands such as pituitary , thymus and adrenal were segmented and volumes computed . the pericardial fat volume is also included . both absolute values as well normalized values by gross animal weights and brain volumes are presented .
the high - resolution color data volumes from the whole mouse that were obtained using the case cryo - imaging system and multiscale viewing provide researchers the ability to seamlessly bridge the requirements of resolution and fov .
most existing small animal imaging systems force the researchers either to scan the mouse at low - resolution to capture the entire animal in a single fov or to choose only a region of the whole mouse for investigation at higher - resolution .
as we have shown in this application , with cryo - imaging , image data is acquired at a very high - resolution , and using multiscale viewing , it is possible to view in 3d or 2d at the whole body level , zoom to a region at higher - resolution , zoom to an organ , and finally zoom to examine tissues at the highest resolution .
skeletal muscle fibers in the mouse , typically 45 m in diameter , were seen very clearly in cryo - images . in earlier work involving fluorescently labeled stem and cancer cells , we showed that one can zoom down to single cell level .
the multiscale approach provides an efficient way of handling extremely large whole mouse data sets . a tiled color bright field acquisition with an adult whole mouse using a 6 4 tiling configuration and 40 m section thickness generates > 60 gb of color image data which is a prohibitively large size for volume rendering on a workstation with a conventional , single graphics processor .
if fluorescence acquisition is added , it imposes an even greater burden due to increased data size . as a remedy to the extreme data problem , we designed the multiscale volume rendering approach which greatly improves the visualization experience .
. some of the important criteria to consider during multiresolution volume rendering are data access time and graphics hardware limitations on the target machine .
data access time can be greatly reduced by employing modern solid state disk ( ssd ) hard drives , which generally have higher data read and write speeds as compared to conventional hard drives . as for graphics hardware , the larger the amount of graphics ram , the smaller the decimation that needs to be applied to produce high - resolution renderings of subregions within the large data sets .
first , it offers a convenient method of scanning an entire mouse since no special preparation involving stains are required .
second , a low - resolution view of the entire mouse with volume visualization of internal organs is available through automatic segmentation of oct and automatic 3d rendering .
third , the organs and tissues are displayed in true color providing a virtual necropsy experience .
fourth , data can be seamlessly explored at every level through user - friendly select - and - view bounding boxes .
semi - automatic segmentation through algorithmic extraction of features followed by interactive manual editing provides visualization and quantification options .
while this application has only utilized brightfield imaging , the system is capable of obtaining molecular fluorescence image data as well , thereby rendering unique versatility to the system .
the featuresets of the system make it useful for applications such as anatomical phenotyping . a particularly interesting approach for identifying mutant outliers was described by kovacevic et al .
where an annotated 3d atlas of the average mouse brain with estimates of average and variability was created from mri images , against which mutants could be compared .
given the ability to segment organs in color , cryo - imaging will be well suited for such analyses for transgenic mice .
the published work on pepck - c transgenic mouse contained histology of excised skeletal muscle tissue and low - resolution whole body mr imaging .
cryo - imaging of this transgenic strain and its control provides the first glimpse at characterizing some of the morphological difference between the pair .
the high amount of total body fat in the control , clearly visible in the 2d sections , is consistent with the published mr results .
however , unlike mri , the high - resolution of cryo - imaging allows distinction between various fat depots and color differences . as an example , we computed and compared the pericardial fat volumes and fractional vascularization in a region of the visceral fat depot in the abdomen .
we were also able to detect various organs and separate the dermal layers similar to histology analysis .
it has been observed that these transgenic mice live longer and are more energetic at an older age .
we chose regions and organs of the two mice to compare few standard ageing biomarkers .
we note that the older pepck mouse has less pericardial fat , less visceral and subcutaneous fat , thinner hypodermis and thicker epidermis as compared to the control animal .
skin thickness measurements are widely accepted as a biomarker of ageing while visceral and pericardial fat deposits are biomarkers of potential metabolic disorders .
the thymus and spleen were chosen as ageing biomarkers since the thymus is a primary lymphoid organ considered to be consistently sensitive to morphological effects and the spleen is a secondary lymphoid organ .
characterization of thymic and splenic weights in age - related studies in nonhuman primates has shown that the thymus becomes smaller with age while the splenic changes were not statistically significant . in this study
, we found the older pepck - c mouse has a larger thymus while the spleen is smaller than noted with the control animal .
we also noted a difference in overall tissue coloration between the pepck mouse and control . in our experience from cryo - imaging wild type mice ,
younger mice have an overall brighter tissue color . in this regard , the pepck - c mouse looks
adipose tissue is pinker , consistent with our observation of increased vascularity . through observations such as these , cryo - imaging can point researchers to examine certain tissues in more detail .
a careful 3d volume measurement was carried out to compare the heart volume of this mouse with the control . while the absolute cubic volume was nearly the same , the transgenic heart volume was 40% greater when normalized by body weight .
when normalized by brain volume this difference reduced . since cardiac volume estimates derived from the cryo - images
are not correlated with the cardiac phase at the time of freezing , we estimated another measure of comparison .
voxels belonging to the blood pool in the cardiac chambers and vessels in the hearts were semiautomatically identified through the color detector scheme and were excluded from cardiac volume measurement .
the resulting cardiac tissue mass , when normalized by body weight , was found to be 22% more for pepck - c mouse than control .
comparison of other organ volumes such as brain and kidney and glands such as pituitary and adrenal showed a general trend of larger organs for the pepck - c mouse . in order to reduce the variability due to animal size and weight ,
organ volume - to - animal weights were computed . to reduce the variability due to lean body mass , organ - to - brain volume normalization was used . in the case of bone lengths ,
digital resectioning along arbitrary planes allowed bone morphometric data to be computed from the cryo - image volume .
measurements such as length and cortical area for tibia and femur were made for two comparisons .
it has been reported that short - term induced exercise in mice leads to differences in bone properties such as length and cortical area for the femur and tibia [ 2628 ] .
further , such changes have also been recorded in ageing studies [ 29 , 30 ] .
the pepck - c mice are highly active and can endure sustained activity at maximal speed like running on mouse treadmills .
the particular mouse we cryo - imaged had participated in numerous unrelated high - activity studies and was chronologically older than the control mouse . although no longitudinal study was performed on either specimen , nonetheless , we were interested in comparing the normalized values .
the pepck - c mouse had a slightly higher value for tibia and lower for femora leading to a significantly higher tibia to femur ratio of 1.5 .
the ratio of 1.2 for the control mouse is similar to ratios derived from published reports .
the ratio of cortical area to bone length is considered to be indicative of bone growth .
this ratio was similar for both the femora and tibia for both animals perhaps suggesting that ageing does not significantly deteriorate bone biomechanics for the transgenic . in this study
, we have characterized the pepck - c supermouse and demonstrated that the case 3d cryo - imaging system can be adopted as a morphological phenotypic screen for transgenic animals .
the multiscale approach offers researchers tissue information at the most appropriate resolution traversing down the scale from mouse to organ to tissue to single cells .
the ability of being able to visualize structures in 3d and extract 2d sections along any arbitrary plane makes comparisons easy and focused .
further , visualizations do not involve laborious manual segmentation and in most cases are achieved semiautomatically through the novel color feature detectors .
the results of such phenotyping effort will provide a roadmap for adopting cryo - imaging for cataloging the plethora of transgenic mouse models . | we have developed , for the case 3d cryo - imaging system , a specialized , multiscale visualization scheme which provides color - rich volume rendering and multiplanar reformatting enabling one to visualize an entire mouse and zoom in to organ , tissue , and microscopic scales . with this system , we have anatomically characterized , in 3d , from whole animal to tissue level , a transgenic mouse and compared it with its control . the transgenic mouse overexpresses the cytosolic form of phosphoenolpyruvate carboxykinase ( pepck - c ) in its skeletal muscle and is capable of greatly enhanced physical endurance and has a longer life - span and reproductive life as compared to control animals .
we semiautomatically analyzed selected organs such as kidney , heart , adrenal gland , spleen , and ovaries and found comparatively enlarged heart , much less visceral , subcutaneous , and pericardial adipose tissue , and higher tibia - to - femur ratio in the transgenic animal .
microscopically , individual skeletal muscle fibers , fine mesenteric blood vessels , and intestinal villi , among others , were clearly seen . |
graves disease is a common autoimmune condition and is commonly associated with other autoimmune conditions such as myasthenia gravis , addison disease , type 1 diabetes mellitus , and vitiligo .
there is also a rare but well - documented association between graves disease and thymic hyperplasia .
clinicians should be aware of the usually benign clinical course of thymic hyperplasia associated with graves disease .
a 30-year - old male presented with intermittent fever , weight loss , and dry cough of 2 months duration .
he presented to our center as a case of pyrexia of unknown origin ( puo ) .
on enquiry , he also gave history of weight loss of 10 kg ( weight 56 kg ) over 2 months despite increased appetite , excessive sweating , restlessness , increased anxiety , and frequent episodes of diarrhea . on evaluation , he was found to have thyromegaly and other features of hyperthyroidism such as digital tremors , warm moist palms , tachycardia ( 110 - 120/min ) , blood pressure 140/68 , eye signs such as exophthalmos , lid lag , staring look with cardiovascular , and other systemic examinations being normal .
his thyroid function tests were suggestive of hyperthyroidism ( t3 2.4 ng / ml , t4 19 g / dl , and tsh 0.09 iu / ml ) .
a cect chest done ( as a part of puo workup ) showed an anterior mediastinal mass ( 5.6 2.1 6.0 cm ) with mild contrast enhancement , suggestive of a thymic mass [ figure 1 ] . in view of close proximity of the mediastinal mass to the great vessels and other important structures in mediastinum
cect scan chest of the patient showing a 5.6 2.1 6.0 cm mass in the anterior mediastinum suggestive of thymic hyperplasia patient was started on carbimazole 20 mg bd , propranolol 20 mg tds , and supportive therapy .
he gradually improved , became afebrile , gained 5 kg weight over the next 12 weeks and had gradual regression of clinical features of hyperthyroidism .
repeat cect scan chest at 12 weeks after initiation of atd showed regression in the size of the mass ( 2.4 1.2 3.0 cm ) [ figure 2 ] . presently , the patient is clinically and biochemically euthyroid .
repeat cect scan chest of the patient after treatment with antithyroid drugs showing regression in size of the thymus ( 2.4 1.2 3.0 cm )
the association between graves disease and thymic hyperplasia was first recognized in 1929 by hammar .
thymic involvement may range from microscopic abnormalities in the thymus consisting of medullary lymphoid follicles to massive enlargement of the thymus .
earlier literature suggested that the hyperplastic thymus played an etiologic role in graves disease as it does in myasthenia gravis by playing a central role in determining self versus non - self recognition by t cells .
however , most patients with graves disease do not have obvious immune dysfunction except predisposition to autoimmunity .
murakami et al . studied 23 untreated patients with graves disease and 38 control subjects and showed the presence of thyrotropin receptor in non - neoplastic thymic tissue by polymerase chain reaction amplification and northern and western blot analysis indicating that thymic thyrotropin receptor may be involved in developing thymic hyperplasia in patients with graves disease by serving as an autoantigen .
treatment with antithyroid drugs ( atds ) can induce rapid , although often incomplete , thymic involution .
murakami et al . studied thymic size and density in 23 untreated patients with graves disease with use of computerized tomography . in comparison with 38 age - matched controls , both thymic size and density were higher in patients with graves disease .
after treatment with atds , both thymic size ( 852 245 mm before and 402 280 mm after treatment ; p < 0.001 ) and density ( 9.1 50.7 hounsfield unit before and 35.5 36.3 hounsfield unit after treatment ; p < 0.01 ) were significantly reduced . the decrease in thymic size and density by atd
could be produced , partly due to decreased circulating thyroid hormone levels by atd and also by immunosuppressive effects of the drugs .
another study involving three patients with graves disease and marked thymic enlargement showed a regression in thymic volumes by 72% and 78% in two of the three patients who presented for follow - up and whose initial volumes were 67 , 81 , and 54 cm .
an association between thymic hyperplasia and graves disease seems well established , but the precise pathophysiology of thymic hyperplasia in graves disease has not yet been determined .
however , treatment of graves disease leads to regression of thymic hyperplasia as was seen in our patient .
the other differential diagnosis of an anterior mediastinal mass which have to be differentiated from thymic mass are lymphomas , germ cell tumors , congenital cysts , intrathoracic thyroid tissue , and parathyroid lesions which may require histological diagnosis for a confirmation .
about 91 cases of thymus hyperplasia and graves disease association have been reported in the literature , of which about 20 were histologically confirmed . among these 91 cases ,
accordingly , surgical procedures are most frequently unnecessary in such associations because of the thymic mass decrease incurred by atd treatment . in our case
too as the thymic enlargement showed a significant reduction in size with antithyroid drugs , any surgical procedure was not planned .
it is important for the clinician to recognize this benign association between thymic hyperplasia and graves disease , and its regression with atd which can spare patients from a major surgical procedure . | graves disease is an autoimmune thyroid condition characterized by the production of autoantibodies against the thyrotropin receptor .
it is known to be associated with autoimmune conditions such as myasthenia gravis , addison 's disease , type 1 diabetes mellitus , and vitiligo .
we present a case of rare autoimmune association of graves disease with thymic hyperplasia which regressed after treatment with antithyroid drugs .
exact pathophysiology of thymic hyperplasia in graves is not well understood ; it is likely to be the result of rather than the cause of graves disease . |
the practice of modern pediatric dentistry requires delivery of quality care in combination with adherence to excellent business and management principles .
dentists traditionally trained in the art and science of dentistry must also become skilled in techniques of sound business management . today 's pediatric dental practitioner should be clinically astute and knowledgeable about consumer needs and demands , government regulations , third party participation , and so forth , aspects which may not have been taught in a dental school .
the primary responsibility of any dentist is to provide quality oral and dental health care to the patient .
however , private practice , like any other business , must make a profit to survive .
the dental practitioner thus should maintain a balance between patient care and business requirements while keeping moral , ethical , legal , and professional responsibilities in proper perspective [ 2 , 3 ] . while maintaining this balance
a pediatric dentist must provide a place where children can feel safe , loved , and well cared for , where parents can be educated about how to help their children have a lifetime of good oral health , where the staff know that they are an integral part of the practice and their opinions are counted for and where the community as a whole knows , respects , and appreciates the practice .
pediatric dentistry is a unique specialty of dentistry as the patient , in the said case a child patient , can not come alone to the dental clinic for his treatment but has to be brought by the parents who seek dental care for their child .
hence , the schedule of appointments and its related processes have to be discussed with the parents .
the parents of children could have different attitudes like overprotective , overindulgent , authoritarian , underaffectionate , rejecting , overanxious , and so forth .
the manner in which the attitudes of parents ( or patients ) affect the appointment scheduling systems is a matter of further research . to guide the parents into choosing the most suited appointment ,
the receptionist should be prepared with the relevant information to justify the time of scheduling .
the length of the appointment should be as short as possible ( not greater than 30 minutes ) .
the children should not be made to wait too long in the waiting room as they tend to get restless with passing time .
a definite schedule , preferably designed in advance for individual patients , based on the magnitude of the dental problem and its subsequent management should be presented to the parents on the first or second appointment .
more importantly , the prescribed schedule should be followed to the best of the professional abilities of the pediatric dentist .
studies have been conducted over the world in an effort to devise and study patient appointment scheduling systems and the effectiveness of these systems in improving the quality of the practice as also the level of patient satisfaction .
taylor studied 639 patients who attended a practice whose surgeries are run on a mixed system of open access and by appointment .
he concluded that the number of practices that run appointment systems has sharply increased ; however such practices may have difficulty in fitting in urgent or extra consultations .
campbell studied the relationship between ( i ) measures of how appointment systems work , ( ii ) patients ' views of the arrangements for seeing their general practitioner , and ( iii ) practice self - referral rates to accident and emergency departments .
these measures of appointment system operation correlated with patient dissatisfaction with the arrangements of seeing a doctor in their practice and with the perceived availability of a doctor to deal with nonurgent problems .
studied a total of 450 children between 3 and 9 years of age to evaluate the age - specific effect of treatment duration on pediatric patient behavior .
the study concluded that treatment duration may affect the behavior of pediatric patients parallel with chronological age and , thus , should be considered in the arrangement of the treatment plan .
the findings of this study suggest appropriate pediatric behavior management should include thoughtful scheduling of appointments according to a treatment plan formulated with consideration of the effects of age and appointment length .
thus appointment scheduling systems lie at the intersection of efficiency and timely access to health services .
the goal of a well - designed appointment system is to deliver timely and convenient access to health services for all patients .
appointment systems also ensure smooth work flow , reduce crowding in waiting rooms , and allow health systems to honour patient and provider preferences while matching supply and demand .
all these factors go a long way in promoting patient satisfaction , at the same time ensuring a high quality of dental care to the child . with these objectives in mind , a study was carried outin the department of pediatric and preventive dentistry , ymt dental college & hospital , with the following aims and objectives .
aim . to assess the co - relation between appointments scheduling and overall patient satisfaction in a pediatric dental setup
.
objective . to understand the parameters related to appointment scheduling and to incorporate necessary changes , if any , to increase patient satisfaction .
the study was carried out in the department of pedodontics and preventive dentistry , ymt dental college & hospital , kharghar , navi mumbai .
a total of 40 patients , ages ranging from 8 to 12 years , who visited the department of pediatric and preventive dentistry , ymt dental college & hospital , for dental treatment were selected on a random basis
. only those patients and their parents were included who had reached or were in the process of reaching the completion of their proposed treatment . to further standardise the process of appointment scheduling and related parameters of patient satisfaction and eliminate bias ,
only those patients under treatment by postgraduate students ( already have completed graduation and hence are registered dental practitioners ) were included as appointments for the said patients were given by the postgraduates themselves and not by the support staff of the department .
necessary permissions were obtained from the institution and consent of the participants was taken prior to the start of the study . a questionnaire with a set of 6 questions with a likert rating scale of ( 1)(5 ) ( the appendix ) to assess the patient satisfaction related to appointment scheduling was prepared .
the parents of the selected patients were interviewed as the patients assessed belonged to the pediatric age group who could not possibly analyse and fill the questionnaire .
the parents of the selected children were explained the meaning of the questions and the criteria of rating .
subsequently , they were asked to fill in the questionnaire and instructed to give the most accurate rating to the prescribed set of questions .
further , they were instructed to fill out the questionnaire in isolation of the operator or another parent to avoid any bias in rating the answers to the questions .
the parents were instructed to abstain from writing their names or putting their signatures on the questionnaire forms in an effort to protect their identity .
the parents were encouraged to give their valuable comments / suggestions for improving the services provided by the department ( in relation to appointment scheduling ) as regards ( q. 1 ) to ( q. 5 ) .
spearman 's correlation coefficient was used for correlating the individual experience with the overall patient satisfaction .
table 1 shows the ratings of the questionnaires that were obtained from the parents of the patients . the average rating for ( q. 1 ) that was related to the procedure of appointment procurement was 3.675 which indicates that the majority of the patients were neutral ( neither happy nor unhappy ) , with inclination towards being happy with the procedure . for ( q. 2 ) that was related to the number of appointments for the procedure , the average rating was 3.45 which indicated that majority of the patients were inclined towards a neutral ( neither happy nor unhappy ) rating . for ( q. 3 ) that was related to the duration of each appointment , the rating of 3.725 indicated that majority of the patients were happy with the same . for ( q. 4 ) that was related to the following of the prescribed schedule , the rating was 3.675 that indicated that
for ( q. 5 ) that was related to waiting experience , a rating of 3.5 was indicative of neutral to happy feeling of the patients towards this parameter . for ( q. 6 ) that was related to overall satisfaction , a rating of 3.775 indicated that there was a degree of happiness towards the entire process of appointment systems .
table 2 shows the distribution of patient satisfaction related to the parameters of questionnaire .
table 3 depicts the association of the overall experience of the patient with the experience of getting the appointment .
there was a statistically significant association between the experience of the patient and the experience of getting the appointment .
table 4 and figure 2 show the association of the overall experience of the patient with the number of appointments .
there was a statistically significant association between the experience of the patient and the number of appointments .
table 5 and figure 3 show the association of the overall experience of the patient with the duration of the appointments .
there was a statistically significant association between the experience of the patient and the duration of appointments .
table 6 depicts the association of the overall experience of the patient with the appointment schedule .
there was a statistically significant association between the experience of the patient and the appointment schedule .
table 7 and figure 5 show the association of the overall experience of the patient with the waiting experience .
there was a statistically significant association between the experience of the patient and the waiting experience .
this is mandatory to deliver quality health care in an effective and efficient manner to the child patients .
in addition , the scheduling of an appointment and the following of the schedule for a child patient is directly correlated to the behavior management plan parallel with chronological age and , thus , should be considered while devising and presenting the treatment plan .
the world over , various systems have been devised and studied to improve the appointment scheduling systems so as to render quality treatment to patients . most of these systems have been studied in reference to the various branches of medicine , but there is a paucity of literature related to pediatric dentistry . most of these systems have concluded that though appointment scheduling does help to improve the quality of health care delivery , not all the systems may be patient friendly and patient satisfaction with these systems varies [ 12 , 13 , 15 , 16 ] .
in our study we found that a majority of the patients were happy with the existent appointment system that was being followed in the department of pediatric dentistry , ymt dental college & hospital , where the present study was carried out .
however , there was a section of the parents who were not at all happy with the existent appointment system , especially related to the process of procuring the appointment and the duration of each appointment ( tables 2 , 3 , and 5 ) .
this unhappiness could possibly be related to not getting the appointments of their choice , which may not be possible every time in a pediatric dental setup owing especially to the fact that the timings of the dental hospital ( where the study was conducted ) entail the child to miss his school .
the unhappiness could also be linked to operator variation , especially related to duration of the appointments , as each operator may not possess the same competency as another operator , who may be more efficient at time management . also ,
in a typical pediatric dental setup , the duration of the appointment can not be kept above a particular duration ( ideal not greater than 30 min ) which could be resented by certain patients , considering the fact that they may have travelled a considerable distance to reach the hospital .
it is also important to note that a number of patients also were unhappy with the following of the appointment schedule ( tables 2 and 6 ) , which could be totally related to the operator being less competent or due to the absence of a particular operator on the day the appointment was scheduled .
many patients chose to remain neutral on the issues of number of appointments , the duration of the appointments , the following of the schedule of appointments , and the waiting experience ( table 2 ) .
this indicated that if the department can incorporate improvements in all aspects of the appointment scheduling systems it will definitely help improve the rating of the overall experience and could elevate this class of patients from the neutral category to the happy or very happy category .
it was also observed that the difference between the happy patients and neutral patients related to parameters like number of appointments , appointment schedule implementation , and waiting experience was not very high ( table 2 ) . in our study ,
an assessment of the descriptions provided by the parents under the subheading of any other comments / suggestions to improve the services provided as regards appointment systems showed that most of the parents were most concerned about the number of appointments and the average waiting time . some parents suggested that improving the infrastructure in the children 's waiting area and reducing the number of appointments would attract more patients to the hospital .
it is probably these areas on which the department should toil so that the level of patient satisfaction could be increased .
the happy overall experience could be attributed to the fact that the appointments were being given by postgraduates themselves under the supervision of the staff .
also , the treatment was being done by individuals who had received adequate training in the subject of pediatric dentistry .
in addition , the treatment plan for each patient was thoroughly discussed and approved by the staff , thereby leading to more efficient delivery and completion of the treatment . however , reduction of the number of appointments to a bare minimum may not be possible given the differences in the nature of oral and dental diseases and differences in the behavioural temperament of each child .
the department should aim at incorporating changes in the existing appointment scheduling to convert that category of patients who were unhappy or neutral with the appointment scheduling system to the happy or extremely happy category .
standardization of the procedure , increasing the competency of all the operators , minimizing the waiting period , extending the working hours so that the child may not miss school , and improving the infrastructure of the department , especially the waiting area , are the areas where the department can concentrate on achieving the above mentioned aim .
more research on appointment scheduling using different parameters like age , gender , occupation of parents , and so forth and their correlation with parent satisfaction needs to be conducted .
the real challenge would be to provide maximum possible levels of satisfaction to the patients in relation to all aspects of the health care delivery system . | introduction .
the practice of modern pediatric dentistry requires delivery of quality care in combination with adherence to excellent business as well as time management principles .
a definite appointment schedule should be presented to the parents on the first or second appointment .
more importantly , the prescribed schedule should be followed to the best of the professional abilities of the pediatric dentist . aims .
the aim of the study was to assess the co - relation between appointment scheduling and patient satisfaction in a pediatric dental setup with the objective of understanding the parameters related to appointment scheduling to increase patient satisfaction .
method .
a total of 40 patients , who visited the department of pediatric and preventive dentistry , ymt dental college & hospital , for dental treatment were selected on a random basis . a questionnaire with a set of 6 questions with a rating scale of 15 to assess the patient satisfaction related to appointment scheduling was prepared .
results .
a significant number of the patients were happy with the existing appointment scheduling system barring a few exceptions . |
endoscopic mucosal resection ( emr ) is widely used in western centers for the treatment of non - pedunculated colorectal adenomas > 2 cm in size .
colonic endoscopic submucosal dissection ( esd ) for the treatment of these adenomas is rarely performed outside of japan and korea .
emr is associated with a low risk of adverse events , rapid procedure times , and relatively low technical complexity ; it is also suitable for outpatient treatment as patients generally only need to be observed for a few hours after the procedure and can go home the same day .
esd is technically complex , involves a long procedure time , and is associated with a significant risk of intraprocedure and delayed colon perforation ; patients require hospitalization for observation after the procedure .
the main advantage of esd is the excellent rate of en bloc resection , which reduces local recurrence rates and ensures precise histopathologic staging if malignancy is detected .
however , advances in endoscopic optics have improved the visual assessment of malignancy , and improvements in endoscopic techniques have significantly reduced the local recurrence rate after emr and facilitated the treatment of recurrences .
emr is therefore likely to continue as the widely used technique for the treatment of large colorectal adenomas in the west .
multiple large observational studies have documented the safety and efficacy of emr for the treatment of non - pedunculated colorectal adenomas > 2 cm in size .
the prospective multicenter australian colonic emr ( ace ) study demonstrated an intention - to - treat rate of 84% of successful emrs with a mean procedure duration of 25 minutes .
submucosal invasive cancer was found in 6.9% of the cases and was generally treated with surgery .
the adverse event rate was 7.7% : 2.1% of the cases required overnight hospitalization for post - procedure pain , 1.5% required a 3 to 5 day hospitalization and antibiotics for serositis , 2.9% required hospitalization for post - procedure bleeding , and 1.3% developed a perforation . a subsequent publication that included more patients and
more extensive follow - up demonstrated a local recurrence rate of 16% at 4-month follow - up , with an additional 4% recurrence rate found at 16 months .
risk factors associated with recurrence include lesion size > 40 mm ; use of argon plasma to ablate residual adenoma not amenable to snare resection ; and prior manipulations leading to fibrosis , such as partial snare polypectomy , tattoo application at the site of the lesion , or aggressive biopsies of the lesion [ 3 - 5 ] . increased experience and improved endoscopic imaging using higher resolution cameras , dual focus design allowing closer inspection , and proprietary imaging techniques ( narrow band imaging , blue laser imaging , and iscan ) show significant promise in improving the endoscopic differentiation of benign and malignant colorectal lesions .
this may influence treatment planning , as piecemeal resection with emr is generally considered acceptable for benign adenomas , but en bloc resection should be performed for malignancies .
avulsion , a newly described technique utilizing hot biopsy forceps with cutting current , enables complete removal of many fibrotic islands of residual adenoma encountered during emr . in a comparative analysis involving argon plasma coagulation ,
the use of avulsion resulted in significantly higher rates of complete removal of all visible neoplasia during emr and markedly reduced recurrence rates .
thermal ablation of the margin of the resection site after emr can potentially reduce the incidence of recurrence by ablating microscopic areas of non - visible residual adenoma .
preliminary results from a multicenter randomized study presented in abstract form in 2016 demonstrated a reduction in adenoma recurrence from 20% to 6% with thermal ablation of the margin .
underwater emr is another recently described technique in which colorectal adenomas are removed using a snare underwater , without submucosal injection .
underwater emr is particularly well suited for non - lifting lesions with fibrosis from prior incomplete resection attempts or other manipulations . in this setting , it increases the complete resection rate of visible neoplasia and reduces recurrence .
successful underwater en bloc snare resection of 2- to 4-cm large adenomas has also been reported , challenging the conventional approach of avoiding en bloc snare resection of lesions > 2 cm due to the fear of perforation .
if ongoing studies confirm the excellent efficacy and safety profile of this initial report , underwater en bloc resection may become more widely adopted .
multiple large observational studies have documented the safety and efficacy of emr for the treatment of non - pedunculated colorectal adenomas > 2 cm in size .
the prospective multicenter australian colonic emr ( ace ) study demonstrated an intention - to - treat rate of 84% of successful emrs with a mean procedure duration of 25 minutes .
submucosal invasive cancer was found in 6.9% of the cases and was generally treated with surgery .
the adverse event rate was 7.7% : 2.1% of the cases required overnight hospitalization for post - procedure pain , 1.5% required a 3 to 5 day hospitalization and antibiotics for serositis , 2.9% required hospitalization for post - procedure bleeding , and 1.3% developed a perforation . a subsequent publication that included more patients and
more extensive follow - up demonstrated a local recurrence rate of 16% at 4-month follow - up , with an additional 4% recurrence rate found at 16 months .
risk factors associated with recurrence include lesion size > 40 mm ; use of argon plasma to ablate residual adenoma not amenable to snare resection ; and prior manipulations leading to fibrosis , such as partial snare polypectomy , tattoo application at the site of the lesion , or aggressive biopsies of the lesion [ 3 - 5 ] .
increased experience and improved endoscopic imaging using higher resolution cameras , dual focus design allowing closer inspection , and proprietary imaging techniques ( narrow band imaging , blue laser imaging , and iscan ) show significant promise in improving the endoscopic differentiation of benign and malignant colorectal lesions .
this may influence treatment planning , as piecemeal resection with emr is generally considered acceptable for benign adenomas , but en bloc resection should be performed for malignancies .
avulsion , a newly described technique utilizing hot biopsy forceps with cutting current , enables complete removal of many fibrotic islands of residual adenoma encountered during emr . in a comparative analysis involving argon plasma coagulation ,
the use of avulsion resulted in significantly higher rates of complete removal of all visible neoplasia during emr and markedly reduced recurrence rates .
thermal ablation of the margin of the resection site after emr can potentially reduce the incidence of recurrence by ablating microscopic areas of non - visible residual adenoma .
preliminary results from a multicenter randomized study presented in abstract form in 2016 demonstrated a reduction in adenoma recurrence from 20% to 6% with thermal ablation of the margin .
underwater emr is another recently described technique in which colorectal adenomas are removed using a snare underwater , without submucosal injection .
underwater emr is particularly well suited for non - lifting lesions with fibrosis from prior incomplete resection attempts or other manipulations . in this
successful underwater en bloc snare resection of 2- to 4-cm large adenomas has also been reported , challenging the conventional approach of avoiding en bloc snare resection of lesions > 2 cm due to the fear of perforation . if ongoing studies confirm the excellent efficacy and safety profile of this initial report , underwater en bloc resection may become more widely adopted .
the majority of recurrences after emr are unifocal and < 5 mm in size . over 90% of the recurrences
a second recurrence is found in 10% to 40% of all patients after treatment of the first recurrence .
underwater emr appears to be superior to conventional emr for treating recurrences , with a higher rate of complete removal of visible neoplasia and a lower second recurrence rate .
even lesions that recur two or more times can be treated successfully with additional emr or underwater emr .
treatment of recurrences with emr or underwater emr appears to be as safe as the treatment of naive lesions , with a low risk of bleeding or perforation .
in expert centers , esd enables complete en bloc resection of > 85% of all colorectal adenomas and superficial cancers .
in contrast , en bloc resection rates for colorectal adenomas > 2 cm in size are typically 25% or less with emr .
adenoma recurrence is significantly more common with emr than with esd : recurrence rates for adenomas > 2 cm in size treated with emr are typically approximately 16% in western centers and 7% in japanese centers , while recurrence rates with esd are 1.4% in japan .
however , esd requires significantly longer time to perform than emr : the mean procedure time for emr in a large western study was 25 minutes while that for esd in a large japanese study was 116 minutes . in the same two studies , perforation occurred in 1.3% of the patients undergoing emr and in 4.9% of the patients undergoing esd .
hospitalization was required in < 10% of the western patients who underwent emr , while 100% of japanese patients undergoing colorectal esd are typically hospitalized for > 3 days . while piecemeal emr of adenomas results in good clinical outcomes in most patients , en bloc resection with esd is preferable for cancers with slight submucosal invasion and favorable histologic features , as it provides curative endoscopic treatment without the need for surgery .
piecemeal resection with emr may reduce the accuracy of histologic analysis and may lead to poor oncologic outcomes due to local recurrence . with current technology ,
endoscopic differentiation between adenoma and cancer is only 70% to 90% accurate , and the accuracy of differentiating between slight and deep submucosal invasion is only 70% to 80% ; therefore , precise histologic staging with an en bloc resection using esd is preferable for lesions that have a significant likelihood of being invasive cancers .
emr is safe and effective for treating colorectal adenomas . compared with esd , emr is significantly faster to perform , has a much lower perforation rate , and does not require post - procedure hospitalization .
advances in emr techniques have led to reduced local recurrence rates , and the modern treatment of recurrences is technically simple , safe , and usually effective .
while esd in expert centers still offers a superior technical result and lower recurrence rate , the procedural complexity , additional unreimbursed procedure time , perforation risk , and requirement of post - procedure hospitalization make it unlikely to be widely adopted in the west for treatment of colorectal adenomas .
improvements in endoscopic visual assessment of early submucosal invasive cancer may enable identification of a limited number of patients who may be suitable candidates for esd in western centers , if additional data showing safety and long - term efficacy comparable to surgery become available . | to discuss the rationale for the widespread application of endoscopic mucosal resection ( emr ) rather than endoscopic submucosal dissection ( esd ) in western centers . in western centers ,
emr is the treatment of choice for most non - pedunculated colorectal adenomas > 2 cm in size .
emr is sufficiently effective and safe to be performed without post - procedure hospitalization .
advances in emr have led to reduced recurrence rates , and recent studies have demonstrated excellent outcomes with endoscopic treatment of recurrent adenomas . while studies from asia have demonstrated lower recurrence rates with esd , concern about the higher perforation risk and lengthy procedure time of esd are two of the barriers preventing widespread adoption of esd in the west .
emr is likely to continue as the dominant method for the treatment of large colorectal adenomas in western centers until the limitations of esd are overcome . |
no definitive treatment has been established for bleeding vesical varices . in symptomatic vesical varices , surgical devascularization , laser sclerosis , and coagulation
are often only temporarily effective . as another treatment option , percutaneous transheapatic obliteration ( 1 ) and endoscopic injection of histoacryl could be performed .
we report a male patient who had a history of bladder substitution with ileal segments and had been treated by balloon - occluded percutaneous transhepatic obliteration of vesical varices that presented with recurrent gross hematuria .
a 50-year - old male presented to the ed with recurrent episodes of gross hematuria .
30 years ago , he was admitted to our hospital and underwent left nephrectomy and orthotopic bladder substitution with ileal segments after radical cystectomy due to renal tuberculosis in the left kidney .
this patient was readmitted in october 2009 with gross hematuria and underwent transurethral cystoscopic coagulation of varices in the urinary bladder ( fig .
he was initially treated with saline bladder irrigation and blood transfusion . in view of his history of recurrent gross hematuria , a contrast enhanced computed tomography ( cect ,
siemens medical systems , erlangen , germany ) scan of the abdomen and pelvis was performed and showed dilated blood vessels along the right side of the bladder wall with drainage into the superior mesenteric vein ( smv ) .
arterial portographies via the superior mesenteric artery showed dilated veins draining into the smv in the right pelvic area ( fig .
percutaneous transhepatic portography ( ptp ) was performed by catheterization of the right anterior branch of the intrahepatic portal vein .
the 10 mm diameter balloon catheter with a length of 40 mm ( cook inc . , bloomington , in , usa ) was then advanced over the wire into the smv .
a retrograde venogram was obtained while the outflow tract was occluded with the balloon ( fig .
a renegade microcatheter ( boston scientific corporation , natick , ma , usa ) was advanced through the balloon catheter into varices using 0.14 transcend microwire ( boston scientific corporation , natick , ma , usa ) .
the balloon catheter was inflated in the smv to prevent the migration of 5% ethanolmaine oleate ( grelan pharmaceutical , tokyo , japan)-lipiodol mixture ( 1 : 1 ) .
about 10 ml of the mixture was injected via the microcatheter until the entire variceal complex was opacified .
after approximately 15 minutes , complete occlusion of the varices was observed during deflation of the balloon catheter ( fig .
the access tract was embolized with one detachable microcoil ( interolocking detachable coil , boston scientfic corporation , natick , ma , usa ) on completion of the procedure .
after 6 weeks , cect showed well obliterated vesical varices and the patient did not complain of gross hematuria ( fig .
vesical varices secondary to portal hypertension are rare since the bladder wall is not the usual collateral route for venous splanchnic blood flow ( 2 ) .
vesical varices may occur in patients with portal hypertension in circumstances where the normal splanchnic collaterals fail to develop due to prior obliteration from treatments such as surgery , sclerotherapy or ligation ( 3 ) .
the second probability is when the anatomy of the venous drainage of the bladder is altered from surgery such as bladder augmentation with a bowel segment . in our case , he underwent left nephrectomy and orthotopic bladder substitution with ileal segments after radical cystectomy due to renal tuberculosis in the left kidney , 30 years ago .
varices secondary to portal hypertension have been known to occur in ectopic areas like duodenum , jejunum , ileum and anorectal regions .
these varices in the gastrointestinal tract have been treated with endoscopic injection sclerotherapy , endosopic band ligation , balloon - occluded retrograde transvernous obliteration , transjugular intrahepatic portosystemic shunts , percutaneous transhepatic obliteration , and surgical procedures such as segmental resection and ligation ( 4 )
. percutaneous transhepatic obliteration of sclerosants is a less invasive procedure when compared to surgical procedure and hence may be the only means of actively arresting bleeding vesical varices in a patient who is unfit or unsuitable for surgery .
the potential disadvantage of sclerosants injection is its embolization which could lead to infarcts at distant sites or even fatality .
, the possibility of vesical varices needs to be considered in the care of patients with recurrent hematuria , especially , those associated with portal hypertension .
balloon - occluded percutaneous transhepatic obliteration could be a treatment option for vesical varices , especially , in a patients who is unfit or unsuitable for surgery . | gross hematuria secondary to vesical varices is an unusual presentation .
we report such a case recurrent gross hematuria in a male patient who had a history of bladder substitution with ileal segments that had been treated by balloon - occluded percutaneous transhepatic obliteration of vesical varices . |
malaria is the most common fatal disease in the world . according to 2013 who malaria report , an estimated 3.4 billion people were at risk of malaria globally and populations living in sub - saharan africa have the highest risk of acquiring malaria .
approximately 80% of cases and 90% of deaths are estimated to occur in the african region . in ethiopia approximately 52 million people ( 68% ) live in malaria risk areas .
plasmodium falciparum and p. vivax are the dominant species of malaria in ethiopia , with 60% and 40% relative frequencies , respectively .
p. falciparum is the most dominant species in endemic areas and causes severe and complicated disease and death . in ethiopia half
a million microscopically confirmed cases of malaria are reported to the federal ministry of health ( fmoh ) in each year from basic health services . however , the actual number of malaria cases in the country is estimated to be more than 5 million each year .
so , choosing an accurate , rapid , and cost - effective diagnostic test is mainstay of efficient clinical and epidemiological management of malaria [ 3 , 4 ] .
clinical diagnosis is the most widely used diagnostic method in rural areas and where laboratory facility does not exist .
however , according to who 2011 guidelines clinical diagnosis of malaria based on signs and symptoms alone is not recommended since it has low specificity and increases the chances of the patient being misdiagnosed and leads to misuse of drugs [ 5 , 6 ] .
a laboratory diagnosis of malaria is one possibility in the management of a patient presenting with fever . to improve the quality care of the patients , many diagnostic procedures have been developed which aim to have accurate diagnosis , to reduce the time of preparation and training needed [ 3 , 4 ] . even though giemsa stain microscopy remains a gold standard method for malaria diagnosis in many developing countries [ 4 , 7 , 8 ] , but it is not 100% sensitive and specific .
the partec rapid malaria test ( pt ) is newly innovative fluorescence microscopy method for the detection of plasmodium species dna in human blood .
it uses readily prepared and ready - to - use slides labeled with an unspecific dna - binding fluorescent dye ( 4-6-diamidino-2-phenylindole ( dapi ) ; emission 365 nm ) that detects plasmodial dna .
it is ultra compact and robust microscope design and has connector for optionally available ccd camera upgrade for visualization of the slides on any pc windows with usb connection [ 10 , 11 ] .
thus this study was conducted to evaluate the performance of cyscope florescence microscopy ( partec rapid malaria test ) in reference to light microscopy in northwest ethiopia .
a study was conducted at gendewuha health center found in metema district , north gondar zone , amhara regional state , from june to july 2013 .
it is far 925 km from addis ababa ( capital city of ethiopia ) and found at 180 km west of gondar town .
the mean annual temperature ranges from 22c to 28c and the daily temperature reaches as high as 43c during the months of march to may .
mean annual rainfall ranges from 850 mm to 1100 mm and has unimodal pattern .
one hundred and eighty malaria suspected patients attending at gendewuha health center during the study period were included and screened for malaria infection using light microscopy and partec rapid malaria test .
patients who had received antimalarial drugs during the past two weeks , children under 1 year , and critically ill patients who were unable to respond to the interview were excluded from the study .
three milliliters of venous blood was collected with edta tubes by laboratory technicians from each study subject for smear preparation of light microscopy and pt . after collecting blood samples , thick and thin blood smears were prepared and stained with 10% giemsa working solution for 10 minutes .
blood films were observed under 100x objectives for the detection of malaria parasites and the result was reported as positive if asexual malaria parasites were seen or negative if malaria parasites were not seen after observing 100 fields of the thick smear .
all blood films were reexamined by an experienced microscopist at university of gondar hospital laboratory who was blinded to initial light microscopy result .
briefly , on the collected edta blood , 10 l blood was placed on the dye labeled area of a slide and cover slipped , incubated at room temperature for a minute , and observed with 40x objective under led uv light ( 365 nm ) .
the presence of bright shiny intracellular tiny dots against a dark background observed under the uv light indicated the presence of malaria parasites in the erythrocytes as shown in figure 1 .
odds ratio , sensitivity , specificity , positive predictive value , negative predictive value , and kappa value were calculated at 95% ci .
ethical clearance was obtained from university of gondar , school of biomedical and laboratory sciences ethical committee , and permission was obtained from gendewuha health office and gendewuha health center authorities to conduct the study . after informing about the objective of the study , written consent was taken from all study participants or parents / guardians .
participants who were positive to malaria parasite were given the appropriate treatment at the health centre .
about 99 ( 55% ) of the study participants were males and 81 ( 45% ) were females .
the mean age of the participants was 19 12.49 years and majority of the participants 60 ( 33.3% ) were within the age range of 2029 years , and 42.8% of the participants were illiterate .
the rural residence has statistically significant association with malaria infection ( aor = 0.437 , ci = 0.0280.835 ) ( table 1 ) .
the overall prevalence of malaria using light microscopy was 81 ( 45% ) . from this plasmodium falciparum accounts for 76 ( 93.8% ) , p. vivax were 4 ( 4.9% ) , and mixed infection ( p. falciparum and p. vivax ) was 1 ( 1.3% ) .
there were 2 discordant results between the first ( study site ) reader and the final reading by experienced microscopist ; thus the final report was based on the final reading .
the parasite positivity of malaria by using partec rapid malaria test was 88 ( 48.9% ) .
seventy - six slides were positive with partec rapid malaria test and the gold standard giemsa stain microscopy .
twelve slides were positive with partec rapid malaria test and negative with light microscopy , while 5 slides were negative with partec rapid malaria test and positive with light microscopy and 87 slides were negative for both ( table 2 ) . using light microscopy as standard test for diagnosing malaria ,
the sensitivity and specificity of partec rapid malaria test were 93.8% ( 95% ci = 87.1100 ) and 87.9% ( 95% ci = 79.796.1 ) , respectively .
there was also an excellent agreement between light microscopy and partec rapid malaria test with kappa value of 0.811 ( 95% ci = 0.6250.996 ) ( table 3 ) .
compared to light microscopy pt is more sensitive , less labor intensive , and faster to use and has a less turnaround time than the lm ( an average of 5 minutes ) .
furthermore , the cyscope florescence microscope is battery operated ; the battery can be kept on for 6 hours after full charge making it best for field work .
it is easier to use and to adjust and can be switched from uv light to bright field .
however , the pt is not suitable for species identification and parasitic load determination and blood films can not be stored for a long period of time .
the high sensitivity and specificity are in agreement with the reports in sudan . however , the sensitivity and specificity are lower than the reports in zimbabwe , ghana [ 11 , 15 ] , sudan , and uganda
thus , a high npv indicates that a person does not have the disease with high certainty , meaning that pt is reliable test method in diagnosing of malaria parasites .
partec rapid malaria test had detected 12 cases which were negative by light microscopy . unlike giemsa stain light microscopy , pt has sensitive fluorescent dye , 4,6-diamidino-2-phenylindole ( dapi ) , which can be detected in such low level of parasites .
in addition , the presence of artifacts such as nonspecific aggregated dapi , immature erythrocytes , or bacterial cells might have been misinterpreted as plasmodium dna . on the other hand , pt produced 5 false negative results which were positive by light microscopy .
this might be due to the fact that unlysed red blood cells may lie on each other or overlap with each other thereby preventing parasites in red blood cells that may be lying beneath other cells .
the performance characteristics of the tests were very similar as indicated in their sensitivities , specificities , ppv , and npv with very good agreements to the light microscopy .
determination of parasitemia with partec rapid malaria test is difficult , because the red blood cell may overlap each other and make the parasites invisible under the uv light .
though , pt is useful as light microscopy thick film for screening purpose in malaria endemic areas .
the overall prevalence of malaria in the study area was very high , as detected by both the lm ( 45% ) and the pt ( 48.9% ) .
this result is higher than the report from other regions in ethiopia [ 18 , 19 ] . the high prevalence could be partly explained by the fact that the study was conducted in malaria transmission season of the country .
partec rapid malaria test showed good sensitivity and specificity with an excellent agreement to the reference light microscopy .
pt has very short turnaround times , requires little training , and is applicable under field conditions .
further studies are needed to determine the cost - effectiveness and long term performance of the partec rapid malaria test in diagnosing malaria . | background . laboratory diagnosis of malaria is the key for effective disease management .
diagnosis of malaria infection requires rapid , sensitive , and specific test methods with an affordable cost .
this study was aimed to assess the diagnostic performance of partec rapid malaria test with reference to light microscopy for the diagnosis of malaria in northwest ethiopia .
methods .
a total of 180 febrile patients were tested for malaria using giemsa stain microscopy and partec rapid malaria test from june to july 2013 at gendewuha health centers , metema district .
data were analyzed using spss version 20 statistical software .
odds ratio with 95% ci was calculated . result . the sensitivity and
specificity of partec rapid malaria test were 93.8% ( 95% ci = 87.1%100% ) and 87.9% ( 95% ci = 79.7%96.1% ) , respectively , while the positive predictive value and negative predictive value were 6.4% ( 95% ci = 77.2%95.5% ) and 94.6% ( 95% ci = 88.7%100% ) , respectively .
there was also an excellent agreement between two tests with kappa value of 0.811 ( 95% ci = 0.6250.996 ) .
conclusion .
partec rapid malaria test showed good sensitivity and specificity with an excellent agreement to the reference light microscopy .
therefore pt can be considered as alternative diagnostic tools in malaria endemic areas . |
sudden out - of - hospital cardiac arrest ( sohca ) remains one of the major causes of death for men and women alike in western societies , accounting for more than 250,000 lives lost annually in the usa alone .
ironically , most cases of sohca are caused by a highly reversible yet time dependent process , namely ventricular fibrillation ( vf ) , which in turn creates a tremendous opportunity for public health intervention [ 1 - 4 ] .
nevertheless , despite well developed emergency medical services ( ems ) with rapid response advanced cardiac life support ( acls ) capabilities , survival rates following sohca have remained very low in most venues , even for out - of - hospital vf [ 1 - 4 ] .
two key interventions have been proven scientifically to improve the chances of survival for those experiencing sohca : immediate performance of basic cardiopulmonary resuscitation ( cpr ) by bystanders ; and immediate delivery of specialized countershock in cases of vf . therefore , poor resuscitation rates in ems systems have been attributed most often to delays in the delivery of basic cpr by witnesses or delays in rapid defibrillation by ems personnel .
however , recent laboratory and clinical data have also begun to suggest that the current standard of immediately providing counter - shock as the first intervention for vf may be detrimental when the vf is prolonged beyond several minutes [ 5 - 9 ] .
the mechanism underlying this is complicated and multifactorial , but , in short , several studies now suggest that when myocardial energy supplies and oxygenation begin to dwindle following prolonged periods of vf , improvements in coronary artery perfusion must first be achieved in order to prime the heart for successful return of spontaneous circulation ( rosc ) after defibrillation [ 5 - 7,10 - 12 ] . along with experimental and supportive clinical evidence ,
histologic and physiologic studies have resulted in an evolving hypothesis that delivery of an electrical countershock to an ischemic heart may be more damaging than when it is delivered immediately ( within the first 23 min ) following the onset of vf [ 13 - 15 ] . in turn , according to this paradigm , certain pharmacologic and/or mechanical interventions should take precedence over electrical countershock during resuscitative efforts if the countershocks can not be delivered within the first few minutes following onset of vf .
the evolving concept of providing ' preshock ' interventions for vf may explain why several teams of investigators were not able to demonstrate the efficacy of so - called ' high dose adrenaline [ epinephrine ] ' ( i.e. > 1 mg / kg doses ) and other acls procedures in previous clinical trials when they were successful in the laboratory [ 16 - 18 ] . in keeping with international guidelines , these study protocols called for the use of the test intervention ( e.g. high - dose adrenaline ) after multiple countershocks in cases of vf [ 17 - 19 ] . in contrast , the successful preclinical studies used the resuscitative drugs before countershock .
this explanation has been substantiated by specific canine experiments conducted by niemann and coworkers that subsequently tested the resuscitation effects of high - dose adrenaline administered before and after countershocks . in such studies ,
rosc was improved by first administering the high - dose adrenaline after 7.5 min of vf .
several other animal models now strongly corroborate this concept of ' drugs first ' in prolonged vf . using a ' cocktail ' ( multiple drug ) regimen , including high - dose adrenaline , antiarrhythmics , and antioxidants , menegazzi and colleagues demonstrated similar effects in terms of resuscitation and short - term survival in swine that experienced 8 min of vf before interventions .
therefore , these experiments may help to explain the relative lack of effectiveness of high - dose adrenaline in clinical trials , particularly in the subset of patients presenting with vf .
in fact , in some of the clinical trials of high - dose adrenaline , on average the first drugs were given as late as 17 min following notification of the sohca event , even when examining cases of witnessed collapse only .
many of the cities studied in the trial conducted by brown and coworkers had excellent response intervals and greater than average survival rates , thus indicating a relative ' best case ' scenario .
thus , it may be speculated further that the need for preshock interventions would generally be indicated in such prolonged periods of vf , particularly when compared with animal studies demonstrating the efficacy of drugs first with much briefer periods of arrest .
therefore , it may very well be that acls drugs ( and high - dose adrenaline in particular ) may be of more value than previously demonstrated , and that their efficacies may have been masked , in part , during clinical trials because of inappropriate timing of administration relative to countershock .
if delivering countershocks first might be harmful , then should other interventions always be delivered first ?
experimental studies such as those cited above appear to demonstrate the need for high - dose adrenaline and other interventions before countershock .
in fact , more than two decades ago , yakaitis and coworkers showed a marked improvement in outcomes using only standard doses of adrenaline ( coupled with basic cpr procedures ) before countershock in a canine model .
therefore , it is possible that higher doses of adrenaline may only be needed after more prolonged periods of vf .
nevertheless , all of these studies still indicate the need for some supportive intervention before defibrillation attempts when several minutes of untreated vf have elapsed .
more recently , preliminary clinical studies have supported this evolving concept in terms of providing basic cpr procedures ( i.e. chest compressions ) for a short period before defibrillation in unmonitored out - of - hospital vf . in such scenarios , there is de facto more than several minutes of vf while the emergency response is being made , even in rapid response ems systems . in one of these studies , conducted in the rapid response seattle ems system ,
there was still a marked improvement in outcomes when first responder firefighter crews provided 90 seconds of basic cpr before defibrillation attempts .
although that study used an historical control ( 2 years of no preshock cpr by the first responders versus a subsequent period using 90 seconds of cpr before defibrillation attempts ) , survival rates were clearly improved .
this finding was particularly compelling when analyzing the subset of patients who received the 90 seconds of cpr first when the ems response intervals were greater than 4 min ( fig .
1 ) . in the cases in which the ems responded in less than 4 min , there was little difference in outcomes but the results were still not worse with 90 seconds of cpr first ( fig .
1 ) . given that this study involved a relatively short ( 4 min average ) response interval , one might surmise that these results are good enough to support a ' intervention first ' approach in all cases , especially because there seemed to be no harm in performing 90 seconds of basic cpr before shock , even in the shorter than average response periods .
however , before drawing final conclusions about this study , it should be noted that even in cases of witnessed collapse there is also a finite amount of time before ems is called after the collapse , and that there is another minute or two required to reach the patient 's side and deliver the shock after on - scene arrival of ems .
therefore , this ' 4 min response interval ' may translate into a 7 or 8 min period of vf , and one should not immediately extrapolate a time frame for ' shock first ' or ' cpr first ' .
in addition , one should note that basic cpr was provided by bystanders in a large percentage of these cases ( in all subgroups ) .
therefore , many patients were already receiving some degree of basic cpr before the counter - shock , even in the historical control period .
although the seattle study may be subject to scrutiny because of the ( historical control ) study design , wik and colleagues in oslo , norway later reported almost identical results but in a controlled clinical trial . in their clinical trial , patients were randomly assigned to either 3 min of chest compressions first or shock first .
again , those patients receiving basic cpr first did much better , particularly in the subgroups of patients with more than 5 min ems response intervals ( i.e. presumably at least 89 min of vf before professional intervention ) . specifically , rosc occurred more often in the group with 3 min of cpr first when response intervals exceeded 5 min ( 58% versus 38% , p < 0.04 ) with an odds ratio of 2.22 and 95% confidence interval of 1.064.63 .
similar to the seattle study , rosc was not significantly different in the groups for whom the response was less than 5 min . more impressively , survival to hospital discharge was improved ( 22% versus 4% , p = 0.006 ; odds ratio 7.42 , 95% confidence interval 1.6134.3 ) , as was 1 year survival ( 20% versus 4% , p = 0.01 ; odds ratio 6.76 , 95% confidence interval 1.4231.4 ) .
almost all ( approximately 90% ) of those discharged alive in the study were either neurologically
normal or had only had minor problems ( with no significant differences noted in the subgroups ) . recognizing that even those patients with a response interval less than 5 min did no worse with ' cpr first ' ( fig .
2 ) , wik and colleagues concluded that 3 min of cpr before defibrillation attempts is always indicated unless the patient collapsed in front of ems .
the strength of these clinical data has added more credibility to the evolving notion that interventions should always be performed before defibrillation attempts . however , this proposed approach does pose problems for current resuscitation policies .
in addition to conflicting with internationally accepted standards of patient management , the deferred counter - shock concept may also pose a potential glitch for current automated defibrillator initiatives , especially certain public access defibrillation initiatives .
in addition , successful defibrillation and rosc can be achieved after relatively prolonged periods of arrest especially with well performed and immediately executed basic cpr . in most cases of successful resuscitation from vf , resuscitative drugs are never needed , even after the countershock .
if the heart remains well perfused , then the shock may still be delivered first .
furthermore , in the early canine experiments conducted by yakaitis and coworkers that demonstrated the superiority of giving adrenaline and cpr before countershocks after 5 min of vf , companion experiments also demonstrated that shocking first was clearly superior following only 1 min of vf .
also , recent studies have indicated very high survival rates when patients are shocked within 5 min , such as a recent study of public access defibrillation at the chicago municipal airports . in that study of public use of automated defibrillators ,
three quarters of the patients were resuscitated and achieved full neurologic recovery when shocked within 5 min of collapse .
in fact , many of the patients were already awakening by the time of ems arrival at the scene . nevertheless , the authors also noted that all survivors received some period of chest compressions and other basic cpr techniques , even if briefly , while awaiting defibrillatory attempts . assimilating all of the studies to date , one might conclude that rapid defibrillation should be a priority in the first few minutes after arrest , but that basic cpr may also be provided as long as it does not delay the defibrillatory attempts .
however , after several minutes of arrest ( perhaps 4 or 5 min ) , basic cpr and perhaps other acls interventions may need to be provided for a finite period of time before the shocks .
it is clear , however , that such judgments and time determinants are all guesswork and that many factors , particularly the rapid provision of well performed early basic cpr , may be confounding variables . therefore , somehow being able to delineate objectively between a hypoxic and nonhypoxic heart might be a critical adjunct to therapeutic decisions .
in addition to defining whether defibrillation should be deferred , it would also be important to define what therapies are required at any given point , be they chest compressions alone , chest compressions and adrenaline , high - dose adrenaline and other drugs , or perhaps new alternative cpr devices .
in addition , it remains unclear as to whether chest compressions alone are indicated after a few minutes of vf or whether drug infusions should also be given .
likewise , it may turn out that , at some point in the protraction of vf , multiple drugs or progressively higher doses of drugs may be needed before countershock .
again , all of these considerations must be addressed within the context of a number of confounding variables such as scenarios involving immediate and well performed bystander cpr or scenarios of chest pain or ventricular tachycardia ( with spontaneous pulses ) deteriorating into vf just before arrival of the rescuers with a defibrillator .
therefore , having the technology or ability to predict the level of ischemia in the heart would be more useful than a stopwatch .
fortunately , successful defibrillation with rosc following a countershock first approach may be more predictable with real - time scoring of the vf waveform signal .
specifically , techniques such as online electrocardiographic median frequency or scaling exponent analysis can be used to predict successful defibrillation [ 12,23 - 27 ] .
conceptually , in a real - time setting , a defibrillator can perform an analysis of the vf waveform and score the electrical signal . if the score is high enough ( or low enough , depending on the analysis ) , then a shock would be advised .
if missing the mark , other therapies would be advised first and perhaps at progressively different levels depending on the severity of the poor score .
studies have shown , for example , that basic cpr and certain pharmacologic interventions can ( but not always ) improve the vf waveform score [ 12,24 - 26 ] .
therefore , one might speculate that , in the future , user - friendly technology with automated algorithms will be developed that will not only guide the type and degree of initial therapeutic interventions , but also the duration of resuscitative efforts . moreover
it has become clear that the timing of certain interventions in sohca is time dependent or , more accurately , dependent upon the duration and degree of the ischemic insult after the onset of vf .
although the overall concept of providing certain therapeutic interventions before countershock in cases of prolonged vf has become very compelling , it must always be appreciated that there are multiple variables that may confound the appropriateness of this approach .
although new technologies may eventually help to overcome these concerns , the dynamics of proposed waveform analyses and their specific relationships to successful rosc and ultimate outcome must be carefully weighed against the clinical circumstances .
in addition , there are also factors related to the intervention used , such as the type of countershock being delivered .
for example , low - energy biphasic shocks and other evolving energy delivery mechanisms may behave differently than traditional high - energy or monophasic shocks [ 12,29 - 31 ] .
there are also new cpr devices that may be found to be more effective than current techniques in providing resuscitation after countershock delivery .
nevertheless , the evolving evidence for preshock therapies following several minutes of vf is very strong . although it will require aggressive , multifaceted studies to delineate the many confounding variables and the specific interventions that should be delivered under specific circumstances , the preliminary data certainly justify further study .
interestingly , in many ways , these data revalidate the important discovery of basic cpr more than four decades ago . in addition , today , with the introduction of various promising resuscitative techniques such as the active compression decompression pump , ' vest ' cpr , the inspiratory threshold device and minimally invasive direct cardiac massage , it is plausible that we may be able to resuscitate many more persons than ever before , particularly if these interventions are applied before defibrillation attempts .
acls = advanced cardiac life support ; cpr = cardiopulmonary resuscitation ; ems = emergency medical services ; rosc = return of spontaneous circulation ; sohca = sudden out - of - hospital cardiac arrest ; vf = ventricular fibrillation .
comparison of survival rates ( successful hospital discharge ) in seattle , usa , during the years when emergency responders made defibrillation attempts their first priority ( 19901993 ) versus subsequent years ( 19941996 ) , when they provided 90 seconds of basic cardiopulmonary resuscitation before defibrillatory attempts for out - of - hospital cases of ventricular fibrillation . survival rates and historical comparisons
are stratified according to those patients receiving an emergency response within 4 min versus those with response intervals greater than 4 min .
the response interval was measured from the time of dispatch of emergency vehicles until the time of arrival at the street address ( not time of collapse to arrival at the patient 's side ) . adapted from cobb and coworkers .
comparison of out - of - hospital ventricular fibrillation survival rates ( successful hospital discharge ) with defibrillation attempts provided first versus cases for which there was provision of 3 min of basic cardiopulmonary resuscitation before defibrillation attempts in oslo , norway .
survival rate comparisons are stratified according to those patients receiving an emergency response within 5 min versus those with response intervals greater than 5 min .
the response interval was measured from the time of dispatch of emergency vehicles until the time of arrival at the street address ( not time of collapse to arrival at the patient 's side ) . adapted from wik and coworkers . | despite well developed emergency medical services with rapid response advanced life support capabilities , survival rates following out - of - hospital ventricular fibrillation ( vf ) have remained bleak in many venues . generally , these poor resuscitation rates are attributed to delays in the performance of basic cardiopulmonary resuscitation by bystanders or delays in defibrillation , but recent laboratory data suggest that the current standard of immediately providing a countershock as the first therapeutic intervention may be detrimental when vf is prolonged beyond several minutes . several studies now suggest that when myocardial energy supplies begin to dwindle following more prolonged periods of vf , improvements in coronary artery perfusion must first be achieved in order to prime the heart for successful return of spontaneous circulation after defibrillation .
therefore , before countershocks , certain pharmacologic and/or mechanical interventions might take precedence during resuscitative efforts .
this evolving concept has been substantiated recently by clinical studies , including a controlled clinical trial , demonstrating a significant improvement in survival when basic cardiopulmonary resuscitation is provided for several minutes before the initial countershock .
although this evolving concept differs from current standards and may pose a potential problem for automated defibrillator initiatives ( e.g. public access defibrillation ) , successful defibrillation and return of spontaneous circulation have been rendered more predictable by evolving technologies that can score the vf waveform signal and differentiate between those who can be shocked immediately and those who should receive other interventions first . |
the skin forms a protective layer around the body against physical , chemical and thermal environmental challenges .
in addition to providing a physical barrier , the skin is considered a sensory organ that allows the body to perceive harmful external stimuli and appropriate behavior or body movement can be initiated thereafter . the peripheral nervous system governs the broadly defined sense of touch perception , communicating with the central nervous system regarding the external environment , allowing for conscious sensations of balance and coordination , pressure and vibration , pain and temperature .
sensory nerves are innervated in the entire skin tissue as well as subcutaneous fat layer .
most nerve fibers and endings are found in the mid - dermis and the papillary dermis . in the epidermis ,
sensory nerves are linked to keratinocytes , melanocytes , langerhans cells ( lc ) and merkel cells .
the epidermal nerves consist of free nerve endings or nerve organs . in the dermis , there are free sensory nerve endings such as pinkus discs , ruffini , meissner , krause and vater - pacini corpuscles [ 14 ] .
peripheral sensory nerves can be classified on the basis of the degree of myelination and the velocity at which action potentials travel through afferent fibers with the fast conducting , myelinated ( large ) a fibers , a fibers , a fibers and the slow conducting , unmyelinated ( small ) c fibers . among them , a and a fibers are mostly mechanical sensitive afferents ( type i ) localized on hairy and glabrous skin . a subpopulation of a fibers on hairy skin are believed to be mechanically insensitive ( type ii ) .
a fibers constitute approximately 80% of primary sensory nerves sprouting from the dorsal root ganglion ( drg ) , whereas c fibers constitute approximately 20% of the primary afferents [ 35 ] .
c fibers are either polymodal nociceptors which can respond to chemical or mechanical stimuli and temperature changes , or more specialized perception . among human peripheral nerves ,
45% of the cutaneous afferent nerves belong to a subset of sensory nerves that are both mechano- and heat - responsive c fibers .
a certain subpopulation of a fibers respond to gentle cooling , whereas selective c fibers become activated during noxious cold .
when we lightly touch with our fingertips , the a fibers respond because they are associated with a variety of cells including keratinocytes , merkel cells and hair follicles . in addition , the c fibers are known for nociceptor action terminated at the target tissue as a free nerve ending .
similarly , specific receptor distribution and biochemical diversity of different sensory nerve subtypes exists , which is important for various perceptions . the information detected from the peripheral sensory nerves is transmitted to the drg and cranial nerve ganglia such as the trigeminal ganglion .
drg are a cluster of sensory neuronal cell bodies located in the vertebral column lateral to the spinal cord .
a functionally specialized zone exists at the drg neurons that can be partitioned according to specific sensory modalities .
based on the stimulus detected , drg neurons are functionally classified as proprioceptors , low - threshold mechanoreceptors , and cells that sense pain , itch and/or temperature .
the drg neuronal branch relays information through the dorsal horn of the spinal cord to the central nervous system to recognize the type of sensation [ 69 ] .
when decreasing the stimuli temperature , the quality of sensation may change from cool to cold , from icy cold to pain .
similarly , increasing the stimuli temperature can cause a change in the sensations from warmth , to heat then to sharp or dull pain .
the reported temperatures that cause these different sensations vary considerably . this variability can be explained by differences in the characteristics of the stimulus used , experimental conditions employed , and individual background differences ( previous experiences of pain , ethnicity , sex , social status or religion ) .
cutaneous thermosensation is mediated by a variety of primary afferent nerve fibers that transduce , encode and transmit thermal information [ 711 ] .
there are 2 populations of thermosensory fibers , one responding to warmth and the other to cold .
specific cutaneous cold and warm receptors have been defined as slowly conducting units that exhibit a steady - state discharge at constant skin temperature and a dynamic response to temperature changes .
cold - specific and warm - specific receptors can be distinguished from nociceptors that respond to noxious low and high temperatures ( < 20c and > 45c ) , and also from thermo - sensitive mechanoreceptors .
in general , the perception of non - painful , cool temperature occurs when the skin is cooled as little as 1c from normal body temperature . in a fiber recording study
, temperatures in the range of 30 - 15c can activate both a and c fibers .
these cold - sensitive afferents fire continuously at normal body temperature , but under cold stimuli the rate of firing increases , while warm temperatures reduce ( or antagonize ) this activity .
however , the peak responsiveness can vary when the temperatures fall below either 25 or 15c .
once the temperature approaches 15c , the perception of cold as well as pain is evoked , described as burning , aching , and pricking sensations .
this delicate thermosensing process is also related to specific receptor proteins that are located within the free nerve endings in the skin , known as transient receptor potential ( trp ) .
advances in understanding the temperature sensation have emerged from the cloning and characterization of trp ion channels whose activity depends on the temperature of their environment .
activation of trp channels depolarize cells from the resting membrane potential and shortens action potential duration .
most trp channels are cation - non - selective and permeable to the signal transduction element ca .
each of these receptors operates over a specific temperature range , thereby providing a potential molecular basis for thermosensation .
these specialized thermal receptors are embedded in the terminals of afferent fibers which end as free nerve endings in the skin .
trps are classified according to their primary amino acid sequence rather than selectivity or ligand affinity , because their properties are heterogenous and their regulation is complex .
trp channels are membrane proteins with 6 putative trans - membrane ( tm ) spans and a cation - permeable pore region formed by a short hydrophobic stretch between tm5 and tm6 ( fig .
trp proteins are essentially cation - permeable ion channels sensitive to a remarkable range of stimuli . in humans ,
28 different trp channels have been identified and are grouped into 6 families . of these , members of 3 families , i.e. the vanilloid trp channels ( trpv ) , the melastatin or long trp channels ( trpm ) , and the ankyrin transmembrane protein channels ( trpa ) are of particular interest as thermoreceptors .
representative mammalian trp channels are gated by heat and capsaicin ( > 43c ; trpv1 ) , noxious heat ( > 52c ; trpv2 ) , and cooling ( < 22c ; trpm8 ) ( table 1 ) .
trpv1 , vanilloid receptor 1 , is the most well - known and characterized trp channel which is activated by noxious stimuli such as capsaicin and excessive heat and ph below 5 .
trpv1 is predominantly expressed in sensory neurons and believed to play a crucial role in temperature sensing and nociception .
therefore , they were used as a molecular target for pain treatment and pruritus over the last several decades . while trpv1 and trpv2 are established as neuronal thermosensors for heat sensation , other trpv subfamily members , trpv3 and trpv4
are also involved in the sensation of warmth . in regards to cool and cold sensation , trpa1 and transient receptor potential melastatin 8 ( trpm8 )
trpa1 is activated to mediate noxious cold sensation while trpm8 mediates cool and fresh mint - like sensation .
recently , the cooling sensation generated especially by trpm8 and/or cold and menthol receptor 1 ( cmr1 ) has attracted attention for its potential usefulness . at steady state temperatures , cold fibers have a characteristic stimulus response function which is bell - shaped , with a maximal steady activity between 20 and 30oc and less activity at either lower or higher temperatures .
the reported temperature range of cold fiber actions varies . at constant temperatures above 40c or below 17c
however , some cold fibers can also be activated by high temperatures in the noxious range , and this may be the basis for the paradoxical cold sensation .
cold afferents respond vigorously when the skin is actively cooled down . however , these dynamic responses are rather transient [ 1820 ] .
in addition , high - threshold cold receptors ( hcrs ) have previously been identified .
these afferents are believed to be sensitive to cold temperatures below 27c , but do not respond to mechanical or heat stimuli and have conduction velocities in the low range of a and c fibers .
rapid cooling of the skin is also able to activate unmyelinated , low threshold mechanoreceptors ( cltms ) .
these c fibers are non - responsive to intense heat stimuli and respond vigorously to gentle touch .
this response to cooling is only transient , and relatively rapid decreasing temperatures are required for long effective activation .
several studies suggest cold sensitivity can also be found in large myelinated afferents which respond vigorously to mechanical stimuli [ 2022 ] .
trp channels sense the innocuous cold through the trpm8 ion channel , which is specifically expressed in the drg and trigeminal ganglion neurons with small nerve fibers , probably c fibers . based on mice studies , the role of trpm8 in thermosensation is well - established .
trpm8 is activated by temperatures below 26c , and is expressed in approximately 15% of small drg neurons .
trpm8 is activated by agents known to produce the sensation of cold in humans ; the well - known agonists are menthol , eucalyptol and icilin .
, sensory reaction showed a heightened sensitivity to temperatures in the innocuous range ( allodynia ) and in the already painful range ( hyperalgesia ) .
colburn et al . assessed the involvement of trpm8 in injury - evoked hypersensitivity to cold , and suggested that trpm8 cooling sensation may modulate the majority of allodynia and hyperalgesia .
in addition , numerous studies have demonstrated trpm8 expressing afferents are related to analgesic effects . the analgesic role mediated by trpm8
cold temperatures can produce the pain sensation with temperatures between 10 and 15c in glabrous skin and approximately 18c in hairy skin .
noxious cold stimuli cause distinct sensations such as pricking , burning , aching and even heat , suggesting different fiber populations exist and are activated in the cold sensation .
mechanosensitive a afferent fibers ( a - msa ) , including those sensitive to heat stimuli , can be also activated by noxious cold . instead of signaling the intensity of a cold stimulus
, a - msa fibers might encode the pricking sensation associated with noxious cold stimuli ; cold - induced pricking sensation is largely reduced when a fibers are blocked .
in addition to a fibers , a subset of c fiber nociceptors can be excited by noxious cold and may therefore contribute to the sensation of cold pain .
similar to a fibers , most of the cold - sensitive c fibers are believed to be poly - modal in nature .
although c fibers are also responsive to mechanical and heat stimuli , their threshold temperatures for activation vary over a wide range . during the block of an a fiber ,
when c afferents are still active , the perception of noxious cold stimulus is experienced as burning or heat .
cool sensation either blocks or modifies the c fiber input into the central nervous system .
therefore , the sensory experience induced by noxious cold stimuli depends on the integration of neuronal activity from both small myelinated and unmyelinated afferents . in noxious cold , around 17oc or below , trpa1 ( ankyrin transmemebrane protein 1 ; anktm ) is activated and known to be insensitive to menthol and capsaicin but activated by icilin .
trpa1 is specifically expressed in a subset of sensory neurons that express the nociceptive markers cgrp and substance p , and may operate as peripheral mechanosensors .
trpa1 are receptors for isothiocyanates , the pungent ingredients in wasabi and yellow mustard , cinnamon , mustard oil and inflammatory peptides and bradykinin .
the expression pattern in nociceptors suggests that trpa1 is involved in nociceptive signaling as well as mediating the distinct pungent sensations .
moreover , trpa1 has been postulated to play an important role in inflammatory hypersensitivity through receptor - operated mechanism by pro - algesic or pro - inflammatory mediators .
however , recently , whether trpa1 is involved in cold sensation needs to be reconciled because data are conflicting regarding trpa1 functioning as a cold sensor in vivo [ 2934 ] .
advances in understanding the temperature sensation have emerged from the cloning and characterization of trp ion channels whose activity depends on the temperature of their environment .
activation of trp channels depolarize cells from the resting membrane potential and shortens action potential duration .
most trp channels are cation - non - selective and permeable to the signal transduction element ca . each of these receptors operates over a specific temperature range , thereby providing a potential molecular basis for thermosensation .
these specialized thermal receptors are embedded in the terminals of afferent fibers which end as free nerve endings in the skin .
trps are classified according to their primary amino acid sequence rather than selectivity or ligand affinity , because their properties are heterogenous and their regulation is complex .
trp channels are membrane proteins with 6 putative trans - membrane ( tm ) spans and a cation - permeable pore region formed by a short hydrophobic stretch between tm5 and tm6 ( fig .
1 ) . trp proteins are essentially cation - permeable ion channels sensitive to a remarkable range of stimuli . in humans ,
28 different trp channels have been identified and are grouped into 6 families . of these , members of 3 families , i.e. the vanilloid trp channels ( trpv ) , the melastatin or long trp channels ( trpm ) , and the ankyrin transmembrane protein channels ( trpa ) are of particular interest as thermoreceptors .
representative mammalian trp channels are gated by heat and capsaicin ( > 43c ; trpv1 ) , noxious heat ( > 52c ; trpv2 ) , and cooling ( < 22c ; trpm8 ) ( table 1 ) .
trpv1 , vanilloid receptor 1 , is the most well - known and characterized trp channel which is activated by noxious stimuli such as capsaicin and excessive heat and ph below 5 .
trpv1 is predominantly expressed in sensory neurons and believed to play a crucial role in temperature sensing and nociception .
therefore , they were used as a molecular target for pain treatment and pruritus over the last several decades . while trpv1 and trpv2 are established as neuronal thermosensors for heat sensation , other trpv subfamily members , trpv3 and trpv4
are also involved in the sensation of warmth . in regards to cool and cold sensation ,
trpa1 is activated to mediate noxious cold sensation while trpm8 mediates cool and fresh mint - like sensation .
recently , the cooling sensation generated especially by trpm8 and/or cold and menthol receptor 1 ( cmr1 ) has attracted attention for its potential usefulness .
at steady state temperatures , cold fibers have a characteristic stimulus response function which is bell - shaped , with a maximal steady activity between 20 and 30oc and less activity at either lower or higher temperatures .
the reported temperature range of cold fiber actions varies . at constant temperatures above 40c or below 17c
however , some cold fibers can also be activated by high temperatures in the noxious range , and this may be the basis for the paradoxical cold sensation .
cold afferents respond vigorously when the skin is actively cooled down . however , these dynamic responses are rather transient [ 1820 ] .
in addition , high - threshold cold receptors ( hcrs ) have previously been identified .
these afferents are believed to be sensitive to cold temperatures below 27c , but do not respond to mechanical or heat stimuli and have conduction velocities in the low range of a and c fibers .
rapid cooling of the skin is also able to activate unmyelinated , low threshold mechanoreceptors ( cltms ) .
these c fibers are non - responsive to intense heat stimuli and respond vigorously to gentle touch .
this response to cooling is only transient , and relatively rapid decreasing temperatures are required for long effective activation .
several studies suggest cold sensitivity can also be found in large myelinated afferents which respond vigorously to mechanical stimuli [ 2022 ] .
trp channels sense the innocuous cold through the trpm8 ion channel , which is specifically expressed in the drg and trigeminal ganglion neurons with small nerve fibers , probably c fibers . based on mice studies , the role of trpm8 in thermosensation is well - established .
trpm8 is activated by temperatures below 26c , and is expressed in approximately 15% of small drg neurons .
trpm8 is activated by agents known to produce the sensation of cold in humans ; the well - known agonists are menthol , eucalyptol and icilin .
, sensory reaction showed a heightened sensitivity to temperatures in the innocuous range ( allodynia ) and in the already painful range ( hyperalgesia ) .
colburn et al . assessed the involvement of trpm8 in injury - evoked hypersensitivity to cold , and suggested that trpm8 cooling sensation may modulate the majority of allodynia and hyperalgesia . in addition , numerous studies have demonstrated trpm8 expressing afferents are related to analgesic effects .
cold temperatures can produce the pain sensation with temperatures between 10 and 15c in glabrous skin and approximately 18c in hairy skin .
noxious cold stimuli cause distinct sensations such as pricking , burning , aching and even heat , suggesting different fiber populations exist and are activated in the cold sensation .
mechanosensitive a afferent fibers ( a - msa ) , including those sensitive to heat stimuli , can be also activated by noxious cold . instead of signaling the intensity of a cold stimulus , a - msa fibers might encode the pricking sensation associated with noxious cold stimuli ; cold - induced pricking sensation is largely reduced when a fibers are blocked .
in addition to a fibers , a subset of c fiber nociceptors can be excited by noxious cold and may therefore contribute to the sensation of cold pain .
similar to a fibers , most of the cold - sensitive c fibers are believed to be poly - modal in nature .
although c fibers are also responsive to mechanical and heat stimuli , their threshold temperatures for activation vary over a wide range . during the block of an a fiber , when c afferents are still active , the perception of noxious cold stimulus is experienced as burning or heat .
cool sensation either blocks or modifies the c fiber input into the central nervous system .
therefore , the sensory experience induced by noxious cold stimuli depends on the integration of neuronal activity from both small myelinated and unmyelinated afferents . in noxious cold , around 17oc or below , trpa1 ( ankyrin transmemebrane protein 1 ; anktm ) is activated and known to be insensitive to menthol and capsaicin but activated by icilin .
trpa1 is specifically expressed in a subset of sensory neurons that express the nociceptive markers cgrp and substance p , and may operate as peripheral mechanosensors .
trpa1 are receptors for isothiocyanates , the pungent ingredients in wasabi and yellow mustard , cinnamon , mustard oil and inflammatory peptides and bradykinin .
the expression pattern in nociceptors suggests that trpa1 is involved in nociceptive signaling as well as mediating the distinct pungent sensations .
moreover , trpa1 has been postulated to play an important role in inflammatory hypersensitivity through receptor - operated mechanism by pro - algesic or pro - inflammatory mediators .
however , recently , whether trpa1 is involved in cold sensation needs to be reconciled because data are conflicting regarding trpa1 functioning as a cold sensor in vivo [ 2934 ] .
a cool and gentle breeze is known to make humans feel better and relieve discomfort and pain .
the chemical sensory / cooling agents that can mimic these sensations without a change in tissue temperatures are promising as a new treatment for various disorders .
menthol , a cyclic terpene alcohol found in the leaves of the genus mentha is representative of these agents and has been used in a variety of products , such as toothpaste , candy and even tobacco . when applied at low concentrations to the skin or mouth ,
the cooling sensation of menthol arises from a molecular site of the cold receptor , trpm8 , as mentioned above .
however , menthol can evoke pain ( or irritation ) through activation and sensitization of c fibers with activation thresholds within the noxious range of temperatures .
therefore , in the case of menthol , both pain ( or discomfort ) and cold sensation appear closely linked .
notably , low concentrations of trpm8 agonists such as menthol or icilin were found to be anti - hyperalgesic in certain conditions .
in addition , cold patches have traditionally been used to induce analgesia or pain relief caused by traumatic injuries .
most recently , trpm8 antagonists were shown to be effective in reversing established pain in neuropathic and visceral pain models .
in addition to menthol , other cooling agents such as icilin , which is considered more efficacious and potent than menthol , activates the trpm8 channel .
we previously showed the effect of topical application of icilin in the management of paroxysmal itching sensation over the vulvae area .
a trpm8 agonist also showed efficacy in the histamine - induced itching model in vivo .
however , limitations may exist with regard to the duration of action because cold receptors must adapt with a prolonged change of membrane potential dependent upon calcium current or because trpm8 requires the presence of phosphatidylinositol 4,5 bisphosphate ( pip2 ) for stable activity .
therefore , prolonged trpm8 activation depletes calcium and pip2 as well as a certain level of cellular adaptation .
recently , several researchers have suggested trpm8 can be a target for treating prostate cancer because an androgen - dependent ca channel is necessary for prostate cancer to survive and grow . as summarized above ,
cold sensation of peripheral afferent sensory is not well understood but is relevant for further exploration .
the cellular and molecular mechanisms involved in the trp family , especially trpm8 , and trpa1 are not well recognized , thus further investigation on the interactive neuronal system of cooling and nociception can extend our knowledge for clinical use in various fields . | a patient recently presented with long - standing severe itching originating from lichen sclerosus et atrophicus at the vulva area .
we successively treated her using a topical formulation of trpm8 agonist which produces a cooling sensation .
the cooling sensation , an afferent sensory perception in various skin neuronal pathways , could be a useful mechanism to relieve an itchy sensation in various skin disorders .
mechanoreceptors are related to touch vibration and pressure sensations and have a special morphology where the nerve endings are optimized to receive sensory inputs .
however , unmyelinated nerve fibers are believed to transfer nociception such as pain , itching , stinging and burning derived from chemical or thermal stimuli . among them , the function of transient receptor potential ( trp ) receptors is very unique because they transfer the signal not only in the neuronal perception pathway but also in the cellular signal pathway where it appears as an ion channel .
this review explains the cooling sensation of skin which has not been evaluated thoroughly , and provides insights for further clinical applications . |
inspired by the power of modularity in synthetic design and the mathematical merits of oligomerization as a central theme for molecular diversification ( 7 , 8) , we hypothesized that a new class of chiral and conformationally constrained synthetic oligomers , where chirality could be readily addressed in concert with local conformational preferences , would define a substantial advance in discovery - oriented science .
being unbound by the desire to mimic three - dimensional motifs seen in biological macromolecules ( 920 ) , we turned to the structure of polyketide - derived small molecule natural products for inspiration ( figure 1b).(21 ) members of this class often contain relatively simple stereochemically - defined structural motifs that , in sum , dictate their global conformational preferences . with this molecular characteristic as a guiding principle ,
we have designed and executed a two - step iterative synthetic pathway to polyketide - inspired oligomers termed copas ( chiral oligomers of pentenoic amides)(figure 1c ) , where a central n - substituted 5-amino-2,4-dialkyl-3-pentenoic amide provides a rigid and chiral environment about each monomeric unit .
the control of conformation resulting from this motif is substantial , and is based on the minimization of non - bonded steric interactions about the -branched trisubstituted alkene and -branched tertiary amide.(22 ) as illustrated in figure 1d , each of these structural motifs imparts substantial rigidification , as the c2 proton is constrained to being in - plane with the c4-alkyl group ( i.e. me ) , and r - amide substituent defining a rigid chiral environment at each monomer , where the amide and alkyl substitution emerging from this core are precisely oriented in three - dimensional space . while the local effect of stereochemistry and substitution within each copa core unit is clear ( figure 1d ) ,
the combined influence of distinct chiral subunits on the gross conformational preferences of a copa oligomer is more difficult to conceptualize / predict . for an initial assessment of the potential unique properties of chiral copa oligomers
, we turned to molecular mechanics calculations offered within spartan-08 ( conformer distribution , mmff model / monte - carlo algorithm ) . at the outset
, we embraced the notion that these calculations would only provide a uniform lens through which to observe differences associated with model oligomers , and would likely not depict relevant solution phase behavior .
that said , analysis of homogeneously substituted oligomer backbones ( copa vs. peptoid ) with this computational method leads to the identification of some striking differences .
first , for the copa oligomer , the three - dimensional structures of the lowest energy conformers found within each isomeric series ( i.e. four random examples are depicted rrrr , rrss , rsrs , and rssr ) are unique with respect to one another , despite their identical polymethylated substitution pattern .
second , the analysis of each stereoisomer resulted in the identification of two conformers within 1.1 kcal / mol of the low energy conformer found .
using the identical molecular mechanics calculation for analysis of a polymethylated peptoid tetramer ( mw = 371.4 ) and the larger peptoid octamer of related molecular weight to the copa tetramers analyzed ( mw = 655.7 ) , seven and thirteen conformers were identified within 1.1 kcal / mol of the low energy conformation found for each ( this analysis includes the mirror images of the conformations depicted ) . while we are mindful of not over - interpreting these results , the analysis described is consistent with the hypothesis that copas are more conformationally restricted than peptoids , and stereoisomeric copa oligomers have disparate conformational preferences .
synthetically , copas have been designed to be accessible using a sub - monomer route akin to that employed in peptoid synthesis ( figure 3a ; 23).(2325 ) this allows simple primary amines , of which hundreds are commercially available , to be employed as one of the diversity elements in split - and - pool library synthesis a critically important characteristic to enable the synthesis of large compound collections necessary to increase the likelihood of finding high affinity hits in unbiased screens.(26 ) furthermore , this design was optimistically thought to be compatible with ms - ms analysis , where compound from a single bead would be sufficient to decode the precise structure of oligomer present . as such , one could avoid employing an encoding strategy to elucidate hit - structure.(27 ) to affect this strategy , we required a practical and scalable synthesis of both antipodes of chloropentenoic acids like 1 ( figure 3b ) .
ideally , this synthesis would also be capable of delivering future analogs of 1 with diverse c2 and c4 substitution .
therefore , a convergent synthetic pathway was targeted to facilitate future analog generation that could avoid chromatographic purification at any step , proceed from readily available starting materials , limit the use of air and moisture sensitive reagents , and deliver the chiral monomers with high levels of stereochemical fidelity .
synthesis of the propionyl oxazolidinone 4 was accomplished without the requirement of a highly reactive base ( 28 ) and subsequent stereoselective aldol reaction with methacrolein ( 6 ) was achieved under reaction conditions that do not require pre - generation of a metal enolate.(29 ) isolated by simple filtration through silica , the tms - ether 7 was converted to the stereodefined allylic chloride 8 by nb - mediated stereoselective halogenation ( e : z 20:1).(30 ) while seemingly difficult to accomplish in a highly selective fashion , hydrolysis of the imide proceeded uneventfully ( without significant hydrolysis of the allylic chloride ) and delivered 1 in 90% ee and 41% overall yield .
notably , this synthesis procedure delivers optically active 1 in acceptable yield and purity through a simple four - step sequence that does not require a single chromatographic operation.(31 ) as an indication of the robust nature of this sequence , routine syntheses of 1 ( 4 g ) were performed in just a few days from 10 g of 4 . as depicted in figure 3c , solution phase amide bond formation with a simple secondary amine ( via the mixed anhydride ) proceeded effectively , in this case delivering the chloroamide 10 in 94% yield . unlike related coupling reactions for the synthesis of peptides ,
subsequent coupling with benzylamine proceeded in a similarly straightforward manner , delivering aminoamide 11 in 82% yield .
this simple two - step sequence validated the central steps of the proposed oligomerization of 1 and confirmed that chiral chloroacids of this and related structure can be functionalized in a similar manner to -bromoacetic acid in peptoid synthesis .
subsequent homologation of 11 with either enantiomer of 1 leads to the production of the corresponding dimers 12 and 13 with similarly high levels of efficiency .
this success indicates that double asymmetric relationships between amine 11 and acid 1 have little impact on chemical efficiency for this bond construction a critically important virtue of this chemistry , as split - and - pool techniques will aim to prepare all combinations of stereochemistry along the growing copa backbone .
with regard to this later consideration associated with the projected application of this chemistry in split - and - pool format , we recognize that oligomerization of chiral monomers of 90% ee will result in the production of minor diastereomers on each bead
. the combinatorial nature of this process however will ensure that the population contains beads that present these minor impurities as major constituents .
in this way , a built in control mechanism exists to aid in the analysis of assay results as the minor component on any particular bead will be present as a major component on a different bead within the collection .
that said , we anticipate that conditions can be found in future studies to crystallize intermediate 7 or 8 as a means to attain isomeric homogeneity .
moving forward to explore the utility of copa oligomers as a potential source of protein ligands , a library of tetramers was prepared by split - and - pool methods . to be compatible with our on - bead screening platform ( 32 ) , we selected 160 m tentagel beads that were functionalized with a tetrameric polyamide ( figure 3e ) , the structure of which was selected to optimize subsequent ms - based structure elucidation.(31 ) targeting a library of 160,000 members , we employed ten primary amines and two pentenoic acids as depicted in figure 3d . since we planned to carry out structural elucidation by mass spectrometry , we employed a heavy atom label ( cd3 at c2 ) to correlate differences in the mass of fragment ions with absolute stereochemistry of the chloropentenoic acid monomer . alongside these efforts ,
a library of peptoid tetramers was prepared with the same amines used for the copa library ( figure 3f ) in an effort to establish a baseline for comparison between these two synthetic oligomer platforms .
maldi mass spectra revealed a single strong peak for the copas released from several individual beads chosen randomly from the library , indicating that each bead displays predominantly a single compound and that each synthetic step proceeded in high yield .
having established that the library was of high quality , it was screened against the dna - binding domain of p53 , an important transcription factor that regulates a variety of genes involved in cell cycle control and apoptosis .
more than half of human cancers express inactive p53 due to the presence of missense mutations in the dna - binding domain ( dbd ) that destabilize the folding of the protein.(33 ) there is considerable interest in the identification of chemical chaperones whose binding to p53 might stabilize the wild - type , functional , folded conformation.(34 ) since transcription factors are generally considered to be extremely challenging targets for small molecules ( 35 ) , we considered p53 recognition a stringent test of the utility of this new class of compounds .
purified , bacterially expressed , flag - tagged p53 dbd ( 10 m ) was incubated with the bead - displayed copa library in the presence of high levels of competitor proteins to suppress non - specific binding events .
the beads were then washed and treated with anti - flag antibody followed , after another washing step , by anti - igg antibodies conjugated to red quantum dots .
several beads with a strong red halo surrounding them , indicating binding of the quantum dot via the p53-flag / anti - flag antibody / anti - igg - qd sandwich complex , were observed ( figure 4a ) .
these , as well as some beads with weaker staining , were picked using a micropipette . in all , 22 beads were collected .
six of these putative hits proved to be ligands for either anti - flag antibody or the secondary antibody - conjugated quantum dots .
the same experiment was done with the peptoid library . in this case , no obvious hits with strong red halos were observed , but several more weakly fluorescent beads were picked .
the beads were separated in wells of a microtiter plate and the oligomer was released from the resin via cnbr - mediated cleavage of a methionine residue in the linker . while strong precursor ion signals were observed in the maldi mass spectrum for the copas , sequence specific product ions were not produced via collision - induced dissociation .
therefore , we attempted to sequence these molecules via tandem esi mass spectrometry with electron transfer dissociation ( etd).(36 ) this etd approach proved reasonably successful and the sequences of 8 of the 16 copa hits could be determined unequivocally ( figure s7 ) .
these eight molecules were re - synthesized with a fluorescein tag ( figure s8 ) and tested for binding to p53 by fluorescence anisotropy .
the best of these , compound 14a shown in figure 4c , bound to the p53 dbd with a kd of approximately 10 m , but did not bind detectably ( kd > 500 m ) to three control proteins . to the best of our knowledge ,
copa 14a constitutes the first non - covalent ( 37 ) small molecule ligand for the wild - type ( 38 ) p53 dna - binding domain .
the addition of an oligonucleotide that binds p53 tightly did not disrupt the p53-copa complex , indicating that the synthetic ligand does not recognize the dna - binding surface of the p53 core domain ( supplementary figure 13 ) .
the same set of experiments was carried out for the peptoids collected as possible hits in the screening experiment .
not surprisingly , given the low intensity of qd fluorescence observed on the beads , none of the peptoids exhibited binding to the p53 dna - binding domain ( kds > 500 m ) .
this is interesting in that the copa and peptoid libraries contained exactly the same amine - derived side chains .
furthermore , controls with independently synthesized copa diastereomers of 14a and an achiral peptoid bearing the identical amine side chains as 14a , all showed a substantial decrease of affinity for the p53 dbd ( figures s11 and s12 ) . while more work will be required before definitive conclusions can be reached , the results of these experiments are consistent with the proposition that conformationally constrained copas may be superior to peptoids as a source of protein ligands .
inspired by the power of modularity in synthetic design and the mathematical merits of oligomerization as a central theme for molecular diversification ( 7 , 8) , we hypothesized that a new class of chiral and conformationally constrained synthetic oligomers , where chirality could be readily addressed in concert with local conformational preferences , would define a substantial advance in discovery - oriented science .
being unbound by the desire to mimic three - dimensional motifs seen in biological macromolecules ( 920 ) , we turned to the structure of polyketide - derived small molecule natural products for inspiration ( figure 1b).(21 ) members of this class often contain relatively simple stereochemically - defined structural motifs that , in sum , dictate their global conformational preferences . with this molecular characteristic as a guiding principle ,
we have designed and executed a two - step iterative synthetic pathway to polyketide - inspired oligomers termed copas ( chiral oligomers of pentenoic amides)(figure 1c ) , where a central n - substituted 5-amino-2,4-dialkyl-3-pentenoic amide provides a rigid and chiral environment about each monomeric unit .
the control of conformation resulting from this motif is substantial , and is based on the minimization of non - bonded steric interactions about the -branched trisubstituted alkene and -branched tertiary amide.(22 ) as illustrated in figure 1d , each of these structural motifs imparts substantial rigidification , as the c2 proton is constrained to being in - plane with the c4-alkyl group ( i.e. me ) , and r - amide substituent defining a rigid chiral environment at each monomer , where the amide and alkyl substitution emerging from this core are precisely oriented in three - dimensional space .
while the local effect of stereochemistry and substitution within each copa core unit is clear ( figure 1d ) , the combined influence of distinct chiral subunits on the gross conformational preferences of a copa oligomer is more difficult to conceptualize / predict . for an initial assessment of the potential unique properties of chiral copa oligomers
, we turned to molecular mechanics calculations offered within spartan-08 ( conformer distribution , mmff model / monte - carlo algorithm ) . at the outset
, we embraced the notion that these calculations would only provide a uniform lens through which to observe differences associated with model oligomers , and would likely not depict relevant solution phase behavior .
that said , analysis of homogeneously substituted oligomer backbones ( copa vs. peptoid ) with this computational method leads to the identification of some striking differences .
first , for the copa oligomer , the three - dimensional structures of the lowest energy conformers found within each isomeric series ( i.e. four random examples are depicted rrrr , rrss , rsrs , and rssr ) are unique with respect to one another , despite their identical polymethylated substitution pattern .
second , the analysis of each stereoisomer resulted in the identification of two conformers within 1.1 kcal / mol of the low energy conformer found . using the identical molecular mechanics calculation for analysis of a polymethylated peptoid tetramer ( mw = 371.4 ) and the larger peptoid octamer of related molecular weight to the copa tetramers analyzed ( mw = 655.7 ) , seven and thirteen conformers were identified within 1.1 kcal / mol of the low energy conformation found for each ( this analysis includes the mirror images of the conformations depicted ) . while we are mindful of not over - interpreting these results , the analysis described is consistent with the hypothesis that copas are more conformationally restricted than peptoids , and stereoisomeric copa oligomers have disparate conformational preferences .
synthetically , copas have been designed to be accessible using a sub - monomer route akin to that employed in peptoid synthesis ( figure 3a ; 23).(2325 ) this allows simple primary amines , of which hundreds are commercially available , to be employed as one of the diversity elements in split - and - pool library synthesis a critically important characteristic to enable the synthesis of large compound collections necessary to increase the likelihood of finding high affinity hits in unbiased screens.(26 ) furthermore , this design was optimistically thought to be compatible with ms - ms analysis , where compound from a single bead would be sufficient to decode the precise structure of oligomer present . as such , one could avoid employing an encoding strategy to elucidate hit - structure.(27 ) to affect this strategy , we required a practical and scalable synthesis of both antipodes of chloropentenoic acids like 1 ( figure 3b ) .
ideally , this synthesis would also be capable of delivering future analogs of 1 with diverse c2 and c4 substitution .
therefore , a convergent synthetic pathway was targeted to facilitate future analog generation that could avoid chromatographic purification at any step , proceed from readily available starting materials , limit the use of air and moisture sensitive reagents , and deliver the chiral monomers with high levels of stereochemical fidelity .
synthesis of the propionyl oxazolidinone 4 was accomplished without the requirement of a highly reactive base ( 28 ) and subsequent stereoselective aldol reaction with methacrolein ( 6 ) was achieved under reaction conditions that do not require pre - generation of a metal enolate.(29 ) isolated by simple filtration through silica , the tms - ether 7 was converted to the stereodefined allylic chloride 8 by nb - mediated stereoselective halogenation ( e : z 20:1).(30 ) while seemingly difficult to accomplish in a highly selective fashion , hydrolysis of the imide proceeded uneventfully ( without significant hydrolysis of the allylic chloride ) and delivered 1 in 90% ee and 41% overall yield .
notably , this synthesis procedure delivers optically active 1 in acceptable yield and purity through a simple four - step sequence that does not require a single chromatographic operation.(31 ) as an indication of the robust nature of this sequence , routine syntheses of 1 ( 4 g ) were performed in just a few days from 10 g of 4 . as depicted in figure 3c , solution phase amide bond formation with a simple secondary amine ( via the mixed anhydride ) proceeded effectively , in this case delivering the chloroamide 10 in 94% yield . unlike related coupling reactions for the synthesis of peptides ,
subsequent coupling with benzylamine proceeded in a similarly straightforward manner , delivering aminoamide 11 in 82% yield .
this simple two - step sequence validated the central steps of the proposed oligomerization of 1 and confirmed that chiral chloroacids of this and related structure can be functionalized in a similar manner to -bromoacetic acid in peptoid synthesis .
subsequent homologation of 11 with either enantiomer of 1 leads to the production of the corresponding dimers 12 and 13 with similarly high levels of efficiency .
this success indicates that double asymmetric relationships between amine 11 and acid 1 have little impact on chemical efficiency for this bond construction a critically important virtue of this chemistry , as split - and - pool techniques will aim to prepare all combinations of stereochemistry along the growing copa backbone .
with regard to this later consideration associated with the projected application of this chemistry in split - and - pool format , we recognize that oligomerization of chiral monomers of 90% ee will result in the production of minor diastereomers on each bead .
the combinatorial nature of this process however will ensure that the population contains beads that present these minor impurities as major constituents . in this way ,
a built in control mechanism exists to aid in the analysis of assay results as the minor component on any particular bead will be present as a major component on a different bead within the collection .
that said , we anticipate that conditions can be found in future studies to crystallize intermediate 7 or 8 as a means to attain isomeric homogeneity .
moving forward to explore the utility of copa oligomers as a potential source of protein ligands , a library of tetramers was prepared by split - and - pool methods . to be compatible with our on - bead screening platform ( 32 ) , we selected 160 m tentagel beads that were functionalized with a tetrameric polyamide ( figure 3e ) , the structure of which was selected to optimize subsequent ms - based structure elucidation.(31 ) targeting a library of 160,000 members , we employed ten primary amines and two pentenoic acids as depicted in figure 3d . since we planned to carry out structural elucidation by mass spectrometry , we employed a heavy atom label ( cd3 at c2 ) to correlate differences in the mass of fragment ions with absolute stereochemistry of the chloropentenoic acid monomer . alongside these efforts ,
a library of peptoid tetramers was prepared with the same amines used for the copa library ( figure 3f ) in an effort to establish a baseline for comparison between these two synthetic oligomer platforms .
maldi mass spectra revealed a single strong peak for the copas released from several individual beads chosen randomly from the library , indicating that each bead displays predominantly a single compound and that each synthetic step proceeded in high yield .
having established that the library was of high quality , it was screened against the dna - binding domain of p53 , an important transcription factor that regulates a variety of genes involved in cell cycle control and apoptosis .
more than half of human cancers express inactive p53 due to the presence of missense mutations in the dna - binding domain ( dbd ) that destabilize the folding of the protein.(33 ) there is considerable interest in the identification of chemical chaperones whose binding to p53 might stabilize the wild - type , functional , folded conformation.(34 ) since transcription factors are generally considered to be extremely challenging targets for small molecules ( 35 ) , we considered p53 recognition a stringent test of the utility of this new class of compounds .
purified , bacterially expressed , flag - tagged p53 dbd ( 10 m ) was incubated with the bead - displayed copa library in the presence of high levels of competitor proteins to suppress non - specific binding events .
the beads were then washed and treated with anti - flag antibody followed , after another washing step , by anti - igg antibodies conjugated to red quantum dots .
several beads with a strong red halo surrounding them , indicating binding of the quantum dot via the p53-flag / anti - flag antibody / anti - igg - qd sandwich complex , were observed ( figure 4a ) . these , as well as some beads with weaker staining , were picked using a micropipette . in all , 22 beads were collected .
six of these putative hits proved to be ligands for either anti - flag antibody or the secondary antibody - conjugated quantum dots .
the same experiment was done with the peptoid library . in this case , no obvious hits with strong red halos were observed , but several more weakly fluorescent beads were picked .
the beads were separated in wells of a microtiter plate and the oligomer was released from the resin via cnbr - mediated cleavage of a methionine residue in the linker . while strong precursor ion signals were observed in the maldi mass spectrum for the copas , sequence specific product ions were not produced via collision - induced dissociation .
therefore , we attempted to sequence these molecules via tandem esi mass spectrometry with electron transfer dissociation ( etd).(36 ) this etd approach proved reasonably successful and the sequences of 8 of the 16 copa hits could be determined unequivocally ( figure s7 ) .
these eight molecules were re - synthesized with a fluorescein tag ( figure s8 ) and tested for binding to p53 by fluorescence anisotropy .
the best of these , compound 14a shown in figure 4c , bound to the p53 dbd with a kd of approximately 10 m , but did not bind detectably ( kd > 500 m ) to three control proteins . to the best of our knowledge ,
copa 14a constitutes the first non - covalent ( 37 ) small molecule ligand for the wild - type ( 38 ) p53 dna - binding domain .
the addition of an oligonucleotide that binds p53 tightly did not disrupt the p53-copa complex , indicating that the synthetic ligand does not recognize the dna - binding surface of the p53 core domain ( supplementary figure 13 ) .
the same set of experiments was carried out for the peptoids collected as possible hits in the screening experiment .
not surprisingly , given the low intensity of qd fluorescence observed on the beads , none of the peptoids exhibited binding to the p53 dna - binding domain ( kds > 500 m ) .
this is interesting in that the copa and peptoid libraries contained exactly the same amine - derived side chains .
furthermore , controls with independently synthesized copa diastereomers of 14a and an achiral peptoid bearing the identical amine side chains as 14a , all showed a substantial decrease of affinity for the p53 dbd ( figures s11 and s12 ) . while more work will be required before definitive conclusions can be reached , the results of these experiments are consistent with the proposition that conformationally constrained copas may be superior to peptoids as a source of protein ligands .
in summary , we have developed a new class of natural product - inspired oligomeric compounds that promises to be a valuable source of protein ligands .
copas are unusual amongst synthetic small molecule oligomers in that their stereodefined structure is readily malleable and their backbone conformations are supported by concepts long utilized in organic synthesis for acyclic stereocontrol [ such as minimization of a(1,3 ) interactions ] these features offer a unique and powerful means of addressing the disposition of all main chain substituents in three - dimensional space.(39 ) in essence , we describe a practical chemical solution to diversity - oriented library construction that couples building block diversity to substantial scaffold diversity .
this is noteworthy , because the desirability of scaffold diversity in natural product - like libraries has been well documented ( 40 ) , and current solutions to this problem require careful synthesis planning to accomplish strategic and divergent reactivity of complex organic intermediates in a library synthesis.(4143 ) further compounding the virtues of copas as a chemical foundation to discovery - oriented science , the synthetic strategy described here is completely compatible with split - and - pool solid phase synthesis , making very large libraries readily accessible .
moreover , the highly practical and scalable synthesis of either optically pure antipode of the chloropentenoic acid building blocks ( i.e. 1 ) , combined with the use of simple primary amines as the source of side chain diversity , allows the synthesis of potentially millions of compounds at a modest cost . finally , copa libraries synthesized on hydrophilic tentagel beads can be employed in a variety of inexpensive , yet powerful , binding screens ( 32 , 4445 ) , hence eliminating the requirement of substantial infrastructure to maintain diverse compound collections for use in traditional high - throughput screening .
we look forward to future developments that explore the power of this chemistry in combination with available on - bead screening technologies as a platform for the discovery of bioactive synthetic molecules .
| the discovery of new compounds for the pharmacological manipulation of protein function often embraces the screening of compound collections , and it is widely recognized that natural products offer beneficial characteristics as protein ligands .
much effort has therefore been focused on natural product - like libraries , yet the synthesis and screening of such libraries is often limited by one or more of the following : modest library sizes and structural diversity , conformational heterogeneity , and the costs associated with the substantial infrastructure of modern high - throughput screening centers . here , we describe the design and execution of an approach to this broad problem by merging principles associated biologically - inspired oligomerization and the structure of polyketide - derived natural products . a novel class of chiral and conformationally - constrained oligomers is described ( termed chiral oligomers of pentenoic amides copas ) that offers compatibility with split - and - pool methods and can be screened en masse in a batch mode .
we demonstrate that a copa library containing 160,000 compounds is a useful source of novel protein ligands by identifying a non - covalent synthetic ligand to the dna - binding domain of the p53 transcription factor . |
the numbers of hypothesis tests in science and genomics , in particular , are increasing at an ever - expanding rate .
total studies and hypothesis tests per study have both increased exponentially since the 1920s when the conventional 0.05 significance level was first adopted .
simultaneously , as technological advances have provided the means to easily measure , store and manipulate huge quantities of data , the need for stronger a priori testing of one hypothesis has become more complex to justify . with almost inevitable large numbers of hypothesis tests in a single experiment comes the well - recognized need to use some type of statistical correction for multiple testing to avoid generating ever - increasing numbers of false - positive results . the more stringent level of evidence required necessarily reduces the power to identify a true - positive finding . as a consequence
, some experts now advocate larger and larger sample sizes for genomic studies that are impractical for many diseases and , even when practical , may require broader , more heterogeneous phenotype definitions and less costly , more imprecise phenotypic measurements to accomplish .
current genotype microarray technology is now approaching a capacity of five million single - nucleotide polymorphisms ( snps ) per study .
as whole genome sequencing becomes widely available , the number of tests will continue to increase .
investigation of a variety of phenotype definitions , genetic models and subsets of individuals also increases the total number of hypothesis tests actually underlying any reported finding .
other fields ( for example , neuroimaging or data mining of medical records ) face a similar burden of multiplicity of tests .
cross - disciplinary studies , such as genomic analyses of neuroimaging or other high - dimensional phenotypes , will only further expand the problem .
there are reasonable arguments as to when it is appropriate to statistically adjust for multiple tests and how best to do so .
the universe of tests subject to adjustment , the level of stringency , underlying assumptions and statistical methods are all subjects of debate .
whatever adjustment is used , extensive multiple testing inevitably leads to some loss of power or the need for a compensatory increase in the detectable effect size and/or sample size .
this study examines the statistical cost ' defined in terms of power , targeted effect size or sample size imposed by large and ever - increasing numbers of hypothesis tests , for the broad class of tests based on statistical estimates that follow a normal distribution in large samples : ( 1 ) we develop a novel formulation of this relationship and ( 2 ) discuss its implications for genomic and other studies in the era of high - throughput technology .
( 3 ) explicit numerical results and a user - friendly calculator for study design and comparison of alternative high - throughput technologies are also provided .
this study extends and expands the basic principles of power analysis found in introductory biostatistics texts .
we consider the class of hypothesis tests based on a statistic , b , such that the standardized z - statistic , , has a standard normal distribution in large samples , where b is the expected value of b and is its standard error in a sample of size n. owing to the central limit theorem and its extensions , this situation encompasses most commonly used statistical tests .
for example , b might be a coefficient in a regression model , a sample proportion , a difference of two means , the log of an odds ratio ( or ) or another maximum likelihood estimate .
one - degree - of - freedom tests are also covered owing to the relationship between the normal and distributions .
let be the significance level or type i error probability and be the type ii error probability , the probability that the null hypothesis is accepted for an alternative value of b0 . by definition ,
the power of the test is 1. a single two - sided hypothesis test is illustrated in figure 1a .
the unit of measurement for the x axis is the standard error for a sample of size n. the curve on the left , centered at zero , is the sampling distribution of b under the null hypothesis in large samples ( that is , b has a normal distribution with mean 0 and standard error ) .
the critical values are c standard errors , where c depends on and is taken from the cumulative normal distribution . for =0.05 , c=1.96 .
the null hypothesis will be rejected if b is more than 1.96 standard errors from zero ( that is , in either shaded region ) .
the curve on the right centered at b is the sampling distribution of b under the alternative hypothesis that the expected value of b = b .
( throughout , we use effect size ' to refer to the expected value of b under the alternative .
our results may not hold for other definitions that are used in the literature . )
the power to reject the null hypothesis is the percentage of area under the alternative distribution inside the bold - outlined area above the upper critical value c. the distance between the critical value c and the effect size b , denoted by d , is also a multiple of the standard error obtained from the normal distribution . for 80% power , d=0.84 .
thus , 80% power is reached for a 0.05 significance test when the effect size is or 2.80 standard errors away from zero . for 90% power , d=1.28 and
formally , the effect size is , where c is the 100th percentile of the normal distribution and d is the 100th percentile of the normal distribution . to evaluate the statistical cost of multiple tests ,
this conservative approach sets the per - test significance level to /h , which guarantees for an experiment comprised of h tests that the probability of one or more false positives ( known as the family - wise error rate ) is no more than . this is true for any possible relationship among the tests .
( see discussion for implications for other less conservative multiple - testing corrections . ) for =0.05 and h=2 , the bonferroni critical value is 2.24 , measured in standard errors .
increasing the critical value reduces the bold - outlined area under the alternative distribution , reducing power from 80 to 71% ( figure 1b ) .
note that the cost of going from a single test with 80% power to two bonferroni - adjusted tests is always a reduction of power to 71% if the initial effect size and sample size are maintained .
the number does not depend on the type of test , statistical model , effect size or sample size .
the cost of multiple tests can also be quantified in terms of the effect size that can be detected with the original , single - test power and sample size .
the new detectable effect size is found by moving the alternative hypothesis to the left by units ( figure 1c ) to accommodate the increase in the critical value . for h=2 , the minimum detectable effect size at 80% power
the detectable effect size for h tests relative to that for a single test is which depends only on h , and and not other aspects of the test , the sample size or the original effect size .
note that the impact of h is only through the critical value and not power , as power is evaluated on a per - test basis . a third way to quantify
the cost of multiple tests is the increase in sample size needed to maintain the original level of power at the original effect size .
if the sample size is increased by a factor m , it has the effect of dividing the standard error by , narrowing the widths of both the null and alternative curves accordingly ( figure 1d ) . to offset the addition of , the distance between the two curves expressed in terms of the new standard error for the larger sample size and h tests should equal the distance expressed in the original standard errors for a single test
thus , m should satisfy or for two tests , =0.05 and 80% power , the sample size should be multiplied by m=1.2 to maintain the original power at the original effect size .
again , m depends only on h , and and not the effect size or original sample size .
note that the sample size multiplier is the square of the effect size multiplier . more generally , suppose a study design with h tests at unadjusted significance level and power 1 is to be compared to a second study design with h * tests at unadjusted significance level and power 1 , then the detectable effect size for the second design relative to the effect size of the first design is where the numbers in the denominator are based on the initial design and the numbers in the numerator are based on the second .
furthermore , a sample size multiplier for the second design relative to the first is given by numerical results were calculated using the normal distribution and expressions ( 1 ) and ( 2 ) in r. all tests are two tailed and , if not otherwise specified , at significance level =0.05 . logs , unless otherwise indicated , are base 10 .
a cost of multiple tests calculator that implements equations ( 1)(4 ) in microsoft excel 2003 for any choices of h , and is also provided ( supplementary table 1 ) .
for a fixed targeted effect size and fixed sample size , power decreases as the number of tests and corresponding critical value increase ( table 1 , figure 2a ) .
if the power for a single test is 80% , the power is approximately 50% for 10 ; 10% for 1000 ; and 1% for 100 000 bonferroni - adjusted tests . to avoid a drop in nominal power without increasing sample size ,
an investigator may target larger effect sizes ( table 1 , figure 2b ) using equation ( 1 ) .
for one million tests , the effect size multiplier is 2.25 at 80% power and 2.08 at 90% power .
suppose it has been determined that a sample of 100 yields 80% power to detect a difference in group means when the mean in group 1 is 10 and the mean in group 2 is 12 .
the original sample size of 100 would also yield 80% power to detect a mean difference of 2.25 2=4.50 , with bonferroni adjustment for one million tests .
the effect size multiplier works on the scale of the underlying normally distributed test statistic .
for example , effect size multiplication for an or should be carried out after conversion to the natural log scale .
suppose 500 total cases and controls provide 90% power to detect an or=1.2 , the effect size multiplier for 90% power and 1000 tests is 1.65 .
since loge(1.2)=0.18 , the same sample of 500 yields 90% power in 1000 tests to detect a loge(or)=1.65 0.18=0.30 or , equivalently , an or = exp(0.30)=1.35 . to compensate for a greater number of tests , a more realistic strategy may be to increase the sample size ( equation 2 ) .
table 1 and figure 2c give sample sizes needed to maintain the original power at the original targeted effect size .
for one million tests , the sample size multiplier is 5.06 for 80% power and 4.33 for 90% power , using equation ( 2 ) . in the first example above
, 506 subjects would be sufficient to reach 80% power to detect that the means differ by 2 in one million bonferroni - adjusted tests . although it might appear counterintuitive , the sample size multiplier is smaller for 90% power because the initial sample size is larger . in the same example
, 132 subjects would be needed to reach 90% power for one test . for 90% power for one million tests , 4.33 132=572 subjects are needed .
noting the nearly linear relationship in figure 2c , we also obtained an approximate rule - of - thumb for the sample size multiplier by fitting zero - intercept linear regression models to the results in figure 2c .
the estimated slopes show that m is approximately , where =1.2 for 50% power , 0.68 for 80% power , 0.55 for 90% power and 0.38 for 99% power .
the rate at which the critical value and , consequently , the effect size and sample size multipliers increase becomes slower and slower as the number of tests becomes larger ( figure 2d ) , owing to the exponential decline in the tails of the normal density .
for example , effect size multipliers for one million vs ten million tests at 80% power are 2.25 and 2.39 , respectively .
, ten million tests require only a 6% increase in the targeted effect size or a 13% increase in the sample size when compared to one million tests .
in contrast , 10 tests require a 30% increase in the targeted effect size or a 70% increase in the sample size as compared with a single test . for 80% power and one billion or one trillion tests , the required sample sizes , respectively , are approximately 7 or 9 times that needed for a single test . see table 1 for some numerical results and the provided excel calculator ( supplementary table 1 ) to explore unreported results and specific study designs . the sample size and effect size multipliers can be used in other ways .
for example , different numbers of tests can be compared by taking the ratio of two sample size multipliers or the effect size multipliers .
the detectable effect size for a larger array with 2.5 million snps would yield equivalent power for a 2% bigger effect size or , alternatively , a 4% bigger sample size .
a smaller array with 560 000 snps would yield equivalent power for a 2% smaller effect size or a 4% smaller sample size . following a data analysis involving multiple tests ,
the sample size multiplier can also be used to find the effective sample size for a single test post hoc .
for example , suppose 1000 hypothesis tests have been carried out by permutation test in a sample of 120 subjects with negative results .
the observation that the relationship between the number of hypothesis tests and the detectable effect size or required sample size depends only on the choice of significance level and power covers most commonly used statistical tests .
this relationship is independent of the original effect size and sample size and other specific characteristics of the data , the statistical model and test procedure .
our results show that most of the cost of multiple testing is incurred when the number of tests is relatively small .
the impact of multiple tests on the detectable effect size or required sample size is surprisingly small when the overall number of tests is large . when the number of tests reaches extreme levels , on the order of a million , doubling or tripling the number of tests has an almost negligible effect .
this is reassuring in light of continuing developments in methods of high - throughput data collection and the trend toward data mining and exploration of a variety of statistical models entailing greater numbers of tests and comparisons .
in addition , we used our results to create a power , effect size and sample size calculator to facilitate the comparison of alternative large - scale study designs .
underlying the results is the fact that the critical value in the context of extreme multiple testing is not affected much by typical changes in the number of tests .
thus , even a rigorous application of the bonferroni correction that accounts for every one of a large number of tests is unlikely to change actual hypothesis test results .
consequently , multiple analyses of alternative genetic or statistical models impose minimal costs in a statistical sense in the context of a one million snp genome - wide association study .
nonetheless , very large sample sizes are still required for large numbers of tests if , as many have proposed , it is necessary to target very small effect sizes that would require a sizable sample even for a single test .
our results also show that , for the range of contemporary genotyping platforms , there is little difference with respect to detectable effect size or required sample size . at 80% power ,
the detectable effect size for a 2.5 million snp platform is only 1.8% bigger than for 1.2 million tests and 4.1% bigger than for 560 000 tests .
this relatively small increase is potentially offset by the fact that , for any risk - related variant in the genome , denser chips are more likely to include a tagging snp with a larger effect size .
thus , consideration of alternative platforms should be primarily based on monetary costs and not the possibility of having too many tests . '
whole genome sequencing theoretically provides data on approximately three billion base pairs , but might reasonably be expected to generate no more than 100 million variant sites .
the sample size required to maintain equivalent power for that many tests is only 30% greater than that required for a 560k snp chip .
newer , denser snp chips do include lower frequency variants than older chips , whereas whole genome sequencing theoretically enables identification of variants appearing at any frequency in the sample .
the effect sizes of these lower frequency variants are likely to differ on average from those of more common polymorphisms , an issue that should also be considered when designing a study .
our results also suggest that the bonferroni correction may not be as conservative as sometimes thought , when compared with multiple testing methods based on the effective number of tests , either explicitly or implicitly through permutation testing or other means . in a one million snp genome - wide association study ,
a precise estimate of the effective number of independent tests ' as , say , 900 000 would not much improve the detectable effect size over the bonferroni correction and significant findings are likely to be the same . in practice
, the effective number of independent tests will usually be larger . in this paper , we have not considered significance levels other than 0.05 . for a very large number of tests , it may be appropriate to use a less stringent standard for family - wise error .
this study is limited in that the calculations are based on the normal approximation and rely on its accuracy .
although this is usual for analytic discussions of power , this caveat should be considered before applying our formulae to small sample sizes .
our results have also not been shown to apply to hypothesis tests based on other statistical distributions , such as the f distribution or the distribution with more than one degree of freedom .
we have also followed common practice in ignoring any possible dependence of the standard error on the true value of b.
lastly , there is a practical limit to the size of data set that can be conveniently analyzed and interpreted even if samples can be generated that theoretically provide adequate power .
our theoretical results do not address problems that can arise when conclusions are based on extremely small p - values .
extreme results sometimes reflect rare data errors , ascertainment biases , confounding or other problems of study design or implementation .
large sample sizes do not reduce bias ; rather , large samples increase the chance of a false positive finding in the presence of bias .
furthermore , a p - value is a random variable , subject to variation from sample to sample .
when tiny p - values are required , only one or two differences in the sample data can change a result from true positive to false negative or false negative to true positive .
this study also assumes that samples are drawn from the same population and effect sizes are the same for different numbers of tests .
larger samples may require less expensive data collection protocols to offset increased costs in time and money .
furthermore , subjects may be recruited from a wider variety of sources and be examined at different sites in order to increase their numbers .
such strategies can introduce greater heterogeneity and effectively reduce the average effect size in larger samples .
choices among alternative chip or sequencing technologies can also implicitly correspond to different effect sizes .
for example , a denser snp chip may have a snp closer to or in stronger linkage disequilibrium with a causal variant .
new , denser chips and sequencing also permit the assessment of lower frequency variants , which may individually account for less of the population variance of a trait , although they also can have stronger effects on individual risk if lower frequencies are the end result of evolutionary selection
. none of these limitations are unique to this study . in conclusion , we have shown that feasible sample sizes can address surprisingly large numbers of hypothesis tests , even when care is taken to control the number of false positives by using an appropriately stringent statistical correction .
in fact , it might be theoretically possible to design a study that achieved a science - wide ' multiple test correction for , say , one trillion tests . despite this finding
, large samples are still needed to target the smaller effect sizes that are often anticipated to be associated with genetic variation .
| advances in high - throughput biology and computer science are driving an exponential increase in the number of hypothesis tests in genomics and other scientific disciplines .
studies using current genotyping platforms frequently include a million or more tests .
in addition to the monetary cost , this increase imposes a statistical cost owing to the multiple testing corrections needed to avoid large numbers of false - positive results . to safeguard against the resulting loss of power ,
some have suggested sample sizes on the order of tens of thousands that can be impractical for many diseases or may lower the quality of phenotypic measurements .
this study examines the relationship between the number of tests on the one hand and power , detectable effect size or required sample size on the other .
we show that once the number of tests is large , power can be maintained at a constant level , with comparatively small increases in the effect size or sample size .
for example at the 0.05 significance level , a 13% increase in sample size is needed to maintain 80% power for ten million tests compared with one million tests , whereas a 70% increase in sample size is needed for 10 tests compared with a single test .
relative costs are less when measured by increases in the detectable effect size .
we provide an interactive excel calculator to compute power , effect size or sample size when comparing study designs or genome platforms involving different numbers of hypothesis tests .
the results are reassuring in an era of extreme multiple testing . |
primary well - differentiated neuroendocrine tumors ( wdnets ) arising in the intracranial portion of the central nervous system ( cns ) are occasional findings . to date , less than 10 cases of cns wdnets have been reported , 3 of which originated from cerebral hemispheres or the skull base and another 3 were paragangliomas [ 1 , 2 , 3 , 4 , 5 , 6 ] .
on the other hand , the cns more frequently represents a metastatic site for primary poorly differentiated net arising elsewhere , most often in the lung ( small - cell lung cancer ) [ 7 , 8 ] . in such cases , other secondary lesions , such as liver , lung and lymph nodes metastases ,
patients with wdnet and brain metastases are rare ; typically they have a poor prognosis , with one study reporting a median survival time of 10.0 months after diagnosis of brain metastases .
overall , brain metastases occur in 1.56% of all wdnets , and their detection is usually preceded by the documentation of the primary , extracranial tumor [ 7 , 8 ] .
in august 2009 , a 38-year - old man referred to the hospital emergency department after a 2-month history of vertigos .
anamnesis and physical examination , including neurological evaluation , were not suggestive of any specific disease .
magnetic resonance ( mr ) imaging of the brain was performed which identified a large , heterogeneous contrast - enhancing lesion in the left cerebellar hemisphere , surrounded by significant edema ( fig . 1 , red arrows ) .
therefore , and given the potential risk for serious complications , immediate radical neurosurgery was performed , without previous systemic staging of the disease .
histology of the surgical specimen was compatible with a grade 2 ( g2 ) , wdnet localization with vaguely lobular architecture and sporadic small clusters of foamy macrophages .
lobules of neoplastic cells were separated by a thin network of fibrous septa enriched in vessels with a few aggregates of mature lymphocytes within them .
neoplastic cells contained polygonal , eosinophilic cytoplasm and ovoid nuclei with irregular profiles , coarse chromatin and one or two eosinophilic nucleoli ( fig .
brisk mitotic activity was detected , with ki67/mib-1 staining positive in around 1520% of tumor cells ( fig .
egfr was expressed on the cytoplasmic membrane of about 30% cells , while vimentin was detectable in the few rhabdoid cells .
p53 , ema , cytokeratin cam5.2 , cytokeratin 7 and cd10 were expressed only in a few neoplastic cells , while staining was consistently negative for neu - n , oligo-1 , myogenin , ttf-1 , psa , smooth muscle actin , melan - a , mart-1 , s-100 protein , cd31 , somatostatin receptor , cytokeratins ae1/ae3 , gfap and progesterone receptors .
given the peculiarity of the case , we asked two other pathologists with proven experience in the field of net to review the slides .
the morphological and immunohistochemical ( ihc ) analysis showed diffuse positive staining for the neuroendocrine markers such as synaptophysin , cga and nse , with a focal staining for cytokeratins 7 and 8/18 .
glioma markers ( gfap , s-100 ) , calcitonin , pituitary ( acth , fsh , gh , lh , prl , tsh , alpha - su ) , and gastroenteropancreatic and pulmonary net hormones ( serotonin , gastrin , insulin , somatostatin , pp , glucagon / glycentin , cdx2 , pdx1 , ttf1 ) were instead undetectable .
a plausible differential diagnosis with pineocytomas with mixed / intermediate differentiation ( ppt ) was proposed because ppt cells often assemble in lobular structures and stain positive for cga , nse and synaptophysin .
however , the absence of pineal localization in our case , and the cytological coarse chromatin aspect , was clearly different from the typically stippled pattern observed in ppts .
both pathologists confirmed the diagnosis of g2 wdnet according to the criteria of the 2010 who classification .
only one ectopic cerebellar localization of a presumptive , primary pineal tumor has been reported in the literature ; in that case , the primary tumor consisted of an immature dysembryoma .
for these reasons , pathologists excluded the hypothesis of an ectopic ppt . given that primary cerebellar wdnets have never been described to date ,
however , extensive imaging studies , including computed tomography ( ct ) , ga pet and fdg - pet , did not show any evidence of extracranial primary tumor . despite this
, we expected that a primary net located outside the brain , or other organ metastases , would become evident over a prolonged follow - up , also since no systemic treatment was administered to inhibit the growth of potentially present extracerebral tumor cells .
however , total - body radiological studies including brain ct and mr imaging regularly performed every 6 months proved negative , and the patient remained asymptomatic and in good general conditions , without evidence of other extracerebral localizations of the disease . in october 2012 , vertigos reappeared and a brain mri examination documented one local recurrence of the previously resected left hemisphere cerebellar lesion and the appearance of a new small lesion in maximum diameter , located at the left cerebellopontine angle ( fig .
3 , red arrows ) . extensive systemic staging including ga pet was performed , but again no extracranial localizations were detected .
partial surgical resection of the cerebellopontine lesion was then performed , followed by stereotactic radiotherapy on residual disease ( 60 gy ) and on left cerebellar hemisphere ( 50 gy ) .
pathological and ihc examination of the recurrence confirmed the primary diagnosis . in june 2014 ,
the patient also underwent total - spine mr imaging and cytological evaluation of cerebrospinal fluid , none of which showed any evidence of disease . at the last control in july 2015 ,
minimal disease persistence at the cerebellopontine angle ( 13 6 9 mm ) was documented by brain mri , with the patient remaining substantially asymptomatic and involved in normal daily and working activities . despite biannual systemic re - evaluations with total - body contrast - enhanced ct and ga pet , according to the clinical course of snc primary tumors ,
extracranial disease has never become evident over a total follow - up of 6 years .
in 1013% of metastatic wdnets , the primary tumor can not be identified at diagnosis and can even remain occult during the entire course of the disease , with the liver being the main site of metastatic spread in such cases .
this can be explained by three different hypotheses : ( a ) if clinical follow - up is long enough , the primary tumor eventually becomes detectable ; ( b ) the primary lesion remains small or even spontaneously regresses due to poor vascularization or clearing by the immune system ; ( c ) the primary tumor arises in the same organ that is erroneously considered the metastatic site ( in most cases the liver ) .
the case described here unlikely represents a metastatic - to - the - cerebellum wdnet of unknown primary origin because of the following reasons .
( 1 ) the putative primary extracranial tumor has not been detected during a prolonged , 6-year - long follow - up .
this strongly contrasts with data in the literature , where detection of brain metastases of elsewhere localized primary wdnets has always been preceded by the detection of the primary neoplasm , most often at the lungs [ 7 , 8 ] .
( 2 ) tumor recurrences occurred in the same area of the brain as the first detected lesion , whereas lesions in sites that are preferential metastatic localizations of extracranial wdnets , such as the liver and thoracic / abdominal lymph nodes , have never been found .
( 3 ) although ihc markers of wdnets are not absolutely specific of any anatomic origin , the lack of ttf1 , cdx2 and pdx1 expression in the tumor makes it unlikely to originate from the lung , gut and pancreas , thus probably excluding the most frequent sites of net origin .
( 4 ) the clinical indolent course of the patient , characterized by long - term survival , is typical of advanced wdnets . ( 5 ) although it can not be definitively excluded that a small , primary extracranial net has metastasized to the brain before becoming quiescent or even being cleared by the immune system , it is hard to believe that the liver , lung and lymph nodes were completely spared , with the cerebellum instead being the only site of subsequent metastatic waves .
( 6 ) plausible differential diagnoses , including ppt and cerebellar medulloblastoma ( mb ) .
we therefore conclude that this case represents the first pathological report of a primary cerebellar wdnet .
recurrences observed during patient follow - up represent , in our opinion , locoregional relapses of the primary cns tumor .
this work aims , above all , to make aware pathologists and clinicians about the existence of primary wdnets of the cns .
these tumors would definitively consist of very rare entities , neglected until now because erroneously considered metastatic localizations of elsewhere located primary neuroendocrine neoplasms .
written informed consent was obtained from the patient for publication of this case report and any accompanying images .
the authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the case report .
this case report did not receive any specific grant from any funding agency in the public , commercial or not - for - profit sector . | we report the case of a 38-year - old patient who was diagnosed with a cerebellar well - differentiated neuroendocrine tumor ( wdnet ) in 2009 . at first glance
, we believed that it was a metastasis from an unrecognized wdnet arising outside the cerebellum .
however , despite a prolonged follow - up of 6 years , an extracranial wdnet has never been found . during this time , the tumor recurred locally twice , and the patient was treated with surgery and radiotherapy . at the moment , he enjoys good general conditions and his tumor is under control . due to the histopathological characteristics and clinical behavior of the tumor
, we believe that this is the first report to date of a primary cerebellar wdnet . |
, rural health issues have attracted much attention , and providing rural health care has become a concern and topic for international discussions .
the health of these societies is a major concern for nurses , midwives , other health care providers and health care systems , and governments .
if half of the world 's population lives in rural areas then considerable services are needed in those places .
the primary health care program is used as a strategy to meet the needs of rural areas across the world .
most of the primary health care programs in low income countries have used community health workers who were not competent for providing health services in poor rural areas .
many of these services have not been widely appreciated , therefore they were mostly considered as unsuccessful . the quality , effectiveness , and performance of health care services are mostly related to the people in charge of providing them .
only skilled and well trained professionals are able to provide high quality health care services .
iran has a population of more than 75 million people , 25% of which are in rural areas according to 2011 census .
after the revolution the priority of rural and underserved areas has been the basic policy .
iran 's primary health care system has been established to improve access to health care for the deprived , and reduce the gap between health consequences of urban and rural areas .
the health house , as the most basic unit of the primary health care network , is usually the only health facility for rural areas in iran .
these health houses are equipped with health staff known as behvarz that are employed from local communities .
behvarz have an essential role in the primary health care system of iran . in the past two decades the health indicators in iran have been significantly improved due to the execution of primary health care programs .
however , the health care system is still facing many challenges in dealing with the society 's needs in rural areas .
studies show that in addition to the primary problems , the primary health care system is facing new challenges .
iran is experiencing rapid changes in all aspects of life : lifestyle changes , demographical changes , environmental , economic , social changes , and changes in the health and disease patterns , which is due to epidemiological transition .
evidence related to international studies in health and health care in rural areas suggest that there are problems and challenges in these fields .
strasser believes that despite the differences between developed and developing countries they all face challenges in terms of primary health care in rural areas .
moreover , despite the claim to the efficiency of primary health care this approach has not been actually evaluated .
sadrizadeh has mentioned some weaknesses in the primary health care system in iran , including weak inter- and intra - sector coordination , dissatisfied clients and suppliers , limited resources , and centralized decisions .
malekafzali has also noted some other issues in iran 's health care system such as lack of attention to primary health care in medical education , incompatibility of the health data collection system with the new technology , lack of an evidence - based decision making culture , and lack of organization and community participation in decision making .
these challenges are mostly related to the structure of the health care system , and are the result of a quantitative perspective toward the issues of health care in rural areas .
there have been no studies with a qualitative approach on health care of rural areas , whereas understanding the process of health care in terms of social and cultural complexities is not possible within the proof oriented paradigm with its scientific and experimental approach .
therefore , the best method to understand it is to study it in its natural environment .
this view is compatible with the nature - oriented approach or an interpretation which attempts to study phenomena and processes in their natural environment .
qualitative research methods have been specified as suitable methods of studying health and other areas of social researches in rural areas .
the qualitative methods have the capacity to produce data that can determine the effects of social context on health .
application of qualitative approaches in primary health care research is essential when the researcher wants to focus on the experiences of the participants .
considering these view points and the limited understanding of health care phenomena , due to its social and cultural complexities , this research aimed to describe the experiences of health care workers and people of the process of health care in rural areas .
this study was a qualitative research with content analysis and aimed to describe the process of health care consequences in rural areas of iran in 2011 - 2012 . research environment was the rural areas of arsanjan in fars province of iran .
26 participants were chosen including 21 health care providers ( 13 behvarz , 2 family physicians , 2 midwives and 4 nurses in the rural area ) , and 5 patients from that area .
they were chosen based on the aim of the study and having experience in providing health care in rural areas ( at least 2 years of experience ) and the tendency to express their experience .
the main participants of the study were behvarzs , who had a key role in the process of health care in rural areas , and according to need their experience was used to complete the data .
data collection was conducted through semi - structured interview with open questions and small group discussion ( one session with four nurses ) .
the interview took place in the health houses , health care centers , and the people 's houses in rural areas .
the questions started with from your experience what are the processes of rural health care ? and
all the ethical considerations were followed including written consent , right to withdraw from the study , maintaining anonymity , and confidentiality .
the form about the mentioned facts was given to the participants before the study and the process of the study was explained , then the forms were signed by the participants .
explanation about recording their voices during the interview were given and permission was obtained . the time and place of the interviews were agreed between them .
data analysis was performed using qualitative content analysis approach based on the descriptive method by granheme and landman .
the interviews were read sentence by sentence and freely coded by the researcher so that all the aspects of the content were described by the respondents .
next the codes which were semantically similar were placed in the same level and these levels were grouped based on bigger categories .
the aim of categorizing them was to reduce the levels , and finally they were interpreted in order to reveal their implications [ table 1 ] .
an example of the data analysis process different methods were used to determine the accuracy of the study .
the researcher performed member checking , prolonged engagement , and triangulation to increase the study 's credibility .
after coding the interview , some parts of it were returned to the participants so they could check its accuracy .
the researcher tried to have more meetings and engage more with the participants to increase his experience . for combining the sources and methods of data collection the researcher tried to choose the samples from all the related groups associated with the process of health care in rural areas .
he also used different methods such as interviews and group discussion to collect the data .
the interviews , codes , and emerged levels were sent to supervisors , consultants , and lecturers , who were familiar with the qualitative approach , to check the accuracy of the analysis and the interpretation for at least one - third of the interviews . to increase the transmission of the study , the researcher tried to make it possible for others to follow the research process by giving detailed and targeted descriptions of the research process .
the participants in this study were 26 people ; including 21 people from health care team and 5 rural patients .
their demographic characteristics are given in table 2 . by analyzing the data a level was formed and named inefficiency of care process in rural area , and this level had sub - classes such as self - arbitrary treatment , slow process of care , burnout and stress , dissatisfaction with care process , superficial view to care , and ineffective care .
participant 's demographical characteristics the results showed that self - treatment and excessive drug use were very common among rural participants .
behvarz number 3 stated that self - treatment can be seen very often in a way that in every house you go to you can see lots of drugs without prescriptions . the participants explained the reason for this was not having enough access to physicians and the inability of the health houses in providing drugs .
the health house does not have medications when there is no physician and people will go to drug stores and get cold stops and painkillers . furthermore , cultural factors are another reason for self - treatment .
people demand medication due to their wrong culture and we do not have that medication , so they go to the drugstore and buy it . the results indicated that another reason for inefficient care process is the slow process of care and this is from both the patients and the health care providers .
the quality of care registration system ( old registration system , recording one problem in many places , and the many forms and frequent replacement ) and behvarz 's inadequate health capabilities are the reasons for delays of health care providers .
the behvarz 's are very slow , for example they would delay a pregnant lady 's checkup by two to three hours just because it is time consuming .
participant number 16 stated that he has to spend a whole day for a simple matter in the health care center since they are very slow in performing their duties .
behvarz number 8 stated that when a child has vaccination they delay coming to the center so we have to go to their house .
in many treatment cases patients do not refer to the health center on time .
a nurse in the group discussion argued that in rural areas people go to the health center very late , for example a man had chest pain for three days and did not know that the pain was related to his heart .
results showed that job stress is another reason for inefficient performance of health care providers .
behvarz number 6 stated that since we have lots of work to do the pressure is very high and it influences our personal life . the large increase in population over time has also affected this matter .
behvarz number 7 said that we have to deal with all kinds of people from their birth to the day they die , and it is really frustrating . other factors such as the patients and
behvarz number 10 stated that we are trapped between the patients and the authorities , on the one hand the patients expect from us and on the other hand the authorities want us to warn them and this causes a lot of discomfort for us .
dissatisfaction is one of the consequences of inefficient care process which can be seen among patients and care providers .
patients dissatisfaction has various reasons such as behvarz 's inadequate capabilities , their disability in meeting patients needs and providing medications , and the quality of existing services and its accessibility .
participant number 18 stated that the health house does not perform what they should do ; they only measure people 's blood pressure and check their height and weight , which we can do ourselves , therefore we are not satisfied .
behvarz number 8 said for a simple illness they refer to the center and they complain why there is no medication in the center .
people are not satisfied with the referral system ; they expect us to do something which we can not since it is set by the ministry .
behvarz 's are also dissatisfied due to the high workload , and authorities not respecting and understanding their work .
behvarz number 1 said : we do all this work and no one even sees and respects it ; we do not even receive any appreciation for what we do .
behvarz number 12 declared : you have to continuously track the patients which is very difficult since the work is too much .
behvarz number 13 stated : the written work is too much and everyone is complaining about it .
results showed that there is a superficial view to the care process and care is not performed the way it should be in rural areas .
the findings showed that another consequence of inefficient care process was quantity oriented and paying attention to the appearances and figurative numbers .
number 1 behvarz stated : as i said it 's all about quantity not quality , we should just make files , our section has become a statistical section .
the care providers feel that the aim of the health care system is not about providing care in its actual meaning , but it is about paying attention to the appearances and extracting statistical and figurative numbers .
a nurse in the group discussion declared that most of the work is paper work and the statistical information does not help the patient .
behvarz number 10 stated : it does not matter how much training we provide ,
behvarz number 2 said : what kind of care is this , everything is paper work , and nothing is effective . findings showed that an effective relationship does not exist in the health care process of rural areas .
behvarz number 2 stated : people do not trust the health house ; therefore , they do not refer to it .
this distrust exists among the people and care providers , and also the authorities do not trust the behvarzs .
midwife number 5 said : all the statistical forms are in my handwriting , i do nt trust the behvarz , and they always have problems .
misunderstanding also exists among people and behvarz , or between the specialist and behvarz .
i keep explaining to the specialist that people in rural areas have problems , they can not even provide food for their families , how can you expect them to have a sanitized bathroom , they wo nt understand .
care providers believe that the authorities do not fully understand the issues that exist in rural areas .
a couple of people in the ministry provide programs without considering the issues , they do not know how rural areas are and they wo nt listen .
the results showed that self - treatment and excessive drug use were very common among rural participants .
behvarz number 3 stated that self - treatment can be seen very often in a way that in every house you go to you can see lots of drugs without prescriptions .
the participants explained the reason for this was not having enough access to physicians and the inability of the health houses in providing drugs .
the health house does not have medications when there is no physician and people will go to drug stores and get cold stops and painkillers . furthermore , cultural factors are another reason for self - treatment .
people demand medication due to their wrong culture and we do not have that medication , so they go to the drugstore and buy it .
the results indicated that another reason for inefficient care process is the slow process of care and this is from both the patients and the health care providers .
the quality of care registration system ( old registration system , recording one problem in many places , and the many forms and frequent replacement ) and behvarz 's inadequate health capabilities are the reasons for delays of health care providers .
the behvarz 's are very slow , for example they would delay a pregnant lady 's checkup by two to three hours just because it is time consuming .
participant number 16 stated that he has to spend a whole day for a simple matter in the health care center since they are very slow in performing their duties .
behvarz number 8 stated that when a child has vaccination they delay coming to the center so we have to go to their house . in many treatment cases patients do not refer to the health center on time .
a nurse in the group discussion argued that in rural areas people go to the health center very late , for example a man had chest pain for three days and did not know that the pain was related to his heart .
results showed that job stress is another reason for inefficient performance of health care providers .
behvarz number 6 stated that since we have lots of work to do the pressure is very high and it influences our personal life . the large increase in population over time has also affected this matter .
behvarz number 7 said that we have to deal with all kinds of people from their birth to the day they die , and it is really frustrating . other factors such as the patients and
behvarz number 10 stated that we are trapped between the patients and the authorities , on the one hand the patients expect from us and on the other hand the authorities want us to warn them and this causes a lot of discomfort for us .
i am tired of this job , every day there is a new problem .
dissatisfaction is one of the consequences of inefficient care process which can be seen among patients and care providers .
patients dissatisfaction has various reasons such as behvarz 's inadequate capabilities , their disability in meeting patients needs and providing medications , and the quality of existing services and its accessibility .
participant number 18 stated that the health house does not perform what they should do ; they only measure people 's blood pressure and check their height and weight , which we can do ourselves , therefore we are not satisfied . behvarz number 8 said for a simple illness they refer to the center and they complain why there is no medication in the center .
people are not satisfied with the referral system ; they expect us to do something which we can not since it is set by the ministry . behvarz 's are also dissatisfied due to the high workload , and authorities not respecting and understanding their work .
behvarz number 1 said : we do all this work and no one even sees and respects it ; we do not even receive any appreciation for what we do .
behvarz number 12 declared : you have to continuously track the patients which is very difficult since the work is too much .
behvarz number 13 stated : the written work is too much and everyone is complaining about it .
results showed that there is a superficial view to the care process and care is not performed the way it should be in rural areas .
the findings showed that another consequence of inefficient care process was quantity oriented and paying attention to the appearances and figurative numbers .
number 1 behvarz stated : as i said it 's all about quantity not quality , we should just make files , our section has become a statistical section . the care providers feel that the aim of the health care system is not about providing care in its actual meaning , but it is about paying attention to the appearances and extracting statistical and figurative numbers .
a nurse in the group discussion declared that most of the work is paper work and the statistical information does not help the patient .
behvarz number 10 stated : it does not matter how much training we provide ,
behvarz number 2 said : what kind of care is this , everything is paper work , and nothing is effective .
the findings showed that another consequence of inefficient care process was quantity oriented and paying attention to the appearances and figurative numbers .
number 1 behvarz stated : as i said it 's all about quantity not quality , we should just make files , our section has become a statistical section . the care providers feel that the aim of the health care system is not about providing care in its actual meaning , but it is about paying attention to the appearances and extracting statistical and figurative numbers .
a nurse in the group discussion declared that most of the work is paper work and the statistical information does not help the patient .
behvarz number 10 stated : it does not matter how much training we provide ,
behvarz number 2 said : what kind of care is this , everything is paper work , and nothing is effective .
findings showed that an effective relationship does not exist in the health care process of rural areas .
behvarz number 2 stated : people do not trust the health house ; therefore , they do not refer to it .
this distrust exists among the people and care providers , and also the authorities do not trust the behvarzs .
midwife number 5 said : all the statistical forms are in my handwriting , i do nt trust the behvarz , and they always have problems .
misunderstanding also exists among people and behvarz , or between the specialist and behvarz .
i keep explaining to the specialist that people in rural areas have problems , they can not even provide food for their families , how can you expect them to have a sanitized bathroom , they wo nt understand .
care providers believe that the authorities do not fully understand the issues that exist in rural areas .
a couple of people in the ministry provide programs without considering the issues , they do not know how rural areas are and they wo nt listen .
the findings of the present study showed the inefficiency of the process of health care in rural areas .
the consequences of this inefficiency can be seen in different aspects of the care process for people and care providers .
one aspect of these consequences was the self - treatment of patients and using excessive medications .
this matter was due to the culture of rural areas and the value of self - reliance and independence .
but the main reason was not having access to care facilities , disability of health houses in providing care , and medication and the patient 's financial problems .
arbitrary self - treatment in rural areas is associated with the structure of the health system and the cultural context of rural populations .
the findings also indicated that there was delay in the process of health care system from the staff and also from the patients .
delay from the patients was related to their not following treatment and failure to refer to the health center on time .
other reasons such as culture , economic status , and health care system 's quality and accessibility were also effective .
these findings are consistent with the study by mason that showed people in rural areas do not follow their treatment , and by the time they refer to the health center their illness has become sever .
rass also indicated in his study that people do not refer to health care center unless their illness is sever and then they need to be hospitalized .
the delay in health care system from the staff was related to the traditional registration of health care system such as the huge amount of files and information that need to be recorded , the time that it takes and being inadequate .
they showed that registering all the information takes most of the health providers time and it is the main obstacle for the service quality .
overall , the results of this study indicated that both health care structure and economic and cultural factors are involved in delaying the people 's treatment follow up .
however , for the care providers this delay is mainly due to the health care system structure especially the health care system registration . according to the study , the outcome of inefficiency in the process of health care in rural areas is the stress and burnout of the staff .
other factors such as the patient 's and authorities expectations from the care providers and the contradictory expectations of the care system also influence behvarzs stress and depression .
their study also showed that many of the health care providers in rural areas had medium to high stress level due to the increase of workload and role confusion .
the stress from workload was associated with the high population that each care provider had to cover compared with the standards , and the new health care programs which were added to their duties over time .
their role confusion was related to the lack of unity of commands from the health centers in rural areas .
lloyd et al . also noted in their study that organizational factors such as work pressure , workload , role confusion , and communication with supervisors are the predictors of social workers stress and exhaustion . on the other hand malakouti
indicated in their study that despite the increase in the behvarzs workload during 30 years their stress and burnout has not increased .
the results of the present study reveal dissatisfaction on both sides of the care process .
these results were consistent with the study of arab et al . which showed that the model of overall job satisfaction of primary health care providers indicated that they are dissatisfied with their job .
stated in their research that the number of care providers who were dissatisfied with their job was more than the number who were satisfied .
the dissatisfaction of the patients in the present study was related to insufficient capabilities of care providers , the quality , and accessibility of services , and the obligation to comply with the referral system .
they believe that people in developing countries , such as iran , will not be satisfied by the health care services due to the care system structure , characteristics of care providers , distribution of resources and payment methods , the mechanism of the referral system , and health information management .
the results of both studies confirm the effects of the characteristics of health care providers and referral system on the people 's satisfaction with the care process .
the results showed that the care process in rural areas was superficial and due to the care system being quantity oriented and its paper works has been detached from its aim which was to improve people 's health . most of the care providers believe that the care actions are not effective .
they also noted in their study that there was a common complaint among the participants about the high amount of paper work ; most of the records were unnecessary and redundant , lack of coordination between different parts of a client 's case , the care providers had to record all the provided services in different parts including the patient 's file , different papers , and report tables . some of the care providers stated that the high amount of recording was the reason that they could not provide their services efficiently and it reduced the quality their work .
lack of provided care in relation to distrust and lack of mutual understanding in the process of care in rural areas was another outcome of the inefficient health care process .
gilson showed in his study that ensuring health and care depends on the patient 's cooperation , and care providers and health system factors .
patient 's confidence in the health care system and health care staff is a prerequisite for the effectiveness of health care system , and an important requirement for a good communication , treatment success , and acceptance .
the results also showed the lack of mutual understanding among caregivers , authorities , and expert supervisors .
they reported conflict between the values of managers and caregivers as a source for stress , and that care givers felt frustration due to the misunderstanding of their role by others , lack of understanding of their skills and values , and not being appreciated for what they do .
overall , the findings of the present study indicated dysfunction and inefficiency in the current health care process of rural areas in iran . this inefficiency has been found in different aspects of the care process such as the care quality , quality of interactions , people and caregivers satisfaction , stress and burnout , and arbitrary self - treatment .
inefficient health care process outcomes showed difficulties in the current health care system in rural areas of iran .
these consequences are rooted in the cultural and social context of rural areas , and are associated with the constituent elements of the health system such as the quality of care system registration , the ability of health care providers , the service quality and accessibility , and the referral system . | background : health care systems in rural areas face numerous challenges in meeting the community 's needs and adequate attention has not been paid to this problem .
the aim of this study was to describe the outcomes of health care process in rural society.materials and methods : twenty - six participants including twenty - one rural health care providers and five clients were selected according to purposive sampling .
the data were collected via semi - structured individual interviews and a mini focus group .
data were analyzed by using qualitative content analysis based on methods described by granheme and landman.results:data analysis eventually led to formation of one category of inefficiency in health care process in rural society including subcategories such as arbitrary self - therapy , slow care process , dissatisfaction with the care process , superficial caring , job stress and burn out of caregivers , and ineffective caring relationship.conclusion:outcomes in health care in rural society of iran represents inefficiency of the current health care process .
these outcomes are related to the cultural and social context of rural communities and the structure of the health system .
these outcomes in health care in the rural society of iran represent impairment of the current health care process .
the necessity of modifying the existing care trend with new models designed to improve the health care process is felt . |
the 1970s and 1980s saw extensive research on microtubules and actin . during this
period , the consensus developed that these cytoskeletal elements were unique to
eukaryotes and that nothing related to tubulin or actin existed in bacteria or
archaeans .
this consensus was overthrown in the 1990s when a series of discoveries
revealed that prokaryotes actually did have homologues of tubulin and actin and that
these assembled into cytoskeletal filaments .
it is now generally accepted that
eukaryotic microtubules and actin filaments originated from these prokaryotic
homologues .
the key discoveries of bacterial tubulin and actin were published in 1992
and are reviewed here on their 25th anniversary .
the discovery of ftsz as a bacterial homologue of tubulin came first and was made
independently by three groups ( de boer
et al .
, 1992 ; raychaudhuri and park , 1992 ; mukherjee
et al . , 1993 ) .
these independent studies each
purified ftsz protein from an escherichia coli expression system and
demonstrated that it bound and hydrolyzed gtp .
they noted that bacterial ftszs were
missing the walker sequence motifs characteristic of g proteins but that they had a
conserved short sequence , gggtgtg , that was almost identical to the g / aggtgsg
sequence conserved in all - , - , and -tubulins .
that sequence ,
known as the tubulin signature sequence , was believed to be involved in the binding
of gtp in the tubulins .
the three groups all concluded that the gtp - binding site of
ftsz appeared to be related to that of tubulins .
a year earlier , before any link to tubulin was known , bi and lutkenhaus ( 1991 ) were the first to propose that ftsz might be a
cytoskeletal protein .
electron microscopy to show that ftsz
localized to the invaginating septum in dividing e. coli : in
our model the role of ftsz is to form a cytoskeletal element that is functionally
analogous to the role of actin in cytokinesis in animal cells .
( 1992 )
noted that the gtpase activity showed a dependence on ftsz concentration
characteristic of the self - assembly of tubulin and actin . a subsequent study by mukherjee and lutkenhaus
( 1994 ) provided two major advances .
first , they demonstrated that purified
ftsz could assemble in vitro into filamentous polymers .
second , they extended the sequence alignment to
identify > 20 amino acids that were highly conserved in all tubulins and ftsz .
they
did this by starting the alignment at the gggtgtg sequence and inserting gaps in the
ftsz to maximize identity .
remarkably most of the gaps coincided with gaps already in
the alignment of - , - , and -tubulins .
i considered this
alignment to be compelling evidence for full homology , but the editors of
cell were more cautious , insisting on adding a question mark to
my 1995 minireview ,
our laboratory took up the question of in vitro assembly and showed that ftsz
assembled in vitro into sheets of protofilaments and mini - rings that were similar to
tubulin polymers ( erickson
et al . , 1996 ) .
any question of homology was
dramatically resolved when the structures of ftsz ( lwe and amos , 1998 ) and tubulin ( nogales et al .
they had an identical complex fold , which is the ultimate
test of homology .
many
archaeans have up to five ftsz homologues , some with very divergent sequences that
likely serve functions other than cell division .
the discovery of bacterial actins was complicated by the homology of actin to other
protein families .
sometimes , this is
indicated by amino acid sequence identity , but often this sequence identity is too
weak to recognize .
the most definitive demonstration of homology is from protein
structure . when the x - ray structure of actin was determined ( kabsch et al . ,
1990 ) , it was seen to have
a complex fold that was identical to that of hexokinase and also to hsp70 , a
chaperonin . because this fold is so complex yet was shared so precisely , it was
concluded that the three shared a common ancestry .
actin , hexokinase , and hsp70 are
homologues and are considered to be members of an actin superfamily .
probably the original protein in this family
was the sugar kinase , which underwent gene duplications that evolved into a
chaperonin and separately into a protein that could assemble cytoskeletal filaments ,
actin .
the first strong suggestion for actin homologues in bacteria was a theoretical study
by bork et al .
they used the recent x - ray structures of actin , hexokinase , and hsp70 to do a
structure - based sequence alignment , which identified four short signature sequences
that were conserved across the superfamily .
they then looked for these signatures in
bacterial proteins and found them in three : ftsa , mreb , and parm ( stba ) .
these
bacterial proteins were closest in sequence to hsp70 and actin rather than the sugar
kinases and were therefore candidates for bacterial actin .
( 2001 ) studied the localization of mreb in bacteria by light microscopy ,
and found helical filaments running through the cell under the membrane .
( 2001 )
isolated mreb protein and showed by electron microscopy that it assembled thin
filaments .
their major discovery was x - ray crystallography , which showed that mreb
had the actin fold and was assembled in the crystals into actin - like filaments .
later
work has discovered multiple prokaryotic actins with a variety of cytoskeletal
functions , although the functions of most are unknown .
it now seems clear that both tubulin and actin were invented in bacteria and/or
archaeans and proliferated into diverse families of cytoskeletal filaments well
before the emergence of eukaryotes ; for various perspectives on the evolution , see
erickson ( 2007 ) , lwe and amos ( 2009 ) , and wickstead and gull ( 2011 ) .
an interesting irony is that roles of tubulin
and actin have somewhat switched from bacteria to eukaryotes .
ftsz forms the
cytokinetic ring in bacteria , whereas actin provides the major cytoskeletal framework
in eukaryotes .
some bacterial ( plasmid ) actins function for nucleoid segregation , a
role performed by microtubules in eukaryotes .
a global conclusion would be that once a protein
has evolved the ability to assemble cytoskeletal filaments , these can be modified to
perform a wide range of useful and sometimes overlapping cellular functions .
the discovery of ftsz as a bacterial homologue of tubulin came first and was made
independently by three groups ( de boer
et al . , 1992 ; raychaudhuri and park , 1992 ; mukherjee
et al . , 1993 ) . these independent studies
each
purified ftsz protein from an escherichia coli expression system and
demonstrated that it bound and hydrolyzed gtp .
they noted that bacterial ftszs were
missing the walker sequence motifs characteristic of g proteins but that they had a
conserved short sequence , gggtgtg , that was almost identical to the g / aggtgsg
sequence conserved in all - , - , and -tubulins .
that sequence ,
known as the tubulin signature sequence , was believed to be involved in the binding
of gtp in the tubulins .
the three groups all concluded that the gtp - binding site of
ftsz appeared to be related to that of tubulins .
a year earlier , before any link to tubulin was known , bi and lutkenhaus ( 1991 ) were the first to propose that ftsz might be a
cytoskeletal protein .
electron microscopy to show that ftsz
localized to the invaginating septum in dividing e. coli : in
our model the role of ftsz is to form a cytoskeletal element that is functionally
analogous to the role of actin in cytokinesis in animal cells .
( 1992 )
noted that the gtpase activity showed a dependence on ftsz concentration
characteristic of the self - assembly of tubulin and actin .
a subsequent study by mukherjee and lutkenhaus
( 1994 ) provided two major advances .
first , they demonstrated that purified
ftsz could assemble in vitro into filamentous polymers .
second , they extended the sequence alignment to
identify > 20 amino acids that were highly conserved in all tubulins and ftsz .
they
did this by starting the alignment at the gggtgtg sequence and inserting gaps in the
ftsz to maximize identity .
remarkably most of the gaps coincided with gaps already in
the alignment of - , - , and -tubulins .
i considered this
alignment to be compelling evidence for full homology , but the editors of
cell were more cautious , insisting on adding a question mark to
my 1995 minireview ,
our laboratory took up the question of in vitro assembly and showed that ftsz
assembled in vitro into sheets of protofilaments and mini - rings that were similar to
tubulin polymers ( erickson
et al . , 1996 ) .
any question of homology was
dramatically resolved when the structures of ftsz ( lwe and amos , 1998 ) and tubulin ( nogales et al . , 1998 )
they had an identical complex fold , which is the ultimate
test of homology .
many
archaeans have up to five ftsz homologues , some with very divergent sequences that
likely serve functions other than cell division .
the discovery of bacterial actins was complicated by the homology of actin to other
protein families .
sometimes , this is
indicated by amino acid sequence identity , but often this sequence identity is too
weak to recognize .
the most definitive demonstration of homology is from protein
structure . when the x - ray structure of actin was determined ( kabsch et al . ,
1990 ) , it was seen to have
a complex fold that was identical to that of hexokinase and also to hsp70 , a
chaperonin . because this fold is so complex yet was shared so precisely , it was
concluded that the three shared a common ancestry .
actin , hexokinase , and hsp70 are
homologues and are considered to be members of an actin superfamily .
probably the original protein in this family
was the sugar kinase , which underwent gene duplications that evolved into a
chaperonin and separately into a protein that could assemble cytoskeletal filaments ,
actin .
the first strong suggestion for actin homologues in bacteria was a theoretical study
by bork et al .
they used the recent x - ray structures of actin , hexokinase , and hsp70 to do a
structure - based sequence alignment , which identified four short signature sequences
that were conserved across the superfamily .
they then looked for these signatures in
bacterial proteins and found them in three : ftsa , mreb , and parm ( stba ) .
these
bacterial proteins were closest in sequence to hsp70 and actin rather than the sugar
kinases and were therefore candidates for bacterial actin .
( 2001 ) studied the localization of mreb in bacteria by light microscopy ,
and found helical filaments running through the cell under the membrane .
( 2001 )
isolated mreb protein and showed by electron microscopy that it assembled thin
filaments .
their major discovery was x - ray crystallography , which showed that mreb
had the actin fold and was assembled in the crystals into actin - like filaments .
later
work has discovered multiple prokaryotic actins with a variety of cytoskeletal
functions , although the functions of most are unknown .
it now seems clear that both tubulin and actin were invented in bacteria and/or
archaeans and proliferated into diverse families of cytoskeletal filaments well
before the emergence of eukaryotes ; for various perspectives on the evolution , see
erickson ( 2007 ) , lwe and amos ( 2009 ) , and wickstead and gull ( 2011 ) .
an interesting irony is that roles of tubulin
and actin have somewhat switched from bacteria to eukaryotes .
ftsz forms the
cytokinetic ring in bacteria , whereas actin provides the major cytoskeletal framework
in eukaryotes .
some bacterial ( plasmid ) actins function for nucleoid segregation , a
role performed by microtubules in eukaryotes . however , some tubz filaments also
function for plasmid segregation .
a global conclusion would be that once a protein
has evolved the ability to assemble cytoskeletal filaments , these can be modified to
perform a wide range of useful and sometimes overlapping cellular functions . | the year 2017 marks the 25th anniversary of the discovery of homologues of tubulin
and actin in prokaryotes . before 1992 , it was largely accepted that tubulin and actin
were unique to eukaryotes .
then three laboratories independently discovered that
ftsz , a protein already known as a key player in bacterial cytokinesis , had the
tubulin signature sequence
present in all - , - , and
-tubulins .
that same year , three candidates for bacterial actins were
discovered in silico .
x - ray crystal structures have since confirmed multiple
bacterial proteins to be homologues of eukaryotic tubulin and actin .
tubulin and
actin were apparently derived from bacterial precursors that had already evolved a
wide range of cytoskeletal functions . |
tropical calcific pancreatitis ( tcp ) is chronic pancreatitis of uncertain etiology , usually presenting with the classical features of pain abdomen and steatorrhea in childhood or adolescence , with evidence of pancreatic calculi along with atrophic pancreas on imaging , found predominantly in the tropical and subtropical countries with the highest prevalence in the indian states of tamil nadu and kerala . when associated with diabetes , representing a more advanced stage of the disease , it is known as fibrocalcific pancreatic diabetes ( fcpd ) .
it has been suggested that the risk of developing pancreatic cancer is the highest in patients with fcpd as compared to all other forms of pancreatitis and is believed to be 100-fold greater than those without fcpd .
this case series intends to highlight the increased occurrence of pancreatic cancer in fcpd , an observation not previously reported from this part of india .
pancreatic cancer was diagnosed in 3 patients during evaluation of patients with fcpd in the diabetic clinic of the department of endocrinology in the last 1-year .
dp , 43-year - old man with diabetes since 24 years age , initially on oral hypoglycemic drugs with switch to premixed insulin use for last 2 years due to persistently uncontrolled blood glucose , presented to the diabetic clinic with anorexia , weight loss for 6 months , and painful swelling of left leg for last 7 days .
examination was significant for low body mass index ( bmi ) 16.9 kg / m , pallor , hepatomegaly , and non - pitting edema of left leg with erythema .
ultrasonography ( usg ) doppler of left leg confirmed thrombosis of popliteal , anterior tibial , posterior tibial , and common peroneal veins of the left leg .
ultrasonography abdomen revealed calcific pancreatitis with mass in the tail of pancreas . computerized tomography ( ct ) abdomen revealed atrophic pancreas with dilated main and accessory pancreatic duct with multiple calculi in the head , body , and tail of pancreas along with 38 mm 38 mm 32 mm mass in the tail of pancreas [ figure 1 ] .
multiple target lesions were seen in the right lobe of liver , largest measuring 1.5 cm .
serum ca19- 9 was elevated ( 828.8 u / l ; normal < 40 ) .
a diagnosis of metastatic pancreatic carcinoma with deep venous thrombosis ( dvt ) was made in a patient with fibrocalcific pancreatic diabetes ( fcpd ) .
computerized tomography abdomen of patient-1 showing atrophic pancreas , dilated main pancreatic duct with multiple calculi in the head , body , and tail of pancreas with a 38 mm 38 mm 32 mm mass in the tail of pancreas .
multiple target lesions can also be seen in liver suggestive of metastasis pb , 46-year - old lady with fibrocalcific pancreatic diabetes for last 25 years , on multiple subcutaneous insulin injections ( msii ) , was detected to have a pancreatic tail mass on usg abdomen done as a part of evaluation for severe pain abdomen , vomiting , weight loss , and worsened glycemic control for last 3 months .
ct abdomen revealed dilated main pancreatic duct with multiple calculi , and a mass in the tail of pancreas ( 46 mm 34 mm 31 mm ) encasing the celiac plexus , superior mesenteric artery , infiltrating the splenic hilum and splenic flexure of colon [ figure 2 ] .
computerized tomography abdomen of patient-2 showing atrophic pancreas , multiple calculi in the tail of pancreas along with a 46 mm 34 mm 31 mm encasing the celiac plexus infiltrating the splenic hilum and splenic flexure of colon sc , 54-year - old lady with fcpd for last 28 years , diagnosed during evaluation for pain abdomen with osmotic symptoms , on premixed insulin , was diagnosed to have mass in the tail of pancreas ( 33 mm 31 mm 22 mm ) , multiple calculi in the head and body of pancreas with multiple target lesion in the liver suggestive of metastasis on ct abdomen during evaluation for weight loss , fatigue , malaise along with worsened glycemic control for the last 1 year .
dp , 43-year - old man with diabetes since 24 years age , initially on oral hypoglycemic drugs with switch to premixed insulin use for last 2 years due to persistently uncontrolled blood glucose , presented to the diabetic clinic with anorexia , weight loss for 6 months , and painful swelling of left leg for last 7 days .
examination was significant for low body mass index ( bmi ) 16.9 kg / m , pallor , hepatomegaly , and non - pitting edema of left leg with erythema .
ultrasonography ( usg ) doppler of left leg confirmed thrombosis of popliteal , anterior tibial , posterior tibial , and common peroneal veins of the left leg .
ultrasonography abdomen revealed calcific pancreatitis with mass in the tail of pancreas . computerized tomography ( ct )
abdomen revealed atrophic pancreas with dilated main and accessory pancreatic duct with multiple calculi in the head , body , and tail of pancreas along with 38 mm 38 mm 32 mm mass in the tail of pancreas [ figure 1 ] .
multiple target lesions were seen in the right lobe of liver , largest measuring 1.5 cm .
serum ca19- 9 was elevated ( 828.8 u / l ; normal < 40 ) .
a diagnosis of metastatic pancreatic carcinoma with deep venous thrombosis ( dvt ) was made in a patient with fibrocalcific pancreatic diabetes ( fcpd ) .
computerized tomography abdomen of patient-1 showing atrophic pancreas , dilated main pancreatic duct with multiple calculi in the head , body , and tail of pancreas with a 38 mm 38 mm 32 mm mass in the tail of pancreas .
pb , 46-year - old lady with fibrocalcific pancreatic diabetes for last 25 years , on multiple subcutaneous insulin injections ( msii ) , was detected to have a pancreatic tail mass on usg abdomen done as a part of evaluation for severe pain abdomen , vomiting , weight loss , and worsened glycemic control for last 3 months .
ct abdomen revealed dilated main pancreatic duct with multiple calculi , and a mass in the tail of pancreas ( 46 mm 34 mm 31 mm ) encasing the celiac plexus , superior mesenteric artery , infiltrating the splenic hilum and splenic flexure of colon [ figure 2 ] .
ca19 - 9 was 179.65 u / l ( normal < 40 u / l ) . the mass was considered inoperable .
computerized tomography abdomen of patient-2 showing atrophic pancreas , multiple calculi in the tail of pancreas along with a 46 mm 34 mm 31 mm encasing the celiac plexus infiltrating the splenic hilum and splenic flexure of colon
sc , 54-year - old lady with fcpd for last 28 years , diagnosed during evaluation for pain abdomen with osmotic symptoms , on premixed insulin , was diagnosed to have mass in the tail of pancreas ( 33 mm 31 mm 22 mm ) , multiple calculi in the head and body of pancreas with multiple target lesion in the liver suggestive of metastasis on ct abdomen during evaluation for weight loss , fatigue , malaise along with worsened glycemic control for the last 1 year .
pancreatic adenocarcinoma is the fourth most common cause of cancer death worldwide with extremely poor prognosis ( 5-year survival < 3% ) .
association of diabetes with pancreatic cancer is controversial with some studies , suggesting a two - fold increase in patients of diabetes of > 5 years duration and others suggesting diabetes to be protective against pancreatic cancer .
all patients with pancreatic cancer in our series had a long duration of diabetes ( 19 years , 25 years , and 28 years , respectively ) .
pancreatic cancer has been reported to be associated with development of diabetes with improvement in glycemia following tumor resection .
ca19 - 9 is elevated in 70 - 80% of patients with pancreatic cancer , and elevated levels ( at the time of diagnosis ) are predictive of distant metastasis , survival , and disease recurrence ( post surgery and/or chemotherapy ) .
consensus statements have recommended screening for pancreatic cancer in any individual with an increased risk of > 10-fold , which includes individuals with 3 first degree relatives with pancreatic cancer , peutz jegher syndrome , familial atypical multiple mole melanoma , and hereditary pancreatitis . since among all forms of pancreatitis ,
patients with fcpd have the highest risk of pancreatic cancer ( absolute risk > 100-fold ) , routine screening of all patients of fcpd with serum ca19 - 9 may help us in detecting the patients with pancreatic cancer at an earlier stage .
further studies are needed , however , in this direction before routine use of ca19 - 9 as a screening test for pancreatic cancer in fcpd .
anorexia , weight loss , worsening of glycemic control in any patient with fcpd should be taken as warning signs , and these patients should be evaluated for any occult pancreatic malignancy , even in the absence of classical signs of pancreatic cancer like obstructive jaundice , palpable gall bladder , and pain abdomen . | fibrocalcific pancreatic diabetes ( fcpd ) is a rare cause of diabetes ( < 1% ) of uncertain etiology associated with > 100-fold increased risk of pancreatic cancer .
we present 3 patients of fcpd with pancreatic cancer who had long duration of diabetes ( 19 years , 25 years , and 28 years , respectively ) , all of whom presented with anorexia , weight loss , and worsened glycemic control .
patient-1 in addition presented with deep venous thrombosis .
all the 3 patients had evidence of metastasis at the time of diagnosis . computerized tomography ( ct )
abdomen revealed atrophic pancreas , dilated pancreatic ducts , and multiple calculi in the head , body , and tail of pancreas in all of them .
patient-1 had 38 mm 38 mm 32 mm mass in the tail of pancreas with multiple target lesions were seen in the right lobe of liver .
patient-2 had a mass in the tail of pancreas ( 46 34 31 mm ) encasing the celiac plexus and superior mesenteric artery infiltrating the splenic hilum and splenic flexure of colon .
patient-3 also had a mass in the tail of pancreas ( 33 31 22 mm ) , with multiple target lesions in the liver , suggestive of metastasis .
all patients had elevated serum ca19 - 9 ( 828.8 , 179.65 , and 232
u / l , respectively ; normal < 40
u / l ) .
patients of fcpd with anorexia , weight loss , worsening of glycemic control should be evaluated to rule out pancreatic cancer .
studies are warranted to evaluate ca19 - 9 as a screening tool for diagnosing pancreatic cancer at an earlier stage in fcpd . |
we included publications on randomized phase 3 clinical trials comparing gefitinib , erlotinib , or afatinib with chemotherapy or one egfr - tki with another as first - line therapy in patients with advanced nsclc whose tumors present with an egfr - activating mutation .
studies were included if they contained only patients with egfr - activating mutations or they reported relative efficacy within the egfr - positive subgroup .
end points of interest were progression - free survival , overall response rate , disease control rate , and overall survival .
publications were included if they provided the most up - to - date analysis of at least one of the above - mentioned end points of interest .
literature search of medline was performed using pubmed to identify published studies using the search ( gefitinib or
erlotinib or afatinib ) and ( non small - cell lung cancer or adenocarcinoma ) and ( phase 3 or phase iii ) . the search results were further limited to clinical trials published within the last 5 years . the medline search was augmented by search of the american society of clinical oncology meeting library , european cancer congress 2013 , chinese clinical trial registry , clinicaltrials.gov , eu clinical trials register , and umin clinical trials registry , using relevant keywords
direct and indirect meta - estimates were generated in the context of log - linear mixed - effects models , similar to the model proposed by dersimonian and laird , with fixed effects for each relative comparison and random effects for each study .
heterogeneity across studies was tested and partially summarized using chi - squared tests and i statistics as proposed by higgins and thompson .
however , tests of heterogeneity and i can be misleading , especially when treatments differ markedly and one treatment can be expected to outperform the other across settings despite non - negligible heterogeneity .
predictive intervals ( pis ) , which provide an interval within which any particular study s relative effectiveness may be expected to fall , were calculated using the study - to - study variance estimates from each mixed - effects model .
adverse event rates ( 95% confidence interval [ ci ] ; 95% pi ) were summarized separately for each first - line therapy in the context of logistic mixed - effects models with a random effect for study . for adverse event summaries ,
the analyses were based on each study s full safety population , potentially a mix of patients with and without egfr - activating mutations .
details of the statistical analysis are given in supplemental digital content 1 ( http://links.lww.com/jto/a562 ) .
we included publications on randomized phase 3 clinical trials comparing gefitinib , erlotinib , or afatinib with chemotherapy or one egfr - tki with another as first - line therapy in patients with advanced nsclc whose tumors present with an egfr - activating mutation .
studies were included if they contained only patients with egfr - activating mutations or they reported relative efficacy within the egfr - positive subgroup .
end points of interest were progression - free survival , overall response rate , disease control rate , and overall survival .
publications were included if they provided the most up - to - date analysis of at least one of the above - mentioned end points of interest .
literature search of medline was performed using pubmed to identify published studies using the search ( gefitinib or
erlotinib or afatinib ) and ( non small - cell lung cancer or adenocarcinoma ) and ( phase 3 or phase iii ) . the search results were further limited to clinical trials published within the last 5 years . the medline search was augmented by search of the american society of clinical oncology meeting library , european cancer congress 2013 , chinese clinical trial registry , clinicaltrials.gov , eu clinical trials register , and umin clinical trials registry , using relevant keywords
direct and indirect meta - estimates were generated in the context of log - linear mixed - effects models , similar to the model proposed by dersimonian and laird , with fixed effects for each relative comparison and random effects for each study .
heterogeneity across studies was tested and partially summarized using chi - squared tests and i statistics as proposed by higgins and thompson .
however , tests of heterogeneity and i can be misleading , especially when treatments differ markedly and one treatment can be expected to outperform the other across settings despite non - negligible heterogeneity .
predictive intervals ( pis ) , which provide an interval within which any particular study s relative effectiveness may be expected to fall , were calculated using the study - to - study variance estimates from each mixed - effects model .
adverse event rates ( 95% confidence interval [ ci ] ; 95% pi ) were summarized separately for each first - line therapy in the context of logistic mixed - effects models with a random effect for study . for adverse event summaries ,
the analyses were based on each study s full safety population , potentially a mix of patients with and without egfr - activating mutations .
details of the statistical analysis are given in supplemental digital content 1 ( http://links.lww.com/jto/a562 ) .
literature search yielded 11 publications on eight randomized phase 3 clinical trials comparing gefitinib , erlotinib , or afatinib with chemotherapy or one egfr - tki with another as first - line therapy in patients with nsclc harboring egfr - activating mutations , during the last 5 years .
selection diagram for studies comparing gefitinib , erlotinib , and afatinib with chemotherapy as first - line therapies for patients with advanced nsclc harboring egfr - activating mutations .
asco , american society of clinical oncology ; nsclc , non small - cell lung cancer ; egfr , epidermal growth factor receptor .
identified studies were ipass , west japan , north - east japan , and first - signal , comparing gefitinib with carboplatin and paclitaxel , cisplatin and docetaxel , carboplatin and paclitaxel , or gemcitabine and cisplatin , respectively .
optimal and eurtac , respectively , compared erlotinib with gemcitabine and carboplatin , and platinum - based doublet chemotherapy .
lux - lung 3 and lux - lung 6 , respectively , compared afatinib with pemetrexed and cisplatin , and gemcitabine and cisplatin .
the most up - to - date analyses of overall survival for ipass , north - east japan , and optimal were respectively reported in the studies by fukuoka et al .
, inoue et al . , and zhou et al . the ipass and first - signal studies both recruited patients from a clinically selected population associated with egfr mutations , but containing patients both with and without egfr - activating mutations
however , both studies reported subgroup analyses focusing on patients whose tumors presented with egfr - activating mutations . summaries of included patient populations , sample sizes , treatment arms , and relative effectiveness , in terms of progression - free survival , overall response rate , disease control rate , and overall survival , are given in table 1 .
summary of studies comparing gefitinib , erlotinib , and afatinib with chemotherapy as first - line therapies for patients with advanced nsclc harboring egfr - activating mutations the test of heterogeneity indicated moderately high study - to - study variability with q = 16.1 on 5 degrees of freedom ( p = 0.007 ) and i of 69% .
the pooled hazard ratio meta - estimate for gefitinib versus chemotherapy was 0.44 ( 95% ci , 0.310.63 ; 95% pi , 0.220.88 ) , erlotinib versus chemotherapy was 0.25 ( 95% ci , 0.150.42 ; 95% pi , 0.110.55 ) , afatinib versus chemotherapy was 0.44 ( 95% ci , 0.260.75 ; 95% pi , 0.200.98 ) , erlotinib versus gefitinib was 0.57 ( 95% ci , 0.301.08 ; 95% pi , 0.241.36 ) , afatinib versus gefitinib was 1.01 ( 95% ci , 0.531.92 ; 95% pi , 0.422.42 ) , and erlotinib versus afatinib was 0.56 ( 95% ci , 0.271.18 ; 95% pi , 0.221.46 ) .
comparisons of gefitinib , erlotinib , afatinib , and chemotherapy as first - line therapies for patients with advanced nsclc harboring egfr - activating mutations individual study hazard ratios along with comparative meta - estimates for progression - free survival in first - line therapy for patients with advanced nsclc harboring egfr - activating mutations .
the test of heterogeneity indicated moderate study - to - study variability with q = 7.32 on 5 degrees of freedom ( p = 0.198 ) and i of 32% .
the pooled odds ratio meta - estimate for gefitinib versus chemotherapy was 4.1 ( 95% ci , 2.76.3 ; 95% pi , 2.37.6 ) , erlotinib versus chemotherapy was 8.2 ( 95% ci , 4.515.1 ; 95% pi , 3.917.5 ) , afatinib versus chemotherapy was 5.5 ( 95% ci , 3.48.8 ; 95% pi , 2.910.5 ) , erlotinib versus gefitinib was 2.0 ( 95% ci , 0.94.1 ; 95% pi , 0.84.7 ) , afatinib versus gefitinib was 1.3 ( 95% ci , 0.72.5 ; 95% pi , 0.62.8 ) , and erlotinib versus afatinib was 1.5 ( 95% ci , 0.73.3 ; 95% pi , 0.63.7 ) .
individual study odds ratios along with comparative meta - estimates for overall response rate in first - line therapy for patients with advanced nsclc harboring egfr - activating mutations .
the test of heterogeneity indicated moderate study - to - study variability with q = 5.26 on 4 degrees of freedom ( p = 0.262 ) and i of 24% .
the pooled odds ratio meta - estimate for gefitinib versus chemotherapy were 2.1 ( 95% ci , 1.33.5 ; 95% pi , 1.23.7 ) , erlotinib versus chemotherapy was 2.5 ( 95% ci , 1.44.7 ; 95% pi , 1.34.9 ) , afatinib versus chemotherapy was 2.9 ( 95% ci , 1.84.6 ; 95% pi , 1.74.8 ) , erlotinib versus gefitinib were 1.2 ( 95% ci , 0.52.7 ; 95% pi , 0.52.8 ) , afatinib versus gefitinib were 1.4 ( 95% ci , 0.72.7 ; 95% pi , 0.72.8 ) , and erlotinib versus afatinib were 0.9 ( 95% ci , 0.41.9 ; 95% pi , 0.42.0 ) .
individual study odds ratios along with comparative meta - estimates for disease control rate in first - line therapy for patients with advanced nsclc harboring egfr - activating mutations .
the test of heterogeneity indicated low study - to - study variability with q = 2.39 on 5 degrees of freedom ( p = 0.793 ) and i of 0% .
the pooled hazard ratio meta - estimate for gefitinib versus chemotherapy was 0.99 ( 95% ci , 0.811.21 ; 95% pi , 0.811.21 ) , erlotinib versus chemotherapy was 1.06 ( 95% ci , 0.821.37 ; 95% pi , 0.821.37 ) , afatinib versus chemotherapy was 1.01 ( 95% ci , 0.781.31 ; 95% pi , 0.781.31 ) , erlotinib versus gefitinib was 1.07 ( 95% ci , 0.771.47 ; 95% pi , 0.771.47 ) , afatinib versus gefitinib was 1.02 ( 95% ci , 0.731.41 ; 95% pi , 0.731.41 ) , and erlotinib versus afatinib was 1.05 ( 95% ci , 0.731.51 ; 95% pi , 0.731.51 ) .
these results are summarized in table 2 . the more common adverse events with tkis were diarrhea , rash or acne , dry skin , and pruritis , whereas anorexia , anemia , fatigue , nausea , vomiting , alopecia , and neutropenia were more common with chemotherapy .
liver enzyme elevations were more common with gefitinib and erlotinib than with chemotherapy , but not reported for afatinib .
broadly , adverse event profiles were similar among tkis although there was some indication that gefitinib was associated with more anemia and afatinib was associated with more stomatitis or mucositis .
adverse event profiles by first - line therapy are summarized in supplemental digital content 2 ( http://links.lww.com/jto/a563 ) .
literature search yielded 11 publications on eight randomized phase 3 clinical trials comparing gefitinib , erlotinib , or afatinib with chemotherapy or one egfr - tki with another as first - line therapy in patients with nsclc harboring egfr - activating mutations , during the last 5 years .
selection diagram for studies comparing gefitinib , erlotinib , and afatinib with chemotherapy as first - line therapies for patients with advanced nsclc harboring egfr - activating mutations .
asco , american society of clinical oncology ; nsclc , non small - cell lung cancer ; egfr , epidermal growth factor receptor .
identified studies were ipass , west japan , north - east japan , and first - signal , comparing gefitinib with carboplatin and paclitaxel , cisplatin and docetaxel , carboplatin and paclitaxel , or gemcitabine and cisplatin , respectively .
optimal and eurtac , respectively , compared erlotinib with gemcitabine and carboplatin , and platinum - based doublet chemotherapy .
lux - lung 3 and lux - lung 6 , respectively , compared afatinib with pemetrexed and cisplatin , and gemcitabine and cisplatin .
the most up - to - date analyses of overall survival for ipass , north - east japan , and optimal were respectively reported in the studies by fukuoka et al .
, inoue et al . , and zhou et al . the ipass and first - signal studies both recruited patients from a clinically selected population associated with egfr mutations , but containing patients both with and without egfr - activating mutations
however , both studies reported subgroup analyses focusing on patients whose tumors presented with egfr - activating mutations . summaries of included patient populations , sample sizes , treatment arms , and relative effectiveness , in terms of progression - free survival , overall response rate , disease control rate , and overall survival , are given in table 1 .
summary of studies comparing gefitinib , erlotinib , and afatinib with chemotherapy as first - line therapies for patients with advanced nsclc harboring egfr - activating mutations
the test of heterogeneity indicated moderately high study - to - study variability with q = 16.1 on 5 degrees of freedom ( p = 0.007 ) and i of 69% .
the pooled hazard ratio meta - estimate for gefitinib versus chemotherapy was 0.44 ( 95% ci , 0.310.63 ; 95% pi , 0.220.88 ) , erlotinib versus chemotherapy was 0.25 ( 95% ci , 0.150.42 ; 95% pi , 0.110.55 ) , afatinib versus chemotherapy was 0.44 ( 95% ci , 0.260.75 ; 95% pi , 0.200.98 ) , erlotinib versus gefitinib was 0.57 ( 95% ci , 0.301.08 ; 95% pi , 0.241.36 ) , afatinib versus gefitinib was 1.01 ( 95% ci , 0.531.92 ; 95% pi , 0.422.42 ) , and erlotinib versus afatinib was 0.56 ( 95% ci , 0.271.18 ; 95% pi , 0.221.46 ) .
comparisons of gefitinib , erlotinib , afatinib , and chemotherapy as first - line therapies for patients with advanced nsclc harboring egfr - activating mutations individual study hazard ratios along with comparative meta - estimates for progression - free survival in first - line therapy for patients with advanced nsclc harboring egfr - activating mutations .
the test of heterogeneity indicated moderate study - to - study variability with q = 7.32 on 5 degrees of freedom ( p = 0.198 ) and i of 32% .
the pooled odds ratio meta - estimate for gefitinib versus chemotherapy was 4.1 ( 95% ci , 2.76.3 ; 95% pi , 2.37.6 ) , erlotinib versus chemotherapy was 8.2 ( 95% ci , 4.515.1 ; 95% pi , 3.917.5 ) , afatinib versus chemotherapy was 5.5 ( 95% ci , 3.48.8 ; 95% pi , 2.910.5 ) , erlotinib versus gefitinib was 2.0 ( 95% ci , 0.94.1 ; 95% pi , 0.84.7 ) , afatinib versus gefitinib was 1.3 ( 95% ci , 0.72.5 ; 95% pi , 0.62.8 ) , and erlotinib versus afatinib was 1.5 ( 95% ci , 0.73.3 ; 95% pi , 0.63.7 ) .
individual study odds ratios along with comparative meta - estimates for overall response rate in first - line therapy for patients with advanced nsclc harboring egfr - activating mutations .
the test of heterogeneity indicated moderate study - to - study variability with q = 5.26 on 4 degrees of freedom ( p = 0.262 ) and i of 24% .
the pooled odds ratio meta - estimate for gefitinib versus chemotherapy were 2.1 ( 95% ci , 1.33.5 ; 95% pi , 1.23.7 ) , erlotinib versus chemotherapy was 2.5 ( 95% ci , 1.44.7 ; 95% pi , 1.34.9 ) , afatinib versus chemotherapy was 2.9 ( 95% ci , 1.84.6 ; 95% pi , 1.74.8 ) , erlotinib versus gefitinib were 1.2 ( 95% ci , 0.52.7 ; 95% pi , 0.52.8 ) , afatinib versus gefitinib were 1.4 ( 95% ci , 0.72.7 ; 95% pi , 0.72.8 ) , and erlotinib versus afatinib were 0.9 ( 95% ci , 0.41.9 ; 95% pi , 0.42.0 ) .
individual study odds ratios along with comparative meta - estimates for disease control rate in first - line therapy for patients with advanced nsclc harboring egfr - activating mutations .
the test of heterogeneity indicated low study - to - study variability with q = 2.39 on 5 degrees of freedom ( p = 0.793 ) and i of 0% .
the pooled hazard ratio meta - estimate for gefitinib versus chemotherapy was 0.99 ( 95% ci , 0.811.21 ; 95% pi , 0.811.21 ) , erlotinib versus chemotherapy was 1.06 ( 95% ci , 0.821.37 ; 95% pi , 0.821.37 ) , afatinib versus chemotherapy was 1.01 ( 95% ci , 0.781.31 ; 95% pi , 0.781.31 ) , erlotinib versus gefitinib was 1.07 ( 95% ci , 0.771.47 ; 95% pi , 0.771.47 ) , afatinib versus gefitinib was 1.02 ( 95% ci , 0.731.41 ; 95% pi , 0.731.41 ) , and erlotinib versus afatinib was 1.05 ( 95% ci , 0.731.51 ; 95% pi , 0.731.51 ) .
the more common adverse events with tkis were diarrhea , rash or acne , dry skin , and pruritis , whereas anorexia , anemia , fatigue , nausea , vomiting , alopecia , and neutropenia were more common with chemotherapy .
liver enzyme elevations were more common with gefitinib and erlotinib than with chemotherapy , but not reported for afatinib .
broadly , adverse event profiles were similar among tkis although there was some indication that gefitinib was associated with more anemia and afatinib was associated with more stomatitis or mucositis .
adverse event profiles by first - line therapy are summarized in supplemental digital content 2 ( http://links.lww.com/jto/a563 ) .
in this meta - analysis , gefitinib , erlotinib , and afatinib out - performed chemotherapy in terms of progression - free survival , overall response rate , and disease control rate . there was no evidence that gefitinib , erlotinib , or afatinib improved overall survival when compared with chemotherapy .
one of the proposed mechanisms of resistance to gefitinib and erlotinib is the t790 m mutation on exon 20 .
this mutation sterically prevents reversible binding of gefitinib or erlotinib , but it can potentially be overcome by tkis such as afatinib , which binds irreversibly to the receptor .
however , our meta - analysis did not show superiority of afatinib over gefitinib or erlotinib in terms of progression - free survival , overall response rate , disease control rate , and overall survival .
as the theoretical advantage of afatinib versus the first - generation egfr - tki did not translate into progression - free survival gains , maybe the clinical relevance of possible inhibition of t790 m is minimal , at least in the first - line setting , when t790m - positive clones are rarely detected .
a limitation of our study is the indirect comparison of gefitinib , erlotinib , and afatinib with one another , which relies on the quality of variance component estimates .
indirect comparisons are increasingly used to make preliminary comparisons when direct head - to - head phase 3 trials are not available .
a strength of our study is the inclusion of predictive estimates that provide an estimate of treatment effect in individual settings .
this is the first meta - analysis to provide evidence comparing gefitinib , erlotinib , and afatinib with standard chemotherapy and indirect comparisons of gefitinib , erlotinib , and afatinib with each other .
currently , the lux - lung 7 phase iib trial is comparing afatinib versus gefitinib for first - line advanced nsclc and is expected to complete late 2014 ( nct01466660 ) .
till then , our study hopes to provide evidence to guide clinical decision making for oncologists when considering first - line therapies for patients with advanced nsclc having egfr - activating mutations . in conclusion ,
gefitinib , erlotinib , and afatinib out - performed chemotherapy in terms of progression - free survival , overall response rate , and disease control rate . | introduction : tyrosine kinase inhibitors gefitinib , erlotinib , and afatinib have been compared with chemotherapy as first - line therapies for patients with advanced non small - cell lung cancer harboring epidermal growth factor receptor activating mutations .
this meta - analysis compares gefitinib , erlotinib , afatinib , and chemotherapy.methods:literature search was performed using relevant keywords .
direct and indirect meta - estimates were generated using log - linear mixed - effects models , with random effects for study .
study - to - study heterogeneity was summarized using i2 statistics and predictive intervals ( pis).results : literature search yielded eight randomized phase 3 clinical trials comparing gefitinib , erlotinib , or afatinib with chemotherapy as first - line therapy in patients with advanced non small - cell lung cancer during the last 5 years .
hazard ratio meta - estimates for progression - free survival were for gefitinib versus chemotherapy 0.44 ( 95% confidence interval [ ci ] 0.310.63 ; 95% pi , 0.220.88 ) , erlotinib versus chemotherapy 0.25 ( 95% ci , 0.150.42 ; 95% pi , 0.110.55 ) , afatinib versus chemotherapy 0.44 ( 95% ci , 0.260.75 ; 95% pi , 0.200.98 ) , erlotinib versus gefitinib 0.57 ( 95% ci , 0.301.08 ; 95% pi , 0.241.36 ) , afatinib versus gefitinib 1.01 ( 95% ci , 0.531.92 ; 95% pi , 0.412.42 ) , and erlotinib versus afatinib 0.56 ( 95% ci , 0.271.18 ; 95% pi , 0.221.46 ) .
results for overall response rate and disease control rate were similar .
there was no evidence that gefitinib , erlotinib , or afatinib improved overall survival compared with chemotherapy.conclusion:gefitinib , erlotinib , and afatinib out - performed chemotherapy in terms of progression - free survival , overall response rate , and disease control rate .
differences among gefitinib , erlotinib , and afatinib were not statistically significant . |
motor neuron innervation is the most relevant maturation event in skeletal muscle development since muscle physiology , also accounting for locomotion and breath , depends on it .
innervation implies sophisticated interactions between motor neuron axons and end plates on the muscle fibers , thus resulting in a development of highly elaborated synapses called neuromuscular junctions ( nmjs ) .
as soon as the action potential reaches the axon terminals , it induces the opening of the voltage - gated ca channels on the presynaptic nerve membrane .
this allows a ca influx that induces synaptic vescicles to fuse with the presynaptic membrane and to release the neurotransmitter acetylcholine ( ach ) in the synaptic cleft . from here , ach diffuses and binds to acetylcoline receptors ( achrs ) localized on the postsynaptic membrane on the muscle fiber .
this binding makes achrs permeable to both na and k and opens the associated voltage - gated na channels on the muscle membrane which , in turn , initiate an action potential causing ca release from the sarcoplasmic reticulum into the cytosol and muscle contraction .
acetylcholinesterase , located on the synaptic portion of the basal lamina that envelops muscle fibers , quickly inactivates ach released from the presynaptic membrane so that the ach concentration in the synaptic cleft decreases rapidly and neurotransmission stops . the correct nerve
muscle impulse transmission requires an intricate network of interacting signaling pathways displaying a certain rate of redundancy , which is , however , necessary to ensure the process s spatial and temporal accuracy . defects in the signaling pathways that regulate nmj differentiation and functioning lead to a variety of congenital neuromuscular disorders termed congenital myasthenic syndromes ( cms ) , typified by muscle weakness and fatigue .
loss of the nerve signaling caused by the degeneration of motor neurons leads to a debilitating loss of muscle mass , atrophy , and paralysis .
also , aging and diseases such as cancer , aids , and chronic heart failure are physical conditions in which nmj normal activity , muscle mass , and function are highly compromised . for these reasons , understanding the molecular basis of neuromuscular synapse functioning is fundamental to such disorders therapeutic approach .
this review will focus on the development of the postsynaptic apparatus and will highlight the role of some defects of this process which are at the onset of neuromuscular disease and muscle wasting - associated pathologies .
nmjs do not develop at random locations in muscles ; rather , they are assembled in a narrow central region of the muscle fiber , so that many nmjs are located in a row , forming an end plate across the fiber .
each nmj consists of an area of apposition between a motoneuron axon branch and a single multinucleated muscle fiber .
achrs localize on the postsynaptic muscle membrane , and their spatial distribution is critical for synaptic function . a high density of achrs at synapses
vice versa , around synapses and in the rest of the fiber , the density of achrs has to be kept low in order to allow a complete maturation of the nmj .
when myoblasts fuse to form myotubes , achr subunits are assembled into the membrane at a very low density ( 1,000 m ) .
in mature synapses , achrs accumulate at a density > 10,000 mdirectly beneath the motor nerve terminal while they drop to < 10 m in the extrasynaptic membrane [ 2 , 3 ] .
a puzzling question about the developing postsynaptic structure is whether the motoneurons or the muscle fibers determine where and how the nmjs are formed .
the motor nerve might initiate achr cluster formation ( neurocentric model ) or clusters might form aneurally and then recognized by the nerve ( myocentric model ) .
some studies indicate that the primitive achr clusterization is nerve independent , while the nerve induces only maturation and enlargement of some of the primitive aneural achr clusters . during early mouse embryogenesis , between embryonic day 12.5 ( e12.5 ) and e14.5
, aneural muscle fibers begin spontaneously to accumulate achrs in a central region where innervation will occur ( prepatterning ) [ 59 ] .
this phenomenon also occurs in mutant mice where motor axons fail to contact skeletal muscle .
following such nerve - independent achr clustering , nerve terminals overlap some prepatterned achr clusters and , at e18.5 , the innervated clusters are enlarged , whereas the other primitive clusters disappear in synaptic and extrasynaptic regions [ 8 , 9 ] .
conversely , other studies in cultured myotubes have indicated that motor neurons induce postsynaptic differentiation ignoring preexisting aneural clusters [ 11 , 12 ] .
it has also been recently hypothesized that some already formed achr clusters are recognized by the nerve on muscle fibers , while others are induced by motor axons .
it is also possible that the myogenic and the neurogenic component role might depend on the species and on the developmental context , although the topic keeps being highly controversial .
the mechanisms that lead to the clusterization of ach receptors within the postsynaptic membrane require a precise interaction of signals among motoneurons and skeletal muscle fibers and occur through different modalities .
it has been described that achrs move from the extrasynaptic region and are trapped to the synaptic pool through a rapid lateral diffusion .
clustering also induces a higher stability of the achrs ; at early stages of development , the half - life of junctional non - clustered achrs is about 1 day , whereas in adult , is increased to 814 days .
nuclei that are associated at the postsynaptic membrane ( synaptic nuclei ) actively transcribe the achr subunit genes and other postsynaptic component genes at a higher rate than the non - synaptic nuclei .
the formation of achr clusters depends not only on positive signals that enhance achr concentration in the synaptic area but also on negative signals necessary to decrease achr concentration outside the synaptic area , along the entire fiber .
the best characterized signaling responsible for achr accumulation at the nmjs is the agrin / lipoprotein receptor - related protein 4 ( lrp4)/muscle - specific tyrosine kinase receptor ( musk ) pathway .
agrin is a large heparan sulfate proteoglycan first isolated from the torpedo electric organ synthesized in the motoneurons , transported along axons , and released in the synaptic basal lamina , which surrounds the muscle fiber .
agrin is sufficient to induce achr ectopic clusters in adult muscle and postsynaptic specialization in denervated fibers [ 22 , 23 ] .
although it is not essential for prepatterning , in agrinmice , nmjs and achrs are uniformly distributed in the muscle fiber and not clustered [ 8 , 9 , 24 ] , and postsynaptic differentiation is inhibited.fig .
1the agrin musk lrp4 and neuregulin erbb pathways induce nmj assembly positive signals .
a agrin is released by the motor axon terminal and induces achr clustering , phosphorylation , and stabilization at the postsynaptic membrane . lrp4 associates with musk in the absence of agrin .
once phosphorylated , musk recruits the adapter protein dok-7 which binds crk and crkl and stimulates further musk phosphorylation and kinase activity .
b neuregulin ( ngr-1 ) is released by the nerve and induces achr transcription in synaptic nuclei .
erbb phosphorylation induced by nrg stimulates erk and jnk kinase activity which phosphorylates gabp- and gabp- transcription factors .
gabp- heterodimerizes with gabp- and binds dna at the n - box thereby enhancing transcription of achr genes the agrin musk
a agrin is released by the motor axon terminal and induces achr clustering , phosphorylation , and stabilization at the postsynaptic membrane .
once phosphorylated , musk recruits the adapter protein dok-7 which binds crk and crkl and stimulates further musk phosphorylation and kinase activity .
b neuregulin ( ngr-1 ) is released by the nerve and induces achr transcription in synaptic nuclei .
erbb phosphorylation induced by nrg stimulates erk and jnk kinase activity which phosphorylates gabp- and gabp- transcription factors .
gabp- heterodimerizes with gabp- and binds dna at the n - box thereby enhancing transcription of achr genes agrin acts by activating another important player in the nmj assembly pathway , musk .
musk is a tyrosine kinase receptor expressed in the postsynaptic membrane of nmjs where it co - localizes with achrs .
the main role of agrin is to initiate musk autophosphorylation and activation ( fig .
however , musk can also be activated in an agrin - independent manner . indeed , in muskfibers , no nmjs are formed , and also , prepatterning before innervation is absent [ 8 , 9 , 29 ] . like achr ,
. however , upon innervation , in the synaptic nuclei , musk is upregulated by different transcriptional pathways .
although agrin stimulates musk phosphorylation , the two proteins do not interact directly , but additional proteins ( coreceptors ) are engaged to allow agrin - mediated signaling in myotubes [ 31 , 32 ] .
lrp4 is concentrated at the nmjs and is necessary for agrin - stimulated musk phosphorylation and achr clustering . according to this model , lrp4 self - associates and interacts with musk in the absence of agrin .
when agrin binds to this preformed complex , it triggers a reorganization of musk , so promoting its transphophorylation and kinase activity .
once phopshorylated , musk activates signaling pathways that lead to synaptic differentiation including clustering of achrs [ 34 , 35 ] ( fig .
agrin activation of musk also leads to concentration at synapses of other proteins such as acetylcholinesterase ( ache ) , rapsyn , and neuregulin ( nrg ) receptors ( erbbs ; see below ) .
moreover , lrp4 has a crucial role in muscle prepatterning since it promotes musk phosphorylation and activation in the absence of agrin .
once phosphorylated , musk recruits docking protein-7 ( dok-7 ) , a non - catalytic adapter protein , which stimulates further musk phosphorylation and increases musk kinase activity ( fig .
1a ) . to properly regulate nmj formation , dok-7 engages at two phosphorylation sites two adapter proteins , crk and crkl .
in mouse models lacking dok-7 , crk , and crkl , neuromuscular synapse fails to form [ 3740 ] .
a coeffector in achr assembly is rapsyn , a membrane protein associated with the cytoplasmic side of the plasma membrane ( fig .
rapsyn anchorates achrs at the muscle membrane from the earliest stages of synaptogenesis , including prepatterning .
rapsyn also interacts with cytoskeletal proteins , thus being essential for achr clustering . in mice lacking rapsyn ,
it has been proposed that musk induces phosphorylation of nascent achr clusters and promotes also the binding of additional rapsyn to achr aggregates , which contributes to achr cluster growth and stability .
rapsyn may function as a scaffolding protein presenting a musk - activated src - related kinase to the achrs . upon
phosphorylation and activation , musk interacts with a large number of effectors and pathways converging toward the regulation of achr clusterization through different mechanisms .
musk indeed controls ( 1 ) achr scaffolding and redistribution via actin cytoskeleton reorganization , ( 2 ) achr stabilization through achr phosphorylation , ( 3 ) its proper endocytosis and turnover are also important for achr clustering , and ( 4 ) enhancement of transcription of achr genes selectively in subsynaptic nuclei ( reviewed in ) .
mrnas encoding other synaptic proteins such as ache , musk , and rapsyn are also concentrated at the synapses .
one candidate activator is neuregulin-1 ( ngr-1 ) , a glycoprotein which works as an extracellular signal that stimulates achr and other synapse - specific components transcription .
nrg-1 acts by binding to membrane - associated tyrosine kinase receptors related to the egf receptor ( erbb proteins 2 - 3 - 4 ) ( fig .
heterozygous mice are viable and fertile and show a decreased number of achrs at the nmjs [ 45 , 46 ] .
however , some recent in vivo studies , in which neuregulin-1 and erbb proteins were conditionally deleted , demonstrated that neuregulin signaling is dispensable [ 47 , 48 ] .
for this reason , the idea that nrg-1 is the main signal responsible for the increased transcription of achrs is highly controversial .
recent evidences also indicate that neuregulin may have a role in achr clustering and trafficking by modulating rather than determining achr expression at the nmjs .
the pathways leading from nrg-1erbb binding to regulation of achr gene transcription have been identified .
both in cultured muscle cells and in vivo , erbb phosphorylation by nrg-1 concomitantly stimulates erk [ 50 , 51 ] and the c - jun n - terminal kinase ( jnk ) activity [ 52 , 53 ] . the activation of these pathways results in increased levels and phosphorylation of the e - twenty - six ( ets)-domain - binding transcription factor ga - binding protein ( gabp)- which binds dna , heterodimerizes with gabp- , and increases achr transcription [ 54 , 55 ] ( fig .
ets transcription factors bind dna at the n - box , a dna element located not only on the achr promoter but also on the promotor of other nmj components ( utrophin and acelylcholinesterase ) [ 5658 ] . in vivo inhibition of gabp results in reduced expression of achrs indicating the gabp signaling pathway , activated directly or indirectly by neuregulin , is necessary for the formation of functional synapses . as stated before ,
the agrin musk pathway also contributes to increase synaptic achr gene transcription by a nrg / erbb - independent but rac - dependent jnk activation ( fig .
1b ) and also by interacting with the nrg / erbb pathway [ 43 , 60 , 61 ] .
the positive signals we described enhance achr accumulation only in the synaptic area . to achieve a correct formation of achr clusters in the nmjs ,
the presence of some negative signals that decrease achr concentration in the rest of the fiber , in non - synaptic areas , is necessary .
it inhibits achr localization , stability , and transcription along the muscle fiber by stimulation of cyclin - dependent kinase 5 [ 63 , 64 ] , protein kinase c ( pkc ) , and ca / calmodulin - dependent kinase ii ( camkii ) .
indeed , mutant mice lacking acetyltransferase , a biosynthetic enzyme for ach , develop faster and larger achr clusters ; they also exhibit hyperinnervation due to a broader distribution of achr clusters along the muscle fiber [ 66 , 67 ] . in detail , through muscle action potential , ach induces downregulation of achr transcription along the entire fiber .
however , since the previously described positive signals enhance achr accumulation only in the synaptic area ( fig . 1 )
, this results in a ach - dependent suppression of achr subunit gene transcription in extrasynaptic nuclei ( fig . 2 ) .
myogenin is a basic helix - loop - helix myogenic transcription factor that activates achr genes in the absence of innervation or by blockade of muscle electrical activity by denervation .
myogenin regulates achr and many other muscle gene transcription by binding the e - boxes located on their promoter / enhancers .
myogenin is downregulated as the muscle matures around the time when innervation is completed , and it is upregulated upon denervation .
forced expression of myogenin in vivo is able to induce achr expression along the entire muscle fiber .
multiple mechanisms have been proposed to explain how neural activity controls myogenin in postnatal muscle . on the basis of one of these mechanisms , following muscle depolarization , extracellular calcium enters through voltage - activated channels and activates different signal transduction cascades mediated by pkc and camkii , leading to myogenin phosphorylation [ 63 , 72 ] .
this posttranslational modification inhibits myogenin binding to e - boxes of achr promoters , resulting in reduced achr expression ( fig .
a second regulatory mechanism restraining myogenin activity involves transcriptional repression of myogenin ( fig . 2 ) .
the posttranslational and the transcriptional mechanisms of myogenin repression could act on a different timescale to ensure that myogenin expression remains blocked as long as the muscle is innervated .
although it is very well known that myogenin is transcriptionally modulated as a consequence of muscle depolarization , the mechanisms that are upstream of this regulation have only recently started to be elucidated .
several studies reported that in myogenin repression during innervation , class ii deacetylases are recruited at the myogenin promoter .
the hystone deacetylase 9 ( hdac9 ) splice variant mitr is a transcriptional repressor induced in innervated muscle .
it contributes to myogenin repression by chromatin acetylation and acts as a corepressor in a complex with the myocyte enhancer factor-2 ( mef2 ) transcription factor ( fig . 2 ) .
another myogenin repressor is dachshund homolog 2 ( dach2 ) ( fig . 2 ) .
it has been shown that , during denervation , dach2 is inhibited by hdac4 , another class ii deacetylase , which induces myogenin expression thereby activating achrs [ 7678 ] .
a third circuit of postnatal myogenin expression regulation is mediated by msy3/csda , a y - box factor that binds the myogenin promoter at a new binding motif ( highly conserved element , hce ) and exerts a repression function ( fig . 2 ) .
msy-3 is therefore partially responsible for the restricted spatiotemporal expression of achr in the subsynaptical regions of muscle fibers . the conserved binding motifs of hdac9/mef2 , dach2 , and msy-3 are adjacent sequences on the myogenin promoter , which suggests a strong interplay between the three cis
trans systems to ensure a proper myogenin and achr repression in adult and innervated muscle.fig .
the myogenic transcription factor myogenin activates muscle - specific genes including achrs by binding the e - box element .
when the action potential reaches the axon terminals , it induces the opening of the voltage - gated ca channels on the presynaptic nerve membrane .
the ca influx into the neuron leads synaptic vesicles to fuse with the presynaptic membrane and to release the neurotransmitter acetylcholine ( ach ) in the synaptic cleft . from here ,
ach diffuses and binds to acetylcoline receptors ( achrs ) localized on the postsynaptic membrane on the muscle fiber .
this binding makes achrs permeable to ions so that na flows into the fiber while k flows out of the muscle cytosol . in this way
, the muscle membrane locally depolarizes , and this local depolarization opens voltage - gated na channels on the membrane , allowing a propagation of the depolarization ( action potential ) that spreads to involve the entire plasma membrane .
the action potential induced by ach release leads to the release of ca from the sarcoplasmic reticulum into the cytosol which causes muscle contraction .
the high concentration of ca inside the cytosol also activates camkii and pkc kinases which mediate signaling pathways leading to myogenin phosphorylation and inactivation . when myogenin is inactive , achr expression is suppressed .
in addition to myogenin phosphorylation , a second mechanism , which represses myogenin , is activated by ca signaling upon innervation and leads to the activation of three transcription repressors ( msy-3 , dach2 , and hdac9 ) .
these repressors bind myogenin promoter and inhibit its transcriptional activation . the binding motifs for msy-3 , dach2 , and hdac9 ( respectively hce , six , and mef2 ) are located on adjacent sequences on the myogenin promoter the ach
the myogenic transcription factor myogenin activates muscle - specific genes including achrs by binding the e - box element . when the action potential reaches the axon terminals , it induces the opening of the voltage - gated ca channels on the presynaptic nerve membrane .
the ca influx into the neuron leads synaptic vesicles to fuse with the presynaptic membrane and to release the neurotransmitter acetylcholine ( ach ) in the synaptic cleft . from here ,
ach diffuses and binds to acetylcoline receptors ( achrs ) localized on the postsynaptic membrane on the muscle fiber .
this binding makes achrs permeable to ions so that na flows into the fiber while k flows out of the muscle cytosol . in this way
, the muscle membrane locally depolarizes , and this local depolarization opens voltage - gated na channels on the membrane , allowing a propagation of the depolarization ( action potential ) that spreads to involve the entire plasma membrane .
the action potential induced by ach release leads to the release of ca from the sarcoplasmic reticulum into the cytosol which causes muscle contraction .
the high concentration of ca inside the cytosol also activates camkii and pkc kinases which mediate signaling pathways leading to myogenin phosphorylation and inactivation . when myogenin is inactive , achr expression is suppressed .
in addition to myogenin phosphorylation , a second mechanism , which represses myogenin , is activated by ca signaling upon innervation and leads to the activation of three transcription repressors ( msy-3 , dach2 , and hdac9 ) .
these repressors bind myogenin promoter and inhibit its transcriptional activation . the binding motifs for msy-3 , dach2 , and hdac9 ( respectively hce , six , and mef2 ) are located on adjacent sequences on the myogenin promoter in conclusion , while during development the first events leading to achr clusterization are musk- and rapsyn - dependent but nerve - independent ( achr prepatterning ) , the stabilization of some prepatterned achr clusters requires the innervation . indeed , once the muscle is contacted by the nerve , ach released by the motor neuron
induces a postsynaptic potential which stabilizes previous achr clusters in the contacted area and prevents achr clustering in non - contacted area .
moreover , agrin released by the neuron also stabilizes the achr clusters and , as well as neuregulin , strongly increases achr transcription in subsynaptic nuclei ( fig . 1 ) .
concomitantly , ca - dependent signals triggered by nerve - induced muscle electrical activity cause myogenin downregulation and inhibition of achr transcription in extrasynaptic regions of the muscle fiber ( fig . 2 ) .
the integration of these two mechanisms makes possible the compartmentalization of achr gene expression in subsynaptic nuclei and the stabilization of achr clusters only at nmjs .
the best characterized signaling responsible for achr accumulation at the nmjs is the agrin / lipoprotein receptor - related protein 4 ( lrp4)/muscle - specific tyrosine kinase receptor ( musk ) pathway .
agrin is a large heparan sulfate proteoglycan first isolated from the torpedo electric organ synthesized in the motoneurons , transported along axons , and released in the synaptic basal lamina , which surrounds the muscle fiber .
agrin is sufficient to induce achr ectopic clusters in adult muscle and postsynaptic specialization in denervated fibers [ 22 , 23 ] .
although it is not essential for prepatterning , in agrinmice , nmjs and achrs are uniformly distributed in the muscle fiber and not clustered [ 8 , 9 , 24 ] , and postsynaptic differentiation is inhibited.fig .
1the agrin musk lrp4 and neuregulin erbb pathways induce nmj assembly positive signals .
a agrin is released by the motor axon terminal and induces achr clustering , phosphorylation , and stabilization at the postsynaptic membrane . lrp4 associates with musk in the absence of agrin .
once phosphorylated , musk recruits the adapter protein dok-7 which binds crk and crkl and stimulates further musk phosphorylation and kinase activity .
b neuregulin ( ngr-1 ) is released by the nerve and induces achr transcription in synaptic nuclei .
erbb phosphorylation induced by nrg stimulates erk and jnk kinase activity which phosphorylates gabp- and gabp- transcription factors .
gabp- heterodimerizes with gabp- and binds dna at the n - box thereby enhancing transcription of achr genes the agrin musk
a agrin is released by the motor axon terminal and induces achr clustering , phosphorylation , and stabilization at the postsynaptic membrane . lrp4 associates with musk in the absence of agrin .
once phosphorylated , musk recruits the adapter protein dok-7 which binds crk and crkl and stimulates further musk phosphorylation and kinase activity .
b neuregulin ( ngr-1 ) is released by the nerve and induces achr transcription in synaptic nuclei .
erbb phosphorylation induced by nrg stimulates erk and jnk kinase activity which phosphorylates gabp- and gabp- transcription factors .
gabp- heterodimerizes with gabp- and binds dna at the n - box thereby enhancing transcription of achr genes agrin acts by activating another important player in the nmj assembly pathway , musk .
musk is a tyrosine kinase receptor expressed in the postsynaptic membrane of nmjs where it co - localizes with achrs .
the main role of agrin is to initiate musk autophosphorylation and activation ( fig .
however , musk can also be activated in an agrin - independent manner . indeed , in muskfibers , no nmjs are formed , and also , prepatterning before innervation is absent [ 8 , 9 , 29 ] . like achr ,
however , upon innervation , in the synaptic nuclei , musk is upregulated by different transcriptional pathways .
although agrin stimulates musk phosphorylation , the two proteins do not interact directly , but additional proteins ( coreceptors ) are engaged to allow agrin - mediated signaling in myotubes [ 31 , 32 ] .
lrp4 is concentrated at the nmjs and is necessary for agrin - stimulated musk phosphorylation and achr clustering . according to this model ,
when agrin binds to this preformed complex , it triggers a reorganization of musk , so promoting its transphophorylation and kinase activity .
once phopshorylated , musk activates signaling pathways that lead to synaptic differentiation including clustering of achrs [ 34 , 35 ] ( fig .
agrin activation of musk also leads to concentration at synapses of other proteins such as acetylcholinesterase ( ache ) , rapsyn , and neuregulin ( nrg ) receptors ( erbbs ; see below ) .
moreover , lrp4 has a crucial role in muscle prepatterning since it promotes musk phosphorylation and activation in the absence of agrin .
once phosphorylated , musk recruits docking protein-7 ( dok-7 ) , a non - catalytic adapter protein , which stimulates further musk phosphorylation and increases musk kinase activity ( fig .
1a ) . to properly regulate nmj formation , dok-7 engages at two phosphorylation sites two adapter proteins , crk and crkl . in mouse models lacking dok-7 , crk , and crkl ,
a coeffector in achr assembly is rapsyn , a membrane protein associated with the cytoplasmic side of the plasma membrane ( fig .
rapsyn anchorates achrs at the muscle membrane from the earliest stages of synaptogenesis , including prepatterning .
rapsyn also interacts with cytoskeletal proteins , thus being essential for achr clustering . in mice lacking rapsyn ,
it has been proposed that musk induces phosphorylation of nascent achr clusters and promotes also the binding of additional rapsyn to achr aggregates , which contributes to achr cluster growth and stability .
rapsyn may function as a scaffolding protein presenting a musk - activated src - related kinase to the achrs . upon
phosphorylation and activation , musk interacts with a large number of effectors and pathways converging toward the regulation of achr clusterization through different mechanisms .
musk indeed controls ( 1 ) achr scaffolding and redistribution via actin cytoskeleton reorganization , ( 2 ) achr stabilization through achr phosphorylation , ( 3 ) its proper endocytosis and turnover are also important for achr clustering , and ( 4 ) enhancement of transcription of achr genes selectively in subsynaptic nuclei ( reviewed in ) .
mrnas encoding other synaptic proteins such as ache , musk , and rapsyn are also concentrated at the synapses .
one candidate activator is neuregulin-1 ( ngr-1 ) , a glycoprotein which works as an extracellular signal that stimulates achr and other synapse - specific components transcription .
nrg-1 acts by binding to membrane - associated tyrosine kinase receptors related to the egf receptor ( erbb proteins 2 - 3 - 4 ) ( fig .
heterozygous mice are viable and fertile and show a decreased number of achrs at the nmjs [ 45 , 46 ] .
however , some recent in vivo studies , in which neuregulin-1 and erbb proteins were conditionally deleted , demonstrated that neuregulin signaling is dispensable [ 47 , 48 ] .
for this reason , the idea that nrg-1 is the main signal responsible for the increased transcription of achrs is highly controversial .
recent evidences also indicate that neuregulin may have a role in achr clustering and trafficking by modulating rather than determining achr expression at the nmjs .
the pathways leading from nrg-1erbb binding to regulation of achr gene transcription have been identified .
both in cultured muscle cells and in vivo , erbb phosphorylation by nrg-1 concomitantly stimulates erk [ 50 , 51 ] and the c - jun n - terminal kinase ( jnk ) activity [ 52 , 53 ] .
the activation of these pathways results in increased levels and phosphorylation of the e - twenty - six ( ets)-domain - binding transcription factor ga - binding protein ( gabp)- which binds dna , heterodimerizes with gabp- , and increases achr transcription [ 54 , 55 ] ( fig .
ets transcription factors bind dna at the n - box , a dna element located not only on the achr promoter but also on the promotor of other nmj components ( utrophin and acelylcholinesterase ) [ 5658 ] . in vivo inhibition of gabp results in reduced expression of achrs indicating the gabp signaling pathway , activated directly or indirectly by neuregulin , is necessary for the formation of functional synapses . as stated before , the agrin
musk pathway also contributes to increase synaptic achr gene transcription by a nrg / erbb - independent but rac - dependent jnk activation ( fig .
1b ) and also by interacting with the nrg / erbb pathway [ 43 , 60 , 61 ] .
the positive signals we described enhance achr accumulation only in the synaptic area . to achieve a correct formation of achr clusters in the nmjs ,
the presence of some negative signals that decrease achr concentration in the rest of the fiber , in non - synaptic areas , is necessary .
it inhibits achr localization , stability , and transcription along the muscle fiber by stimulation of cyclin - dependent kinase 5 [ 63 , 64 ] , protein kinase c ( pkc ) , and ca / calmodulin - dependent kinase ii ( camkii ) .
indeed , mutant mice lacking acetyltransferase , a biosynthetic enzyme for ach , develop faster and larger achr clusters ; they also exhibit hyperinnervation due to a broader distribution of achr clusters along the muscle fiber [ 66 , 67 ] . in detail , through muscle action potential , ach induces downregulation of achr transcription along the entire fiber .
however , since the previously described positive signals enhance achr accumulation only in the synaptic area ( fig . 1 )
, this results in a ach - dependent suppression of achr subunit gene transcription in extrasynaptic nuclei ( fig . 2 )
myogenin is a basic helix - loop - helix myogenic transcription factor that activates achr genes in the absence of innervation or by blockade of muscle electrical activity by denervation .
myogenin regulates achr and many other muscle gene transcription by binding the e - boxes located on their promoter / enhancers .
myogenin is downregulated as the muscle matures around the time when innervation is completed , and it is upregulated upon denervation .
forced expression of myogenin in vivo is able to induce achr expression along the entire muscle fiber .
multiple mechanisms have been proposed to explain how neural activity controls myogenin in postnatal muscle . on the basis of one of these mechanisms , following muscle depolarization , extracellular calcium enters through voltage - activated channels and activates different signal transduction cascades mediated by pkc and camkii , leading to myogenin phosphorylation [ 63 , 72 ] .
this posttranslational modification inhibits myogenin binding to e - boxes of achr promoters , resulting in reduced achr expression ( fig .
a second regulatory mechanism restraining myogenin activity involves transcriptional repression of myogenin ( fig . 2 ) .
the posttranslational and the transcriptional mechanisms of myogenin repression could act on a different timescale to ensure that myogenin expression remains blocked as long as the muscle is innervated .
although it is very well known that myogenin is transcriptionally modulated as a consequence of muscle depolarization , the mechanisms that are upstream of this regulation have only recently started to be elucidated .
several studies reported that in myogenin repression during innervation , class ii deacetylases are recruited at the myogenin promoter .
the hystone deacetylase 9 ( hdac9 ) splice variant mitr is a transcriptional repressor induced in innervated muscle .
it contributes to myogenin repression by chromatin acetylation and acts as a corepressor in a complex with the myocyte enhancer factor-2 ( mef2 ) transcription factor ( fig . 2 ) .
another myogenin repressor is dachshund homolog 2 ( dach2 ) ( fig . 2 ) .
it has been shown that , during denervation , dach2 is inhibited by hdac4 , another class ii deacetylase , which induces myogenin expression thereby activating achrs [ 7678 ] .
a third circuit of postnatal myogenin expression regulation is mediated by msy3/csda , a y - box factor that binds the myogenin promoter at a new binding motif ( highly conserved element , hce ) and exerts a repression function ( fig . 2 ) .
msy-3 is therefore partially responsible for the restricted spatiotemporal expression of achr in the subsynaptical regions of muscle fibers .
the conserved binding motifs of hdac9/mef2 , dach2 , and msy-3 are adjacent sequences on the myogenin promoter , which suggests a strong interplay between the three cis trans systems to ensure a proper myogenin and achr repression in adult and innervated muscle.fig .
the myogenic transcription factor myogenin activates muscle - specific genes including achrs by binding the e - box element .
when the action potential reaches the axon terminals , it induces the opening of the voltage - gated ca channels on the presynaptic nerve membrane .
the ca influx into the neuron leads synaptic vesicles to fuse with the presynaptic membrane and to release the neurotransmitter acetylcholine ( ach ) in the synaptic cleft . from here , ach diffuses and binds to acetylcoline receptors ( achrs ) localized on the postsynaptic membrane on the muscle fiber .
this binding makes achrs permeable to ions so that na flows into the fiber while k flows out of the muscle cytosol . in this way
, the muscle membrane locally depolarizes , and this local depolarization opens voltage - gated na channels on the membrane , allowing a propagation of the depolarization ( action potential ) that spreads to involve the entire plasma membrane .
the action potential induced by ach release leads to the release of ca from the sarcoplasmic reticulum into the cytosol which causes muscle contraction .
the high concentration of ca inside the cytosol also activates camkii and pkc kinases which mediate signaling pathways leading to myogenin phosphorylation and inactivation .
in addition to myogenin phosphorylation , a second mechanism , which represses myogenin , is activated by ca signaling upon innervation and leads to the activation of three transcription repressors ( msy-3 , dach2 , and hdac9 ) .
these repressors bind myogenin promoter and inhibit its transcriptional activation . the binding motifs for msy-3 , dach2 , and hdac9 ( respectively hce , six , and mef2 ) are located on adjacent sequences on the myogenin promoter the ach
the myogenic transcription factor myogenin activates muscle - specific genes including achrs by binding the e - box element .
when the action potential reaches the axon terminals , it induces the opening of the voltage - gated ca channels on the presynaptic nerve membrane .
the ca influx into the neuron leads synaptic vesicles to fuse with the presynaptic membrane and to release the neurotransmitter acetylcholine ( ach ) in the synaptic cleft . from here ,
ach diffuses and binds to acetylcoline receptors ( achrs ) localized on the postsynaptic membrane on the muscle fiber .
this binding makes achrs permeable to ions so that na flows into the fiber while k flows out of the muscle cytosol . in this way
, the muscle membrane locally depolarizes , and this local depolarization opens voltage - gated na channels on the membrane , allowing a propagation of the depolarization ( action potential ) that spreads to involve the entire plasma membrane .
the action potential induced by ach release leads to the release of ca from the sarcoplasmic reticulum into the cytosol which causes muscle contraction .
the high concentration of ca inside the cytosol also activates camkii and pkc kinases which mediate signaling pathways leading to myogenin phosphorylation and inactivation . when myogenin is inactive , achr expression is suppressed .
in addition to myogenin phosphorylation , a second mechanism , which represses myogenin , is activated by ca signaling upon innervation and leads to the activation of three transcription repressors ( msy-3 , dach2 , and hdac9 ) .
these repressors bind myogenin promoter and inhibit its transcriptional activation . the binding motifs for msy-3 , dach2 , and hdac9 ( respectively hce , six , and mef2 )
are located on adjacent sequences on the myogenin promoter in conclusion , while during development the first events leading to achr clusterization are musk- and rapsyn - dependent but nerve - independent ( achr prepatterning ) , the stabilization of some prepatterned achr clusters requires the innervation .
indeed , once the muscle is contacted by the nerve , ach released by the motor neuron induces a postsynaptic potential which stabilizes previous achr clusters in the contacted area and prevents achr clustering in non - contacted area .
moreover , agrin released by the neuron also stabilizes the achr clusters and , as well as neuregulin , strongly increases achr transcription in subsynaptic nuclei ( fig . 1 ) .
concomitantly , ca - dependent signals triggered by nerve - induced muscle electrical activity cause myogenin downregulation and inhibition of achr transcription in extrasynaptic regions of the muscle fiber ( fig . 2 ) .
the integration of these two mechanisms makes possible the compartmentalization of achr gene expression in subsynaptic nuclei and the stabilization of achr clusters only at nmjs .
in such disorders , defects in neuromuscular transmission cause a fatigable weakness in limb , ocular , bulbar , truncal , and respiratory muscles . generally , weakness becomes more severe with exercise and improves with rest .
cms have been identified worldwide and occur at a frequency of < 1/500,000 people , although this incidence is constantly growing .
most patients present symptoms during infancy , although in some syndromes , symptoms are not manifested until childhood or adult life . depending on the neuromuscular transmission defect , cms can be classified as presynaptic , synaptic , or postsynaptic , even though it is not always possible to identify accurately the cause of the pathology . over the past few years
, many causative genes and mutations for cms have been identified , although in many patients , no causative mutations can be found . in most cases ,
cms causative mutations are missense , truncation , or splice - site mutations and result in structural changes of achrs or in achr low affinity for ach .
cms - associated genetic mutations have been mapped in achr subunits , choline acetyltransferase , the collagen tail subunit of acetylcholinesterase , rapsyn , musk , and skeletal muscle
phenotype that mainly affects proximal muscles rather than the distal ones with ptosis present from early age .
mutations in the dok-7 locus were identified in groups of patients who had small eps , a reduced number of achrs at the eps , and a limb - girdle phenotype with no mutations in rapsyn or achrs .
dok-7 mutants generally have abnormally small and simplified nmjs but show normal achr and ache functions , correct musk activation , and achr clusterization , although dok-7 acts in concert with musk in activating rapsyn to concentrate achrs at the junctional folds .
it has therefore been suggested that the altered size and integrity of nmjs observed were probably the consequence of a high and widespread degeneration and remodeling of the eps [ 86 , 87 ] .
together with genetic alterations in the coding region of components of the synaptic apparatus , also mutations in their regulative regions can account for the congenital myasthenic phenotype .
a mutation in the n - box of the achr subunit promoter , resulting in a decreased gabp transcription factor binding activity , causes a reduction in the achr number which is the cause of cms clinical symptoms in human [ 89 , 90 ] . besides being congenital , myasthenia
can also be caused by a complex autoimmune disorder of neuromuscular transmission ( myasthenia gravis , mg ) .
mg is characterized by a fluctuating weakness of the head , neck , and upper extremity muscles that worsens with activity and improves with rest [ 91 , 92 ] .
when the bulbar and the respiratory muscle deteriorate , the disease becomes life - threatening , and ventilation with medication is required .
approximately 80% of mg patients have autoantibodies against achr and , consequently , they show a decreased number of achrs at the postynaptic membrane . in addition , the deposit of the complement causes a structural and functional damage of the postsynaptic membrane , resulting in a neuromuscular transmission failure .
in some mg patients , autoantibodies against musk have also been found , but their pathogenic role remains unclear since the number of achrs is not reduced and no deposits of complement are found at the postsynaptic membrane .
it has been therefore suggested that , since musk can be required for retrograde signal , it is possible that musk autoantibodies interfere with the presynaptic apparatus organization [ 94 , 95 ] .
the age - related muscle loss ( sarcopenia ) strongly impairs the quality of life in the elderly , and it is associated with increased risk of morbidity and mortality .
however , many studies suggest that nmj structural and functional impairment plays a key role in muscle wasting during aging .
nmj degeneration is a feature of sarcopenia , and it has been documented both in animal models and in humans .
changes in nmj morphology depend on the type of muscle and include variation of the nerve terminal area size , of the end plate size , of the number of synaptic vesicles , of mitochondrial content , and of smooth endoplasmic reticulum content ( reviewed in ) . by in vivo
live animal imaging , it has been revealed that at 1218 months , in mice , a few nmjs start to display loss of motor neuron terminal branches and achrs . at this point , probably in order to compensate for loss of some synaptic sites , motor neuron sprouting increases and accounts for the formation of new nmjs .
therefore , by 2436 months , most nmjs are completely denervated , and postsynaptic eps are highly fragmented .
age - related changes in the morphology of nmjs can be related to alteration of the axonal transport , which impairs the availability of trophic factors and organelles , such as mitochondria , essential for neuronal survival .
age - related changes in myelinated schwann cells have also been reported and might be associated with nmj degeneration .
it has interestingly been shown that neurotrophic and myotrophic factors such as bdnf , nt-3 , insulin - like growth factor ( igf)-i , and igf - ii , which are necessary for the maintenance of presynaptic and postsynaptic apparatus at the nmj , also play a modulating role in aging [ 101 , 102 ] .
for example , igf-1 injections into muscle inhibit motor neuron and nmj degeneration and prevent age - related force decline in mice .
recent studies suggest that motor neuron degeneration and consequent denervation of myofibers are a major cause of muscle mass loss [ 104 , 105 ] . in aged people ,
some myofibers are lost , and others , which were innervated by fast motor neurons ( type ii fibers ) , are reinnervated by slow ones ( type i fibers ) .
this results in an increased percentage of type i fibers and atrophy , which characterizes sarcopenia .
it has also been demonstrated that chronic exercise training and higher neuromuscular activity counteract the alterations in motor neurons and delay the onset of denervation and sarcopenia in aged people [ 104106 ] .
although the causes of age - associated nmj degeneration remain unclear , other mechanisms might contribute to compromised muscle neurotransmission and sarcopenia in aging .
presynaptic terminals and postsynaptic eps contain a high concentration of mitochondria , which are critical for nmj function since they account for energy support , ca buffering , synaptic transmission , and apoptosis .
mitochondrial dysfunctions might lead to altered calcium buffering , less atp generation and more ros production , and oxidative damage during aging , which impairs nmjs [ 108 , 109 ] .
in particular , the impaired buffering of ca determines an increased concentration of cytosolic ca , which might activate some proteases called calpains .
it has been shown that calpains interact with rapsyn thereby disrupting achr clusters . through this mechanism ,
moreover , a direct correlation between increased oxidative stress and neuromuscular function has been recently proposed .
mice lacking the antioxidant enzyme cuznsod ( cu / zn superoxide dismutase , sod 1 ) show high oxidative damage and rapid sarcopenia .
the morphology of nmjs at 11 months in sod1mice is altered , and it is comparable to nmj degeneration in 33-month - old , wild - type mice .
furthermore , denervated nmjs and fragmentated achrs observed in young sod1mice notably reduce muscle contractile force [ 111 , 112 ] .
these observations suggest that nmj degeneration during aging might be caused also by age - related oxidative stress , which usually is produced by mitochondrial dysfunctions .
oxidative stress also induces damage in myelinated peripheral nerves and consequent increased inflammatory cytokines during aging ( reviewed in ) .
interestingly , it has been recently reported that calorie restriction slows down the aging process and , in particular , decreases deterioration of peripheral nerve by upregulating autophagy .
it has also been shown that calorie restriction reduces oxidative damage , nmj degeneration , and muscle atrophy in sod1mice , and this further suggests that oxidative damage affects nmjs [ 111 , 114 ] .
the complexity of the neuromuscular junction apparatus and of the regulative systems responsible for its organization indicates that the functional modulation of the connection between neurons and muscle fibers is crucial .
the right concentration of the acetylcholine receptors in the area where the axon terminals juxtaposes the postsynaptic membrane accounts for the nmj s ability to generate the action potential that provokes muscle contraction and consequently movements .
for this reason , multiple levels of regulation are committed to accurately control this process .
animal models show that impairing only one of these regulative pathways can cause severe phenotypes characterized by compromised coordinate movements and breath capacity which usually prove fatal .
we have also seen that transcriptional control plays an important role in muscle physiological response to nerve activity .
cms and myasthenia gravis are human pathologies characterized by an extreme fatigability and muscle weakness and in which insufficiency of a single component of nmj frequently accounts for their pathophysiology .
studying nmj transmission in aging and in those pathological conditions characterized by different levels of muscle weakness may reveal new mechanisms that contribute to the fine - tuning of the interplay between motor neurons and muscles .
understanding the mechanisms underlying age- and disease - associated atrophy and alteration of nmjs should open up new potential therapeutic avenues to these skeletal muscle dysfunctions . | skeletal muscle innervation is a multi - step process leading to the neuromuscular junction ( nmj ) apparatus formation .
the transmission of the signal from nerve to muscle occurs at the nmj level .
the molecular mechanism that orchestrates the organization and functioning of synapses is highly complex , and it has not been completely elucidated so far .
neuromuscular junctions are assembled on the muscle fibers at very precise locations called end plates ( ep ) .
acetylcholine receptor ( achr ) clusterization at the end plates is required for an accurate synaptic transmission .
this review will focus on some mechanisms responsible for accomplishing the correct distribution of achrs at the synapses .
recent evidences support the concept that a dual transcriptional control of achr genes in subsynaptic and extrasynaptic nuclei is crucial for achr clusterization .
moreover , new players have been discovered in the agrin
musk pathway , the master organizer of postsynaptical differentiation .
mutations in this pathway cause neuromuscular congenital disorders .
alterations of the postynaptic apparatus are also present in physiological conditions characterized by skeletal muscle wasting .
indeed , recent evidences demonstrate how nmj misfunctioning has a crucial role at the onset of age - associated sarcopenia . |
health care delivery in germany is highly fragmented , resulting in poor vertical and horizontal integration and a system that is focused on curing single diseases instead of managing patient populations . while it is now widely accepted that a strong primary care system can help to improve coordination and responsiveness in health care , with the endorsement of the government , primary care in the german system
traditionally there has been no gatekeeper function ; patients can freely choose and directly access both primary and secondary care providers , making coordination and cooperation within and across sectors difficult .
since 2000 , in an unusually long phase of programmatic and personal continuity in health care policy in germany , the federal ministry of health prepared several decisive legislative moves to improve care continuity with primary care as a hub .
it promoted more integrative forms of care via disease management programmes and medical care centres , it induced competition via selective contracting among providers and payers , it fostered gatekeeping and introduced patient registries for the chronically ill , and it began to align financial incentives for physicians , insurers , and patients . in this article , we will first give a brief working definition of integrated primary care and then outline the current status of the german health care system from an integrated primary care perspective .
all these reforms have placed primary care in the centre , strengthening its role as the patient s navigator through the health care system .
as far as evaluation results are available implementation of most of the reforms is ongoing and systematic evaluation is not always a requirement we will discuss the impact of the new forms of care on coordination and health outcomes . in the concluding section we will assess future implications for policy makers :
have these reforms pulled the right levers for promoting stronger coordination and strengthening the primary care system s role as navigator through the health system ? what other barriers must be addressed by future reforms ?
a strong primary care system can help improve continuity and responsiveness in health care especially for specific population groups such as frail elderly or people with complex conditions , but also for the population in general [ 1 , p. 15 ] . according to starfield , primary care has four main functions .
a primary care system should enable first - contact access for each new need ; provide long - term person - focused care ; ensure comprehensive care for most health needs , and it should coordinate care , both horizontally and vertically , when services from other providers are needed .
this is because person - focused , comprehensive care can only be provided when primary care is supported by other levels of care , including community services and hospital care . for our purposes , we define integrated primary care as a system that fulfils all these four functions and especially the coordinative function . in the following section
, we will assess how well the current german system is prepared to fulfil this coordinative function and thus to provide integrated primary care .
how well does cooperation between different professions work within the primary care sector , between the primary care sector and other sectors of health care , and between health and social care ?
what role does primary care play in the coordination process , and what are the barriers to a more integrated role of primary care ?
primary care in germany includes all ambulatory care services provided by office - based , mostly single - handed , private for - profit general practitioners / family doctors , general internists or paediatricians .
the other half are specialists almost all specialities are offered in germany by office - based secondary care providers .
individuals can freely choose their primary care provider , and patients have free choice of specialists , psychotherapists ( since 1998 ) , dentists , pharmacists and emergency care [ 4 , p. 5 ] .
since office - based primary and secondary care physicians work in solo practice , health care is often not coordinated .
doctor hopping is a well - known phenomenon and consequence from the way the system is set up .
sixty - eight percent of primary care physicians in germany work in solo practice ; 31% work in small group practices with 24 full - time equivalent doctors sharing office space but not patients or patients health care files .
midlevel of training ( like nurse practitioners or physician assistants in the us , canada , or the netherlands ) does not exist in german primary care .
traditionally doctors have worked with medical assistants ( arzthelferin ) who complete a three - year vocational training , and whose role in physicians practices combines administrative and some clinical tasks . in a medical assistant s daily work administrative tasks prevail , their clinical responsibilities are limited to minor tasks like taking blood pressure , giving injections or taking and analysing blood samples
. there have been efforts to develop the medical assistants profession into something closer to a nurse practitioner ( see below in the section on community medicine nurses ) , but most doctors in germany ( 56% ) oppose the idea of expanding the role of non - physicians in delivering care to patients .
health information technology is not very advanced in primary care practice and still mostly used for administrative purposes , not for clinical decision support or patient management .
the most common feature is electronic prescribing of medication ( used routinely by 59% of primary care physicians in germany ) . in the use of electronic medical records ( used by 42% ) , electronically ordering tests or accessing test results or hospital records , germany lags behind other countries ( see table 1 percentage of primary care physicians using electronic support ) .
care coordination between the ambulatory sector and the hospital is a challenge for the german health care system .
hospitals have legally been restricted to focus on inpatient care and to provide outpatient emergency care ; only university hospitals have formal outpatient facilities [ 4 , p. 16
] . health care reforms in 2004 and 2007 have granted hospitals additional competencies to provide outpatient services to patients .
today , the main forms of ambulatory care provided by hospitals are day surgery , highly specialised outpatient care , and outpatient care as part of disease management programmes and integrated care contracts .
if inpatient treatment is needed , office - based physicians refer their patients ( but do not follow them during their hospital stay ) and usually but not systematically receive them back after discharge .
not surprisingly is diverging pharmaceutical treatment , prior , during and post hospitalization , hard to explain to patients , and it is a typical bone of contention between hospitals and primary care providers . more than 50% of primary care physicians report that it takes more than 14 days for them to receive a full report from a hospital once their patient has been discharged ; for 15% it takes more than a month .
electronic access to their patients hospital records is available only for 14% of primary care physicians .
seventy percent of the respondents stated that better integration of information systems between office - based physicians and hospitals would be an effective way to improve quality of care .
poor linkages between the health care system and services in the community like long - term care , social services , self - help or patient groups , family and lay carers , are also notorious in germany , and constitute another obstacle to more holistic care and better care coordination .
social care in germany is provided by a myriad of mainly private organizations that complement family and lay support for people with special needs and various levels of dependency , i.e. the elderly , children with special needs , mentally ill and the physically or mentally handicapped [ 4 , p. 16
one major reason for poor coordination between health and social care is financing : services in these sectors are financed by different funding streams and insurance regimes .
thus , mainstream health care in germany is still far from being an integrated system with primary care at its centre .
current access rules , i.e. free choice of providers , do not provide incentives for coordination through a primary care provider
. moreover , the financing and the organisational set - up of the system are two additional barriers to stronger cooperation .
also , with the spatial separation of care providers and poor use of health information technology , providers administrative costs for coordinating care are still rather high and are not appropriately reimbursed in the doctors fee schedule .
further , the development of new professions such as academically trained nurses who could complement gp services has only just begun [ 8 , 9 ] .
aware of these problems , the german government has introduced a number of reforms during the last nine years , which address the various barriers identified above .
a strong primary care system can help improve continuity and responsiveness in health care especially for specific population groups such as frail elderly or people with complex conditions , but also for the population in general [ 1 , p. 15 ] . according to starfield , primary care has four main functions .
a primary care system should enable first - contact access for each new need ; provide long - term person - focused care ; ensure comprehensive care for most health needs , and it should coordinate care , both horizontally and vertically , when services from other providers are needed .
this is because person - focused , comprehensive care can only be provided when primary care is supported by other levels of care , including community services and hospital care . for our purposes , we define integrated primary care as a system that fulfils all these four functions and especially the coordinative function . in the following section
, we will assess how well the current german system is prepared to fulfil this coordinative function and thus to provide integrated primary care .
how well does cooperation between different professions work within the primary care sector , between the primary care sector and other sectors of health care , and between health and social care ?
what role does primary care play in the coordination process , and what are the barriers to a more integrated role of primary care ?
primary care in germany includes all ambulatory care services provided by office - based , mostly single - handed , private for - profit general practitioners / family doctors , general internists or paediatricians .
the other half are specialists almost all specialities are offered in germany by office - based secondary care providers .
individuals can freely choose their primary care provider , and patients have free choice of specialists , psychotherapists ( since 1998 ) , dentists , pharmacists and emergency care [ 4 , p. 5 ] .
since office - based primary and secondary care physicians work in solo practice , health care is often not coordinated .
doctor hopping is a well - known phenomenon and consequence from the way the system is set up .
sixty - eight percent of primary care physicians in germany work in solo practice ; 31% work in small group practices with 24 full - time equivalent doctors sharing office space but not patients or patients health care files .
midlevel of training ( like nurse practitioners or physician assistants in the us , canada , or the netherlands ) does not exist in german primary care . traditionally doctors have worked with medical assistants ( arzthelferin ) who complete a three - year vocational training , and whose role in physicians practices combines administrative and some clinical tasks . in a medical assistant s daily work administrative tasks prevail , their clinical responsibilities are limited to minor tasks like taking blood pressure , giving injections or taking and analysing blood samples .
there have been efforts to develop the medical assistants profession into something closer to a nurse practitioner ( see below in the section on community medicine nurses ) , but most doctors in germany ( 56% ) oppose the idea of expanding the role of non - physicians in delivering care to patients .
health information technology is not very advanced in primary care practice and still mostly used for administrative purposes , not for clinical decision support or patient management .
the most common feature is electronic prescribing of medication ( used routinely by 59% of primary care physicians in germany ) . in the use of electronic medical records ( used by 42% ) , electronically ordering tests or accessing test results or hospital records , germany lags behind other countries ( see table 1 percentage of primary care physicians using electronic support ) .
care coordination between the ambulatory sector and the hospital is a challenge for the german health care system .
hospitals have legally been restricted to focus on inpatient care and to provide outpatient emergency care ; only university hospitals have formal outpatient facilities [ 4 , p. 16
] . health care reforms in 2004 and 2007 have granted hospitals additional competencies to provide outpatient services to patients .
today , the main forms of ambulatory care provided by hospitals are day surgery , highly specialised outpatient care , and outpatient care as part of disease management programmes and integrated care contracts .
if inpatient treatment is needed , office - based physicians refer their patients ( but do not follow them during their hospital stay ) and usually but not systematically receive them back after discharge .
not surprisingly is diverging pharmaceutical treatment , prior , during and post hospitalization , hard to explain to patients , and it is a typical bone of contention between hospitals and primary care providers .
more than 50% of primary care physicians report that it takes more than 14 days for them to receive a full report from a hospital once their patient has been discharged ; for 15% it takes more than a month .
electronic access to their patients hospital records is available only for 14% of primary care physicians .
seventy percent of the respondents stated that better integration of information systems between office - based physicians and hospitals would be an effective way to improve quality of care .
poor linkages between the health care system and services in the community like long - term care , social services , self - help or patient groups , family and lay carers , are also notorious in germany , and constitute another obstacle to more holistic care and better care coordination .
social care in germany is provided by a myriad of mainly private organizations that complement family and lay support for people with special needs and various levels of dependency , i.e. the elderly , children with special needs , mentally ill and the physically or mentally handicapped [ 4 , p. 16
one major reason for poor coordination between health and social care is financing : services in these sectors are financed by different funding streams and insurance regimes .
thus , mainstream health care in germany is still far from being an integrated system with primary care at its centre .
current access rules , i.e. free choice of providers , do not provide incentives for coordination through a primary care provider
. moreover , the financing and the organisational set - up of the system are two additional barriers to stronger cooperation .
also , with the spatial separation of care providers and poor use of health information technology , providers administrative costs for coordinating care are still rather high and are not appropriately reimbursed in the doctors fee schedule .
further , the development of new professions such as academically trained nurses who could complement gp services has only just begun [ 8 , 9 ] .
aware of these problems , the german government has introduced a number of reforms during the last nine years , which address the various barriers identified above .
sixty - eight percent of primary care physicians in germany work in solo practice ; 31% work in small group practices with 24 full - time equivalent doctors sharing office space but not patients or patients health care files .
other health care workers with a midlevel of training ( like nurse practitioners or physician assistants in the us , canada , or the netherlands ) does not exist in german primary care .
traditionally doctors have worked with medical assistants ( arzthelferin ) who complete a three - year vocational training , and whose role in physicians practices combines administrative and some clinical tasks . in a medical assistant s daily work administrative tasks prevail , their clinical responsibilities are limited to minor tasks like taking blood pressure , giving injections or taking and analysing blood samples
. there have been efforts to develop the medical assistants profession into something closer to a nurse practitioner ( see below in the section on community medicine nurses ) , but most doctors in germany ( 56% ) oppose the idea of expanding the role of non - physicians in delivering care to patients .
health information technology is not very advanced in primary care practice and still mostly used for administrative purposes , not for clinical decision support or patient management .
the most common feature is electronic prescribing of medication ( used routinely by 59% of primary care physicians in germany ) . in the use of electronic medical records ( used by 42% ) , electronically ordering tests or accessing test results or hospital records , germany lags behind other countries ( see table 1 percentage of primary care physicians using electronic support ) .
care coordination between the ambulatory sector and the hospital is a challenge for the german health care system .
hospitals have legally been restricted to focus on inpatient care and to provide outpatient emergency care ; only university hospitals have formal outpatient facilities [ 4 , p. 16
] . health care reforms in 2004 and 2007 have granted hospitals additional competencies to provide outpatient services to patients .
today , the main forms of ambulatory care provided by hospitals are day surgery , highly specialised outpatient care , and outpatient care as part of disease management programmes and integrated care contracts .
if inpatient treatment is needed , office - based physicians refer their patients ( but do not follow them during their hospital stay ) and usually but not systematically receive them back after discharge .
post - surgical care is usually also done by office - based physicians . not surprisingly is diverging pharmaceutical treatment , prior , during and post hospitalization , hard to explain to patients , and it is a typical bone of contention between hospitals and primary care providers .
more than 50% of primary care physicians report that it takes more than 14 days for them to receive a full report from a hospital once their patient has been discharged ; for 15% it takes more than a month .
electronic access to their patients hospital records is available only for 14% of primary care physicians .
seventy percent of the respondents stated that better integration of information systems between office - based physicians and hospitals would be an effective way to improve quality of care .
poor linkages between the health care system and services in the community like long - term care , social services , self - help or patient groups , family and lay carers , are also notorious in germany , and constitute another obstacle to more holistic care and better care coordination .
social care in germany is provided by a myriad of mainly private organizations that complement family and lay support for people with special needs and various levels of dependency , i.e. the elderly , children with special needs , mentally ill and the physically or mentally handicapped [ 4 , p. 16
one major reason for poor coordination between health and social care is financing : services in these sectors are financed by different funding streams and insurance regimes .
thus , mainstream health care in germany is still far from being an integrated system with primary care at its centre .
current access rules , i.e. free choice of providers , do not provide incentives for coordination through a primary care provider
. moreover , the financing and the organisational set - up of the system are two additional barriers to stronger cooperation . also , with the spatial separation of care providers and poor use of health information technology , providers administrative costs for coordinating care
further , the development of new professions such as academically trained nurses who could complement gp services has only just begun [ 8 , 9 ] .
aware of these problems , the german government has introduced a number of reforms during the last nine years , which address the various barriers identified above .
since the year 2000 , the german government has introduced a variety of managed care tools and structures , through three subsequent reforms [ 1012 ] .
the most recent reform act of 2007 has broadened opportunities of care coordination between providers and across sectors .
gatekeeping , disease management programmes , integrated care contracts , medical care centres and community medicine nurses all can lead to a stronger role for primary care .
receiving previously unknown political support , primary care providers can now fulfil a more integrating function and act as patient navigators through the health care system .
other objectives pursued in the series of reforms mentioned above are quality improvement and cost control , as care coordination is expected to contribute to a more efficient use of health care .
to make new forms of care possible , the government changed the rules of contracting between health insurance funds and providers . prior to 2000 , contracting between ambulatory care practitioners and health insurance funds had been compulsory and indirect for physicians contracting with statutory health insurance funds , membership in a regional association of statutory health insurance physicians has been ( and still is ) mandatory .
these regional associations negotiate collective contracts for ambulatory care with the health insurance funds that operate in their region .
they receive a total budget from the health insurance funds based on historical data and distribute it among their physician members on a fee - for - service basis . the reform act of statutory health insurance 2000 for the first time broke with the strict system of collectively negotiated contracts and budgets , introducing the possibility for physicians to selectively sign contracts with health insurance funds for integrated care schemes , gatekeeper models and disease management programmes .
the 2000 reform thus established the legal basis for health insurance funds and providers to enter selective integrated care contracts , besides the above - mentioned unitary but mandatory collective contractual system . under integrated care contracts ,
care is provided in provider networks that can be managed by independent management organizations . but uptake of integrated care contracts was initially very slow . a key measure toward accelerating care coordination was the offer of financial incentives for providers , introduced by law but for a limited period of time : from 2004 to 2008 , one percent of the total statutory health insurance budget available for ambulatory and hospital care has been earmarked to initially fund integrated care contracts . in total , the start - up financing scheduled until the end of 2008 amounted to approximately e 800 million . from just over 600 contracts in early 2005 , by december 2008 their number had risen to more than 6000 with about four million patients being treated under this contractual form of integrated care ( see also figure 1 fast expansion of integrated care ) . the statutory health insurance competition strengthening act of 2007 established further integrated care opportunities . since then , long - term care providers can be included in contracts , and non - medical professionals can become the main contractual partner to health insurance funds , a position formerly restricted to physicians .
also since 2007 , integrated care contracts now are to focus on population - oriented integrated care , a term not defined by the lawmaker to allow for creativity in designing integrated care models .
it is usually understood as proactive , patient - centred health care for a defined population with providers taking responsibility for the coordination of care and for improving or maintaining the health status of the insured population , thereby putting a focus on health promotion or prevention .
so far , however , disease- or procedure - oriented contracts continue to constitute the bulk of the integrated care contracts signed . only a few companies are developing ambitious models of population - oriented integrated care in germany [ 16 , p. 129223 ] .
particularly the move towards population - oriented integrated care can imply a strengthening of primary care as the coordinating agent in a patient s care process .
population - oriented care implies a more comprehensive concept of health care , in which a multidisciplinary group of providers is not only responsible for curing illness but also for maintaining or improving the health status of the population .
this comes very close to starfield s model of integrated primary care as an ongoing , person - focused , comprehensive and coordinating system of care .
existing models of population - oriented integrated care in germany use either a primary care physician or team as the coordinating agent for participating patients . gatekeeping based on primary care physicians was introduced in 2000 .
gatekeeping in primary care also exists in other countries with shi systems like the netherlands and has recently been implemented in france in 2004 . in germany
, patients are free to choose a family physician who then serves as gatekeeper and guide through the health care system .
once a patient has subscribed to a gatekeeping scheme , specialists can only be seen upon referral , although exceptions apply for gynaecologists , paediatricians and ophthalmologists . since 2007 legislation requires health insurance funds to offer gatekeeper contracts . for patients ,
enrolment in gatekeeping arrangements is voluntary and can be rewarded through financial incentives by their health insurance fund .
about six million patients had signed up for the gatekeeping scheme by the end of 2007 .
family physicians willing to enter a gatekeeper contract with a health insurance fund have to fulfil certain criteria : participate in quality circles , follow evidence - based treatment guidelines , run a quality management programme in their practice , and attend trainings in areas like patient - oriented communication , basic treatment and diagnostics of mental disorders , palliative or geriatric care .
gatekeeping is a very forthright provision of the lawmaker to strengthen primary care , installing the primary care physician as the coordinating agent in patient health care and restricting the patient s free choice of specialists . through better care coordination and the above - mentioned criteria for participating physicians
, gatekeeping contracts are to enhance the quality of care and to reduce costs by preventing unnecessary specialist visits .
however , a survey among health insurance members conducted by the bertelsmann stiftung between 2004 and 2007 revealed that in their current set - up , gatekeeping arrangements do not achieve their aims of controlling the number of patient visits to specialists or of improving health outcomes .
patients enrolled in gatekeeper contracts do not report better health outcomes than patients who are not enrolled , and the number of visits to specialists does not seem to go down . in future contracts , more incentives for physicians to improve the quality of care seem to be necessary if gatekeeping models are to actually reach their goals .
disease management programmes ( dmps ) were introduced in germany in 2002 , in continuation of an earlier reform . in 1996 , a risk equalization scheme based on average spending by age and sex was introduced between statutory health insurance funds .
however , the costs of providing care for chronically ill patients had not been taken into account adequately , which led to health insurance funds particularly targeting young , healthy insurees . in 2004 , a separate high - risk structure compensation scheme for patients enrolled in disease management programmes was added . under the new scheme ,
dmp participants no longer generate a deficit : health insurance funds receive an additional lump sum from the risk equalization scheme for each person enrolled .
there are six requirements for dmp accreditation by the german federal insurance authority : treatment according to evidence - based guidelines with respect to the relevant sectors of care ; quality assurance measures ; required procedure for enrolment of insured , including duration of participation ; training and information for care providers and patients ; electronic documentation of diagnostic findings , applied therapies and outcomes ; evaluation of clinical outcomes and costs .
disease management programmes currently exist for six major chronic conditions : diabetes type 1 , diabetes type 2 , coronary heart disease , breast cancer , asthma and chronic obstructive pulmonary disease . in june 2008 ,
more than 5.2 million patients were enrolled , the largest share ( 2.7 million ) of which participate in diabetes type 2 dmps [ personal conversation with representative of the german federal insurance authority ( bundesversicherungsamt ) on february 6 , 2009 ] . for
incentives exist for both : patients are exempt from out - patient fees and co - payments ; physicians receive a lump sum payment for coordination and documentation activities .
usually , primary care physicians take on the role of coordinating care for dmp patients over time , referring them to specialists when necessary and documenting the care process . a growing number of dmp evaluations
participants experience less complications and emergency hospital admissions ; instead , the number of early - stage hospitalization is higher . compared to non - enrolled control group patients , diabetes type 2 patients enrolled in dmps self - report higher quality of life and a better physical and mental health status ;
similarly , a representative case - control study published in mid-2008 reported less relapses , less pain , better results for blood pressure and cholesterol for patients participating in a coronary heart disease dmp . among physicians ,
acceptance is also rising , although initially documentation requirements were perceived as an extra burden .
most of the time in the implementation of disease management programmes , primary care takes a central coordinating position . among diabetes type 2 patients , the largest patient group enrolled in dmps ,
90% have a primary care physician as their partner in the programme . when developing its disease management programmes , germany had looked at managed care models in the usa .
meanwhile , with their clearly defined requirements for documentation , evaluation and treatment guidelines and their careful mix of incentives for payers , providers , and patients , german dmps have themselves become a model for other countries .
most chronically ill patients suffer from concurrent chronic conditions a fact slowly taken into account in disease management .
one of the first dmps to address this problem is the programme on coronary heart disease to which recently a module on chronic heart failure has been added .
while integrated care contracts ( see the previous section on integrated care contracts ) allow for contracts between providers of inpatient and outpatient care , medical care centres are legally required to only provide ambulatory care .
medical care centres , also referred to as polyclinics , build upon a state - run primary care delivery model that was well established in former east germany . by law , they are defined as interprofessional institutions , headed by physicians , with other registered physicians working as employees .
medical care centres usually offer a primary care delivery system that brings together general practitioners and specialists under one roof .
the average centre still only employs four physicians just about the size of a small group practice in other countries .
today ownership and management arrangements may vary medical care centres can be run by hospitals or medical groups ; legislation also permits the integration of pharmacies and non - medical health care services ( e.g. physiotherapy , ergotherapy ) .
medical care centres offer physicians the possibility to work as salaried employees in ambulatory care , an option that did not exist prior to 2004 .
it is a particularly attractive option to the rising number of women physicians looking for a better work - life balance , or to doctors who prefer team work over single - handed practice .
medical care centres furthermore provide the opportunity to practice in ambulatory care without taking the financial risk of a solo practice , enabling physicians to concentrate on clinical work without having to deal with practice administration or documentation requirements , and allow for flexible work hours [ 5 , 33 ] . for patients ,
medical care centres are supposed to improve the quality of care through fewer visits ( the larger ones offering one - stop - shop services ) , faster diagnosis using electronic medical records , standardized processes , coordinated care according to treatment guidelines and better access to specialists .
however , since medical care centres are not systematically evaluated in germany , very little data exists to affirm these assumptions .
a patient survey published in 2007 showed that patients treated in medical care centres gave better ratings for quality of care , accessibility and service , infrastructure and organisational structures than patients treated in solo practices .
ninety - five percent of patients stated that they would return to the medical care centre for receiving care and also 95% stated that they would recommend the centre to others .
some concerns regarding the introduction of medical care centres were stated by an english study discussing the introduction of
polyclinics in the uk : bringing professions together under one roof does not necessarily lead to integrated care infrastructure and processes have to be in place to assist integration .
however , about 50% of german medical care centres still have no shared electronic patient record in place .
integration of ambulatory care providers within a medical care centre does not yet imply a good coordination of care between the centre and the hospitals .
lack of personal continuity of care can be a problem if a patient can not choose a personal primary care physician in a medical care centre .
the existence of primary and specialist care within the same institution might encourage overuse of specialty care , thereby increasing costs .
whether medical care centres in less populated regions of germany lead to access barriers because of a concentration of physicians in one place or to improved access
flexible working conditions may as well attract further physicians is still being disputed . with medical care
centres the lawmaker gave physicians in ambulatory care and hospitals the option of a new form of cooperation that allows for a shared use of resources .
although the law does not include mandatory participation of primary care providers , many medical care centres offer primary care services .
since 2004 , more than 1000 medical care centres have been set up ( see figure 2 medical care centres growing numbers ) , with ca .
4800 staff physicians compared to the total of 130,000 doctors who work in ambulatory care in germany . among them
are about 793 general practitioners and 488 internists making primary care physicians the largest specialty group working in this type of health care delivery system [ 32 , p.3 , 7 ] . as depicted in the previous section on a brief working definition of integrated primary care , medical care in the german primary care setting is exclusively provided by physicians . under the name of agnes the institute of community medicine at the university of greifswald started several pilots in 2005 to test
if nurse practitioners , so - called community medicine nurses , can support primary care physicians in sparsely populated areas in prevention , nursing and assistance during routine home visits .
they are expected to ensure regular access to basic health care services for elderly patients .
they visit patients at home , run basic diagnostic tests , apply new bandages or take blood samples and they serve as contact persons for mostly elderly patients , supervise their medication , consider preventive action , and offer advice and support .
they are provided with a tablet pc that enables them to transfer medical data from the patient s home to the doctor s practice immediately and to reach the physician via video communication at any time .
pilots in four states were evaluated for the first time in july 2008 by surveying participating physicians and patients .
ninety - eight percent of the patients perceived the nurse practitioner as a competent partner in health care , 94% supported the delegation of regular home visits to nurse practitioners . a large majority of primary care physicians stated that the nurse practitioners provided valuable support ( 38 of 42 physicians ) and had a positive effect on patient compliance ( 37 of 42 physicians ) . for 92% of patients , physicians
perceived the delegation of task as having no negative effect on the quality of care . to qualify physician assistants or traditional nurses for the work profile of a community medicine nurse the university of greifswald developed an advanced training programme .
the profession of community medicine nurses implies the redistribution of some of the tasks that today are the sole responsibility of physicians . if implemented on a larger scale , this would have a rather fundamental impact on german health care structures
however , the open question of how to include the new profession into financing structures in ambulatory care is still a major obstacle to a large - scale implementation . the main goal of the agnes project is not to improve care coordination but to establish a new structure of support for primary care physicians in rural areas .
still , the introduction of nurse practitioners into the german health care system and the current discussion about the delegation of clinical tasks to non - physician staff might over time turn out to be a step towards integrated primary care in germany . to deliver continuing , person - focused , comprehensive care and fulfil a coordinating role ,
a multidisciplinary primary care team is better fitted than a physician in solo practice with little support [ 36 , 37 ] .
the discussion around agnes nurse practitioners might open the door for new forms of cooperation within primary care practices .
the 2000 reform thus established the legal basis for health insurance funds and providers to enter selective integrated care contracts , besides the above - mentioned unitary but mandatory collective contractual system . under integrated care contracts ,
care is provided in provider networks that can be managed by independent management organizations . but uptake of integrated care contracts was initially very slow . a key measure toward accelerating care coordination was the offer of financial incentives for providers , introduced by law but for a limited period of time : from 2004 to 2008 , one percent of the total statutory health insurance budget available for ambulatory and hospital care has been earmarked to initially fund integrated care contracts . in total , the start - up financing scheduled until the end of 2008 amounted to approximately e 800 million . from just over 600 contracts in early 2005 , by december 2008 their number had risen to more than 6000 with about four million patients being treated under this contractual form of integrated care ( see also figure 1 fast expansion of integrated care ) . the statutory health insurance competition strengthening act of 2007 established further integrated care opportunities . since then
, long - term care providers can be included in contracts , and non - medical professionals can become the main contractual partner to health insurance funds , a position formerly restricted to physicians . also since 2007
, integrated care contracts now are to focus on population - oriented integrated care , a term not defined by the lawmaker to allow for creativity in designing integrated care models .
it is usually understood as proactive , patient - centred health care for a defined population with providers taking responsibility for the coordination of care and for improving or maintaining the health status of the insured population , thereby putting a focus on health promotion or prevention .
so far , however , disease- or procedure - oriented contracts continue to constitute the bulk of the integrated care contracts signed .
only a few companies are developing ambitious models of population - oriented integrated care in germany [ 16 , p. 129223 ] .
particularly the move towards population - oriented integrated care can imply a strengthening of primary care as the coordinating agent in a patient s care process .
population - oriented care implies a more comprehensive concept of health care , in which a multidisciplinary group of providers is not only responsible for curing illness but also for maintaining or improving the health status of the population .
this comes very close to starfield s model of integrated primary care as an ongoing , person - focused , comprehensive and coordinating system of care .
existing models of population - oriented integrated care in germany use either a primary care physician or team as the coordinating agent for participating patients .
gatekeeping in primary care also exists in other countries with shi systems like the netherlands and has recently been implemented in france in 2004 . in germany , patients are free to choose a family physician who then serves as gatekeeper and guide through the health care system . once a patient has subscribed to a gatekeeping scheme , specialists can only be seen upon referral , although exceptions apply for gynaecologists , paediatricians and ophthalmologists . since 2007 legislation requires health insurance funds to offer gatekeeper contracts . for patients ,
enrolment in gatekeeping arrangements is voluntary and can be rewarded through financial incentives by their health insurance fund .
about six million patients had signed up for the gatekeeping scheme by the end of 2007 .
family physicians willing to enter a gatekeeper contract with a health insurance fund have to fulfil certain criteria : participate in quality circles , follow evidence - based treatment guidelines , run a quality management programme in their practice , and attend trainings in areas like patient - oriented communication , basic treatment and diagnostics of mental disorders , palliative or geriatric care .
gatekeeping is a very forthright provision of the lawmaker to strengthen primary care , installing the primary care physician as the coordinating agent in patient health care and restricting the patient s free choice of specialists . through better care coordination and the above - mentioned criteria for participating physicians , gatekeeping contracts are to enhance the quality of care and to reduce costs by preventing unnecessary specialist visits .
however , a survey among health insurance members conducted by the bertelsmann stiftung between 2004 and 2007 revealed that in their current set - up , gatekeeping arrangements do not achieve their aims of controlling the number of patient visits to specialists or of improving health outcomes .
patients enrolled in gatekeeper contracts do not report better health outcomes than patients who are not enrolled , and the number of visits to specialists does not seem to go down . in future contracts , more incentives for physicians to improve the quality of care seem to be necessary if gatekeeping models are to actually reach their goals .
disease management programmes ( dmps ) were introduced in germany in 2002 , in continuation of an earlier reform . in 1996 , a risk equalization scheme based on average spending by age and sex was introduced between statutory health insurance funds .
however , the costs of providing care for chronically ill patients had not been taken into account adequately , which led to health insurance funds particularly targeting young , healthy insurees . in 2004 , a separate high - risk structure compensation scheme for patients enrolled in disease management programmes was added . under the new scheme ,
dmp participants no longer generate a deficit : health insurance funds receive an additional lump sum from the risk equalization scheme for each person enrolled .
there are six requirements for dmp accreditation by the german federal insurance authority : treatment according to evidence - based guidelines with respect to the relevant sectors of care ; quality assurance measures ; required procedure for enrolment of insured , including duration of participation ; training and information for care providers and patients ; electronic documentation of diagnostic findings , applied therapies and outcomes ; evaluation of clinical outcomes and costs .
disease management programmes currently exist for six major chronic conditions : diabetes type 1 , diabetes type 2 , coronary heart disease , breast cancer , asthma and chronic obstructive pulmonary disease . in june 2008 ,
more than 5.2 million patients were enrolled , the largest share ( 2.7 million ) of which participate in diabetes type 2 dmps [ personal conversation with representative of the german federal insurance authority ( bundesversicherungsamt ) on february 6 , 2009 ] . for patients and physicians dmp participation
incentives exist for both : patients are exempt from out - patient fees and co - payments ; physicians receive a lump sum payment for coordination and documentation activities .
usually , primary care physicians take on the role of coordinating care for dmp patients over time , referring them to specialists when necessary and documenting the care process . a growing number of dmp evaluations
participants experience less complications and emergency hospital admissions ; instead , the number of early - stage hospitalization is higher .
compared to non - enrolled control group patients , diabetes type 2 patients enrolled in dmps self - report higher quality of life and a better physical and mental health status ; their abilities for self - management of their condition are strengthened .
similarly , a representative case - control study published in mid-2008 reported less relapses , less pain , better results for blood pressure and cholesterol for patients participating in a coronary heart disease dmp . among physicians ,
acceptance is also rising , although initially documentation requirements were perceived as an extra burden .
most of the time in the implementation of disease management programmes , primary care takes a central coordinating position . among diabetes type 2 patients , the largest patient group enrolled in dmps ,
when developing its disease management programmes , germany had looked at managed care models in the usa .
meanwhile , with their clearly defined requirements for documentation , evaluation and treatment guidelines and their careful mix of incentives for payers , providers , and patients , german dmps have themselves become a model for other countries .
most chronically ill patients suffer from concurrent chronic conditions a fact slowly taken into account in disease management .
one of the first dmps to address this problem is the programme on coronary heart disease to which recently a module on chronic heart failure has been added .
while integrated care contracts ( see the previous section on integrated care contracts ) allow for contracts between providers of inpatient and outpatient care , medical care centres are legally required to only provide ambulatory care .
medical care centres , also referred to as polyclinics , build upon a state - run primary care delivery model that was well established in former east germany . by law , they are defined as interprofessional institutions , headed by physicians , with other registered physicians working as employees .
medical care centres usually offer a primary care delivery system that brings together general practitioners and specialists under one roof .
the average centre still only employs four physicians just about the size of a small group practice in other countries .
today ownership and management arrangements may vary medical care centres can be run by hospitals or medical groups ; legislation also permits the integration of pharmacies and non - medical health care services ( e.g. physiotherapy , ergotherapy ) .
medical care centres offer physicians the possibility to work as salaried employees in ambulatory care , an option that did not exist prior to 2004 .
it is a particularly attractive option to the rising number of women physicians looking for a better work - life balance , or to doctors who prefer team work over single - handed practice .
medical care centres furthermore provide the opportunity to practice in ambulatory care without taking the financial risk of a solo practice , enabling physicians to concentrate on clinical work without having to deal with practice administration or documentation requirements , and allow for flexible work hours [ 5 , 33 ] . for patients ,
medical care centres are supposed to improve the quality of care through fewer visits ( the larger ones offering one - stop - shop services ) , faster diagnosis using electronic medical records , standardized processes , coordinated care according to treatment guidelines and better access to specialists . however , since medical care centres are not systematically evaluated in germany , very little data exists to affirm these assumptions . a patient survey published in 2007 showed that patients treated in medical care centres gave better ratings for quality of care , accessibility and service , infrastructure and organisational structures than patients treated in solo practices .
ninety - five percent of patients stated that they would return to the medical care centre for receiving care and also 95% stated that they would recommend the centre to others .
some concerns regarding the introduction of medical care centres were stated by an english study discussing the introduction of
polyclinics in the uk : bringing professions together under one roof does not necessarily lead to integrated care infrastructure and processes have to be in place to assist integration .
however , about 50% of german medical care centres still have no shared electronic patient record in place .
integration of ambulatory care providers within a medical care centre does not yet imply a good coordination of care between the centre and the hospitals .
lack of personal continuity of care can be a problem if a patient can not choose a personal primary care physician in a medical care centre .
the existence of primary and specialist care within the same institution might encourage overuse of specialty care , thereby increasing costs .
whether medical care centres in less populated regions of germany lead to access barriers because of a concentration of physicians in one place or to improved access
flexible working conditions may as well attract further physicians is still being disputed . with medical care
centres the lawmaker gave physicians in ambulatory care and hospitals the option of a new form of cooperation that allows for a shared use of resources .
although the law does not include mandatory participation of primary care providers , many medical care centres offer primary care services .
since 2004 , more than 1000 medical care centres have been set up ( see figure 2 medical care centres growing numbers ) , with ca .
4800 staff physicians compared to the total of 130,000 doctors who work in ambulatory care in germany . among them
are about 793 general practitioners and 488 internists making primary care physicians the largest specialty group working in this type of health care delivery system [ 32 , p.3 , 7 ] .
as depicted in the previous section on a brief working definition of integrated primary care , medical care in the german primary care setting is exclusively provided by physicians . under the name of agnes the institute of community medicine at the university of greifswald started several pilots in 2005 to test
if nurse practitioners , so - called community medicine nurses , can support primary care physicians in sparsely populated areas in prevention , nursing and assistance during routine home visits .
they are expected to ensure regular access to basic health care services for elderly patients .
they visit patients at home , run basic diagnostic tests , apply new bandages or take blood samples and they serve as contact persons for mostly elderly patients , supervise their medication , consider preventive action , and offer advice and support .
they are provided with a tablet pc that enables them to transfer medical data from the patient s home to the doctor s practice immediately and to reach the physician via video communication at any time .
pilots in four states were evaluated for the first time in july 2008 by surveying participating physicians and patients .
ninety - eight percent of the patients perceived the nurse practitioner as a competent partner in health care , 94% supported the delegation of regular home visits to nurse practitioners .
a large majority of primary care physicians stated that the nurse practitioners provided valuable support ( 38 of 42 physicians ) and had a positive effect on patient compliance ( 37 of 42 physicians ) . for 92% of patients , physicians
perceived the delegation of task as having no negative effect on the quality of care . to qualify physician assistants or traditional nurses for the work profile of a community medicine nurse the university of greifswald developed an advanced training programme .
the profession of community medicine nurses implies the redistribution of some of the tasks that today are the sole responsibility of physicians . if implemented on a larger scale , this would have a rather fundamental impact on german health care structures
however , the open question of how to include the new profession into financing structures in ambulatory care is still a major obstacle to a large - scale implementation . the main goal of the agnes project is not to improve care coordination but to establish a new structure of support for primary care physicians in rural areas .
still , the introduction of nurse practitioners into the german health care system and the current discussion about the delegation of clinical tasks to non - physician staff might over time turn out to be a step towards integrated primary care in germany . to deliver continuing , person - focused , comprehensive care and fulfil a coordinating role ,
a multidisciplinary primary care team is better fitted than a physician in solo practice with little support [ 36 , 37 ] .
the discussion around agnes nurse practitioners might open the door for new forms of cooperation within primary care practices .
the reforms described in the previous section can be considered as the first careful steps towards a better integrated care system in which primary care takes on a stronger role as coordinator and navigator .
the government , assuming a leading role throughout a year - long reform process , negotiated with all stakeholders in the system , introduced legal changes , making possible selective contracting between health insurance funds and providers .
health insurance funds are obliged by law to offer gatekeeping programmes to their insured , thus strengthening the role of general practitioners in the system .
the role of primary care is also strengthened through dmps , the majority of which is coordinated by general practitioners . to increase uptake of the new schemes , financial incentives for providers and patients were introduced . for patients ,
the rather complex developments of several new forms of care in germany are hard to comprehend as a general trend towards more coordination and more competition in health care .
however , patients participating in the new primary care arrangements most often approve of the more patient - oriented care delivery .
the increasing number of integrated care contracts , dmps , gatekeeping programmes and medical care centres as well as the increasing number of enrolled patients indicate that these new forms of care slowly gain acceptance in the german system .
nevertheless , the reforms have also met with considerable resistance and implementation of primary care - focused care has not yet been achieved on a large scale .
one reason is that it is difficult to change long - established traditions , expectations of providers and patients , practice habits and structures .
german physicians feel threatened in many ways by the structural changes that policy makers have initiated .
tools for transparency and benchmarking are by some physicians seen as an attack on their independence and professionalism , as are new forms of care and care management , such as larger medical care centres , or the staff physician status in ambulatory care . however , younger physicians and female doctors are more likely to consider the advantages in new workplace and contractual arrangements in more professionally managed settings , and of ( peer ) evaluation and feedback .
future reforms and a constant dialogue between policy makers and health professionals will have to address the following challenges : primary care as the foundation of the health care system and as a public good needs continuing regulatory endorsement and political protection ; shared leadership : interdisciplinary and horizontal cooperation between providers from different specialties and sectors needs support and shi - endorsed incentives , particularly in regional negotiations about budgetary redistribution with shi physician associations where primary care providers are often outweighed by specialists ; population orientation : dmps and most integrated care contracts still are predominantly single disease - oriented and lack a broader population - centred approach that embraces both prevention and multimorbidity . in short , in germany the debate about granting a stronger role to primary care as a lever for better care coordination and integration has only just begun .
continuing political support , a rare window of opportunity in the form of prolonged personal continuity at the head of the federal ministry of health , and a visionary leadership willing to learn from primary care experiences elsewhere have been instrumental throughout the reform years . or as marc danzon
has put it more broadly when commenting on similar developments across europe : these types of fundamental organizational adjustments are , by their very nature , long - term endeavours .
progress must be counted in years and requires focused and persistent efforts from key actors [ 38 , p. xvii ] .
, executive director centre for primary health care and equity , faculty of medicine , university of new south wales , sydney , australia geoff meads , hon .
professor of international health studies , warwick medical school , university of warwick , uk petra riemer - hommel , prof . , | problem statementhealth care delivery in germany is highly fragmented , resulting in poor vertical and horizontal integration and a system that is focused on curing acute illness or single diseases instead of managing patients with more complex or chronic conditions , or managing the health of determined populations . while it is now widely accepted that a strong primary care system can help improve coordination and responsiveness in health care ,
primary care has so far not played this role in the german system .
primary care physicians traditionally do not have a gatekeeper function ; patients can freely choose and directly access both primary and secondary care providers , making coordination and cooperation within and across sectors difficult.description of policy developmentsince 2000 , driven by the political leadership and initiative of the federal ministry of health , the german bundestag has passed several laws enabling new forms of care aimed to improve care coordination and to strengthen primary care as a key function in the german health care system .
these include on the contractual side integrated care contracts , and on the delivery side disease management programmes , medical care centres , gatekeeping and community medicine nurses.conclusion and discussionrecent policy reforms improved framework conditions for new forms of care .
there is a clear commitment by the government and the introduction of selective contracting and financial incentives for stronger cooperation constitute major drivers for change .
first evaluations , especially of disease management programmes , indicate that the new forms of care improve coordination and outcomes . yet
the process of strengthening primary care as a lever for better care coordination has only just begun .
future reforms need to address other structural barriers for change such as fragmented funding streams , inadequate payment systems , the lack of standardized it systems and trans - sectoral education and training of providers . |
our previous analysis for cis - regulatory elements was unable to elucidate the mechanism regulating the gene expression pattern in 1-cell
embryos , except for the gc - rich nature of regions upstream of active genes [ 11 , 12 ] .
since there is no cis - regulatory element specific to 1-cell embryos , it is possible that the chromatin structure is involved in regulating gene
expression in 1-cell embryos .
the chromatin structure is essentially repressive for transcription ,
necessitating the presence of enhancers to help transcription factors access the gene promoters .
however , it has been
shown by reporter gene assays that enhancers do not increase transcriptional activity in 1-cell embryos , suggesting that transcription is regulated independently
of enhancers at this stage [ 2 , 14 , 15 ] .
furthermore , we demonstrated that transcription occurs only via the core promoter in 1-cell embryos , but not in 2-cell embryos .
suggested that enhancer - independent transcription is caused by the
loosened chromatin structure in 1-cell embryos , since this type of transcription was still observed at the 2-cell stage when the embryos were treated with
butyrate ( an inhibitor of histone deacetylase ) , which increases histone acetylation to loosen the chromatin structure .
using fluorescence recovery after
photobleaching analysis , we have recently shown that preimplantation embryos at the 1-cell stage form the loosest chromatin structure .
a recent analysis of the genome - wide landscape of chromatin accessibility to dnase i demonstrated that the number of dnase
i - hypersensitive sites ( dhss ) is the fewest at the 1-cell stage and increases during preimplantation development ,
which appears to contradict the hypothesis made by the above - mentioned reports [ 2 , 14,15,16,17 ] .
however , in the
analysis using dnase i , the dhss do not necessarily correspond to the sites of the genome with a loosened chromatin structure . in sites with a loosened chromatin
structure
, transcription factors can easily access the dna and bind to it , thereby obstructing the dnase i access to the dna . in this case , the sites of chromatin
with a loosened structure are insensitive , rather than sensitive , to dnase i. furthermore , since the chromatin structure is extremely loose in 1-cell embryos ,
dnase i might have cleaved an enormously large number of sites to make small fragments of dna that were removed by size selection ( > 50 bp ) for high - throughput
sequencing , which would underestimate the number of dhss .
taken together , we propose that 1-cell embryos have a loose chromatin structure that does not require
enhancers for gene expression .
if this is the case , we should expect a large number of genes to be promiscuously expressed , and the variation in their expression level should be relatively
low . since many transcription factors would be able to easily access the promoters , enhancers should not have a strong effect on transcription .
indeed , the
analysis of our rna - seq data bears out this hypothesis .
when genes are classified as active or inactive based on kernel density estimation , more than 90% of genes
are active in 1-cell embryos , whereas only around 70% of genes are active in embryos at other stages and in oocytes ( fig .
the kernel density estimation was calculated to classify all known genes as active or
inactive in oocytes , 1-cell and 2-cell embryos , and blastocysts .
horizontal and vertical axes are shown as the log2 of reads per kilobase per million ( rpkm )
and gene density , respectively .
reads in exons were used to calculate the rpkm values in oocytes , 2-cell embryos , and blastocysts , whereas reads in introns
were used for 1-cell embryos .
this analysis also reveals that the variation in expression level is much lower in 1-cell embryos than in cells of other stages ; the slope of the
histogram is steeper for 1-cell embryos .
therefore , we suggest that a low level of enhancer - independent transcription occurs promiscuously in a large proportion
of genes , which is probably caused by a loosened chromatin structure in 1-cell embryos ( fig . 2fig .
gene expression
is facilitated by the enhancers , which also regulate the gene expression levels .
the chromatin structure is loosened genome - wide , which allows a low level of transcription in most genes independently of enhancers . ) .
the kernel density estimation was calculated to classify all known genes as active or
inactive in oocytes , 1-cell and 2-cell embryos , and blastocysts .
horizontal and vertical axes are shown as the log2 of reads per kilobase per million ( rpkm )
and gene density , respectively .
reads in exons were used to calculate the rpkm values in oocytes , 2-cell embryos , and blastocysts , whereas reads in introns
were used for 1-cell embryos .
gene expression
is facilitated by the enhancers , which also regulate the gene expression levels .
the chromatin structure is loosened genome - wide , which allows a low level of transcription in most genes independently of enhancers .
we hope that this hypothesis sheds light on the mechanism that regulates the initiation of the gene expression program in embryos . | after fertilization , the genome of zygotes is transcriptionally silent .
the timing of the initiation of transcription is species - specific and occurs at the
mid-1-cell stage in mice .
recent analyses using high - throughput sequencing ( hts ) have identified thousands of genes transcribed at the 1-cell stage , and the
pattern of expression among these genes appears to be unique . in this article
, we show the result of an additional analysis using hts data from a previous
study , and present the hypothesis that an extremely loose chromatin structure causes promiscuous gene expression in 1-cell embryos . |
infections are important causes of mortality and morbidity in the world . out of 4 million annual neonatal deaths ,
they also cause anxiety and stress in parents and other family members . therefore , infection control is of high importance to save patients and provide hospitalized infants with optimal life conditions .
invasive skin pathogens play an important role in occurrence of infections and mortalities in infants .
an infant 's skin may be infected during labor through the birth canal , labor team , or medical instruments .
it might also be infected by relatives , care providers , and the surrounding environment after birth .
these microorganisms could become the normal flora of the infants and damage their immune systems later .
hence , skin is an important potential place for entrance of infectious agents and the cause of neonatal infections .
this risk manifests as the increased incidence of bacterial sepsis in diseased infants who require longer hospital stay and invasive procedures such as blood sampling , peripheral and central catheter insertion .
various antiseptic solutions are used before any invasive procedure to remove microorganisms from the skin .
povidone - iodine ( pvp - i ) and chlorhexidine are the most common antiseptics .
they are both accessible as solutions mixed with water or alcohol and are used in neonatal wards .
for instance , many studies have shown that the incidence of infection at catheter insertion site was lower after disinfection by chlorhexidine than by pvp - i .
( 2010 ) used chlorhexidine - alcohol versus pvp - i for surgical site antisepsis and suggested that the infection rate of surgical site was significantly lower ( p = 0.004 ) in the chlorhexidine - alcohol group compared to the pvp - i group ( 9.5% vs. 16.1% ) , while the study of garland et al .
( 2009 ) on infants showed no difference in bacterial colonization rate of inserted catheters between two antiseptic methods with chlorhexidine and pvp - i .
one of the responsibilities of trained nurses is performing research about nosocomial infections and their control procedures , diagnosing underlying infectious agents , and providing suggestions to eliminate such factors .
they are also required to supervise and control the clinical and care methods employed in various wards , particularly neonatal intensive care units ( nicus ) . on the other hand , pvp - i
since it may not be the best option , we decided to conduct the present study to compare the effects of chlorhexidine and pvp - i on reducing the amount of skin bacterial flora among hospitalized neonates .
in a single - blind clinical trial with a single - group pretest posttest design , 98 eligible infants were enrolled . after obtaining written consents from the parents , the infants who met the inclusion criteria
the inclusion criteria were birth weight over 1 kg , gestational age over 28 weeks , not having infections and dermal diseases , and a minimum nicu stay of 24 h. patients were selected from nicus of two university hospitals affiliated to isfahan university of medical sciences , isfahan , iran .
subjects were excluded and replaced by others if the blood sampling site was used for therapeutic procedures or disinfected by another solution or if the infants died before taking all required cultures . before taking skin cultures , four pieces of cotton balls ( iran , sahand co ) were placed in graft paper and sealed . they were then sterilized in an autoclave .
blood sampling and catheterization are invasive procedures which can increase the risk of infection by skin flora , and are usually performed on areas behind the hand and anterior middle areas of elbow and ankle .
so , of these areas , we assigned the site of skin culture sampling for each infant randomly .
afterward , one side of the body was determined randomly by coin flipping to be disinfected by pvp - i and the contralateral site by chlorhexidine .
skin cultures were done as below : one of the researchers used a 2 2 cm template to draw a square on the selected area of the skin .
she scrubbed her hands with epi - max hand scrub solution ( iran , emad pharmaceutical products ) , soaked cotton in antiseptic solution , and disinfected each area from center away to the sides .
skin cultures were taken from the selected area before disinfection and immediately after drying the antiseptic solution . however , due to difference in color of the solutions , blinding during the skin culture sampling was not possible .
all skin cultures were taken through rubbing a sterile swab wetted in normal saline solution ( samen co. , mashhad , iran ) gently on the mapped area five times horizontally and twice vertically .
the cultures were immediately placed in amies transport medium ( amies transport medium with charcoal , staphylococcus aureus atcc 25923 , escherichia coli atcc 25922 ) and were immediately sent to the laboratory of al - zahra hospital , isfahan , iran .
a microbiologist unaware of the study cultured the swabs on sheep blood agar ( atcc 25922 , atcc 33400 , atcc 8668 ) and eosin methylene blue agar ( atcc 25922 , ptcc 1609 ) plates . using a standard method ,
they were maintained at 37c for 2 days to identify the count and types of bacteria .
afterward , bacteria type and count of colonies were determined for nine common pathogens which are the main causes of neonatal sepsis in developing countries ( klebsiella , s. aureus , acinetobacter , enterobacter , salmonella , candida , pseudomonas , e. coli , and coagulase - negative staphylococci ) .
the cultured skin that contained one or more of these pathogens was considered as positive .
the data were recorded in an information sheet including a demographic part and another part for culture results .
paired t - test was used to compare mean counts of microorganisms before and after disinfection with each of the solutions . on the other hand , independent t - test
was employed to compare the two groups in terms of mean counts of microorganisms before and after disinfection .
finally , it should be noted that the researchers had no bias toward any of the solutions since no financial benefit was gained from the companies .
in a single - blind clinical trial with a single - group pretest posttest design , 98 eligible infants were enrolled . after obtaining written consents from the parents , the infants who met the inclusion criteria
the inclusion criteria were birth weight over 1 kg , gestational age over 28 weeks , not having infections and dermal diseases , and a minimum nicu stay of 24 h. patients were selected from nicus of two university hospitals affiliated to isfahan university of medical sciences , isfahan , iran .
subjects were excluded and replaced by others if the blood sampling site was used for therapeutic procedures or disinfected by another solution or if the infants died before taking all required cultures . before taking skin cultures , four pieces of cotton balls ( iran , sahand co ) were placed in graft paper and sealed . they were then sterilized in an autoclave .
blood sampling and catheterization are invasive procedures which can increase the risk of infection by skin flora , and are usually performed on areas behind the hand and anterior middle areas of elbow and ankle .
so , of these areas , we assigned the site of skin culture sampling for each infant randomly .
afterward , one side of the body was determined randomly by coin flipping to be disinfected by pvp - i and the contralateral site by chlorhexidine .
skin cultures were done as below : one of the researchers used a 2 2 cm template to draw a square on the selected area of the skin .
she scrubbed her hands with epi - max hand scrub solution ( iran , emad pharmaceutical products ) , soaked cotton in antiseptic solution , and disinfected each area from center away to the sides .
skin cultures were taken from the selected area before disinfection and immediately after drying the antiseptic solution . however , due to difference in color of the solutions , blinding during the skin culture sampling was not possible .
all skin cultures were taken through rubbing a sterile swab wetted in normal saline solution ( samen co. , mashhad , iran ) gently on the mapped area five times horizontally and twice vertically .
the cultures were immediately placed in amies transport medium ( amies transport medium with charcoal , staphylococcus aureus atcc 25923 , escherichia coli atcc 25922 ) and were immediately sent to the laboratory of al - zahra hospital , isfahan , iran .
a microbiologist unaware of the study cultured the swabs on sheep blood agar ( atcc 25922 , atcc 33400 , atcc 8668 ) and eosin methylene blue agar ( atcc 25922 , ptcc 1609 ) plates . using a standard method ,
they were maintained at 37c for 2 days to identify the count and types of bacteria .
afterward , bacteria type and count of colonies were determined for nine common pathogens which are the main causes of neonatal sepsis in developing countries ( klebsiella , s. aureus , acinetobacter , enterobacter , salmonella , candida , pseudomonas , e. coli , and coagulase - negative staphylococci ) .
the cultured skin that contained one or more of these pathogens was considered as positive .
the data were recorded in an information sheet including a demographic part and another part for culture results .
paired t - test was used to compare mean counts of microorganisms before and after disinfection with each of the solutions . on the other hand ,
independent t - test was employed to compare the two groups in terms of mean counts of microorganisms before and after disinfection .
finally , it should be noted that the researchers had no bias toward any of the solutions since no financial benefit was gained from the companies .
the results showed that out of 98 infants who were enrolled in the study , 60.2% were males and 39.8% were females .
the mean ( sd ) gestational age of the subjects was 33 ( 3.5 ) weeks .
the mean age of the infants at the time of sampling was 9.94 ( 8.66 ) days ( 1 - 28 days ) .
their mean birth weight was 2005 ( 833 ) g. during sampling , two infants were excluded since the sampling site was used to take peripheral vein , one case was transferred to neonatal surgery department , and two other subjects were excluded because the sampling site was infected by the staff .
all excluded cases were replaced . in total , 392 skin cultures , including 196 skin cultures from pvp - i antiseptic solution 10% and 196 from chlorhexidine , were taken .
comparisons of mean counts of skin microorganisms before and after disinfection in each group of pvp - i 10% and chlorhexidine 2% as well as intergroup comparisons are presented in table 1 .
percentages of positive skin cultures before and after disinfection with each solution are shown in figure 1 .
comparisons of mean counts of skin microorganisms before and after disinfection ( based on colony count ) in each group and intergroups percentage of positive skin cultures before and after disinfection with povidone - iodine 10% and chlorhexidine 2% solutions paired t - test showed that mean counts of skin microorganisms reduced significantly after disinfection with both chlorhexidine 2% and pvp - i 10% solutions ( p < 0.001 ) . according to independent t - test , mean counts of skin microorganisms in the two groups were not significantly different before disinfection ( p = 0.93 , t = 0.08 ) .
however , significant differences were observed after disinfection ( p = 0.049 , t = 1.97 ) .
the present study showed that both pvp - i and chlorhexidine were effective on reducing skin bacterial flora in infants .
however , the effect of pvp - i 10% was significantly more compared with chlorhexidine 2% .
( 2008 ) reported 33% of skin cultures to be negative after pvp - i shower , in the present study , 94.9% of skin cultures were negative after disinfection with pvp - i .
therefore , it seems that compared to pvp - i shower method , using pvp - i through skin preparation is more effective in reducing microorganismscounts .
( 2010 ) studied the effects of pvp - i 5% on removing bacteria in the upper lid .
they showed that mean counts of skin microorganisms reduced significantly ( by 82.6% ) after the intervention ( p = 0.001 ) .
however , in the present study , the reduction in mean count of skin microorganisms was 94.9% after antisepsis with pvp - i .
this difference can be due to using a solution with a higher concentration of pvp - i ( 10% ) in the present study .
difference in areas of sampling might have been another reason since we took skin cultures from behind the hand and the anterior middle areas of elbow and ankle , while bekibele et al .
( 2007 ) assessed the safety and efficacy of chlorhexidine skin cleaning on skin flora of neonates in bangladesh .
they suggested that in skin cultures taken from the armpits , around the navels , and the groins of infants , mean counts of microorganism had significant differences 2 h after cleaning the areas with chlorhexidine compared with before the intervention . according to the results of the above - mentioned research and the present study , the microorganism counts would obviously be reduced after disinfecting with any antiseptic solution .
however , comparison of efficacy of chlorhexidine 2% and pvp - i 10% in reducing microorganisms by garland et al .
( 2009 ) revealed a higher ( although insignificantly ) colonization rate at the tip of catheters in the chlorhexidine group compared to the pvp - i group .
similarly , in the present study , the amount of positive skin cultures in the chlorhexidine group was significantly higher than the pvp - i group .
( 98 vs. 48 infants ) , and thus our results might be more reliable .
( 2008 ) reported that the incidence of catheter colonization in the chlorhexidine 2% group was significantly lower than the pvp - i 10% group .
however , the incidence of catheter - related bacteremia was similar in both groups . besides , they showed that chlorhexidine was more effective than pvp - i in preventing catheter colonization due to gram - positive bacteria .
on the contrary , in the present study , the effects of the both solutions were similar on gram - positive and gram - negative bacteria .
it can therefore be concluded that the difference between the results of the two studies originated from the types of microbial flora in the environment .
in addition , the study of valles et al . was conducted in an intensive care unit for adults while our research was performed in nicus .
could not find significant differences in the mean counts of microorganisms after disinfection with chlorhexidine and pvp - i .
however , in the present study , the pvp - i was more effective than chlorhexidine in reducing microorganisms .
this inconsistency might have been caused by the higher concentration of chlorhexidine in the study of khera et al .
( 2010 ) used chlorhexidine - alcohol versus pvp - i for surgical - site antisepsis .
they indicated that the infection rate of surgical site was significantly lower in the chlorhexidine - alcohol group compared to the pvp - i group ( p = 0.004 ) . likewise , mimoz et al .
( 1999 ) showed that skin preparation with chlorhexidine - alcohol was more efficient than pvp - i in reducing contamination of blood cultures . in a similar study ,
2008 ) reported that the contamination rate of blood cultures was significantly lower after disinfecting the venipuncture site with chlorhexidine - alcohol 2% than with pvp - i 10% ( p < 0.001 ) .
the results of the present study were not in accordance with the results obtained by darouiche et al . , mimoz et al . , and suwanpimolkul et al .
the difference can be due to the form of chlorhexidine - alcohol solution in the above studies , i.e. , perhaps alcohol enhances the germicidal properties of chlorhexidine . in the present study , we did not use the alcoholic compound due to potential unknown complications of chlorhexidine - alcohol on infants .
our results showed that skin disinfection with pvp - i 10% compared to chlorhexidine 2% was more effective on reducing bacterial skin colonies .
therefore , pvp - i 10% solution can be suggested to be used in nicus for skin disinfection before implementing invasive procedures . in this study , investigating the stability of the solutions was not possible due to time limitation . the stability of these two antiseptic solutions ( chlorhexidine and pvp - i ) is hence recommended to be assessed in further studies . | background : infection control should be an integrated part of patient care , especially to ensure safety and survival in hospitalized neonates . although povidone - iodine ( pvp - i ) solution has been used as the most common antiseptic in hospitals of iran , chlorhexidine is currently used in some wards for skin disinfection . however , there is no evidence about the superiority of either antiseptic solution over the other one .
this clinical trial carried out in two university hospitals affiliated to isfahan university of medical sciences , isfahan , iran , aimed to compare the effects of chlorhexidine and pvp - i solutions on skin bacterial colonization in hospitalized neonates.materials and methods : the participants were 98 hospitalized infants . in each infant , one area on the skin was disinfected by chlorhexidine while the contralateral site was disinfected by pvp - i .
skin cultures were taken before and after disinfection .
microorganisms were determined and colony count was performed based on a standard method .
the collected data were analyzed using descriptive and inferential statistical methods in spss v. 14.results:the mean of microorganism colony count before and after disinfection by either solution was statistically different ( p < 0.001 ) .
there was no significant difference between the two groups in terms of mean microorganism colony count before disinfection .
however , a significant difference was observed after disinfection.conclusions:pvp-i is more efficacious than chlorhexidine for skin disinfection .
consequently , it seems better to use pvp - i for skin preparation before invasive procedures . |
in the last two decades motivation theory has reached developments of paramount importance for psychology and neuroscience .
incentive motivation theory was a crucial crossroads along the way the led to such important developments .
incentive motivation concepts rose in the 1960s when several new realizations about brain and motivation led many psychologists and behavioral neuroscientists to reject simple drive and drive - reduction theories .
specific alternative theories were developed in the form of incentive motivation theories ( bolles , 1972 ; bindra , 1978 ; toates , 1986 , 1994 ; panksepp , 1998 ; berridge , 2001 ) .
bolles ( 1972 ) proposed that individuals were motivated by incentive expectancies , not by drives or drive reduction .
s associations , were essentially learned expectations of a hedonic reward , indistinguishable from cognitive predictions .
accordingly , a predictive neutral stimulus ( s ) , such as a light or a sound , became associated by repeated pairing with a hedonic reward that followed ( s ) , such as a palatable food .
the s caused an expectancy of the s. the s was , in terms of pavlovian learning processes , a conditioned stimulus ( cs or cs+ ) , and the s an unconditioned stimulus ( ucs ) .
bindra ( 1974 , 1978 ) acknowledged that expectations might well be important to cognitive strategies to obtain the reward , but suggested that a cs for a reward actually evokes the same incentive motivational state normally caused by the reward itself , as a consequence of classical conditioning .
it also causes the individual to perceive the cs as a hedonic reward , and lets the cs elicit incentive motivation just as would the original hedonic reward .
that means that the cs takes on specific motivational properties that normally belong to the s itself , and these motivational properties are specifically incentive properties . note that this was true not only for reward s , but also for painful s motivation , that would be based on fear or punishment properties .
bindra views suggesting that physiological depletion states could enhance the incentive value of their goal stimuli .
this leads to a multiplicative interaction between physiological deficit and external stimulus , which determined the stimulus ' incentive value . however , physiological deficit signals do not drive motivated behavior directly , but they are able to magnify the hedonic impact and incentive value of the actual reward ( s ) , and also the hedonic / incentive value of predictive stimuli for the reward ( css ) . around 1990 the incentive salience model was proposed ( berridge et al . , 1989 ; berridge and valenstein , 1991 ) that followed bindra
toates rules for incentive conditioning but identifies separable brain substrates for liking a reward versus wanting the same reward .
liking is essentially hedonic impact the brain reaction underlying sensory pleasure - triggered by immediate receipt of reward , for instance , a sweet taste ( unconditioned liking ) . wanting , or incentive salience , is the motivational incentive value of the same reward ( berridge and robinson , 1998 ) , the incentive motivational value of a stimulus , not its hedonic impact .
the important point is that liking and wanting normally go together , but they can be split apart under certain circumstances , especially by certain brain manipulations .
liking without wanting can be produced , and so can wanting without liking .
motivation can be conceptually described as a continuum along which stimuli can either reinforce or punish responses to other stimuli .
behaviorally , stimuli that reinforce are called rewarding and those that punish aversive ( skinner , 1953 ) . reward and aversion
describe the impact a stimulus has on behavior , and provided of motivational properties , thus able to induce attribution of motivational salience .
the incentive salience model underscored the main role of dopamine ( da ) function as brain mechanism of motivational processes . indeed da
suppression leaves individuals nearly without motivation for any pleasant incentive at all : food , sex , drugs , etc . , ( ikemoto and panksepp , 1999 ; naranjo et al . , 2001 ; berridge , 2004 ; salamone et al . , 2005 ) .
thus , disruption of mesolimbic da systems via neurochemical lesions of the da pathway that projects to nucleus accumbens ( nac ) or by receptor - blocking drugs , dramatically reduces incentive salience or
wanting to eat a tasty reward , but does not reduce affective facial expressions of liking for the same reward ( pecina et al .
one type of da neuron encodes motivational value , excited by rewarding events and inhibited by aversive or stressful events ( bromberg - martin et al .
these neurons support brain systems for seeking goals , evaluating outcomes , and value learning .
indeed , most da neurons are activated by reward - predicting stimuli and code bidirectional reward prediction errors ( i.e. , better than expected / worse than expected ) in humans , monkeys , and rats ( ikemoto and panksepp , 1999 ; ikemoto , 2007 ; schultz , 2007 ) . although discrete aversive stimuli such as air puffs , hypertonic saline , and electric shock induce activating responses in a small proportion of da neurons in awake animals ( guarraci and kapp , 1999 ; joshua et al .
, 2008 ; matsumoto and hikosaka , 2009 ) , most da neurons are depressed by aversive stimuli ( ungless et al . , 2004 ; jhou et al . , 2009
this response variability indicates that recorded cells are part of different , independent circuits ( margolis et al . , 2006 ; ikemoto , 2007 ; bromberg - martin et al . ,
a second type of da neuron encodes motivational salience , excited by both rewarding and aversive events ( bromberg - martin et al . , 2010 ) .
evidences suggest that different groups of da neurons convey motivational signals in distinct manners ( matsumoto and hikosaka , 2009 ) and the mesocorticolimbic da system may be comprised of distinct circuits , each modified by distinct aspects of motivationally relevant stimuli , based on da projections to nac medial shell mediating positive stimuli , on da projections to mpfc affected by aversive stimuli , and projections to nac lateral shell affected by both rewarding and aversive stimuli , presumably reflecting saliency ( lammel et al . , 2011 ) .
it has been shown how the vta da neurons may employ the convergent encoding strategy for processing both positive and negative experiences , intimately integrating with cues and environmental context ( wang and tsien , 2011 ) . the mesolimbic dopaminergic system , that projects from the ventral tegmental area ( vta ) neuronal cell bodies rostrally to the nac , is a primary link in the reward pathway ( wise , 1996 , 2004 ) .
however , da release is not necessary for all forms of reward learning and may not always be liked in the sense of causing pleasure , but it is critical for causing goals to become wanted in the sense of motivating actions to achieve them ( robinson and berridge , 1993 , 2003 ; berridge and robinson , 1998 ; palmiter , 2008 ) .
a line of evidence supporting a role for da in the motivational properties of stimuli comes from the place - conditioning paradigm ( mucha and iversen , 1984 ; van der kooy , 1987 ; carr et al . , 1989 ) .
this paradigm treats the increase in amount of time spent in an environment that has been paired with an ucs ( either drugs or natural reinforces ) as an index of the stimulus 's reward properties .
by contrast , if animals are repeatedly exposed to an environment paired with an aversive stimulus they will avoid the environment . in the first case we speak of conditioned place preference ( cpp ) , in the second of conditioned place aversion ( cpa ) .
da antagonists administered before each conditioning session with amphetamine block amphetamine - conditioned place preferences ( nader et al . , 1997 for review ) .
these results are not interpretable in terms of a general learning deficit because animals have been shown to be able to form normal cs - us associations in place conditioning with other us ( shippenberg and herz , 1988 ) .
these findings suggest that normal da transmission is necessary for the rewarding properties of stimuli to occur . if the dopaminergic pathway from the vta to the nac is a primary link in the pathways mediating the motivational properties of stimuli ( tsai et al . , 2009 ; adamantidis et al . , 2011 ) , then examples of da - independent reward should be nonexistent .
there are a number of examples , however , of stimuli that possess reinforcing properties independent of da .
thus , behavioral pharmacological experiments indicate that although increasing mesolimbic da transmission plays an important role in the reinforcing effects of abused substances , there are also da - independent processes that contribute significantly to the reinforcing effects of these compounds ( joseph et al . , 2003 ; pierce and kumaresan , 2006 for review ) .
for instance , da antagonist pretreatment or 6-ohda lesions of the nac have been reported to have no effect on morphine or heroin self - administration ( ettenberg et al . , 1982 ; pettit et al .
, 1984 ; dworkin et al . , 1988 ) , and on ethanol oral self - administration ( rassnick et al . , 1993 ) .
, 1982 ; mackey and van der kooy , 1985 ) has been reported following systemic or intra - accumbens administration ( koob and bloom , 1988 ; hemby et al . , 1992 ; caine and koob , 1993 ) . under some conditions ,
da - independent opiate place preferences has been demonstrated ( mackey and van der kooy , 1985 ; bechara et al . , 1992 ;
moreover , da - deficient mice display a robust conditioned place preference for morphine in specific experimental conditions ( hnasko et al . , 2005 ) , and da is not involved in opiate naive state ( laviolette et al . , 2004 ; vargas - perez et al . , 2009 ) .
a da - independent reward mechanism for caffeine has been shown ( sturgess et al .
dopamine d2 receptor knockout mutation in c57bl/6 mice failed to block ethanol - conditioned place preferences in ethanol - dependent and withdrawn mice ( ting - a - kee et al . , 2009 ) .
in a more naturalistic conditions operant place conditioning by male chemo - signal in female mice was not affected by d1 or d2 receptor antagonists ( agustin - pavon et al . , 2007 ) .
it is worth noting that vta - mediated but da - independent positive reinforcement has been demonstrated ( fields et al . , 2007 ) .
these examples of da - independent motivated behavior seriously question the original da hypothesis that suggested da is a final common pathway in the processes mediating reinforcement .
about a couple of decade ago , research has pointed to prefrontal catecholamine ( ca ) regulation of mesoaccumbens da transmission in response to pleasant or aversive stimuli ( le moal and simon , 1991 ) . in particular
, da transmission in subcortical structures , such as the nac , seems to be modulated by the da mesocortical system in an inhibitory way ( ventura et al .
, 2004 , for review ) , thus strongly suggesting that the mesoaccumbens da response is inversely related to the mesocortical da response .
mesoaccumbens da transmission has been suggested to be regulated by prefrontal transmission via glutamatergic projections ( carr and sesack , 2000 , for review ) , through activation of excitatory prefrontal - cortical projection to the vta ( sesack and pickel , 1990 ) , and/or through activation of a corticoaccumbens glutamatergic projection ( taber and fibiger , 1995 ) .
thus , besides possible direct cortico - accumbal circuit , a cortico-(vta)- accumbal da network involving different brain areas , such as amygdala ( jackson and moghaddam , 2001 ; mahler and berridge , 2011 ) , has been proposed to have an important role on accumbal da modulation . in late nineties , a french study ( darracq et al . ,
1998 ) showed that prefrontal cortical norepinephrine ( ne ) had a pivotal role in the increased accumbal da release induced by systemic amphetamine administration .
till that moment , brain noradrenergic system involvement in behavior control was mostly focused on locus coeruleus ( lc ) functions ( aston - jones et al . , 1999 ) or on emotional memory regulation by the amygdala ( mcgaugh , 2006 ) .
the pioneer work of darracq and coworkers , suggested implicitly that da transmission in the nac could be controlled by and directly related to ne in the medial prefrontal cortex ( mpfc ) .
this view , together with the established inhibitory role of prefrontal da on dopaminergic activity in the accumbens , suggested a possible opposite action of the two amines in the prefrontal cortex on subcortical da transmission . experimental evidence from our lab on mice of c57bl/6 ( c57 ) and dba/2 ( dba ) inbred strains supported this hypothesis .
comparative studies of neurotransmitter activity and behavior in different genetic backgrounds make available a major strategy for investigating the neural basis of drug effects related to individual differences .
mice of the dba background have been shown to be poorly responsive to the enhancing extracellular da induced by the psychostimulant in the nac ( shell ) as well as to the stimulating / reinforcing effects of amphetamine , that are dependent on increased accumbal da release .
the opposite occurs in mice of the c57 background , which have been shown to be highly responsive to stimulating / reinforcing effects of amphetamine , as shown by increased locomotor activity or to amphetamine - induced cpp ( zocchi et al . , 1998 ;
, amphetanine produces low mpfc da and high da in the nac , the opposite occurs in dba mice that show lower locomotor activity than c57and no cpp or even cpa .
moreover , selective da depletion in the mpfc of dba mice makes this strain similar to highly responsive c57 mice leading to high da outflow in the nac and hyper locomotion .
nevertheless , no differences in structure or expression of da transporter in the nac between c57 and dba strains have been reported ( womer et al . , 1994 ) .
these results showed that the different effects of amphetamine on accumbal da outflow in the two backgrounds do not depend on differences in dat related mechanisms .
however , microdialysis experiments showed that amphetamine increased ne and da outflow in the mpfc of c57 and dba mice in a different way . while c57 showed higher ne increase than da , dba mice present an opposite pattern , thus indicating that ne / da ratio induced by amphetamine is higher in c57 versus dba .
since da is inhibitory on da nac , while ne was suggested to be enabling ( darracq et al .
, 1998 ) , we hypothesized that imbalanced ne / da in the mpfc controlled da in the nac and related behavioral outcomes , making the c57 strain more responsive than dba .
such a hypothesis was confirmed by subsequent experiments showing that selective prefrontal cortical ne depletion abolished the effects of amphetamine on da in accumbens and cpp in c57 mice ( ventura et al .
, 2003 ) , while selective prefrontal da depletion ( sparing ne ) led to da outflow in the nac and behavioral outcomes in dba mice entirely similar to those of c57 ( ventura et al .
these data suggested strongly that da in the nac is controlled by prefrontal cortical ne that enables it , and by da that inhibits it .
moreover , our data indicated that prefrontal ne transmission is critical for attribution of motivational salience , as demonstrated by the impairment of amphetamine - induced cpp in mpfc ne depleted c57 mice ( ventura et al . , 2003 ) .
, 2002 for review ) and results on stress obtained in our lab on c57 and dba mice had shown that this was true also for aversive experiences ( restraint , forced swimming ) , at least as far prefrontal da control over da in the nac was concerned .
indeed , we found that restraint stress produced inhibition of mesoaccumbens da release accompanied by a very fast and strong activation of mesocortical da metabolism in c57 mice , and the opposite in mice of the dba strain , showing a genetic control over the balance between mesocortical and mesoaccumbens da responses to stress ( ventura et al .
. moreover , c57 mice but not mice of the dba strain showed an extremely high level of immobility on their first experience with the forced swimming test ( fst ) as well as immediate and strong activation of mesocortical da metabolism and inhibition of mesoaccumbens da metabolism and release . in addition , the behavioral and the mesoaccumbens da responses to fst in c57 mice were reduced and reversed , respectively , by selective dopamine da depletion in the mpfc ( ventura et al . , 2002 ) .
prefrontal ne transmission was known to play a critical role in regulating many cortical functions , including arousal , attention , motivation , learning , memory , and behavioral flexibility ( sara and segal , 1991 ; tassin , 1998 ; feenstra et al . , 1999 ; arnsten , 2000 ; robbins , 2000 ; bouret and sara , 2004 ; dalley et al . , 2004 ; mingote et al . , 2004 ;
tronel et al . , 2004 ; aston - jones and cohen , 2005 ; rossetti and carboni , 2005 ; lapiz and morilak , 2006 ; van der meulen et al . , 2007 ; robbins and arnsten , 2009 ) .
moreover , both rewarding / reinforcing and aversive stimuli have been shown to increase ne release in pfc ( finlay et al .
, 1995 ; dalley et al . , 1996 ; goldstein et al . , 1996 ; jedema et al . , 1999 ;
, 1999 ; mcquade et al . , 1999 ; feenstra et al . , 2000 ;
these evidences suggested that ca prefrontal transmission could control da in the accumbens also in stressful conditions , a hypothesis that deserved to be assessed .
these studies showed that novel stressful experiences enhance da release in the nac through activation of prefrontal cortical alpha-1 adrenergic receptors ( ars ) by high levels of released ne ( nicniocaill and gratton , 2007 ; pascucci et al . , 2007 ) .
indeed , the experience with a novel stressor promotes a rapid , massive , and transient increase in ne release within the mpfc which parallels the enhancement of mesoaccumbens da release ( pascucci et al . , 2007 ) . a selective depletion of prefrontal cortical ne prevents both the cortical ne response and the increase in accumbal da , leaving stress - induced enhancement of prefrontal cortical da release as well as basal cas levels unaffected ( pascucci et al . , 2007 )
moreover , applications of the alpha-1 ar selective antagonist benoxathian into the mpfc inhibits stress - induced da release in the nac dose - dependently ( nicniocaill and gratton , 2007 ) .
( 2007 ) also confirmed that stress - induced enhanced nac da release is constrained by activation of mpfc da .
indeed , either da depletion ( deutch et al . , 1990 ; doherty and gratton , 1996 ; king et al . , 1997 ; pascucci et al . ,
2007 ) or blockade of d1 receptors by infusion of a selective antagonist in the mpfc ( doherty and gratton , 1996 ) enhances stress - induced da release in the nac . it is known that da in the mpfc exerts an inhibitory influence on da release in the nac and depletion of mesocortical da facilitates stress - induced activation of mesoaccumbens da release ( deutch et al . , 1990 ; doherty and gratton , 1996 ; king et al . ,
however , our results demonstrated that during novel stressful experiences the mpfc determines mesoaccumbens da response through the opposing influences of ne and da .
indeed , the balanced action of the two cas in the mpfc may be required for healthy coping , whereas unbalanced action may promote hyper- or hypo - responding by mesoaccumbens da , leading to different and even opposite behavioral disturbances . the opposite influence exerted by mpfc ne and da on da transmission in the nac during stressful experiences points to possible opposite modulation of frontal cortical glutamate ( glu ) by the two cas . since blockade of mpfc
alpha-1 ars or d1 receptors has opposite effects on stress - induced glu increase ( lupinsky et al . , 2010 ) , it is likely that frontal cortical ne and da exert opposite effects on mpfc output , possibly through glutamatergic stimulation of gaba interneurons in the mpfc ( del arco and mora , 1999 ; homayoun and moghaddam , 2007 ) .
the involvement of alpha1-ars in the prefrontal ne control of da release in the nac during stress is consistent with evidence that a sustained increase of prefrontal cortical ne ( as the one induced by stress ) is capable to activate these low - affinity receptors subtypes , while mild increase is able to activate high affinity alpha2- or beta1- ars ( ramos and arnsten , 2007 ) .
however , the main role of alpha1-ars in the mesoaccumbens da activation by stress or by amphetamine ( darracq et al . , 1998 ; ventura et al . , 2003 ; nicniocaill and gratton , 2007 ) , and the crucial role of prefrontal ne in attribution of motivational salience to stimuli related to amphetamine , as shown by cpp study in the mouse ( ventura et al . , 2003 ) , point to a main role of these receptors in motivated behavior and coping .
mpfc and nac receive da afferents from different populations of vta da cells and these are controlled by different circuits ( joel and weiner , 1997 ; carr and sesack , 2000 ; lewis and o'donnell , 2000 ; margolis et al . , 2006 ; lammel et al .
vta also receives afferents from the central nucleus of amygdala ( cea ) ; the inhibition of cea , and hence of its inhibitory input to vta , leads to an increase of nac da ( ahn and phillips , 2003 ; phillips et al . , 2003a ) , suggesting that this input is part of a double inhibition mechanism ( fudge and haber , 2000 ; ahn and phillips , 2002 ; floresco et al . , 2003 ; fudge and emiliano , 2003 ) .
ne afferents in the mpfc originate from the relatively small group of cells of lc ( aston - jones et al .
, 1999 ; valentino and van bockstaele , 2001 ; berridge and waterhouse , 2003 ) .
lc receives strong convergent projections from the orbito - frontal and cingulate cortex , which have been suggested to drive transitions between phasic and tonic modes in ne neurons to fit the behavioral / cognitive states with environmental conditions ( aston - jones and cohen , 2005 ) .
lc activity is also modulated by cea ( curtis et al . , 2002 ) through innervation of the pericoerulear region ( berridge and waterhouse , 2003 ) and through the excitatory corticotropin - releasing hormone ( van bockstaele et al . , 2001 ; bouret et al . ,
ne has different effects on target cortical areas depending on its concentration and on the distribution of alpha1 and alpha2 receptors ( briand et al .
, 2007 ; arnsten , 2009 ) . indeed , different levels of tonic neuromodulator release affects receptors that are differentially located among cortical layers , so that a neuromodulator may differently affect its target subregions depending on the receptors it activates .
the evidences considered till now indicate that a prefrontal ca system controls da release in the nac , a sub - cortical area known to be involved in all motivated behavior , independently of the valence of the stimuli or experiences .
thus , a similar prefrontal - accumbal regulation has been shown for rewarding ( amphetamine ) or aversive ( stress ) stimuli .
further studies provided substantial support to this view , through experimental evidences that prefrontal cortical ne is crucial in the effects of other addictive drugs , of palatable food , and of aversive pharmacological or physical stimuli .
moreover , they demonstrated that prefrontal ne through its action on nac da is essential in attribution of motivational salience in specific conditions , as it will be shown in the next paragraph .
other addictive drugs , in addition to amphetamine , increase da release in the nac through prefrontal ne , as shown by experiments based on intracerebral microdialysis in the mouse and on selective ne depletion in the mpfc .
selective ne depletion was carried - out by the neurotoxin 6-hydroxydopamine and pre - treatment with the selective da transporter blocker gbr-12909 that produced about 90% ne afferents destruction , with no significant effects on da . to avoid substantial changes in receptor regulation , neurochemical and behavioral testing
morphine ( ventura et al . , 2005 ) , cocaine ( ventura et al . , 2007 ) , ethanol ( ventura et al . , 2006 , in preparation ) have been shown to induce dose - dependent increase of ne in the mpfc and a parallel increase of da in the nac .
selective prefrontal ne depletion abolished the outflow increase of both prefrontal ne and da in the nac , thus confirming the crucial role of ne in mpfc in accumbal da activation induced by different classes of drugs of abuse .
it is worth noting that all the assessed drugs increased da outflow in the mpfc , that was not affected by ne depletion .
however , one may hypothesize that , based on the known inhibitory role of prefrontal da on da release in the nac observed in animals receiving drugs ( e.g. , amphetamine ) or stress , the failure of da increase in the nac of ne mpfc depleted subjects receiving drugs was due to the prevalent inhibitory action of prefrontal da in the absence of ne . such a view , would affirm the crucial promoting role of prefrontal ne on accumbal da , pointing , however , to a complementary role of da in mpfc that would exert an inhibitory role leading to flatting accumbal da when cortical ne is depleted .
this possibility was ruled - out by complementary experiments showing that concomitant depletion of ne and da in mpfc does not change the impaired accumbal da release in mice receiving amph in comparison with animals subjected to selective ne depletion .
a body of evidence suggests that da in the prefrontal cortex is co - released with ne from noradrenergic terminals ( devoto et al . , 2001 , 2002 ) .
moreover , it has been reported that da in this brain area is normally cleared by ne transporter ( tanda et al . , 1997 ; moron et al . , 2002 ) .
different set of data obtained in both mice and rats , showed a lack of effects of the ne depletion on basal extracellular da , suggesting that the likely reduction of da released from destructed noradrenergic terminals is compensated by augmented availability of da due to its reduced uptake from these terminals ( ventura et al .
, 2005 ; pascucci et al . , 2007 ) . however , ne - depleted mice showed an increase of morphine - induced da release similar to that exhibited by sham animals , thus suggesting that prefrontal noradrenergic and dopaminergic projections are functionally uncoupled . in agreement with this observation ,
selective prefrontal ne depletion in rats did not affect stress - induced da release and selective da depletion did not affect stress - induced ne release . taken together , these data indicate that , in both reinforcing ( morphine injection ) and aversive ( stressful situation ) conditions , ne and da release in the mpfc are independent .
this evidence suggests that ne is a common regulating element responding to different class of stimuli to induce da activation in the nac , irrespective of the specific pharmacological or physiological properties of stimuli .
, it is worth to point out that different class of pleasant stimuli as well as aversive experiences such stress are likely to activate a common prefrontal cortical - subcortical network .
however , whether also a system , involving prefrontal ne and accumbal da , has a role , needs experimental support . to study incentive learning and incentive motivation ,
place conditioning is commonly exploited in rats and mice , but in the last species is prevalent since operant procedures that are mostly used for studying drug self - administration in rats , present a number of difficulties in mice .
anyway , this method allows attribution of motivational salience to stimuli related to either pleasant ( appetitive ) or aversive stimuli ( us ) . in the first case pairings between stimuli and environment ( cs )
lead to place preference ( cpp ) , while in the second produce place aversion ( cpa ) .
the process of attribution of motivational salience is measured by the preference ( or the aversion ) shown when a subject has to choose between the environment previously paired with the us and a neutral environment ( tzschentke , 1998 ; mueller and stewart , 2000 ) .
this method is also useful to assess relapse to previously preference ( or aversion ) after extinction , and is a choice method in modeling addiction ( lu et al .
indeed , a before mentioned study had shown that selective prefrontal cortical ne depletion besides impairing amphetamine - induced da outflow increase in the nac , impaired cpp induced by the stimulant .
these effects were not due to motor deficits , or learning impairments , since depleted animals were not different from sham controls in motor behavior , and , most importantly , were able of associative learning as shown by avoidance test ( ventura et al . , 2003 ) .
moreover , these results indicate that intact prefrontal cortical ne is necessary for cpp induced by morphine , cocaine , or ethanol as well as for reinstatement ( relapse ) of extinguished morphine - induced cpp , and for ethanol intake in a choice test .
thus , they demonstrate that prefrontal ne is crucial for da release in the nac induced by addictive drugs and for attribution of motivation salience to drug - related stimuli .
however , the results concerning aversive experiences demonstrate that the noradrenergic control of accumbal da activation is evident also for stress , suggesting a common network involved in processing both pleasant ( rewarding ) and aversive stimuli . to assess this hypothesis we planned two experiments . in the first one we observed that a pharmacological aversive stimulus such lithium chloride administered systemically in mice induced a clear - cut increase of ne in the mpfc and da in the accumbens that was abolished by selective prefrontal ne depletion .
moreover , lithium induced a cpa that was abolished by prefrontal ne depletion , thus confirming that prefrontal ne is crucial for attribution of motivational salience to stimuli related to aversive experience ( ventura et al . , 2007 ) .
next step was suggested by preliminary results obtained when we decided to assess the role of prefrontal - accumbal ca system in attribution of motivational salience to natural non - pharmacological stimuli .
previous data in the literature allowed to hypothesize that appetitive or aversive stimuli produce a graded activation of prefrontal noradrenergic transmission , thus more salient is a stimulus stronger the prefrontal ne release will be ( feenstra et al .
if this were the case , then prefrontal ne release could be considered an index of stimuli salience . to further support
that prefrontal ne - accumbal da system is crucial for attribution of motivational salience also for aversive stimuli we used as aversive non - pharmacological experience a stressor ( intermittent lights ) that could be graded in order to provide parallel effects to those of pleasant ( rewarding ) stimuli like palatable food before described . in place
conditioning preliminary tests , in which the two stressors were compared we observed that they differ in the conditioned aversive effects , the pulsating intermittent lights being more aversive than intermittent non - pulsating lights .
this result paralleled the effects of the two aversive conditions on prefrontal cortical ne release .
both lighting conditions increased prefrontal ne release , but pulsating lighting produced more pronounced increase than non - pulsating lighting .
moreover , noradrenergic response in mpfc was paralleled by graded increase of da in the nac ( ventura et al .
, in preparation ) . then , we assessed if appetitive non - pharmacological stimuli , used as us in place conditioning , required intact prefrontal ne - accumbal da functioning for attribution of motivational salience .
we have observed that mice preferred white chocolate ( wch ) to milk ( mch)-chocolate in a free choice test , a preference that was confirmed in a cpp paradigm where mice chose the environment paired with wch in comparison with that paired to mch - chocolate .
consistently , intracerebral microdialysis showed that exposure to wch intake produces a higher ne release in mpfc than mch ( ventura et al .
, 2008 , in preparation ) accompanied by a more sustained da outflow in the nac .
these results demonstrate that prefrontal ne and accumbal da respond to different salient stimuli , either pleasant or aversive , in a graded manner .
incentive motivation theory has pointed to the major role of the motivational status of the organism ( hungry , thirsty , tired , alert etc . ) when it is confronted with a stimulus or experience .
stress has received much attention in motivation related studies , particularly those concerning addiction models , for neuro - adaptation it can produce in the brain systems involved in the response to drug priming , incentive learning processes , and relapse .
we wondered if pre - exposure to stressful experience could affect the perceived salience of the stimulus and the response of prefrontal - accumbal ca system , and if such changes could affect attribution of motivational salience in our experimental conditions .
we used a food - restriction regimen as chronic stress that was also shown to change the behavioral response to amphetamine and , affect attribution of motivational salience in mice ( cabib et al .
food - restriction ( fr ) led to higher ne release in mpfc and higher da release in the nac in comparison with control mice .
this increase was similar to that shown by free - fed ( non - fr ) mice exposed to wch , thus showing that the state of the organism , as expected , influenced the response to appetitive stimuli .
this effect may be ascribed obviously to the food deprivation that would make it more palatable .
however , our data indicate that fr regimen is an environmental condition that affects the perceived salience , independently of food - related mechanism .
indeed , we observed that fr made the effects induced by the less salient stressor ( intermittent light ) similar to the effects produced in non - fr mice by the more salient stressor ( pulsating intermittent light ) .
this means that fr is capable to increase the salience of both pleasant ( rewarding ; food ) and aversive ( stressful lighting ) stimuli , regardless of hunger - related mechanisms .
note that in additional experiments sham and ne depleted mice subjected to a different no food - related chronic stressful experience ( social isolation ) showed similar effects to those of fr animals , thus indicating that the effect of prefrontal ne depletion on mch - induced cpp can not be ascribed to the homeostatic response to dietary restriction ( ventura et al . , 2008 ) .
food - restriction can also be considered as leading to a generalized drive effect ( niv et al . , 2006 ;
our results , however , indicate that a generalized drive effect produced by food - restriction regimen before exposure to specific stimuli affects not only appetitive alimentary stimuli but also aversive stimuli .
in fact , aversive effects of intermittent light are stronger in food - restricted than in free - fed mice .
thus , a generalized drive effect should involve common neural mechanisms regulating both appetitive and aversive experiences .
taken together , these results show that the prefrontal - accumbal ca response is an index of the emotional / motivational impact of differently salient stimuli depending on the stimuli characteristics or on the state of the organism . the graded response of prefrontal ne was in agreement with previous results and suggested to us to ascertain the role of prefrontal - accumbal ca system in attribution of motivational salience related to differently salient stimuli . using the experimental paradigms of other studies on the same topics , we assessed the effects of selective prefrontal ne depletion on the ca response and on attribution of motivational salience measured by place conditioning .
surprisingly , we observed that ne depletion abolished the increase of prefrontal cortical ne release and of accumbal da , consistently with previous experiments .
however , it prevented place preference ( cpp ) in animals exposed to wch and in food - restricted ( fr ) animals exposed to milk chocolate ( mch ; both conditions of high salience ) but not in non - fr ( free - fed ) animals exposed to mch ( low salience ) .
moreover , it prevented place aversion ( cpa ) in animals exposed to intermittent pulsating light ( ipl ) and in the fr animals exposed to intermittent light ( il ; high salience ) but not in non fr animals exposed to il ( low salience ; figure 1 ) .
effects of prefrontal cortical norepinephrine depletion on conditioned place preference ( cpp ) induced by chocolate ( milk chocolate in control , mch ; milk chocolate in food restricted mch+fr ; white chocolate in control , wch ) and conditioned place aversion ( cpa ) induced by light ( intermittent light in control , il ; intermittent light in food restricted , il+fr ; intermittent - pulsating light in control ; ipl ) [ sham , norepinephrine - depleted in medial prefrontal cortex ( mpfc ne depleted ) ] .
all data are expressed as mean ( sec se ) time spent in paired , and unpaired chambers .
p < 0.05 in comparison with the unpaired chamber ; p < 0.005 in comparison with the unpaired chamber ( from ventura et al . , 2008 , modified ) .
these results show that that pfc ne depletion affects attribution of motivational salience only when the salience of the ucs is high enough to induce sustained ca activation , thus indicating that prefrontal - accumbal ca system is involved in processing motivational salience attribution selectively when intense motivational salience is processed .
salience refers to the capability of stimuli to be arousing ( horvitz , 2000 ) .
salient stimuli cause the reallocation of available cognitive resources in order to produce an attentional or a behavioral switch ( zink et al . , 2006 ) .
the more salient the stimulus , the more likely it will lead to an attentional or a behavioral switch .
recent reports in humans have shown that striatum has a major role in prompting the reallocation of resources to salient stimuli ( zink et al .
however prefrontal cortex , due to its supervisory functions , has an unquestionable central role in attentional and motivational processing of salient stimuli .
moreover , data indicate that ventral striatum ( or nac ) and prefrontal cortex constitute a common substrate for processing both rewarding and aversive stimuli ( berridge and robinson , 1998 ; darracq et al . , 1998 ;
, 2001 ; jensen et al . , 2003 ; kensinger and schacter , 2006 ; borsook et al . , 2007 ) , and
neuroimaging studies in humans suggest that different areas of prefrontal cortex ( o'doherty et al . , 2001 ;
small et al . , 2001 ; killgore et al . , 2003 ; wang et al . , 2004 ) and of striatum ( jensen et al . , 2003
again , we have previously demonstrated that intact ne prefrontal transmission is necessary for motivational salience attribution to both natural ( in food restricted animals ) and pharmacological reward related stimuli as well as to pharmacological aversion related stimuli through modulation of da in nac ( ventura et al . , 2007 ) .
therefore , it is likely that the effects of prefrontal ne depletion on cpp and cpa in animals exposed to highly salient stimuli depend on the impaired response of prefrontal - accumbal ca system , whose activation by unconditioned rewarding and aversive highly salient stimuli is a substrate for motivational salience .
since amygdala is involved in pavlovian conditioning of emotional responses and plays a specific role in modulating memory for arousing experiences ( balleine , 2005 ; balleine and killcross , 2006 ; mcgaugh , 2006 ) , and given the complex anatomical and functional connections between this brain area and prefrontal cortex ( cardinal et al . , 2002
; holland and gallagher , 2004 ; roozendaal et al . , 2004 ) a role of a prefrontal cortex - amygdala system in the effects of highly salient stimuli reported here must be considered ( belova et al . , 2007 ) .
attribution of motivational salience is related to the salience of an ucs ( dallman et al . , 2003 ; pecina et al . ,
thus , the more salient an ucs the more likely a neutral ( to - be - conditioned ) stimulus will be associated with it through motivational salience attribution .
prior experience is a major determinant of the motivational impact of any given stimulus ( borsook et al . ,
2007 ) and emotional arousal induced by motivational stimuli increases the attention given to stimuli influencing both the initial perceptual encoding and the consolidation process ( anderson et al . , 2006 ; mcgaugh , 2006 ) .
we provided evidence that prefrontal - accumbal ca transmission is necessary for motivational salience attribution to both reward- and aversion - related stimuli only under those conditions able to induce stronger increases of ca outflow in response to highly salient unconditioned natural stimuli , independently of valence .
thus , selective prefrontal ne depletion abolished the place conditioning induced by highly salient stimuli ( i.e. , wch and ipl ) in control animals and by mildly salient stimuli ( i.e. , mch and il ) in stressed groups , but had no significant effects in control animals exposed to mild salient stimuli .
these results demonstrate that prefrontal - accumbal ca transmission is necessary for the acquisition of conditioned properties to stimuli paired with highly salient natural rewarding or aversive events in a place - conditioning procedure .
many different factors have major regulatory role in motivated behaviors , including the internal variables of the organism ( i.e. , motivational state , stress response ) and stimulus properties ( i.e. , salience or intensity ) , both of which affect motivational salience attribution processes ( berridge and robinson , 1998 ; richard and berridge , 2011 ) .
( belova et al . , 2007 ) , thus suggesting that a common neural system might be involved in processing stimuli salience , irrespectively of valence .
moreover , arousing pleasant or aversive stimuli that elicit valence - specific responses have been suggested to enhance attention and memory formation through a common , valence - insensitive pathway ( belova et al . , 2007 ) and
prefrontal cortex has been involved in processing both rewarding and aversive stimuli ( rolls , 2000 ; o'doherty et al . , 2001 ;
dopaminergic transmission within nac is considered to mediate the hedonic impact of reward or some aspects of reward learning ( everitt and robbins , 2005 for review ) .
our results , in agreement with a different view ( berridge and robinson , 1998 ) , show that da transmission in nac plays a role in both positively and aversively motivated behavior ; most importantly , however , they demonstrate that this motivational process is governed by prefrontal cortical ne .
norepinephrine in mpfc might activate mesoaccumbens da release through excitatory prefrontal cortical projection to vta da cells ( sesack and pickel , 1992 ; shi et al . , 2000 ) and/or through corticoaccumbal glutamatergic projections ( darracq et al . , 2001 ) .
moreover , a role for mpfc projections to the lc in exerting an excitatory influence can be envisaged because this nucleus has been shown to activate vta da neurons ( grenhoff et al . , 1993 ; jodo et al . , 1998 ;
2004 ) , which could lead to increased da release in nac . however , since amygdala is involved in pavlovian conditioning of emotional responses and plays a specific role in modulating memory for arousing experiences ( balleine and killcross , 2006 ; mcgaugh , 2006 ) , and given the complex anatomical and functional connections between this brain area and prefrontal cortex ( cardinal et al . , 2002 ; roozendaal et al . ,
2004 ) , a role of the prefrontal cortex - amygdala system in the effects of the highly salient stimuli reported here must be considered ( belova et al . , 2007 ; mahler and berridge , 2011 ) .
note that nac and dopaminergic transmission are considered to play major role in motivation processes besides the role that da plays in other aspects of incentive motivation and instrumental learning ( salamone et al . , 2005 ) .
indeed , based on the view that is doubtful that accumbens da performs only one function , substantial evidences support the hypothesis that da is involved in the exertion of effort or effort - related decision - making ( salamone et al .
2009 ) , that is not incompatible with the involvement of this system in instrumental learning , incentive motivation , or pavlovian - instrumental transfer .
animal and human studies seem to converge in that , together with animal studies focused on effort related functions of accumbal da , clinical findings are consistent with the hypothesis that da systems are involved in behavioral activation , pointing to a striking similarity between the brain systems implicated in effort - related processes in animals and those involved in energy dysfunctions in humans ( salamone et al . , 2007 ) .
according to this view nac functioning is to be considered , along with prefrontal cortex and the amygdala , as a component of the brain circuitry regulating effort - related functions . in this framework ,
the prefrontal / accumbal ca system we have envisaged may conceivably be a part of a complex network involving cortical and subcortical brain areas involved in regulation of effort - related functions controlling motivation outcomes , and possibly linking salience intensity to effort intensity .
in our view the impact of salient stimuli is crucial in the processes that lead to attribution of motivational salience , due to the salience perceived .
that means that the impact of stimuli produce an emotional response that tunes the association processes leading to motivation outcome , thus pointing to the basic role of emotional salience as the individual is exposed to the ucs .
the prefrontal / accumbal system we propose to control motivational salience processes depending on salience intensity should be considered included in complex networks regulating perceived emotion ( phillips et al . , 2003b ) .
emotion perception , according to the appraisal theories ( arnold , 1960 ; lazarus , 1991 ) has been suggested to stems from three processes : the identification of the emotional significance of a stimulus , the production of an affective state in response to the stimulus , and the regulation of the affective state .
, 2003b for review ) , these processes depend on different brain emotional systems involving brain areas including mesencephalic , cortical , and subcortical such as the amygdala , insula , ventral striatum , ventral , and dorsal anterior cingulate gyrus , the septo - hippocampus system , the prefrontal cortex , all characterized by reciprocal functional relationships ( salzman and fusi , 2010 ) .
the septo - hippocampus system has been considered as a general - purpose comparator , with a central role in determining the extent of conflict between different goal - directed behaviors ( gray and mcnaughton , 2000 ) .
amygdala has a well - known role in emotion and in memory consolidation processes depending on emotional arousal .
indeed , the amygdala can evoke conditioned responses capable of exerting a dominant effect on choice , and perceived emotional values in pavlovian conditioning are exploited by instrumental ( habit - based and goal - directed ) learning mechanisms through connectivity with other brain regions such as the striatum and prefrontal cortex ( seymour and dolan , 2008 ) .
it is worth noting that values are affected by the action of stress hormones , such as glucocorticoids , on amygdala , and these effects control memory consolidation pointing to a link between emotional salience and the strength of memories ( roozendaal , 2000 ; setlow et al .
moreover glucocorticoids have been shown to be biological substrate of reward ( piazza and le moal , 1997 ) and substantial evidences show that they play a role in the modulation of both appetitive and aversive emotional memories indicating that modulation of appetitive and aversive discrete - cue learning may be subserved by a common mechanism ( zorawski and killcross , 2002 ) .
we have provided evidence that prefrontal - accumbal ca system is involved in processing motivational salience attribution selectively when intense motivational salience is processed , thus pointing to an allegedly different neural system involved in the attribution of motivational salience related to mildly salient stimuli .
our results are consistent with those that had shown da transmission is not always involved in motivation ( nader et al . , 1997 , for review ) . in contrast with the da hypothesis that is based on a single - system model of reward , a non - deprived /
deprived model has been proposed in the late nineties that claims that two separate neurobiological reward systems can be double dissociated , each of which makes a significant contribution to motivated behavior depending on deprivation state .
note that the model is supported by experiments where drug naive animals are considered similar to food - sated ( i.e. , non - deprived ) , differently from drug - dependent animals in withdrawal or food - restricted animals that are considered deprived ( nader et al .
a state of deprivation inhibits the non - deprived system [ involving the peduncolo - pontine nucleus ( tpp ) ] .
thus , the differential activation of the two systems is predicated specifically on whether animals are , for instance , in a state of withdrawal or not ( nader et al . , 1997 ) .
the second implication is that a state of deprivation engages a second neurobiologically distinct motivational system , a component of which is da .
the obvious question that arises from this model is whether all motivated behaviors can be considered to have a non - deprived and a deprived component . as it was questioned by the proponents ( nader et al .
, 1997 , for review ) : do some stimuli only work through one of the two systems ?
although this discussion is out of the aim of our present work , we ca n't help noting the parallelism between our findings on prefrontal - accumbal ca system and the non - deprived / deprived system , in that our system is crucial in attribution of motivational salience when the stimuli salience is high and characterized by high emotional impact ( either positive or negative ) . in this case , another system involved in low salience processing is inhibited or off - line , and this system that is on - line when low salience is processed ( and that we did not envisaged yet ) , parallels the non - deprived system , characterized by low emotional impact .
our results also suggest strongly that , like suggested for the non - deprived / deprived model , the system processing high salience ( prefrontal - accumbal ca system ) and the supposed one involved in low salience are mutually exclusive . in terms of the neural dynamics involved in the selective and exclusive engagement of these systems
, we can tentatively pose that the graded increase of ne outflow in mpfc depending on low or high salience of stimuli , may involve different ar - subtypes , that , in turn , depending on a given threshold level of released ne , will engage different circuits , and , in the case of high salience , including da in the nac .
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 .
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 . | motivational salience regulates the strength of goal seeking , the amount of risk taken , and the energy invested from mild to extreme .
highly motivational experiences promote highly persistent memories .
although this phenomenon is adaptive in normal conditions , experiences with extremely high levels of motivational salience can promote development of memories that can be re - experienced intrusively for long time resulting in maladaptive outcomes .
neural mechanisms mediating motivational salience attribution are , therefore , very important for individual and species survival and for well - being .
however , these neural mechanisms could be implicated in attribution of abnormal motivational salience to different stimuli leading to maladaptive compulsive seeking or avoidance .
we have offered the first evidence that prefrontal cortical norepinephrine ( ne ) transmission is a necessary condition for motivational salience attribution to highly salient stimuli , through modulation of dopamine ( da ) in the nucleus accumbens ( nac ) , a brain area involved in all motivated behaviors .
moreover , we have shown that prefrontal - accumbal catecholamine ( ca ) system determines approach or avoidance responses to both reward- and aversion - related stimuli only when the salience of the unconditioned stimulus ( ucs ) is high enough to induce sustained ca activation , thus affirming that this system processes motivational salience attribution selectively to highly salient events . |
since their introduction in 1972 , glass ionomer ( gi ) cements have been widely used as dental restorative materials , in particular in restoration of cervical lesions . despite the advantages associated with the use of conventional gi ( cgi ) cements as ; their chemical bonding efficacy to tooth substrate , long - term fluoride ion release , low coefficient of thermal expansion and relatively ease of use , yet they suffer from several problems as ; moisture sensitivity , low mechanical strength and impaired translucency . in an attempt to overcome these problems , several innovations were done . in 1988 resin modified gi ( rmgi )
this material gained wider acceptance in the dental professions as adding light polymerizable ( hydroxyethyl methacrylate [ hema ] ) resin to the gi enhanced its handling characteristics , increased its working time and improved its esthetic properties .
however , rmgi cements exhibit shrinkage and substantial water sorption owing to the hydrophilic nature of its contents .
recently a nanofilled rmgi restorative material ( nano - ionomer [ ni ] ) was launched into the market .
apart from user - friendliness , the major innovations of these materials involve incorporation of nanotechnology , which allowed a highly packed filler composition ( approximately 69% ) in the form of nano - sized fillers and nanoclusters .
gi cements are the material of choice when dealing with root caries or noncarious cervical lesions . in such areas ,
this substratum is a nonuniform human cementum , which can be represented as a woven fabric - like material that provides tissue porosity and permeability .
one of the surface features that distinguish cementum from dentin is the lack of patent tubule orifices , which may alter its bonding capability . searching the literature ,
it was evident that almost all research work was concerned with bonding efficacy to enamel and dentin , however , the bonding efficacy to the cementum was scarcely addressed .
thus , this study was carried out to determine the bonding efficacy of the nanofilled rmgi ( ni ) restorative material to enamel , dentin , and cementum and compare it to conventional rmgi restorative material and packable conventional cgi cements .
the null hypotheses examined that ( 1 ) there is no difference in the bonding performance between the three tested gi cements ; cgi , rmgi , and ni when applied to enamel , dentin and cementum .
( 2 ) each tested gi cement bond equally to enamel , dentin and cementum substrates .
forty - five extracted , noncarious lower first molars were collected , cleaned from debris and stored in distilled water with 0.5% thymol .
three gi - based restorative materials were tested : conventional high viscosity gi cement ( fuji ix , gc corporation , tokyo , japan ) , rmgi cement ( fuji ii lc , gc corporation , tokyo , japan ) and ni cement ( ketac n 100 , 3 m espe , ni , st paul , mn , usa ) .
10 molars were used to test their bond strength to different tooth substrates ; enamel , dentin , and cementum while 5 teeth were utilized for the ultrastructural examination of the interface .
material 's composition and manufacturer each molar was sectioned 1 mm coronal to the cervical line to separate the crown from the roots using a diamond disk ( k6974 , komet , germany ) in a low - speed handpiece under copious water cooling .
the roots were separated , and each root was embedded in self - cured resin ( rapid repair , degudent gmbh , hanau , germany ) .
the flat cementum regions from the mesial and distal surfaces of the mesial and distal roots respectively , at cervical root third , were used as a bonding substrate , after grinding with wet 600 grit silicon carbide paper .
the presence of cementum was previously verified under a stereomicroscope , ( carl zeiss , germany ) at 25 magnification .
the coronal portion of each tooth was embedded vertically in self - cured resin that the occlusal surface was used for testing .
the occlusal enamel was then removed , and 1 mm of dentin was ground , thus exposing superficial occlusal dentin surrounded by an enamel rim .
the thickness of the enamel should be at least 1.5 mm wide to be utilized for microshear bond strength ( sbs ) testing .
a uniform smear layer was created by abrading the occlusal surface ( enamel and dentin ) with 600 grit silicon carbide paper .
ketac dentin conditioner was applied to enamel , dentin or cementum substrates for 20 s using a microbrush ( shofu , japan ) .
it was then rinsed thoroughly with air - water spray and the excess water was blotted with cotton pellet . for ni group , ketac
n100 primer was applied for 15 s. the primer was then air - thinned with an air syringe for 10 s and light cured with a light curing unit ( db 686 - 1 , coxo , china ) at an intensity of 1200 mw / cm for 20 s. after mixing of cgi and rmgi capsules using an amalgamator ( silamat plus , vivadent , austria ) and manual mixing of equal volumes of ni , the materials were injected into polyethylene tubes of 0.9 mm in diameter and 0.7 mm in height . for rmgi and ni group ,
the restoration was light - cured for 20 s. light intensity was periodically checked using a radiometer ( kerr ; orange , ca , usa ) .
then , ketac glaze was painted on the top of cgi and rmgi cylinders only and light - cured for 10 s. all polyethylene tubes were then removed , and the bonded specimens were stored in distilled water for 24 h. in coronal specimens , enamel and dentin substrates received four microcylinders for each . in each root specimen , each surface received two microcylinders ( each molar received four microcylinders for enamel , dentin , and cementum ) .
microshear bond strength testing was done using a universal testing machine ( model lrx - plus , lloyd instruments ; fareham , uk ) .
each specimen with its bonded microcylinders was secured with tightening screws to the lower fixed compartment of the universal testing machine .
a loop of orthodontic stainless steel wire ( 0.014 inch in diameter ) was wrapped around the bonded microcylinder as close to its base as possible and aligned with the loading axis of the upper movable compartment of the testing machine .
the specimens were stressed in shear using a load cell of 5 kn at a crosshead speed of 0.5 mm / min . the shear force at failure was recorded and converted to shear stress in mpa units using computer software ( nexygen - mt lloyd instruments , fareham , uk ) . for each molar ,
the four readings obtained from enamel , dentin and cementum substrates were averaged to obtain one reading for each substrate from each molar ( n = 10 ) .
data were analyzed using two - way analysis of variance ( anova ) to determine the effect of restorative material , tooth substrate and their interaction on mean sbs values . comparing between the three materials for each tooth substrate and between the three substrates for each restorative material
was carried out using one way anova followed by tukey 's post - hoc test .
statistical analysis was performed with spss 16.0 , inc . , ( spss , inc . ,
following microshear testing , specimens were examined using a stereomicroscope ( carl zeiss , germany ) at 25 magnification to evaluate the fracture mode .
the mode of failure was identified as : type i ( adhesive failure at the interface ) , type ii ( mixed adhesive failure at the interface + cohesive in the restorative material ) , type iii ( cohesive in the restorative material ) .
fifteen molar were utilized for ultrastructural analysis of the interface ( five for each restorative material ) .
the restorative materials ( fuji ix , fuji ii lc and ketac n100 ) were applied to the different substrates as described before but in one layer of 2 mm in thickness .
a transparent mylar strip ( moyco , usa ) was placed on top of the restorative material until setting .
teeth were then sectioned bucco - lingually using a diamond disc at a slow speed under copious water cooling .
the sectioned specimens were finished with soflex discs ( 3m - espe dental products , st .
the specimens were then placed into an ultrasonic cleaner ( elma - transsonic 460 hz , germany ) in distilled water for 30 min to remove the smear layer .
specimens were examined under low vacuum without gold sputtering using environmental field emission scanning electron microscopy ( quanta field emission gun 250 , holland 115/230 v , inspect sfei , phillips , holland ) , and representative enviromental scanning photomicrographs of enamel- , dentin- and cementum - restoration interface were obtained at 500 magnification .
forty - five extracted , noncarious lower first molars were collected , cleaned from debris and stored in distilled water with 0.5% thymol .
three gi - based restorative materials were tested : conventional high viscosity gi cement ( fuji ix , gc corporation , tokyo , japan ) , rmgi cement ( fuji ii lc , gc corporation , tokyo , japan ) and ni cement ( ketac n 100 , 3 m espe , ni , st paul , mn , usa ) .
10 molars were used to test their bond strength to different tooth substrates ; enamel , dentin , and cementum while 5 teeth were utilized for the ultrastructural examination of the interface .
each molar was sectioned 1 mm coronal to the cervical line to separate the crown from the roots using a diamond disk ( k6974 , komet , germany ) in a low - speed handpiece under copious water cooling .
the roots were separated , and each root was embedded in self - cured resin ( rapid repair , degudent gmbh , hanau , germany ) .
the flat cementum regions from the mesial and distal surfaces of the mesial and distal roots respectively , at cervical root third , were used as a bonding substrate , after grinding with wet 600 grit silicon carbide paper .
the presence of cementum was previously verified under a stereomicroscope , ( carl zeiss , germany ) at 25 magnification .
the coronal portion of each tooth was embedded vertically in self - cured resin that the occlusal surface was used for testing .
the occlusal enamel was then removed , and 1 mm of dentin was ground , thus exposing superficial occlusal dentin surrounded by an enamel rim .
the thickness of the enamel should be at least 1.5 mm wide to be utilized for microshear bond strength ( sbs ) testing .
a uniform smear layer was created by abrading the occlusal surface ( enamel and dentin ) with 600 grit silicon carbide paper .
ketac dentin conditioner was applied to enamel , dentin or cementum substrates for 20 s using a microbrush ( shofu , japan ) .
it was then rinsed thoroughly with air - water spray and the excess water was blotted with cotton pellet . for ni group ,
ketac n100 primer was applied for 15 s. the primer was then air - thinned with an air syringe for 10 s and light cured with a light curing unit ( db 686 - 1 , coxo , china ) at an intensity of 1200 mw / cm for 20 s. after mixing of cgi and rmgi capsules using an amalgamator ( silamat plus , vivadent , austria ) and manual mixing of equal volumes of ni , the materials were injected into polyethylene tubes of 0.9 mm in diameter and 0.7 mm in height . for rmgi and ni group , the restoration was light - cured for 20 s. light intensity was periodically checked using a radiometer ( kerr ; orange , ca , usa ) .
then , ketac glaze was painted on the top of cgi and rmgi cylinders only and light - cured for 10 s. all polyethylene tubes were then removed , and the bonded specimens were stored in distilled water for 24 h. in coronal specimens , enamel and dentin substrates received four microcylinders for each . in each root specimen , each surface received two microcylinders ( each molar received four microcylinders for enamel , dentin , and cementum ) . microshear bond strength testing was done using a universal testing machine ( model lrx - plus , lloyd instruments ; fareham , uk ) . each specimen with its bonded microcylinders was secured with tightening screws to the lower fixed compartment of the universal testing machine .
a loop of orthodontic stainless steel wire ( 0.014 inch in diameter ) was wrapped around the bonded microcylinder as close to its base as possible and aligned with the loading axis of the upper movable compartment of the testing machine .
the specimens were stressed in shear using a load cell of 5 kn at a crosshead speed of 0.5 mm / min . the shear force at failure was recorded and converted to shear stress in mpa units using computer software ( nexygen - mt lloyd instruments , fareham , uk ) . for each molar
, the four readings obtained from enamel , dentin and cementum substrates were averaged to obtain one reading for each substrate from each molar ( n = 10 ) .
data were analyzed using two - way analysis of variance ( anova ) to determine the effect of restorative material , tooth substrate and their interaction on mean sbs values . comparing between the three materials for each tooth substrate and between the three substrates for each restorative material
was carried out using one way anova followed by tukey 's post - hoc test .
statistical analysis was performed with spss 16.0 , inc . , ( spss , inc . ,
following microshear testing , specimens were examined using a stereomicroscope ( carl zeiss , germany ) at 25 magnification to evaluate the fracture mode .
the mode of failure was identified as : type i ( adhesive failure at the interface ) , type ii ( mixed adhesive failure at the interface + cohesive in the restorative material ) , type iii ( cohesive in the restorative material ) .
fifteen molar were utilized for ultrastructural analysis of the interface ( five for each restorative material ) .
the restorative materials ( fuji ix , fuji ii lc and ketac n100 ) were applied to the different substrates as described before but in one layer of 2 mm in thickness .
a transparent mylar strip ( moyco , usa ) was placed on top of the restorative material until setting .
teeth were then sectioned bucco - lingually using a diamond disc at a slow speed under copious water cooling .
the sectioned specimens were finished with soflex discs ( 3m - espe dental products , st .
the specimens were then placed into an ultrasonic cleaner ( elma - transsonic 460 hz , germany ) in distilled water for 30 min to remove the smear layer .
specimens were examined under low vacuum without gold sputtering using environmental field emission scanning electron microscopy ( quanta field emission gun 250 , holland 115/230 v , inspect sfei , phillips , holland ) , and representative enviromental scanning photomicrographs of enamel- , dentin- and cementum - restoration interface were obtained at 500 magnification .
each molar was sectioned 1 mm coronal to the cervical line to separate the crown from the roots using a diamond disk ( k6974 , komet , germany ) in a low - speed handpiece under copious water cooling .
the roots were separated , and each root was embedded in self - cured resin ( rapid repair , degudent gmbh , hanau , germany ) .
the flat cementum regions from the mesial and distal surfaces of the mesial and distal roots respectively , at cervical root third , were used as a bonding substrate , after grinding with wet 600 grit silicon carbide paper .
the presence of cementum was previously verified under a stereomicroscope , ( carl zeiss , germany ) at 25 magnification .
the coronal portion of each tooth was embedded vertically in self - cured resin that the occlusal surface was used for testing .
the occlusal enamel was then removed , and 1 mm of dentin was ground , thus exposing superficial occlusal dentin surrounded by an enamel rim .
the thickness of the enamel should be at least 1.5 mm wide to be utilized for microshear bond strength ( sbs ) testing .
a uniform smear layer was created by abrading the occlusal surface ( enamel and dentin ) with 600 grit silicon carbide paper .
ketac dentin conditioner was applied to enamel , dentin or cementum substrates for 20 s using a microbrush ( shofu , japan ) .
it was then rinsed thoroughly with air - water spray and the excess water was blotted with cotton pellet . for ni group ,
ketac n100 primer was applied for 15 s. the primer was then air - thinned with an air syringe for 10 s and light cured with a light curing unit ( db 686 - 1 , coxo , china ) at an intensity of 1200 mw / cm for 20 s. after mixing of cgi and rmgi capsules using an amalgamator ( silamat plus , vivadent , austria ) and manual mixing of equal volumes of ni , the materials were injected into polyethylene tubes of 0.9 mm in diameter and 0.7 mm in height . for rmgi and ni group ,
the restoration was light - cured for 20 s. light intensity was periodically checked using a radiometer ( kerr ; orange , ca , usa ) .
then , ketac glaze was painted on the top of cgi and rmgi cylinders only and light - cured for 10 s. all polyethylene tubes were then removed , and the bonded specimens were stored in distilled water for 24 h. in coronal specimens , enamel and dentin substrates received four microcylinders for each . in each root specimen , each surface received two microcylinders ( each molar received four microcylinders for enamel , dentin , and cementum ) .
microshear bond strength testing was done using a universal testing machine ( model lrx - plus , lloyd instruments ; fareham , uk ) .
each specimen with its bonded microcylinders was secured with tightening screws to the lower fixed compartment of the universal testing machine .
a loop of orthodontic stainless steel wire ( 0.014 inch in diameter ) was wrapped around the bonded microcylinder as close to its base as possible and aligned with the loading axis of the upper movable compartment of the testing machine .
the specimens were stressed in shear using a load cell of 5 kn at a crosshead speed of 0.5 mm / min . the shear force at failure was recorded and converted to shear stress in mpa units using computer software ( nexygen - mt lloyd instruments , fareham , uk ) . for each molar
, the four readings obtained from enamel , dentin and cementum substrates were averaged to obtain one reading for each substrate from each molar ( n = 10 ) .
data were analyzed using two - way analysis of variance ( anova ) to determine the effect of restorative material , tooth substrate and their interaction on mean sbs values . comparing between the three materials for each tooth substrate and between the three substrates for each restorative material
was carried out using one way anova followed by tukey 's post - hoc test .
statistical analysis was performed with spss 16.0 , inc . , ( spss , inc . ,
following microshear testing , specimens were examined using a stereomicroscope ( carl zeiss , germany ) at 25 magnification to evaluate the fracture mode .
the mode of failure was identified as : type i ( adhesive failure at the interface ) , type ii ( mixed adhesive failure at the interface + cohesive in the restorative material ) , type iii ( cohesive in the restorative material ) .
fifteen molar were utilized for ultrastructural analysis of the interface ( five for each restorative material ) .
the restorative materials ( fuji ix , fuji ii lc and ketac n100 ) were applied to the different substrates as described before but in one layer of 2 mm in thickness .
a transparent mylar strip ( moyco , usa ) was placed on top of the restorative material until setting .
teeth were then sectioned bucco - lingually using a diamond disc at a slow speed under copious water cooling .
the sectioned specimens were finished with soflex discs ( 3m - espe dental products , st .
the specimens were then placed into an ultrasonic cleaner ( elma - transsonic 460 hz , germany ) in distilled water for 30 min to remove the smear layer .
specimens were examined under low vacuum without gold sputtering using environmental field emission scanning electron microscopy ( quanta field emission gun 250 , holland 115/230 v , inspect sfei , phillips , holland ) , and representative enviromental scanning photomicrographs of enamel- , dentin- and cementum - restoration interface were obtained at 500 magnification .
two - way anova revealed that restorative materials , substrates examined , and their interaction had a statistically significant effect on mean sbs values at p values ; < 0.0001 , 0.0108 and 0.0037 respectively [ table 2 ] .
it was evident that rmgi showed the highest statistical significant sbs to all tested substrates compared to cgi and ni .
meanwhile , ni showed statistically significant lower sbs values to enamel compared to dentin and cementum .
meanssd of sbs values in mpa of all tested groups fracture mode analysis results are presented in percentages in table 4 .
it was evident that the highest percent of cohesive failure ( type iii ) was present in the cgi group .
the percentages of fracture modes of all tested groups all tested groups showed a gap - free junction with the existence of a filler - free zone of variable thickness , except at the ni - enamel interface at which a gap was evident [ figure 1 ] .
the greatest thickness of the filler - free zone was observed for the ni - enamel interface .
environmental field emission scanning electron microscope photomicrograph representing tooth - restoration interface of all tested groups at 500 magnification .
it revealed the existence of a filler free zone at the interface of all tested groups .
two - way anova revealed that restorative materials , substrates examined , and their interaction had a statistically significant effect on mean sbs values at p values ; < 0.0001 , 0.0108 and 0.0037 respectively [ table 2 ] .
it was evident that rmgi showed the highest statistical significant sbs to all tested substrates compared to cgi and ni .
meanwhile , ni showed statistically significant lower sbs values to enamel compared to dentin and cementum .
meanssd of sbs values in mpa of all tested groups fracture mode analysis results are presented in percentages in table 4 .
it was evident that the highest percent of cohesive failure ( type iii ) was present in the cgi group .
all tested groups showed a gap - free junction with the existence of a filler - free zone of variable thickness , except at the ni - enamel interface at which a gap was evident [ figure 1 ] .
the greatest thickness of the filler - free zone was observed for the ni - enamel interface .
environmental field emission scanning electron microscope photomicrograph representing tooth - restoration interface of all tested groups at 500 magnification .
it revealed the existence of a filler free zone at the interface of all tested groups .
this study was conducted to elaborate the bonding efficacy of gi cements to different tooth substrates ( enamel , dentin , and cementum ) via the use of sbs test and environmental field emission scanning electron microscope ( efesem ) .
three gi cements were evaluated ; high viscosity chemical - cure cgi cement , rmgi , and ni .
ni , ketac n 100 , is light curing rmgi modified using bonded nanofillers , and nanocluster fillers , along with fluoroaluminosilicate glass .
a rmgi ( fuji ii lc ) was chosen to elucidate the value of the latter modification .
a cgi cement ( fuji ix ) was compared as control . in this study enamel , dentin and
cementum specimens were obtained from the same tooth for more standardization of the chemical composition and the degree of mineralization when comparing the three substrates with each other . in the present study ,
this test is considered a relatively simple test that permits efficient screening of adhesive systems , regional and depth profiling of a variety of substrates , with conservation of teeth .
furthermore , it could be a viable test when evaluating brittle materials , having low modulus of elasticity as gi cements as it lowers the probability of having a crack opening relative to the load applied .
fractographic analysis of the debonded specimens gives clues about some of the reasons of failure , the usage and limitations of each material .
efesem photomicrographs may give clues about the bond quality at the interface , which can be a complementary tool for bond strength evaluation .
it is a more suitable tool for evaluation of gi cements restoratives which are sensitive to the vacuuming , processing and gold sputtering step which are mandatory in the conventional scanning electron microscope .
results of the present study revealed that the rmgi showed the highest statistically significant sbs values compared to cgi and ni irrespective to substrate examined , thus the first null hypothesis is rejected .
this result is in agreement with several authors whom found that the bond strength of rmgi was significantly higher than cgi cements to enamel . and to dentin .
in addition , in agreement with coutinho et al . , whom found that rmgi showed significantly higher bond strength values compared to ni to both enamel and dentin substrates .
however , the bonding of gi restoratives to cementum was not previously addressed in literature .
the better bonding performance of the rmgi compared to cgi could be attributed to several theories acting simultaneously ; first ; to the difference in the rates of adsorption on the calcified tissue surface and crosslinking in the bulk regions of the material .
light - curing of rmgi causes an initial increase in the ionization rate due to the production of acid ( via the photoinitiator ) , and this results in a very strong adsorbed layer . at the same time
thus , the unsaturated methacrylate groups will polymerize and co - polymerize with the modified polyacrylic acid , forming a polyacrylic acid network entangled within the collagen web .
second ; to the resinous component in rmgi cements that can form a covalently bonded matrix , allowing the material to have greater fracture strengths than cgi cements .
third , this might be attributed to the carboxylic acid copolymers contained in the cement , which provide a more reactive and acidic liquid with increased number of carboxyl groups per chain unit thus increasing its chemical bonding potential . forth ; to the high hema concentration in rmgi cement , which improves its wetting ability and results in better bonding .
the hema concentration in the rmgi cement used is 35% ; meanwhile , it is completely absent in cgi cement .
nano - ionomer primer contains the same hema concentration as rmgi , but it showed lower bonding performance with all tooth substrates , despite the existence of a thick filler - free zone with gap - free junction with dentin and cementum substrates .
this observation is in line with coutinho et al . , whom attributed this to the difference in the technique of application and the conditioning step .
in rmgi cement polyacrylic acid conditioning is applied and rinsed prior to the material application which allows for partial demineralization and removal of the smear layer , giving the opportunity for the hema component to enhance the wetting of the surface and the production of microporosity in the different tooth substrates .
this could contribute to either increased surface area for chemical bonding with residual hydroxyl apatite or micromechanical bonding through micromechanical interlocking .
meanwhile , with ni , the polyacrylic acid is incorporated in the primer which is applied onto the smear layer and is not rinsed away .
in addition , ni primer has a relatively higher ph ( 3 ) than polyacrylic acid ph ( 1.5 ) .
this difference could be reflected in the ability of polyacrylic acid to remove the smear layer resulting in distinct performance .
coutinho et al . , reported that the the filler - free zone at the ni interface most likely represented remnants of the primer that did not polymerize well due to the presence of oxygen , and therefore only showed a nonhomogeneous layer which might have not contributed to the bonding effectiveness . although the existence of filler free zone is important to act as a stress breaking layer , the increase in its thickness above a certain level appears to be a negating factor similar to the scenario of the undesired increase in the hybrid / adhesive layer thickness .
however , this fesem observation is in contrary to el - askary and nassif , whom reported the existence of a gap between the ni and the dentin substrate , when the material was applied according to the manufacturer instructions .
this contradiction could be attributed to the differences in the technique adopted for specimen preparations for fesem examination .
the results also revealed that there was no statistically significant difference between enamel , dentin and cementum substrates with both cgi and rmgi cements .
meanwhile , ni showed significantly lower bond strength values to enamel compared to dentin and cementum .
this result is in partial agreement with fritz et al . , and coutinho et al . , whom found no statistically significant difference between enamel and dentin substrates .
on the other hand , sbs values revealed that ni showed significantly lower bond strength values to enamel compared to dentin and cementum .
this result is in line with fesem photomicrograph , which revealed the existence a gap at ni - enamel interface .
this result is in agreement with uysal et al . , who found a significant decrease in bond strength ni was bonded to the enamel .
compared to the low organic content in the enamel ( 1% by weight ) , the organic content in dentin comprises 20% by weight while cementum comprises 33% by weight .
this might explain the low bonding capacity of ni to enamel . as the high ph of ketac primer , which acts as a mild self - etch adhesive ,
these results are in contrary to korkmaz et al . , whom found that there was no significant difference observed between enamel and dentin at the ni group . in the present investigation , no direct relationship between the shear bond strength and the mode of failure observed .
this substantiates other results in which high bond strength values were not necessarily related to a cohesive type of fracture .
, air voids , cracks , defects or geometric features act as stress raisers when an interface is loaded , which lead to rupture of the joint at stresses very much lower than that which should theoretically occur .
this was confirmed by the fesem micrograph which showed many cracks in the cgi extending to the interface which was also observed with several authors .
the presence of cracks in gi cement might reflect the high percentage of type iii failure present in this group .
this would suggest that the bonding configuration surpassed the inherent strength of the gi strength , denoting that the weak point in this interface is the mechanical properties of the material .
further studies should be carried out to test the effect of the complex oral environmental conditions on the mechanical and chemical adhesion of different gi cement categories to the different tooth substrates .
under the conditions of the present study , the following conclusions could be derived :
cgi and rmgi cement showed equal performance to the different tooth substrates , meanwhile ni showed lower bonding performance to enamel compared to dentin and cementumrmgi cement showed prompt bonding to enamel , dentin and cementum compared to cgi and ni .
cgi and rmgi cement showed equal performance to the different tooth substrates , meanwhile ni showed lower bonding performance to enamel compared to dentin and cementum rmgi cement showed prompt bonding to enamel , dentin and cementum compared to cgi and ni . | objective : this study was conducted to evaluate the microshear bond strength ( sbs ) and ultramorphological characterization of glass ionomer ( gi ) cements ; conventional gi cement ( fuji ix , cgi ) , resin modified gi ( fuji ii lc , rmgi ) and nano - ionomer ( ketac n100 , ni ) to enamel , dentin and cementum substrates.materials and methods : forty - five lower molars were sectioned above the cemento - enamel junction .
the occlusal surfaces were ground flat to obtain enamel and dentin substrates , meanwhile the cervical one - third of the root portion were utilized to evaluate the bonding efficacy to cementum substrate .
each substrate received microcylinders from the three tested materials ; which were applied according to manufacturer instructions .
sbs was assessed using a universal testing machine .
the data were analyzed using two - way analysis of variance ( anova ) and tukey 's post - hoc test .
modes of failure were examined using stereomicroscope at 25 magnification .
interfacial analysis of the bonded specimens was carried out using environmental field emission scanning electron microscope.results:two-way anova revealed that materials , substrates and their interaction had a statistically significant effect on the mean sbs values at p values ; 0.0001 , 0.0108 and 0.0037 respectively .
rmgi showed statistically significant the highest sbs values to all examined tooth substrates .
cgi and rmgi show substrate independent bonding efficiency , meanwhile ; ni showed higher sbs values to dentin and cementum compared to enamel.conclusion:despite technological development of gi materials , mainly the nano - particles use , better results have not been achieved for both investigations , when compared to rmgi , independent of tooth substrate . |
surgical treatment of ossified posterior longitudinal ligament ( opll ) is riddled with confusion and debate .
a number of possible etiological factors have been incriminated that possibly initiate the formation and growth of opll and maturation of clinical symptoms .
the vertical , cross - sectional , and horizontal extents of opll have implications in surgical decision - making .
while a successful clinical outcome may be encouraging , any complication can be devastating for the patient and the family . despite the number of proposed forms of treatment
we recently proposed an alternative form of surgical treatment of opll that involved only multisegmental spinal fixation as the mode of treatment .
this form of treatment was deployed as spinal instability and was identified to be a major or primary factor responsible for the generation of opll . in this presentation
, we identify and analyze the role of atlantoaxial instability in the management of opll .
during the period of november 2014 to august 2015 , we treated seven patients having cervical opll that extended in the high cervical region , up to or above the lower border of c3 vertebra on sagittal magnetic resonance imaging ( mri ) imaging .
all patients were males and their ages were 48 years , 50 years , 53 years , 55 years , 55 years , 63 years , and 65 years .
the presenting clinical symptoms , radiological features , and outcomes are enumerated in tables 1 - 4 . a 5-point clinical grading scale discussed by us recently to assess disability related to craniovertebral junction region myelopathy , the japanese orthopedic association ( joa ) score , and visual analog scale ( vas )
dynamic ( flexion and extension views ) plain radiography , computed tomography ( ct ) scan and mri were done before and after surgery and at follow - up [ figures 1 and 2 ] .
all patients had characteristic multilevel opll that resulted in varying degrees of cord compression that led to crippling symptoms and severe neurological deficits .
the rostral extent of the opll extended at least up to the lower border of c3 vertebra .
as per our recently described classification , we identified that there was type b facetal instability in all cases , meaning thereby that on sagittal imaging the facet of the atlas was dislocated posterior to the facet of axis .
it was not possible to identify radiological instability at any other facets joint in the subaxial spine .
but all joints were functional and active as could be appreciated from the radiological evidences of intact articular cavity and smooth articular cartilage .
table showing the demographics of the patients , types of opll , and number of spinal levels affected distribution as per clinical grading system table showing the preoperative and postoperative clinical assessments as per joa scoring system visual analog scale ( 0 no pain , 10 maximum pain ) images of a 65-year - old male patient .
( a ) sagittal images of ct scan shows multilevel opll ( b ) sagittal images through the facets show type b facetal instability ( c ) t2-weighted mri shows the cord compression and alteration in the cord signal ( d ) postoperative ct scan image showing fixation implant ( e ) lateral plain radiograph showing the implant ( f ) ap view of plain radiograph showing the implant ( g ) postoperative t2-weighted image showing the persistence of cord compression and altered cord signal images of a 55-year - old male .
( a ) sagittal image of ct scan showing multisegmental opll ( b ) sagittal image of ct scan with the cut through the facets showing type b facetal instability ( c ) t2- weighted mri showing multilevel cord compression by the opll ( d ) postoperative ct scan showing the multilevel spinal facetal fixation ( e ) anteroposterior view of the ct scan showing the fixation the patient was placed in the standard prone position with the head held in cervical gardner wells traction that kept the head in a
the surgical position was adopted from our earlier described position used for surgery for atlantoaxial fixation .
traction was deployed essentially to keep the head and neck stable during surgery and the face off the head holder , avoiding any pressure injury to the eyes and facial skin .
midline incision was taken and employing subperiosteal dissection , the articular joints and facets of the cervical spine were exposed widely on both sides .
the spinal segments that were exposed depended on the number of cervical levels involved by opll .
apart from treating the levels of cervical spine involved by opll , direct observation of the facetal articulation and manual manipulation were used to assess the levels of spinal instability .
atlantoaxial fixation was done first and subsequently subaxial spinal fixation was carried out , essentially by similar surgical strategy .
c2 ganglion was resected on both sides in one patient to widely expose the atlantoaxial joint .
the articular capsule of the c1 - 2 joint and subaxial joints was sharply cut and the joints were exposed .
varying sizes of osteotomes were used and were introduced with their flat , sharp edge and then turned 90 in a screwing motion to affect denuding of the articular capsule and cartilage endplate .
c1 - 2 facetal fixation was done employing plate and screw technique described by us earlier in 1988 .
the titanium material screws used for the fixation of facets of both atlas and axis measured 2.8 mm in thickness and ranged 26 - 28 mm in length .
transarticular facet fixation by the technique described earlier by roy - camille was deployed for subaxial spinal stabilization . for the transarticular method of fixation ,
bone graft obtained following sectioning of the adjoining spinous processes and that obtained from the iliac crest was placed over the cut surface of the base of the spinous process and the host bone area of the laminae was appropriately prepared by drilling its outer cortex .
the patient was then advised limited cervical movement using external orthosis for a period of 8 weeks . after this period and confirmation of preliminary evidences of spinal fusion ( that included a stable and satisfactory screw positioning on dynamic imaging ) , all routine activities were permitted .
the patient was placed in the standard prone position with the head held in cervical gardner wells traction that kept the head in a floating position .
the surgical position was adopted from our earlier described position used for surgery for atlantoaxial fixation .
traction was deployed essentially to keep the head and neck stable during surgery and the face off the head holder , avoiding any pressure injury to the eyes and facial skin .
midline incision was taken and employing subperiosteal dissection , the articular joints and facets of the cervical spine were exposed widely on both sides .
the spinal segments that were exposed depended on the number of cervical levels involved by opll .
apart from treating the levels of cervical spine involved by opll , direct observation of the facetal articulation and manual manipulation were used to assess the levels of spinal instability .
atlantoaxial fixation was done first and subsequently subaxial spinal fixation was carried out , essentially by similar surgical strategy .
c2 ganglion was resected on both sides in one patient to widely expose the atlantoaxial joint .
the articular capsule of the c1 - 2 joint and subaxial joints was sharply cut and the joints were exposed .
varying sizes of osteotomes were used and were introduced with their flat , sharp edge and then turned 90 in a screwing motion to affect denuding of the articular capsule and cartilage endplate .
c1 - 2 facetal fixation was done employing plate and screw technique described by us earlier in 1988 .
the titanium material screws used for the fixation of facets of both atlas and axis measured 2.8 mm in thickness and ranged 26 - 28 mm in length .
transarticular facet fixation by the technique described earlier by roy - camille was deployed for subaxial spinal stabilization . for the transarticular method of fixation ,
bone graft obtained following sectioning of the adjoining spinous processes and that obtained from the iliac crest was placed over the cut surface of the base of the spinous process and the host bone area of the laminae was appropriately prepared by drilling its outer cortex .
the patient was then advised limited cervical movement using external orthosis for a period of 8 weeks .
after this period and confirmation of preliminary evidences of spinal fusion ( that included a stable and satisfactory screw positioning on dynamic imaging ) , all routine activities were permitted .
the follow - up period ranged 3 - 12 months , the average being 8 months .
the clinical evaluation included the location and degree of preoperative and postoperative pain and myelopathy symptoms using an analog scale , subjective and objective alterations in sensation and weakness , walking difficulty , and bowel or bladder changes .
all patients improved in symptoms in the immediate postoperative period following surgery . during the period of follow - up ,
all patients underwent evaluation on static and dynamic cervical spine radiographs and ct scanning and mri .
static neutral lateral radiographs were used to assess cervical sagittal balance , whereas anteroposterior radiographs were used to exclude preoperative abnormal coronal alignment [ figure 1 ] .
all patients had loss of cervical lordosis and straightening of the cervical spine before surgery that persisted after the operation .
the lordotic angle was measured using cobb 's method of measurement and there was no significant difference between the preoperative and postoperative values .
fusion of the spinal segment was defined as an absence of all kinds of motion and alterations in the interlaminar and intervertebral body distances on flexion - extension radiographs .
bone formation was additionally observed inside and across the facets and over the laminae . on follow - up
although identified throughout the world , the number of scientific articles from asia outnumbers papers from other continents .
dietary , environmental , and physical body constitution - related factors , apart from a host of other possible etiological factors , have been suggested to be the possible causes . in general , the patients harboring opll are marginally or significantly obese and have a relatively sedate lifestyle .
the devastating nature of complications of surgery makes the treatment of patients having opll one of the most feared surgical issues .
the surgical treatment is difficult to conceptualize essentially because the exact pathogenesis of the abnormal bone formation is unclear .
opll that extends in the high cervical region is particularly more formidable to surgical treatment .
anterior c2 corpectomy , removal of the arch of the atlas and odontoid process , and decompression of the cord by removal of opll in the craniovertebral junction have only rarely been advocated . in general , a majority of the surgeons dealing with lesions at this location have advocated laminectomy or laminoplasty for indirect decompression of the spinal cord in an attempt to increase the volume of the spinal canal and accommodate the additional and uninvited intrusion . there have been a number of reports in the literature that indicate the need of a wide removal of the opll as it has been observed that any treatment that does not remove the primary cause of compression can be wrought with failure and does not stall the progressive and relentless growth of the opll . any surgical treatment that involves removal of the high cervical and craniovertebral junction oplls is not only technically difficult but a wide exposure and radical removal of opll is only rarely possible and has never been demonstrated by imaging . moreover
the search of an ideal surgical treatment of long and multisegmental oplls , particularly those that extend into the high cervical region continue .
bone formation along the ligaments is considered to be an additionally stabilizing factor . as the lesion is considered to be stable , the treatment involves decompression of the cord , removal of the opll , or widening of the spinal canal size by removing bone anterior , posterior , or circumferentially around the cord . the surgical procedures that involve stabilization are aimed to prevent the destabilizing effects of the decompressing bone removal surgery .
the twin operative strategy that involves decompression and fixation , either by anterior or by posterior surgical route , has been generally adopted .
however , despite the surgical experience and expertise of the surgeon , such a surgical procedure and the maneuver of resection of oplls , particularly those that extend into high cervical region , can be tedious , time - consuming , and potentially hazardous .
we had recommended oblique corpectomies for exposure and resection of the opll and had suggested that such a conservative bone removal procedure does not affect the stability of the spine and additional stabilization procedures can be avoided .
craniovertebral junction instability has been identified to be associated with secondary effects on the cervical spine and even in the rest of the spine .
short neck , torticollis , and bone fusion have been associated with craniovertebral junction instability .
we recently identified the association of facetal instability with longstanding or relentlessly progressive pathologic phenomenon at the craniovertebral junction of chiari 1 malformation and syringomyelia .
we proposed that chiari 1 malformation could simulate nature 's air bag that is positioned in place to provide a cushion for critical neural structures and avoid their pinching between bones in the event of potential or manifest instability .
we also identified that facetal instability can be associated with group b basilar invagination . in this group of basilar invagination , there are evidences of longstanding and progressive pathological phenomenon .
we recently identified an association of atlantoaxial facetal instability in cases with multilevel cervical spondylotic disease and recommended atlantoaxial fixation in such cases .
our literature search did not reveal any article that related craniovertebral instability to cervical opll .
conventionally , instability at the craniovertebral junction has been identified by alteration in the atlantodental interval .
we recently identified facetal malalignment as an important measure of instability at the craniovertebral junction .
facetal instability may or may not be associated with odontoid process - related cord compression or symptoms . as cord compression is not a prominent or major issue , the symptoms related to facetal instability can be subtle and longstanding .
although there is no morphological study that evaluates the relationship of the facets of the atlas and axis during dynamic movements , it does seem that some degree of facetal malalignment can be within the range of normal variation . in the presented series of patients
apart from instability observed on imaging , direct intraoperative observations confirmed the presence of instability .
the role of spinal instability in the generation and growth of opll has been only been infrequently discussed in the literature .
we identified that stabilization of the spinal segments alone without any form of bone or soft tissue decompression can have a defining role in the treatment of opll .
identification of subaxial facetal instability on radiological imaging is difficult due to their oblique profile .
however , facetal instability at the atlantoaxial joint can be identified relatively easily due to their more horizontal lie and rectangular box - like alignment . on the basis of our 30-year experience in dealing with the atlantoaxial instability and with direct handling of the facets of the atlas and axis , we realize that the atlantoaxial joint in cases with opll that extends into the high cervical region is unstable .
this fact is also suggested by the presence of facetal malalignment on imaging and by the presence of an active and functioning joint with well - defined articular surface despite the multilevel presence of additional abnormal bone of opll . additionally , we observed instability of spinal segments involved by opll by direct observation of the status of articular cavity and facets and direct manual handling and manipulations .
however , as the scale is not validated as yet , we additionally used the standard and recognized joa score .
our satisfactory clinical outcome also reinforces our proposed concept that atlantoaxial instability may have a defining role in the generation and progression of oplls in general and particularly those that extend into the high cervical region .
it also appears that facetal malalignment on imaging may not be the only criterion to detect atlantoaxial instability . in type c facetal instability ,
although the procedure of atlantoaxial fixation is technically demanding , it is relatively simpler when compared to other possible surgical options in such cases .
our surgical outcome analysis suggests that missing or ignoring the presence of atlantoaxial facetal instability can be an important cause of suboptimal result or failure of surgery for high cervical opll . in all our seven cases ,
atlantoaxial fixation was done in addition to other cervical level fixation . from the study , it is unclear if fixation of other spinal segment could have been avoided .
the contribution of atlantoaxial dislocation in the overall spinal instability is also difficult to assess .
the negative aspect of the study is that the surgical decision - making had a subjective influence that was based on the personal operative experience of manual handling of facets of the spine .
although the total study period and duration of follow - up are relatively small , the remarkable clinical recovery following the treatment has prompted the reporting of observations .
it appears that multilevel spinal stabilization may have a leading role to play in the treatment of opll .
atlantoaxial fixation may be done is all cases where the opll extends into the high cervical region , up to or superior to the inferior border of c3 .
atlantoaxial fixation should be done irrespective of the presence or absence of facetal malalignment on imaging .
| background : multilevel ossified posterior longitudinal ligaments ( oplls ) , particularly those that extend into the high cervical region , are formidable and challenging surgical problems . the aim of the presentation is to analyze the results of surgical treatment of seven consecutive patients having high cervical opll with atlantoaxial and subaxial facetal fixations.objectives:we analyze the role of atlantoaxial instability in the management of opll that extended into the high cervical region , above the lower border of c3 vertebra.materials and methods : all patients in the series were males .
the age of the patients ranged 48 - 65 years .
clinical evaluation was done by a 5-point clinical grading scale described by us , japanese orthopedic association ( joa ) score , and visual analog scale ( vas ) .
all patients were identified to have relatively subtle but definite atlantoaxial facetal instability on sagittal imaging and the instability was confirmed by direct handling of the facets during surgery .
all patients were treated by multilevel facetal fixation that included fixation of atlantoaxial facets .
the aim of surgery was stabilization and arthrodesis of the involved spinal segments , as instability was considered to be the prime pathogenetic factor of opll .
spinal canal decompression , either by anterior corpectomy or discoidectomy or by posterior laminectomy or laminoplasty was not done and no attempts were made to remove the opll . at an average follow - up of 8 months , all patients showed progressive symptomatic recovery.conclusion:atlantoaxial facetal instability can be a cause or an association of high cervical opll .
stabilization of the atlantoaxial joint forms a remarkably effective method of treatment . |
recent advances made in nanotechnology and in molecular and biomolecular chemistry have resulted in the design of supramolecular and biologically inspired systems capable of showing inventive related functions that fuel areas such as bioengineering , biosensing , and bionanotechnology into new horizons.1 in this context , some new systems are based on the use of hybrid materials , obtained by anchoring organic molecules or supermolecules to certain inorganic scaffoldings.2 in this field , one appealing application is the development of gated nanodevices for controlled delivery .
in fact the development of stimuli - responsive nanoscopic hybrid gated materials , able to release an entrapped cargo when external stimuli are applied , has attracted much attention.3 these gated materials usually contain a switchable gate - like ensemble capable of being opened or
closed when certain external stimuli are applied , as well as a suitable inorganic support that acts as a nanocontainer ( for loading the carrier ) . for the support
, different - sized mesoporous silicas have been selected and used as inorganic scaffolds in gated ensembles thanks to their suitable properties.4 in particular , silica mesoporous supports ( smss ) can be obtained in different sizes , from micrometric to nanometric , with tailor - made homogeneous pores of around 2 - 10 nm .
they also show high inertness , are easy to functionalize using well - known chemistries , and have a very large specific surface area and specific volume , and therefore have remarkable load capacity.5 while dealing with the gate - like ensemble , smss have been functionalized with a large collection of switchable molecular , supramolecular and biomolecular pore - capping ensembles to develop gated particles capable of showing a zero release of cargo and of delivering it upon the application of specific chemical , physical or biochemical stimuli.6 as stated above , these capped materials have been used mainly in drug delivery applications . in contrast , very few examples of their use in sensing protocols are available .
the core concept for applying smss into sensing protocols is to load the support with a reporter and to design the capping mechanism in such a way that a target analyte is able to selectively trigger uncapping and the delivery of the cargo ( a reporter).7 this new sensing paradigm conceptually differs from classic supramolecular binding site signaling subunit systems because the new protocol disconnects the recognition step from the signaling event , which therefore makes signaling independent of the host
for instance , it has been reported that the presence of relatively few molecules of a certain analyte may induce the release of a relatively large quantity of entrapped dye molecules.9 this sensing approach using gated systems offers great potential for the preparation of new sensing systems with enhanced features compared with classical signaling probes .
in particular , it is possible to select , with minimum effort , different porous supports , a large range of gate - like systems , and a number of reporters which display chromogenic , fluorogenic , or electrochemical responses . when using gated concepts for sensing , two possible situations are envisioned ( see figure 1 ) . in one
, pores are open and the reporter is delivered to the solution , whereas in the presence of a given analyte , this molecule can bind to receptors on the external surface of the sms and close the gate . as capping of the mesoporous support
is selectively achieved in the presence of a target analyte , the design of a probe can be envisioned . in the second approach ,
the starting material is capped , and the presence of a target guest induces pore opening and dye delivery due to competitive binding .
of both these approaches , the second is perhaps the most interesting because it is able to show a signal when the reporter is released ( i.e. off on behavior ) , which is generally easier to measure than dye delivery inhibition ( i.e. on off behavior ) .
however , examples of the first approach were reported first , whereas examples of the second have been reported more recently.7 scheme of the recognition paradigm using nanoscopic gate - like scaffoldings : a ) inhibition of dye release due to analyte coordination with grafted binding sites ; b ) uncapping of the pores by an analyte - induced displacement reaction .
this review aims to act as a complete compilation of published examples of the design of probes based on capped silica mesoporous supports .
examples of stochastic sensors ( based on nanometer - sized pores in insulating membranes),10 simple probes based on dyes on silica mesoporous supports ( using uncapped systems),11 or electrochemical sensors constructed with mesoporous membranes12 are not reported herein .
the examples detailed below showed capped smss with applications in recognition protocols divided into four main sections : sensing of anions , sensing of cations , sensing of small neutral molecules , and sensing of biomolecules .
our aim was to also include capped smss that could be opened in the presence of certain molecules and which , in our opinion , may find use in sensing , even though they were designed for a different application in the original work .
as far as we know , the first time it was suggested that gated smss could be used for sensing applications was reported by our group in 2006 , and it was based on the on off behavior shown in figure 1 a. the system consisted of an sms loaded with ru(bipy)3 dye as the reporter and functionalized on the external surface with 3-[2-(2-aminoethylamino)ethylamino]propyl trimethoxysilane . at a neutral ph , in which the experiments were carried out , the gate was open , and delivery of the ruthenium complex was detected chromo - fluorogenically .
however , presence of adenosine triphosphate ( atp ) , and of adenosine diphosphate ( adp ) to a lesser extent , allowed the selective inhibition of indicator delivery by the formation of strong complexes between tethered polyamines and atp anions through hydrogen bonding and electrostatic interactions ( figure 2 ) .
the system presented good selectivity for atp in the presence of other anions , such as chloride , sulfate , or guanosine monophosphate ( gmp ) , which were either too small or formed complexes that were too weak to effectively cap pores.13 in a complementary study , the same authors studied the behavior of a similar amine - functionalized material obtained by anchoring suitable polyamines to pore outlets of the sms loaded with ru(bipy)3.14 a ph - driven open / close mechanism was observed that emerged from a hydrogen - bonding interaction between amines at a neutral ph ( open gate ) and coulombic repulsions at an acidic ph between closely located polyammoniums at pore openings ( closed gate ) .
in addition to the ph - driven protocol , the opening / closing of the gate - like ensemble could also be modulated by an anion - controlled mechanism .
choice of a certain anionic guest resulted in a different gate - like ensemble behavior , which ranged from basically no action ( chloride ) to complete ( atp ) or partial pore blockage , depending on ph ( sulfate and phosphate ) .
the authors explained the remarkable anion - controllable response of the gate - like ensemble in terms of anion complex formation with tethered polyamines .
micrometric silica mesoporous support functionalized with polyamines for the detection of atp . in another work , which also used an on off behavior ,
as shown in figure 1 a , the authors developed a hybrid sms capable of chromo - fluorogenically detecting long - chain carboxylates by using mesoporous silica microparticles loaded with ru(bipy)3 and functionalized on the external surface with imidazolium binding sites.15 the release profile of the entrapped dye from the described material was tested in aqueous media at a neutral ph in the presence of different linear carboxylates ( ch3(ch2)nco2 , n=0 , 2 , 4 , 6 , 8 , and 10 ) .
it was found that short carboxylates , such as acetate , butanoate , hexanoate , and octanoate , were unable to induce pore blockage , whereas presence of larger carboxylates , such as decanoate and dodecanoate inhibited dye release , most likely due to the electrostatic interaction of these carboxylates with the imidazolium binding sites on the surface and the formation of a dense hydrophobic monolayer around pore outlets ( see figure 3 ) . in particular , dodecanoate completely inhibited dye delivery at the millimolar level . in a similar study ,
the same authors16 developed new sensing materials , also for the optical detection of long - chain carboxylates , based on the use of smss loaded with ru(bipy)3 and functionalized on the external surface with thiourea or urea binding sites .
the release profile of the entrapped dye from these materials was also tested in aqueous media in the presence of different linear carboxylates ( ch3(ch2)nco2 , n=0 , 2 , 4 , 6 , 8 , and 10 ) . in general , a similar result to that found for the solid functionalized with imidazolium was observed .
micrometric silica mesoporous support functionalized with imidazolium binding sites for the detection of long - chain carboxylates .
a final example , based on the open closed protocol shown in figure 1 a for anion sensing , was developed for the detection of borate . using the well - known reaction between polyalcohols and borate anion to form boronate esters
, the authors developed a mesoporous mobil composition of matter 41 ( mcm-41 ) support loaded with ru(bipy)3 dye and functionalized with a saccharide derivative on the external surface.17 presence of borate ( ph 7.0 ) induced pore closure , which inhibited dye delivery due to the formation of boroesters through the reaction of borate with the hydroxyl moieties of the anchored saccharides ( figure 4 ) .
the control of mass transport by borate anion was very selective ; for instance , other anions ( e.g. co3 , so4 , cl , br , no3 , po4 ) or cations ( e.g. aluminum , copper , iron , sodium , potassium , and calcium ions ) showed no effect . based on this simple protocol , a limit of detection ( lod ) for borate of about 70 ppb in water ( hepes , ph 7.0 ) was achieved .
a common characteristic of the above examples is the use of uncapped materials , which were selectively capped in the presence of certain anions by coordination of the anion with relatively small binding sites attached to the external surface of smss , which generally resulted in an on off sensing protocol .
in contrast to this approach , in an off on sensing systems it is normally necessary to use large molecules to allow to prepare capped materials , which could be opened in the presence of target analytes . one method to achieve this goal used aptamers .
an example of detection of anions using this protocol was reported in 2011 , by zalp and co - workers , who paid attention to the use of aptamers to develop an atp - responsive gated material ( see figure 5).18 in their work , they used an amino - terminated oligonucleotide sequence ( 5-cac ctg ggg gag tat tgc gga gga agg ttc cag gtg - nh2 - 3 ) , which contained the well - known atp aptamer ( 5-cac ctg ggg gag tat tgc gga gga agg tt-3 ) , and a short extra fragment ( 5-cca ggt g - nh2 - 3 ) to induce a hairpin - like structure that blocked pores .
an sms in the form of nanoparticles , loaded with fluorescein and functionalized on the external surface with sulfhydryl groups , was prepared and the amino functionalized aptamer was covalently attached using sulfo - n - succinimidyl 4-maleimidobutyrate as a crosslinker .
these authors observed that the hairpin aptamer blocked pores , while presence of atp triggered the delivery of the entrapped fluorescein dye .
this was explained by atp binding , which induced a conformational change from a duplex to a single - stranded dna near the aptamer region , which was close to the sms surface and resulted in fluorescein release .
these authors also found that nanoparticles capped with a mutated hairpin did not respond to atp , and that guanosine 5-triphosphate ( gtp ) was also unable to trigger fluorescein release .
later the same authors extended their previous work by focusing on monitoring the performance of aptamer - gated smss using circular dichroism.19 in this new study , the same atp aptamer as that described above was employed , but in this case a different hairpin - forming extension was used , which resulted in the final capping sequence 5-cac ctg ggg gag tat tgc
gga gga agg ttt ttt tcc agg tg-(ch2)6-nh2 - 3. nanometric silica mesoporous support loaded with fluorescein and capped with atp aptamers for the detection of atp .
wang and co - workers also developed atp - selective delivery systems using silica mesoporous nanoparticles capped with the same atp aptamer,20 but based on a different configuration of the gated ensemble to that used by zalp et al .
in particular , these authors functionalized smss with 3-cloropropyltrimethoxysilane and then transformed the chloride atom of the attached organic groups into an azide moiety by a reaction with sodium azide . on the other hand ,
the atp aptamer was hybridized with two different single - stranded dna ( ssdna ) sequences ( e.g. 5-alkyne - ttc ctc cgc a-3 and 5-alkyne - ata ctc cc-3 ) yielding a sandwich - type dna structure .
the pores of the inorganic scaffold , functionalized with azide moieties , were loaded with ru(bipy)3 , and then the system was capped with the sandwich - type dna structure containing the atp aptamer sequence through a click chemistry reaction between the azide group in the solid and alkyne groups in the dna ensemble .
this allowed pores to be blocked , which inhibited dye release ( figure 6 ) .
cargo delivery was studied in tris - hcl buffer at different atp concentrations , from 0 to 20 mm , and was monitored by fluorescence spectroscopy .
the atp - induced response was attributed to the competitive displacement of the atp aptamer from the sandwich - type dna via the formation of the corresponding atp - aptamer complex .
the ru(bipy)3 reporter release was observed only when atp was present , whereas cytosine 5-triphosphate ( ctp ) , gtp , and uridine 5-triphosphate ( utp ) at 20 mm were unable to induce dye delivery .
nanometric silica mesoporous support loaded with ru(bipy)3 and capped with atp aptamers for the detection of atp .
yang and co - workers developed atp - responsive gated materials using aptamer - containing gold nanoparticles ( aunps ) as caps ( figure 7).21 in their work , these authors functionalized the external surface of an sms with amino groups and , in further steps they anchored adenosine-5-carboxylic acid moieties ( via an amidation reaction ) and loaded the porous network of the support with fluorescein isothiocyanate ( fitc ) . on the other hand , aunps derivatized with atp aptamer 5-cct ggg gga gta ttg cgg agg aag gtt - sh-3 by forming an aus bond
the final sensory material was obtained by capping the loaded support pores with aptamer - functionalized aunps .
phosphate - buffered saline ( pbs , ph 7.4 ) suspensions of the capped solid showed negligible dye leaching , whereas presence of atp triggered the release of the fitc reporter .
the authors also found that the amount of released fitc was dependent on atp concentration ( with a maximum dye release at the 8 mm atp concentration ) .
the uncapping process was ascribed to a competitive displacement reaction of aunps from the solid surface upon the coordination of atp with the grafted aptamer .
finally , selectivity of the system using atp analogs , such as ctp , gtp , and utp , was studied .
these authors also examined the uncapping ability of an oligonucleotide complementary to the atp aptamer and a random sequence . in this case , they found remarkable dye release with the complementary sequence , which was even better than with atp given its higher binding constant ( kd oligonucleotide : 1 nm ; kd atp : 0.70.8 m ) , whereas presence of the random sequence induced negligible dye delivery .
nanometric silica mesoporous support loaded with fluorescein isothiocyanate and capped with atp aptamer containing gold nanoparticles ( aunps ) for the detection of atp .
tang and co - workers recently developed an sms capped with aunps to detect atp by using a conventional glucometer readout and the same selective atp aptamer as that used above by zalp , wang , and yang ( see figure 8).22 for this purpose , the authors functionalized the external surface of silica mesoporous nanoparticles with aminopropyl moieties and then linked a single - stranded sequence ( e.g. 5-nh2-ttt tta cct tcc tcc gca a-3 , dna1 ) to amino groups using glutaraldehyde as linker .
, aunps were coated with another single - stranded sequence ( 5-tac tcc ccc agg ttt ttt - sh-3 , dna2 ) .
both dna1 ( grafted on the outer surface of the mesoporous support and dna2 ( used as a coating of aunps ) are complementary to the adjacent areas of the atp aptamer sequence . in the presence of the atp aptamer , immobilized dna1 and dna2 hybridized ( and formed a three - stranded complex ) , with the subsequent pore capping by bulky aunps . buffered suspensions of the capped system at ph 7.3 showed no entrapped glucose release .
however in the presence of atp , glucose release was clearly detected and was measured by a commercially available glucometer .
the observed response was due to atp binding with the aptamer , which resulted in a separation of aunps from the sms and cargo delivery .
the capped material was able to detect atp within a linear range from 0.01 to 0.8 mm with an lod of 8 m .
presence of other nucleotides , such as ctp , gtp , and utp , was unable to uncap pores .
a similar strategy was adopted to detect cocaine ( linear range from 0 to 1 mm ) .
a ) nanometric silica mesoporous support loaded with glucose and capped with atp aptamer containing aunps for the detection of atp .
b ) selectivity release profile for the sensing material triggered by atp , ctp , gtp and udp ( 0.1 mm after 6 h ) . pgm = personal glucometer .
capped mesoporous silica nanoparticles designed for the detection of small cations are scarce . as far as we know
, the first example reported was designed to sense the presence of cation ch3hg.23 this was , as far as we can tell , also the first example to have used an off on approach based on the general paradigm shown in figure 1 b. the gated material consisted of a mesoporous support loaded with dye safranin o and capped with 2,4-bis-(4-dialkylaminophenyl)-3-hydroxy-4-alkylsulfanylcyclobut-2-one ( apc ) groups .
apc moieties were readily formed by the reaction of a squaraine dye and thiol units , which were previously anchored to the external surface of the mesoporous support .
when ch3hg was added to the acetonitrile : toluene 4:1 v / v suspension of the apc - capped support , safranin o release was observed .
this uncapping process derived from the reaction of methylmercury with the thiol group on apc moieties , which resulted in the coordination of the cation to thiols and , in both , the release of the bulky squaraine chromophore and the delivery of the entrapped safranin o reporter ( see figure 9 ) .
the chromogenic indication reaction allowed the detection of ch3hg down to 0.5 ppm , whereas the use of standard fluorometric methods reduced the lod to below 2 ppb .
experiments were done in acetonitrile : toluene 4:1 v / v mixtures to achieve discrimination from hg given its low solubility in this medium .
this procedure was successfully tested to optically determine methylmercury in fish samples by a simple extraction procedure with toluene and ch3hg detection with the apc - capped solid .
these real fish samples were also spiked with cations na , k , ca , mg , cu , ni , zn , ag , pd , cd , au , and tl and various organic species , for example , sodium lauryl sulfate , cysteine , histamine , ethanol , heptylamine , and hexanethiol . however , none of these species affected the response of the capped material to ch3hg .
micrometric silica mesoporous support loaded with safranine and capped with 2,4-bis-(4-dialkylaminophenyl)-3-hydroxy-4-alkylsulfanylcyclobut-2-one ( apc ) groups for the detection of methylmercury .
more recently , tan , zhang , and co - workers proposed a mercury probe based on a dna - capped sms . mcm-41-type
amino - modified oligonucleotide 5-gaa gaa caa caa aaa - nh2 - 3 was then reacted with the isocyanate groups . a second larger strand ( 5-gtt gtt ctt cct ttg ttt ccc ctt tct ttg gtt gtt cttc-3 ) , complementary to
this second oligonucleotide strand was very rich in thymine groups and showed a high affinity to hg .
in presence of this ion , the authors found that the strand was displaced from the surface to form the corresponding hg aptamer complex , which resulted in dye delivery ( figure 10 ) .
quantification studies , which measured the delivered rhodamine dye fluorescence , showed that the system was able to detect hg in water with a lod of 4 ppb .
the system was selective and no important cargo delivery was observed in the presence of other cations , for example , ni , pd , fe , fe , ba , zn , ca , mg , cu , co , and cd.24 a ) nanometric silica mesoporous support loaded with rhodamine and capped with aptamers for the detection of hg cations .
b ) emission intensity in the presence of increasing quantities of hg ( inset : linear fit of the fluorescence signal ) .
copyright 2012 , american chemical society ) . in an independent work wen , song , and
co - workers developed smss capped with a k - selective aptamer and demonstrated that cargo delivery can be achieved when this cation is present.25 however , these authors did not specifically carry out sensing studies , but used the system to demonstrate the potential use of this aptamer for the design of logic gate systems .
reported the design of capped silica mesoporous nanoparticles capable of being opened in the presence of k using macrocycles.26 these authors used a sms in the form of nanoparticles , which was loaded with curcumin dye and anchored a 18-crown-6 derivative to the external surface .
nanoparticles were finally capped upon the addition of the cs cation , which formed sandwich complexes with the grafted 18-crown-6 moieties .
the systems showed a zero release until the k cation was added , which induced the release of the entrapped curcumin .
the uncapping protocol was due to the cs exchange , owing to the formation of 1:1 macrocycle - k complexes with a higher stability constant ( see figure 1 ) .
however the authors did not specifically use this system to sense applications , and no opening studies of macrocycle - capped nanoparticles with other metal cations were carried out .
nevertheless this study suggested the potential use of macrocycle - capped smss for sensing specific metal cations .
nanometric silica mesoporous support loaded with curcumin and capped with cs-18-crown-6 complexes for the detection of k cations .
metal - ion - dependent catalytic nucleic acids ( dnazymes ) were used as caps by willner et al . to prepare gated smss , which were selectively opened in the presence of cations mg and uo2 ( see figure 2).27 to prepare the mg - selective capped material , mesoporous silica nanoparticles were selected as an inorganic scaffold . in a first step , nanoparticles were functionalized with aminopropyl moieties .
then the amino groups reacted with a crosslinker agent , which yielded maleimide moieties on the external surface .
afterward , a thiolated ribonuclease , containing two complementary single - stranded dna sequences ( 5-hs-(ch2)6-cag tga att ragg aca tag aag aag aag-3 ) of mg - dependent dnazyme , was linked . in order to prepare the final sensing material ,
the pores were loaded with methylene blue and were then capped by the addition of the mg - dependent dnazyme sequence 5-ctt ctt ctt cta tgt cag cga ttc cgg aac gga cac cca tgt att cac tg-3 , through hybridization with the previously linked dna sequence .
the same experimental procedure was used to prepare the uo2-selective capped material , but thionine was employed as the loading molecule instead of methylene blue , and the pores capped with the uo2-dependent dnazyme sequence 5-ctt ctt ctt
cta tgt cag ccg gaa cgg cct tgc aat tca ctg-3. aqueous suspensions at ph 7.2 of mg - selective capped nanoparticles showed negligible methylene blue release in the absence of mg , whereas remarkable dye release was observed in the presence of this cation . the observed release was ascribed to the coordination of a mg cation with linked dnazyme , which induced cleavage of caps .
the optical response was highly selective since the other divalent cations tested ( zn , pb , ca , sr , ba , cu , co , mn , ni , fe , hg ) were unable to induce pore opening and subsequent dye release . the same behavior , but at ph 5.2 , and in the presence of cation uo2 , was observed with the nanoparticles capped with the uo2-dependent dnazyme sequence .
nanometric silica mesoporous support loaded with methylene blue or methionine and capped with dnazyme for the detection of mg and uo2 cations .
the same authors used dnazymes as caps to prepare two materials which were opened in the presence of mg and zn cations.28 mesoporous silica nanoparticles were loaded with methylene blue or thionin and then functionalized with two different thiolated single strands of dna ( 5-ctt ctt ctt cta tgt cag cga tcc gga acg gca ccc atg ttg ttg ttg-3 for mg - dependent dnazyme and 5-ctt ctt ctt cta tgt ctc cga gcc ggt cga aat gtt gtt g-3 for zn - dependent dnazyme ) , using n - - maleimidicaproyl - oxosulfosuccinimide ester as crosslinker .
the capped materials were able to release the entrapped dye in the presence of mg and zn cations , respectively , whereas other divalent metal cations ( pb , ca , sr , ba , cu , co , mn , ni , fe , and hg ) were unable to induce cargo delivery .
a similar approach was adopted by tang and co - workers to prepare dnazyme - capped mesoporous silica nanoparticles , which were selectively opened in the presence of cation pb.29 accordingly , silica mesoporous nanoparticles were selected as inorganic support and the external surface was functionalized with 3-glycidyloxipropyl trimethoxysilane . then by an epoxy amino reaction , pb - dnazyme ( 5-nh2-ttt cat ctc ttc tcc gag ccg gtc gaa ata gtg agt-3 )
finally , the system was capped by the addition of a single - stranded dna sequence ( 5-act cac tat ragg aag aga tg-3 ) that hybridized with the grafted dnazyme sequence ( figure 3 ) .
pbs ( ph 7.4 ) suspensions of the capped nanoparticles showed negligible glucose release , whereas remarkable glucose delivery took place upon the addition of cation pb , measured by a personal glucometer .
cargo delivery was related to the cleavage of caps upon pb coordination with the linked dnazyme .
the response was highly selective , and other cations tested ( cu , co , cd , mg , zn , fe , ag , and hg ) were unable to induce glucose release .
nanometric silica mesoporous support loaded with glucose and capped with dnazyme for the detection of pb cations .
lu and co - workers developed a gated sms that was able to deliver an entrapped reporter using glucose as a trigger.30 mesoporous silica nanoparticles were selected as the inorganic scaffold , and their external surface was functionalized with prop-2-yn-1-yl(3-(triethoxyxilyl)propyl)carbamate . following this functionalization ,
then pores were loaded with rhodamine b and capped by the addition of the glucose oxidase enzyme ( gox ) through the formation of a complex with the grafted inhibitor ( see figure 4 ) .
pbs suspensions of the capped material showed negligible rhodamine b release , whereas the addition of glucose induced marked dye release .
the observed release , which was proportional to the amount of glucose added , was the result of a displacement reaction of gox from the pore outlets due to the formation of the corresponding glucose
the uncapping protocol was highly selective and the authors confirmed that other tested monosaccharides ( i.e. fructose , mannose , and galactose ) were unable to induce any rhodamine b release .
a ) nanometric silica mesoporous support loaded with rhodamine b and capped with glucose oxidase enzyme ( gox ) for the detection of d - glucose .
b ) selectivity release profile for the capped nanoparticles triggered by saccharides ( 1 mm glucose , 10 mm others ) . ( reproduced with permission from ref . 30 .
villalonga and co - workers developed a new gated sms in which an enzyme also acted as a cap ( figure 5 ) .
however in this case , the uncapping process was triggered by the product obtained by the enzymes activity on glucose.31 in their work , the authors loaded silica mesoporous nanoparticles with reporter ru(bipy)3 and functionalized the external surface with 3-iodopropyltrimethoxysilane , which was transformed into 1-propyl-1-h - benzimidazole moieties via a nucleophilic substitution reaction using benzimidazole .
mesopores were then capped with active cyclodextrin - modified glucose oxidase ( cd - gox ) through the formation of an inclusion complex between the cyclodextrins and the propylbenzymidazole groups anchored to the solid support . in their study
, these authors confirmed that dye delivery was induced when glucose was present in the solution due to the displacement of cd - gox as a result of the cd - gox - induced oxidation of glucose to gluconic acid , which induced the subsequent protonation of the benzimidazole groups .
the authors found a linear glucose response within the 110110 m range and a lod of 1.510 m which fell within the range of other glucose detection systems .
the capped material was also tested in the presence of others saccharides , such as mannose , fructose , galactose , maltose , and saccharose , at the 110 mol l concentration ; however , no cargo delivery was observed .
nanometric silica mesoporous support loaded with ru(bipy)3 and capped with cyclodextrin - modified glucose oxidase ( cd - gox ) for the detection of d - glucose . in another work,32 the same authors demonstrated that the gating mechanism and different effector ensembles can be integrated into a single system based on the use of janus - type nanoparticles with opposing gold and mesoporous silica faces ( see figure 6 ) .
in particular , the porous network of the silica face was loaded with ru(bipy)3 and the external surface was grafted with 3-(2-aminoethylamino)propyltrimethoxysilane . aside from this , the aunp side was functionalized with thiol - modified urease enzyme .
aqueous solutions ( acetate buffer ph 5.0 ) of the janus nanoparticles showed negligible dye release , because polyamines were protonated , and the molecular gate was closed . in the presence of urea , delivery of the entrapped complex was marked .
this release resulted from the urease - catalyzed hydrolysis of urea into co2 and nh3 , which induced an increase in the local environments ph . with this increase , polyamines became deprotonated , and gates subsequently opened .
silica mesoporous nanoparticle support loaded with ru(bipy)3 and capped with polyamines for the detection of urea .
another gated system using janus - type nanoparticles has been recently described.33 the authors loaded the mesoporous silica face of janus nanoparticles with ru(bipy)3 , and 3-iodopropyltrimethoxysilane was grafted onto the external surface . through a nucleophilic substitution reaction , benzimidazole moieties were attached to anchored 3-iodopropyl residues , which resulted in 1-propyl-1h - benzimidazole groups .
the pores were then blocked by the formation of inclusion complexes between -cd moieties and the propylbenzimidazole groups .
experimental results confirmed that the presence of either d - glucose or ethyl butyrate , or a combination of both , brought about a drop in ph locally through an enzyme - catalyzed substrate conversion into gluconic acid or butyric acid , which induced the opening of the -cd - gated nanovalves and entrapped dye release ( see figure 7 ) .
silica mesoporous nanoparticle support loaded with ru(bipy)3 and capped with -cd for the detection of d - glucose and ethyl butyrate .
kim and co - workers developed an sms that was selectively opened in the presence of fructose and galactose .
mesoporous silica nanoparticles were grafted with amine moieties and coupled with p - dihydroxyborylbenzoic acid .
hybrid material was loaded with calcein , and the boronic acid units on the surface were indistinctly coordinated to - , - , or -cd ( figure 8) .
fluorescence spectrometry measurements showed clear dye delivery in the presence of fructose and galactose , whereas no delivery for mannose or glucose occurred .
probe uncapping was explained by the displacement of bulky cyclodextrins by the competitive interaction of monosaccharides with boronic acid moieties.34 it is also worth mentioning that , despite not having been designed specifically for sensing purposes , other authors have developed selective glucose - responsive systems , and have used them for the glucose - induced release of insulin , which was related directly with the amount of glucose present in media.35,36 nanometric silica mesoporous support loaded with calcein and capped with - , - or -cd for the detection of fructose or galactose . martnez mez et al37 reported the design of a capped sms for the selective chromo - fluorogenic detection of glutathione ( gsh ) in both pure water and human serum ( see figure 9 ) .
this system consisted of mesoporous silica nanoparticles , which were first loaded with safranin o as a reporter .
then the external surface was functionalized with ( 3-mercaptopropyl)trimethoxysilane , and the thiol - functionalized solid was further reacted with 2,2-dipyridyl disulfide .
the signaling mechanism was based on a selective gsh - induced reduction of the disulfide bond , which resulted in pore opening and entrapped dye release . a correlation between the delivered dye and the amount of glutathione
these authors obtained a lod of 0.1 m for glutathione in both pure water and human serum .
the gated system was demonstrated to be highly selective for glutathione against similar sulfur - containing derivatives ( cysteine , homocysteine , s - methyl cysteine , hs ) , anions ( f , br , cl , i , cn , oh , hpo4 , aco , citrate , n3 , no3 , so3 , so4 , and s2o4 ) , and oxidants ( h2o2 ) .
nanometric silica mesoporous support loaded with safranin o and capped with poly(ethylene glycol ) for the detection of glutathione ( gsh ) .
the same group reported a family of capped sms for the fluorimetric sensing of nitroaromatic explosives ( figure 20 a).38 in a first example , the authors designed an sms loaded with ru(bipy)3 as a fluorophore and functionalized on the surface with 3-(azidopropyl ) triethoxysilane groups .
afterward , a suitable pyrene derivative was attached to azido moieties by a copper(i)-catalyzed huisgen azide / alkyne 1,3-dipolar cycloaddition click reaction , which led to the formation of a 1,2,3-triazole heterocycle and yielded the final sensing material .
pores were blocked by the presence of a dense network of bulky pyrene moieties on the outer surface . as a result , dye delivery in acetonitrile was inhibited .
presence of nitroaromatic explosives 2,4,6-trinitrophenylmethylnitramine ( tetryl ) and 2,4,6-trinitrotoluene ( tnt ) induced the formation of pyrene
nitroaromatic complexes , which pushed apart the bulky pyrene from pore voids to thus unblock pores , and allowed dye release .
lods of 1.4 and 11.4 ppm for tetryl and tnt , respectively , were calculated by fluorogenic titration curves .
in addition , a lod was also calculated from the nitroaromatic - induced quenching of pyrene emission . with this procedure ,
finally , selectivity studies performed in the presence of other aromatic derivatives ( e.g. 2,4-dinitrotoluene , n - methylaniline , 2-nitrotoluene , and nitrobenzene ) , nonaromatic explosives ( e.g. hexahydro-1,3,5-trinitro-1,3,5-triazine and pentaerythritol tetranitrate ) , methylene blue , and naphthalene were carried out .
these studies showed that 2,4-dinitrotoluene ( dnt ) was also able to induce partial entrapped - fluorophore release , but to a lesser extent .
micrometric silica mesoporous support loaded with ru(bipy)3 and capped with pyrene or tetrathiafulvalene ( ttf ) derivatives for the detection of nitroaromatic explosives . in a later work,39 the same authors reported the use of a tetrathiafulvalene ( ttf)-capped sms for the detection of certain nitroaromatic explosives ( figure 20 b ) .
the sms was loaded with ru(bipy)3 as the reporter and the ttf derivative was anchored to the surface by a copper(i)-catalyzed huisgen azide / alkyne 1,3-dipolar cycloaddition click reaction .
release studies into acetonitrile revealed that presence of tetryl , tnt , and to a lesser extent of 1,3,5-trinitrobenzene ( tnb ) , induced pore opening and dye release . from uv - vis titration studies , lods of 28 m ( 8 ppm ) and 66 m ( 15 ppm ) for tetryl and tnt , respectively , were calculated . with fluorescence measurements , lods of 3.5 m ( 1 ppm ) and 26 m ( 6 ppm ) for tetryl and tnt , respectively , were found .
these authors also reported a similar ttf - capped sms , but in this case they used different ttf derivatives of varying sizes and shapes , and incorporated different numbers of sulfur atoms ( figure 20 c , d).40 in all cases , sensing supports were loaded with the ru(bipy)3 reporter .
the slight differences found in the chemical structures of ttf - capping molecules resulted in different responses of solids . of all the explosives tested , only tetryl , tnt , and tnb were capable of inducing , to some extent , the dye release from sensing materials . by using color or emission measurements , the lods within the 110 ppm range for the detection of these explosives were calculated . by using one of these ttf - capped smss ,
the authors were able to detect low levels of tetryl in soil samples with good results .
capped silica mesoporous supports have also been used to detect nerve agent simulants ( figure 1).41 nerve agents ( such as sarin , soman , and tabun ) are highly toxic organophosphorous compounds that have severe effects on human health and have been used in terrorist attacks .
given the high toxicity of these chemicals , simulants diethyl chlorophosphate ( dcp ) , diisopropylfluorophosphate ( dfp ) , and diethyl cyanophosphate ( dcnp ) , which present similar reactivity to nerve agents , but are much less toxic , were used in this work .
the sensing system was based on an sms loaded with ru(bipy)3 and capped with the bis(2-hydroxyethyl)aminopropyltriethoxysilane ( het ) groups .
these het moieties formed a thick hydrogen - bonding network around pore outlets , especially in aprotic solvents ( e.g. acetonitrile ) and inhibited dye delivery .
these authors observed that addition of nerve agent simulants induced pore opening , which resulted in selective entrapped dye release .
this release was attributed to the reaction between the nerve agent simulants and oh groups in het , which induced the rupture of the dense hydrogen - bonding network to result in dye release .
simple chromogenic titrations allowed the detection of dcp down to about 15 ppm , whereas the use of fluorimetric procedures permitted the detection of dcp at concentrations as low as 0.1 ppm .
similar lods were obtained for dfp and dcnp , whereas other tested organophosphorus compounds induced no response .
this material was also used to detect nerve agent mimics in the gas phase with good results .
micrometric silica mesoporous support loaded with ru(bipy)3 and capped with hydroxyamino groups for the detection of nerve agent simulants .
the design of gated systems involving biomolecules as capping agents for the detection of small molecules has also been recently explored .
one of the first works in this area used a polyclonal antibody for capping pores . in this first work ,
an antibody for sulfathiazole was selected ( see figure 2 a).42 specifically , the authors loaded the pores of the sms with reporter ru(bipy)3 and anchored the silane derivative 4-(4-aminobenzenesulfonylamino)benzoic acid on the pore outlets of the inorganic support .
finally the material was capped with a polyclonal antibody for sulfathiazole , which also showed good affinity for the anchored molecule .
the authors found that the ruthenium complex was released when the antigen for the capping antibody ( e.g. sulfathiazole ) was present in the solution .
the system was highly selective , and dye delivery was observed only for sulfatiazole , whereas it remained closed in the presence of a family of related compounds .
nanometric silica mesoporous support loaded with ru(bipy)3 and capped with different antibodies for the detection of sulfathiazole , finasteride and triacetone triperoxide ( tatp ) .
mez et al.43 further developed an antibody - capped sms for the selective detection of finasteride with a similar approach ( see figure 2 b ) . in this case , the mesoporous support was loaded with rhodamine b and functionalized with n-(tert - butyl)-3-oxo-(5 a,17 b)-4-aza - androst-1-ene-17-carboxamide groups ( a similar molecule to finasteride ) on the external surface .
addition of polyclonal antibodies for finasteride induced capping of pores due to the interaction with the anchored hapten - like derivative .
addition of finasteride to water suspensions of the antibody - capped material led to the antibody being displaced , pores being uncapped , and the entrapped dye being released .
the amount of delivered dye was found to be proportional to the finasteride concentration , and a typical noncompetitive immunoassay response curve with a lod of 20 ppb was displayed .
selectivity studies showed that only finasteride , among all the other steroids ( testosterone , metenoloneand 16--hydroxystanozolol ) , was able to induce a significant uncapping process .
the material was isolated and stored to be finally tested to detect finasteride in spiked urine samples .
recovery ranges from 94 % to 118 % were observed . by following a similar protocol ,
a new antibody - gated dye - delivery system for the detection of peroxide - based explosive triacetone triperoxide ( tatp ) by test - strip assays was developed ( see figure 2 c).44 in this work , an sms in the form of nanoparticles was loaded with dye sulforhodamine b. then the external surface was functionalized with an appropriate hapten - like molecule , and the system was capped using a tatp - selective polyclonal antibody .
the uncapping of pores with the subsequent dye delivery in the presence of tatp was remarkable and highly selective in pbs ( ph 7.4 ) . with this protocol , tatp was detected at concentrations as low as 12.5 ppb .
the authors also demonstrated that the capped sms can be integrated into a lateral - flow assay . using this latter procedure , a lod of 15 ppb for tatp was determined .
common explosives , like tnt , hexogen , nitropenta , octogen , nitroguanidine , and hexamethylene triperoxide diamine , were unable to uncap pores .
a probe that also employed antibodies , but based on a different configuration , has been recently developed by zhang et al .
for the detection of biotoxin brevetoxin b ( pbtx-2 ) , a neurotoxin produced by algae that can cause intoxication ( see figure 3).45 these authors used mesoporous silica nanoparticles of 150 nm , which were first functionalized with aminopropyl groups .
afterward , an antibody for brevetoxin b was attached to the amino moieties by using glutaraldehyde as a linker . in order to avoid the detachment of the linked antibody , the formed imine bonds were further reduced by the addition of sodium cyanoborohydride .
finally , the pores of the nanoparticles were loaded with methylene blue and capped upon the addition of aminated polystyrene spheres with a diameter of 25 nm .
the capping protocol was carried out in pbs at ph 6.5 . at this ph , strong electrostatic interactions occurred between the grafted antibody , which was negatively charged , and the aminated polystyrene spheres , which were positively charged .
addition of brevetoxin b induced a remarkable methylene blue release , detected by voltammetry due to the selective interaction of this toxin with the grafted antibody and the subsequent displacement of the polystyrene spheres from the surface of the nanoparticles .
linear dependence from 10 pg ml to 3.5 ng ml between the peak current and the pbtx-2 concentration was observed .
these authors also calculated a lod of 6 pg ml for pbtx-2 , which was significantly lower than that of a commercially available pbtx-2 enzyme - linked immunosorbent assay ( elisa ) kit .
the capped system showed high cross - reactivity for pbtx-1 and pbtx-3 , explained by the non - specificity of the anti - pbtx-2 antibody used for these analytes .
the system responded to neither okadaic acid , aflatoxin b1 , and microcystin - lr , nor the presence of several possible components in seawater , including na , ca , mg , cl , so4 , hco3 , and f. the sensing system was tested for the detection of pbtx-2 in different food samples . in another work done in 2014 , the same authors applied a similar antibody - gated design to detect pbtx-2 coupled with a portable personal glucometer.46 the authors loaded a magnetic mesoporous nico2o4 nanostructure with glucose , and pores were capped by adopting a similar protocol to that described above . in the presence of pbtx-2 ,
gated nanoparticles were uncapped to release glucose , which was quantitatively determined by a simple glucometer .
this particular system displayed a signal that was proportional to pbtx-2 up to a concentration of about 20 ng ml .
the authors also confirmed the potential use of capped nanoparticles to detect pbtx-2 in real samples .
nanometric silica mesoporous support loaded with methylene blue or glucose and capped with antibody polystyrene microspheres for the detection of brevetoxin b. tang et al.47 used a similar approach to detect the mycotoxin aflatoxin b1 ( afb1 ) . for this purpose ,
the pores of polyethyleneimine - coated silica mesoporous nanoparticles were loaded with glucose ( figure 4 ) .
afterward , pores were capped upon the addition of aunps functionalized with antibodies for afb1 ( through electrostatic interactions between the positively charged polyethyleneimine shell and the negatively charged antibodies ) .
buffered suspensions ( ph 7.3 ) of capped nanoparticles showed no signal , whereas marked glucose release was observed in the presence of afb1 .
this release was ascribed to the coordination of afb1 with the antibodies and the subsequent detachment of aunps .
linear dependence from 0.01 ppb to 15 ppb between the personal glucometer readout and the afb1 concentration was observed .
the same authors presented a new fluorescence immunoassay to detect afb1.48 in order to build the designed immunosensing probe , mannose - terminated silanes were covalently attached to the external sms surface .
subsequently , pores were loaded with rhodamine b and capped with biotinylated concanavalin a ( con a ) via multivalent carbohydrate protein interactions .
biotinylated con a and biotinylated anti - afb1 were conjugated with streptavidin . at the same time
, aunps were functionalized with invertase and with a bovine serum albumin ( bsa)afb1 conjugate .
when target afb1 was present , a competitive immunoreaction was carried out for the immobilized anti - afb1 antibody on the sms between the target analyte ( afb1 ) and the labeled afb1bsa on aunp .
therefore , the amount of functionalized aunps on smss decreased when the target afb1 concentration increased in the sample . upon sucrose addition , an invertase on aunp hydrolyzed sucrose into glucose and fructose .
the generated glucose competed with mannose for con a coordination and unblocked pores , and released the entrapped dye ( see figure 5 ) .
the release experiments of the immunosensing probe in tris buffer ( ph 6 ) in the presence of sucrose showed linear dependence between the fluorescence signal and the afb1 concentration within the 10 pg ml5 ng ml range .
a lod of 8 pg ml ( 8 ppt ) was calculated for afb1 .
these authors also tested the systems response in the presence of zn , na , k , cl , hco3 , no3 , collagens , mucins , thyroid - stimulating hormone , and -fetoprotein .
nanometric silica mesoporous support loaded with rhodamine b and capped with functionalized aunps for the detection of aflatoxin b1 ( afb1 ) .
wen and co - workers prepared aunp - aptamer - capped smss for the detection of adenosine ( figure 6).49 for this purpose , mesoporous silica nanoparticles were functionalized with 3-aminopropyl moieties and amino groups were transformed into carboxylic acids by a reaction with succinic anhydride .
then the ssdna sequence 3-nh2c6-tct ctt gga ccc cct-5 was grafted on the outer surface by an amidation reaction . at this point , pores were loaded with rhodamine b. finally , pores were capped with aunps functionalized with a complementary aptamer for adenosine ( e.g. aunp - s - aga gaa cct ggg gga gta ttg cgg agg aag gt-3 ) .
however , addition of adenosine induced pore opening and entrapped rhodamine b release due to the formation of aptamer adenosine complexes that detached aunps from the sms surface .
the observed response was selective , and the authors found that addition of cytidine , guanosine , and uridine induced negligible rhodamine b delivery .
nanometric silica mesoporous support loaded with rhodamine b and capped with adenosine aptamer aunps for the detection of adenosine .
a first example of a capping uncapping protocol for the detection of biomolecules , in particular a certain oligonucleotide , was reported by martnez mez et
al ( figure 7).50 the system was based on an sms in the form of nanoparticles loaded with fluorescein and functionalized with ( 3-aminopropyl)triethoxysilane on the outer surface .
oligonucleotide 5-aat gct agc taa tca atc ggg-3 was used to cap pores via electrostatic interactions with the anchored amines partially protonated in water at a neutral ph .
these authors demonstrated that delivery of dye from the solid at ph 7.5 was selectively triggered in the presence of the complementary single strand of the capping oligonucleotide due to the hybridization of both single oligonucleotide sequences with the concomitant cargo release .
they also tested dye delivery in the presence of other oligonucleotides with a single or two - base mismatch sequence(s ) , but delivery was poor in these cases .
nanometric silica mesoporous support loaded with fluorescein and capped with oligonucleotide for the detection of the complementary strand . by taking the system one step forward
, these authors designed a similar capped material to detect genomic dna ( figure 8).51 for this purpose , an sms , also in the form of nanoparticles , was loaded with rhodamine b and functionalized with ( 3-aminopropyl)triethoxysilane on the external surface . as the capping oligonucleotide , a highly conserved sequence in the mycoplasma genome , which corresponds to a fragment of the 16s ribosomal rna subunit , was used ( e.g. 5-ggg agc aaa cag gat tag ata ccc t-3 ) .
the system remained closed until the genomic dna of mycoplasma fermentans was added , which had been previously dehybridized by thermal treatment .
the capped sms was unable to deliver the cargo in the presence of genomic dna from other bacteria , such as candida albicans or legionella pneumophilla .
capped nanoparticles were used to detect mycoplasma contamination in real contaminated cell culture media without applying pcr techniques .
nanometric silica mesoporous support loaded with rhodamine b and capped with a single - stranded oligonucleotide for the detection mycoplasma fermentans genomic dna . in a more recent work , the same authors developed a similar system to detect genomic dna . in this case , they used nanoparticles loaded with rhodamine b and capped with covalently attached dna ( see figure 9).52 two single - stranded oligonucleotides were selected for the gating mechanism : 1 ) a short dna sequence ( nh2-(ch2)6 - 5-gac tac gac ggt atc-3 ) , which was covalently anchored to the sms via the formation of urea bonds and 2 ) a single - stranded oligonucleotide selective for mycoplasma ( e.g. 5-aag cgt ggg gag caa aca gga tta gat acc ctg gta gtc-3 ) .
the probe was able to detect mycoplasma fermentas genomic dna at a concentration as low as 70 dna copies l . the authors also found that candida albicans and legionella pneumophilla genomic dna were unable to induce dye release .
nanometric silica mesoporous support loaded with rhodamine b and capped with covalently anchored oligonucleotides for the detection mycoplasma fermentans genomic dna .
he , wang , and co - workers combined the catalytic properties of platinum nanoparticles ( ptnps ) and capped mesoporous silica to develop a colorimetric detection system for oligonucleotides ( see figure 30).53 for this purpose , and as an inorganic scaffold , ptnps were synthesized and then coated with a mesoporous silica shell . in order to prepare the final gated material ,
the external silica shell was functionalized with aminopropyl moieties , and pores were capped through electrostatic interactions with the single oligonucleotide strand 5-tct ttc ctt gat ttt ctt cct ttt gtt cac-3 , which contained a mutation of gene brca1 , which is related to breast cancer .
buffered ( acetate , ph 4.7 ) suspensions of capped nanoparticles in the presence of 3,3,5,5-tetramethylbenzidine ( tmb ) remained colorless because the molecules of the indicator were unable to access the platinum core . however , in the presence of the complementary strand , a clear blue color developed due to the displacement of the capping oligonucleotide and the pt - induced catalytic oxidation of tmb .
the systems response time was very short ( between 3 and 10 min ) , and the lod was calculated to be 3 nm . aside from this , other strands with one , two , or three mismatched bases induced negligible color changes .
mez54 and yang55 simultaneously developed aptamer - gated smss based on a similar synthetic approach , but with different analytical techniques , to detect thrombin .
martnez - mez et al . used mesoporous silica nanoparticles loaded with dye rhodamine b ( see figure 1 ) , while yangs system consisted of fe3o4 magnetic nanoparticles coated with a mesoporous silica shell loaded with the ru(bipy)3 complex . in both cases ,
external sms surfaces were grafted with 3-aminopropyltriethoxysilane , and the 15-mer thrombin - binding aptamer 5-ttt tttggttggtgtggt tgg-3 ( tba ) was used as a gatekeeper .
the aptamer was absorbed on the surface through electrostatic interactions with the positively charged amines on sms surfaces . in the presence of -thrombin ,
the tba aptamer was displaced from the surface , due to the formation of tba protein complexes , and the entrapped reporter was delivered .
detected presence of thrombin by measuring the fluorescence emission of the rhodamine b dye in the solution .
these authors tested their system successfully in simulated human blood plasma and achieved a lod of 2 nm , and a lod of 4 nm in pbs buffer with 10 % human serum .
yang et al . studied the presence of thrombin by detecting the electrochemiluminescence of the ru(bipy)3 reporter .
this allowed them to calculate a lod of 0.5 pm in tris - hcl buffer .
found that a mixture of other nonexclusive binding proteins , such as ovalbumin and bsa , was unable to induce the uncapping of pores .
similarly , yang et al demonstrated that bsa , lysozyme , and gox possessed negligible uncapping capacity .
a ) nanometric silica mesoporous support loaded with rhodamine b and capped with thrombin binding aptamer for the detection of thrombin .
b ) release profile of rhodamine b from the capped solid in the presence ( i ) and in the absence ( ii ) of thrombin in simulated human blood plasma .
dna - capped mesoporous silica nanoparticles have also been used for the optical and electrochemical detection of the glycoprotein enzyme prostate - specific antigen ( psa).56 the sms was functionalized with aminopropyl moieties , and pores were loaded with methylene blue .
finally , pores were capped upon the addition of a single dna strand ( 5-gta atc ctc agc aac ctc agc-3 ) by electrostatic interactions with the positively charged nanoparticle surface .
the uncapping protocol was related with the use of two antibodies capable of coordinating psa , which were labeled with two ssdna fragments ( dna1 : 5-gct gag gtt atc aag act ttt ttt atc aca tca ggc tct agc gta tgc tat tg - sh-3 and dna2 : 5-sh - tac gtc cag aac ttt acc aaa cca cac cct ttt ttt gtc ttg gct gag gat-3 ) , which were complementary to that used for capping mesoporous nanoparticles . in the presence of both dna - labeled antibodies and psa , a proximate complex was formed and was able to hybridize with the ssdna used as the cap , which resulted in methylene blue release ( see figure 2 ) .
psa was sensitively detected with a linear range from 0.002 to 100 ng ml , and a lod of 1.3 pg ml was determined .
no interference was observed in the detection of psa when the carcinoembryonic antigen ( cea ) was present in the solution .
nanometric silica mesoporous support loaded with methylene blue and capped with dna for the detection of prostate - specific antigen ( psa ) . regarding the use of biomolecules for uncapping protocols , certain examples
have been reported where delivery has been selectively observed in the presence of a given enzyme.57 however , most of these examples have explicitly been prepared for drug delivery , and very few examples for sensing applications have been described . in this context , ju and co
- workers have recently developed a telomerase - responsive gated system for the sensitive in situ tracking of telomerase activity in living cells which , for instance , can be used to distinguish cancer cells from normal cells ( see figure 3).58 in their work , the authors prepared an sms in the form of nanoparticles , which were functionalized with a fluorescence quencher on inner walls and were loaded with fluorescein .
the system was capped with dna sequence 5-(ccctaa)n aatccg tcgagc agagtt-3 , which contains a telomerase primer , via electrostatic interactions with the amino groups attached to the sms . in the presence of telomerase and deoxynucleoside triphosphate monomers ( dntps ) ,
the oligonucleotide sequence was extended and formed a rigid hairpin - like dna structure , which moved away from the sms surface to allow entrapped fluorescein release .
the telomerase - driven response was studied by incubating the enzyme , dntps , and capped nanoparticles , and by monitoring fluorescein release by fluorescence and uv / vis spectroscopy . a gradual increase in fluorescein intensity with a prolonged incubation time
these authors also conducted an extended in vitro study on hela cells ( cervical cancer cells ) using the gated nanoparticles .
they concluded that fluorescence due to fluorescein release was activated by the action of intracellular telomerase and suggested that the system could be used for tracking intracellular telomerase activity .
these authors also performed different assays with other cell lines , such as bel-7402 cells ( liver cancer cells ) and qsg-7701 cells ( liver normal cells ) , which confirmed that the designed strategy can be applied to distinguish cancer cells from normal cells .
following a similar approach , lu and co - workers used the same gated design , but loaded the sms with glucose to monitor telomerase activity with a glucometer.59 in this case , linear dependence between the glucometer readout and the number of hela cells used in the experiment was achieved within the range of 1005000 cells .
the lod was set at 80 hela cells ml . finally , cui and co - workers also prepared a similar material , but used core
shell nanoparticles ( in which au@ag nanorods were coated with mesoporous silica ) and doxorubicin as the payload.60 these authors also demonstrated the telomerase - induced release of the loaded drug and were able to trace the uptake of the prepared nanoparticles by hela cells using surface - enhanced raman spectroscopy ( sers ) measurements .
willner and co - workers prepared dna - capped mesoporous silica nanoparticles for the recognition of nucleic acid biomarkers.61 the external surface of the nanoparticles was functionalized with aminopropyl moieties which were then reacted with a single - stranded dna sequence ( 5-caa ggg aag tct tca ctg ccc ttg cac act 3 ) , using n - maleimidocaproyl - oxysulfosuccinimide ester ( emcs ) as covalent crosslinker .
the grafted dna included a tailored base sequence that generates at room temperature a hairpin structure ( blocking the pores ) and also included a single - stranded loop able to recognize the nucleic acid biomarker 5-agt gtg caa ggg cag tga aga ctt gat tgt-3. the pores were loaded with rhodamine b. treatment of the capped solid with the biomarker induced the opening of the hairpin forming a duplex structure that also blocked the pores inhibiting dye release .
however , in this case , addition of exo iii induced the hydrolysis of the 3-end of the capping sequence allowing release of the entrapped dye . using the same approach ,
other solids functionalized with 5-aac gaa gct gag gat gtg ttc gtt-3 for the biomarker 5-atc ctc agc ttc g-3 was prepared . both dna strands formed a duplex structure that was able to cap the pores and inhibit dye release . in this case
upon addition of < the nicking endonuclease nb.bbvcl , rhodamine b release was observed .
the design of gated mesoporous materials has proved to be a promising starting point for applying the versatility of molecular - based ideas to design nanoscopic gating solids and a way to study factors that can influence the design of molecular gating functions based on molecular / biomolecular / supramolecular concepts .
this review provides an account of examples based on silica mesoporous supports ( smss ) for sensing applications .
these new sensing systems are related to the interest in designing gated nanoscopic hybrid materials with the ability to release a previously entrapped cargo when certain external stimuli are applied .
as stated above , most of these functional materials have been used for drug delivery studies . in sensing applications however , the main idea is that the coordination or reaction of a target analyte with the gate could modulate the delivery of a reporter from pores to solution , which would result in a suitable easy - to - measure response . one major characteristic in capped sensing smss is that cargo delivery should be fast .
whereas delivery should often be sustained over time in most therapeutic applications of gated materials , delivery in sensing systems should be as fast as possible . in this review ,
we include examples that have been specifically designed to act as sensing systems by the selective delivery of a reporter . in this context , we have described examples of the sensing of anions , cations , small molecules , and biomolecules . moreover , we have included some selected examples that , although originally designed for controlled cargo delivery , have displayed some features in terms of selectivity and sensitivity , making them suitable for sensing applications . capped supports for sensing applications
is a developing research area , and a number of new advances in this field are envisioned .
in particular , it should be noted that only minor changes are required to transform a controlled - release system , for instance specifically designed for drug delivery , into a sensing material .
the crucial issues include achieving selectivity against other analytes , sensitivity , and a fast response time .
in particular , response time is one important issue for the use of these capped materials in real analytical systems , such as test - strip assays or microfluidic devices .
most of the described systems have response times ranging from several minutes ( most of them ) to hours .
a decrease in response time to seconds or very few minutes is thus crucial for real applications . as far as we know , there is only one example of the implementation of capped smss in a lateral - flow assay,44 and further studies need to be carried out regarding response times of these materials in order to advance their use in analytical detection / determination protocols .
moreover , the existence of amplification features , the possibility of using a wide variety of mesoporous supports , and the potential employment of a wide range of reporters able to display a signal ( optical , electrochemical , etc . ) upon delivery all these make the possibilities for this approach practically unlimited and highly appealing for the design of new advanced sensing systems for a variety of guests .
however , in order to achieve this goal , certain barriers have to be overcome .
further systems to be developed need to be highly selective , have low limits of detection , and display a very short response time . | silica mesoporous supports ( smss ) have a large specific surface area and volume and are particularly exciting vehicles for delivery applications .
such container - like structures can be loaded with numerous different chemical substances , such as drugs and reporters .
gated systems also contain addressable functions at openings of voids , and cargo delivery can be controlled on - command using chemical , biochemical or physical stimuli .
many of these gated smss have been applied for drug delivery . however , fewer examples of their use in sensing protocols have been reported .
the approach of applying smss in sensing uses another concept that of loading pores with a reporter and designing a capping mechanism that is selectively opened in the presence of a target analyte , which results in the delivery of the reporter . according to this concept ,
we provide herein a complete compilation of published examples of probes based on the use of capped smss for sensing .
examples for the detection of anions , cations , small molecules and biomolecules are provided .
the diverse range of gated silica mesoporous materials presented here highlights their usefulness in recognition protocols . |
the tridimensional structure of the transmembrane core of some kv channels has been solved at high resolution ( jian et al . , 2003 ; long et al . , 2005a , 2007 ) .
given the overall identity and primary structure conservation of these protein segments , it is reasonable to assume that all of the kv family members would share a common arrangement in these regions .
however , solving the high - resolution structure of a complete kv channel still remains a major challenge and the tridimensional structure and/or complete architecture of the cytoplasmic domains of the kv channels are unknown .
in fact , these regions show far more divergence than the rest of the molecule does , even among closely related channels .
nevertheless , specific regions of these domains have been structurally characterized using a divide and conquer approach ( gaudet , 2009 ) , thus providing partial information to be joined together and integrated with biochemical and electrophysiological data to yield a better picture of the channel organization on a domain by domain basis ( biggin et al . , 2000 ) .
this approach has led to the proposal of two general assemblies for the kv channel cytoplasmic components ( figure 3 ; yellen , 2002 ) : ( a ) the pattern typically exhibited by the kv1kv4 cluster ( yu et al . , 2005 ; pischalnikova and sokolova , 2009 ) , in which , hanging centrally below the transmembranal core there is an n - terminal t1 tetramerization domain that determines , among other functional characteristics , the specificity of subunit assembly .
( b ) the pattern shared by the kv7 and kv10kv12 channels , characterized by the absence of a t1 domain and the presence of a c - terminal domain located centrally below the transmembrane channel core .
interestingly , this second overall organization also pertains to other relatives from the voltage - gated ion channel superfamily ( gutman et al .
, 2005 ; yu et al . , 2005 ) , including those gated coordinately by voltage and ligand binding [ such as the hyperpolarization - activated and cyclic nucleotide - gated ( hcn ) and bk / kca1 channels ] and those gated exclusively by intracellular ligands [ cyclic nucleotide - gated ( cng ) and sk / kca2 ] .
besides these cytoplasmic cores , a panoply of protein domains and motifs ( figure 4 ) can be present in the cytoplasmic regions that , in addition to some accessory subunits , add diversification in structure , function , and regulation to the different channel subfamilies and even to different members of the same subfamily . basic patterns of kv channel cytoplasmic domains structural organization . ( a ) general organization of the kv1kv4 channels group with the t1 tetramerization domains hanging centrally below the transmembranal core and attached to it through four linkers continued from the first transmembrane helices . in this case
the c - terminal structures probably track to the periphery surrounding t1 and extending to its bottom .
( b ) general cytoplasmic architecture of the kv7 and kv10kv12 channels characterized by a carboxy terminus ( i.e. , the c - linker / cnbd region of the kv10kv12 channels or the a
d helical regions of the kv7 channels ) forming a compact tetrameric structure in a central position immediately below the cytoplasmic pore opening . in this case
the amino terminus probably surrounds the c - terminus and extends to its bottom establishing extensive contacts with its top and side surfaces .
note that in both models the initial amino terminal structures ( ball - like and eag / pas domains for kv1kv4 and kv10kv12 , respectively ) are likely to interact with the gate surroundings in the transmembrane core .
schematic view of different structural and/or functional domains recognized at the cytoplasmic ends of kv channels . in the amino terminus
these include the ball - like structure responsible for fast n - type inactivation , the eag / pas domain of the eag - like channels , the nip domain that protects kv1.6 channels against rapid inactivation , the secondary inactivation domain reported for kv1.4 channels , the double sh3 binding domain of kv1.5 , and the t1 tetramerization domain ( also called nab ) of the kv1kv4 channels . in the carboxy terminus the domains shown
correspond to the c - linker and cnbd encountered in the eag - like channels , the localization domain and the c - terminal activation ( cta ) domain of kv2.1 and the post - synaptic density protein ( psd-95)-binding domain of some kv1 channels .
a more detailed view of the kv7 channels carboxy terminus organization is shown schematically in figure 6 .
examples of cytoplasmic domains for which the tridimensional structure has been solved , include the cited t1 domain ( kreusch et al . , 1998 ; bixby et al . , 1999 ;
, 2004 ; long et al . , 2005a ; pioletti et al . , 2006
) , the distal n - terminus inactivation structures from kv3.4 , shakerb , and kv1.4 ( antz et al . , 1997 ; schott et al . , 1998 ; wissmann et al . , 2003 ) , the n - terminal pas domain and its initial distal segments from kv11.1 ( morais - cabral et al . , 1998
; li et al . , 2010 ; muskett et al . , 2011 ; ng et al . ,
2011 ) , and the c - terminal coiled - coil segment from kv7.4 and kv7.1 ( howard et al . , 2007
it is also interesting to note the current availability of high - resolution tridimensional structures for the c - terminal c - linker / cnbd regions of the kv - related hcn and cng channels ( zagotta et al .
2011 ) , providing a valuable reference for homologous regions present in some kv channels such as those of the eag family .
additionally , several regions putatively involved in different aspects of channel behavior other than gating ( e.g. , oligomerization and assembly , trafficking , surface expression , protein protein interactions , modulation , or drug channel interactions ) have been recognized in the kv cytoplasmic channel regions ( figure 4 ; hopkins et al .
, 1994 ; xu et al . , 1995 ; ponce et al . , 1997 ;
, 1998 ; bentley et al . , 1999 ; cukovic et al . , 2001 ; yang et al . , 2001
, 2005 ; choi et al . , 2005 ; xu et al . , 2010 ;
zheng et al . , 2010 ) , that in many cases have not been associated to a known tridimensional structure .
finally , the availability of low - resolution images for some voltage - dependent k channels yields interesting possibilities to fit with partial x - ray data , and provides significant new insights into the overall architecture of the channel molecule ( sokolova et al . , 2001 , 2003 ;
orlova et al . , 2003 ; parcej and eckhardt - strelau , 2003 ; kim et al . , 2004 ; adair et al . , 2008 ; pischalnikova and sokolova , 2009 ; wang and sigworth , 2009 )
a pioneering indication that some cytoplasmic region(s ) could participate in the control of gating behavior was provided by analyzing the process of inactivation .
thus , the specific abolition of voltage - dependent na current inactivation by intracellular treatment with proteolytic enzymes ( armstrong et al
. , 1973 ) subsequently led to the proposal of a ball - and - chain mechanism as the determinant of this inactivation process ( armstrong and bezanilla , 1977 ) .
however , a better molecular explanation of this mechanism , in which a cytoplasmic blocking particle tethered to the channel protein ( the ball , see figures 1 and 4 ) binds rapidly to the pore and plugs it after the activation gate opens , was obtained in 1990 by studying the fast / n - type - inactivation of the kv - type shaker k channel of drosophila .
thus , deletion of the first 20 amino acids of the amino terminus eliminated rapid inactivation ( hoshi et al . ,
1990 ) , whereas a soluble peptide containing that sequence restored inactivation in a concentration - dependent manner ( zagotta et al . , 1990 ) .
it was subsequently observed that the soluble peptide ( or n - terminal inactivation domain ) competes with intracellularly applied channel blockers ( e.g. , quaternary amines , choi et al . , 1991 ) , that it is expelled to the cytoplasm by potassium flow from the extracellular side of the membrane ( demo and yellen , 1991 ) , and that the inactivation process traps the channel in an open - inactivated state that prevents it from closing , making the recovery of inactivation typically via the open state ( demo and yellen , 1991 ; ruppersberg et al . , 1991 ) .
moreover , the inactivation domain was shown to interact with a binding site in the pore cavity , which overlaps with the binding site for quaternary amine blockers
some aspects of the ball - and - chain inactivation mechanism are worthy of further consideration , since the structural arrangements of the chain are not as clear as those of the ball .
thus , shortening the length of the shaker chain accelerated inactivation rates ( hoshi et al . , 1990 ) as if this region were acting as a flexible linker to tether the ball to the channel .
however , some kv1.4 deletions that reduce the length of the chain not only do not increase the rate of inactivation , but actually slow inactivation as if the specific structure of this region could affect the movement and/or orientation of the inactivating particle ( rasmusson et al . , 1998 ) .
it has also been suggested that rather than being a simple tether , this chain
region may act in concert with other cytoplasmic structures to orientate and constrain the movement of the inactivation particle ( isacoff et al .
, 1991 ; jern and covarrubias , 1997 ; sankaranarayanan et al . , 2005 ;
in this sense , it has been proposed that interactions of the kv1.1 t1 domain with the more distal n - terminal structures play an important role in defining the strength of the ball - triggered n - type inactivation ( baker et al . , 2006 ) and it has also been recognized that ball interaction(s ) with the t1 domain and the s4s5 linker seem to be crucial for proper development of the fast / n - type inactivation ( isacoff et al .
thus , in contrast with the well defined position of the ball plugging the conduction pathway inside the pore to inactivate the channel , the resting position of the chain segments and their dynamic rearrangements during the closed to open and inactive transitions are not well known .
there is indirect evidence supporting the interpretation that these structures might wrap around the scaffold provided by the t1 tetramer hanging below the channel core ( figure 3a ) , so that upon depolarization they would snake through the windows lined by the s1t1 linkers to reach a pocket on the upper t1 domain surface and subsequently the channel cavity below the selectivity filter ( gulbis et al .
, 2000 ; kobertz et al . , 2000 ; zhou et al . , 2001 ;
however , it has also been proposed that the ball - and - chain combination is confined to the space between the t1 domain and the transmembrane portion of the channel ( varhsney et al . , 2004 ) .
in addition to the ball , a second inactivating domain with the ability to endow the channel with rapid inactivation has been identified in a more proximal part of the n - terminus of some kv channels ( e.g. , kv1.4 ; kondoh et al . , 1997 ; hllerer - beitz et al . , 1999
; wissmann et al . , 2003 ) , and an n - type inactivation - prevention ( nip ) domain ( figure 4 ) has been mapped close to the kv1.6 n - terminal end ( roeper et al . , 1998 ) .
as an alternative to the ball and length - dependent chain mechanism , a fast inactivation mechanism has been proposed for kv1.4 , based on electrostatic interactions between oppositely charged n - terminal segments and between the positively charged inactivation ball and acidic residues of the t1s1 linker , that shorten the distance between the inactivating particle and its binding site , helping to orientate the ball and causing the channel to inactivate more efficiently ( fan et al . , 2012 ) .
it is important to mention that even though a similar architecture is maintained around the gating machinery , the functional effects of the cytoplasmic domains can vary among channels even belonging to the same subfamily .
this is the case of the fast inactivating a - type kv4 channels , in which the existence of a n - type inactivation mechanism and a ball - like inactivating peptide at the n - terminus have been recognized ( gebauer et al . , 2004 ) , although it seems to play a minor role since the kv4 most prominent inactivation is based in a temporary loss of coupling between the voltage sensor and the cytoplasmic gate ( barghaan et al . , 2008 ; barghaan and bhring , 2009 ) .
remarkably , the development of kv4 inactivation is determined by the presence of amphipathic n - terminal sequences and a positively charged domain at the c - terminus , that may interact with each other ( jern and covarrubias , 1997 ; hatano et al .
as indicated above ( see figure 1 ) , collapsing of the selectivity filter during depolarization steps can lead to a second type of inactivation named
c - type inactivation ( or selectivity filter gating ; wang et al . , 2011 ) .
recent data from some prototypical k channels ( e.g. , kcsa and kir ) have provided a structural basis for allosteric coupling between activation at the level of the internal membrane surface and this type of inactivation at the channel selectivity filter , and about the influence exerted on them by intracellular domains ( uysal et al .
, 2011 ; clarke et al . , 2010 ; cuello et al . , 2010 ; zhou and jan , 2010 ) .
this yields some new ideas about how the structural modifications in the cytosolic domains can be allosterically transmitted to the intracellular gate and even to more distant gating elements in the channel core .
it is important to note that selectivity filter gating not only pertains to kcsa and kir voltage - independent k channels containing exclusively a pore domain ( norton and gulbis , 2010 ) , but also to a wide range of kv channels , to other highly selective k channels such as sk and bkca , and to non - selective cation channels ( e.g. , cng channels ) related to the kv family ( bruening - wright et al .
, 2007 ; cox and hoshi , 2011 ; wang et al . , 2011 ) in which the gating motions around the s6 bundle cross and hence the coupled selectivity filter rearrangements , can also be influenced by conformational changes in the cytoplasmic domains .
the kv channel s activation gate is located at the intracellular end of the pore at the c - terminus of the s6 transmembrane segment ( armstrong , 1971 ; holmgren et al . , 1997 , 1998 ; liu et al . , 1997 ; perozo et al . , 1999 ;
del camino et al . , 2000 ; del camino and yellen , 2001 ; hackos et al .
, 2002 ; webster et al . , 2004 ; wynia - smith et al . ,
although other contacts and interactions between different domains could be involved ( ding and horn , 2003 ; li et al . ,
2008 ; labro et al . , 2011 ) , coupling of the voltage sensor movement to operation of the gate mainly involves the s4s5 linker which seems to act as a structural link , influencing gate dynamics via physical interaction(s ) with the intracellular end of s6 and/or the cytoplasmic c - linker region ( figure 4 ) following it ( lu et al . , 2002 ;
, 2005b ; ferrer et al . , 2006 ; boulet et al . , 2007 ;
interestingly , a similar coupling via s4s5 linker interaction with the cytoplasmic end of s6 has been proposed for hcn and kat1 channels ( chen et al .
, 2001 ; decher et al . , 2004 ; prole and yellen , 2006 ) , that show an overall structure very similar to that of kv channels , but in which opening is triggered by membrane hyperpolarization rather than depolarization ( mnnikk et al .
, 2002 ; latorre et al . , 2003 ; vemana et al . , 2004 ; grabe et al . , 2007 ) .
this opposite polarity of gating is perhaps achieved by a different binding of the s4s5 linker to the extreme of s6 only in the open state whereas in the kv channels this interaction takes place in the closed state ( choveau et al . , 2011 ) . since the gate and the sensor gate coupler
( i.e. , the s4s5 linker ) locate at the cytoplasmic channel surface , these structures become accessible targets for modulation from the cytoplasmic milieu , providing attractive means of channel activity regulation not only by amino- and /or carboxy - located channel domains but also by soluble products of signal transduction cascades .
early work with kv2.1 indicated that the activation gating could be strongly affected by the cytoplasmic amino and carboxyl termini ( vandongen et al . ,
1990 ) , and the influence of the channel amino terminus in voltage - dependent gating and modulation has been subsequently confirmed ( pascual et al . , 1997 ) .
it has been demonstrated that interactions between the n- and c - terminal regions of kv2.1 lacking auxiliary subunits determine its activation properties , trafficking , and phosphorylation - dependent modulation ( ju et al . , 2003 ; scholle et al . , 2004 ; mohapatra et al . ,
2008 ) , in a way that resembles the interaction between the and the auxiliary subunits in other kv channels ( mohapatra et al . , 2008 ) .
another case is that of kv1.2 , in which an interaction between the c - terminus and the s4s5 linker of a neighboring subunit which is able to regulate channel activation has also been suggested ( zhao et al . , 2009 ) .
it has also been reported that the polar t1 surfaces of the mammalian kv1.2 and the aplysia kv1.1 channels play a key role in the conformational changes that lead to channel opening ( cushman et al .
more recently , use of fluorescence resonance energy transfer ( fret ) microscopy combined with voltage - clamp recording has allowed the detection of conformational rearrangements in the relative orientation of kv1.2 n- and c - termini upon depolarization ( kobrinsky et al . , 2006 ; lvov et al . , 2009
) , a mechanism that could be related to gating regulation and that may also pertain to other kv channels . interestingly ,
similar gating - related motions of the n- and c - termini have been detected in kv7.1 and the cnga1 channel using an equivalent approach ( taraska and zagotta , 2007 ; haitin et al .
, 2009 ) , although both channels belong to the subgroup characterized by the absence of the t1 domain ( see above ) .
finally , in the case of kv4 channels , it has been demonstrated that the t1t1 interface is functionally active and dynamic during gating ( wang et al . , 2005 ) and ,
based on work with kv4.1 channels , it has been concluded that the complex voltage - dependent gating rearrangements of eukaryotic kv channels are not limited to the membrane - spanning core , but must include the intracellular t1t1 interface ( wang and covarrubias , 2006 ) .
an interesting contribution to the topic of gating control by cytoplasmic domains is provided by members of the eag family , that comprises the kv10kv12 subfamilies ( warmke and ganetzky , 1994 ) .
overall sequence similarity between eag- and shaker - type kv1kv4 channels is quite low except for the pore region , but a much higher degree of homology is found between eag - type channels and cng , hcn , and inwardly rectifying plant k channels ( anderson et al . , 1992 ;
sentenac et al . , 1992 ; warmke and ganetzky , 1994 ; yu et al . , 2005 ) .
kv10 ( ether - - go - go or eag ) channels are involved in setting the membrane potential in several cell types and appear to be related to processes like cell - cycle , proliferation , and tumor progression ( felipe et al . , 2006 ; arcangeli et al . , 2009 ;
several studies have demonstrated the involvement of cytoplasmic domains in the control of eag channel gating .
thus , it has been observed that the most distal region and the pas domain at the n - terminus of the eag channels are involved in determining gating kinetics ( terlau et al . , 1997 ; stevens et al . , 2009
; wray , 2009 ) , and that alterations in eag gating following truncation of the initial portion of the n - terminus can be compensated by mutation of the voltage sensor s4 segment ( terlau et al . ,
it has also been shown that the voltage dependence of eag currents is not determined solely by the membrane - spanning domains ( lrinczi et al . , 2009 ) , and that the n- and c - terminal regions could be involved in controlling a gating step after movement of the voltage sensor , as well as in regulating biophysical properties of the channel ( li et al . , 2011 ) . also
, voltage - dependent gating characteristics of plant kat1 channels seem to be influenced by the n- and c - termini of the protein ( marten and hoshi , 1997 , 1998 ) .
the relationship between cytoplasmic structures and gating properties has been exhaustively investigated in the kv11.1 ( human ether - - go - go - related gene or herg ) channel , a remarkable entity both from the pathophysiological and the biophysical points of view .
, 1995 ; trudeau et al . , 1995 ) and mutations in the herg gene and drug inhibition of herg channels underlie inherited and acquired type 2 long - qt syndrome ( sanguinetti et al . , 1995 ; viskin , 1999 ; keating and sanguinetti , 2001 ; redfern et al . , 2002 ; thomas et al . , 2003 ; finlayson et al . , 2004 ; roden and viswanathan , 2005 ; goldenberg and moss , 2008 ) .
on the other hand , herg channels play a key role in setting the electrical behavior of a variety of non - cardiac cell types ( barros et al . , 1994 , 1997 ;
these physiological roles of herg derive from its unusual gating properties , characterized by slow activation kinetics and a very fast inactivation on depolarization .
thus , herg currents increase during repolarization due to a fast recovery from inactivation followed by a much slower deactivation for which the channels remain open for longer periods of time at negative voltages , giving rise to the typical herg tail currents . in the case of the heart
, this contributes to the repolarization of the cardiac action potential and to the prevention of arrhythmias induced by early after - depolarizations or ectopic beats ( smith et al .
, 1996 ; lu et al . , 2001 ) . as for other voltage - gated potassium channels , voltage sensors and gating elements
have been mapped to the transmembrane core of herg ( smith and yellen , 2002 ; liu et al . , 2003 ; piper et al .
, 2003 ; subbiah et al . , 2004 , 2005 ; zhang et al . , 2004 , 2005 ;
a volume larger than the core region hangs toward the cytoplasm since the cytoplasmic regions account for around 80% of the channel protein ( figure 5 ) , but details about the structural architecture of these extensive regions are still lacking ( miranda et al . , 2008 ) .
what has been clearly demonstrated is that the distinctive gating properties of herg are strongly influenced by some cytoplasmic protein domains .
thus , the conserved eag n - terminal domain at the beginning of the herg n - terminus and the exclusive proximal domain following it up to the s1 segment ( figure 5 ) were identified as important determinants of the remarkably slow deactivation and activation gating kinetics , respectively ( schnherr and heinemann , 1996 ; spector et al . , 1996 ; morais - cabral et al . , 1998
, 2008 ; gustina and trudeau , 2009 ; ng et al . , 2011 ) .
in these studies , it has been repeatedly demonstrated that the n - terminal eag domain determines the slow herg deactivation , since channels with particular deletions of the amino terminus , or with point mutations in the eag domain , show rapid deactivation kinetics .
the fact that short deletions at the beginning of the n - terminus mimic the effect of more extensive amino terminal removal , points to the initial segment of the eag domain ( residues 126 ) as the essential regulator of herg deactivation gating ( wang et al . , 1998 , 2000 ;
the regions corresponding to the eag / pas ( residues 1135 ) and the proximal domains up to the first transmembrane helix are shown as striped and solid red bars , respectively .
the transmembranal core region containing the six transmembrane helices and the carboxy terminus are shown colored in gray and blue , respectively .
tcc indicates the proposed location of a tetramerization coiled - coil at the c - terminus .
the size of every domain is represented on a horizontal scale proportional to the total length of the protein .
( b ) schematic representation of a herg channel -subunit showing the proposed relative positioning of the pas region in the amino terminus ( dotted line ) , and the flexible tail of the amino end ( initial solid red line segment ) and the amphipathic -helix separating them ( red rectangle ) pointing toward the s4s5 linker on the cytoplasmic surface of the channel core .
the proximity between the initial regions of the n - terminus and the c - linker / cnbd domains that are directly linked to the cytoplasmic gate at the bottom of helix s6 is also used to illustrate the possible existence of additional interactions between these channel structures . for more explanations ,
functional reconstitution of mutant channels with a recombinant eag n - terminal fragment , combined with tirf fret spectroscopy , have recently been used to provide some additional insights into the regulation of herg deactivation ( gustina and trudeau , 2009 ; fernndez - trillo et al . , 2011 ) .
thus , it has been recognized that normal slow deactivation gating involves an interaction between the initial n - terminal flexible segment ( figure 5b ; li et al . , 2010 ;
, 2011 ) and the gating machinery , likely at the level of the s4s5 linker ( fernndez - trillo et al . , 2011 ) .
indeed , it has been shown that physical proximity exists between that unstructured and flexible segment of the herg amino terminus and the n - terminal portion of the s4s5 linker ( de la pea et al . , 2011 ) , further supporting this interpretation . in this context , the pas sub - domain from the herg eag domain and the amphipathic -helix ( residues 1323 ) could act as a scaffold and a spacer helping to correctly orientate the initial flexible tail of the amino end ( residues 29 ) toward the s4s5 linker to modulate herg gating ( de la pea et al .
, 2011 ; fernndez - trillo et al . , 2011 ; ng et al . ,
additionally , the extensive contacts of these structures with the top and side surfaces of the c - linker / cnbd domains , that hang centrally below the pore domain in this type of channel ( figures 3b and 5b ) , could help to allosterically modulate the operation of the cytoplasmic gate at the c - terminus of the s6 segment , which is directly attached to the c - linker ( muskett et al .
in fact , the involvement of some residues of the c - linker / cnbd regions of the herg carboxy terminus in the control of deactivation gating through their interaction with the n - terminal - most region has also been proposed ( al - owais et al . , 2009 ;
on the other hand , it has also been demonstrated that the herg proximal domain ( figure 5 ) plays an important role in the activation channel properties and their modulation by hormones ( viloria et al . , 2000 ; gmez - varela et al . , 2002 , 2003 ; alonso - ron et al . , 2008 , 2009 ) .
a small cluster of basic residues within this proximal domain close to the s1 segment has been subsequently recognized as a determinant of the herg activation behavior through an electrostatic influence on the gating machinery ( saenen et al . , 2006 ) .
however , although the modulation of herg activation gating by a g protein - coupled hormone receptor is impaired after deletion of the proximal domain , it is observed in the absence of the cluster of basic residues ( gmez - varela et al . , 2003 ; alonso - ron et al . ,
interestingly , kinetic measurements of herg activation under steady - state conditions , indicated that the initial region of the eag domain also affects the activation behavior ( alonso - ron et al . , 2008 ) , suggesting that an interaction of this region with the channel core may also be involved in the modifications of gating caused by the deletion of the proximal domain ( viloria et al . , 2000 ;
nevertheless , the exact regions of the gating machinery at which the regulatory effects on activation are exerted remain to be established , although the bottom part of s4 , the s4s5 linker , the c - terminal portion of s6 and the c - linker / cnbd regions have been considered as possible candidates ( viloria et al .
, 2000 ; tristani - firouzi et al . , 2002 ; saenen et al . , 2006 ; alonso - ron et al . , 2008 ; de la pea et al . , 2011 ; gustina and trudeau , 2011 )
therefore , the long cytoplasmic domains of herg exert important contributions to its unusual gating properties , supporting the interpretation that physical interactions between cytoplasmic domains and/or between them and the transmembrane channel core may also constitute an essential component of the gating machinery in other kv channels .
due to their accessibility to inner cell components , the cytoplasmic domains of kv channels are obvious targets for cell - physiological control of channel function .
some illustrative cases are the regulation of kv1.1 inactivation by concurrent direct binding of syntaxin and g protein subunits ( michaelevski et al . , 2002 ) , the inhibition of human kv10.1 channels by ca / calmodulin binding to the n- and c - terminal structures ( ziechner et al . , 2006 ) , the dependence of kv11.1 rerg hormonal modulation on the presence of the n - terminal splicing variant erg1b ( kirchberger et al . , 2006 ) , and
the modulation of kv2.1 gating by binding of the snare complex components to the cytoplasmic channel tails ( he et al . , 2006 ) .
also , two paradigmatic examples , the calmodulin modulation of kv7 channels via their c - terminus , and the widespread regulation of channel function by protein phosphorylation , are considered next .
kv7 ( kcnq ) channels are a subfamily of kv channels of great physiological importance in the kidney , gastro - intestinal tract , brain , and heart .
in fact , there are several mutations in human kv7 genes that lead to cardiac and neurological disorders such as long - qt syndrome and neonatal epilepsy ( reviewed in ashcroft , 2006 ; haitin and attali , 2008 ; maljevic et al . , 2010 ) .
kv7 channels exhibit , as a distinctive feature , a c - terminus much longer than that of other kv channels , which is critical for kv7 assembly and trafficking , and also gating ( reviewed in haitin and attali , 2008 ) , and thus some kv7 associated channelopathies involve mutations or alterations in this domain ( wen and levitan , 2002 ; haitin and attali , 2008 ; zheng et al . , 2010 ) .
the kv7 c - terminus contains several structural motifs including coiled - coils , calmodulin - binding structures , and basic amino acid clusters ( figure 6 ) .
also , calmodulin interacts with the c - terminus of kv7.1kv7.5 ( yus - njera et al . , 2002 ;
haitin and attali , 2008 ) , an association that has also been demonstrated by tirf / fret techniques to take place in living mammalian cells ( bal et al . , 2008 ) . although calmodulin can be constitutively tethered to kv7 channels both in the absence or presence of ca ( wen and levitan , 2002 ; shahidullah et al . , 2005 ) , these interactions are likely to have ca - dependent and ca - independent components ( bal et al . , 2008 ; alaimo et al . ,
thus , it has been proposed that the calmodulin - binding module at the kv7 c - terminus is endowed with a dual regulatory function : a ca sensor function that affects gating , and a second role in folding and trafficking mediated by a binding site with high affinity for calmodulin ( haitin and attali , 2008 ) .
interestingly , the calmodulin - binding module at the kv7 c - termini seems to physically and functionally overlap with a phosphatidylinositol 4,5-bisphosphate ( pip2)-binding site , allowing a more complex and sophisticated modulation of channel properties ( haitin and attali , 2008 ; hernandez et al .
m current , channel activity is enhanced by pip2 and suppressed by ca / calmodulin ( gamper and shapiro , 2003 , 2007 ; zhang et al . , 2003 ; gamper et al . , 2005 ; suh and hille , 2005 ; hernandez et al . , 2008 ) .
in contrast , both pip2 and ca / calmodulin seem to activate kv7.1 channels mediating the cardiac iks current ( loussouarn et al . , 2003 ; shamgar et al . , 2006 )
remarkably , disruption of ca / calmodulin binding to kv7.2 is associated with neonatal epilepsy ( etxeberria et al . , 2007 ; haitin and attali , 2008 ) , and mutations impairing pip2 or calmodulin binding to kv7.1 lead to certain forms of cardiac long - qt syndrome ( park et al .
, 2005 ; ghosh et al . , 2006 ; shamgar et al . , 2006 )
schematic representation of the carboxy - terminal tail structures and/or interaction sites present in kv7 channels .
formation of coiled - coil assemblies at the level of helices c and d is indicated .
the proposed location of the conserved interaction sites with calmodulin ( cam ) and phosphatidylinositol 4,5-bisphosphate ( pip2 ) , with a - kinase anchoring protein 79/150 ( akap79/150 ) in kv7.2 , with the auxiliary subunit kcne1 in kv7.1 , with akap - yotiao in kv7.1 , with ankyrin - g in kv7.2kv7.3 and with ubiquitin - protein ligase nedd4 - 2 in kv7.1 and kv7.2kv7.3 , are also shown .
phosphorylation sites by src kinase in helix a and by protein kinase c in helix b are indicated by an encircled p. phosphorylation constitutes the most common covalent post - translational modification in eukaryotes ( cohen , 2001 ) and it can be an important modulatory mechanism for ion channel function , since the presence of phosphorylation targets for one or more kinases potentially allows for sensitive , dynamic , and reversible tuning of the structural and functional status of the protein ( levitan , 1994 ; jonas and kaczmarek , 1996 ; park et al . , 2008 ; cerda and trimmer , 2010 ) .
the exposure of the cytoplasmic domains to the internal cell environment makes them suitable molecular targets for phosphorylation - mediated modulation of channel activity .
they can also act as sites of interaction with modulatory partners able to recruit targeting proteins such as phosphatases and kinases leading to the phosphorylation - dependent regulation of gating ( kass et al . ,
a c - terminally located leucine zipper is involved in recruiting , via an adaptor protein , a signaling complex including protein kinase a ( pka ) and protein phosphatase 1 , that are required for -adrenergic receptor modulation of cardiac kv7.1 ( marx et al . ,
indeed , several phosphorylated residues located in the n- and c - termini have been recognized as targets for regulation of kv gating properties .
thus , the c - terminal kv2.1 cytoplasmic domain can act as an autonomous domain sufficient to transfer kv2.1-like clustering , voltage - dependent activation , and cholinergic modulation to diverse kv channels ( mohapatra and trimmer , 2006 ) , and direct phosphorylation of its distal end alters channel voltage - dependent activation ( murakoshi et al . , 1997 ) . interestingly ,
differential phosphorylation at a specific subset of sites , instead of the utilization of cell - specific phosphorylation sites , can explain differences in gating properties of kv2.1 in different cell types under basal conditions , and in the same cell type under basal versus stimulated conditions ( park et al . , 2007 ) .
some other cases of gating regulation by phosphorylation of cytoplasmic domains have been less studied but are also illustrative : ( i ) tyrosine kinase - dependent suppression of kv1.2 by phosphorylation of an n - terminal residue has been observed , and antagonization of this effect by recruiting a protein tyrosine phosphatase to the n- and c - termini of the channel has also been shown ( huang et al . , 1993 ; tsai et al . , 1999 ) .
( ii ) inactivation gating parameters of shaker and kv3.4 channels have been reported to be modulated by pka - dependent phosphorylation of the c - terminus and protein kinase c ( pkc)-dependent phosphorylation of the n - terminal inactivation particle , respectively ( covarrubias et al . , 1994 ; drain et al . , 1994 ; beck et al . , 1998
( iii ) it is known that some n- and c - terminal kv4.2 residues can be phosphorylated by pka and mapk , respectively , and that some cross - talk between these phosphorylation loci can exist ( adams et al . , 2000
; anderson et al . , 2000 ; schrader et al . , 2009 ) , but the functional consequences of such an interaction still remain obscure .
( iv ) ca / calmodulin - dependent protein kinase ii phosphorylates c - terminally located sites of drosophila
eag channels and modulates their function ( whang et al . , 2002 ) .
( v ) it has been shown that phosphorylation of the s4s5 linker of neuronal kcnq ( kv7 ) channels inhibits them ( surti et al . , 2005 ) .
( vi ) the state of phosphorylation of the kv1.1 c - terminus regulates the extent of inactivation conferred by the kv1.1 subunit through a mechanism related to the interaction of the channel complex with the microfilaments via a psd - like protein ( jing et al . , 1997 ) .
all these data constitute overwhelming evidence that phosphorylation of cytoplasmic domains is a widespread mechanism of reversible gating modulation in kv channels .
a promising addition to the study of channel modulation by phosphorylation is the use of phosphoproteomic approaches to identify the channel target residues of the phosphorylation events ( reviewed in cerda and trimmer , 2010 ; cerda et al . , 2010 ;
this method has allowed identification of n- and c - terminal phosphosites on subunits of kv1.1kv1.6 , kv2.1kv2.2 , kv3.1kv3.4 , kv4.1kv4.3 , and kv7.2kv7.5 , although the functional significance of most of these phosphosites has not yet been determined .
this aspect is quite relevant , since even though a phosphorylation mechanism may be involved in channel regulation , it is possible that the phosphate modification does not take place in the channel protein itself ( vacher and trimmer , 2011 ) . on the other hand ,
an agonist - dependent change in the basal phosphorylation level of the channel molecule may not cause a concomitant modification of the gating or other channel properties .
an illustrative case of some of these concerns is provided by the hormonal modulation of the kv11.1 ( erg ) channel gating .
thus , early studies about the mechanism of erg channels inhibition by thyrotropin - releasing hormone in adenohypophysial cells , pointed to a phosphorylation event as determinant of the hormonal induced response ( delgado et al . , 1992 ; barros et al . , 1993 , 1998 ) .
further work demonstrated that the cytoplasmic n - terminal proximal domain was required for hormonal modulation of herg gating ( gmez - varela et al . , 2003 ) .
subsequently , it has been proposed that the pkc - dependent modulation of herg is exerted either independently of direct phosphorylation of the channel protein itself in xenopus oocytes ( thomas et al . , 2003 , 2004 ) , or via a direct phosphorylation of the pore forming herg subunits in mammalian cells ( cockerill et al . , 2007 ) .
in this sense , the possibility that the cell types differentially supply molecular components of the signaling pathways involved in the channel modulation ( miranda et al . , 2005 ) , should also be considered .
interestingly , a direct phosphorylation of the herg cytoplasmic domains by pka is involved in the regulation triggered by activation of -adrenergic receptors ( thomas et al . , 1999 ;
cui et al . , 2000 , 2001 ; kiehn , 2000 ; kagan et al . , 2002 ;
karle et al . , 2002 ) , and some signaling cross - talk between pkc and pka can not be excluded . in summary , either by directly affecting the gating machinery or by influencing it in response to cellular modulators , kv cytoplasmic regions constitute important regulators of voltage - dependent channel gating .
unfortunately , the striking advances characterizing the tridimensional structures of some channel core constituents have not been paralleled by similar success regarding the architecture of the cytoplasmic regions . whereas physical interactions between the cytoplasmic domains and/or between them and the transmembranal channel core clearly constitute essential components of the gating machinery , details about dynamic conformational rearrangements in these regions during channel functionality remain mostly unknown .
combining structural studies with other fluorimetric , spectroscopic , and biophysical functional approaches could provide a better understanding of the dynamic steps connecting the discrete conformations resolved in the crystallographic structures .
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 . | the basic architecture of the voltage - dependent k+ channels ( kv channels ) corresponds to a transmembrane protein core in which the permeation pore , the voltage - sensing components and the gating machinery ( cytoplasmic facing gate and sensor gate coupler ) reside .
usually , large protein tails are attached to this core , hanging toward the inside of the cell .
these cytoplasmic regions are essential for normal channel function and , due to their accessibility to the cytoplasmic environment , constitute obvious targets for cell - physiological control of channel behavior . here
we review the present knowledge about the molecular organization of these intracellular channel regions and their role in both setting and controlling kv voltage - dependent gating properties .
this includes the influence that they exert on kv rapid / n - type inactivation and on activation / deactivation gating of shaker - like and eag - type kv channels . some illustrative examples about the relevance of these cytoplasmic domains determining the possibilities for modulation of kv channel gating by cellular components
are also considered . |
parkinson 's disease ( pd ) is the second - most common neurodegenerative disease , and its motor characteristics include resting tremors , rigidity , bradykinesia , and postural instability
it has been suggested that losses in proprioception may result from a lack of proper regulation of motor control and body reflexes .
for example , proprioception of the knee joint is essential for neuromotor control of the lower limbs .
therefore , assessing proprioception may play an important role in evaluating changes in postural instability , gait , and risk of falls .
few studies have assessed proprioception in individuals with pd compared to healthy individuals [ 413 ] .
the relationship between proprioception and the severity and laterality of motor symptoms and the type of predominant symptom ( akinetic rigid versus tremor ) has not been evaluated in previous studies . the present study aimed to evaluate knee proprioception in individuals with pd and the association between proprioception and motor symptoms , cognitive status , postural instability , and disease severity .
this controlled , cross - sectional study was approved by the ethics committee of the universidade federal de cincias da sade de porto alegre , number 988/12 , and was performed according to the code of ethics of the world medical association ( declaration of helsinki ) .
this study consisted of individuals with parkinson 's disease group ( pg ) and a control group ( cg ) made up of healthy age- and sex - matched individuals .
the number of participants needed for this study was determined using a calculation based on the results of the study by duman et al .
( 2012 ) , which evaluated kinetic postural proprioception in individuals with knee osteoarthritis and established a target angle of 45 for knee flexion .
the authors found that the standard deviation for the angular error was 1.60 for the left knee and 1.24 for the right knee .
we adopted a margin of error of 0.5 for each standard deviation and assumed a significance level of 0.05 ; therefore , the total number of individuals needed for this study was 20 participants in each group .
all participants who agreed to participate in the study signed an informed consent form and were scheduled for the procedure .
the pg participants included individuals of both sexes with a clinical diagnosis of pd according to the london brain bank criteria and were ranked between stages 1 and 3 according to the hoehn and yahr staging scale . for both groups ,
the inclusion criteria were as follows : age older than 45 years , ability to walk alone without the assistance of devices , a score of 26 points or higher on the montreal cognitive assessment scale , and the cognitive ability to understand the tasks .
individuals with a history of knee surgery or lower limb fractures in the past 6 months or amputation of any part of the leg , who presented with any restricting pain at the time of the assessment , who were not right - handed , or who had any other neurological disease or diabetes mellitus were excluded from both groups .
pd patients were classified into the tremulous form or akinetic rigid type depending on the predominant symptoms , which were assessed using the updrs scale .
all ratings were performed 1 hour after taking antiparkinsonian medication for patients in the on phase .
the participants in the pg were assessed through an interview and scales to assess cognitive ability ( montreal cognitive assessment , moca ) , motor function ( unified parkinson 's disease rating scale , updrs ) , postural instability ( pull test and stabilometric analysis ) , and disease stage ( hoehn and yahr scale ) .
kinetic postural proprioception of knee flexion and knee extension were assessed using an isokinetic dynamometer ( biodex multi - joint system 4 pro ) .
the researcher who conducted the proprioceptive assessment of the participants was blinded to the other ratings , which were performed by a second evaluator .
the cg participants responded to an anamnesis and moca scale , and they were subsequently evaluated for kinetic postural proprioception and postural instability .
static balance was assessed by analyzing oscillations of the center of pressure ( cop ) using a baropodometer ( footwork ist / am3 intermetique ) .
the baropodometer consists of a pressure platform that uses capacitive sensors with a thickness of 4 mm , an active area of 490 mm 490 mm , and a sampling rate of 40 hz .
the participants were instructed to remain immobile for 30 seconds using self - selected lateral spacing of the feet , a quasistatic posture on the baropodometer platform , and a constant gaze at a point on the wall located 3 m in front of the person at eye level .
three measurements were performed with a 1 min interval between each measurement , and an average cop value was calculated for the three assessments .
kinetic postural proprioception was evaluated with an isokinetic dynamometer according to the manufacturer 's guidelines ; the same method was used in both groups .
specifically , to test the position sense of the knee , the participants remained seated in a chair tilted to 70 with their hips and knees flexed , and one leg was attached at the ankle using specific support equipment . for testing , the lower limb was stabilized , and the knee remained aligned with the mechanical axis of the dynamometer through a point marked on the lateral femoral condyle in the sagittal plane ( figure 1 ) .
a single examiner , who was blinded to the other tests , performed the experiments , and the assessment orientations remained the same for all the participants .
the equipment was programmed for the proprioceptive assessment of the right and left knees with 45 and 75 of flexion as the testing parameters .
initially , the lower limb was positioned at the reference angle ( 90 for knee flexion ) .
subsequently , the target angle was reached with the help of a device at a speed of 10/s , and the leg remained in this position for 15 seconds to allow the patient to memorize it .
once the patient memorized the position , the limb was passively returned to the reference angle and remained in this position for 10 seconds to rest .
then , the participant was asked to place the limb at the memorized target angle and press the stop button on the equipment to record the angle reached .
the first round of this process was performed for the participants to become familiar with the test .
after the training period , the participants performed three additional attempts , and we calculated the difference between the original target angle and the angle reached , the angular error , and the average proprioceptive error in both tests ( 45 and 75 ) for the three attempts for each lower limb and for bilateral proprioception .
only nonparametric tests were used for the data analysis , eliminating the requirement of a normal distribution . to compare the control and patient groups
, we used the wilcoxon test for paired samples . to compare the variables within the group of patients
, we used the mann whitney and kruskall - wallis tests , for comparisons between two or more variables , respectively .
the spearman correlation was used to assess the relationship between two quantitative characteristics of the patients .
the sample consisted of 40 participants ( 20 pd patients and 20 age- and gender - matched controls ) .
there were no statistically significant differences between the pd and control group in terms of cognition ( p = 0.072 ) .
the mean duration of disease among the individuals with pd was 6.1 years ( table 1 ) .
half of the pd participants ( 10 ) presented with initial symptoms on the right side and the other half presented with initial symptoms on the left .
all participants in the pg were undergoing some type of physiotherapy treatment for motor disorders arising from pd , whereas none of the individuals in the cg were undergoing physiotherapy because they were healthy subjects .
the lateral predominance of motor symptoms at the time of assessment remained in the same proportion .
based on the participants ' reports during the anamnesis , the first noticeable symptom of pd was rigidity in the majority of the participants ( 55% ) . with respect to the analysis of the center of pressure ,
oscillations of the cop were higher in patients with pd compared to controls ( p = 0.002 ) .
pd patients had higher angular errors in the proprioceptive assessments than the participants in the control group ( table 2 and figure 2 , p = 0.002 ) .
pd patients had impaired proprioception performance compared with the control participants , which was observed by analyzing both the side less affected by the disease ( p = 0.050 ) and the side more affected by pd ( p = 0.004 ) .
the significant differences between the pg and the cg were observed even when the side with the initial symptoms of pd was analyzed ( p = 0.040 for the right side and p = 0.006 for the left side ) .
the analysis of the factors correlated with angular proprioceptive errors in pd is shown in table 3 and described separately in the following .
predominant motor symptoms . regarding the type of parkinsonian symptom ( tremor , rigidity , or bradykinesia ) that most interfered with proprioception , patients with tremor as the predominant symptom had larger angular proprioceptive errors ( p = 0.017 ) .
we verified that there is a statistically significant correlation between proprioceptive deficits and the degree of staging of the more affected side ( r = 0.461 , p = 0.041 ) and in the bilateral analysis ( r = 0.054 , p = 0.011 ) .
impaired proprioception was observed in both sides and was significantly correlated with the degree of functionality as assessed using the updrs ( p = 0.027 in the bilateral analysis ) .
other variables .
the duration of the disease , presence of dyskinesia , on / off phenomenon , oscillations in the cop , and pull test results did not influence the proprioceptive angular errors .
in the present study , pd patients had higher angular errors in proprioception than healthy individuals .
pd patients also presented with higher postural instability when evaluated using oscillations of the cop .
pd patients showed greater impairment in reaching the target knee angle than did the controls .
this deficit in reaching known targets has also been found in other studies [ 46 , 8 ] .
these results support the existence of a proprioceptive deficit in pd patients . the proprioception impairment in pd patients was present even in the side less affected by the disease . at the time of the proprioceptive assessment ,
some authors have suggested that antiparkinsonian medications may negatively affect the proprioceptive system in pd [ 4 , 17 , 18 ] .
however , some studies have suggested that proprioception impairment in pd patients could be related to levodopa - induced dyskinesia .
we observed that participants who had tremor as the first noticeable symptom of pd had the largest proprioceptive errors . to date , no previous studies have linked the first motor symptom of pd with proprioceptive changes caused by the disease .
bradykinesia and postural instability are the most common motor symptoms associated with deficits in proprioception [ 7 , 10 , 11 , 19 , 20 ] .
it is important note that rigidity is always a sign and not a symptom although patients reported this as a symptom and most likely this represents a misperception of bradykinesia .
determining the most common , disabling symptoms or the symptoms that may influence other motor disorders is important for planning physiotherapy treatment , which , according to the literature , should be focused on the main motor disorders reported by the patient .
one possibility for the absence of a correlation between the cop results and the pull test with angular errors of proprioception could be the compensation of the visual system in the postural stability tests .
it is known that the visual system aids in minimizing proprioceptive deficits and postural reactions because vision compensates for instabilities , preventing falls .
therefore , the results of the postural instability test would likely have been different if the tests were conducted without the influence of vision , in which case there might have been a relationship between postural instability and the proprioceptive alteration of parkinsonian participants .
postural instability in pd is not fully understood ; however , it is believed to be the result of a complex interaction between compensatory strategies and the impairment caused by the disease at different levels of the nervous system [ 21 , 22 ] .
several posturographic studies investigating cop [ 23 , 24 ] in both static and dynamic conditions have shown that pd patients sway significantly more than healthy subjects because they tend to exceed their limits of stability to a much greater extent .
these results are in accordance with our results , which showed that patients with pd had greater oscillations of the cop than the control subjects .
the impaired motor ability of the participants in the pg ( based on the updrs - iii scale ) and more severe disease staging was significantly correlated with higher angular errors in the proprioceptive assessment .
this relationship between impaired motor symptoms and higher deficits in proprioception was also observed in previous studies [ 4 , 12 , 20 ] .
although the severity of the disease influenced the proprioceptive aspects in the sample , the time of diagnosis was not significantly associated with proprioception .
this finding may be because sensory deficits increase with duration of disease , and the mean disease duration of the study participants was relatively low . from the findings of this study and
based on previous studies , we observed that there was a proprioceptive deficit in pd and , therefore , in the joint position sense .
however , the real origin of this alteration is still unknown because the function of the muscle spindles , particularly the receiver of the joint position sense , appears to be normal in pd .
in addition , there is no evidence of the existence of an alteration in the ascending somatosensory pathways in pd .
some authors argue that proprioceptive deficits in pd may be associated with dysfunction in the sensory integration of cues used to guide movements within the basal nucleus ( bn ) because intact bn circuitry is essential for the perception of joint position and movement , and damage to this circuit can result in kinesthetic deficits .
therefore , the bn could be responsible for alterations in proprioception in individuals with pd .
however , if the bn was responsible for the proprioceptive alterations in pd , studies that evaluate the effect of dopaminergic medications on proprioceptive deficits should find similar results for all patients , regardless of disease severity .
therefore , some authors [ 11 , 12 , 26 ] have suggested that dopaminergic dysfunction within the bn is not primarily responsible for dysfunction of the proprioceptive system . to corroborate this hypothesis ,
jacobs and horak hypothesized that disturbances in proprioception in pd may be primarily due to a dysfunction in the supplementary motor area ( sma ) because this is one of the main projections of the bn and is sensitive to dopamine and significantly contributes to the planning and direction of movement .
furthermore , the cells of the sma showed a loss of selectivity in the response to passive joint movement in parkinsonian monkeys , which demonstrates the importance of the sma in the proper processing of proprioceptive inputs . in this study , we did not find any significant relationship between proprioceptive errors and disease duration , the presence of dyskinesia and motor fluctuations , oscillations of the cop , and the pull test .
the present study showed that the participants with pd have higher angular proprioceptive errors , which supports the hypothesis that pd affects the proprioceptive system .
there were also significant differences in fluctuations of the center of pressure between individuals with pd and healthy controls , indicating that individuals with pd tend to have greater postural instability than healthy people .
the analysis of the factors that have a relationship with proprioception showed that tremors were the dominant symptom and that impaired motor ability significantly influenced the presence of proprioceptive alterations in the parkinsonian individuals . in this study , the postural instability in individuals with pd and the relationship between proprioceptive deficits and motor skills and the severity of pd highlight the importance of focusing on these aspects during the rehabilitation process .
based on our results , we suggest that physiotherapy for pd should include proprioceptive and balance exercises to improve motor skills and , in turn , improve the functionality and quality of life of individuals with pd . | background . changes in proprioception may contribute to postural instability in individuals with neurological disorders .
objectives . evaluate proprioception in the lower limbs of patients with parkinson 's disease ( pd ) and the association between proprioception and cognitive ability , motor symptoms , postural instability , and disease severity . methods .
this is a cross - sectional , controlled study that evaluated proprioception in pd patients and healthy age- and sex - matched individuals .
kinetic postural proprioception of the knee was evaluated using an isokinetic dynamometer ( biodex multi - joint system 4 pro ) .
participants were evaluated using the montreal cognitive assessment ( moca ) , the hoehn and yahr rating scale and postural instability ( pull test and stabilometric analysis ) , and motor function ( updrs - iii ) tests . results .
a total of 40 individuals were enrolled in the study : 20 pd patients and 20 healthy controls ( cg ) .
the pd patients had higher angular errors on the proprioceptive ratings than the cg participants ( p = 0.002 ) .
oscillations of the center of pressure ( p = 0.002 ) were higher in individuals with pd than in the controls .
proprioceptive errors in the pd patients were associated with the presence of tremors as the dominant symptom and more impaired motor performance . conclusion .
these findings show that individuals with pd have proprioceptive deficits , which are related to decreased cognitive ability and impaired motor symptoms . |
health care is becoming more patient - centered , or at least that goal is being more widely articulated .
patients ' preferences and rights to play a central role in health care decisions are being more widely discussed .
one area in which patient values are extremely important deals with the relative emphasis placed on the alternative outcomes of care .
whether judging how successful treatment has been or in weighing the risks and benefits of alternative treatment strategies , some means of summarizing the overall effects of care is necessary .
as chronic disease becomes the dominant form of illness , functional status , which may encompass a wide variety of domains ( including physical , emotional , and social functioning ) , assumes an ever - larger role as a means of describing outcomes .
interest is growing in examining the more global effects of disease and treatment on overall functioning , and therefore attention has been focused on activities of daily living ( adls ) and instrumental activities of daily living ( iadls ) and the need for a summary measure . in general , there is wide appreciation that such a measure should cover a broad spectrum , ranging from the basic activities needed to ensure independent function ( adls ) to more complex activities ( iadls ) ( katz et al . , 1963 ; lawton and brody , 1969 ) .
these two levels of function have been shown to form a general hierarchy , with iadls being lost first ( spector et al . , 1987 ) .
the basic adls have similarly been shown to exhibit guttman properties of ordinality ( katz and stroud , 1989 ) .
the most commonly used summary measures of these functions have relied on simple sums of areas of dependency .
for example , the inability to carry out 3 of 5 specified adls was proposed as the basis for eligibility for long - term care ( ltc ) benefits under the unsuccessful clinton health proposal ( stone and murtaugh , 1990 ) . however , a simple summing , although administratively facile , loses substantial information .
such a step relies on the assumption that all included activities are of equal weight , or that they can only be lost in a fixed ordinality , such that the sum represents a cumulative report .
even a casual examination of one 's own experience and beliefs would reveal that different adls make quite distinct contributions to overall disability .
for example , some activities occur several times a day ( e.g. , using the toilet or eating ) , whereas others occur less often ( e.g. , transferring or dressing ) .
some iadl activities occur less often than daily ( e.g. , managing money or shopping ) .
the traditional approach to simply summing dependencies presents at least two types of problems : as already noted , it assumes that each type of functional loss is equivalent , and it requires that some arbitrary cut point be established to distinguish dependent from independent . for example , one might decide that needing a little assistance to perform an activity constitutes dependency , or , alternatively , the threshold for dependency might be set as needing a great deal or complete assistance .
these various thresholds would result in quite different patterns of inclusion and exclusion for the counts of dependencies .
not only may different levels of dependency be treated as equivalent , but also , useful information can be lost , especially if the threshold is set high . to create a summary adl measure that would combine different levels of dependency within each adl and iadl item
, we conducted a survey of 27 professionals , representing the variety of disciplines involved in ltc .
the professional respondents included 2 physicians ( a psychiatrist and a geriatrician ) , 10 ltc administrators , 3 nurses , 2 social workers , a physical therapist , and a psychologist .
we asked each respondent to rate the relative importance of both adl and iadl domains and the levels of function within each domain , in terms of their contribution to overall disability ( finch , kane , and philp , 1995 ) .
this ratio scale was used as the dependent variable in studies of the outcomes of post - hospital care ( kane et al . , 1996a , 1996b ) .
we chose to poll professionals on the basis that they were the ones who would be held accountable .
however , a reasonable criticism could be raised about the importance of ascertaining the values from older consumers as well . to address this concern , we interviewed frail older persons , using the same approach originally used with the ltc professionals .
our original goal was to recruit a sample of persons living in nursing homes , but too few of these individuals were able to perform the necessary cognitive tasks .
instead , we used a sample of older persons living in sheltered housing settings attached to nursing homes .
these people were living on their own but receiving congregate meals and assistance and were judged to be at risk for ltc . although members of this group were not incapacitated to the point of requiring institutionalization , they were no longer living independently in the community .
also , several of the adl and iadl items used , such as house cleaning , do not apply to those living in a total institution such as a nursing home .
residents in two sheltered housing programs attached to larger ltc campuses in minneapolis were invited to participate in this study .
written notices were posted in several places in each setting , and staff helped to recruit volunteers .
older persons opting to participate met with project staff in each housing program 's common area at a designated time . at each meeting the purpose of the study
the respondents completed the forms independently during the session with a project staff member available to answer any questions that arose .
a total of 61 at - risk elderly participated in the research ( 30 in one sheltered housing site and 31 in the other ) . out of the 61 forms , 10 had to be discarded because of incomplete data , yielding a total of 51 completed forms .
to preserve anonymity no demographic data on the participants were collected . the form used in the study
the first section of the form contained a list of 13 adl and iadl items ( table 1 ) .
as a reference point , the inability to bathe oneself was assigned an arbitrary score of 500 .
participants were then instructed to rate the remaining 12 items in terms of their severity relative to the base established in the bathing item .
if they believed that debilitation resulting from a given adl or iadl presented was more severe than the bathing item , they were instructed to give it a higher score . alternatively ,
this first section provides a comparative weighting for all of the adls and iadls ( similar to guttman scaling ) .
the only limitation placed on the values that would be assigned was that nothing would be less than zero .
the second section of the form asked the participant to assign a value between 0 and 100 to various dependency levels associated with each of the 13 adl and iadl items . for each adl and iadl item ,
the extreme categories were preassigned values ; for complete independence , e.g. , needing no assistance to bathe oneself , a value of zero was provided ; alternatively , for complete dependence , e.g. , unable to bathe oneself at all , a value of 100 was provided . between these extremes , one or two levels of assistance ( e.g. , needing a little help to bathe oneself , needing a lot of help to bathe oneself )
participants were instructed to assign a value between 0 and 100 to these two levels of assistance .
this portion of the questionnaire provided a relative weight for the dependence level for each adl and iadl .
measuring dependence in this manner takes into account that needing a little help for one activity is not the same as needing a little help for another activity , thus the measure of dependence is specific to each adl and iadl . to compute the scoring to be used in the analysis ,
the value assigned in the first section was multiplied by the dependency value assigned in the second .
this produces three or four scores for each adl and iadl ( one for each level of performance ) . in the state of complete independence for each adl and iadl ,
one of these scores was always zero and hence was not used in the analysis .
the remaining 34 scores can be ordered to generate a ranking for each of the adls and iadls that incorporates an assessment of the importance of each item relative to the others ( section one ) , as well as the item - specific weighting associated with dependence ( section two ) .
the analysis of the distribution of the 34 adl variables among the groups drew upon three separate statistical techniques .
a non - parametric test , wilcoxon sign rank test ( hollander and wolfe , 1973 ) , and two parametric tests : bonferroni means test and ordinary least - squares regression were used to evaluate the differences between groups . to compare the relative value of items developed through this process with another widely used weighted scale , the barthel index ( mahoney and barthel , 1965 ) , we used seven of the items from the barthel index that approximated those used in the current scale .
the maximal value for these seven items was obtained and the relative value of the intermediate levels was calculated by dividing each by the total . because the barthel assigns greater weights to independence , and our approach assigns higher scores to greater dependence , we had to reverse the barthel scoring approach to facilitate comparison .
they are quite different . in 10 instances the consumer ratings are significantly different from the professional ratings . in four instances
the consumer ratings are higher for the iadl items , and the professional ratings higher for the more disabled levels of the adls
. this pattern can be better seen when the two sets of ratings are displayed graphically as in figure 1 .
the level of agreement can also be explored in terms of the relative ranking given each rating by the two groups .
was plotted against the mean rank of the other group assigned to the corresponding item . the overall slope ( i.e.
, the correlation coefficient calculated ) is quite high ( 0.816 ) . to get a better idea of where the differences in the two groups ' ratings lay , we compared the ratings for the importance of complete disability in each major domain .
the level of agreement with regard to rank order is quite high , although once again the values assigned by consumers tend to be higher for iadls , whereas the experts assign higher scores for the adls .
one way to reduce the apparent variation between these groups might be to norm the two sets of scores , reducing each to a comparable scale by dividing each set of ratings by the total score assigned by that group .
however , such a step would not likely affect the distributions , because the total scores for the two groups are well within the same order of magnitude .
to estimate the effects of using one or the other set of weightings , we applied the weights to three hypothetical patients .
one was chosen to represent a mildly disabled person with only iadl needs ; the second was a severely physically disabled person ; and the third was someone with functional losses associated with dementia .
as shown in table 3 , the consumer - based score is much higher than that using the experts ' weightings for the mildly disabled case ( consistent with the earlier observed bias ) .
the scores are also identical for the severely disabled person , despite the experts ' higher weightings for several adls .
the consumers ' weightings also led to higher scores for the dementia case , again likely reflecting the iadl dependencies . another way to compare the effects of using consumers ' or experts ' values
perhaps the best known disability scale is the barthel index , which was created to measure the effectiveness of rehabilitation ( granger , albrecht , and hamilton , 1979 ) .
just the opposite of the approach we have used , it assigns maximal points to states of independence in each domain .
thus , its point values distinguish levels of independence but render total dependency in each dimension equivalent ( i.e. , by assigning all values of zero ) .
( by contrast , we would argue that it makes more clinical sense to treat independence in all domains as equivalent and discriminate among causes of dependence . ) only 7 of the 10 barthel items correspond to items in the current scale .
( grooming , ascending or descending stairs , and bowel control are not included in our domains . ) to create a more comparable scale , we reversed the barthel weights , assigning the maximum value used by mahoney and barthel ( 1965 ) for each item to the most dependent state .
table 4 presents a comparison of the item values generated from consumer and expert ratings and the corresponding value from the modified barthel score .
both are created such that complete dependence in all domains yields a value of 75 ( i.e. , the value of the original 7 barthel items ) .
using either set of values produces quite different relative emphasis from those based on the barthel weights for complete dependence .
compared with the barthel values , feeding , using the toilet , bathing , and incontinence receive more importance , whereas transferring and walking receive less .
this difference may reflect the rehabilitation context for which the barthel index was originally developed , as opposed to the more general geriatric context of the new scale .
the barthel state for needing help seems to correspond closer to needing a little help in the new scale .
the same distinctions between professional and consumer values noted earlier apply here as well , although they are muted by the extensive rounding .
the samples of respondents for both the professional and consumer groups were chosen on the basis of convenience and can not be said to accurately represent any larger population .
ideally , one might have wished to tap the values of persons already actively involved in receiving ltc , but the rating task proved too complex for most of the nursing home residents we first approached .
instead , we were left to work with older persons who were somewhat more intact but nonetheless were living in adjacent facilities in situations that afforded at least some components of assistance with daily living . to address concerns about representativeness
it should also be noted that the samples of professionals and consumers were totally independent .
this is not a study of agreement about the rating of dependency for specific individuals .
rather , it addresses the emphasis that different constituencies place on the different elements and the levels of dependence within each .
scores that span a range from 0 to 1.5 million are too large to be clinically useful . for practical purposes of communication
, it may prove easier to take advantage of the ratio scale properties to convert the scores developed into a more familiar range of 0 to 100 .
because the emphasis was on dependence , higher scores were assigned to reflect greater dependence .
( 1963 ) , but , as noted in the text , another widely used scoring system , developed by mahoney and barthel ( mahoney and barthel , 1965 ) does just the opposite , assigning higher values to greater independence .
the results from these admittedly convenient samples suggest that older consumers and aging experts place quite different importance on various aspects of disability .
consumers viewed dependency in iadls as more of a loss than did the professionals ; the professionals were more concerned about inability to perform basic adls .
these findings raise serious red flags about the ability of those providing care to assume that the recipients share their values about what is important .
providers who feel comfortable about exercising judgments on behalf of their clients may be motivated to give some additional thought before undertaking this task .
the major importance of using some approach to value weighting and being sensitive to whose values are incorporated lies in developing measures that reflect the importance of outcomes .
outcomes should reflect the importance of the individual components to those who must live with the consequences .
although adl scores may also be used as criteria for allocation of ltc , value weighting is less relevant in that pursuit .
the relative importance of individual adl and iadl items in determining the need for ltc is best estimated by examining the statistical relationship of each to maintaining an independent existence or receiving regular assistance .
although the concepts seem closely related , the elements that are believed to be most important to a person may not necessarily be those that lead to a need for institutional care . in an era of consumer - centered care , it behooves health professionals to understand more about the preferences of their clients .
if patients and providers of care hold different views about what types of losses are most important , it seems unlikely that both parties will agree on the best course of treatment , if that treatment has any specific effects .
if one assumes that ltc produces only diffuse results with little specificity , then the differences in preferences may matter little .
indeed , if such an assumption is correct , little measurement is needed , because one type of effect will be much like another . the only determining feature will be the nature of the order of functional loss , which must presumably occur independently of disease or treatment .
however , if one adopts a more focused model , in which treatment is intended to address particular problems , then the major benefits will be measured by the relevant outcomes , but consideration needs to be given to the effects on overall function as well . some method for summarizing this state should prove useful .
if the summary is sensitive to the source of the values , as indicated here , this effect should be carefully considered in developing the summary procedure . from a policy perspective ,
an outcome system that incorporates the values of the appropriate constituency will be more likely to detect relevant changes .
the finding of substantial differences in professional and consumer ratings suggests at the least that a great deal more discussion is needed .
the results of this study suggest that professionals can not adequately represent the values of consumers .
the question then is how to best integrate consumer values . at one level , some argue that consumers should make the ultimate choices about what is done to or for them . in most instances
however , making such a decision correctly involves a number of complicated steps and a great deal of information .
for each one , the values assigned to each risk and benefit must be assessed . in practice , few decisions are made this precisely or deliberately . even those who might wish to pursue a structured decision process
will find themselves missing many critical elements of information about the efficacy of alternative approaches .
one step in that process is helping consumers to identify what they truly hope to accomplish as a result of the care they seek . to estimate these goals , consumers have to first articulate what they seek and then assign some type of importance weights to the various competing ends .
the underlying premise of this effort is that one important role for consumers lies in establishing the relative priority assigned to various outcome states .
if health care professionals are to become more active advocates of better decisionmaking for consumers and facilitators , they will need some sort of structured approach to the process .
the techniques described here represent one step in that direction . although one may want or need to go through the entire rating exercise each time an important decision is made , some method of deliberately considering alternatives and estimating their relative importance is warranted .
pressures of time and money will make it unlikely that deliberate decisionmaking is employed often .
consumers will have to accept more responsibility for the results of decisions in which they participate actively . whether expressed in terms of increasing satisfaction or avoiding malpractice litigation
the differences in the means are useful in pointing to discrepancies that should be addressed when considering the extent to which professional judgment can be used in lieu of specifically eliciting consumer preferences .
the ultimate role for consumer preferences would be to allow each individual to identify the aspects of outcomes that he or she wants to maximize in a given situation .
rather than assigning some uniform set of weights based on the average values of a dominant party or some type of compromise , it might be preferable to allow each patient to establish his or her own values about the relative importance of different outcomes .
such an approach would make it difficult to compare successes among different patients unless one were prepared to assess outcomes specifically in terms of the extent to which they met patient preferences .
an issue worthy of future research would be the extent to which consumers ' values vary among themselves .
the present sample is too small and too limited to test the effects of varying levels of consumer disability on perceptions of importance .
other work that examines patients ' utilities suggests that these can differ widely between those who have a given problem and those who seek to avoid contracting it ( torrance , 1987 ) .
similarly , those already quite dependent may view the elements contributing to dependency differently from those who are less impaired . | as the population ages and chronic disease becomes the more dominant form of illness , measures of functional loss and disability assume greater importance in the assessment of both quality of life and the cost - effectiveness of care .
the authors studied the responses of consumers and health care professionals regarding the impact on dependency of various levels of disability .
striking differences in perception were noted , raising concerns about the ability of those providing care to assume that the recipients share their values about what is important .
this study makes clear the need for more research on functional outcome measurements that incorporate the values of consumers . |
unintentional spinal block occurs if the medication intended to be placed in the epidural space enters the intrathecal space.1 this is a serious complication that requires immediate diagnosis and prompt cardiopulmonary resuscitation.2,3 in the obstetric population , this can lead to severe fetal compromise necessitating immediate cesarean section . in a retrospective study of maternal mortality in 19792002 ,
death due to anesthesia was found to be the sixth leading cause of pregnancy - related mortality in the us , and 9% of these deaths were attributed to high spinal blocks following epidural anesthesia.4 currently none of the clinical tests used can reliably differentiate cerebrospinal fluid ( csf ) from other substances in all situations .
chemical analysis of fluid for glucose and protein is unreliable as a means of determining if the fluid is csf.5,6 radiographic studies , with or without intrathecal injection of dye or radioisotope , are not always successful in demonstrating small or delayed csf leaks , and are technically difficult and time - consuming . at present , electrophoresis of fluid from a suspected csf leak combined with immunofixation to detect beta-2 transferrin , a unique csf protein , is a highly specific and widely used method of detecting csf.79 however , this technique is cumbersome , time - consuming , and very expensive , with few clinical laboratories being able to perform this test .
recent studies have revealed that prostaglandin d2 ( pgd ) , an endogenous sleep - promoting substance , accumulates in csf.1012 prostaglandin d2 synthase in the brain catalyzes the isomerization of prostaglandin h2 to pgd , which circulates in the csf.13 although pgd is present in other body fluids , due to the presence of high levels in csf , pgd could be a valuable tool as a marker for the detection of csf traces .
the aim of this study was to determine whether a specific csf marker like pgd has any predictive value when csf is diluted with local anesthetic , saline , and serum .
an in vitro study was performed to identify pgd in csf when diluted with local anesthetic , saline , or serum in a 1:5 and 1:10 dilution .
using an enzyme - linked immunosorbent assay , pgd was reliably identified in all csf samples independent of dilution or diluent .
these results will aid in the development of a detection kit , which will be beneficial for clinicians requiring a rapid and reliable bedside test to detect csf leak .
after obtaining institutional review board approval and patient consent , csf samples were obtained from five healthy patients undergoing spinal anesthesia in the labor and delivery unit of university hospital and analyzed for pgd .
csf samples diluted with various volumes of saline , local anesthetic , and serum were analyzed for the presence of pgd .
the assay was performed by enzyme - linked immunosorbent assay ( cayman chemicals , ann arbor , mi ) .
all chemicals used were purchased from sigma chemicals ( st louis , mo ) . in this assay
, csf samples were diluted with local anesthetic ( bupivacaine ) , normal saline , and serum in the ratios of 1:5 and 1:10 .
pgd levels in the straight and diluted csf samples were analyzed using a pgd - methoxime enzyme - linked immunosorbent assay kit ( cayman chemicals ) .
this enzyme - linked immunosorbent assay is based on the conversion of pgd to a stable methoxime derivative , where treatment of pgd in csf with methoxime hydrochloride will convert pgd into pgd - methoxime .
this stable conjugate is analyzed with rabbit antiserum binding sites specific for pgd - methoxime in the enzyme - linked immunosorbent assay .
because the concentration of the pgd - methoxime tracer is held constant while the concentration of pgd - methoxime varies , the amount of pgd - methoxime tracer that is able to bind to the rabbit antiserum will be inversely proportional to the concentration of pgd - methoxime in the well .
this rabbit antiserum pgd - methoxime ( either free or tracer ) complex binds to the mouse monoclonal antirabbit igg that has been previously attached to the well .
the plate is washed to remove any unbound reagents and then ellman s reagent ( which contains the substrate to ache ) is added to the well .
the product of this enzymatic reaction has a distinct yellow color and absorbs strongly at 412 nm .
the intensity of this color , determined spectrophotometrically , is proportional to the amount of pgd - methoxime tracer bound to the well , which is inversely proportional to the amount of free pgd - methoxime present in the well during the incubation .
csf samples obtained from five healthy patients undergoing spinal anesthesia in the labor and delivery unit of university hospital were diluted in different ratios with saline and with different concentrations of drugs and analyzed for pgd . to evaluate the relative concentrations of pgd in csf samples , initially different concentrations of pure pgd - methoxime conjugate ( see methods section ) were analyzed by methoxime and a standard curve was derived ( figure 1 ) .
pgd in csf and csf with diluents in different ratios was determined using a pgd - methoxime enzyme - linked immunosorbent assay protocol and the values were extrapolated onto the standard curve .
our results show a well defined correlation for the presence of pgd in csf samples .
the pgd marker was also easily identified in csf samples diluted with different eluates ( figure 2 ) .
the fact that pgd was detected in such small amounts of csf shows that this test is highly sensitive .
in this in vitro analysis , pgd was identified in all the csf samples . during the epidural procedure when the dura is punctured with an epidural needle
however , when saline is used for loss of resistance there may be a need to use a test that confirms the presence of csf .
the various physicochemical bedside tests that are currently used to detect the presence of csf in this situation , ie , rate of flow , temperature , tests for glucose and protein , and ph , can not provide reliable identification.5,14,15 when fluid is aspirated from the catheter , even before contamination with local anesthetic , a combination of tests like glucose , protein , and ph may be used .
however , contamination with blood and interstitial fluid can result in positive glucose and protein , as well as a change in ph close to that of csf which may lead to false positive identification of csf and needless repetition of epidural procedures.6 when the epidural aspirate contains local anesthetic , a thiopental precipitation test will form precipitate with local anesthetic but not with csf .
this test was used in the past to differentiate reliably between local anesthetic and csf.6,16 recently , this test has proven to be unreliable with the current change of practice whereby a mixture of dilute local anesthetics and opioids are administered and both false positive and false negative results have been noted , highlighting the potential for serious complications.6,16 the issue is even more complex when the aspirate is obtained during the course of continuous epidural anesthesia . in this situation , the aspirate is a mixture of local anesthetic , opioid , interstitial fluid , blood , and possibly csf .
thus , the bedside test available at present can be misleading . the standard test dose with 1.5% lidocaine and epinephrine 5 g / cc is routinely used to detect subarachnoid placement by producing clinically detectable sensory and motor blockade that is not unsafe for patients .
however , this test dose can be dangerous and difficult to interpret in patients with pre- existing partial block .
furthermore , in parturients , it can produce a dangerously high block requiring aggressive treatment .
richardson et al in their report of high block following a test dose in five parturients raise the question of its safety.17 in our study using the enzyme - linked immunosorbent assay technique , pgd was reliably identified in csf when diluted with local anesthetic , saline , and serum .
although pgd was reported to be present in other biological fluids , our results showed no significant pgd in urine and saliva samples when compared with high levels of this marker in csf . when analyzed by the pgd - methoxime enzyme - linked immunosorbent assay protocol , pgd levels were found to be lower in csf diluted with either serum or local anesthetic , suggesting that serum factors and bupivacaine interfere with the spectrophotometric analysis .
however , the enzyme - linked immunosorbent assay method was able to identify pgd in these diluted samples , also suggesting that this test can be used effectively to determine csf leaks when admixed with different entities , like drugs and serum .
detection of pgd , particularly in epidural eluates , will be of immense value in indicating a potential csf leak to the anesthesiologist . through the development of a rapid detection technique to detect pgd
there are many complications associated with epidural anesthesia . in order to reduce serious complications , such as unintentional spinal blockade , and to ensure safe epidural procedures ,
a rapid detection method is necessary to detect csf in epidural aspirate . in this in vitro study ,
although this kit is not yet clinically available as a bedside test , the possibility of development of such test in the near future is very likely .
furthermore , such a test is of great value not only for anesthesiologists but also in other instances where fluid needs to be identified as csf . | backgroundit is accepted that there is a severe risk of dural puncture in epidural anesthesia . of major concern to anesthesiologists
is unintentional spinal block .
reliable identification of cerebrospinal fluid ( csf ) from the aspirate is crucial for safe epidural anesthesia .
the aim of this study was to determine whether prostaglandin d2 could be clinically used as a marker for the detection of csf traces.methodsafter obtaining institutional review board approval and patient consent , csf was obtained from patients undergoing spinal anesthesia , and blood , urine , and saliva were obtained from normal subjects and analyzed for prostaglandin d2 ( pgd ) .
csf ( n=5 ) samples were diluted with local anesthetic ( bupivacaine ) , normal saline and blood in the ratios of 1:5 and 1:10 .
pgd levels in the csf samples were analyzed with a pgd - methoxime ( mox ) eia kit ( cayman chemicals , mi ) .
this assay is based on the conversion of pgd to a stable derivative , which is analyzed with antiserum specific for pgd-mox.resultsdifferent concentrations of pure pgd - mox conjugate were analyzed by eia and a standard curve was derived .
pgd levels in csf and csf with diluents were determined and the values were extrapolated onto the standard curve .
our results show a well - defined correlation for the presence of pgd both in straight csf samples and in diluted csf ( dilution factor of 1:5 and 1:10).conclusionprostaglandin d2 was reliably identified in csf by enzyme - linked immunosorbent assay when diluted with local anesthetic , saline , and serum , and can be used as a marker to identify the presence of csf in epidural aspirates . |
neuroendocrine tumours ( net ) of the ampulla of vater are considered to be an exceptional pathology .
a total of 139 net of the ampulla of vater ( carcinoids and high - grade neuroendocrine carcinomas ) were identified between 1973 and 2006 in a recent analysis of the surveillance , epidemiology and end results program of the national cancer institute . to date , data in the literature regarding net of the ampulla of vater consist only of case reports [ 2 , 3 ] or very small case series [ 48 ] . currently , there is no standard of care for net of the ampulla of vater , mostly due to their poorly defined natural history .
therapy for these tumours varies from local resection ( endoscopic or surgical ampullectomy ) [ 912 ] to aggressive surgery ( i.e. pancreaticoduodenectomy with lymph node dissection ) [ 4 , 5 , 13 ] .
the prognostic value of locoregional lymph node metastasis or other factors , such as depth of invasion or tumour diameter , are not clearly established [ 1 , 4 ] .
this study reports a single - centre experience of six patients diagnosed with net of the ampulla of vater , staged according to the 2010 world health organisation ( who ) classification and tumour - nodes - metastasis ( tnm ) staging [ 15 , 16 ] ( table 1 ) .
the clinical and pathological findings and the surgical treatment of the patients included in the study are discussed .
furthermore , an analysis of potential factors influencing survival after curative intent surgery for net of the ampulla of vater was performed based on data from the present study and available data from the literature.table 1staging systems for net of the ampulla of vater : who , enets and uicc who classificationnn grade 1 ( low grade)<2 mitoses/10 hpf and < 3 % ki67nn grade 2 ( intermediate grade)220 mitoses/10 hpf or 3 % 20 % ki67nc grade 3 ( high grade ) > 20 mitoses/10 hpf or > 20 % ki67enets staging systemuicc staging systemt primary tumour txprimary tumour can not be assessed t0no evidence of primary tumour t1invasion of lamina propria or submucosa and size 1 cmlimited to ampulla of vater or sphincter of oddi t2invasion of muscularis propria or size > 1 cminvasion of the duodenum wall t3invasion of the pancreas or retroperitoneuminvasion of the pancreas t4invasion of the peritoneum or other organsinvasion in peripancreatic soft tissues or other adjacent organs or structuresn regional lymph nodes nxregional lymph nodes can not be assessed n0no regional lymph node metastasis n1regional lymph node metastasis presentm distant metastasis mxdistant metastasis can not be assessed m0no distant metastasis m1distant metastasis presentstagingstage it1n0m0stage iat1n0m0stage iiat2n0m0stage ibt2n0m0stage iibt3n0m0stage iiat3n0m0stage iiiat4n0m0stage iibt1,2,3n1m0stage iiibany tn1m0stage iiit4any nm0stage ivany tany nm1stage ivany tany nm1who world health organisation classification , enets european neuroendocrine tumour society staging system , uicc international union against cancer staging system , nn neuroendocrine neoplasm , nc neuroendocrine carcinoma , hpf high power fields staging systems for net of the ampulla of vater : who , enets and uicc who world health organisation classification , enets european neuroendocrine tumour society staging system , uicc international union against cancer staging system , nn neuroendocrine neoplasm , nc neuroendocrine carcinoma , hpf high power fields
all patients with a final pathological diagnosis of net of the ampulla of vater were retrospectively identified from a prospectively gathered electronic database established at our department . between 2002 and 2011 (
august 1 ) , 128 patients with malignant tumours of the ampulla of vater underwent pancreaticoduodenectomy at the center of general surgery and liver transplantation , fundeni clinical institute , bucharest , romania . out of these
, 120 tumours were diagnosed as adenocarcinoma ( 93.8 % ) , 2 tumours were diagnosed as gastrointestinal stromal tumours ( 1.62 % ) and the remaining 6 patients were diagnosed with net ( 4.6 % ) .
patient data included age at diagnosis , sex , presenting symptoms , associated pathology , preoperative imaging and biopsy results , type of operation , pathology and follow - up with survival status . computed tomography ( ct ) and/or a magnetic resonance imaging ( mri )
were the main preoperative imaging work - up modalities , with the aim of characterizing the tumour and assessing the presence of involved locoregional lymph nodes or distant metastases .
the pathological data were assessed by the same pathologist ( vh ) , considering the who 2010 classification , the european neuroendocrine tumour society ( enets ) and international union against cancer ( uicc ) staging systems .
all the tumours were immunostained for chromogranin a , synaptophysin and neuron - specific enolase to confirm the diagnosis of net . for grading ,
the ki67 index was assessed using the mib1 antibody as a percent of 2,000 tumour cells in areas where the highest nuclear labelling was noticed .
follow - up protocol after surgery included clinical examination and ct or mri every 3 months in the first year and every 6 months after the first year .
because the number of the patients in the present series was too small to perform a survival analysis and to assess the potential factors influencing the long - term outcome after curative intent surgery for net of the ampulla of vater , we searched the literature for additional available data ( i.e. who classification , enets and uicc staging systems , tumour diameter , t stage , lymph node involvement , survival status , overall and disease - free survival ) .
a total of 37 patients who underwent pancreaticoduodenectomy for net of the ampulla of vater were introduced in a univariate analysis , including our cases ( table 2 ) [ 4 , 5 , 8 , 1721].table 2data of 37 patients with pancreaticoduodenectomy for net of the ampulla of vater , taken into consideration for survival analysisnoreferencewhoenetsuiccdt ( enets)ndfsosstatus1nassar h et al .
nt g2iiibiib0.7t2n12424alive32present studynt g2iiibiib0.8t1n18080alive33present studync g3iia1t1n01824dead34present studync g3iiibiib1.5t2n15252alive35present studynt g1iiaia2t2n03232alive36present studynt g1iiibiib2t3n166alive37present studynt g1iiibiib1t1n144alivewho world health organisation classification , enets european neuroendocrine tumour society staging system , uicc international union against cancer staging system , d tumour maximal diameter ( cm ) , t tumour status , n locoregional lymph nodes status , dfs disease - free survival ( months ) , os overall survival ( months ) , nt neuroendocrine tumour , nc neuroendocrine carcinoma data of 37 patients with pancreaticoduodenectomy for net of the ampulla of vater , taken into consideration for survival analysis who world health organisation classification , enets european neuroendocrine tumour society staging system , uicc international union against cancer staging system , d tumour maximal diameter ( cm ) , t tumour status , n locoregional lymph nodes status , dfs disease - free survival ( months ) , os overall survival ( months ) , nt neuroendocrine tumour , nc neuroendocrine carcinoma numeric data are expressed as median ( range ) .
survival curves were estimated using the kaplan meier method and were compared using the log - rank test . a p value < 0.05 was considered to be statistically significant .
survival curves were estimated using the kaplan meier method and were compared using the log - rank test .
the patients were predominantly male ( male / female ratio = 4:2 ) , with a median age of 54 years ( range , 3268 years ) .
the main presenting symptom was jaundice ( four patients ) , while two patients presented with epigastric pain .
five patients presented with cholestasis syndrome ( minor cholestasis in one patient , major cholestasis in four patients ) . in the patients with jaundice , the total bilirubin level ranged from 3.0 to 17.0 mg / dl ( median 13 mg / dl ) .
preoperative imaging included ct ( two patients ) , mri ( three patients ) or both ( one patient ) .
the ct and/or mri showed the ampullary tumour in all cases ; in patients with cholestasis syndrome , on noticed dilatation of the intra- and extrahepatic bile ducts , along with dilatation of wirsung 's duct ( fig .
no distant metastases to the liver , lung , peritoneum or other organs were observed .
1a magnetic resonance cholangiopancreatography showing an ampullary soft tissue mass ( t ) , with secondary dilatation of the common bile duct ( c ) and main pancreatic duct ( arrow head ) ; b endoscopic retrograde cholangiopancreatography showing an irregular , fusiform stenosis of the common bile duct toward the ampulla of vater , with secondary dilatation ; c endoscopic ultrasound revealing the ampulla with a 8 7 mm hypoechoic lesion ( t ) , not invading the muscularis propria and with secondary dilatation of the common bile duct ( c ) and wirsung 's duct ( w ) ; d histological features ( hematoxylin - eosin , original magnification 40 ) revealing metastases into a locoregional lymph node of a g1 neuroendocrine neoplasm nests of uniforms , polygonal tumour cells with round nuclei and salt and pepper chromatin a magnetic resonance cholangiopancreatography showing an ampullary soft tissue mass ( t ) , with secondary dilatation of the common bile duct ( c ) and main pancreatic duct ( arrow head ) ; b endoscopic retrograde cholangiopancreatography showing an irregular , fusiform stenosis of the common bile duct toward the ampulla of vater , with secondary dilatation ; c endoscopic ultrasound revealing the ampulla with a 8 7 mm hypoechoic lesion ( t ) , not invading the muscularis propria and with secondary dilatation of the common bile duct ( c ) and wirsung 's duct ( w ) ; d histological features ( hematoxylin - eosin , original magnification 40 ) revealing metastases into a locoregional lymph node of a g1 neuroendocrine neoplasm nests of uniforms , polygonal tumour cells with round nuclei and salt and pepper chromatin endoscopic ultrasound was performed on one patient , showing a hypoechoic tumour mass at the level of the ampulla of vater ( 0.8 cm , not invading the muscularis propria ) , with secondary dilatation of the common bile duct and wirsung 's duct .
preoperative tumour biopsy diagnosed a net in three patients ; in one patient , although malignancy was suspected , it was not possible to differentiate the tumour histopathologically .
endoscopic retrograde cholangiography was performed in one patient , showing a dilated common bile duct with distal stenosis ( fig .
1b ) . none of the patients had symptoms of an endocrine hypersecretion syndrome , but one patient had associated neurofibromatosis ( von recklinghausen 's disease ) .
all the operative specimens were assessed as r0 resections ( without microscopic residual tumour ) .
median size of the tumour was 1.25 cm ( range , 0.8 to 2 cm ) .
all of the tumours were found to be diffusely positive for chromogranin a , synaptophysin and neuron - specific enolase with immunohistochemistry , confirming their neuroendocrine origin .
the ki67 index was 2 % in three tumours , 320 % in one tumour and > 20 % in two tumours .
locoregional lymph node metastases were observed in four patients . lymph node ratio ( i.e. ratio of positive to excised lymph nodes ) ranged from 0.06 to 0.68 .
the presence of lymph node metastases was assessed by histopathological examination using hematoxylin - eosin staining ( fig .
tumours assessments according to the enets , uicc staging systems and the who 2010 classification are shown in table 2 .
complete follow - up data were available for all patients , as shown in table 2 .
no patient had adjuvant chemotherapy . at the last follow - up ( november 1 , 2011 ) , five patients were alive and disease - free , while one patient died at 24 months after surgery with liver metastases .
interestingly , the deceased patient 's tumour was assessed as a stage i tumour according to enets / uicc tnm classification ( 1-cm tumour size , confined to the submucosa and no lymph node metastases ) , although it was a high - grade neuroendocrine carcinoma .
patients included in the survival analysis had the following features ( table 2 ) : g3 neuroendocrine carcinoma was found in 17 patients ( 63 % ) and g1/g2 neuroendocrine neoplasms were found in 10 patients .
twenty patients had lymph node metastases ( 60 % ) and 13 patients had negative lymph nodes .
tumours were limited to the ampulla ( t1/t2 enets ) in 15 patients ( 60 % ) and were locally invasive ( t3/t4 enets ) in 10 patients .
tumour diameter was < 2 cm in 12 patients ( 48 % ) and was 2 cm in 13 patients .
enets stage iii disease was found in 24 patients ( 73 % ) and enets stages i and ii disease was found in 9 patients .
uicc stage iii disease was found in 3 patients , while uicc stage i and ii diseases were found in 8 patients and 22 patients ( 66 % ) , respectively . who classification was found to be a significant factor for predicting both overall and disease - free survival ( table 3 ) , as shown in fig .
2a , b. interestingly , there were no significant differences in terms of overall and disease - free survival in patients with lymph node metastases , as compared with patients who had negative lymph nodes ( table 3 ) .
depth of the tumour invasion ( t status ) appeared to be a significant factor influencing overall and disease - free survival ( table 3 ) , as shown in fig .
3a , b. regarding tumour diameter , the overall and disease - free median survival for patients with tumours < 2 cm was significantly higher than in patients with tumours 2 cm ( table 3 ) , as shown in fig .
3c , d. there were no significant differences in overall and disease - free survival among patients with enets stage i / ii vs. stage iii disease ( table 3 ) , as shown in fig .
4a , b. no differences of both overall and disease - free survival rates were observed between patients with uicc stages i , ii and iii ( table 3 ) , as shown in fig . 4c , d.table 3univariate predictors of overall and disease - free survival in patients with pancreaticoduodenectomy for net of the ampulla of vatermedian os ( months)p valuemedian dfs ( months)p valuewho classification g1/g2na0.006na0.004 g3nanalymph nodes n0420.760 , ns370.0745 , ns n15050enets t1/t2630.008630.004 t3/t42217tumour diameter < 2 cm650.030660.032 2 cm2723enets stage stage i / ii420.169 , ns410.137 , ns stage iii4543uicc stage stage i420.073 , ns370.177 , ns stage ii5149 stage iii1610os overall survival , dfs disease - free survival , who world health organisation classification , enets european neuroendocrine tumour society staging system , uicc international union against cancer staging systemdata for median survival were not available because all of the patients in the g1/g2 group were censoredfig .
2overall ( a ) and disease - free ( b ) survival curves according to who classification for 27 patients undergoing pancreaticoduodenectomy for net of the ampulla of vaterfig .
3overall ( a ) and disease - free ( b ) survival curves according to local invasiveness of the tumour for 25 patients undergoing pancreaticoduodenectomy for net of the ampulla of vater ( tumours limited to ampulla t1/t2 vs. local invasive tumours
t3/t4 ) ; overall ( c ) and disease - free ( d ) survival curves according to tumour diameter for 25 patients undergoing pancreaticoduodenectomy for net of the ampulla of vater ( < 2 cm vs. 2 cm)figure 4overall ( a ) and disease - free ( b ) survival curves according to enets staging system for 33 patients undergoing pancreaticoduodenectomy for net of the ampulla of vater ( stage i / ii vs. stage iii ) ; overall ( c ) and disease - free ( d ) survival curves according to uicc staging system for 33 patients undergoing pancreaticoduodenectomy for net of the ampulla of vater ( stage i vs. ii vs. stage iii ) univariate predictors of overall and disease - free survival in patients with pancreaticoduodenectomy for net of the ampulla of vater os overall survival , dfs disease - free survival , who world health organisation classification , enets european neuroendocrine tumour society staging system , uicc international union against cancer staging system data for median survival were not available because all of the patients in the g1/g2 group were censored overall ( a ) and disease - free ( b ) survival curves according to who classification for 27 patients undergoing pancreaticoduodenectomy for net of the ampulla of vater overall ( a ) and disease - free ( b ) survival curves according to local invasiveness of the tumour for 25 patients undergoing pancreaticoduodenectomy for net of the ampulla of vater ( tumours limited to ampulla t1/t2 vs. local invasive tumours
t3/t4 ) ; overall ( c ) and disease - free ( d ) survival curves according to tumour diameter for 25 patients undergoing pancreaticoduodenectomy for net of the ampulla of vater ( < 2 cm vs. 2 cm ) overall ( a ) and disease - free ( b ) survival curves according to enets staging system for 33 patients undergoing pancreaticoduodenectomy for net of the ampulla of vater ( stage i / ii vs. stage iii ) ; overall ( c ) and disease - free ( d ) survival curves according to uicc staging system for 33 patients undergoing pancreaticoduodenectomy for net of the ampulla of vater ( stage i vs. ii vs. stage iii )
the patients were predominantly male ( male / female ratio = 4:2 ) , with a median age of 54 years ( range , 3268 years ) .
the main presenting symptom was jaundice ( four patients ) , while two patients presented with epigastric pain .
five patients presented with cholestasis syndrome ( minor cholestasis in one patient , major cholestasis in four patients ) . in the patients with jaundice , the total bilirubin level ranged from 3.0 to 17.0 mg / dl ( median 13 mg / dl ) .
preoperative imaging included ct ( two patients ) , mri ( three patients ) or both ( one patient ) .
the ct and/or mri showed the ampullary tumour in all cases ; in patients with cholestasis syndrome , on noticed dilatation of the intra- and extrahepatic bile ducts , along with dilatation of wirsung 's duct ( fig .
no distant metastases to the liver , lung , peritoneum or other organs were observed .
1a magnetic resonance cholangiopancreatography showing an ampullary soft tissue mass ( t ) , with secondary dilatation of the common bile duct ( c ) and main pancreatic duct ( arrow head ) ; b endoscopic retrograde cholangiopancreatography showing an irregular , fusiform stenosis of the common bile duct toward the ampulla of vater , with secondary dilatation ; c endoscopic ultrasound revealing the ampulla with a 8 7 mm hypoechoic lesion ( t ) , not invading the muscularis propria and with secondary dilatation of the common bile duct ( c ) and wirsung 's duct ( w ) ; d histological features ( hematoxylin - eosin , original magnification 40 ) revealing metastases into a locoregional lymph node of a g1 neuroendocrine neoplasm nests of uniforms , polygonal tumour cells with round nuclei and salt and pepper chromatin a magnetic resonance cholangiopancreatography showing an ampullary soft tissue mass ( t ) , with secondary dilatation of the common bile duct ( c ) and main pancreatic duct ( arrow head ) ; b endoscopic retrograde cholangiopancreatography showing an irregular , fusiform stenosis of the common bile duct toward the ampulla of vater , with secondary dilatation ; c endoscopic ultrasound revealing the ampulla with a 8 7 mm hypoechoic lesion ( t ) , not invading the muscularis propria and with secondary dilatation of the common bile duct ( c ) and wirsung 's duct ( w ) ; d histological features ( hematoxylin - eosin , original magnification 40 ) revealing metastases into a locoregional lymph node of a g1 neuroendocrine neoplasm nests of uniforms , polygonal tumour cells with round nuclei and salt and pepper chromatin endoscopic ultrasound was performed on one patient , showing a hypoechoic tumour mass at the level of the ampulla of vater ( 0.8 cm , not invading the muscularis propria ) , with secondary dilatation of the common bile duct and wirsung 's duct .
preoperative tumour biopsy diagnosed a net in three patients ; in one patient , although malignancy was suspected , it was not possible to differentiate the tumour histopathologically .
endoscopic retrograde cholangiography was performed in one patient , showing a dilated common bile duct with distal stenosis ( fig .
1b ) . none of the patients had symptoms of an endocrine hypersecretion syndrome , but one patient had associated neurofibromatosis ( von recklinghausen 's disease ) .
all the operative specimens were assessed as r0 resections ( without microscopic residual tumour ) .
median size of the tumour was 1.25 cm ( range , 0.8 to 2 cm ) .
all of the tumours were found to be diffusely positive for chromogranin a , synaptophysin and neuron - specific enolase with immunohistochemistry , confirming their neuroendocrine origin .
the ki67 index was 2 % in three tumours , 320 % in one tumour and > 20 % in two tumours .
locoregional lymph node metastases were observed in four patients . lymph node ratio ( i.e. ratio of positive to excised lymph nodes ) ranged from 0.06 to 0.68 .
the presence of lymph node metastases was assessed by histopathological examination using hematoxylin - eosin staining ( fig .
tumours assessments according to the enets , uicc staging systems and the who 2010 classification are shown in table 2 .
complete follow - up data were available for all patients , as shown in table 2 .
no patient had adjuvant chemotherapy . at the last follow - up ( november 1 , 2011 ) , five patients were alive and disease - free , while one patient died at 24 months after surgery with liver metastases .
interestingly , the deceased patient 's tumour was assessed as a stage i tumour according to enets / uicc tnm classification ( 1-cm tumour size , confined to the submucosa and no lymph node metastases ) , although it was a high - grade neuroendocrine carcinoma .
patients included in the survival analysis had the following features ( table 2 ) : g3 neuroendocrine carcinoma was found in 17 patients ( 63 % ) and g1/g2 neuroendocrine neoplasms were found in 10 patients .
twenty patients had lymph node metastases ( 60 % ) and 13 patients had negative lymph nodes .
tumours were limited to the ampulla ( t1/t2 enets ) in 15 patients ( 60 % ) and were locally invasive ( t3/t4 enets ) in 10 patients .
tumour diameter was < 2 cm in 12 patients ( 48 % ) and was 2 cm in 13 patients .
enets stage iii disease was found in 24 patients ( 73 % ) and enets stages i and ii disease was found in 9 patients .
uicc stage iii disease was found in 3 patients , while uicc stage i and ii diseases were found in 8 patients and 22 patients ( 66 % ) , respectively . who classification was found to be a significant factor for predicting both overall and disease - free survival ( table 3 ) , as shown in fig . 2a , b. interestingly , there were no significant differences in terms of overall and disease - free survival in patients with lymph node metastases , as compared with patients who had negative lymph nodes ( table 3 ) .
depth of the tumour invasion ( t status ) appeared to be a significant factor influencing overall and disease - free survival ( table 3 ) , as shown in fig .
3a , b. regarding tumour diameter , the overall and disease - free median survival for patients with tumours < 2 cm was significantly higher than in patients with tumours 2 cm ( table 3 ) , as shown in fig .
3c , d. there were no significant differences in overall and disease - free survival among patients with enets stage i / ii vs. stage iii disease ( table 3 ) , as shown in fig .
4a , b. no differences of both overall and disease - free survival rates were observed between patients with uicc stages i , ii and iii ( table 3 ) , as shown in fig . 4c , d.table 3univariate predictors of overall and disease - free survival in patients with pancreaticoduodenectomy for net of the ampulla of vatermedian os ( months)p valuemedian dfs ( months)p valuewho classification g1/g2na0.006na0.004 g3nanalymph nodes n0420.760 , ns370.0745 , ns n15050enets t1/t2630.008630.004 t3/t42217tumour diameter < 2 cm650.030660.032 2 cm2723enets stage stage i / ii420.169 , ns410.137 , ns stage iii4543uicc stage stage i420.073 , ns370.177 , ns stage ii5149 stage iii1610os overall survival , dfs disease - free survival , who world health organisation classification , enets european neuroendocrine tumour society staging system , uicc international union against cancer staging systemdata for median survival were not available because all of the patients in the g1/g2 group were censoredfig .
disease - free ( b ) survival curves according to who classification for 27 patients undergoing pancreaticoduodenectomy for net of the ampulla of vaterfig .
3overall ( a ) and disease - free ( b ) survival curves according to local invasiveness of the tumour for 25 patients undergoing pancreaticoduodenectomy for net of the ampulla of vater ( tumours limited to ampulla t1/t2 vs. local invasive tumours
t3/t4 ) ; overall ( c ) and disease - free ( d ) survival curves according to tumour diameter for 25 patients undergoing pancreaticoduodenectomy for net of the ampulla of vater ( < 2 cm vs. 2 cm)figure 4overall ( a ) and disease - free ( b ) survival curves according to enets staging system for 33 patients undergoing pancreaticoduodenectomy for net of the ampulla of vater ( stage i / ii vs. stage iii ) ; overall ( c ) and disease - free ( d ) survival curves according to uicc staging system for 33 patients undergoing pancreaticoduodenectomy for net of the ampulla of vater ( stage i vs. ii vs. stage iii ) univariate predictors of overall and disease - free survival in patients with pancreaticoduodenectomy for net of the ampulla of vater os overall survival , dfs disease - free survival , who world health organisation classification , enets european neuroendocrine tumour society staging system , uicc international union against cancer staging system data for median survival were not available because all of the patients in the g1/g2 group were censored overall ( a ) and disease - free ( b ) survival curves according to who classification for 27 patients undergoing pancreaticoduodenectomy for net of the ampulla of vater overall ( a ) and disease - free ( b ) survival curves according to local invasiveness of the tumour for 25 patients undergoing pancreaticoduodenectomy for net of the ampulla of vater ( tumours limited to ampulla t1/t2 vs. local invasive tumours
t3/t4 ) ; overall ( c ) and disease - free ( d ) survival curves according to tumour diameter for 25 patients undergoing pancreaticoduodenectomy for net of the ampulla of vater ( < 2 cm vs. 2 cm ) overall ( a ) and disease - free ( b ) survival curves according to enets staging system for 33 patients undergoing pancreaticoduodenectomy for net of the ampulla of vater ( stage i / ii vs. stage iii ) ; overall ( c ) and disease - free ( d ) survival curves according to uicc staging system for 33 patients undergoing pancreaticoduodenectomy for net of the ampulla of vater ( stage i vs. ii vs. stage iii )
although net ( initially assessed as carcinoid tumour ) was first described over 100 years ago at the university of munich by siegfried oberndorfer , these tumours still raise many issues regarding classification , prognosis and choice of the best therapeutic approach .
the incidence and prevalence of net seems to have increased in recent years , most likely due to an improvement in diagnostic techniques [ 23 , 24 ] .
however , among gastroenteropancreatic net , the ampulla of vater represents an uncommon site for the disease .
nevertheless , the number of papers addressing the ampulla of vater net seems to have increased in recent years [ 1 , 46 , 19 ] .
gastroenteropancreatic net are , by far , less frequent than adenocarcinomas , a statement that is also true for net of the ampulla of vater [ 19 , 25 ] . in a recent analysis of 450 patients with ampullary neoplasms
other series reported an incidence of net that ranged from less than 1 to 8.8 % ( of all patients undergoing resections of malignant tumours of the ampulla of vater ) [ 1 , 5 , 19 , 25 ] .
ct , mri , ultrasound endoscopy and endoscopic retrograde cholangiopancreatography with biopsy are the main tools for preoperatively assessing net of the ampulla of vater .
accuracy rates of biopsy for the preoperative diagnosis of net range from to 14 to 66 % [ 36 ] . in this series , a correct diagnosis of a net was established preoperatively in three patients ( out of the four patients who had a preoperative biopsy ) .
. currently , several options are available to treat net of the ampulla of vater .
endoscopic local resection and surgical ampullectomy have been considered to be safe for small net of the ampulla of vater ( less than 2 cm ) or in patients with severe comorbidities .
endoscopic ampullectomy is considered to be safe only in very selected cases of well - differentiated net ( t1 n0 , negative resection margins , ki67 < 2 % ) .
a major limitation of these procedures is the inability to address locoregional lymph node metastases , possibly leading to an inadequate oncological operation and potentially jeopardizing the patient 's prognosis .
pancreaticoduodenectomy has been generally considered the procedure of choice for net of the ampulla of vater that are larger than 2 cm and for cases of neuroendocrine carcinomas [ 3 , 6 ] . in a review of 105 reported cases from the literature ,
pancreaticoduodenectomy was performed in more than 50 % of the patients ; a preference for this surgical approach was also noticed in the most recent reported series [ 46 ] . in a recent multi - institutional review of patients with resected small neuroendocrine pancreatic and periampullary net ,
local resection had similar results to pancreaticoduodenectomy in terms of overall survival but was associated with significantly lower morbidity .
similar data were reported by previous studies . however , this study addressed a selected group of patients ( with tumours less than 3 cm and no involved lymph nodes or liver metastases ) .
moreover , in experienced centres , the morbidity and mortality rates of the transduodenal local excision of ampullary lesions are comparable to pancreaticoduodenectomy . in clinical practice , it is impossible to differentiate a neuroendocrine neoplasm from a neuroendocrine carcinoma intraoperatively .
sometimes , it is very difficult to differentiate net from an adenocarcinoma of the ampulla of vater on frozen sections .
moreover , preoperative imaging tools or intraoperative exploration may not identify liver metastases that are smaller than 0.5 cm or lymph node metastases .
the lack of an accurate assessment of small liver or lymph node metastases may jeopardize the choice for an adequate surgical approach .
a question that arises is : does the therapeutic approach to the net of the ampulla of vater have the same principles of adenocarcinoma ?
pancreaticoduodenectomy represents the procedure of choice for adenocarcinoma of the ampulla of vater and has proved to be curative in the majority of patients without lymph node metastases .
ampullectomy is recommended only in highly selected cases tis / t1 n0 , g1g2 tumours [ 25 , 28 ] . moreover , even in t1 tumours , the rate of lymph node metastases is approximately 10 % ; thus , in patients with a ptis or pt1 carcinoma , ampullectomy should be accomplished by a local lymph node dissection to ensure an oncological resection . for ampullary adenocarcinomas ,
the presence of metastases in locoregional lymph nodes is a poor prognostic factor that is encountered in 47 to 65 % of the resected patients [ 25 , 26 , 28 , 32 ] .
an increased rate of metastases in the locoregional lymph nodes ( up to 80 % ) was also observed in patients with net of the ampulla of vater resected by pancreaticoduodenectomy , especially in patients with high - grade carcinomas [ 1 , 8 ] , but no correlation with survival was found [ 4 , 6 ] .
interestingly , even in small tumours ( less than 2 cm ) , the presence of lymph node metastases is higher than 50 % [ 4 , 7 , 30 , 33 ] .
however , a recent study from asan medical center showed an incidence of positive lymph nodes of only 20 % after curative intent resection of net of the ampulla of vater . in this series of patients , a high incidence of locoregional lymph node metastases
was observed , although none of the tumours were larger than 2 cm ( median size1.25 cm ) .
the four patients with histologically proven positive lymph nodes survived without recurrence for up to 80 months , while a patient without lymph node metastases died 2 years after surgery with recurrent disease in the liver .
interestingly , preoperative imaging failed to detect metastases in the locoregional lymph nodes in most patients and in patients with pathologically confirmed lymph node metastases ; intraoperative exploration did not show macroscopically enlarged lymph nodes .
conversely , the enlarged lymph nodes detected on preoperative ct in one patient were not pathologically confirmed as metastases .
the same features were reported by other studies . in summary , although an increased rate of positive lymph nodes was reported in both adenocarcinomas and net of the ampulla of vater , the clinical impact of this finding appears to be different in the two pathologies .
net of the ampulla of vater has a very similar prognosis to the prognosis of gastroenteropancreatic net in general , with a 5-year survival rate of approximately 70 % .
some studies reported a worse prognosis for net of the ampulla of vater compared to duodenal net . according to data from the surveillance , epidemiology and end results program ,
survival rates at 5 and 10 years are 82 and 71 % for net , respectively , while for high - grade neuroendocrine carcinomas of the ampulla of vater , the prognosis is significantly worse ( 15 % at 5 and 10 years ) [ 1 , 8 ] .
the recurrence rate is approximately 40 to 50 % [ 3 , 5 ] , with the liver being the most common site of initial metastases .
assessment of prognosis in patients with net of the ampulla of vater is difficult due to the small number of patients reported in the literature .
the who proposed a new pathological classification for gastroenteropancreatic net and the enets previously ( 2006 ) proposed a grading system along with a tnm staging .
the main goal of these classifications was to better assess the prognosis of patients with different tumour locations .
although enets provides a tnm staging system for net of the ampulla of vater , its clinical value is unknown because there are no large series of patients to provide survival data . in the present study , tumour grading ( who 2010 ) classification seems to accurately predict the prognosis of patients with net of the ampulla of vater , while the enets or uicc staging systems does not appear to be correlated with survival .
previous studies showed that the enets staging system is not a good predictor of prognosis for patients with net of the ampulla of vater ; however , high - grade neuroendocrine carcinoma has been associated with a poor prognosis and a median survival between 10.3 and 14.5 months [ 1 , 6 , 8 , 19 ] .
thus , an advanced tnm stage is not necessarily correlated with a worse survival rate .
the present study showed a correlation of the tumour size with the recurrence rate and overall survival , contrary to data from previous studies .
tumour diameter ( more than 2 cm ) and tumour extension beyond the ampulla were also found to be associated with a high risk for recurrence upon univariate analysis in a recent study .
although certain factors , such as younger age [ 1 , 6 ] , small tumour size [ 1 , 6 , 8 ] and the presence of type i neurofibromatosis seem to be more frequent in patients with g1/g2 net of the ampulla of vater , these data can not be used in clinical decision making .
angiolymphatic , perineural or venous invasion was recently found to be more frequent in high - grade carcinomas , but no correlation with survival was demonstrated .
the analysis performed in the present paper showed that the presence of positive locoregional lymph nodes has no impact on overall or disease - free survival .
this feature may explain the previously reported good oncological results obtained with local resection of net of the ampulla of vater [ 29 , 30 ] .
the only factors that seem to be negatively correlated with patient prognosis upon univariate analysis are local invasiveness of the tumour ( into the pancreas , retroperitoneum , serosa and other adjacent organs ) , large tumours ( over 2 cm ) and high - grade / g3 neuroendocrine carcinoma .
first , the survival analysis is limited by the small number of patients , precluding a multivariate analysis .
furthermore , a relatively large number of patients had high - grade neuroendocrine carcinomas , a feature that could explain a worse long - term prognosis ( compared to previous reports ) [ 1 , 7 ] .
additionally , heterogeneity in the follow - up of the patients included in the survival analysis may be a confounding factor , mainly in regard to the long - term estimates of disease - free and overall survival .
although net of the ampulla of vater represents a rare pathology , it has been increasingly reported in the last years .
the who classification seems to accurately predict prognosis , while the enets or uicc staging systems have limited value ( especially in regard to lymph node metastases ) . however , net limited to the ampulla of vater ( without local invasion ) seems to have a better prognosis , as do tumours smaller than 2 cm .
radical surgery ( i.e. pancreaticoduodenectomy ) should be the standard approach in most patients with net of the ampulla of vater , due to the high incidence and poor accuracy of preoperative and intraoperative assessments of lymph node metastases .
the low accuracy of preoperative biopsies in the diagnosis of net and the difficulty of differentiating net from adenocarcinomas by frozen section analysis are also strong arguments for an aggressive surgical approach .
although the overall and disease - free survival rates do not appear to be influenced by the presence of lymph node metastases , locoregional lymphadenectomy should be performed routinely because this procedure removes all the potential tumour - bearing tissue .
this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited . | background / aimsneuroendocrine tumours occur very rarely in the ampulla of vater and their clinical behaviour is unknown .
the aim of this study is to assess the clinico - pathological features , surgical approach and prognosis of these patients.methodssix patients with neuroendocrine tumours of the ampulla of vater treated with curative intent surgery at a single centre were retrospectively analysed .
a univariate analysis of potential prognostic factors was also performed ( data provided from the present study and literature review).resultspancreaticoduodenectomy was curative in all the patients .
overall and disease - free survival rates were significantly better for g1/g2 tumours ( p = 0.006 and p = 0.004 , respectively ) . although frequent , lymph node metastases did not influenced both overall ( p = 0.760 ) and disease - free survival rates ( p = 0.745 ) .
no significant differences of survival were observed in patients with enets stage i / ii disease , as compared to enets stage iii disease ( p = 0.169 and p = 0.137 , respectively ) .
no differences were observed according to uicc staging system ( p = 0.073 and p = 0.177 , respectively ) .
tumours that are less than 2 cm or limited to the ampulla appear to have a better prognosis.conclusionthe who 2010 classification appear to accurately predict patient prognosis , while the enets or uicc staging systems have a limited value ( especially in regard to lymph node metastases ) .
radical surgery ( i.e. pancreaticoduodenectomy with lymphadenectomy ) should be the standard approach in most patients with net of the ampulla of vater because this procedure removes all the potential tumour - bearing tissue . |
the rat genome has been sequenced and assembled , creating a need for rat gene nomenclature .
the obvious source of gene nomenclature for the rat would seem to be the mouse genome .
this logic has led to an automated naming of rat genes -- leading to problems of two kinds .
genes from both rat and mouse were sequenced and named for nearly 20 years before the genomes were being sequenced . in the example of cytochromes p450 , the first mammalian sequences cyp2b1 and cyp2b2 were determined in the rat .
the established nomenclature for cyp genes has been in place since 1987 [ 5 - 7 ] , and these names have been used in publications for several years . because the names were assigned independently , mostly in chronological order , orthologues do not always carry the same name . the second nomenclature problem has to do with divergence over time between species ' genomes . here
, mouse and rat will be discussed , but the same applies to other species such as human and rhesus monkey .
when similar genes appear in gene clusters , the one to one relationship of the genes between mouse and rat is often broken , meaning that the orthology is broken . compared with the 57 cyp genes of the human , the mouse has greatly expanded its set of cyp genes to 102 full - length genes ; the rat has been a little more conservative , with 87 cyp genes .
the solo genes in a mammalian cyp subfamily -- those that occur without related neighbours -- are strict orthologues , and so nomenclature by best reciprocal blast hit between mouse and rat is a viable strategy .
eighty - seven rat genes can not be matched up to 102 mouse genes as orthologue pairs , however , and this nomenclature method can be seen to fail in the gene clusters .
not all cyp gene clusters are disordered between mouse and rat . for example , the cyp4f gene cluster has nine genes in both species and there is a clean 1:1 mapping between orthologous pairs ( table 1 ) .
in fact , there are 33 such pairs in the cyp gene clusters ( table 1 ) ; two of these pairs involve matches to pseudogenes in the other species . after these 64 pairs are subtracted and a correction is made in the count for pseudogenes , there are still 40 mouse genes remaining to pair with 24 rat genes .
these genes either have no orthologues ( paired with an ' x ' in table 1 ) or they are in paralogous gene sets ( shaded and boldened in table 1 ) .
orthology between mouse and rat cyp genes paralogues are boldened the discrepancy is explained by extra duplications , mostly in the mouse but , in at least some cases , there is duplication in the rat that is not seen in the mouse .
figures 1 and 2 illustrate a comparison of two such gene clusters between rat and mouse in detail .
cyp2d5 and cyp2d1 in the rat are most similar by blast searches to the five mouse genes -- cyp2d11 , cyp2d10 , cyp2d9 , cyp2d12 and cyp2d34 -- that are boxed in the mouse cluster ; these represent paralogous sets of genes mouse cyp2d13 and cyp2d40 are almost equally similar to cyp2d3 in the rat . in between these genes
this whole cluster of genes and pseudogenes may have been derived from a cyp2d3-like ancestor that expanded in the mouse .
these rat genes have had official cyp names for more than 15 years ; in fact , they were the first five genes in the cyp2d subfamily to be identified .
this is often a feature of genes in gene clusters : the edges of the cluster are more likely to be conserved .
notice how the rat cyp4a1 gene has given rise to three cyp4a genes in the mouse .
by contrast , mouse cyp4a14 has duplicated , making cyp4a2 and cyp4a3 in the rat , based on blast similarities .
the mouse cluster is further complicated by an approximately 100 kilobase duplication involving the cyp4a12 and cyp4a30 genes .
this did not happen in that rat and there does not seem to be a cyp4a30 equivalent in that animal -- unless it might be the rat cyp4a33-ps pseudogene .
there are seven cyp gene clusters in the rat , some being even more complex than that described for the cyp2d and cyp4abx clusters .
the example of mouse versus rat cyp genes that has been chosen in this paper are by no means the only gene sets that will have this problem . in the 5th december , 2002 issue of nature , in which the mouse genome was reported , table 11 ( p. 542 ) shows the top 50 interpro domain families in mouse compared with that in human , fish , worm and fly .
the 45 other families that are more abundant than cyp in the mouse will potentially have similar nomenclature issues .
fortunately , some of these groups ( eg the homeobox genes ) have a firmly established nomenclature and will not be renamed .
it is not so clear what confusion will descend on the atpase , kinase , zinc finger protein and the many other gene families .
the point made by these figures and tables is that : naming genes can not be an automatic process , unless one wishes to create confusion .
best reciprocal blast hits can be used in assigning names , but they should not be used indiscriminately -- if they are , the result presented in figure 3 might occur .
figure 3 is a screenshot of the university of california santa cruz ( ucsc ) browser showing the rat cyp2d cluster , with its five genes .
note that these genes are named cyp2d22 , cyp2d10 , cyp2d9 , cyp2d13 and cyp2d26 . from figure 2
, it can be seen that these rat genes had already been named cyp2d4 , cyp2d5 , cyp2d1 , cyp2d3 and cyp2d2 .
these names were assigned between 1987 and 1989 by the committee on standardized cytochrome p450 nomenclature and are official names used in many dozens or hundreds of publications .
the two outside ' rat genes cyp2d22 and cyp2d26 ' ( figure 3 ) are , in fact , orthologues of rat cyp2d4 and cyp2d2 ( table 1 ) , but the other three rat genes in between cyp2d22 and cyp2d26 in figure 3 -- cyp2d10 , cyp2d9 and cyp2d13 -- are not orthologous pairs .
thus , rat cyp genes that already have official names have been renamed to match seemingly orthologous mouse cyp genes . on other views in the ucsc browser , rat cyp genes that already have official names
these names are wrong , yet because they appear in the genbank database they will probably be used by companies making microarrays and by genome browsers like ucsc and ensembl .
the rat cyp2d locus , as shown by the university of california santa cruz genome browser .
note that the gene nomenclature being used follows the existing mouse gene nomenclature , which is incorrect .
gene nomenclature committees have been established to impose order on gene families and in whole genomes to prevent duplication of names and multiple uses of the same root symbol .
ignoring the existence of naming systems in order to assign hundreds , or thousands , of names quickly to rat genes to match genes in other genomes will come with a price , and the price will be in failed communication and widespread confusion .
these problems are not so different from those that must occur when a carefully constructed language is corrupted . | the current proliferation of mammalian genomes is creating a nomenclature issue caused by naming genes based on their best blast hit to a gene in another annotated genome .
the rat genome is relying heavily on the mouse genome for nomenclature , but not all rat genes have direct orthologues in the mouse ; often , there are paralogous groups of genes -- due to expansions of that gene subfamily in one or the other genome . many of these genes have already been assigned names in the rat , so that renaming them based on blast scores leads to duplicate sets of names .
the supposed orthology created by name sharing across genomes is not always found .
these inaccurate names are appearing in frequently used sites , such as the university of california santa cruz genome browser .
the example of rat cytochrome p450 ( cyp ) genes is presented here , but other gene families are also likely to be affected . |
aging of the population is a worldwide phenomenon that is accompanied by a series of modifications to several physiological parameters , such as a progressive increase in fat mass and a decrease in lean body mass.1 however , these alterations are not linear and must be constantly monitored.24 in elderly individuals , changes in body composition result in the prevalence of overweight and obesity combined with a loss of muscle mass and strength ; this has recently been defined as sarcopenic obesity.57 sarcopenic obesity is associated with functional limitations and increased mortality.8 among the consequences of obesity in elderly individuals are increased risk of cardiometabolic complications , physical incapacity , sexual dysfunction , urinary incontinence , depression , type 2 diabetes , arthritis , decreased cognitive function , dementia , and compromised health - related quality of life.912 apart from this , the loss of muscle mass associated with the aging process results in muscle weakness , increased fall risk , and fat infiltration in the skeletal muscle.13,14 in this sense sarcopenia is defined as a decline in muscle mass associated with spinal shortening and a decrease in muscle strength and functionality.5,15 these deleterious effects seem to result from a complex interaction of innervation disturbances , decreased hormonal levels , and increased inflammatory mediators during aging.1,16,17 the reduction in muscle mass and strength is responsible for the decrease in mobility , decreased functional capacity , and increase in dependency ; there are also economic and social costs.4,6,14 the impact of sarcopenic obesity on the health of older individuals is poorly understood .
thus , the present study aimed to investigate the associations between body composition profile and sarcopenia prevalence in elderly sedentary brazilian women .
our initial hypothesis was that a proper diagnosis of sarcopenic obesity would be independent of the nutritional state .
this transversal and analytical study consisted of 272 elderly women from a local community located in the federal district , brazil .
participants were not specifically representative of the brazilian population and were recruited on a voluntary basis from the local community from posters and lectures about the study .
individuals visited the laboratory on two occasions . on the first visit they completed an anamnesis form and physical activity questionnaire and
the following day they were subjected to dual - energy x - ray absorptiometry ( dxa ) analysis .
inclusion criteria were age 60 years , sedentary females , and completion of all anthropometric testing .
individuals with inflammatory , rheumatic , or autoimmune conditions or use of medications ( beta blockers and metformin ) that could modulate body composition were excluded .
the local ethics committee for human research of the catholic university of brasilia approved the methodology of the present study , and all participants signed an informed consent document .
the procedures were in accordance with guidelines for experimentation with human participants . additionally , the study met the ethical standards proposed by the international journal of sports medicine.18 the determination of body composition was completed using dxa ( general electric - ge model 8548 bx1l , year 2005 , lunar dpx type , software encore 2005 ; rommelsdorf , germany ) .
the appendicular fat - free mass ( affm ) was determined by the sum of the fat - free mass from the lower and upper body .
sarcopenic obesity was determined by body composition measured by the dxa method according to oliveira et al.19 individuals with residual values 3.4 were classified as presenting an inadequate ffm in reference to the body surface .
the identification of sarcopenia was based on the values proposed by baumgartner et al,3 which define female individuals as sarcopenic with an affm < 5.45 kg / m .
body fat percentage values were distributed according to the recommendations of the national institute of diabetes and digestive and kidney diseases,20 assuming a cut - off point of 32% for women .
obesity levels determined by bmi ( body mass / height ) followed the proposal of lipschitz,21 assuming a cut - off point of 27 kg / m for women .
based on differences between individuals aged 60 to 69 years and those above 70 years , the participants were divided into two age groups .
smirnov normality test and a homoscedasticity test ( mauchly ) were used to test the normal distribution of the data .
whitney test for variables with two levels , and kruskal wallis test for variables with more than two levels .
the statistical package for the social sciences ( [ spss ] v.19 ; ibm corporation , armonk , ny ) was used for analyses .
the determination of body composition was completed using dxa ( general electric - ge model 8548 bx1l , year 2005 , lunar dpx type , software encore 2005 ; rommelsdorf , germany ) .
the appendicular fat - free mass ( affm ) was determined by the sum of the fat - free mass from the lower and upper body .
sarcopenic obesity was determined by body composition measured by the dxa method according to oliveira et al.19 individuals with residual values 3.4 were classified as presenting an inadequate ffm in reference to the body surface .
the identification of sarcopenia was based on the values proposed by baumgartner et al,3 which define female individuals as sarcopenic with an affm < 5.45 kg / m .
body fat percentage values were distributed according to the recommendations of the national institute of diabetes and digestive and kidney diseases,20 assuming a cut - off point of 32% for women .
obesity levels determined by bmi ( body mass / height ) followed the proposal of lipschitz,21 assuming a cut - off point of 27 kg / m for women . based on differences between individuals aged 60 to 69 years and those above 70 years
smirnov normality test and a homoscedasticity test ( mauchly ) were used to test the normal distribution of the data .
whitney test for variables with two levels , and kruskal wallis test for variables with more than two levels .
the statistical package for the social sciences ( [ spss ] v.19 ; ibm corporation , armonk , ny ) was used for analyses .
characterization of the nutritional state by bmi revealed that 65.1% ( n = 177 ) were eutrophic , while all women were classified as obese by dxa ( n = 272 ) with different degrees of severity ; most of them exhibited morbid obesity ( table 2 ) .
the majority of women ( 61.76% ) were classified as morbid obesity , while 34.2% ( n = 93 ) presented sarcopenic obesity . among this group
women between 60 to 69 years exhibited a higher body fat percentage ( p = 0.004 ) and obesity determined by body composition ( p = 0.001 ) measured by dxa as compared with women above 70 years , according to the classification of the national institute of diabetes and digestive and kidney diseases.20 there was no statistically significant difference between age groups in the parameters determined by bmi ( p = 0.07 ) , sarcopenic obesity ( p = 0.40 ) , or sarcopenia ( p = 0.40 ) .
thirty - nine percent of the eutrophic elderly women determined by bmi exhibited sarcopenic obesity , while 23.1% of those classified with mild and moderate obesity by dxa presented sarcopenic obesity ( table 3 ) .
there was no association with sarcopenic obesity independent of obesity severity determined by dxa ( table 3 ) .
however , there was a negative association between sarcopenia and sarcopenic obesity , hence most of the elderly women with sarcopenia did not present sarcopenic obesity ( table 3 ) .
independent of obesity or eutrophy , there was an association between sarcopenia and bmi ( table 4 ) .
apart from this , there was an association between sarcopenia and obesity determined by dxa , and as obesity increased in severity the prevalence of sarcopenia increased ( table 4 ) .
the combination of sarcopenia and obesity ( known as sarcopenic obesity ) is an important public health problem that induces fragility in the elderly.2,22,23 the current incidence of sarcopenic obesity in elderly brazilian women suggests it plays a role as an important negative factor that counteracts a successful aging process . in comparison ,
an elderly mexican population showed a higher incidence of sarcopenic obesity ( 48%)3 compared with our results .
the reduction in muscle mass ( sarcopenia ) that accompanies the normal aging process is also associated with increments in fat mass . with the concurrent increment in elderly people as reported by the brazilian institute of geography and statistics24 and the prevalence of sarcopenic obesity in brazilians , a higher fragility could be expected in this population.25,26 our results suggest the necessity of proper diagnosis of sarcopenic obesity independent of the nutritional state , such as muscle cross - sectional area , because the eutrophic status or the different obesity levels do not influence the diagnosis of sarcopenic obesity .
our study indicates that a sedentary lifestyle may be an additional risk factor for both overweight and obesity , highlighting the concurrent muscular force loss as previously suggested.27 this is in accordance with previous literature.28 the diagnosis for obesity requires the utilization of various methods because bmi has been suggested to be inappropriate for this purpose in the elderly.10 moreover , the current results suggest some discrepancies between bmi and dxa methods regarding the nutritional status of participants .
thus , eutrophic elderly women as determined by bmi were classified as obese with various severity levels with the employment of dxa , confirming the previous observations of pahor et al.29 in this regard , bmi has demonstrated some limitations because of the concurrent lean body mass loss and fat mass gain observed in the elderly.10 furthermore , all participants of the present study exhibited various levels of obesity , thus confirming the findings of previous studies in brazilian populations .
30,31 although bmi has been widely used in population studies , this method considers only height and body mass . on the other hand
adipose tissue is currently recognized as an important metabolic and hormonal secretory organ with more than 50 product molecules identified.1 subsequently , when there is an increase in adipose tissue , the impact of these molecules is also augmented , thus favoring the development of insulin resistance , type ii diabetes , atherosclerosis , and metabolic syndrome and therefore impacting the health of older individuals.8,32,33 with the current results it was possible to verify an overestimation of muscle mass as a consequence of not taking into account the true differences in water , bone mass , and ffm , as the elderly have demonstrated an excessive extracellular fluid accumulation.29 this syndrome is associated with a progressive loss of muscle force and mass with subsequent lower physical capacity and quality of life , and hence the current results should be considered for further interventions in this population .
the premise is based on the fact that individuals with high fat mass also demonstrate a high ffm .
therefore , individuals with high fat mass and subsequently high ffm would not be diagnosed as sarcopenic , independently of its influence on total body mass or functional capacity . in order to adjust ffm with fat mass ,
newman et al34 proposed a method based on the residuals of a regression equation that predicts affm from fat mass and height ; this was helpful in the diagnosis of sarcopenic obesity in the current study .
the limitations of the present study are the reduced number of participants and lack of additional measures , such as calf circumference and other metabolic parameters , that would certainly be of interest .
in summary , the present study observed a critical relationship between sarcopenia and obesity in an elderly female population . the combination of sarcopenic obesity may have a dynamic negative impact on the aging process in the elderly , and thus the ability to correctly diagnose this condition becomes important .
further studies are needed for the diagnosis of sarcopenic obesity as well for understanding its etiology and clinical impact , specifically in the elderly population of developing countries where public health systems are not prepared for the demands of this population sector . | the aim of the present study was to investigate the prevalence of sarcopenic obesity and its association with obesity and sarcopenia in elderly brazilian women .
two hundred and seventy - two sedentary women with a mean age of 66.75 5.38 years were recruited for participation in this study .
obesity was determined by both body mass index and dual - energy x - ray absorptiometry ( dxa ) evaluations .
sarcopenic obesity diagnosis was established from the ratio between fat - free mass and body surface area as obtained by dxa .
there was no association of obesity with sarcopenic obesity ( p = 0.424 ) .
in contrast , sarcopenia was significantly related to sarcopenic obesity ( p < 0.001 ) , although most of the elderly women with sarcopenia ( n = 171 ) did not exhibit sarcopenic obesity .
these results highlight the importance of diagnosing sarcopenic obesity as elderly women exhibiting sarcopenia could be either eutrophic or obese . |
during the past 5 years , great strides have been made in unraveling the complex interplay of factors that are responsible for initiating and maintaining atrial fibrillation ( af ) . according to our present understanding of af ( which is still in its infancy )
, af is initiated by a number of possible supraventricular tachyarrhythmias , most commonly premature atrial contractions or rapid , often nonsustained , atrial tachycardias that arise from the pulmonary veins and , less commonly , nonpulmonary vein sites [ [ 1 - 3 ] ] .
these are generally ' focal ' arrhythmias ; that is , they can be mapped to relatively small areas of less than a few millimeters .
these focal triggering arrhythmias may initiate true af , which is due to a complex series of multiple wavefronts that simultaneously activate both the right and left atria in a non - repeating pattern or in minimally repeating patterns . the ability to maintain af
is dependent on a number of factors , such as atrial size , mass , stretch and fibrosis , heterogeneity of activation and repolarization , continued activation of triggering arrhythmias to prevent organization and termination , and autonomic tone . if the appropriate substrate is not present , af once initiated will not be maintained and will spontaneously terminate .
thus , the interaction between initiators and substrate determine whether the clinical presentation is that of paroxysmal or persistent af .
in addition , a host of factors serve to modulate both the initiators and substrate of af , including autonomic tone , volume status , body position , drugs , and electrical and structural remodeling that occur as a direct consequence of af [ [ 5 - 7 ] ] .
thus , af can be considered as a complex interplay of initiators , substrate , and modulating factors .
palliative procedures are aimed at nonpharmacological rate control by creation of complete heart block ( av node ablation with permanent pacemaker placement ) or high - grade av block ( av nodal modification without pacemaker ) .
these approaches obviously do not prevent af from continuing or recurring , not do they eliminate the need for antiarrhythmic drugs or anticoagulation .
these curative ablation approaches for af are aimed at either eliminating or isolating af initiators , and/or alterating of atrial substrate in order to prevent the maintenance of af .
what limits all of these attempts at af ablation is that we do not understand the full range of events that initiate and/or maintain af .
nonetheless , progress has been made toward successful ablation of af in a number of clinical conditions , such as af in the setting of no or minimal heart disease .
the question presented by this rapid growth in our knowledge is whether af ablation is sufficiently mature to serve as frontline therapy for af , with widespread application within the community .
the greatest advances have been made in the ablation of the focal atrial arrhythmias that are responsible for initiating and , in some cases , maintaining af . although regular rapid atrial tachyarrhythmias were shown in animal models to be possible causes of af as long as 50 years ago and
were observed to be the initiating events of af in humans 30 years ago , the precise nature of these initiators in humans remained poorly understood until recently .
haissaguerre and coworkers and others recently showed that atrial tachycardias , which arise most typically within the residual left atrial musculature that surrounds the pulmonary veins as they approach the left atrium , are responsible for initiating most af , at least in those patients with no or minimal heart disease [ [ 1 - 3 ] ] .
ablation of these atrial tachycardias has been shown to cure af in patients with frequent atrial ectopy and insignificant cardiac disease .
a number of retrospective and uncontrolled studies [ [ 1 - 3 ] ] have shown that focal af ablation may be curative in 60 - 85% of these patients .
variables that are related to success rates include patient inclusion criteria , ablation approaches , severity of underlying atrial disease , number of initiator sites , and the duration of af .
although none of these studies had parallel control groups , most of the patients had high frequencies of af before ablation , and the lack of symptomatic episodes after successful ablation strongly suggests that the procedure is effective .
despite the encouraging initial reports , ablation of focal initiators of af is still in its infancy .
typically , these groups have included younger patients with no or minimal heart disease , and with paroxysmal af , high - density atrial ectopy , and a priori assessment that they had ' focal af ' .
the efficacy of these procedures applied to unselected patient populations with paroxysmal af is not known , and only limited data are available regarding efficacy for patients with persistent and chronic af .
preliminary studies that evaluated patients with persistent af have demonstrated more modest results , and preliminary results for patients with chronic af are very disappointing .
furthermore , there is little available data regarding the efficacy of the procedure in the setting of significant cardiac disease .
more information regarding optimal patient selection for focal af ablation approaches is needed . in reported studies regarding the ablation of af initiators ,
follow up is limited to several months , or at best a couple of years .
insensitive follow - up techniques may result in considerable errors of omission , and may overestimate long - term cure rates .
in addition , conversion of symptomatic to asymptomatic af could occur as a consequence of these procedures and not be detected on simple symptom reviews during follow up .
appropriate means for long - term follow up in these trials needs to be determined . however , the limitations of random 24-h holter recordings once every month or a few seconds of event recordings every week are obvious in a disease process that may manifest itself only for a few hours every other month .
recently developed arrhythmia - triggered ( as well as patient - activated ) event monitors may be useful in this situation for more precise determination of whether the end result is truly curative or just quantitative suppressive of af events . given the relatively short duration of available follow up for focal af ablation approaches , we do not know whether these procedures will continue to protect patients over years and decades .
importantly , we do not understand either the pathogenesis or the natural history of these initiating arrhythmias . whether additional initiators will evolve over the years is not known , and will remain unknown until long - term follow - up study results are available . in addition ,
although most of the initiating arrhythmias appear to arise within the pulmonary veins , the applicability of this assumption to larger , more varied groups of patients with af remains to be tested .
thus , the efficacy of empiric pulmonary vein isolation procedures in the diversity of patients with af is unknown . of major concern with pulmonary vein isolation or ablation procedures
symptomatic stenosis is detected in fewer than 5% of patients , and is due to complete or near - complete stenosis of one or more pulmonary vein [ [ 1 - 3 ] ] .
however , up to 42% of patients may have some degree of pulmonary vein stenosis .
although tolerance of these mild degrees of stenosis is excellent , the long - term sequelae are not known .
cases of pulmonary hypertension as a consequence of pulmonary vein ablation and stenosis have been reported .
although stenting of the stenosis may be effective acutely , the long - term patency of these stents is unknown .
even less is known about procedures that are aimed at eliminating the ability of the atria to maintain af .
curative ablation of af can be achieved during open - heart surgery by surgically isolating the pulmonary veins from the atria , dividing the right and left atria into several connected , dead - end corridors in continuity with the sinus node , and excising the atrial appendages .
the surgically created lines of conduction block created by the connected dead - end corridors prevent the wandering wavelets of re - entry during af from sustaining .
the surgical maze procedures have been shown to be effective treatments for paroxysmal , persistent , and chronic af .
although open - chest procedures are required , the cure rate is approximately 85% , with substantial morbidity and some mortality attendant to the surgical procedures .
following these principles , multiple investigators have shown [ [ 13 - 16 ] ] that creation of multiple long lines of conduction block in both the right and left atria to simulate in part the surgical maze procedure may cure some af patients .
although the initial retrospective analyses looked exceptionally promising , with up to 87% cure rates , two ongoing prospective studies of biatrial lesion sets have been less optimistic , with success rates of approximately 50% or less .
although a greater number of patients have less frequent af on antiarrhythmic drugs , this end - point is subjective , is difficult to quantify , and is subject to bias .
more importantly , rates of major complications in the prospective studies have been unacceptably high , occurring in approximately one - quarter of patients . although symptomatic control with continued antiarrhythmic drug therapy can be achieved in the majority of patients , the high rates of serious complications significantly limit application of these procedures to only a few , heavily symptomatic patients , and hamper enrollment of patients in studies evaluating new technologies that are aimed at simplifying the procedure .
limitation of the linear lesions to the right atrium greatly simplifies the procedure and avoids the potential risks associated with creation of linear lesions in the left atrium .
right - atrium - only linear lesion sets rarely cure patients with paroxysmal af , however , and are ineffective in chronic af .
selected patients with paroxysmal af may have high rates of cure , but the criteria for selection of these patients are not well defined .
by eliminating af with ablation of either the initiators or substrate , it is assumed that the long - term need for chronic anticoagulation for stroke prophylaxis is avoided .
although uncontrolled data from patients after surgical maze procedures appears to support this contention , there are no long - term data regarding af ablation procedures
these may be due to emboli from a number of sources , including the following : pre - existing left atrial thrombi ; lesion - related thrombus in the left atrium or pulmonary veins ; thrombus from diminished left atrial transport ; paradoxic emboli across persistence flow communications from trans - septal access sites ; continued undetected atrial fibrillation ; and valvular or peripheral vascular disease .
further data from the individual procedures are needed in order to determine the advisability of withdrawal of anticoagulation after af ablation .
as with all issues regarding nonpharmacologic therapy , prospective , controlled trials comparing various ablation approaches with traditional antiarrhythmic drug therapy or atrioventricular nodal ablation and pacemaker placement need to be performed .
these trials can address such issues as comparative efficacy , complication rates , quality of life indices , and cost - effectiveness .
because mortality rates for conventional and new ablation approaches are very low , it is unlikely that trials of sufficient power will ever be performed to determine whether mortality benefits exist .
these include ablation directed at spontaneously discharging foci , isolation of pulmonary veins with spontaneously occurring arrhythmias , and empiric pulmonary vein isolation of all pulmonary veins with a muscular sleeve , regardless of whether an arrhythmogenic focus is identified .
various technologies have been or are being developed to ablate initiators and modify substrate . as these approaches are developed , prospective comparisons are needed to determine which approaches and technologies have the greatest efficacy and safety .
it is clear from the available information that curative ablation approaches for af are rapidly advancing , but too little is known at present to advocate widespread implementation . in particular , given the potential for serious complications , even with ablation of af initiators in healthy individuals , it is difficult to advocate ablation as frontline therapy in advance of traditional pharmacologic approaches .
furthermore , the procedures are technically difficult , arduous , time - consuming , and are not cost - effective .
given the risks of serious complications , even in experienced hands , a loud note of caution must be expressed concerning widespread application by individuals who are inexperienced in technical aspects of the procedure , such as trans - septal catheterization , pulmonary vein ablation , and linear lesion creation in the left atrium .
appropriate educational guidelines need to be established in order to ensure safe application of these approaches in the community .
| various approaches for catheter ablation of focal initiators of atrial fibrillation or the substrate for maintaining atrial fibrillation have evolved over the past 5 years . despite these advances , there are still a large number of unresolved issues regarding the efficacy and safety of these procedures as well as optimal patient selection for the different approaches .
these uncertainties raise questions about the applicability at the present time of atrial fibrillation as front - line therapy within the community . |
fixation - off sensitivity ( fos ) is an interesting electroencephalography ( eeg ) phenomenon elicited by the elimination of central vision and fixation .
fixation - off sensitivity can act as a seizure trigger in patients with panayiotopoulos syndrome ( ps ) and more frequently in patients with idiopathic childhood occipital epilepsy of gastaut .
rarely , it can be manifested in symptomatic occipital lobe epilepsy or within the spectrum of benign childhood seizure susceptibility syndrome .
it can also be seen in eyelid myoclonia with absences , in atypical benign partial epilepsy ( abpe ) , and even in asymptomatic children without epilepsy .
the reverse of fos , inverted fos ( ifos ) , in which the epileptiform activity is suppressed by the absence of central vision or fixation and activated by central vision or fixation , has been described in only two patients . in one patient with lafora disease
whose myoclonus was suppressed by passive eye closure , neurophysiologic studies disclosed that fixation was the most important enhancer of myoclonus .
magnetoencephalographic studies of visual evoked fields revealed abnormal activation of the visual corticocortical pathway via the insular cortex not seen in controls .
the authors hypothesize that abnormal activation of the insular cortex may be involved in triggering the mechanism of fixation - sensitive myoclonus .
the second patient with ifos was diagnosed with abpe , in which a decrease of epileptiform activity was evident upon closure of the eyes and upon inhibition of fixation by the use of frenzel goggles .
here we describe the first reported case of coexistence of fos and ifos , documented in an emirati girl with ps whose eegs unexpectedly and repeatedly showed a combination of fos and ifos .
an emirati girl with no significant pre- or postnatal history and normal motor and cognitive development presented to our pediatric neurology clinic at the age of 5 years and 3 months with a history of 4 attacks of screaming while asleep .
the first seizure occurred at the age of 4 years and 4 months of age . at that time
, she developed recurrent retching and vomiting , her eyes turned to one side , and she then became flaccid like a rag doll , with minimal motor activity for 23 min and following that was partially unresponsive for about 30 min .
she was rushed to a local hospital where magnetic resonance imaging of the head was done and reported as normal .
the second and third attacks occurred 2 and 4 months later , respectively , with similar semiology and lasting for 12 min .
the fourth attack occurred 2 weeks before the patient was presented to us , during which versive seizures with eye deviation to one side continued for about 30 min .
a diagnosis of ps was made , based on the symptomatology in addition to the eeg results , and the patient was started on oxcarbazepine orally .
ten days later , she developed a rash on the skin and mucous membranes associated with a mild fever and lethargy .
johnson syndrome was suspected , and oxcarbazepine was discontinued and the patient was started on levetiracetam .
the parents are first - degree cousins who have 5 other children , 3 girls and 2 sons .
an older sister , the third sibling , an 8-year - old child , has a history of recurrent febrile seizures from the age of 5 months up to 4 years .
three near relatives on the father 's side have had epilepsy since early childhood . a male cousin to both parents who is now 12 years old had a history of frequent nocturnal seizures beginning at the age of 5 years .
he was treated initially with traditional remedies with no improvement , then was treated abroad with levetiracetam and has been seizure - free since the age of 9 . at the age of 7 years , it was determined that he has learning difficulties but is now in a regular school with the help of a shadow teacher .
another paternal cousin , a girl , now 8 years of age , had a history of prolonged , most probably focal seizures associated with severe vomiting , 2 to 3 times per month , since the age of 5 .
she was put on oxcarbazepine and has been seizure - free for the last 18 months .
the third relative with seizures is a distant paternal cousin , a 14-year - old boy , who had a history of seizures , usually during sleep , since the age of 11 .
he is now on levetiracetam and has been seizure - free for the last year .
a 20-channel scalp video - eeg was recorded at our eeg laboratory over a 30-min period on 2 separate occasions .
the first test was performed at the time of the patient 's initial presentation at the age of 5 years and 3 months .
all recordings were performed according to the protocol suggested by panayiotopoulos , which consisted of spontaneous eye opening and closing in a well - illuminated room , with and without the use of dark underwater goggles covered with opaque tape , underwater goggles covered with semitransparent tape , and frenzel lenses to eliminate central fixation and vision .
the patient was asked to open and close her eyes every 6 s for a total of 6 times .
hyperventilation and intermittent photic stimulation were performed in all records , and a natural - sleep eeg was also obtained .
both eegs revealed a normal background , and ifos was suspected when continuous right - sided , moderate - voltage occipital paroxysms appeared when the patient opened her eyes in a lit room and disappeared immediately when she closed her eyes ( fig .
continuous bilateral occipital epileptiform discharges were seen when the patient closed her eyes , and this persisted for as long as her eyes were closed , which suggested fos ( fig .
the diagnosis of fos was unequivocally established by demonstrating that these occipital discharges are activated by impeding central vision using the panayiotopoulos protocol mentioned earlier ( fig . 4a and b ) .
the parents are first - degree cousins who have 5 other children , 3 girls and 2 sons .
an older sister , the third sibling , an 8-year - old child , has a history of recurrent febrile seizures from the age of 5 months up to 4 years .
three near relatives on the father 's side have had epilepsy since early childhood . a male cousin to both parents who is now 12 years old had a history of frequent nocturnal seizures beginning at the age of 5 years .
he was treated initially with traditional remedies with no improvement , then was treated abroad with levetiracetam and has been seizure - free since the age of 9 . at the age of 7 years , it was determined that he has learning difficulties but is now in a regular school with the help of a shadow teacher .
another paternal cousin , a girl , now 8 years of age , had a history of prolonged , most probably focal seizures associated with severe vomiting , 2 to 3 times per month , since the age of 5 .
she was put on oxcarbazepine and has been seizure - free for the last 18 months .
the third relative with seizures is a distant paternal cousin , a 14-year - old boy , who had a history of seizures , usually during sleep , since the age of 11 .
he is now on levetiracetam and has been seizure - free for the last year .
a 20-channel scalp video - eeg was recorded at our eeg laboratory over a 30-min period on 2 separate occasions .
the first test was performed at the time of the patient 's initial presentation at the age of 5 years and 3 months .
all recordings were performed according to the protocol suggested by panayiotopoulos , which consisted of spontaneous eye opening and closing in a well - illuminated room , with and without the use of dark underwater goggles covered with opaque tape , underwater goggles covered with semitransparent tape , and frenzel lenses to eliminate central fixation and vision .
the patient was asked to open and close her eyes every 6 s for a total of 6 times .
hyperventilation and intermittent photic stimulation were performed in all records , and a natural - sleep eeg was also obtained .
both eegs revealed a normal background , and ifos was suspected when continuous right - sided , moderate - voltage occipital paroxysms appeared when the patient opened her eyes in a lit room and disappeared immediately when she closed her eyes ( fig .
, continuous bilateral occipital epileptiform discharges were seen when the patient closed her eyes , and this persisted for as long as her eyes were closed , which suggested fos ( fig .
the diagnosis of fos was unequivocally established by demonstrating that these occipital discharges are activated by impeding central vision using the panayiotopoulos protocol mentioned earlier ( fig .
fixation - off sensitivity refers to forms of epilepsy or eeg discharges characterized by continuous focal or generalized spikes , spike wave , or sharp - wave focal discharges , usually in occipital regions , that consistently occur within 13 s of eye closure , persist throughout the eye - closed state ( i.e. , when central vision and fixation are eliminated ) , and disappear immediately with eye opening ( fig .
3 ) . the diagnosis of fos is confirmed only by demonstrating that the same abnormalities also occur by blocking central vision and fixation using semitransparent goggles ( which , by reducing incoming light , obscure any visual input ) , frenzel lenses ( which produce refractive errors ) , translucent spherical lenses ( which distort light refraction ) , or ganzfeld stimulation ( using a homogeneous visual field that gives the subjective visual experience of the surface fading away ) in uncooperative patients .
described a patient with persistent left - sided fos following perinatal insult in which brain magnetic resonance imaging revealed gliosis and ulegyria over the left posterior occipital cortex corresponding to the topographic representation of the macula .
they suggested that the extensive denervation of the area representing the macula along with the presence of hyperexcitable ulegyric cortex is responsible for the phenomenon of fos .
fixation - off sensitivity should be differentiated from pure forms of scotosensitivity , in which eeg discharges or epileptic seizures are elicited by darkness , and from eye - closure sensitivity , in which the epileptiform discharges are triggered by eye closure , occur within 1 to 3 s after the positive deflection of the eye closure artifact , are self - limited and of brief duration , typically relate to photosensitivity , and are suppressed by complete darkness .
we document for the first time , to the best of our knowledge , two opposite phenomena in a female child with ps .
the first was the reverse of fos , a condition called ifos , where occipital discharges were activated during eye opening and fixation and were suppressed by the elimination of central vision and fixation ; in addition , during the same eeg recordings , fos was unequivocally documented .
kumada et al . described an adolescent with lafora disease whose myoclonus was suppressed by passive eye closure .
immediate inhibition of epileptiform activity was evident upon closure of the eyes and upon inhibition of fixation by the use of frenzel lenses .
the explanation of these different mechanisms is difficult , but it seems that ifos may be a variant phenomenon of fos . in lafora disease , the presence of giant somatosensory - evoked potentials , indicating hyperexcitability of the sensorimotor cortex , is well known .
this hyperexcitability of the sensorimotor cortex in response to afferent stimuli indicates a severe impairment of inhibitory mechanisms in lafora disease .
the authors stated that they did not know whether the ifos represented an exaggeration of photosensitivity because of the impaired inhibitory mechanism or was based on another mechanism .
this patient was diagnosed with ps , a benign , age - related focal seizure disorder occurring in early and mid - childhood .
it is characterized by seizures , often prolonged , with predominantly autonomic symptoms , manifested mainly by emesis and by an eeg that shows shifting or multiple foci or both , often with occipital predominance . because of frequent eeg occipital spikes seen in patients with ps , it has been erroneously considered as occipital epilepsy . in the present patient , the eegs showed mainly occipital discharges .
indicated that the eeg foci in most patients with ps easily shift location , multiply , and propagate diffusely with age rather than persistently localizing in the occipital region and that the occipital eeg focus is seen most frequently between 2 and 5 years , as is the case with the present patient .
the present patient has 1 sibling with a history of febrile seizures in addition to 3 more distant relatives diagnosed with epilepsy ( fig .
although it is difficult to identify the classification of seizure types in these distant relatives , it is known that siblings and relatives of those with ps may , in rare instances , have the same type of seizures or rolandic and idiopathic childhood occipital epilepsy .
interestingly , fos has eeg features that are the opposite of those observed during photosensitivity . despite the conflicting corresponding mechanisms underlying these two phenomena , both fos and photosensitivity can occur in the same patient , and one phenomenon may evolve into the other . in conclusion ,
fos is a rare phenomenon , and its variant ifos has been diagnosed previously in only 2 patients .
we document , for the first time , the coexistence of the two conditions in a child with ps .
fosfixation - off sensitivityifosinverted fixation - off sensitivitypspanayiotopoulos syndromeabpeatypical benign partial epilepsyeegelectroencephalography fixation - off sensitivity inverted fixation - off sensitivity panayiotopoulos syndrome atypical benign partial epilepsy electroencephalography
parental written informed consent was obtained for publication of this case report and any accompanying images .
| fixation - off sensitivity ( fos ) is a rare phenomenon elicited by elimination of central vision and fixation , which even in the presence of light induces occipital paroxysms or generalized paroxysmal discharges .
it is most commonly encountered in patients with idiopathic childhood occipital epilepsies but may also be observed in cases of symptomatic focal and generalized epilepsies .
we describe a female emirati child with panayiotopoulos syndrome who exhibited fos in addition to the reverse phenomenon called inverted fixation - off sensitivity , in which the electroencephalographic discharges were suppressed by the absence of central vision or fixation and activated by central vision or fixation . |
pelvic insufficiency fractures are common in female patients with osteoporosis and mostly occurred by trivial injury or slip down . as elderly population increases
if pelvic insufficiency fractures occur in elderly population , it leads to increase in dependency by mobility reduction in patients and then mortality rate also increases due to aggravation of medical comorbidities .
however , diagnosis and treatments are easily missed for the pelvic insufficiency fractures occurred in elderly population .
the aim of treatments for pelvic insufficiency fractures is to let patients walk early with bed rest and medication for pain relief . as we obtained outstanding clinical outcome such as early pain relief and walk utilizing parathyroid hormone ( pth ) 1 - 34(teriparatide ) in pelvic insufficiency fractures , we present the cases as well as a literature review .
the patient was a 76 year old female and visited outpatient clinic due to the left hip pain that was occurred after a fall ( slip down ) .
fractures in left pubic ramus were observed on simple radiograph at the admission and fractures in sacrum left side were found in bone single photon emission computed tomography ( spect ) and computed tomography ( ct ) scan ( fig .
1 ) . in bone mineral density examination utilizing dual - energy x - ray absorptiometry ( dxa ; prodigy advance , ge - lunar corp . , madison , wi , usa ) , osteoporosis was diagnosed with t - score -5.2 ( l1 - 4 ) but the patient did n't have history of diagnosis and treatments in osteoporosis before the injury . although teriparatide was only allowed for the treatment of osteoporosis in korea , the patient
was given 20 g of teriparatide ( 1 - 34 pth , forsteo ) subcutaneously once a day in order to promote fracture healing as well as treat osteoporosis .
bed rest and medication for pain relief was combined at the same time ; tramadol 50 mg ( tridol ) was injected intravenously twice , morning and night , a day for seven days and transdermal fentanyl patch 12 g / hr ( durogesic dtrans ) was administered every 72 hours for 4 weeks .
pain visual analogue scale ( vas ) was 7 at the time of admission and then decreased to 3 two weeks after the administration .
the patient was able to move using a wheelchair a week after the injury and walked using a walker 10 days after the injury .
then , the patient was able to walk herself without pain six weeks after the injury . on simple radiograph a month after the injury , callus formation was observed in left pubic ramus and then it was even definitely denser on 3 months follow - up x - ray ( fig .
eighty two years old female visited outpatient clinic due to the pain on the right buttock that was occurred after a fall ( slip down ) on her behind .
simple radiograph at the time of her visiting indicated fractures in right pubic ramus and ct scan showed fractures in sacrum left side ( fig .
3 ) . osteoporosis with t - score -3.3 ( l1 - 4 ) was observed , but the patient had never diagnosed and treated for osteoporosis before the injury .
the patient was injected with 20 g of teriparatide ( 1 - 34 pth , forsteo ) subcutaneously once a day for 8 weeks and bed rest and medication for pain relief was combined at the same time ; tramadol 50 mg ( tridol ) was injected intravenously twice , morning and night , a day for two weeks and transdermal fentanyl patch 12 g / hr ( durogesic dtrans ) was administered every 72 hours for three weeks .
vas was 7 at the time of admission and then decreased to 2 two weeks after the administration ( three weeks after the injury ) .
the patient was able to ambulate with a wheelchair three weeks after the injury and walked independently without a walker four weeks after the injury .
on simple radiograph four weeks after the injury , callus formation was observed in right pubic ramus and then it was even definitely denser on 3 months follow - up x - ray ( fig .
the patient was a 76 year old female and visited outpatient clinic due to the left hip pain that was occurred after a fall ( slip down ) .
fractures in left pubic ramus were observed on simple radiograph at the admission and fractures in sacrum left side were found in bone single photon emission computed tomography ( spect ) and computed tomography ( ct ) scan ( fig .
1 ) . in bone mineral density examination utilizing dual - energy x - ray absorptiometry ( dxa ; prodigy advance , ge - lunar corp . , madison , wi , usa ) , osteoporosis was diagnosed with t - score -5.2 ( l1 - 4 ) but the patient did n't have history of diagnosis and treatments in osteoporosis before the injury . although teriparatide was only allowed for the treatment of osteoporosis in korea , the patient
was given 20 g of teriparatide ( 1 - 34 pth , forsteo ) subcutaneously once a day in order to promote fracture healing as well as treat osteoporosis .
bed rest and medication for pain relief was combined at the same time ; tramadol 50 mg ( tridol ) was injected intravenously twice , morning and night , a day for seven days and transdermal fentanyl patch 12 g / hr ( durogesic dtrans ) was administered every 72 hours for 4 weeks .
pain visual analogue scale ( vas ) was 7 at the time of admission and then decreased to 3 two weeks after the administration .
the patient was able to move using a wheelchair a week after the injury and walked using a walker 10 days after the injury .
then , the patient was able to walk herself without pain six weeks after the injury . on simple radiograph a month after the injury , callus formation was observed in left pubic ramus and then it was even definitely denser on 3 months follow - up x - ray ( fig .
eighty two years old female visited outpatient clinic due to the pain on the right buttock that was occurred after a fall ( slip down ) on her behind .
simple radiograph at the time of her visiting indicated fractures in right pubic ramus and ct scan showed fractures in sacrum left side ( fig .
3 ) . osteoporosis with t - score -3.3 ( l1 - 4 ) was observed , but the patient had never diagnosed and treated for osteoporosis before the injury .
the patient was injected with 20 g of teriparatide ( 1 - 34 pth , forsteo ) subcutaneously once a day for 8 weeks and bed rest and medication for pain relief was combined at the same time ; tramadol 50 mg ( tridol ) was injected intravenously twice , morning and night , a day for two weeks and transdermal fentanyl patch 12 g / hr ( durogesic dtrans ) was administered every 72 hours for three weeks .
vas was 7 at the time of admission and then decreased to 2 two weeks after the administration ( three weeks after the injury ) .
the patient was able to ambulate with a wheelchair three weeks after the injury and walked independently without a walker four weeks after the injury . on simple radiograph four weeks after the injury ,
callus formation was observed in right pubic ramus and then it was even definitely denser on 3 months follow - up x - ray ( fig .
fatigue fractures occur when excessive stress is applied to normal osseous tissues and insufficiency fractures occur when normal stress is applied to abnormal osseous tissues such as osteoporosis .
vertebral fractures , hip fractures , wrist fractures , and humerus fractures are typical osteoporotic fractures . although pelvic insufficiency fractures which are not paid attention so far have occurred more frequently as elderly population increases , they have been still overlooked compared to vertebral fractures or hip fractures .
it was reported that incidence rate of pelvic insufficiency fractures is one third to one fourth of hip fractures . according to an recent epidemiological study , however , the incidence rate of pelvic insufficiency fractures increased from 20 every 100,000 people in 1970 to 92 every 100,000 people in 1997 for patients more than 60 years old and from 1997 to 2002 , it increase 5.3 times .
in addition to the incidence rate , mortality rate of pelvic insufficiency fractures is also comparable with hip fractures ; recent studies reported that annual mortality rate of pelvic insufficiency fractures is 16.3% on average which is similar to annual mortality rate of hip fractures .
as pelvic insufficiency fractures hardly have secondary displacement or nonunion , bed rest and medication for pain relief are typical treatment . however
, when the medication for pain relief is performed for a long time in elderly patients , side effects can occur commonly ; gastrointestinal side effects can occur when administering nonsteroidal anti - inflammatory drugs ( nsaids ) or constipation and further cognitive disorders can occur when administering opioids .
if pains are not controlled sufficiently , patients can not recover their mobility in early stage and risk of complications increase due to the long - term bed rest .
therefore , in case where sufficient medication is hard to be carried out , surgical treatments such as ramoplasty or sacroplasty can be performed in order to relieve pains in early stage thereby improving mobility of patients .
recently , many animal studies have proved that when pth was injected , enchondral ossification and callus formation occur rapidly as accelerating cartilage differentiation , indicating that pth facilitates fracture healing . it is reported that callus developed by administration of pth possesses superior bone quality and mechanical strength compared to the callus observed when administering bisphosphonate agents . for osteoporosis patients with pelvic insufficiency fractures ,
we performed typical treatment bed rest and analgesics for pelvic insufficiency fractures and administered pth 1 - 34(teriparatide ) for osteoporosis . in the present cases , we experienced that pth 1 - 34(teriparatide ) was effective in callus formation as well as treatment of osteoporosis .
this is also in agreement with a clinical study that when pelvic insufficiency fractures was treated with pth derivatives , fracture healing were promoted and duration of bed rest was shortened as treatment duration was reduced . in present two cases ,
pth 1 - 34(teriparatide ) was administered for two months and then replaced with bisphosphonate due to financial reasons .
decrease in callus formation or reccurrence of pains did n't happen after terminating the pth .
weber et al . reported that pains were continued for 9 weeks when pelvic insufficiency fractures were treated with bed rest and analgesics and the authors also had similar results with the same treatment .
we confirmed that pain relief and callus formation were relatively faster when analgesic was administered with pth 1 - 34(teriparatide ) in our cases .
effectiveness of analgesics such as fentanyl patch ca n't be ruled out for rapid pain relief and two cases are not enough to conclude that the treatment was facilitated .
therefore , additional studies as control groups are required in order to conclude the effectiveness of treatment with pth 1 - 34(teriparatide ) .
pth 1 - 34(teriparatide ) should not be administered in the patients at high risk of osteosarcoma including patients with paget 's disease , patients who showed increase in alkaline phosphatase without any reasons , infants , and patients with radiation . as side effects such as dizziness due to orthostatic hypotension and leg pains
were reported , more attention needs to be paid . knowing that typical treatment of pelvic insufficiency fractures shortens the duration of bed rest with sufficient pain relief , while the treatment with pth 1 - 34(teriparatide ) is anticipated to reduce the duration of bed rest by facilitating fracture healing .
considering that pelvic insufficiency fractures are fragility fractures caused by osteoporosis , it can be a fundamental treatment method of pelvic insufficiency fractures .
moreover , fewer amounts of drugs may be administered for pain control as fracture healing are accelerated so that the side effects due of drug in elderly patients may be decreased additionally . in conclusion ,
pth 1 - 34(teriparatide ) treatment is considered to be useful to relieve pain rapidly , to allow walk in early stage , and to reduce side effects caused by drug administration in pelvic insufficiency fracture patients with osteoporosis . | as a result of aging population , the incidence of pelvic insufficiency fracture has been increasing . pain - related immobility caused by pelvic insufficiency fractures may result in a serious dependency and high mortality with preexisting comorbidities .
we present two cases of pelvic insufficiency fracture in elderly patients which had good clinical outcome by parathyroid hormone 1 - 34(teriparatide ) treatment as well as a literature review . |
hydrogels with predictable degradation
are highly desired for biomedical
applications where timely disintegration of the hydrogel ( e.g. , drug
delivery , guided tissue regeneration ) is required .
however , precisely
controlling hydrogel degradation over a broad range in a predictable
manner is challenging due to limited intrinsic variability in the
degradation rate of liable bonds and difficulties in modeling degradation
kinetics for complex polymer networks .
more often than not , empirical
tuning of the degradation profile results in undesired changes in
other properties . here we report a simple but versatile hydrogel platform
that allows us to formulate hydrogels with predictable disintegration
time from 2 to > 250 days yet comparable macroscopic physical properties .
this platform is based on a well - defined network formed by two pairs
of four - armed polyethylene glycol macromers terminated with azide
and dibenzocyclooctyl groups , respectively , via labile or stable linkages .
the high - fidelity bioorthogonal reaction between the symmetric hydrophilic
macromers enables robust cross - linking in water , phosphate - buffered
saline , and cell culture medium to afford tough hydrogels capable
of withstanding > 90% compressive strain .
strategic placement of
labile
ester linkages near the cross - linking site within this superhydrophilic
network , accomplished by adjustments of the ratio of the macromers
used , enables broad tuning of the disintegration rates precisely matching
with the theoretical predictions based on first - order linkage cleavage
kinetics .
this platform can be exploited for applications where a
precise degradation rate is targeted . | |
fragile x syndrome ( fxs ) is the most common inherited form of intellectual disability ( i d ) affecting ~1/4000 males and ~1/5000 to 1/8000 females ( refs 1 , 2 ) and is caused by the full - mutation ( fm , > 200 repeats ) hyperexpansion of ( cgg)n triplet - repeat sequence in the 5-untranslated region of the x - linked fragile x mental retardation 1 ( fmr1 ) gene ( refs 3 , 4 , 5 , 6 ) .
fxs is characterised by i d , seizures and other behavioural issues such as anxiety , hyperactivity and autism spectrum disorder ( asd ) .
symptoms are usually milder in affected females due to x - inactivation and cellular mosaicism ( ref . 7 ) .
premutation ( pm ) expansions ( 55200 repeats ) , which have been associated with the late - onset fmr1-related conditions fragile x - associated tremor / ataxia syndrome ( fxtas ) and fragile x - associated primary ovarian insufficiency ( fxpoi ) , occur more frequently , with three recent studies reporting pm prevalence rates of 1/209 females and 1/430 males ( ref . 8) , 1/148 females and 1/290 males ( ref .
approximately 40% of male and 816% of female pm carriers will develop fxtas ( refs 11 , 12 ) , with female pm carriers having an additional 20% risk for fxpoi ( refs 13 , 14 ) .
in addition , a spectrum of medical co - morbidities such as thyroid dysfunction , fibromyalgia , neuropathy , sleep apnea , seizures and hypertension have been observed among pm carriers , particularly in females with fxtas ( refs 15 , 16 , 17 , 18 , 19 , 20 ) . developmental delay ,
attention problems , asd and anxiety have also been reported in some pm individuals ( refs 21 , 22 ) .
furthermore , the meiotically unstable nature of expanded fmr1 alleles predisposes female pm carriers to an increased risk of having fxs - affected offspring , with almost 100% fm transmission risk when the transmitted allele is > 90 cgg repeats ( ref .
this marked decline in repeat stability among expansion carriers is strongly correlated with the reduction or absence of agg interruptions within the cgg repeat tract ( refs 24 , 25 ) .
normal ( nl , 544 repeats ) and intermediate ( i m , 4554 repeats ) alleles do not expand to fm upon transmission , although some i m alleles can gradually expand to fm after multiple generations ( refs 26 , 27 ) .
however , the key question of whether i m individuals are at risk for pm - related conditions still remains to be addressed ( ref .
2 ) . according to the fxs testing guidelines , only individuals with a family history of fxs , asd , developmental delay , learning disabilities or clinical features suggestive of fxs , fxtas or fxpoi , qualify for fmr1 molecular testing ( refs 1 , 28 ) .
there has been enormous interest in evaluating the possibilities of extending fragile x genetic tests to newborns and women of reproductive age so as to detect expansion carriers who are most likely to benefit from early identification ( refs 1 , 21 , 29 , 30 , 31 , 32 )
. however , for such large - scale screenings to be practicable , cost - effective and technically simpler fmr1 screening tools are required .
southern blot ( sb ) , the gold standard fxs testing method is still the preferred diagnostic tool for characterising expanded fmr1 alleles in fm individuals .
however , owing to the laborious and low - throughput nature of sb , several polymerase chain reaction ( pcr)-based assays have been developed as alternative procedures primarily to minimise or eliminate the need to perform sb on every sample ( refs 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 ) . most commonly , these studies have employed the traditional repeat - spanning pcr and/or the triplet - repeat primed pcr ( tp - pcr ) . although the initial limitation of amplifying large gc - rich pm and fm expansions has been overcome ( refs 39 , 40 ) , the reliability and upper limit of fm sizing ability of repeat - spanning pcr is contentious .
in contrast , tp - pcr methods can reliably detect all fm alleles regardless of the actual fm repeat size .
tp - pcr assays facilitate accurate repeat size determination of up to ~200 cgg repeats by virtue of their assay design that incorporates capillary electrophoresis ( ce ) for analysing the heterogeneous tp - pcr amplicon fragments ( refs 37 , 38 ) .
however , for newborn and carrier screening applications that involve processing large numbers of samples , methods requiring ce are not ideal due to the high - cost factor associated with the selected approach of amplicon analysis .
we had previously proposed a cost - effective screening platform that coupled tp - pcr with melting curve analysis ( mca ) for rapid identification of fmr1 expansions in both males and females ( ref .
a higher return to baseline temperature was used as an indicator to confirm the presence of an expanded allele .
therefore , for unambiguous determination of expansion status in samples carrying fmr1 alleles in the high - im to low - pm size range , precise discernment of return to baseline temperature is imperative .
reliance on this analysis mode is prone to producing ambiguous results when samples do not display an obvious drop in df / dt .
the assay did not generate defined melt peaks , which prohibited identification of melt peak temperature ( tm ) and necessitated dependence on return to baseline temperature .
we now describe an improved direct tp - pcr ( dtp - pcr ) mca assay that yields remarkably well - defined melt profiles , thus enabling utilisation of the highly reliable melt peak temperature ( tm ) instead of the return to baseline temperature to identify expansion carriers .
furthermore , to show the agreement between dtp - pcr mca classification and the actual fmr1 genotypes of tested samples , we simultaneously performed a dtp - pcr ce - based sizing assay that was optimised to detect all fmr1 allelic classes .
in addition , we have also demonstrated the sensitivity of both assays in detecting low - level mosaicism for fm allele using artificial dna mixtures .
29 lymphoblastoid cell line - derived genomic dna samples ( listed in table 1 ) obtained from the coriell cell repositories ( ccr ; coriell institute for medical research , camden , nj ) were used for the initial optimisation and subsequent evaluation of direct triplet - primed pcr ( dtp - pcr ) assays . additionally , a panel of archived and previously characterised peripheral blood - derived patient dnas consisting of 54 nl , 25 pm and 28 fm fmr1 carriers was tested in a blinded fashion .
approval to perform this study was granted by the national university of singapore institutional review board ( 07 - 123e ) and the singhealth centralized institutional review board ( 2013/-73/a ) .
table 1.genomic dna samples from the coriell cell repositories used for direct tp - pcr mca and cecoriell idsexcoriell fmr1 sizeresults from a consensus study ( ref .
47)direct tp - pcrmca resultallele sizeallele structuregm 07175f23/30n.a.nl23/3013 + 9/10 + 9 + 9gm 06890m30n.a.nl3010 + 9 + 9na 07174m3030nl3010 + 9 + 9gm 04479fn.a.n.a.nl29/309 + 9 + 9/10 + 9 + 9na 07538f29/2929/29nl29/299 + 9 + 9/9 + 9 + 9gm 04738mn.a.n.a.nl3010 + 9 + 9na 20243f29/4129/41nl29/419 + 9 + 9/10 + 9 + 20na 20238f29/3029/30nl29/309 + 9 + 9/9 + 9 + 10na 20244m4141nl419 + 9 + 21na 20234f31/4631/46in31/4610
+ 9 + 10/9 + 9 + 13 + 12na 20232m4646in469 + 36na 20235f29/4529/45in29/459 + 9 + 9/10 + 34na 20236f31/5331/53in31/5410 + 9 + 10/purena 20230m5353in54purecd 00014m5656in569 + 9 + 36na
20242f30/7330/73ex30/7410 + 9 + 9/9 + 9 + 54gm 06892m9386ex9310 + 82na 20240f30/8030/80ex30/8710 + 9 + 9/puregm 06907f29/85n.a.ex29/919 + 9 + 9/10 + 80gm
06896f23/95 - 120 - 140n.a.ex23/11513 + 9/10 + 104gm 06891m118no consensusex~160puregm 06852m>200n.a.ex>200puregm 07862m501550n.a.ex>200puregm 07294mn.a.n.a.ex>200puregm 20239f20/18319320/no consensusex20/~20010 + 9/puregm 07063fn.a.n.a.ex32/>2009 + 22/puregm 05855fn.a.n.a.ex34/>20010 + 23/puregm 07537f2829/>200n.a.ex29/>2009 + 9 + 9/purenl normal ; ex expanded ; in indeterminate ; pure uninterrupted cggs .
not available genomic dna samples from the coriell cell repositories used for direct tp - pcr mca and ce nl normal ; ex expanded ; in indeterminate ; pure uninterrupted cggs .
not available the dtp - pcr mix containing 1 x pcr buffer ( qiagen , hilden , germany ) , 2.5 x q - solution ( qiagen ) , 0.1 x sybr green i nucleic acid dye ( roche applied science , penzberg , germany ) , 5 u hotstartaq dna polymerase ( qiagen ) , dntp mix at a final concentration of 2 mm with a 5:1 dgtp and dctp to datp and dttp ratio ( roche applied science ) , 100 ng of genomic dna , and 0.60 m each of primers ,
3 ) , tail ( 5tgctctggaccctgaagtgtgccgttgata3 ) , and a 1000-fold diluted tp - primer ( 5tgctctggaccctgaagtgtgccgttgata[cgg]53 ) was prepared in a 15-l reaction volume .
an initial denaturation at 95c for 15 min was followed by 40 cycles of 99c for 45 s , 55c for 45 s and 70c for 8 min , and then a final extension at 72c for 10 min on the geneamp pcr system 9700 ( applied biosystems - life technologies , carlsbad , ca ) .
subsequently , the amplicons were transferred into a 96-well plate for mca on the lightcycler 480 real - time pcr system ( roche applied science ) with a similar mca program setting as described previously ( ref .
the dtp - pcr mca results were analysed on the lightcycler 480 software and the melt peak temperature or tm ( the temperature at which the maximum df / dt value is recorded ) obtained for each sample was rounded off to the nearest 0.05c . to identify fmr1 expansion carriers among the male and female samples tested ,
two i m reference male ( na20232 , 46 repeats and na20230 , 54 repeats ) and two i m reference female ( na20234 , 31/46 repeats and na20236 , 31/54 repeats ) samples from ccr were included in each mca plate / run as internal reference controls , respectively . while samples that generated mca profiles with low tm relative to the 46-repeat internal reference control were classified as
nl , those that produced mca profiles with higher tm relative to the 54-repeat internal reference control were classified as expansion carriers , who require confirmatory assessment using reference method(s ) . in comparison , samples that displayed tm identical to either one of the internal reference controls and those that generated mca profiles in the
indeterminate zone ( defined as the melting domain between the 46-repeat and the 54-repeat internal reference controls ) are presumed to be
the dtp - pcr mix for sizing assay had a reaction setup identical to that described for mca , except that sybr green i dye was excluded ; 5 end of the flanking f primer was labelled with 6-carboxyfluorescein ( fam ) ; and 5 u of taq extender pcr additive ( agilent technologies , santa clara , ca ) was included .
an initial denaturation at 95c for 15 min was followed by 40 cycles of 99c for 45 s , 55c for 45 s and 70c for 8 min with a 15 s auto - extension at each cycle , and a final extension at 72c for 10 min on the geneamp pcr system 9700 ( applied biosystems ) .
a 4-l aliquot of fam - labelled tp - pcr amplicon was mixed with 0.5 l of mapmarker1000 ( bioventures , murfreesboro , tn ) and 9 l of hi - di formamide ( applied biosystems ) , denatured at 95c for 5 min and subjected to ce ( 36 cm , pop-7 , 18 s 1.2 kv injection , 50 min 15 kv run ) on the 3130xl genetic analyzer ( applied biosystems ) .
the dtp - pcr electropherograms generally reflect both fmr1 cgg repeat size and the distribution pattern of agg interruptions .
for instance , in case of nl samples with agg - interrupted fmr1 alleles , discrete fam - labelled dtp - pcr peak clusters separated by clear zones of ~18 bp are expected owing to the poor annealing of tp - primers over agg interruptions . in marked contrast
, samples carrying pm and fm expansions display a continuous series of uninterrupted fam - labelled dtp - pcr peaks differing by 3-bp from each other , a pattern characteristic of fmr1 alleles with one or no agg interruption .
for fm samples , in addition to the uninterrupted fam - labelled dtp - pcr peaks exceeding 700 bp , we also observed a fm - peak around 1050 bp .
the dtp - pcr peaks in the range of 700750 bp had signal intensities of 1020 rfu , whereas the fm - peaks had signal intensities ranging from 3080 rfu .
we chose four male and four female coriell cell repository ( ccr ) samples with nl , i m , pm and fm fmr1 alleles for the initial optimisation of dtp - pcr assay conditions ( fig .
carriers and noncarriers of fmr1 expansion generated mca profiles with distinctive melt peak temperatures ( tm ) ; both nl ( gm06890 and gm07175 ) and i m ( na20232 and na20235 ) samples produced mca profiles with tms that ranged from 84.25 to 87.55c , while expanded pm ( gm06892 and gm06907 ) and fm ( gm07862 and gm06852 ) samples yielded mca profiles with a pronounced shift in their tm to 89.25c or above ( fig . 1 , left )
moreover , the dtp - pcr mca profiles showed good correlation with the ce estimated cgg repeat size and structure , which is well - illustrated across both male and female samples with different fmr1 genotypes ( fig .
1 , right ) .
figure 1.dtp-pcr mca profiles ( left ) and genescan electropherograms ( right ) of eight fmr1 genotype - known reference male and female dna samples .
coriell ids and cgg repeat sizes of the samples are indicated on the left and the melt peak temperatures ( tm ) are indicated on the mca profile of each sample .
the df / dt values are shown on the y - axis and the temperatures ( c ) are shown on the x - axis .
distribution pattern of agg interruptions within the cgg repeat region are shown on the top right corner of each dtp - pcr genescan electropherogram , where a
red arrowheads in the inset panels indicate the base - pair ( bp ) size , and the red peaks in the main panel are from a rox - labelled internal size calibrator , whose bp sizes are indicated at the bottom of the electropherogram panel .
dtp - pcr mca profiles ( left ) and genescan electropherograms ( right ) of eight fmr1 genotype - known reference male and female dna samples .
coriell ids and cgg repeat sizes of the samples are indicated on the left and the melt peak temperatures ( tm ) are indicated on the mca profile of each sample .
the df / dt values are shown on the y - axis and the temperatures ( c ) are shown on the x - axis .
distribution pattern of agg interruptions within the cgg repeat region are shown on the top right corner of each dtp - pcr genescan electropherogram , where a
red arrowheads in the inset panels indicate the base - pair ( bp ) size , and the red peaks in the main panel are from a rox - labelled internal size calibrator , whose bp sizes are indicated at the bottom of the electropherogram panel .
furthermore , to determine the ability of dtp - pcr assays in accurately categorising samples carrying fmr1 alleles of different cgg repeat sizes and agg interruption patterns , we carried out a preliminary performance assessment study on 21 additional ccr reference dnas , and also re - evaluated the eight samples used for initial assay optimisation ( listed in table 1 ) .
figure 2 presents the dtp - pcr mca data of 13 reference males and 16 reference females as normalised melt curves ( a , b ) and melt peaks ( c , d ) , followed by the genescan electropherograms of representative samples . using mca we ascertained the fmr1 genotype class of each sample based on its tm relative to reference controls that mark the lower and upper limits of the
indeterminate zone. for the analysis of the reference males , the melt curve and peak temperatures of the i m male samples na20232 and na20230 marked the indeterminate zone boundary , whereas the i m female samples na20234 and na20236 were selected to define the
as expected , all nl males carrying fmr1 alleles ranging in size from 30 to 41 cgg repeats , displayed lower tms compared with that of na20232 carrying a 46-repeat fmr1 allele with a 9 + 36 repeat - pattern , where + indicates the position of agg interruption relative to cgg repeats .
in marked contrast , most pm and all fm males generated right - shifted mca profiles that displayed higher tms compared with that of na20230 carrying a 54-repeat fmr1 allele with no agg interruptions .
a pm male ( cd00014 ) generated a tp - pcr melt peak tm in the
indeterminate zone. cd00014 carries a sequence - verified 56-repeat allele ( the second smallest pm allele size ) with a 9 + 9 + 36 interruption pattern , which was also confirmed by its ce pattern . with the exception of this pm male , all nl , pm and fm reference males
figure 2.direct tp - pcr normalised melt curves ( a , b ) and the derivative melt peaks ( c , d ) of 13 ccr reference male and 16 ccr reference female samples , followed by the genescan electropherograms of representative samples ( bottom ) .
genescan electropherograms of samples marked with asterisk ( * ) are shown in figure 1 .
direct tp - pcr normalised melt curves ( a , b ) and the derivative melt peaks ( c , d ) of 13 ccr reference male and 16 ccr reference female samples , followed by the genescan electropherograms of representative samples ( bottom ) .
genescan electropherograms of samples marked with asterisk ( * ) are shown in figure 1 .
the tms derived from the mca profiles of reference female samples generally showed good correlation with the cgg repeat size of the largest fmr1 allele present in the sample , except in case of gm07175 , a nl female whose two alleles differ by seven cggs .
consistently , this nl female displayed a melt peak tm consistent with the shorter 23-repeat allele rather than the 30-repeat allele .
however , an analysis of the peak pattern indicates the presence of a shoulder with a peak tm consistent with the 30-repeat allele . whether the tm of the tallest melt peak or the higher tm of the peak shoulder is chosen , this sample was classified appropriately .
importantly , the fmr1 genotypes of all reference female samples were accurately classified by mca . all nl females generated mca profiles of lower tms compared with that of na20234 carrying a 46-repeat fmr1 allele with a 9 + 9 + 13 + 12 repeat - pattern , whereas expanded pm or fm females produced mca profiles of higher tms compared with that of na20236 carrying a 54-repeat fmr1 allele with no agg interruptions .
a 45-repeat i m female ( na20235 ) displayed a slightly higher tm relative to na20234 and generated mca profile in the
indeterminate zone , presumably due to the presence of only 1 agg interruption compared with the three aggs in na20234 .
in addition , we have also demonstrated the utility of normalised melt curves with temperature differentials that are slightly different from the tm of melt peaks . similar to melt peaks
, each normalised melt curve displayed a clear and distinct profile pattern of high resolution with respect to temperature and melt curve position relative to other cgg repeat alleles . to determine the reliability of dtp - pcr ce assay in accurately characterising agg interruption patterns
, we verified the repeat sequences of several reference males ( gm06890 , na20244 , na20232 , na20230 , cd00014 , na20231 and gm06892 ) , a homozygous reference female ( na07538 ) and the i m alleles of two reference females na20235 and na20234 by sanger sequencing ( data not shown ) . generally , we observed complete concordance between most sanger sequencing and dtp - pcr ce results .
although the assay displayed superior performance in male samples , characterising the distribution pattern of agg interruptions of some heterozygous female samples was challenging ; this is best - illustrated in case of na20234 carrying a 31-repeat nl allele and a 46-repeat i m allele with 10 + 9 + 10 and 9 + 9 + 13 + 12 repeat - patterns , respectively ( shown in the electropherograms in fig .
, we also examined the mca profiles of reference samples with identical cgg repeat size , but different agg interruption patterns . in the i m samples , na20230 and na20236 carrying a pure 54-repeat fmr1 allele , only a negligible tm variation of 0.05c was observed .
in contrast , we observed a tm difference of 0.30c between na20232 and na20234 , both of which carried an fmr1 allele with 46 cgg repeats , with lower tm recorded for na20234 harbouring 3 agg interruptions compared with na20232 harbouring only one agg interruption .
this observation is consistent with our previous study , wherein we reported varied melt profiles among samples differing only in their repeat - patterns ( ref .
the sensitivity of dtp - pcr assays in detecting low - level mosaicism for an expanded fm allele was tested using artificial dna mixtures that contained 1 , 2 , 3 , 4 , 5 , 10 and 20% of the fm allele . to generate these nl / fm dna mixtures , we pooled the genomic dnas of gm06890 ( nl male , 30 repeats ) and gm07862 ( fm male , ~501550 repeats ) in different proportions with the total dna input maintained constant at 100 ng .
the dtp - pcr mca of nl / fm dna mixtures revealed efficient amplification of fm allele in the presence of predominant levels of nl allele , and enabled accurate detection of fm mosaicism from as low as 1% ( fig .
the low - level mosaicism for fm allele allowed visualisation of both nl and fm mca peaks with discrete tm in 110% nl / fm dna mixtures .
the availability of more fm template in the 20% nl / fm dna mixture resulted in a much more prominent fm mca peak that closely resembles the melt profile of a typical fm female .
we noticed a slight reduction in the sensitivity of the dtp - pcr ce assay compared with mca , with fm mosaicism detected down to 4% ( fig .
figure 3.direct tp - pcr melt peaks ( left ) and genescan electropherograms ( right ) of nl / fm dna mixtures .
presence of fm allele in the nl / fm dna mixtures was confirmed by the identification of mca peaks with higher tm in the melting domains highlighted in pink .
direct tp - pcr melt peaks ( left ) and genescan electropherograms ( right ) of nl / fm dna mixtures .
presence of fm allele in the nl / fm dna mixtures was confirmed by the identification of mca peaks with higher tm in the melting domains highlighted in pink .
a total of 107 clinical samples , previously characterised by pcr and/or southern analysis , were included in the blinded validation study of dtp - pcr assays .
the patient cohort comprised 54 nl and 56 pm and/or fm expansion carriers , and had no samples with fmr1 alleles in the i m size range .
in addition , we also included two sets of internal reference controls from ccr , na20232 and na20230 , and na20234 and na20236 to define the cut - off temperature ranges for male and female samples , respectively .
normalised melt curves and melt peaks of all clinical samples with internal reference controls , followed by the genescan electropherograms of representative nl , pm and fm carriers are shown in figure 4 . as expected , in both males and females , two clusters of mca profiles , one from nl samples exhibiting lower tm compared with the respective 46-repeat reference control , and another from samples carrying expanded fmr1 alleles with tm higher than that of the respective 54-repeat reference control , were generated .
consistent with the lack of i m samples , no mca profiles were detected in the
indeterminate zone. in general , we observed complete agreement between mca and ce results for all clinical samples and achieved 100% sensitivity , 100% specificity , 100% positive predictive value and 100% negative predictive value for both dtp - pcr assays .
additionally , we verified the cgg repeat size and agg interruption pattern of the fmr1 alleles of 17 nl males by sanger sequencing and detected absolute concordance with dtp - pcr ce results ( data not shown ) .
figure 4.direct tp - pcr normalised melt curves ( a , b ) and the derivative melt peaks ( c , d ) of 107 archived patient dna samples , followed by the genescan electropherograms ( bottom ) of selected samples .
mca profiles of samples carrying nl and expanded fmr1 alleles are clustered to the left and right of the indeterminate zones ( highlighted in grey ) , respectively .
direct tp - pcr normalised melt curves ( a , b ) and the derivative melt peaks ( c , d ) of 107 archived patient dna samples , followed by the genescan electropherograms ( bottom ) of selected samples .
mca profiles of samples carrying nl and expanded fmr1 alleles are clustered to the left and right of the indeterminate zones ( highlighted in grey ) , respectively .
one common current approach to fxs molecular diagnosis involves a combinatorial strategy of employing simple pcr and ce to identify putative expansion carriers in the first instance , followed by the sb analysis or the most recent mpcr - ce ( ref .
these first - tier pcr - based fragile x testing methods perform remarkably well in identifying fmr1 expansions and significantly reducing the need for labour - intensive approaches such as the gold standard sb analysis ( refs 37 , 38 )
. however , for large - scale universal newborn and carrier screening applications , methods relying on ce might not be ideal first - line screening tools .
35 ) proposed a two - stage sequential approach of conventional repeat - spanning pcr followed by a chimeric pcr that utilises a cgg - repeat annealing primer , whose utility in newborn screening was recently demonstrated ( ref .
the first round pcr screen identified males with fmr1 alleles of up to ~330 cgg repeats and apparent heterozygous females of up to a pm size range of at least ~160 cgg repeats ( ref .
42 ) , but a second round pcr screen was necessary to distinguish nl homozygous females from heterozygous females carrying a nonamplifiable fm allele .
this two - step strategy ultimately minimised the number of samples that required sb analysis . in this study , we have presented an alternative fxs screening strategy that relies on the analysis of amplicon melt characteristics .
the proof - of - principle of this strategy was first demonstrated in 2012 ( ref .
this strategy utilises direct triplet - primed pcr melting curve analysis ( dtp - pcr mca ) , which is both cost - effective and reliable in identifying fmr1 expansions . a major improvement over the method of teo et al .
( 2012 ) is that our improved assay identifies pm and fm carriers based on melt peak temperature ( tm ) instead of the resumed baseline temperature , thus significantly improving accuracy of temperature determination .
in addition , we also evaluated the concordance between mca results and the actual genotypes of samples through a direct comparison of tm with fmr1 cgg repeat size .
initially , we assessed the performance of the improved mca - based screening tool on 29 ccr ( coriell cell repositories ) reference dna materials using four internal reference controls of known cgg - repeat size .
male and female reference samples were analysed relative to two different sets of controls , each with a 46-repeat and a 54-repeat reference sample , which roughly defined the melting domain of i m fmr1 alleles or the
indeterminate zone. in these reference samples with fmr1 alleles spanning the entire spectrum of fragile x mutation , the first - line mca screen identified pm and fm expansion carriers with high sensitivity and specificity . among the reference males , one pm carrier ( cd00014 ) harbouring a 56-repeat fmr1 allele with a 9 + 9 + 36 repeat - pattern was identified in the
this allele harbours two agg interruptions which may have lowered its tm relative to the 54-repeat control sample , which lacks agg interruptions .
this emphasises the need to have a cut - off temperature range rather than relying on the cut - off temperature of a single internal reference control .
for instance , if the analysis was only based on the cut - off temperature of the 54-repeat control , this small pm male would have been wrongly classified as nl or i m , resulting in a false - negative call and reduced assay sensitivity .
conversely , in the presence of the 46-repeat control alone , most i m samples may appear as expanded , which would result in high false - positive calls and reduced assay specificity .
remarkably , there was complete agreement between the mca melt peak temperatures and ce sizing results of the dtp - pcr amplicons with respect to the classification of samples as nonexpanded or expanded.
the direct tp - pcr ce assay enabled sizing of fmr1 alleles and facilitated determination of agg interruption patterns , both of which are crucial for predicting the risk of unstable fmr1 cgg - repeat expansions during maternal transmission ( ref .
24 ) , several studies have highlighted the utility of maternal cgg - repeat size ( refs 23 , 43 , 44 ) and the density of agg triplets ( refs 27 , 45 , 46 ) in ascertaining the risk of fmr1 allelic expansions .
these findings argue for mapping agg interruptions within the cgg - repeat tract , information that can be potentially used to predict the likelihood of expansion events among women carrying at - risk fmr1 alleles in the i m and pm size ranges .
the dtp - pcr ce assay has potential applications in studies aiming to characterise agg interruption patterns , although interpretation of the repeat structures can be slightly difficult in heterozygous females with complex repeat structures due to the overlap of amplicon peaks from both the fmr1 alleles .
we generally observed good agreement between the fmr1 allele sizes determined by dtp - pcr ce and the sizes reported by ccr , except for discrepancies in the pm samples gm06891 and gm20239 .
we detected a pm allele of ~160 cgg repeats in the cell - line derived genomic dna of gm06891 , contrary to the previously reported size of 118 cgg repeats in this pm male ( ref .
however , our observation is in agreement with a recent study that reported instability and expansion of the pm allele in the same lymphoblastoid cell line ( ref . 48 ) . in gm20239
, we observed a slightly larger expanded allele of ~200 cgg repeats instead of the reported pm size range of 183193 cgg repeats .
again , our observation is consistent with another report that sized the largest expanded allele as ~206 cgg repeats ( ref .
38 ) . in the artificial nl / fm dna mixtures , the mca assay detected fm mosaicism down to 1%
this high degree of sensitivity is critical for a first - line screening tool , without which samples with low - level mosaicism for pm or fm might not be detected . in the blinded patient dnas , including nl and expanded pm and fm samples , the mca screen accurately classified all samples with 100% sensitivity and specificity .
such an exceptional performance of the screening assay may be partly attributed to a lack of i m samples in the patient cohort , which was not intentional but reflects a very low frequency of i m alleles in asian populations ( refs 49 , 50 , 51 , 52 , 53 ) .
it is thus important to note that in populations with higher frequency of i m alleles , the specificity ( and even possibly sensitivity ) of this screening tool might be lower than 100% .
the mca method has potential applications as a first - line tool in newborn and carrier screening programs owing to its simple design and flexibility to tailor the approach for different purposes . for nbs of fxs
, utilising i m internal reference controls would mean identifying both pm and fm carriers .
identification of pm babies might raise ethical concerns related to detection of risk for late - onset disorders , although recent reports of pm carriers with developmental delay suggest a possible benefit to pm screening in babies , that of early developmental intervention ( ref .
if the aim is to identify both pm and fm , the demonstrated approach with i m internal reference controls will ensure high sensitivity and specificity in identifying expansion carriers .
alternatively , for identifying only fm , an appropriate pm reference control can be used to minimise the number of false positives .
nevertheless , all the false positives identified will be eventually excluded at the allele sizing second stage .
if the population pm allele frequency is high , a higher false - positive fm rate can be expected following the first - tier mca screen .
nonetheless , this method would still compare well with a first - tier screen employing flanking pcr and agarose gel electrophoresis ( ref .
35 ) , whereby ~40% of females will require second - tier testing due to apparent homozygosity for the nl allele . using pm reference controls , it should also be possible to avoid identification of i m carriers , whose involvement with disease are still in contention . for population carrier screening programs aimed at identifying pm women of reproductive age , the use of i m internal reference controls would enable identification of virtually all but the smallest pm allele carriers , who are most likely to display melt profiles in the
indeterminate zone. this method also has potential applications as a first - line screen for fxs in a diagnostic setting .
conceivably , in a population with low prevalence of fm alleles , a majority of tested samples will be negative for fm expansions even in high - risk groups such as children with intellectual / behavioural disabilities , and this screen could be a cost - effective first - tier tool to exclude nonexpanded individuals .
however , in an extreme hypothetical population wherein 1 of every two to three samples tested is positive for a fm allele , a screening assay such as this would obviously be unwarranted .
we calculated the reagent cost for fluorescent dtp - pcr , and the reagent plus consumables cost for amplicon ce on the abi 3130xl , to be ~us$ 4.65 and ~us$ 2.30 per sample , respectively .
in contrast , it costs ~us$ 3.05 and ~us$ 0.15 per sample to perform a nonfluorescent dtp - pcr and subsequent mca assay on the roche lc480 , respectively .
although ce - based amplicon analysis allows for precise repeat sizing , employing it as a first - tier screening tool may not be cost - effective , especially in a large - scale population - based screening setting wherein a vast majority of the screened samples will be negative for fmr1 expansions .
also , while some recent studies have demonstrated the technical feasibility of using ce to analyse hundreds of samples ( refs 9 , 10 ) , it is important to consider that such an approach might become very expensive when tens to hundreds of thousands of samples are tested annually in each jurisdiction .
based on the known prevalence rates of fmr1 alleles in the general population ( refs 9 , 10 ) , it can be estimated that for every 50 000 samples screened , ~48 500 samples ( 97% ) will yield a negative result when tested for the presence of fmr1 alleles of > 45 cgg - repeats .
it would cost us$ 160 000 to screen 50 000 samples using dtp - pcr mca as a first - tier screening tool to exclude
nonexpanded samples , and another us$ 10 425 for dtp - pcr and ce confirmation analysis on an estimated 1500 screen - positive samples . if all 50 000 samples were to be screened directly by fluorescent dtp - pcr and ce analysis , it would cost us$ 177 075 more , or ~us$ 3.55 more per sample screened . therefore , in a large - scale screening setting , exclusion of the majority of samples as
nonexpansion carriers using the dtp - pcr mca assay , with more precise sizing confirmation of only the screen - positive samples by fluorescent dtp - pcr and ce analysis , could be a more cost - effective alternative to direct fluorescent dtp - pcr and ce analysis of all samples . even in an unlikely hypothetical situation
in which half the population carries an fmr1 expansion , performing dtp - pcr mca screening followed by the repeat - size confirmation of screen - positive samples using fluorescent dtp - pcr ce would still cost less than directly performing fluorescent dtp - pcr ce on all samples . unlike the approach of performing the dtp - pcr and mca on the same real - time pcr instrument described in our previous study ( ref .
41 ) , the current dtp - pcr assay was optimised on a regular thermal cycler followed by melt analysis on lightcycler 480 real - time pcr system ( lc480 ) .
the rationale for proposing this strategy was to enable a higher throughput and reducing the turnaround time per sample .
for instance , it is possible to process 16 plates of 96 samples by performing dtp - pcr on multiple inexpensive thermal cyclers in parallel and performing the amplicon melting for the 16 plates on a single lc480 instrument in ~24 h. in contrast , for 16 plates to be amplified and analysed completely on the lc480 would require ~6 days for one instrument or six lc480 s for the results to be out in 24 h. however , this method can also be carried out completely on the lc480 if desired ( data not shown ) . in summary ,
the dtp - pcr mca method has potential applications as a first - line test to identify fmr1 expansions in both males and females .
in large - scale universal screening programs , ideally this screening test should be accompanied by a reference method , either sb analysis or mpcr - ce , for detailed characterisation of cgg - repeat size and methylation state of samples that test positive for an expansion in the first round mca screen . | premutation and full - mutation hyperexpansion of cgg - triplets in the x - linked fragile x mental retardation 1 ( fmr1 ) gene have been implicated in fragile x - associated tremor / ataxia syndrome , fragile x - associated primary ovarian insufficiency , and fragile x syndrome ( fxs ) , respectively .
the currently available molecular diagnostic tests are either costly or labour - intensive , which prohibits their application as a first - line fmr1 test in large - scale population - based screening programs . in this study , we demonstrate the utility of a simplified closed - tube strategy for rapid first - line screening of fxs based on melt peak temperature ( tm ) analysis of direct triplet - primed polymerase chain reaction amplicons ( dtp - pcr mca ) . in addition , we also evaluated the correlation between tm and cgg - repeat size based on capillary electrophoresis ( ce ) of dtp - pcr amplicons .
the assays were initially tested on 29 fmr1 reference dna samples , followed by a blinded validation on 107 previously characterised patient dna samples .
the dtp - pcr mca produced distinct melt profiles of higher tm for samples carrying an expanded allele . among the samples tested
, we also observed a good correlation between tm and cgg - repeat size . in the blinded validation study ,
dtp - pcr mca accurately classified all normal and expansion carriers , and the fmr1 genotypic classification of all samples was completely concordant with the previously determined genotypes as well as the dtp - pcr ce results .
this simple and cost - effective mca - based assay may be useful as a first - line fxs screening tool that could rapidly screen out the large majority of unaffected individuals , thus minimising the number of samples that need to be analysed by southern blot analysis . |
primary producers play critical structural and functional roles in aquatic ecosystems ; therefore , it is imperative that the potential risks of toxicants to aquatic plants are adequately assessed in the risk assessment of chemicals .
the standard required macrophyte test species is the floating ( non - sediment - rooted ) duckweed lemna spp .
this macrophyte species might not be representative of all floating , rooted , emergent , and submerged macrophyte species because of differences in the duration and mode of exposure ; sensitivity to the specific toxic mode of action of the chemical ; and species - specific traits ( e.g. , duckweed 's very short generation time ) .
these topics were addressed during the workshop entitled aquatic macrophyte risk assessment for pesticides ( amrap ) where a risk assessment scheme for aquatic macrophytes was proposed .
four working groups evolved from this workshop and were charged with the task of developing tier 1 and higher - tier aquatic macrophyte risk assessment procedures .
subsequently , a setac advisory group , the macrophyte ecotoxicology group ( ameg ) was formed as an umbrella organization for various macrophyte working groups .
the purpose of ameg is to provide scientifically based guidance in all aspects of aquatic macrophyte testing in the laboratory and field , including prospective as well as retrospective risk assessments for chemicals . as ameg expands
, it will begin to address new topics including bioremediation and sustainable management of aquatic macrophytes in the context of ecosystem services .
aquatic macrophytes are important primary producers of aquatic ecosystems that must be protected from adverse chemical effects in order to maintain important ecosystem structure and functions .
macrophytes fulfil several critical functions in aquatic ecosystems including conversion of solar energy and carbon dioxide into organic matter ; production of oxygen ; sequestration of carbon dioxide ; cycling and retention of nutrients ; stabilization of sediments ; and provision of food and habitat resources for aquatic fauna ( wetzel 2001 ) .
thus , aquatic macrophytes , and the ecosystem services that they provide , must be protected on a local and global basis .
currently , the only aquatic macrophyte used in risk assessment of plant protection products ( e.g. , herbicides and plant growth regulators ) under the european regulation ( ec 2009 ) is the standardized growth inhibition test with duckweed ( lemna spp . ;
additional data with other aquatic plant species may be required on a case - by - case basis ( ec 2002 ) . in other regulatory frameworks , macrophytes are not obligatory in the risk assessment , e.g. , in the risk assessment of chemicals in the context of the water framework directive ( lepper 2005 ) .
lemna is a small , floating , non - sediment - rooted monocotyledon with a short generation time and is rather atypical in growth form and traits compared with many other aquatic macrophytes ; therefore , concerns have arisen that risk assessments based on lemna and its endpoints may not be protective of other macrophyte species due to potential differences in exposure route , recovery rate , or sensitivity to chemicals with specific toxic modes of action ( brock et al .
, 41 macrophyte experts from academia , regulatory authorities , and business from europe and north america discussed the current state and potential options for refinement of aquatic macrophyte risk assessment procedures at the workshop
( amrap ) , which was organized under the auspices of setac - europe ( society of environmental toxicology and chemistry ) . during the workshop ,
an overview of the current european regulatory framework for the risk assessment of aquatic macrophytes was discussed , and the uncertainties and areas for improvement within the regulatory framework were identified .
four working groups evolved from the workshop and were charged with the following tasks :
developing and testing a proposal for a modified aquatic macrophyte tier 1 risk assessment scheme for plant protection products in the eu , including criteria when an additional macrophyte must be tested ( fig .
( myriophyllum spicatum and myriophyllum aquaticum ) to be ring - tested in 2010;compiling an overview of laboratory toxicity methods for testing aquatic macrophytes other than lemna;compiling data and developing guidance for the use of species sensitivity distributions ( ssds ) in aquatic macrophyte risk assessment.fig .
( 2010 ) ) ( pgr = plant growth regulator ; ec = effect concentration ; pec = predicted environmental concentration ) developing and testing a proposal for a modified aquatic macrophyte tier 1 risk assessment scheme for plant protection products in the eu , including criteria when an additional macrophyte must be tested ( fig .
1 ) ; drafting and testing a protocol for a laboratory toxicity test with myriophyllum spp .
( myriophyllum spicatum and myriophyllum aquaticum ) to be ring - tested in 2010 ; compiling an overview of laboratory toxicity methods for testing aquatic macrophytes other than lemna ; compiling data and developing guidance for the use of species sensitivity distributions ( ssds ) in aquatic macrophyte risk assessment .
( 2010 ) ) ( pgr = plant growth regulator ; ec = effect concentration ; pec = predicted environmental concentration ) a setac advisory group on aquatic macrophytes was considered to be the optimal platform to facilitate the ongoing discussions regarding these tasks ; the future dissemination of results of these working groups ; and to provide a forum for participation by all those interested in these matters . in general ,
setac advisory groups have been and still are successful as a scientific forum ( see as an example of another recently established setac advisory group : preuss et al .
in addition , an advisory group on aquatic macrophytes allows the original scope of amrap on the registration of plant protection products in europe to be extended to other risk assessment schemes and legislative backgrounds in other countries ( e.g. , the water framework directive ; usa clean water act ) .
the setac advisory group in aquatic macrophyte ecotoxicology ( ameg ) was established in june 2009 to provide scientifically based guidance in all aspects of chemical risk assessments for aquatic macrophyte testing across an array of test conditions including laboratory , outdoor semi - controlled and actual field conditions .
the group will serve as a forum for communication , discussion , and collaboration among scientists from academia , business , and government . within ameg ,
aquatic macrophytes are considered to be all freshwater higher plants including free - floating , rooted floating - leaved , submerged and emerged forms ( including hydrophytes , amphiphytes , and helophytes ) , in addition to freshwater macro - algae like chara , salt - water plants like sea - grasses , and salt - water macro - algae like brown algae .
the objectives of ameg are related to three areas :
development of science for the benefit of aquatic macrophyte risk assessments including :
monitoring , co - ordinating , and communicating the ongoing follow - up activities of the amrap workshop;aquatic macrophyte testing for registration of plant protection products , biocides , and chemicals in general ( e.g. , eu , us , and canadian directives ) ; andincorporation of aquatic macrophytes more fully in retrospective risk assessment ( e.g. , european water framework directive and usa clean water act);development of guidance and tools for aquatic macrophyte risk assessment in support of current and future legislation , e.g. , for the revision of the guidance documents that support the risk assessment of plant protection products in the eu ; andsupport communication and education to build and extend a global network of macrophyte experts from academia , government , and business entities for various needs including :
provision of an overview of ongoing activities and new initiatives in the subject area via the setac website and an email distribution list;organization of sessions ( e.g. , platform or poster ) and short courses at setac and other society and scientific meetings ; andorganization and active participation in expert workshops related to risk assessments with aquatic macrophytes .
development of science for the benefit of aquatic macrophyte risk assessments including :
monitoring , co - ordinating , and communicating the ongoing follow - up activities of the amrap workshop;aquatic macrophyte testing for registration of plant protection products , biocides , and chemicals in general ( e.g. , eu , us , and canadian directives ) ; andincorporation of aquatic macrophytes more fully in retrospective risk assessment ( e.g. , european water framework directive and usa clean water act ) ; monitoring , co - ordinating , and communicating the ongoing follow - up activities of the amrap workshop ; aquatic macrophyte testing for registration of plant protection products , biocides , and chemicals in general ( e.g. , eu , us , and canadian directives ) ; and incorporation of aquatic macrophytes more fully in retrospective risk assessment ( e.g. , european water framework directive and usa clean water act ) ; development of guidance and tools for aquatic macrophyte risk assessment in support of current and future legislation , e.g. , for the revision of the guidance documents that support the risk assessment of plant protection products in the eu ; and support communication and education to build and extend a global network of macrophyte experts from academia , government , and business entities for various needs including :
provision of an overview of ongoing activities and new initiatives in the subject area via the setac website and an email distribution list;organization of sessions ( e.g. , platform or poster ) and short courses at setac and other society and scientific meetings ; andorganization and active participation in expert workshops related to risk assessments with aquatic macrophytes .
provision of an overview of ongoing activities and new initiatives in the subject area via the setac website and an email distribution list ; organization of sessions ( e.g. , platform or poster ) and short courses at setac and other society and scientific meetings ; and organization and active participation in expert workshops related to risk assessments with aquatic macrophytes .
short - term deliverables resulting from the amrap 's activities include a ring - tested protocol for myriophyllum spp . ;
recommendations for aquatic macrophyte species and aquatic macrophyte testing other than lemna spp . ; and development of a proposal for use of ssds with aquatic macrophytes as a tool for the purpose of refined higher - tier risk assessments .
topics for long - term activities of ameg may include the conduct and evaluation of mesocosm studies with macrophytes in order to provide guidance on the use of macrophytes in field experiments ; the validation of laboratory tests performed with floating , submerged , and emergent macrophytes ; the assessment of macrophyte recovery in laboratory and mesocosm tests as an endpoint within the risk assessment process ; comparison of laboratory to mesocosm data to environmental monitoring data ; retrospective analysis of aquatic macrophyte risk assessments in the context of the wfd and the clean water act ; the use of aquatic macrophytes for bioremediation and their restoration in impacted habitats ; and the sustainable management of aquatic macrophytes in the context of the global spread of exotic and invasive species .
the idea of a global setac advisory group on aquatic macrophyte ecotoxicology developed after the amrap workshop .
a number of amrap participants formulated a proposal that was approved by the setac world council in june 2009 in order to start this advisory group .
the steering committee of ameg consists of 12 scientists from europe and north america representing academia , business , and government ( see list of authors ) .
the first ameg meeting was open to all participants interested in aquatic ecotoxicology and was held in gothenburg during the setac eu meeting in may 2009 . over 40 scientists used this opportunity to learn about the background and the objectives of ameg including the proposal and discussion of plans for a ring - test with myriophyllum in a laboratory - based water - sediment test system .
the next meeting of the group will be held during the setac - europe annual meeting planned for may 2327 , 2010 , in seville , spain , where ameg is organizing a poster - session on the use of aquatic macrophytes in ecological risk assessment . in addition , the objectives and plans of ameg were introduced during the setac laurentian chapter meeting in ottawa , canada ( june 2009 ) , the setac german language branch in freising - weihenstephan , germany ( october 2009 ) , and the setac north america meeting in new orleans usa ( november 2009 ) .
a further presentation is planned during the setac asia - pacific joint conference in vietnam , march 2010 . | introduction and backgroundprimary producers play critical structural and functional roles in aquatic ecosystems ; therefore , it is imperative that the potential risks of toxicants to aquatic plants are adequately assessed in the risk assessment of chemicals .
the standard required macrophyte test species is the floating ( non - sediment - rooted ) duckweed lemna spp .
this macrophyte species might not be representative of all floating , rooted , emergent , and submerged macrophyte species because of differences in the duration and mode of exposure ; sensitivity to the specific toxic mode of action of the chemical ; and species - specific traits ( e.g. , duckweed 's very short generation time).discussion and perspectivesthese topics were addressed during the workshop entitled
aquatic macrophyte risk assessment for pesticides ( amrap ) where a risk assessment scheme for aquatic macrophytes was proposed .
four working groups evolved from this workshop and were charged with the task of developing tier 1 and higher - tier aquatic macrophyte risk assessment procedures .
subsequently , a setac advisory group , the macrophyte ecotoxicology group ( ameg ) was formed as an umbrella organization for various macrophyte working groups .
the purpose of ameg is to provide scientifically based guidance in all aspects of aquatic macrophyte testing in the laboratory and field , including prospective as well as retrospective risk assessments for chemicals . as ameg expands
, it will begin to address new topics including bioremediation and sustainable management of aquatic macrophytes in the context of ecosystem services . |
the insulin - producing -cells are the most abundant cell type residing in the islets of langerhans , which are microorgans that are scattered throughout the exocrine tissue and represent only 1 to 2% of the total organ mass .
-cell replacement is considered the best therapeutic option , given the capacity of this particular cell type to accurately respond to highly variable changes in blood glucose level and as such to maintain glucose homeostasis .
islet transplantation via the portal vein of the liver is a promising approach and considerably less invasive than total organ transplantation .
however , among other difficulties , the scarcity of donor material will always remain a major hurdle [ 1 , 2 ] .
adult -cell mass is known to be dynamic and to be able to respond to physiological changes in insulin demand such as obesity , pregnancy , and starvation . in theory ,
-cell mass adaptation can occur via modification of cell size ( hypertrophy versus atrophy ) or modification of cell number ( proliferation of existing mature -cells or formation of new -cells from progenitor cells versus apoptosis ) .
the origin of newly formed -cells has long been debated [ 39 ] , part of the observations reported remaining controversial and partial .
in addition , many statements rely on studies performed in animal models or animal cell(s ) ( lines ) .
as the ultimate goal is translation to the clinic , the aim of this paper will be to compare rodent and human data regarding postnatal cell types envisaged as potential sources to generate -cells during the past decade .
we will focus on the replication capacity of the -cell itself then address the unexpected recent plasticity of differentiated cell types developmentally close to -cells . for many years
the dogma was upheld that terminally differentiated cells were committed to a specific function and could no longer change their identity .
in contrast , progenitor / stem cells are expected to retain some multipotency capacities and therefore be more suitable for tissue replacement strategies .
nowadays more and more examples of efficient transdifferentiation have been reported , without an apparent need for a progenitor cell intermediate stage .
finally , given their various capacities , adult mesenchymal stem cells ( msc ) are increasingly considered for clinical use in many different applications .
we will review the recent reports about both pancreatic and extrapancreatic sources of msc and the possible application of these cells for type 1 diabetes treatment .
several groups reported on a successful derivation of both murine and human embryonic stem cells ( ess ) to -cell - like cells able to revert hyperglycemia in diabetic mouse models ( for detailed review see ) .
mimicking normal pancreatogenesis appears to be the best strategy to differentiate es cells toward the endocrine lineage , although large differences in differentiation capacity exist between different es cell lines using similar protocols .
importantly , the risk of teratomas formation in vivo , inherent to es cells when they remain undifferentiated , as well as ethical principles about any therapeutical use of human es cells currently hold back extensive clinical applications of these cells .
recent developments on technology to generate induced pluripotent stem cells ( ips ) hold great promise [ 1216 ] . however , safety issues due to genetic and epigenetic abnormalities during reprogramming or in subsequent cell culture are a major hurdle for clinical transplantation .
a more immediate application of es and ips cells is more likely to be their use in modeling human disease .
therefore , in this paper , we chose to limit ourselves to alternative postnatal cell sources the data of this part are summarized in table 1 .
during adult life , -cell replication appears to be a predominant mechanism of -cell mass expansion in healthy mice ( figure 1 ) .
dor et al . demonstrated that preexisting -cells , rather than multipotent stem cells , are the major source of new -cells during adult life and after partial pancreatectomy .
the study relies on a transgenic mouse model carrying a rat insulin gene promoter - controlled expression cassette encoding a tamoxifen - dependent cre recombinase .
a pulse treatment of tamoxifen irreversibly induced the expression of a reporter gene ( human placental alkaline phosphatase ( hpap ) ) through cre - mediated excision of a stop sequence specifically in -cells .
intriguingly , the percentage of hpap - positive -cells immediately after tamoxifen treatment and 4 , 6 , 9 , and 12 months later remained very similar , indicating that during normal turnover in murine -cells originate exclusively from preexisting -cells . in islet injury models ,
either by pancreatectomy or by -cell ablation using -cell specific expression of diphtheria toxin a resulting in 70% and 80% reduction in -cell mass , respectively [ 18 , 19 ] , new -cells appeared to be formed largely from genetically labeled preexisting -cells and not from neogenesis or from expansion of non--cell precursors .
furthermore , the question was addressed whether all -cells contribute equally to growth and maintenance of -cell mass or if distinct subpopulations exist .
performed an in vivo pulse - chase labeling experiment using histone 2b - gfp as reporter gene .
next , a clonal analysis of dividing -cells revealed that all clones were of similar sizes .
altogether this suggests that , in mice , the pool of -cells is homogenous and all cells are able to replicate at the same rate . therefore all -cells can be candidate for in vitro expansion .
similar conclusions were obtained from a parallel study , in which a dna analog - based lineage tracing method was developed to detect sequential cell division in vivo .
recently dor and colleagues further investigated the replication dynamics of adult murine -cells and showed that replicated -cells are able to reenter the cell division cycle shortly after mitosis .
this short quiescence period of several days was found to be lengthened with advanced age and shortened during injury - driven -cell regeneration and following treatment with a pharmacological activator of glucokinase [ 25 , 26 ] .
meier et al . investigated the -cell mass from infancy to adulthood by performing immunohistochemistry and morphometric analyses on pancreas obtained at autopsy from 46 donors ( from 2 weeks to 21 years of age ) and determined pancreas volume by ct scan in 135 individuals of similar age .
the authors concluded that the predominant expansion of -cell mass in humans occurs in early childhood ( 2.6% of -cells positive for ki67 ) , without secondary growth phase of -cell mass during adolescence , in contrast to rodents .
interestingly , the islets appear to increase in size rather than in number . moreover , although the authors identified some -cells ( both individual cells and in small clusters ) near the ducts , the number of these insulin - positive cells increased to a similar extent as the overall expansion of -cell numbers in the islets , consistent with the hypothesis of -cell replication .
it is important to note that an efficient -cell mass regeneration approach in a therapeutic perspective would require a similar rate of -cell replication .
kassem et al . reported that -cell replication decreased progressively from 3.2% at 1732 weeks of gestation to 1.1% perinatally .
after birth , levels of -cell replication would drop further to reach less than 0.1% in young adults .
these data also correlate with the findings that -cell mass is established by the first two or three decades of human life as determined by measuring accumulation of lipofuscin bodies as a marker to estimate -cell longevity or by in vivo thymidine analog incorporation and radiocarbon dating .
remarkably , the adaptive increase of -cell mass in adult humans appears to be modest in response to obesity ( 2- versus 10-fold ) , as well as during pregnancy ( 1.4 fold versus 2 to 5-fold ) compared to rodents [ 33 , 34 ] .
in contrast to rodents again , in both obese subjects and pregnant women , the adaptive increase in -cell number was accompanied by an increased number of small new islets , indicative of neogenesis ( cf .
paragraph 2.3 ) , rather than an increase in islet size or number of -cells per islet , characteristic for -cell replication .
finally a study performed on human pancreatic tissue collected from 13 patients who underwent a partial pancreatectomy showed that , unlike in rodents , a 50% pancreatectomy does not trigger any -cell regeneration in adult humans .
this corroborates with the high incidence of diabetes after partial pancreatectomy . in summary , in vivo-cell replication capacity in humans
appears to be mostly limited to the very early postnatal period , and the triggers of such a process are still unknown .
data available on -cell proliferation in vitro are very limited when restricted to primary -cells .
an early report claimed that human -cells were able to replicate efficiently ( 69% of brdu / insulin double - positive cells ) when exposed to a specific matrix ( matrix produced by the rat bladder carcinoma cell line 804 g ) in the presence of hepatocyte growth factor / scatter factor ( hgf / sf ) .
this work was rapidly challenged by another report showing that the defined culture conditions were favorable for replication of ductal cells and not for -cells . in a comparative study between in vitro proliferation of purified human and rat -cells , parnaud et al . also failed to detect any -cells that were positive for ki67 or had incorporated brdu even after 10 days of exposure . however a clear proliferation of purified rat -cells was observed and could be further enhanced by defined coatings or growth factors .
proliferation of human -cells in ( intact ) isolated human islets was also assessed in the same study and remained undetectable .
therefore it appears that culture conditions for efficient human -cell replication ex vivo have not been clearly identified .
differentiation of endocrine non--cells to -cells is an interesting alternative mechanism for increasing the -cell mass .
. showed in various transgenic mouse models that forced expression of pax4 at different stages ( in pancreatic progenitor cells , endocrine precursor cells , or in mature cells ) resulted in a shift of all endocrine lineages toward a -cell fate .
a bicistronic vector ( pax4-ires--galactosidase ) was used in order to follow cells of interest by staining for -galactosidase activity .
importantly , the authors observed an age - dependent increase in islet size and in number of insulin/-galactosidase double - positive cells and a concomitant decrease in -cell content .
these observations indicate that ectopic pax4 expression can also force conversion of adult glucagon - expressing cells into -cells .
remarkably , the subsequent decrease in glucagon was found to activate the differentiation of ducts - associated progenitor cells cells ( via an intermediate stage of ngn3 positive cells ) .
however the newly formed cells failed to correct the hypoglucagonemia since they were shown to be rapidly converted into -cells upon pax4 ectopic expression .
notably , the expression of pax4 in glucagon - positive cells has been reported to be sufficient to restore a functional -cell mass in diabetic mice , although only in the young animals .
thorel et al . developed a transgenic mouse model of near total -cell ablation that relies on the specific expression of diphtheria toxin receptor ( dtr ) in pancreatic -cells ( expression driven by the rat insulin promoter ) .
after administration of diphtheria toxin , a rapid and extreme ( > 99% ) -cell destruction by apoptosis was observed resulting in a characteristic diabetes within a couple of weeks . if given insulin , the mice survived and showed a slow -cell mass regeneration . only after 5 months
the regenerated -cell mass was able to maintain glucose homeostasis without exogenous insulin administration . using a doxycycline inducible -cell lineage tracing system , the authors established that one month after -cell ablation , a large but variable ( 32 to 81% ) fraction of newly formed -cells resulted from the transdifferentiation of about 5 to 10% of cells .
importantly , almost all ( ~90% ) were bihormonal ( still expressing glucagon even as far as 10 months after ablation ) .
therefore , in contrast to previous studies , the -cell mass regeneration was attributed to neogenesis through -cell transdifferentiation , rather than to the slow self - replication mechanism of preexisting mature -cells .
the authors suggested that the amount of -cell loss and the type of injury would determine the mechanism of regeneration .
this theory of unexpected pancreatic cell plasticity triggered by extreme -cell loss was supported by a parallel study done by chung and colleagues .
the model used was a combination of pancreatic duct ligation ( pdl ) with specific elimination of preexisting -cells by alloxan .
no lineage tracing method was used in this work , and results are based on immunohistochemistry stainings performed at 7 and 14 days .
the authors reported a rapid regeneration of -cell mass within weeks and suggested that the newly formed -cells resulted mainly from conversion of adult cells .
interestingly the authors also observed that under injury conditions , cells were able to replicate .
importantly the regenerated -cell mass was not sufficient to revert hyperglycemia , possibly due to the persistent inflammation caused by the pdl . in summary ,
three independent studies performed in transgenic mouse models showed that - to -cell conversion can occur .
however , herrera et al . demonstrated that , during murine embryogenesis , mature glucagon- and insulin - producing cells share a common precursor but belong to separate developmental lineages . in human cells , the embryologic situation appears to be different , since insulin - positive cells are the first to be detected during development .
therefore it is not known whether this extraordinary islet cell plasticity exists in adult human cells in vitro or in vivo .
if so , cells , especially if their capacity to replicate under injury condition is confirmed , could be an ideal intraislet source for in vivo regeneration of -cells .
pancreatic ductal cells have long been thought to be the main source for progenitor cells in the pancreas ( cf . reviews [ 3 , 8 ] ) .
several rodent studies based on immunohistochemical observations in animal models , suggested a possible mechanism of islet neogenesis via recapitulation of the embryological development after activation and differentiation of ductal progenitors [ 4650 ] . recently
a number of lineage tracing studies in transgenic mouse models have been performed in order to identify putative endocrine progenitors in adults , but provided contradictory conclusions .
bonner - weir and collaborators made use of the human carbonic anhydrase ii ( caii ) promoter , specific for differentiated ductal cells , combined to a tamoxifen inducible cre - er / lox recombination system .
caii - expressing cells within the pancreas acting as precursor cells gave rise to both pancreatic endocrine and exocrine tissues after birth and after injury .
the authors proposed that ductal cells represent a pool of homogenous cells from which a fraction can dedifferentiate into progenitor cells that are able to regenerate both endocrine and exocrine tissues . in a parallel study , heimberg and
colleagues suggested a slightly different model : a rare subpopulation of endocrine progenitor cells , located in the ductal lining , could be activated upon pdl in adult mouse pancreas and subsequently started to express an embryonic key endocrine developmental factor , ngn3 .
differentiation of an ngn3-positive subpopulation gave rise to all islet cell types , including glucose responsive -cells both in situ and when cultured in embryonic pancreatic explants .
however , in a subsequent study a lineage tracing of hnf1-positive ductal cells was performed .
the authors show that the ductal epithelium does not make a significant contribution to acinar or endocrine cells during neonatal growth ( 6-month observation period ) or upon regeneration condition ( pdl or alloxan followed by egf / gastrin treatment ) . of interest ,
earlier lineage studies by melton 's group pointed out on the heterogeneity in developmental potential among duct - like structures in early embryo .
lineage tracing experiments suggested that ngn3-positive cells are indeed islet progenitors but distinct from duct progenitors . on the other hand
, the authors could not rule out the possibility that a minor population of mature ductal cells is able to transiently activate ngn3 gene expression and subsequently contributes to islets neogenesis .
lineage tracing of muc1 ductal / acinar cells confirmed that muc1-positive cells give rise to endocrine cells in utero , in line with embryological development .
however , after birth , muc1 lineage - labeled cells were found to be restricted to the exocrine compartment , with no detectable contribution to islet cells .
no injury or regeneration models have been tested in that particular model . along the same line ,
another independent lineage labeling using the sox9 promoter ( active in early pancreatic progenitors ) and resulting in 70% of labeled pancreatic ductal cells established that very few nonendocrine cells continue to arise from sox9-positive precursors in early postnatal life , but no endocrine or acinar cell neogenesis from sox9-positive cells occurs during adulthood .
intriguingly , in contrast with an earlier study , the authors found that after pdl , sox9-positive cells give rise to ngn3-positive cells but these cells do not contribute to islets .
thus , although the function of ductal cells as endocrine progenitors in embryonic development is commonly accepted , more insight is needed into the potential of cells from the ductal compartment as an alternative source for the formation of new -cells in adult rodents .
immunohistochemistry stainings performed on tissues obtained at autopsy or after pancreatectomy are obviously the only possible technique to evaluate the -cell mass in humans .
the occurrence of neogenesis is suggested by the presence of insulin - positive cells in the ductal area combined to the absence of replication of this cell population ( ruling out the expansion of a preexisting insulin - positive ductal cell population ) ( figure 2 ) .
analyzed pancreatic tissues obtained at autopsy from 46 children aged from 2 weeks to 21 years and observed about 0.5% of insulin - positive ductal cells .
a similar percentage has been reported by reers et al . that studied pancreatic tissues from 20 donors aged from 7 to 66 years .
importantly , butler et al . observed that ductal cells positive for insulin were increased by 2- and 3-fold during obesity and pregnancy , respectively [ 32 , 33 ] .
therefore it appears that in humans islet neogenesis from ductal cells is stimulated during metabolic situations that require an increase in insulin level .
it should be noted that during both obesity and pregnancy the adaptive increase of -cell mass is much more limited in humans compared to rodents .
in pathophysiological condition such as chronic pancreatitis , phillips et al . also observed a significant increase of insulin - positive ducts in adult human pancreas of 11 patients compared to control group . in another study
, the question was addressed whether -cell neogenesis occurs in the transplanted pancreas of type 1 diabetic patients who had received a simultaneous pancreas - kidney transplant ( spk ) .
pancreatic tissues from 9 spk patients and 16 nondiabetic organ donors were examined by immunohistochemistry .
remarkably , high numbers 33 to 90% of ductal cells were found to be insulin positive , and 17% to 95% of the ducts harboured insulin - positive cells in spk patients with recurrent autoimmunity and diabetes
. a high degree of neogenesis was observed in patients with the most severe -cell loss , whereas a low degree of neogenesis was observed in normoglycaemic patients , suggesting that in the particular context pancreas transplantation , hyperglycemia and chronic inflammation may strongly stimulate -cell neogenesis from ductal cells .
intriguingly , a study by bouwens et al . analyzing pancreatic tissues from nine adult donors revealed that 15% of all -cells were located in units with a diameter less than 20 mm and without associated glucagon- , somatostatin- , or pancreatic polypeptide cells .
these single -cell units were situated in or along ductules , from which they appear to bud as previously noticed in fetal and neonatal pancreas .
furthermore , simultaneous presence of ki67-positive ductal cells ( 0.05% ) and absence of ki67-immunoreactive budding -cells suggested that -cell neogenesis depends on ductal cell proliferation and differentiation in humans .
about ten years ago murine ductal cells cultured in vitro were reported to form 3d clusters that differentiate to functional islet cells , which are able to respond to a glucose challenge and to reverse diabetes in mice .
an interesting strategy for the prospective isolation of putative progenitors from an enriched ductal cell population is also being pursued by taniguchi and colleagues [ 61 , 62 ] .
the approach combines immunohistochemical analysis of mouse pancreas to define new phenotypic markers and flow cytometry cell sorting to isolate clonal cell populations that are able to differentiate toward the endocrine lineage in vitro or in vivo .
the data suggest that a population of progenitor cells was present among cd133-positive ductal cells .
koblas et al . confirmed that a subpopulation of cd133-positive cells in human islet - depleted tissue was able to differentiate into functional insulin - producing cells in vitro and to secrete insulin in a glucose - dependent manner .
furthermore , bonner - weir and collaborators showed that human primary ductal cells could be isolated from islet - depleted pancreatic tissue , expanded in culture , and triggered to differentiate towards glucose responsive islet - like clusters .
these results were confirmed by gao et al . who further characterized the nature of these pancreatic progenitor cells .
during monolayer expansion , two subpopulations of proliferating cells were observed , ck19-positive ductal cells at an early time point ( day 3 ) and nestin - positive cells at a later time point ( day 7 ) . under serum - free conditions and matrigel covering of the cells ,
the ck19-positive cells , but not the nestin - positive cells , were able to form islet - like clusters that contain insulin- and glucagon - positive cells .
when transplanted under the kidney capsule of nude mice , one out of five grafts demonstrated further growth with foci of both endocrine and exocrine cells .
next , bonner - weir and colleagues used magnetic cell sorting and antibodies raised against the ductal surface marker ca19 - 9 to isolate ductal cells from islet - depleted tissue .
transplantation experiments of purified ductal cells versus unpurified preparations ( 56% ck19-positive cells only ) into normoglycemic nod / scid mice revealed that differentiation of ductal cells to insulin - producing cells was dependent on the presence of nonductal cells , probably pancreatic stromal cells as suggested by the authors . of interest , in vitro islet - to - duct plasticity has also been reported for human cells [ 67 , 68 ] .
although some lineage tracing studies in rodents have provided contradictory results , most in vivo and in vitro data from both human studies indicate that cells from the ductal compartment are an attractive putative cell source for -cell replacement strategies . besides the ductal origin hypothesis , a number of studies show that acinar cells might also display a certain degree of plasticity . melton and colleagues suggested that acinar cells and endocrine cells share a common progenitor after the ductal cell lineage has already separated . on the other hand ,
bouwens and colleagues proposed a variant of the ductal origin hypothesis , in which acinar cells would be able to transdifferentiate towards -cells , through an intermediate dedifferentiated ductal - like stage [ 49 , 69 ] .
-cell mass regeneration has been studied after streptozotocin treatment in a transgenic mouse model expressing interferon gamma under the control of the insulin promoter .
new -cells appeared to result primarily from the formation of new islets from small pancreatic ducts .
however , interestingly , some putative transitional cells were identified harboring both exocrine and endocrine granules , indicative of acinar cells as possible precursor cells .
furthermore , in order to visualize better possible intermediate stages during acinar - to - ductal transdifferentiation in a pdl injury model , lardon et al . took advantage of the fact that dexamethasone treatment inhibits the loss of amylase from acinar cells .
putative transitional cells coexpressing acinus - specific ( amylase ) and duct - specific ( ck20 ) markers were identified in vivo .
furthermore , acinus - to - islet conversion was confirmed in vitro after isolation of acini and identification of putative transitional cells coexpressing acinus - specific ( amylase ) and -cell - specific ( insulin ) markers .
several groups recently performed some in vivo lineage tracing analyses using acinus - specific promoters ( amylase and elastase ) .
replication of preexisting acinar cells is seen as the major mechanism for regeneration of the acinar tissue .
moreover , acinus - to - duct transdifferentiation has been reported to occur in vivo , although at a very low frequency , in mouse models for pancreatitis , and in a mouse model that develops insulin - positive cell - containing hyperplastic ducts in response to the growth factor tgf .
however , the same authors also showed that the insulin positive cells adjacent to acinus - derived ductal cells arose from preexisting insulin - positive cells and not from acinar cells .
along the same line , stoffers and collaborator failed to observe any acinus - to--cell transdifferentiation in adult mice under normal conditions and after 70% pancreatectomy , pdl , or caerulein - induced pancreatitis . on the other hand , melton et al .
reported that adenovirus - mediated coexpression of pdx1 , ngn3 , and mafa in vivo in pancreas from adult mice was sufficient to induce the transdifferentiation of mature exocrine cells into -cells that are indistinguishable from endogenous islet -cells in size , shape , and ultrastructure .
however the question remains open whether acinar cells or other possible precursor cells were reprogrammed in these experiments .
in vitro ,
in contrast , when cultured in low serum medium ( 1% ) in the presence of egf and lif , rat exocrine cells can transdifferentiate to functional -cells . in that particular study ,
10% of dedifferentiated acinar cells expressed insulin , with a total insulin content of 40 to 90% of primary -cells , and transplantation of about 100,000 of these insulin - positive cells was sufficient to revert hyperglycemia in a diabetic nude mouse model .
in an independent study , minami et al . showed that suspension culture in presence of egf and nicotinamide converted 5% of adult murine acinar cells to glucose responsive insulin - producing cells .
an adenovirus mediated in vitro lineage tracing study using the acinus - specific amylase or elastase promoter confirmed the identity of the starting population .
in addition , acinus - to - duct transdifferentiation was shown to occur , in response to egf - receptor signalling , through an intermediate nestin - positive stage in an in vitro culture of pancreatic explants . regarding human cells ,
there are no data available about a possible acinus - to - islet cell plasticity .
one possible explanation is the selective death by apoptosis of human acinar cells when cultured in vitro .
in contrast , human ductal cells can survive , adhere to plastic culture dish , and proliferate .
nevertheless the possible acinus - to - duct transdifferentiation suggested by others can not be ruled out in this study .
liver and pancreas have a common embryological origin , both arising from the primitive gut .
therefore liver cells have been hypothesized to constitute a potential cell source for -cell generation .
ferber and collaborators demonstrated that an adenovirus - mediated expression of the key pancreatic transcription factor pdx1 in mouse liver ( intravenous infusion ) resulted in transdifferentiation of hepatocytes to -cell - like cells .
pdx1 expression found in 60% of hepatocytes resulted in a 3-fold increase of plasma insulin levels , 59% as insulin and 41% as proinsulin .
these data indicate that proinsulin was processed which was substantiated by expression of the prohormone convertase 1/3 .
the amount of insulin produced was sufficient to reduce hyperglycemia in a diabetic mouse model .
a similar approach was successfully reported with adenovirus - mediated expression of pdx1 or ngn3 and the coexpression of neurod1/beta2 and betacellulin in the liver . of note , although the overexpression of pdx1 was able to revert the elevated blood glucose of diabetic mice , the animals died from liver inflammation most likely due to the exocrine - differentiating activity of pdx1 .
interestingly yechoor et al . showed that gene transfer of a pancreatic key transcription factor ( ngn3 in this study ) in liver leads to long - term diabetes reversal in mice .
however the authors demonstrated that , although insulin expression was transiently induced in terminally differentiated hepatocytes , the long - term diabetes reversal obtained in these mice was resulting from the differentiation of hepatic progenitors able to generate islet - like clusters .
transdetermination , that is , lineage switching in lineage - determined , but not terminally differentiated , cells .
human adult liver cells were shown to expand in vitro and to transdifferentiate towards an endocrine pancreatic lineage after pdx1 overexpression .
pdx1-expressing human liver cells were found to express insulin that is stored in secretory granules , which are released in a glucose - regulated manner . when transplanted under the kidney capsule of diabetic immunodeficient mice ,
these cells ameliorated hyperglycemia for prolonged periods of time . similar studies using human fetal progenitor liver cells were reported [ 88 , 89 ] .
since harvesting and propagating significant numbers of primary hepatocytes from patients with diabetes would be theoretically feasible , the liver can be considered as an interesting extrapancreatic source for -cell replacement therapy .
mesenchymal stem cells ( msc ) were originally identified in bone marrow by friedenstein in 1976 as a rare , heterogeneous , non hematopoietic , and multipotent stromal population able to differentiate to mesenchymal lineages including bone , fat , and cartilage .
msc are virtually present in all organs , including the pancreas and the islets of langerhans .
it is now generally accepted that the perivascular area harbors the msc , explaining the ubiquitous distribution of these cells in the body .
interestingly , msc can be obtained from live donors ( and potentially from the patient itself ) and are easily expandable in vitro . therefore ,
despite the fact that their identity and their exact role in vivo have not been clearly defined yet , mesenchymal stem cells have been envisaged for a broad range of therapeutic applications including type 1 diabetes [ 91 , 92 ] .
we and others reported on the expansion of msc - like cell population from isolated human islets : human islet - derived precursor cells ( ipc ) [ 9395 ] , nip / nestin - positive islet - derived progenitors , pidm / pancreatic islet - derived mesenchymal cells , phid / proliferating human islet - derived cells , and more [ 99 , 100 ] .
interestingly , most of the groups reported a common characteristic , which is aggregation into clusters ranging in sizes between 50 and 200 m ( similar range as primary islets ) under serum starvation and a subsequent increase in expression of endocrine markers .
however others failed to reproduce these data of partial differentiation [ 101 , 102 ] . in any case , the endocrine markers remained at very low level when compared to freshly isolated human islets .
reported a further capacity of ipc to differentiate into functional -cells that secrete human c - peptide in response to glucose after reimplantation of 4-day - old aggregates in mice .
initially gershengorn and colleagues proposed an epithelial - to - mesenchymal transition ( emt ) to occur from human -cells .
however this idea was rapidly refuted by the same group and others after lineage tracing experiments performed in transgenic mouse models that failed to show any emt of murine -cells [ 103106 ] .
the overall conclusion of these four studies was that the proliferative cell population derived from cultured murine islets was not originating from -cells , since no -cell specific markers were identified in these cells .
efrat and collaborators recently developed a lineage tracing system similar to the techniques applied in transgenic mouse models , but now applied to human cells in vitro using lentiviral vectors .
the dual viral system relies on the -cell specific expression of the cre recombinase in one vector and a cmv - gfp reporter vector in another vector in which gfp expression is restricted by a
as lentiviral vectors integrate into the genome of the transduced cells , the reporter gene will remain expressed in all cells originating from the initial pool of labeled cells . in their follow - up study , russ et al .
slightly modified the system by using a tamoxifen - inducible cre / er recombinase , restricting the labeling period to the duration of a short ( overnight ) tamoxifen pulse .
human -cells were efficiently ( 50% ) and specifically labeled by the dual lentiviral system .
this powerful technique provided evidence that human -cells can dedifferentiate to an msc - like cell population and proliferate when cultured ex vivo , in contrast to mouse -cells , as revealed earlier from the transgenic mice studies [ 103106 ] .
intriguingly , 40% of cells exhibiting msc markers in culture ( likely to be the cell population previously named ipc , nip , pidm , or phid by others ) resulted from an emt of -cells . on the other hand , several groups suggested , but never unequivocally demonstrated , the presence of a mesenchymal stem cell population within human islets [ 94 , 96 , 97 ] . in a recent study , we investigated the presence of msc(-like cells ) in freshly isolated human islets , and we identified a double - positive cd90/cd105 population representing approximately 2% of the total islet cell population . the presence of these cells inside freshly isolated human islets was confirmed by confocal microscopy .
an independent study validated the presence of pancreatic msc in the periacinar , perivascular , and periductal space of human pancreas .
the functional significance of the presence of these cells in the islets and the possible interplay between islet - msc , endocrine cells , and the vascular system in human islets remain to be further clarified .
altogether these data suggest that the msc - like cell population derived from human islets in culture results from subpopulations of at least two origins : proliferation of islet - msc and proliferation of dedifferentiated -cells .
more studies , in particular detailed lineage - tracing experiments will be needed to confirm this model . at the moment
it remains unclear whether all mesenchymal stem(-like ) cells are equal and whether msc originating from islets would be more prone to differentiate toward the endocrine lineage .
two groups determined the gene expression profiles in human islet - derived msc [ 110 , 111 ] .
both studies confirmed the common mesenchymal character of the population with the archetypical bone marrow - msc .
remarkably cultured islet - derived ipcs are different from bone marrow - msc ( bm - msc ) in that they express a set of islet - specific genes , although at low level . upon differentiation ,
following the rather basic differentiation protocols developed so far for these cells , gene expression data showed that ipcs are able to go further along the endocrine pathways than bm - msc . in summary ,
islet-(derived ) msc could be of a valuable therapeutic significance since they appear to retain some genetic characteristic making them closer to endocrine cells than other sources of msc such as bone - marrow - derived msc .
furthermore , the observation that human -cells can dedifferentiate ex vivo into a mesenchymal phenotype in contrast to murine -cells illustrates again the discrepancy in plasticity of these cells between rodents and humans .
similarly to human islet cells cultured ex vivo , two independent studies reported that a population of msc(-like ) cells could be derived from human pancreatic exocrine tissue . under defined conditions
, these cells could show some sign of differentiation toward the endocrine lineage [ 112 , 113 ] .
epithelial - to - mesenchymal transition from exocrine cells has been proposed by seeberger et al . .
described coexpression of ductal markers and mesenchymal markers both in cultured human ductal - like cells in vitro and in ductal cells in one pancreas from a nondiabetic subject and three pancreata from patients with type 2 diabetes .
shin et al . demonstrated that the transdifferentiation capacity of human ductal cells was reduced after emt . on the other hand ,
sordi et al . suggested that the mesenchymal stem cell population growing out of cultured pancreatic tissue ( both endocrine and exocrine fractions were tested ) would be mostly originating from proliferating
total bone marrow transplantations from donor gfp transgenic mice in lethally irradiated recipient mice were performed .
surprisingly , after 12 weeks , gfp - labeled bone - marrow - derived cells were found to be localized preferentially in two organs , pancreas ( 4.82 4% of total ) and lung ( 4.43 2.3% of total ) .
18.5 4% of msc derived in culture from these pancreata were found to be gfp positive .
however once again , in absence of data on pancreatic tissues after bone marrow transplantation in humans , we can wonder how these results would be translatable to the human situation . in a study of a pancreas allograft removed 8 months after transplantation
, it was found that part of pancreatic msc expressed recipient hla , suggesting an extrapancreatic origin of these cells .
reported that pancreatic msc extracted from both endocrine and exocrine tissue and cotransplanted in mice with a minimal pancreatic islet mass facilitated the restoration of normoglycemia and neovascularization of the islet graft . in conclusion , msc can be derived from human exocrine tissue but show limited capacity of differentiation towards the endocrine pathway . similarly to islet(-derived ) msc , the origin and role of these cells are unclear .
bone marrow , adipose tissue , and umbilical cord blood are the main sources of bm - msc reported so far .
reported that transplantation of murine bone marrow cells in streptozotocin - treated mice was able to reduce hyperglycemia by initiating endogenous pancreatic regeneration .
a majority of bone - marrow - derived cells were found to be localized near ductal and islet structures .
quantitative analysis of the pancreas revealed a very low frequency of donor insulin - positive cells .
however the presence of donor cells was accompanied by a rapid proliferation of recipient pancreatic -cells and by neogenesis of insulin - positive cells of recipient origin within a week after transplantation .
the authors suggested that bone - marrow - derived endothelial cells could be involved by secreting factors that enhance tissue repair .
this model was confirmed by others . along the same line , lechner et al .
observed no evidence for significant transdifferentiation of labeled ( gfp transgenic mice ) murine bone marrow into pancreatic -cells in vivo . by using a mouse model for impaired bone - marrow - derived cell mobilization ( nos3 / mice ) , hasegawa et al .
demonstrated that homing of donor bone - marrow - derived cells in recipient bone marrow and subsequent mobilization into the injured periphery were required for -cell regeneration .
interestingly , simple bone marrow cell infusion without preirradiation had no effects , suggesting that injury signals are involved in triggering this process . finally , in similar transplantation experiments , urban et al .
revealed that murine - bone - marrow derived mesenchymal stem cells cooperate with bone marrow cells since neither bone marrow cells nor msc transplantation was effective alone .
in contrast to all these studies and using a lineage tracing system ( ins / cre - loxp / egfp ) , ianus et al . claimed that transplanted murine bone marrow cells were able to differentiate themselves into functional -cells .
four to six weeks after transplantation from male mice into lethally irradiated recipient female mice , recipient mice contained y chromosome and egfp double - positive cells in their pancreatic islets .
of note , -cell specificity was verified since neither bone marrow cells nor circulating peripheral blood nucleated cells of donor or recipient mice had any detectable egfp .
regarding human cells , prockop and colleagues evaluated the competence of bone marrow msc in a similar type of experiment .
human bone marrow msc were delivered via intracardiac infusions in diabetic nod / scid mice .
although rare -cells were found to be of human origin ( i.e. , bm - msc derived ) , blood glucose levels were found to be decreased after some weeks .
the authors suggested that human bm - msc were able to home to and promote repair of pancreatic islets and renal glomeruli in a diabetic mouse model . in a parallel study ,
sordi et al . showed that human bm - msc express a restricted set of functionally active chemokine receptors ( cxcr4 , cx3cr1 , cxcr6 , ccr1 , ccr7 ) capable of promoting migration to pancreatic islets . butler and
collaborators studied 31 human pancreases obtained at autopsy from patients who had received a bone marrow - derived graft ( 26 cases ) , a peripheral blood - derived graft ( 4 cases ) , or a combination of both peripheral blood- and bone marrow - derived stem cells ( 1 case ) .
more than 4000 islets were examined in this relatively large cohort , and no pancreatic -cells were found to be derived from donor cells ( including two cases of patients with type 2 diabetes ) .
therefore , it appears that in humans the bone marrow compartment does not contribute to pancreatic -cell mass maintenance in healthy individuals .
nevertheless promising clinical results on glucose metabolism were recently reported . a clinical trial in 11 patients with type 1 diabetes
was designed to test the safety and efficacy of intraportal coinfusion of insulin - producing adipose tissue - derived msc and bone marrow cells .
differentiation of adipose tissue - derived msc was initiated in vitro by a 3-day culture period in a defined medium described earlier .
a mean follow - up of about two years showed significant improvements of all clinical parameters related to diabetes ( a decrease in insulin requirements , an increase in c - peptide levels , and absence of diabetic ketoacidosis ) . in a clinical trial involving 25 patients with type 2 diabetes , ricordi and collaborators observed reduced insulin requirements and significant improvements of all metabolic variables ( 12-month follow - up ) after an intrapancreatic infusion of autologous bone marrow cells .
however no data are available about the possible differentiation of msc after implantation , and it is more likely that improved glucose metabolism is related to paracrine effects of msc in this last study .
in vitro , several groups reported the successful derivation of functional -cells from rodent bone marrow cells in the presence or absence of serum and with addition of growth factors like nicotinamide and -mercaptoethanol and conophylline and betacellulin - delta4 .
also murine adipose tissue - derived msc could be efficiently converted reaching up to 48% of cells that expressed c - peptide , following a 3-stage and 10-day differentiation protocol involving activin a , sodium butyrate , -mercaptoethanol , taurine , glp-1 , nicotinamide , and nonessential amino acids .
the approach is slightly different in human cells , involving the combination of defined medium and virus - mediated ectopic expression of proendocrine transcription factors .
karnieli et al . reported that overexpression of pdx1 in bm - msc from 9 of 14 donors can trigger their differentiation to a -cell - like phenotype displaying about 1% of the regular insulin content and able to control the insulin release in a glucose - dependent manner in vitro .
however transplantation into streptozotocin - treated mice resulted in further differentiation , including induction of beta2/neurod1 and reduction of hyperglycemia .
msc isolated from the wharton 's jelly of the umbilical cord were differentiated to islet - like cell clusters through stepwise culturing in neuron - conditioned medium .
the clusters were found to express islet - specific genes and to be glucose responsive in vitro .
functionality was further verified after transplantation into the liver of streptozotocin - induced diabetic rats via laparotomy .
the presence of characteristic secretory granules was observed by electron microscopy 12 weeks after transplantation . in summary , the capacity of extrapancreatic mesenchymal stem cells to differentiate to -cells in vivo appears to be very limited .
nevertheless , several groups reported a successful differentiation to functional -cell - like cells in vitro especially from human msc .
msc appear to display unique migratory and secretory properties ( growth factors , cytokines ) that make them attractive as helper cells for tissue repair ( improve engraftment , viability , function ) ( for review see [ 92 , 136 ] ) .
a crucial aspect common to all putative cell sources will be to uncover the mechanisms that preserve and control cell identity in order to enable successful manipulation of adult cell plasticity in clinical settings .
up to now , most efforts to influence the cell identity were focused on direct gene expression ( overexpression / downregulation of key transcription factors ) and growth - factor - mediated activation of specific signaling pathways .
a new era has started aiming at better understanding the processes that regulate gene expression .
cell identity is regulated by epigenetic factors that tightly regulate the activation or repression of genes including genomic dna methylation , histone modifications , and noncoding rna regulation ( for review please see [ 137 , 138 ] ) .
recently demonstrated that pancreatic -cell identity is maintained by dna methylation - mediated repression of arx .
investigated the patterns of histone modifications over the insulin gene in human islets and ipc ( the msc - like population derived ex vivo from human islets ) compared to hela and bm - msc .
although neither ipc nor hela nor bm - msc express insulin , ipc showed significant levels of active chromatin modifications , similarly to human islets although at a more moderate level .
however these epigenetic marks absent in the unrelated cell types ( hela and bm - msc ) might be part of a general mechanism whereby tissue - derived precursor / stem cells are committed to a distinct specification .
non - coding rna ( such as sirna ( short interference rna ) , mirna ( microrna ) , and lncrna ( long non - coding rna ) ) are emerging as key players in regulation of development .
demonstrated that the mir-30 family of mirnas contribute to the regulation of the dedifferentiation of human fetal -cells through epithelial - to - mesenchymal transition by negatively regulating the translation of mesenchymal genes .
epigenetic reprogramming of cell types with shared developmental history could be an effective strategy for pancreatic -cell replacement therapies .
the cells may display some intrinsic commitment to become islets even during adulthood and might thus require fewer triggers to differentiate / transdifferentiate towards a -cell lineage .
along this line , in the field of reprogramming to ips , the notion of epigenetic memory inherited from the parental cell is coming forward .
these new insights in gene regulation should help to exploit the full potential of adult cell plasticity in the perspective of cell replacement therapies to treat diseases such as type 1 diabetes .
in conclusion , it emerges that extreme caution should be taken when translating the findings obtained from rodent studies to the human situation . regarding in vivo regeneration investigated under physiological conditions or under injury
, it appears that -cell replication is the predominant mechanism occurring in mice . in humans , this process seems to be restricted to the very early postnatal life , whereas during adulthood , neogenesis from ( a subpopulation of ) ductal and/or acinar cells seems to be responsible for the increase of -cell mass required in physiological situations of higher insulin demands like obesity or pregnancy .
nevertheless , the unanswered question remains which cell type could be involved : progenitor cells present in the ductal area , or fully differentiated adult cells able to transdifferentiate , or a distinct subpopulation that is able to dedifferentiate to a progenitor - like intermediate stage followed by redifferentiation to an endocrine lineage .
remarkably , both the animal model and the degree of destruction appear to be key points , as the regeneration processes can be different : -cell mass regeneration from cells has been observed in models of near total ablation only .
regarding in vitro replicative capacity of -cells , human -cells can not efficiently replicate , in contrast to rodent cells .
however , contrary to murine -cells again , human -cells can dedifferentiate to a mesenchymal - like phenotype and proliferate .
these discrepancies between findings from human versus rodent studies are not as unexpected as they seem , given the major differences observed in islet cell biology field such as timing of pancreatic developmental stages , islet architecture and composition [ 144 , 145 ] , and islet innervation .
. finally , cultured human primary cells are more prone to replicative aging than murine cells as telomere shortening limits cell replication and leads to senescence .
however , up to now , there is no way to accurately evaluate the -cell mass in humans , neither by imaging techniques nor by physiological measurements .
studies are limited to histological analysis performed on organs at autopsy or after pancreatectomy generating static pictures that could be misinterpreted , even if careful quantification and repeated observations in different contexts can still result in strong indications .
therefore , animal models become essential offering access to a broad range of technologies obviously not applicable to humans . among others ,
the recently developed genetic approaches ( such as lineage tracing systems , comprising of a tissue - specific promoter and a ( tamoxifen-)inducible recombination system and a reporter gene ) are valuable tools to follow a dynamic process and reinforce ( or sometimes challenge ) earlier theories suggested by more descriptive immunohistochemistry data .
nevertheless several limitations have to be taken into consideration : a possible leakiness of the recombination system , the relative specificity of a given promoter that can display some ( transient ) activity in nonspecific cells , and a limited penetrance ( usually less than half of the cells are actually labeled ) [ 147 , 148 ] .
altogether these elements might contribute to the discrepancies between results obtained by different labs about the origin of -cell regeneration for instance , and hopefully validation of some of the models by separate labs in different experimental contexts will clarify the situation in a near future .
since stimulation of human -cells replication is still elusive , other cell sources have been envisaged .
studies from the last decade revealed an unexpected aspect of plasticity from mature differentiated cells by dedifferentiation or transdifferentiation . of interest
, it appears that regeneration does not always require recapitulation of the embryological development as needed for efficient differentiation of es cells .
for instance , the recent discovery of -to--cell plasticity does not appear to correlate with any developmental process .
murine and human ductal cells , rodent acinar cells ( human ones can not be maintained in culture ) , and human liver cells could be efficiently converted to functional -cells able to revert hyperglycemia in a diabetic mouse model . although the potential contribution of msc to islet regeneration in a physiological situation remains unclear and their origin and function are still elusive , islet - derived mesenchymal stem cells seem to display specific genetic and epigenetic marks that could make them more prone to differentiation towards the endocrine compartment than extrapancreatic sources of msc .
this suggests that all mesenchymal stem cells ( -like cells ) are not equal . in addition
msc , in particular from humans , revealed additional properties ( like homing to injury site and secretion of favorable growth factors ) that may be of clinical use .
therefore , further investigations will be required to determine if islet-(derived- ) msc can be stimulated to contribute in any way to -cell mass regeneration .
another approach successfully tested has been to force expression of proendocrine transcription factors in vivo in the liver and in the exocrine tissue
. however no lineage tracing experiments have been performed in these studies , and the origin of the newly formed -cells needs to be identified . finally , there is currently no unique optimal alternative cell source for -cell ( re)generation .
therefore a crucial aspect common to all putative cell sources will be to further uncover the mechanisms that preserve and control cell identity in order to enable successful manipulation of adult cell plasticity for clinical application . | -cell replacement by allogeneic islet transplantation is a promising approach for patients with type 1 diabetes , but the shortage of organ donors requires new sources of cells .
islet regeneration in vivo and generation of -cells ex vivo followed by transplantation represent attractive therapeutic alternatives to restore the -cell mass . in this paper
, we discuss different postnatal cell types that have been envisaged as potential sources for future -cell replacement therapy .
the ultimate goal being translation to the clinic , a particular attention is given to the discrepancies between findings from studies performed in rodents ( both ex vivo on primary cells and in vivo on animal models ) , when compared with clinical data and studies performed on human cells . |
this paper proposes theoretical work that introduces powerful algorithms of molecular computation that could potentially compromise the security that is afforded by certain cryptography algorithms .
molecular
computation involves
biochemistry and dna rather than silicon chips to tackle formidable
computations .
theoretical aspects of this interdisciplinary field are important
to develop the potential and interest in this form of computation .
elliptic curve cryptography ( ecc ) is a mathematical
approach to public key cryptography using elliptic curves that are typically
defined over finite fields .
elliptic curves [ 4 , 5 ] constitute a major area of current research that is
particularly important to number theory , for example , elliptic curves had a
role in the recent proof of fermats last theorem .
as applied to cryptography , not only has ecc become applied in
diffie - hellman key exchange but also in the digital signature algorithm ( dsa ) ,
a us federal government standard for digital signatures .
it is known as the
elliptic curve dsa ( ecdsa ) or that variant of the dsa operating on elliptic
curve groups .
the security of these cryptosystems relies on the difficulty of solving the elliptic curve discrete logarithm problem [ 6 , 7 ] .
if p is a point with order m on an elliptic curve , and q is some other point on the same curve , then the elliptic curve discrete logarithm problem is
to determine an l such that q = lp , where l is an integer and
0 l m 1 .
if this problem can be solved efficiently , then
elliptic curve - based cryptosystems can be broken efficiently . in order to tackle such a problem ,
adleman succeeded in solving an instance of the hamiltonian path problem in a test tube , simply by the manipulation of dna strands .
following this , lipton demonstrated that the adleman techniques offered a solution to the satisfiability problem ( the first considered np - complete problem ) .
recent advances in molecular biology [ 10 , 11 ] have made it possible to produce roughly 10 dna strands in a test tube .
those 10 dna strands can be made to represent 10 bits of information . in a distant future ,
if biological operations may be run error
free using a test tube with 10 dna strands , then it would be possible to process 10 bits of
information simultaneously .
the objective for biological computing technology is to provide this enormous amount of parallelism for dealing with computationally intensive real world problems [ 1214 ] .
advancement in dna computing has already been made in many areas . in the field of cryptology ,
boneh et al . have cracked des using identical principles to those of adleman 's solution of the travelling salesman problem
they proposed three dna - based algorithms : parallel subtractor ; parallel comparator ; and parallel modular arithmetic unit [ 15 , 16 ] . in this paper , we take a step further with respect to chang 's work in
order to solve the elliptic curve discrete logarithm problem .
we develop dna - based algorithms for a parallel adder ; a parallel multiplier ; a parallel divider over gf(2 ) ( i.e. , a galois field of characteristic
2 ) ; and a parallel adder for adding points on elliptic curves .
we accomplish all of these by means of basic biological operations . we also showed that cryptosystems based on elliptic curves can be broken .
our work presents clear evidence of
molecular computing abilities to accomplish complex mathematical operations .
section 3 introduces the dna computing that solves the elliptic curve discrete logarithm problem , for solution spaces of dna strands .
dna ( deoxyribonucleic acid ) is the molecule that plays the main role in dna - based computing .
distinct
nucleotides are detected only with their bases , which come in two sorts : purines and pyrimidines .
purines include adenine and guanine , abbreviated a and g. pyrimidines contain cytosine and thymine , abbreviated c and t. a dna strand is essentially a sequence ( polymer ) of four types of nucleotides detected by one of four bases they contain . two strands of dna can form ( under appropriate conditions ) a double strand , if the respective bases are the watson - crick complements of each other a matches t and c matches g. hybridization is a
special technology term for the pairing of two single dna strands to make a double helix and also takes advantages of the specificity of dna base pairing for the detection of specific dna strands ( for more discussions of the relevant
biological background , refer to [ 10 , 11 ] ) . in the past decade
, there have been revolutionary advances in the field of biomedical engineering , particularly in recombinant
dna and rna manipulating . due to the industrialization of the biotechnology field
basic principles about recombinant dna can be
found in [ 1720 ] . in the following ,
we describe five biological operations that are useful for solving the elliptic curve discrete logarithm
problem .
a ( test ) tube is a set of molecules of dna ( a multiset of finite strings over the alphabet { a , c , g , t } ) .
extract . given a tube p and a short single strand of dna , s , the operation produces two tubes + ( p , s ) and (p , s ) , where + ( p , s ) is all of the
molecules of dna in p which contain s as a substrand and (p , s ) is all of the molecules of dna in p which do not contain s.merge . given tubes p1 and p2 , yield (p1 , p2 ) , where (p1 , p2 ) = p1 p2 .
this operation is to pour two tubes into one ,
without any change in the individual strands.amplify . given a tube p , the operation amplify ( p , p1 , p2 ) , will produce two new tubes p1 and p2 so that p1 and p2 are totally a copy of p ( p1
and p2 are now identical ) and p becomes an empty tube.append . given a tube p containing a short strand of dna z , the operation will append z onto the end of every strand in p.append-head .
given a tube p containing a short strand of dna , z , the operation will append z onto the head of every strand in p.
extract .
given a tube p and a short single strand of dna , s , the operation produces two tubes + ( p , s ) and (p , s ) , where + ( p , s ) is all of the
molecules of dna in p which contain s as a substrand and (p , s ) is all of the molecules of dna in p which do not contain s. merge .
given tubes p1 and p2 , yield (p1 , p2 ) , where (p1 , p2 ) = p1 p2 .
this operation is to pour two tubes into one ,
without any change in the individual strands .
amplify . given a tube p , the operation amplify ( p , p1 , p2 ) ,
will produce two new tubes p1 and p2 so that p1 and p2 are totally a copy of p ( p1
and p2 are now identical ) and p becomes an empty tube .
, the operation will append z onto the end of every strand in p. append - head .
given a tube p containing a short strand of dna , z , the operation will append z onto the head of every strand in p.
an elliptic curve is defined to be the set of solutions ( x , y ) gf(2 ) gf(2 ) to the equation
( 1)y2+xy = x3+ax2+b ,
where a , b gf(2 ) and b 0 , together with the point on the curve at infinity o , ( with homogeneous coordinates ( 0 , 0 ) ) . the points on an elliptic curve form an abelian group under a well - defined group operation .
the identity of the group operation is the point o. for p = ( x1 , y1 ) a point on the curve , we define p to be ( x1 , y1 + x1 ) , so p + ( p ) = ( p ) + p = o. now suppose p and q are not o , and p q. let p be as above and q = ( x2 , y2 ) , then
p + q = ( x3 , y3 ) , where
( 2)x3=2++x1+x2+a , y3=(x1+x3)+x3+y1,={y2+y1x2+x1if pq , x12+yx1if p = q ,
( refer to ) . in this paper , for convenience , use bn1bn2 b1b0 to denote the value of bn1 + bn2 + + b1 + b0 over gf(2 ) .
let e be an elliptic curve defined over gf(2 ) , and let g e be a fixed and publicly known point .
the receiver b chooses
a randomly and publishes the key ag , while keeping a itself secret .
to transmit a message m to b ,
user a chooses a
random integer k and sends the pair of points ( kg , pm + k(ag ) ) . to read the message ,
b multiplies the first point in the pair by his secret a , and then subtracts the result from the second point in the pair .
so , if a breaker can compute a from public key
g and ag , he can decrypt any encryption sent to b ( refer to ) . over gf(2 ) , the additive operation on two numbers is just doing xor on each bit , respectively , without any carry .
for instance , ( 1101 ) + ( 1001 ) = ( 0100 ) . for every bit xj
one represents the value zero for xj and the other
represents the value one for xj . for convenience , we assume that xj denotes the value of xj to be one and xj denotes the
value of xj to be zero .
the following algorithm is used for parallel adding two n bits binary
number in a strand with one starts from the pth bit and the other one starts from the
qth bit and appending the result from rth bit ( see algorithm 1 ) .
consider that n = 3 , p = 1 , q = 4 , and r = 7 .
that is , two binary numbers to be added in parallel are both 3 bits , while one from the 1st bit and the other one from the 4th bit
in a strand , and
we then suppose , tube t0 = { 110001 , 010101 , 001111 , 100011 } which is regarded as an input tube for the algorithm paralleladder
( t0 , n , p , q , r ) . because the value of
n is 3 , step ( 1a ) to step ( 1f ) will be run 3 times .
after the first execution of step ( 1a ) is finished , t1 = { 110001 , 100011 } and t2 = { 010101 , 001111}. next , after the first execution of step ( 1b ) and
step ( 1c ) is performed , t3 = , t4 = { 110001 , 100011 } , t5 = { 010101 , 001111 } and t6 = , while t0 = t1 = t2 = . after first execution of step ( 1d ) is run , tube t7
= { 110001 , 010101 , 001111 , 100011 } and t8 = . after first execution of step ( 1e ) and step ( 1f ) is run , t0 = { 1100011 , 0101011 , 0011111 , 1000111}. then , after the rest operations are performed , the
result of tube t0 is shown in table 1 . the algorithm paralleladder ( t0 , n , p , q , r ) is applied to finish the function of a parallel adder .
the algorithm paralleladder ( t0 , n , p , q , r ) is implemented by means of the extract , merge , and append operations . each execution of step
( 1a ) is used to produce two tubes t1 and t2 , where all of the molecules of dna in t1 contain xp+j and all of the molecules of dna in t2 contain xp+j . each execution of step ( 1b ) and step ( 1c ) is used to produce four tubes t3 , t4 , t5 , t6 , where all dna strands in t3 contain xp+j and xq+j , all dna strands in t4 contain xp+j and xq+j , all dna strands in t5 contain xp+j and xq+j , and all dna strands in t6 contain xp+j and xq+j . according to the additive theorem over gf(2 ) , the jth bit of the sum in t4 and t5 is 1 and the
jth bit of the sum in t3 and t6 is 0 . from paralleladder ( t0 , n
, p , q , r ) , it takes
3n extract operations , 3n merge operations , 2n append operations , and 9 test tubes to finish parallel addition .
therefore , the algorithm will add
15n bases to all dna strands in tube t0 . over gf(2 ) , the multiplicative operation runs as follows :
( 3)(bn1n1++b1+b0)(bn1n1++b1+b0 ) = bn1n1(bn1n1++b1+b0 ) + +b0(bn1n1++b1+b0 ) = h2n22n2+h2n32n3++h22+h1+h0,h2n2=bn1bn1,h2n3=bn1bn2+bn2bn1,
,hn1 = bn1b0+bn2b1++b0bn1, ,h1=b1b0+b0b1,h0=b0b0. the algorithm parallelmultiplier ( t0 , n , p , q ) is used to
multiply two nbit binary numbers on every strand in parallel with one starts from the pth bit and the
other one starts from the qth bit .
it runs as follows : at first , employ extract operation to form two tubes :
t1 and t2 .
the first tube t1 includes all of
the strands on which xp = 1 and the second tube t2 includes all of
the strands on which xp = 0 .
then , we copy the bits from qth to ( q + n 1)th to the end of every strand in tube t1 and append n bits 0 to the
end of every strand in tube t2 .
after these operations , the ( q + n)th bit to
( q + n + n 1)th bit show the coefficients of to . using the same principle , we get the coefficients of to , to , , to . at last , call algorithm paralleladder ( t0 , n , p , q , r ) to compute the sum of coefficients of , the sum of coefficient of , , and so on . as a result ,
the length of every strand will increase n n + 2n 1 bits . from parallelmultiplier ( t0 , n
, p , q ) , it takes o(n ) extract operations , o(n ) merge
operations , o(n ) append operations , and o(1 ) test tubes to
finish the function of a parallel multiplier .
because over gf(2 ) , the exponent of can not be beyond n 1 , the result of parallel multiplying should be shifted by a certain irreducible polynomial , called primitive polynomial : + bn1 + + b1 + b0 = 0 = bn1 + + b1 + b0 . the algorithm parallelshifter ( t0 , n , p )
, p representing that the multiplicative result starts from the pth bit , is used to parallel shift the multiplicative result b2n2 + b2n3 + + b1 + b0 to legal form bn1 + bn2 + + b1 + b0 over gf(2 ) which can be designed as follows : appends the primitive polynomial 's coefficients from to to the end of every strand at first . employ the extract operation to form two test tubes t1 and t2 .
tube t1 includes all of the strands on which xp = 1 and tube
t2 includes all of the strands on which xp = 0 .
then , we add the coefficients , from item to item , to the coefficients of irreducible polynomial in parallel in tube t1 .
this forms the new coefficients from to and has deleted the item .
the t2 includes all of the strands that have xp = 0 , so just copy the coefficients from to without any change .
after all the executions before are run , the highest exponent of is reduced to
2n 3 .
when this algorithm is run out , the highest exponent of is reduced to n 1 .
this algorithm will totally append n n + ( n 1)(n 2)/2 bits more to every strand . from parallelshifter ( t0 , n
, p ) , it takes o(n ) extract operations , o(n ) merge operations , o(n ) append operations , and o(1 ) test tubes to
finish the function of a parallel shifter . over gf(2 ) , to do a division operation for a dividend and a divisor , one should get the divisor 's inverse first and then multiply the dividend . for the primitive polynomial
+ bn1 + + b1 + b0 is irreducible , there exists a polynomial g( ) and a polynomial f( ) that fit the
equation ( according to euclid algorithm ) :
( 4)g()divisor+f()(n+bn1n1++b1+b0)=1 . because + bn 1 + + b1 + b0 = 0 , g( ) divisor = 1 , which is to say g( ) is the inverse of the divisor . to find
g( ) and f( ) ,
one can do as follows , which is called euclid
algorithm , also called division algorithm ; first , + bn1 + + b1 + b0 is divided by the divisor .
if the value of the remainder is 1 , that is to say ,
( + bn1 + + b1 + b0 ) + g( ) divisor = 1 with g( ) is the division result and the inverse of divisor has found which is g( ) ; else let remainder be
r( ) , let the divisor be the dividend and r( ) be the divisor
and do division operation again .
because the highest exponent of
of remainder reduces by 1 in each repeat , it is at most repeating n 1 times .
so in
the first time division , the dividend is n + 1 bits and the divisor is n bits and the
remainder is at most n 1 bits , and in the second time division the dividend is n bits and the
divisor is n 1 bits and the remainder is at most n 2 bits , , and
in last time division the dividend is 3 bits and the divisor is 2 bits and the remainder is 1 bit .
at first compare the first two bits of dividend with divisor to determine
that addition operation between first two bits of dividend and divisor should be done or not , then compare the first three bits of result with divisor ,
, compare the first n bits of result with divisor , the last time ( finally ) , compare the last
n bits of result with divisor for this time the first bit of result is 0 .
the following
algorithm is used to compare the divisor which is from pth bit to qth bit with the
bits from jth bit to kth bit in parallel , and forms tube t1 and t2 .
t2 contains all strands on which add execution will be done and
t1 contains all strands on which add execution will not be done ( see algorithm 2 ) . the algorithm parallel comparator ( t0 , p , q , j , k , t1
if q p > k j , that means the bits of divisor is more than the bits of the result which are intended to compare this time .
step ( 1 ) considers the
excessive bits of divisor and if any one bit is 1 , which means the divisor is bigger than the bits of the result which are intended to compare this time and pour the strands to t1 .
step ( 2 ) considers the rest of the bits of divisor and the bits of result which are intended to compare this time .
t3 contains all strands on which certain bit of divisor is 1 and corresponding bit of the result is 1 ; t4 contains all
strands on which certain bit of divisor is 1 and corresponding bit of the result is 0 ; t5 contains all
strands on which certain bit of divisor is 0 and corresponding bit of the result is 0 ; t6 contains all
strands on which certain bit of divisor is 0 and corresponding bit of the result is 1 .
so add execution can be done over strands in
t3 and t6 , which can not be done over strands in
t4 , and strands in t5 need more consideration .
from parallelcomparator ( t0 , p , q , j , k , t1 , t2 ) , it takes o(n ) extract operations , o(n ) merge operations , and o(1 ) test tubes to finish the function of a parallel comparator .
suppose the divisor is n bits and is from pth bit and thedividend is n + 1 bits and is
from the qth bit in each strand .
to do the long division , first compare the divisor with first two bits
of dividend using parallelcomparator to get the first bit of the result of division and note down the result of first time addition result .
then , compare the divisor with the first three bits of the addition result last time to get the second bit of the result of the long division , and note down the addition result . finally , compare the divisor with the last
n bits of addition result last time to get the last bit of division result and the
remainder ( see algorithm 3 ) .
the algorithm similardiv ( t0 , n , p , q ) is applied to
finish the function of parallel long division .
proof .
each execution of step ( 1 ) is to get each bit of the long division result .
consider the first cycle of step ( 1 ) , step ( 1a ) compares the divisor with the first two bits of
the dividend , and form two tubes t1 and t2 that t2 contains all strands on which add execution can be done , contrarily the t1 .
step ( 1b ) appends 0 to all strands in t1 and appends 1 to all strands in t2 .
step ( 1d ) adds the divisor and the first two bits of the dividend in t2 .
step ( 1e ) copies the rest of the bits of the dividend
in t2 .
step ( 1f ) pours t1 and t2 together and
finishes the first execution of step ( 1 ) to get the first bit of the division result and the first time addition result .
the second execution of step ( 1 ) is to compare the divisor with the first three bits of the addition result last
time and get the second bit of the division result .
the principle of other cycles and the rest of the steps are similar to the principle above .
the length of each strand will reach to q + n + ( n + 2)n bits when this algorithm is run out.from similardiv ( t0 , n , p , q ) , it takes o(n ) extract operations , o(n ) append operations , o(n ) merge operations , and o(1 ) test tubes to finish the function of a parallel long division .
the algorithm similardiv ( t0 , n , p , q ) is applied to
finish the function of parallel long division . each execution of step ( 1 ) is to get each bit of the long division result .
consider the first cycle of step ( 1 ) , step ( 1a ) compares the divisor with the first two bits of
the dividend , and form two tubes t1 and t2 that t2 contains all strands on which add execution can be done , contrarily the t1 .
step ( 1b ) appends 0 to all strands in t1 and appends 1 to all strands in t2 .
step ( 1d ) adds the divisor and the first two bits of the dividend in t2 .
step ( 1e ) copies the rest of the bits of the dividend
in t2 .
step ( 1f ) pours t1 and t2 together and
finishes the first execution of step ( 1 ) to get the first bit of the division result and the first time addition result . the second execution of step ( 1 ) is to compare the divisor with the first three bits of the addition result last
time and get the second bit of the division result .
the principle of other cycles and the rest of the steps are similar to the principle above .
the length of each strand will reach to q + n + ( n + 2)n bits when this algorithm is run out . from similardiv ( t0 , n
, p , q ) , it takes o(n ) extract operations , o(n ) append operations , o(n ) merge operations , and o(1 ) test tubes to finish the function of a parallel long division .
suppose the irreducible polynomial a( ) = + bn1 + + b1 + b0 and suppose g( ) and f( ) satisfy that g( ) divisor + f()a( ) = 1 , for the purpose of finding the divisor 's inverse , g( ) , we need to do sometimes long division introduced in
section 3.7 ; let a( ) be the dividend
and divisor mentioned above be the divisor in first time and suppose the result is g1( ) , and if the remainder is 1 , then the division result g1( ) is g( ) .
else , let the divisor last time be the dividend and let the remainder last time be the divisor and do the long division .
suppose the result is p( ) and g2( ) = p( ) g1( ) + 1 , if the remainder is 1 , the g2( ) is g( ) .
else , let the divisor last time be the dividend and let the remainder last time be the divisor and do the long division .
suppose the result is
p( ) and g3( ) = p( ) g2( ) + g1( ) , if the remainder is 1 , the g3( ) is g( ) .
generally speaking , we need to trace back after long division each time : first time , the tracing result is the division 's result ; the second time , the tracing 's result is the sum of 1 and the product
of the division 's result and the tracing 's result last time ; from the third
time , the tracing 's result is the sum of the tracing 's result last second time
and the product of the division 's result and the tracing 's result last time .
the following algorithm is used to do tracing operation from the third time in
which t , d , and p mean that the tracing 's result last second time is from the
tth bit and the last tracing 's result is from the dth bit and the
division result is from the pth bit ( see algorithm 4 ) .
the algorithm traceback ( t0 , t , d , p , n ) is used to trace back after long division from the third time in order to get the divisor 's inverse .
proof . in this and following procedures ,
m = n + 2n 1 + n + ( n 1)(n 2)/2 , which represent the total number of increased bits when parallelmultiplier ( t0 , n , p , q ) and
parallelshifter ( t0 , n , p ) are called . step ( 1 )
is used to multiply the last tracing 's result from dth bit to the long division result from pth bit in parallel .
the result will be from the ( p + n + n n)th bit to ( p + n +
n n + 2n 1)th bit . step ( 2 ) is to shift the result of multiplication to legal form which will append ( ( n 1)(n 2)/2 + n ) bits to every strand . and
its result is from ( p + m)th bit to ( p + n + m 1)th bit .
step ( 3 ) is used to add the result to the last second time 's tracing 's result in parallel.from traceback ( t0 , t , d , p , n ) , it takes
o(n ) extract operations , o(n ) append operations , o(n ) merge operations , and o(1 ) test tubes to
finish the function of tracing back .
the algorithm traceback ( t0 , t , d , p , n ) is used to trace back after long division from the third time in order to get the divisor 's inverse . in this and following procedures ,
m = n + 2n 1 + n + ( n 1)(n 2)/2 , which represent the total number of increased bits when parallelmultiplier ( t0 , n , p , q ) and
parallelshifter ( t0 , n , p ) are called . step ( 1 )
is used to multiply the last tracing 's result from dth bit to the long division result from pth bit in parallel .
the result will be from the ( p + n + n n)th bit to ( p + n + n n + 2n 1)th bit .
step ( 2 ) is to shift the result of multiplication to legal form which will append ( ( n 1)(n 2)/2 + n ) bits to every strand . and
its result is from ( p + m)th bit to ( p + n + m 1)th bit .
step ( 3 ) is used to add the result to the last second time 's tracing 's result in parallel . from traceback ( t0 , t ,
d , p , n ) , it takes
o(n ) extract operations , o(n ) append operations , o(n ) merge operations , and o(1 ) test tubes to
finish the function of tracing back . from the algorithms introduced above , we can find divisors ' inverses in parallel as follows : first pick out the strands on which divisor equals to 1 .
then , let the primitive polynomial be the dividend and the
divisor be the divisor and do long division .
trace back to get the tracing 's
result first time and pick up the strands on which the remainder equals to 1 and store them in tube t1 . then , let the divisor last time be the dividend and the remainder last time be the divisor and do long division .
pick up the strands on which the remainder equals to 1 and store them in tube t2 , , these executions , including long division , collecting every bit of the quotient and tracing back , are run n 1 times at most . in the following algorithm ,
the parameters n and
p mean that the divisor is n bits and it starts from pth bit in every
strand .
the last parameter r is used to represent that each strand in tube is
r 1 bit long and we begin append operation from rth bit of every strand . among the algorithm , the procedure picking ( t0 , n , p , ts ) is used to pick out the strands on which the pth bit to the ( p + n 2)th bit are all 0 and the ( p + n 1)th bit is 1 and store them in
ts .
it is easy to program , so just omitted here ( see algorithm 5 ) .
the algorithm parallelinverse ( t0 , n , p , r ) is applied to
find inverses over gf(2 ) in parallel .
proof .
step ( 2 ) is used to append n + 1 bits irreducible polynomial , which is the dividend in first long division , to every
strand .
the execution of step ( 3 ) calls the algorithm similardiv ( t0 , n , p , r ) to finish long division . now
the length of strands is added up to r + n + ( n + 2)n bits .
step ( 4 ) finishes the function of collecting every bit of quotient and will add
n bits to every strand .
step ( 5 ) calls the procedure
picking ( t0 , n , p , ts ) to pick out the
strands on which the remainder is 1 and store them in tube t1 .
step ( 6 ) finishes the operation of appending the dividend , which is divisor last time , to the strands .
step ( 7 ) calls the algorithm similardiv ( t0 , n , p , q ) to accomplish the second time long division .
step ( 8) employs the append operation to append a bit 0 in order to make the quotient to be n bits .
step ( 9 ) finishes the function of collecting every bit of quotient and will add n 1 bits to every strand . steps ( 10 ) and ( 11 ) call the algorithm parallelmultiplier
( t0 , n , p , q ) and parallelshifter
( t0 , n , p ) .
these
three steps accomplish the function of tracing back of the second time . now
step ( 13 ) calls the algorithm picking ( t0 , n , p , ts ) to pick out the strands on which the remainder is 1 and store them in tube
t2
. one execution of step ( 14 ) finishes the function of long division and tracing back and picking out the strands on which the remainder is 1 .
the step will be looped n 3 times , because
the long division should be done n 1 times to make sure that the remainder of each strand equals to 1 and long division has been done twice before . note that while j 2 , sj = n + 2 j + ( n + 3 j)(n + 1 j ) + n + m + n. step ( 15a ) is used to append certain bits 0 to each
tube tj to make sure that strands in t1 to tn1 are all r+s1 + + sn 1 1 bits long .
step ( 15b ) is used to append the inverse gotten before to the last of every strand in t1 to tn1 .
step ( 18 ) pours strands in all tubes together to one tube t0 . from all above , getting inverse one time needs
increasing s1 + s2 + + sn1 + n bits to every strand.from parallelinverse ( t0 , n , p , r ) , it takes
o(n ) extract operations , o(n ) append
operations , o(n ) merge operations , and o(n ) test tubes to
finish the function of finding inverse in parallel .
the algorithm parallelinverse ( t0 , n , p , r ) is applied to
find inverses over gf(2 ) in parallel .
step ( 2 ) is used to append n + 1 bits irreducible polynomial , which is the dividend in first long division , to every
strand .
the execution of step ( 3 ) calls the algorithm similardiv ( t0 , n , p , r ) to finish long division . now
the length of strands is added up to r + n + ( n + 2)n bits .
step ( 4 ) finishes the function of collecting every bit of quotient and will add
n bits to every strand .
step ( 5 ) calls the procedure
picking ( t0 , n , p , ts ) to pick out the
strands on which the remainder is 1 and store them in tube t1 .
step ( 6 ) finishes the operation of appending the dividend , which is divisor last time , to the strands .
step ( 7 ) calls the algorithm similardiv ( t0 , n , p , q ) to accomplish the second time long division . step ( 8) employs the append operation to append a bit 0 in order to make the quotient to be n bits .
step ( 9 ) finishes the function of collecting every bit of quotient and will add n 1 bits to every strand . steps ( 10 ) and ( 11 ) call the algorithm parallelmultiplier
( t0 , n , p , q ) and parallelshifter
( t0 , n , p ) .
these
three steps accomplish the function of tracing back of the second time . now
step ( 13 ) calls the algorithm picking ( t0 , n , p , ts ) to pick out the strands on which the remainder is 1 and store them in tube
t2 .
one execution of step ( 14 ) finishes the function of long division and tracing back and picking out the strands on which the remainder is 1 .
the step will be looped n 3 times , because
the long division should be done n 1 times to make sure that the remainder of each strand equals to 1 and long division has been done twice before . note that while j 2 , sj = n + 2 j + ( n + 3 j)(n + 1 j ) + n + m + n. step ( 15a ) is used to append certain bits 0 to each
tube tj to make sure that strands in t1 to tn1 are all r+s1 + + sn 1 1 bits long . step ( 15b ) is used to append the inverse gotten before to the last of every strand in t1 to tn1 .
step ( 18 ) pours strands in all tubes together to one tube t0 . from all above , getting inverse one time needs
increasing s1 + s2 + + sn1 + n bits to every strand . from parallelinverse ( t0 , n
, p , r ) , it takes
o(n ) extract operations , o(n ) append
operations , o(n ) merge operations , and o(n ) test tubes to
finish the function of finding inverse in parallel .
to do a division operation on gf(2 ) , first one should calculate divisor 's inverse using above mentioned algorithm then multiply the dividend .
the following algorithm is used to finish the function of parallel division over gf(2 ) where the dividend begins with the
qth bit and the divisor begins with the pth bit , and current bit is the
rth bit ( see algorithm 6 ) .
the algorithm paralleldivision ( t0 , n , p , q , r ) is used to finish the function of parallel division over gf(2 ) .
proof .
step ( 1 ) calls the algorithm parallelinverse ( t0 , n , p , r ) to get the divisor 's inverse of every strand .
the length of every strand is r + s1 + + sn1 + n 1 bits now with the inverse from ( r + s1 + + sn1)th bit to ( r + s1 + + sn1 + n 1)th bit .
step ( 2 ) calls parallelmultiplier ( t0 , n , p , q ) to finish the
function of the inverse being multiplied by the dividend every strand with the dividend starting from the qth bit .
now the length of each strand adds up to r + s1 + + sn1 + n + n n + 2n 1 1 bits .
step ( 3 ) shifts the product by calling parallelshifter ( t0 , n , p ) and will add ( n 1)(n
doing parallel division one time need increasing the strands d = s1 + + sn1 + n + m bits.from paralleldivision ( t0 , n , p , q , r ) , it takes o(n ) extract operations , o(n ) append
operations , o(n ) merge operations , and o(n ) test tubes to
finish the function of parallel division .
the algorithm paralleldivision ( t0 , n , p , q , r ) is used to finish the function of parallel division over gf(2 ) . step ( 1 ) calls the algorithm parallelinverse ( t0 , n , p , r ) to get the divisor 's inverse of every strand .
the length of every strand is r + s1 + + sn1 + n 1 bits now with the inverse from ( r + s1 + + sn1)th bit to ( r + s1 + + sn1 + n 1)th bit .
step ( 2 ) calls parallelmultiplier ( t0 , n , p , q ) to finish the
function of the inverse being multiplied by the dividend every strand with the dividend starting from the qth bit . now
the length of each strand adds up to r + s1 + + sn1 + n + n n + 2n 1 1 bits .
step ( 3 ) shifts the product by calling parallelshifter ( t0 , n , p ) and will add ( n 1)(n
2)/2 + n n bits to every
strand . generally speaking , doing parallel division one time
n + m bits . from paralleldivision ( t0 , n , p , q , r ) , it takes o(n ) extract operations , o(n ) append
operations , o(n ) merge operations , and o(n ) test tubes to
finish the function of parallel division . by far the addition , subtraction ( the same to addition ) , multiplication , and division operations over
gf(2 ) have been solved .
now consider how to execute addition of two points on an elliptic curve y + xy = x + ax + b in biological ways .
we should consider five different cases : case 1 , the first point is
o and the point of sum equals to the second point ; case 2 , the second point is o and the point of sum equals to the first point ; case 3 , one point is the inverse of the other one , and the point of sum is o ; case 4 , one point equals to the other one , and computes the sum as the formula in part 3.1 ; case 5 , computes the sum using the formula in part 3.1 .
the algorithm addtwonode ( t0 , n , x1 , y1 , x2 , y2 , r ) is used to add two points .
the first one 's position
x starts from the ( x1)th bit and position y starts from the
( y1)th bit , and the second one 's position x starts from the ( x2)th bit and
y starts from the ( y2)th bit .
, it calls picking01 ( t0 , n , x1 , y1 , x2 , y2 , t11 ) to pick out the strands on which the first point is
o and store them in t11 , picking02 ( t0 , n , x1 , y1 , x2 , y2 , t12 ) to pick out the
strands on which the second point is o and store them in t12 , pickinginverse
( t0 , n , x1 , y1 , x2 , y2 , r , t2 ) to pick out the strands on which one point is the inverse of the other one and store them in
tube t2 , and pickingequal ( t0 , n , x1 , y1 , x2 , y2 , t3 ) to pick out the strands on which one point equals to the other one and store them in tube t3
note that pickinginverse ( t0 , n , x1 , y1 , x2 , y2 , r , t2 ) will increase n bits to every strand in
t0 ( see algorithm 7 ) .
the algorithm addtwonode ( t0 , n , x1 , y1 , x2 , y2 , r ) can compute the sum of two points on elliptic curve .
step ( 5 ) employs append operation to append m bits 0 to all
strands in tube t0 .
step ( 6 ) to step ( 8) are operations on tube t0 to get
of strands in t0 .
step ( 9 ) to step ( 13 ) are operations on tube
t3 to get of strands in t3 .
step ( 14 ) pours
t0 and t3 to t0 and the length
of every strand in t0 now is r + n + n + m + n + d 1 bits .
step ( 15 ) to step ( 21 ) accomplish to compute the position
x of point of sum and the result is from ( r + x)th bit .
the
execution of steps ( 22 ) to ( 26 ) is used to get the position y of the sum
which equals to (x1 + x3 ) + x3 + y1 . and now , the position y of the sum is from ( r + x + y)th bit and the length is
r + n + x + y 1 bits.steps ( 27 ) and ( 28 ) are applied to append the value of positions x and y of the sum of
two points to the last of every strand .
now , the length of every strand in t0 is r + n + x + y + 2n 1 bits with the
value x from the ( r + n + x + y)th bit and
y from the ( r + n + x + y + n)th bit.from addtwonode ( t0 , n , x1 , y1 , x2 , y2 , r ) , it takes o(n ) extract operations , o(n ) append operations , o(n ) merge operations , and o(n ) test tubes to finish the function of a parallel adder for points on elliptic curve .
the algorithm addtwonode ( t0 , n , x1 , y1 , x2 , y2 , r ) can compute the sum of two points on elliptic curve .
step ( 5 ) employs append operation to append m bits 0 to all
strands in tube t0 .
step ( 6 ) to step ( 8) are operations on tube t0 to get
of strands in t0 .
step ( 9 ) to step ( 13 ) are operations on tube
t3 to get of strands in t3 .
step ( 14 ) pours
t0 and t3 to t0 and the length
of every strand in t0 now is r + n + n + m + n + d 1 bits .
step ( 15 ) to step ( 21 ) accomplish to compute the position
x of point of sum and the result is from ( r + x)th bit .
the
execution of steps ( 22 ) to ( 26 ) is used to get the position y of the sum
which equals to (x1 + x3 ) + x3 + y1 . and
now , the position y of the sum is from ( r + x + y)th bit and the length is
r + n + x + y 1 bits . steps ( 27 ) and ( 28 ) are applied to append the value of positions x and y of the sum of
two points to the last of every strand .
now , the length of every strand in t0 is r + n + x + y + 2n 1 bits with the
value x from the ( r + n + x + y)th bit and
y from the ( r + n + x + y + n)th bit . from addtwonode ( t0 , n , x1 , y1 , x2 , y2 , r ) , it takes o(n ) extract operations , o(n ) append operations , o(n ) merge operations , and o(n ) test tubes to finish the function of a parallel adder for points on elliptic curve .
we have constructed the algorithm above for parallel computing the point of the sum of two points .
then , we can solve elliptic curve discrete logarithm problem as follows : consider point p and q are given , and l is what we want to get which matches q = lp .
first , we amplify p into two tubes
and add p in one tube . check if 2p equals to q ; if not , note down the value of
2p and pour two tubes together .
then , amplify the tube into two tubes and add
2p in one tube . check if any point equals to q ; if not , note down the value of
4p and pour two tubes together , or we get the value of l. then , amplify the tube into two tubes and add 4p in one tube , , while this loop executes n times , the value from 1 to
2 for l will have been checked , and the elliptic curve cryptosystem has been broken by
the solved elliptic curve discrete logarithm problem .
this paper is the first effort in literature that
demonstrates that the difficult problem for elliptic curve discrete logarithm
can be solved on a dna - based computer .
while chang 's work makes great progress
in application of dna computing in cryptoanalysis , which is breaking rsa by
factoring integer , this paper proposes application of dna computing in another
popular cryptosystem , ecc , which is more complex and has more challenge in
cryptoanalysis .
though the algorithm is somewhat complex , it takes a series of
steps that is polynomial in the input size , so it is feasible in theory and
inspirits the development of dna computing .
simultaneously , the paper also
shows that humans ' complex mathematical operations can directly be performed
with basic biological operations .
the property for the difficulty of elliptic
curve discrete logarithm is the basis of elliptic curve cryptosystems . however ,
this property seems to be incorrect on a molecular computer .
this indicates
that the elliptic curve cryptosystems are perhaps insecure if the technique of
dna computing is skillful enough to run the algorithms efficiently as discussed
in this paper . | elliptic curve cryptographic algorithms convert input data to unrecognizable encryption and the unrecognizable data back again into its original decrypted form .
the security of this form of encryption hinges on the enormous difficulty that is required to solve the elliptic curve discrete logarithm problem ( ecdlp ) , especially over gf(2n ) , n z+ .
this paper describes an effective method to find solutions to the ecdlp by means of a molecular computer .
we propose that this research accomplishment would represent a breakthrough for applied biological computation and this paper demonstrates that in principle this is possible .
three dna - based algorithms : a parallel adder , a parallel multiplier , and a parallel inverse over gf(2n ) are described .
the biological operation time of all of these algorithms is polynomial with respect to n. considering this analysis , cryptography using a public key might be less secure . in this respect ,
a principal contribution of this paper is to provide enhanced evidence of the potential of molecular computing to tackle such ambitious computations . |
in north american and european studies , cognitive impairment occurs in 40%65% of patients with multiple sclerosis ( ms).1 cognitive decline appears in ms patients in the early stage of the disease and impacts social functioning and employment,2,3 but rarely progresses to dementia.4 attention deficit may be an important indicator of cognitive decline35 and may occur in the early stages of the disease.3 recent magnetic resonance imaging ( mri ) studies revealed that cognitive impairment in ms is dependent on brain atrophy rather than on the total lesion load.68 ms in asians is characterized by a low prevalence rate , a preferential involvement of the optic nerve and the spinal cord , and few plaques in the brain.9 whether asian ms patients with less brain lesion burdens have cognitive impairment and brain atrophy , as is the case with ms patients in western populations , has never been evaluated .
we investigated cognitive impairment in japanese relapsing - remitting ms patients with a focus on attention deficits and correlated them to mri measures of brain atrophy .
of 26 consecutive patients with clinically definite ms10 who attended showa university hospital , 22 patients were classified as having relapsing - remitting ms .
excluding one patient who had a psychiatric illness , we included 21 patients ( six men and 15 women ) in this study .
the study protocol was approved by the institutional ethics committee , and all patients gave written informed consent prior to participation .
the level of physical disability was measured with the expanded disability status scale ( edss).11 edss scores ranged from 1.0 to 5.0 .
significant disabilities were limited to lower limb functions , and all the patients had well - preserved upper limb and visual functions ( edss functional system grade 2 ) .
no patient fulfilled the diagnostic criteria of neuromyelitis optica.12 one patient ( patient 3 ) tested positive for the serum anti - aquaporin 4 channel antibody .
attention deficits were assessed systematically using the clinical assessment for attention ( cat ) developed and standardized by the japan society for higher brain dysfunction .
13 cat assesses attention with respect to reaction time , vigilance , short - term storage capacity , mental tracking ( working memory ) , and complex attention.14 individual tasks include digit and visual tapping spans , a visual cancellation task , an auditory detection task,15 a symbol digit modalities test ( sdmt),16 a memory - updating test,17 a paced auditory serial addition test ( pasat),18 a position stroop test,19 and a continuous performance test ( cpt).20 the digit span test consists of digits forward and digits backward and was given to the subjects in the manner of the wechsler batteries .
the visual tapping span test uses the corsi block - tapping task , and subjects were asked to copy the tapping pattern forward and backward following the examiner s taps in the same prearranged sequence as the digit span test .
the visual cancellation task is a simple test measuring processing speed and accuracy . using a pencil ,
subjects were asked to cross out a target stimulus dispersed within rows of random , interfering stimuli displayed on a sheet .
two sets of the stimulus sequence were used : digits ( part i ) and kana - letters ( part ii ) . the time required to complete the task ( completion time ) and the ratio of correct answers to the total number of stimuli were assessed .
the auditory detection task is a continuous performance test that presents a series of kana - letters read at the rate of one per second on a compact disc .
increasing the phonetic similarity between the target and background stimuli heightens the task difficulty . both the ratio of correct answers and the accuracy ( the number of accurate answers compared to the number of total responses ) were assessed .
the sdmt is a visuographic task that preserves the substitution format of wechsler s digit symbol test but reverses the presentation of the material so that symbols are printed and numbers are written out .
the ratio of completion ( the number of correct answers compared to the number of items ) was assessed .
the memory - updating test requires subjects to listen to strings of digits of unknown length from the subject s perspective and then to recall the three or four most recent digits .
this means that subjects were required to remember the first three or four items presented and then , if there were more than three or four items in a list , to update the contents of their memory by dropping the oldest item and adding the most recent to the string .
the pasat requires the subjects to add 60 pairs of randomized digits so that each is added to the digit immediately preceding it .
the position stroop test uses a japanese - kanji version of the high - mid - low format developed by sohlberg.19 subjects were asked to call out the position of kanji that mean high , mid , or low , instead of their meaning .
one hundred and fourteen items were used in the trials , and were presented in six rows .
the cpt is a computerized vigilance test that presents stimuli briefly and provides reaction times as well as accuracy data . in the simple version
, subjects responded to the digit 7 that appeared briefly in the center of the screen at random intervals . in the x version
, digits appeared in random order , and subjects were asked to respond to every 7 . in the more difficult ( ax ) version , subjects were asked to respond to 7 only if it followed 3 .
each subject s reaction time ( rt ) , ratio of correct answers , and accuracy were assessed for each test .
several institutes associated with the japan society for higher brain dysfunction studied 297 volunteers who had neither a history of brain damage nor significant medical conditions and who ranged in age from 20 to 79 years.13 the mean and standard deviation ( sd ) for each assessment item , except for the cpt , were calculated for patients in each decade of life from their twenties to their seventies , with more than 40 subject numbers for each decade .
the cpt was studied in 109 volunteers ranging in age from 23 to 79 years , and the normal value was calculated for patients in each decade of life from their twenties through their fifties ( n = 52 ) . a z score was calculated for each assessment item for individual patients .
it was derived from the normal value of the corresponding age group and was defined as abnormal if it was < 1.5 sd .
all patients underwent mri of the brain and the spinal cord using a siemens magnetom vision operating at 1.5 tesla ( siemens , erlangen , germany ) .
the brain mr protocol included transverse t2-weighted images [ repetition time / echo time : 3800/96 milliseconds ( ms ) ] with 5 mm thick contiguous slices and coronal fluid - attenuated inversion recovery ( flair ) images ( repetition time / echo time / inversion time : 7000/110/2000 ms ) with 6 mm thick contiguous slices . for the spinal cord mri , t2-weighted , and t1-weighted images were obtained in the sagittal and axial planes .
mri measurements were analyzed by a neurologist blinded to the patients identity using java image software ( sun microsystems , available at : http://rsb.info.nih.gov/ij/ ) .
white matter lesion load was calculated from t2-weighted images using a semiautomated segmentation technique.21,22 the width of the third ventricle ( wtv ) and the bicaudate ratio ( bcr)7,8,23 were measured from coronal flair images in 16 patients and 13 gender- and age - matched controls . to obtain wtv
, a line was drawn through the long axis of the ventricle parallel to the interhemispheric fissure in the section where the third ventricle was most visible .
the width was measured by drawing a second line perpendicular to the first at its midpoint .
the bcr was the minimum intercaudate distance divided by brain width along the same line and was measured in the slice where the heads of the caudate nuclei were most visible and closest to one another .
il ) was used for the analysis . to rank the degree of attention deficits ,
we did not employ crude data of each assessment in the cat but instead used the number of abnormal assessment items to exclude the effects of age .
of 26 consecutive patients with clinically definite ms10 who attended showa university hospital , 22 patients were classified as having relapsing - remitting ms .
excluding one patient who had a psychiatric illness , we included 21 patients ( six men and 15 women ) in this study .
the study protocol was approved by the institutional ethics committee , and all patients gave written informed consent prior to participation .
the level of physical disability was measured with the expanded disability status scale ( edss).11 edss scores ranged from 1.0 to 5.0 .
significant disabilities were limited to lower limb functions , and all the patients had well - preserved upper limb and visual functions ( edss functional system grade 2 ) .
no patient fulfilled the diagnostic criteria of neuromyelitis optica.12 one patient ( patient 3 ) tested positive for the serum anti - aquaporin 4 channel antibody .
attention deficits were assessed systematically using the clinical assessment for attention ( cat ) developed and standardized by the japan society for higher brain dysfunction . 13 cat assesses attention with respect to reaction time , vigilance , short - term storage capacity ,
mental tracking ( working memory ) , and complex attention.14 individual tasks include digit and visual tapping spans , a visual cancellation task , an auditory detection task,15 a symbol digit modalities test ( sdmt),16 a memory - updating test,17 a paced auditory serial addition test ( pasat),18 a position stroop test,19 and a continuous performance test ( cpt).20 the digit span test consists of digits forward and digits backward and was given to the subjects in the manner of the wechsler batteries .
the visual tapping span test uses the corsi block - tapping task , and subjects were asked to copy the tapping pattern forward and backward following the examiner s taps in the same prearranged sequence as the digit span test .
the visual cancellation task is a simple test measuring processing speed and accuracy . using a pencil ,
subjects were asked to cross out a target stimulus dispersed within rows of random , interfering stimuli displayed on a sheet .
two sets of the stimulus sequence were used : digits ( part i ) and kana - letters ( part ii ) .
the time required to complete the task ( completion time ) and the ratio of correct answers to the total number of stimuli were assessed .
the auditory detection task is a continuous performance test that presents a series of kana - letters read at the rate of one per second on a compact disc .
increasing the phonetic similarity between the target and background stimuli heightens the task difficulty . both the ratio of correct answers and the accuracy ( the number of accurate answers compared to the number of total responses ) were assessed .
the sdmt is a visuographic task that preserves the substitution format of wechsler s digit symbol test but reverses the presentation of the material so that symbols are printed and numbers are written out .
the ratio of completion ( the number of correct answers compared to the number of items ) was assessed .
the memory - updating test requires subjects to listen to strings of digits of unknown length from the subject s perspective and then to recall the three or four most recent digits .
this means that subjects were required to remember the first three or four items presented and then , if there were more than three or four items in a list , to update the contents of their memory by dropping the oldest item and adding the most recent to the string .
the pasat requires the subjects to add 60 pairs of randomized digits so that each is added to the digit immediately preceding it .
the position stroop test uses a japanese - kanji version of the high - mid - low format developed by sohlberg.19 subjects were asked to call out the position of kanji that mean high , mid , or low , instead of their meaning .
one hundred and fourteen items were used in the trials , and were presented in six rows . the completion time and the ratio of correct answers were assessed .
the cpt is a computerized vigilance test that presents stimuli briefly and provides reaction times as well as accuracy data . in the simple version
, subjects responded to the digit 7 that appeared briefly in the center of the screen at random intervals . in the x version
, digits appeared in random order , and subjects were asked to respond to every 7 . in the more difficult ( ax ) version , subjects were asked to respond to 7 only if it followed 3 .
each subject s reaction time ( rt ) , ratio of correct answers , and accuracy were assessed for each test .
several institutes associated with the japan society for higher brain dysfunction studied 297 volunteers who had neither a history of brain damage nor significant medical conditions and who ranged in age from 20 to 79 years.13 the mean and standard deviation ( sd ) for each assessment item , except for the cpt , were calculated for patients in each decade of life from their twenties to their seventies , with more than 40 subject numbers for each decade .
the cpt was studied in 109 volunteers ranging in age from 23 to 79 years , and the normal value was calculated for patients in each decade of life from their twenties through their fifties ( n = 52 ) . a z score was calculated for each assessment item for individual patients .
it was derived from the normal value of the corresponding age group and was defined as abnormal if it was < 1.5 sd .
all patients underwent mri of the brain and the spinal cord using a siemens magnetom vision operating at 1.5 tesla ( siemens , erlangen , germany ) .
the brain mr protocol included transverse t2-weighted images [ repetition time / echo time : 3800/96 milliseconds ( ms ) ] with 5 mm thick contiguous slices and coronal fluid - attenuated inversion recovery ( flair ) images ( repetition time / echo time / inversion time : 7000/110/2000 ms ) with 6 mm thick contiguous slices .
for the spinal cord mri , t2-weighted , and t1-weighted images were obtained in the sagittal and axial planes .
mri measurements were analyzed by a neurologist blinded to the patients identity using java image software ( sun microsystems , available at : http://rsb.info.nih.gov/ij/ ) .
white matter lesion load was calculated from t2-weighted images using a semiautomated segmentation technique.21,22 the width of the third ventricle ( wtv ) and the bicaudate ratio ( bcr)7,8,23 were measured from coronal flair images in 16 patients and 13 gender- and age - matched controls . to obtain wtv
, a line was drawn through the long axis of the ventricle parallel to the interhemispheric fissure in the section where the third ventricle was most visible .
the width was measured by drawing a second line perpendicular to the first at its midpoint .
the bcr was the minimum intercaudate distance divided by brain width along the same line and was measured in the slice where the heads of the caudate nuclei were most visible and closest to one another .
spss software ( v 10.0 ; spss inc , chicago , il ) was used for the analysis . to rank the degree of attention deficits
, we did not employ crude data of each assessment in the cat but instead used the number of abnormal assessment items to exclude the effects of age .
the number of low performance items with a z score < 1.5 sd in patients ranged from zero to twelve ( mean , 4.1 ; sd , 3.0 ) .
the number of abnormal items did not correlate with any parameters , including age , duration of illness , number of relapses , edss , or mini - mental status examination ( mmse ) .
with respect to individual assessment items on cat ( table 2 ) , completion time of the visual cancellation task and rts for cpt , pasat , and sdmt were frequently abnormal .
in contrast , digit and tapping spans either forward or backward , the memory updating test , the percentage of correct answers on the visual cancellation , the auditory detection tasks , and cpt , and both the ratio of correct answer and the completion time of position stroop test were relatively spared or impaired in a small number of patients . because a completion time is equivalent to a sum of rts , lengthening of rt was the most prominent abnormality found by cat .
a prolonged completion time for the visual cancellation task was the prevailing abnormality in our patients . however , most patients maintained the normal level of accuracy on this task .
the completion time for the visual cancellation tasks and/or the rt for cpt was prolonged in 14 patients ( 66.7% ) .
cat has an advantage in examining individuals deviations from normal values in each assessment , excluding the effects of age . in cpt
the number of patients with a z score < 1.5 sd was six in the simple version , four in the x version , and three in the ax version ( table 2 ) .
rts for the simple and the x or ax versions correlated positively , but the z score was smaller for the simple version rt compared to the x or ax versions ( figure 1 ) .
the ratio of correct answers and the accuracy of responses were well preserved throughout the cpt .
the number of patients with a z score < 1.5 sd was nine ( 42.9% ) in either version of the pasat , and five ( 23.8% ) in either version of the memory updating task . in each test , the z scores for the two versions correlated with increasing difficulty , and there was no further decline associated with increasing difficulty .
the z score for individual patients was lower for the 2-second interval version than the 1-second interval version for pasat and was lower in the three digits version compared to the four digits version for the memory - updating test ( figure 2 ) .
the achievement rates for sdmt and the completion times for the position stroop test correlated positively to the completion times of the visual cancellation test ( figure 3 ) .
the z scores were lower in the visual cancellation task than in sdmt or the position stroop test
< 1.5 sd was eight ( 38.1% ) for sdmt , and four ( 19.0% ) for the position stroop test .
white matter lesion load ranged from 0.0 to 3.42 ml ( mean , 0.57 ; sd , 0.92 ; table 1 ) .
the mean wtv was greater in patients ( 5.3 mm ; sd , 1.6 ) than in controls ( 4.0 mm ; sd , 1.3 ; p < 0.05 ) .
there was no bcr difference between patients ( mean , 0.14 ; sd , 0.02 ) and controls ( mean , 0.12 ; sd , 0.02 ) . in the patient group
, there was no correlation between the number of low performance items in cat or the z score of individual items and white matter lesion load , wtv , or bcr .
the wtv correlated positively with duration of illness and bcr ( p < 0.05 ) , but not with the number of relapses or white matter lesion load .
the number of low performance items with a z score < 1.5 sd in patients ranged from zero to twelve ( mean , 4.1 ; sd , 3.0 ) .
the number of abnormal items did not correlate with any parameters , including age , duration of illness , number of relapses , edss , or mini - mental status examination ( mmse ) .
with respect to individual assessment items on cat ( table 2 ) , completion time of the visual cancellation task and rts for cpt , pasat , and sdmt were frequently abnormal .
in contrast , digit and tapping spans either forward or backward , the memory updating test , the percentage of correct answers on the visual cancellation , the auditory detection tasks , and cpt , and both the ratio of correct answer and the completion time of position stroop test were relatively spared or impaired in a small number of patients . because a completion time is equivalent to a sum of rts , lengthening of rt was the most prominent abnormality found by cat .
a prolonged completion time for the visual cancellation task was the prevailing abnormality in our patients . however , most patients maintained the normal level of accuracy on this task .
the completion time for the visual cancellation tasks and/or the rt for cpt was prolonged in 14 patients ( 66.7% ) .
cat has an advantage in examining individuals deviations from normal values in each assessment , excluding the effects of age . in cpt
the number of patients with a z score < 1.5 sd was six in the simple version , four in the x version , and three in the ax version ( table 2 ) .
rts for the simple and the x or ax versions correlated positively , but the z score was smaller for the simple version rt compared to the x or ax versions ( figure 1 ) .
the ratio of correct answers and the accuracy of responses were well preserved throughout the cpt .
the number of patients with a z score < 1.5 sd was nine ( 42.9% ) in either version of the pasat , and five ( 23.8% ) in either version of the memory updating task . in each test , the z scores for the two versions correlated with increasing difficulty , and there was no further decline associated with increasing difficulty .
the z score for individual patients was lower for the 2-second interval version than the 1-second interval version for pasat and was lower in the three digits version compared to the four digits version for the memory - updating test ( figure 2 ) .
the achievement rates for sdmt and the completion times for the position stroop test correlated positively to the completion times of the visual cancellation test ( figure 3 ) .
the z scores were lower in the visual cancellation task than in sdmt or the position stroop test
< 1.5 sd was eight ( 38.1% ) for sdmt , and four ( 19.0% ) for the position stroop test .
white matter lesion load ranged from 0.0 to 3.42 ml ( mean , 0.57 ; sd , 0.92 ; table 1 ) .
the mean wtv was greater in patients ( 5.3 mm ; sd , 1.6 ) than in controls ( 4.0 mm ; sd , 1.3 ; p < 0.05 ) .
there was no bcr difference between patients ( mean , 0.14 ; sd , 0.02 ) and controls ( mean , 0.12 ; sd , 0.02 ) . in the patient group
, there was no correlation between the number of low performance items in cat or the z score of individual items and white matter lesion load , wtv , or bcr .
the wtv correlated positively with duration of illness and bcr ( p < 0.05 ) , but not with the number of relapses or white matter lesion load .
using a standardized test that probes various domains of attention , we found that in japanese relapsing - remitting ms patients slowed information processing was prevalent , whereas coherence to the task and accuracy of responses were preserved .
several factors including motor slowing , perceptual deficits , attention deficits , minor cognitive deficits , and overall mental slowing may account for this slowed information processing.24 in our patients , lengthening of the rt in cpt was larger for the simple version than for the tasks that required selective attention ( x and ax versions ) .
this finding suggests that overall mental slowing or minor cognitive deficits are unlikely to be causing the slowed information processing in our patients because disproportionate lengthening of the rt with increasing difficulty has been reported in general slowness or cognitive impairment due to diffuse brain damage.25 we were unable to exclude a slowing of motor acts and visual perceptions in this study , although our patients did not have significant deficits in motor function of the upper limbs or in visual acuity .
however , we do not think that only motor or visual perceptual impairment contributes to the slowing of rt .
tasks that required information processing speed and were examined with auditory perception of stimuli , and verbal output , such as pasat and the memory - updating test , were impaired as well ( figure 2 ) .
kujala et al divided information processing into automatic and controlled processing in ms patients with or without cognitive deficits.5 they found that in cognitively preserved patients , automatic processing that requires relatively effortless recognition of simple visual stimuli was impaired , whereas controlled processing that demands working memory was preserved . in patients with mild cognitive deterioration ,
the results of multiple tasks performed by our patients were comparable to the characteristics of information processing in the cognitively preserved patients . in pasat and the memory - updating task ,
the increasing difficulty of the tasks was not accompanied by a decrease in performance ( figure 2 ) .
the deviations from normal values in sdmt and the position stroop test were smaller than in the visual cancellation task ( figure 3 ) .
the position stroop test requires not only selective attention , but also inhibition / suppression of distracting presentations.26 these results suggest that controlled information processing is preserved in spite of impaired automatic information processing .
although it is difficult to explain the even smaller deviation from normal with increasing difficulty of the task , we hypothesize that the further burden of working memory demands could have evoked the attentional system in our patients . in comparison with western ms patients ,
lesion load in the brain , estimated from the total volume of plaques on mri , was much less than that reported for european patients.21,22 attention deficits were present in patients within months after onset of the clinical disease and were also present in patients who had no visible plaques in their brain .
furthermore , the degree of attention deficits measured by number of abnormal items from cat did not correlate with the duration of illness , physical disabilities , or brain lesion score .
these findings in japanese ms patients with minor brain lesion load support the idea that a neurodegenerative process may be a pathological substrate of the attention deficits in ms.6,27,28 in this study , we did not find any correlation between attention deficits and duration of illness , number of plaques , or mri measures of brain atrophy .
the wtv , one of the indices of brain atrophy,7,8 was increased in patients and correlated positively to illness duration but not to the number of low performance items from cat .
this finding is contrary to previous studies showing a relationship between cognitive function and mr measures of brain atrophy.7,8,22,28 this discrepancy may be due to the fact that the number of subjects examined in this study is low .
the presence of attention deficits early in the disease course of our patients , partly attributable to cat sensitivity , may also contribute to the absence of correlation .
a longitudinal study in individual patients6 or a larger cohort group is required to determine whether attention deficits and brain atrophy correlate in japanese ms patients with minor brain lesion loads .
using a standardized test that probes various domains of attention , we found that in japanese relapsing - remitting ms patients slowed information processing was prevalent , whereas coherence to the task and accuracy of responses were preserved .
several factors including motor slowing , perceptual deficits , attention deficits , minor cognitive deficits , and overall mental slowing may account for this slowed information processing.24 in our patients , lengthening of the rt in cpt was larger for the simple version than for the tasks that required selective attention ( x and ax versions ) .
this finding suggests that overall mental slowing or minor cognitive deficits are unlikely to be causing the slowed information processing in our patients because disproportionate lengthening of the rt with increasing difficulty has been reported in general slowness or cognitive impairment due to diffuse brain damage.25 we were unable to exclude a slowing of motor acts and visual perceptions in this study , although our patients did not have significant deficits in motor function of the upper limbs or in visual acuity .
however , we do not think that only motor or visual perceptual impairment contributes to the slowing of rt .
tasks that required information processing speed and were examined with auditory perception of stimuli , and verbal output , such as pasat and the memory - updating test , were impaired as well ( figure 2 ) .
kujala et al divided information processing into automatic and controlled processing in ms patients with or without cognitive deficits.5 they found that in cognitively preserved patients , automatic processing that requires relatively effortless recognition of simple visual stimuli was impaired , whereas controlled processing that demands working memory was preserved . in patients with mild cognitive deterioration ,
the results of multiple tasks performed by our patients were comparable to the characteristics of information processing in the cognitively preserved patients . in pasat and the memory - updating task ,
the increasing difficulty of the tasks was not accompanied by a decrease in performance ( figure 2 ) .
the deviations from normal values in sdmt and the position stroop test were smaller than in the visual cancellation task ( figure 3 ) .
the position stroop test requires not only selective attention , but also inhibition / suppression of distracting presentations.26 these results suggest that controlled information processing is preserved in spite of impaired automatic information processing .
although it is difficult to explain the even smaller deviation from normal with increasing difficulty of the task , we hypothesize that the further burden of working memory demands could have evoked the attentional system in our patients .
in comparison with western ms patients , our patients were clinically characterized by predominantly spinal cord or brain stem involvement .
lesion load in the brain , estimated from the total volume of plaques on mri , was much less than that reported for european patients.21,22 attention deficits were present in patients within months after onset of the clinical disease and were also present in patients who had no visible plaques in their brain .
furthermore , the degree of attention deficits measured by number of abnormal items from cat did not correlate with the duration of illness , physical disabilities , or brain lesion score .
these findings in japanese ms patients with minor brain lesion load support the idea that a neurodegenerative process may be a pathological substrate of the attention deficits in ms.6,27,28 in this study , we did not find any correlation between attention deficits and duration of illness , number of plaques , or mri measures of brain atrophy .
the wtv , one of the indices of brain atrophy,7,8 was increased in patients and correlated positively to illness duration but not to the number of low performance items from cat .
this finding is contrary to previous studies showing a relationship between cognitive function and mr measures of brain atrophy.7,8,22,28 this discrepancy may be due to the fact that the number of subjects examined in this study is low .
the presence of attention deficits early in the disease course of our patients , partly attributable to cat sensitivity , may also contribute to the absence of correlation .
a longitudinal study in individual patients6 or a larger cohort group is required to determine whether attention deficits and brain atrophy correlate in japanese ms patients with minor brain lesion loads .
japanese relapsing - remitting ms patients with minor brain lesion loads frequently have attention deficits beginning in the initial phase of their disease .
their attention deficits are characterized by a slowness of automatic information processing , which spares controlled information processing . a correlation between cognitive functioning and brain atrophy | backgroundto investigate whether japanese multiple sclerosis ( ms ) patients with minor brain lesion loads have attention deficits and brain atrophy , and to correlate their circumstance.methodtwenty-one japanese patients with relapsing - remitting ms were included in this study .
attention deficits were evaluated using clinical assessment for attention ( cat ) standardized according to age groups .
lesion load in the brain was assessed by tallying the total volume of plaques visible on brain magnetic resonance imaging ( mri ) .
the width of the third ventricle and the bicaudate ratio were measured.resultsthe completion time for the visual cancellation tasks and/or the reaction times for the continuous performance test were prolonged in 14 patients ( 66.7% ) .
the accuracy of responses was preserved throughout the cat .
deviation from the normal value was not exaggerated based on the increasing difficulty of the task .
the total volume of plaques on brain mri was small .
the width of the third ventricle was significantly increased in patients with ms when compared to controls , but was not correlated with the low performance on the cat.conclusionsjapanese ms patients with minor brain lesion loads frequently had attention deficits characterized by slowness of automatic information processing , but controlled processing that requires working memory demands was spared . |
we established and sampled 10 mark - recapture sites within and adjacent to rnbm during 20032007 .
sampling of grids depended upon weather conditions , the purpose of the grid , and transitory human settlements .
each mark - recapture grid consisted of an 11 11 array of trap stations spaced 10 m apart , each of which had 1 standard live trap ( h.b .
sherman traps , tallahassee , fl , usa ) placed on the ground , and another in branches or vines 23 m above ground to capture arboreal species .
grids were sampled for 8 nights , with at least 2 months between sampling sessions .
rodents captured in the mark - recapture grids were individually marked with passive integrated transponder tags , and 100 l of blood was collected from the retroorbital sinus ( once per trapping session ) .
animals were identified to species , age class , reproductive condition , sex , and weight and released .
rodents were also collected in a series of traplines , each of which contained 50 traps placed 10 m apart .
animals collected in traplines were killed , standard collecting information was recorded , and liver , lung , heart , kidney , muscle tissues , and blood specimens were collected .
all samples were snap - frozen in liquid nitrogen , transported to the museum of texas tech university ( ttu ) , and stored at 80c .
standard voucher specimens were prepared from these animals , and have or will be deposited in the museum of ttu or the museo nacional de historia natural del paraguay .
all field protocols followed american society of mammologists guidelines for the use of wild mammals in research ( 6 ) , and were reviewed and approved by the ttu animal care and use committee .
a total of 1,150 small mammals from > 20 species were captured , including 13 sigmodontine rodent species ( 1,140 animals ) ( technical appendix ) .
the dominant rodent species in mbaracay were a. montensis ( 55.7% ) , necromys lasiurus ( hairy - tailed akodont ) ( 10.8% ) , c. callosus ( big laucha ) ( 6.5% ) , and o. fornesi ( fornes colilargo ) ( 6.3% ) .
antibodies to hantavirus antigens were detected in blood specimens by using an indirect immunofluorescent antibody assay and irradiation - sterilized slides of vero e6 cells infected with andes virus as described ( 4 ) .
seven species were antibody positive : a. montensis , n. lasiurus , o. fornesi , o. nigripes , oligoryzomys sp . , oryzomys megacephalus ( azara s broad - headed oryzomys sp . ) , and oxymycterus delator ( paraguayan hocicudo ) .
antibodies to hantavirus antigens were 3 more abundant in blood samples from males than females ( technical appendix ) .
total rna was extracted from antibody - positive blood clots from mark - recapture samples or lung tissue from killed animals . nested reverse transcription
pcr was performed to amplify a 371-nt small ( s ) hantavirus rna segment ( 4 ) .
hantavirus rna was detected in 23 a. montensis , 5 o. fornesi , 1 o. nigripes , and 1 oligoryzomys sp .
of these animals , all but 2 a. montensis were males , which indicated that male rodents play the primary role in maintenance and transmission of hantavirus . a representative sample ( 15 a. montensis , 3 o. fornesi , 1 o. nigripes , and 1 oligoryzomys sp .
pcr - amplified cdnas of a 1,014-nt amino terminus region of the s segment were purified by agarose gel electrophoresis and cloned into pcrii ( invitrogen , carlsbad , ca , usa ) ( 4,5 ) .
m13 forward and reverse primers were used for sequencing . for sequence comparison and phylogenetic analysis ,
phylogeny reconstruction was conducted by using modeltest version 3.6 analysis ( http://darwin.uvigo.es/software/modeltest.html . ) , maximum - likelihood estimation , and bayesian inference ( figure 2 ) .
phylogenetic tree based on bayesian analysis of the small ( s ) segment of american hantaviruses , paraguay , 20032007 .
the tree is based on 1,014 nucleotides of partial s segment of north and south american hantaviruses .
clades ( upper case letters ) , subclades ( numbers ) , and groups ( lower case letters ) are indicated on the left .
alignment and editing of nucleotide sequences were conducted by using vector nti version 10.3.1 ( invitrogen , carlsbad , ca , usa ) .
estimation of the suitable model of nucleotide substitution and phylogenetic analysis were conducted by using modeltest version 3.6 ( http://darwin.uvigo.es/software/modeltest.html ) , paup * 4.0b10 ( http://paup.csit.fsu.edu ) , and mrbayes version 3.1.2 ( http://mrbayes.csit.fsu.edu ) .
two runs of 4 chains each ( 1 cold , 3 heated , temperature 0.20 ) were run for 1 million generations , and trees were sampled every 100 generations .
convergence was assessed by using the average standard deviation in partition frequency values across independent analyses with a threshold value of 0.01 ; burn - in was set to 25% .
nucleotide sequences of specimens collected at reserva natural del bosque mbaracay , departamento canindey , are indicated by acronyms of related viruses , rodent species , and collection numbers .
are aai ( ape aime ) virus from paraguay [ dq345764 ] ; alpa ( alto paraguay ) virus from paraguay [ dq345762 ] ; andv_ah1 ( andes virus , strain ah-1 ) from argentina [ af324902 ] ; andv_chile ( andes virus , strain chile 9717869 ) [ af291702 ] ; andv_nort ( andes virus , strain nort ) from argentina [ af325966 ] ; arav ( araraquara virus ) from brazil [ af307325 ] ; arauv ( araucaria virus ) from brazil [ ay740633 ] ; bayv ( bayou virus ) from the united statesa [ l36929 ] ; bccv ( black creek canal virus ) from the united states [ l39949 ] ; bmjv ( bermejo virus ) from argentina [ af482713 ] ; bmjv - ebu ( bermejo virus , strain eembuc ) from paraguay [ dq345763 ] ; cajuru ( araraquara - like virus , strain cajuru ) from brazil [ ef571895 ] ; catv ( catacamas virus ) from honduras [ dq256126 ] ; cadv ( cano delgadito virus ) from venezuela [ dq285566 ] ; choclo virus from panama [ dq285046 ] ; elmcv ( el moro canyon virus ) from the united states [ u11427 ] ; htn-007 virus from peru [ af133254 ] ; ip37 ( itapa virus , strain 37 ) from paraguay [ dq345765 ] ; ip38 ( itapa virus , strain 38 ) from paraguay [ dq345766 ] ; jabor ( jab ) virus from brazil [ ef492471 ] ; juqv ( juquitiba virus ) from brazil [ ef492472 ] ; lecv ( lechiguanas virus ) from argentina [ af482714 ] ; mulv ( muleshoe virus ) from the united states [ u54575 ] ; lanv ( laguna negra virus ) from paraguay [ af005727 ] ; lscv ( limestone canyon virus ) from the united states [ af307322 ] ; mclv ( maciel virus ) from argentina [ af482716 ] ; maporal virus from venezuela [ ay267347 ] ; mglv ( monongahela virus ) from the united states [ u32591 ] ; nyv ( new york virus ) from the united states [ u47135 ] ; orov ( playa de oro virus ) from the mexico [ ef53407779 ] ; ornv ( oran virus ) from argentina [ af482715 ] ; paranoa virus from brazil [ ef576661 ] ; phv ( prospect hill virus ) from the united states [ u47136 ] ; prgv ( pergamino virus ) from argentina [ af482717 ] ; puuv from finland [ x61035 ] ; riomv ( rio mamore virus ) from bolivia [ u52136 ] ; rios ( rio segundo ) virus from costa rica [ genbank u18100 ( s segment ) ] ; snv_cc107 ( sin nombre virus , strain cc107 ) from the united states [ l33683 ] ; snv_nmr11 ( sin nombre virus , strain nmr11 ) from the united states [ l37904 ] ; and tulv ( tula virus ) from russia [ z30941 ] .
olfo , oligoryzomys fornesi ; olni , o. nigripes ; olsp , oligoryzomys sp . ; akmo , akodon montensis ) . bayesian analysis based on the 1,014-nt sequence showed that all sequences from a. montensis formed a strongly supported clade , which included aaiv - related hantaviruses from itapa department , jabor virus ( jabv ) from southern brazil , and strains from rnbm in paraguay ( figure 2 , clade c1 ) .
phylogenetic analyses identified 3 subclades representing virus sequences from animals in the rnbm , itapa , and southern brazil .
this type of geographic clustering is similar to sin nombre related viruses in deer mice in north america ( 7 ) .
all s segment sequences from a. montensis were closely related , with nucleotide sequence differences between rnbm strains and aaiv and jabv of 4% and 12% , respectively , and derived amino acid differences of 0% or 1% , respectively ( table 1 ) .
* values above the diagonal are percentage nucleotide sequence similarities , and values below the diagonal are percentage amino acid sequence similarities .
lanv , laguna negra virus ; riomv , rio mamore virus ; alpa , alto paraguay virus ; jabv , jabor virus ; aaiv , ape aime virus ; andv , andes virus ; bmjv - nebu , beremjo virus from eembuc ; ipv37 , itapa virus strain 37 ; juqv , juquitiba virus ; prgv , pergamino virus ; arav , araraquara virus .
in contrast , all virus sequences from o. fornesi , o. nigripes , and oryzomys sp . at rnbm
formed a strongly supported clade with viruses related to juquitiba virus ( juqv ) from brazil and itapa virus strain 37 , which was originally detected in o. nigripes from itapa department in eastern paraguay ( figure 2 , clade c2d ) .
nucleotide sequence differences between juqv strains from rnbm and itapa virus strain 37 or juqv ( brazil ) were 5% or 4% , respectively , and derived amino acid differences were 0% ( table 1 ) .
this clade is phylogenetically distinct from viruses that form the akodon
montensis clade at rnbm and more closely related to andes ( clade c2b ) and bermejo - eembuc ( clade c2a ) viruses .
this finding suggests that spillover infection of juqv - related viruses is actively occurring among oryzomyine rodent species at rnbm , as reported for other hantaviruses in oryzomyines ( 8) and other rodent hosts of old world hantaviruses ( 9,10 ) .
additional data are needed to determine the primary oryzomyine reservoir of juqv and to better understand mechanisms by which spillover occurs .
in addition to spillover infection of juqv among oryzomyine rodents , we identified 2 virus strains ( juqv and jabv ) in close proximity ( collected 20 m apart on the same grid in the same sampling session ) on 2 occasions , in sites separated by 30 km ( figure 1 ; table 2 ) .
thus , these 2 distinct hantaviruses appear to be maintaining a sympatric status across a considerable expanse of landscape , rather than reflecting a temporary or localized phenomenon .
we use the term sympatric to underscore that these viruses are in the same community and are near enough ( their rodent reservoirs ) to interact . *
coexistence of hantaviruses in 2 rodent species at mark - recapture sites has been observed ( 1113 ) .
serologic analyses in these studies would not have differentiated whether distinct strains of hantaviruses were co - circulating or active spillover infection was occurring among sympatric rodents at collection sites .
recently , raboni et al . reported juqv circulating in 3 sympatric rodent species in southern brazil and 2 distinct hantaviruses ( jabora and juqv ) in 1 rodent species ( a. montensis ) ( 14 ) .
we have not detected juqv in a. montensis in paraguay . to address host - jumping of hantaviruses among sympatric rodent species in rnbm and other regions in south america ,
such studies are critical to understanding evolutionary adaptation of hantaviruses in rodents in south america , the ability of these viruses to adapt to new rodent reservoirs , and their emergence and maintenance in the environment .
| to explore geographic and host - taxonomic patterns of hantaviruses in paraguay , we established sampling sites in the mbaracay biosphere reserve .
we detected jabor virus and itapa37/juquitiba related virus in locations 20 m apart in different years , which suggested sympatry of 2 distinct hantaviruses . |
self - medication is defined as the consumption of medicinal products with the purpose of treating diseases or symptoms , or even promoting health , without a prescription provided by a medical professional.(12 ) the prevalenceof the practice of self - medication depends on many factors like nature of the disease , educational qualification of the person , nonavailability of the specialized person ( during traveling or such conditions ) , etc . in one study
it was seen that 87% of the chest symptomatics initiated medication on their own for getting relief and this percentage was significantly more in urban people ( 93.9% ) compared to rural subjects ( 80.6%).(3 ) in another survey conducted in hong kong , 97% of the patients practiced self - medication to a variable extent.(4 ) self - medication was found to be significantly low in breast - feeding mothers ( 3.2%).(5 ) inappropriate self - medication results in irrational use of drugs , wastage of resources , increased resistance of pathogens , entails serious health hazards such as adverse reactions and prolonged suffering.(6 ) if action is not taken , the danger of drug interactions and side effects could increase .
safety in self - medication depends on four factors drug itself ( inherent properties of the drug , dose and its duration ) , formulation , information available with all purchases , and patient compliance.(7 ) a number of reasons could be enumerated for the rise of self - medication .
one of the reasons being the increase in chronic diseases and their incidence has raised from 30% to 80% in the last 40 years.(68 ) other reasons which are responsible for self - medication in developing countries are urge of self - care , feeling of sympathy toward family members in sickness , lack of health services , poverty , ignorance , misbeliefs , extensive advertisement , use of drugs from informal sectors such as open markets and quacks , illegal purveyors of drugs ( nonlicensed sellers in the market ) , etc.(68 ) some authors are of the view that self - medication can be practiced and they consider it appropriate for short - term relief of symptoms where accurate diagnosis is unnecessary , uncomplicated cases of some chronic and recurrent disease ( medical diagnosis having been made and advice given).(7 ) however , people should be properly educated about the practice of self - medication in order to prevent the harmful effects caused by the practice .
the increasing self - medication will require more and better education of both the public and health professionals to avoid the complications arising from this practice.(910 ) there has not yet been any systematic research conducted to assess the prevalence of self - medication in the community .
thus self - medication in modern pharmaceuticals seems to be a field in which information is scarce.(8 ) to the best of our knowledge there is no research conducted to reveal the extent of this problem in haryana .
therefore we believe that this research may show the magnitude of problem in haryana so as to initiate intervention by the concerned authorities and the community as well .
the study site is one of the major cities in northern india , i.e. , rohtak .
there is one health university , one civil hospital , many dispensaries , more than 100 pharmacy shops , traditional healers , private and other nongovernmental organizations ( ngo ) clinics .
the houses were selected by systemic random sampling and every fifthhouse was taken for the data collection .
the collected variables includedsociodemographic data , economical condition of the house , questions on self - medication such as which medicines were kept in the house , any specific place for keeping the medications , whether they were kept in a classified manner or not , how often self - medication was practiced , cautions taken during self - administration of the drugs , and use of other type of medications other than allopathy .
data were collected from the lady of the house and one housewife was selected for interviewing from each house .
five trained candidates , post - mbbs residents , were trained to collect the data which were authenticated by the investigators .
data were entered into the computer with statistical package for social sciences ( spss ) software and were analyzed usingthis software .
the study site is one of the major cities in northern india , i.e. , rohtak .
there is one health university , one civil hospital , many dispensaries , more than 100 pharmacy shops , traditional healers , private and other nongovernmental organizations ( ngo ) clinics .
the houses were selected by systemic random sampling and every fifthhouse was taken for the data collection .
the collected variables includedsociodemographic data , economical condition of the house , questions on self - medication such as which medicines were kept in the house , any specific place for keeping the medications , whether they were kept in a classified manner or not , how often self - medication was practiced , cautions taken during self - administration of the drugs , and use of other type of medications other than allopathy .
data were collected from the lady of the house and one housewife was selected for interviewing from each house .
five trained candidates , post - mbbs residents , were trained to collect the data which were authenticated by the investigators .
data were entered into the computer with statistical package for social sciences ( spss ) software and were analyzed usingthis software .
a total of 132 houses were surveyed , out of which proformas were filled by 100 housewives .
ninety - seven percent were found to be having drugs in their house but only 73% practiced self - medication .
education levels of the housewives were as follows : illiterate 9% , under matriculate 4% , matriculate
all used the allopathic system of medicine ; the other systems used were homeopathic 27% , and ayurvedic 21% .
advertisement on newspaper and television 26% , friends 17% , and others 8% .
antacids 44% , antibiotics 33% , nutritional supplements 23% , cough syrups 16% ,
only 80% housewives were in the habit of checking the expiry date before self - medication .
the correct indications of the stored drugs were known to 28% and partial indications to 53% . however 19% of them had wrong impression about the indication of the stored drugs .
the instructions by the physicians regarding dose , frequency , and duration of treatment were strictly adhered to by 41% housewives .
only 49% were aware of the fact that the drugs can cause adverse effects if taken without medical advice .
seventy - one percent of the housewives were of the opinion that self - medication is not justified but felt that it had to be continued because of temporal or financial constraints .
education levels of the housewives following the practice of self - medication common medications used in the practice of self - medication
self - medication is a significant problem in relation to the rational use of drugs and unwanted adverse effects caused by the drugs .
the prevalence of self - medication in this study was found to be 73% compared to that of 27.6% in a study done in jimma town,(11 ) 30% in a study done in mexico,(10 ) and 97% in a study conducted in hong kong.(4 ) one of the studies conducted in the portugese people showed this prevalence to be 26.3%.(12 ) it was also found that prevalence of self - medication was found to be higher in well - educated patients compared to the illiterate or people with low education .
one of the studies published by an indonesian journal also highlighted the fact that education level is the dominant factor for the practice of self - medication.(13 ) a majority of the patients practiced self - medication based on their previous prescription .
in addition to this , friends also constituted 17% of the proportion who guided this irrational practice . in china the most common reason for self - medication
was found to be that people thought they know much to take care of themselves.(11 ) in our study , we found that 71% of the subjects practiced self - medication because they felt that going for repeated consultations to doctor put financial restraint on their budget and rest of them felt that they did not have sufficient time to consult the doctor .
so , they consulted the physician only when the condition of the patient was serious enough or was not relieved by the self - medication . in one of the studiesthe patient 's own knowledge and suggestions from family and
friends contributed the same percentage ( 40% ) while the salesman influenced the decision in 20% of the patients.(4 ) comparing the results of this study with the various studies done , it was found that the results are not in accordance with the various studies being done .
the most commonly used medication in this study was found to be analgesics followed by antacids , antibiotics , nutritional supplements , cough syrups , and antispasmodic .
the commonest conditions that led to self - medication in the study done by worku , et al . were headache , fever , cough , and diarrhea.(11 ) similar results were also seen in the studies done in brazil and france . in one of the studies conducted in the hong kong the reasons for self - medication included the conditions like musculoskeletal pain , minor burns or bruises , gastrointestinal upset , headache , sore throat , skin problems , cough , and dyspepsia.(4 ) in one of the studies conducted in the portugal
it was found that the major preparations used for self - medication included throat preparations , cough and cold preparations , antibiotics and corticosteroids for local oral treatments , laxatives , analgesics , dermatological preparations , vitamins , mineral supplements , and other alimentary tract products.(12 ) in addition to allopathy , this study showed that 27% of the patients practiced homeopathy and 21% of the patients favored herbal drugs . in a study done by hilary and his colleagues , it was found that the most commonly used complementary and alternative modalities in 2002 were herbal therapy ( 18.6% ) followed by relaxation techniques ( 14.2% ) and chiropractic ( 7.4%).(14 ) herbal medicines were the most frequently used medicines ( 51.8% ) amongst the nigerian people.(15 ) one of the studies showed that 25% of the people used herbal drugs and 12.5% of the patients of the people practiced homeopathy.(16 ) also it was seen in this study that a large number of people were not aware of the potential damaging nature of the different medications self - administered by them .
it was quoted in that study : there is an alarming deficiency in the patients knowledge of the possible side - effects of selfmedication ; they should therefore be warned about the danger.(4 ) however some authors consider that self - medicationis appropriate.(7 ) also world health organization is nowadays promoting the self - medication for trivial diseases to save the time and resource wasted in treating the minor diseases.(8 ) in this study along with studying the self - medication patterns and drug use behavior in housewives , some guidelines were also given for the proper use of medicines and their storage and these guidelines are provided as appendix 2 .
this study shows that self - medication is quite prevalent among the housewives belonging to the middle income group and that most of them are educated too .
a majority of housewives are not aware of the fact that adverse effects can occur if medication is taken without doctor 's advice and it is necessary to check the expiry date of medicine .
so more and better education of both the public and health professionals is required to avoid complications arising from this practice .
this study has been conducted on a small group of people , but provides the useful information regarding the self - medication patterns in the community belonging to the middle income group .
this study has been conducted on a small group of people , but provides the useful information regarding the self - medication patterns in the community belonging to the middle income group . | objectives : the objective was to assess the self - medication patterns and drug use behavior in housewives belonging to the middle income group in a city of haryana state in northern india.materials and methods : a detailed questionnaire designed to assess the self - medication patterns and drug use behavior and interview technique was used to elicit the requisite information .
one hundred housewives of the middle income group were interviewed in rohtak.results:most of the housewives were in the habit of keeping the medicines though only 73% of them were in the habit of using it without any prescription . also it was seen that those housewives who were taking self - medication were better educated than those not indulged in self - medication .
all of them were using allopathic drugs on a regular basis while other modes of medications were less used .
the self - medication was most commonly based on the previous prescriptions issued by the doctors followed by the suggestions from friends , advertisement on the television , and newspapers . for most of them the reasons for self - medication were financial restraints and lack of time to go to the medical practitioner.conclusions:the study delineates the difference in the self - medication patterns and drug use behavior in housewives in a city of northern india .
the results emphasize the need for comprehensive measures for intervention strategies to promote rational drug therapy by improving prescribing patterns and influencing self - medication . |
obesity is understood to be the result of complex socioecological factors and interventions that target multiple levels of the socioecological framework , including individual , social , built environment , and policy factors , are more likely to be successful [ 13 ] .
however , the effectiveness of civic engagement projects and/or coalition work to address obesity is difficult to measure , primarily because goals and activities differ between communities . in
a comprehensive review examining community coalitions focused on a range of health promotion initiatives , zakocs and edwards report inconsistent results when examining factors associated with coalition effectiveness ; some factors associated with success were group cohesion , leadership , agency collaboration , member participation , and diversity .
similarly , roussos and fawcett reported that while findings are insufficient to make strong conclusions , community - level coalitions aimed at changing systems and/or behavior hold potential in affecting population - level health outcomes .
other research indicates that individuals engaged in community coalitions perceived benefits including improved physical fitness , nutrition , self - confidence , self - esteem , sense of personal empowerment , and social relationships .
overall , the literature suggests that civic engagement is appropriate for initiating changes that can improve community health , but there is still much to be learned .
the socioecological model was the foundational theory for the design of the strongwomen change club ( swcc ) project 's curriculum which aimed to engage individuals to identify an issue relevant to them in their community context and facilitate implementation of an action plan that would affect social , cultural , environmental , and political factors within each of their seven communities .
the aim of this paper is to describe the processes by which the research team ( ras , scf , ms , men , ey , mf , and bj ) engaged community partners ( each swcc and its leader ) to improve their nutrition and physical activity environments . we provide a starting point for understanding best practices related to civic engagement as a means of creating communities in which the healthy choice is the easier choice .
this study was derived from a partnership of approximately ten years of collaboration between researchers at tufts university 's strongwomen program ( swp ) and community health educators / leaders with the national institute of food and agriculture ( nifa ) cooperative extension programs [ 811 ] .
the research team designed the swcc project and supporting curriculum as a vehicle for an academic - community civic engagement partnership focused on creating local change in active living and healthy eating .
the research team partnered with nifa cooperative extension educators , who are active and well - connected local leaders , to most effectively identify both relevant community issues and to realistically assess community resources to best facilitate interventions .
we chose existing strongwomen exercise groups as the partner for the swcc initiative because they demonstrated a baseline level of interest and readiness to engage in issues of community health and wellness . to identify the seven communities , we worked with state level strongwomen ambassadors and focused on finding small communities ( ideally , fewer than 15,000 residents ) where there was a strongwomen leader ( all cooperative extension agents ) who would be interested and willing to serve as a swcc project leader . once identified
, we corresponded with these leaders over several months to prepare for in - person workshops by outlining expectations , starting to discuss the community 's particular challenges , identifying potential community stakeholders , and providing guidance on formation of the change clubs .
the swcc was defined as a group of approximately 815 committed community members , often current and former swp participants , who were interested in undertaking a project to create positive environmental change relevant to nutrition or physical activity within their respective communities .
study procedures were reviewed and approved by the institutional review boards at fred hutchinson cancer research center and tufts university ( note : ras was formerly of tufts university and is now at cornell university ) . written consent for the survey was obtained by swcc leaders and participants at the in - person workshop ; oral consent was obtained by phone during follow - up interviews .
individuals did not receive a research stipend / participant fee , but each swcc group was provided $ 1,000 to help support their group 's identified issue of focus .
as a first step , leaders received a formal introduction packet that outlined the roles and responsibilities of the leader , the change club rationale and purpose , tentative timeline and schedule , and sample agenda for the three - day workshop that would be conducted at each site .
get to know your town interview conducted via telephone by a research team member ( men , ey , and bj ) , designed to illuminate potential barriers and facilitators to healthy eating and/or physical activity within each town , including features such as the presence or lack of sidewalks and crosswalks or the availability of fast food versus fresh produce .
swcc leaders also received a packet of information on recruiting members , holding interviews with community stakeholders , holding the first meeting , and choosing their area of change / focus . for a period of eight weeks
, two or more members of the research team ( rs , ey , men , and sf ) traveled to seven small communities located in alaska , arkansas , kansas , missouri , montana , pennsylvania , and wisconsin .
the research team facilitated interactive workshops with the swcc leader and all participants in each community that lasted three to four days .
the researchers ' primary role during these visits was to facilitate a structured planning and visioning process and guide team building exercises with the newly formed swcc . during the workshops
, researchers provided educational materials and conducted awareness activities focusing on food and physical activity environments , personal habits , and potential areas for improvement .
each swcc defined an overarching project goal ( called a noble purpose ) , identified strategic community stakeholders , and developed action steps .
once this visioning process was complete , researchers assisted swcc members in establishing specific , measurable benchmarks for goal attainment in the coming year .
the swcc curriculum , developed by the research team , guided each step in the process .
the year following the workshops represented the implementation and follow - up period for the swccs , during which the research team was minimally involved in the interventions .
researchers conducted brief monthly calls with the swcc leader over six months to provide guidance and problem solving as necessary .
a research coordinator conducted an hour - long key informant interview with the swcc leader .
the purpose of the interview was to ( 1 ) gauge the groups ' progress on individual quantitative benchmarks , ( 2 ) understand any unintended consequences or ripple effects in the community as part of this project , and ( 3 ) to assess the leaders ' self - efficacy and feelings of empowerment as community change agents as a result of this project .
the second round of outcome assessments was conducted at 12 months post - visit , again using telephone interviews .
in addition to being asked to rate progress on each benchmark using a 5-point scale ranging from did not accomplish to successfully accomplished , these interviews also captured more extensive qualitative data on leaders ' perceptions of the implementation process , overall perceptions of success , and facilitators and barriers to achievement . by way of summary ,
( i ) achievement of benchmark(s )
example question . in your opinion , has the change club accomplished ( this ) benchmark ?
what have been the major barriers that your change club has experienced when engaging in this type of work ?
please tell me about any other activities or actions ( ripple effects ) that you can think of that have happened since we left your town ?
( v ) leader 's perception of ability to effect change using the change club model
example question . on a scale of 0 to 10 , where 0 to 2
stand for none or a little and 8 to 10 stand for a great amount , to what extent do you consider yourself a change agent role model for others ?
transcript files were entered and coded using nvivo ( qsr international , version 9.0 , 2011 ) , led by ms with a subset of double coding and agreement checks between ms and sf .
manifest content analysis was used to analyze the data , employing the following process : verbatim transcription ; transcription review ; development of and connection between codes and themes ( based upon the main interview topics ) ; and data interpretation and review by the research team in an iterative process .
each swcc initially included 815 female participants ( n = 107 ) , mean age 59 years , sd 14 ( range = 2285 years ) . demographic characteristics of swcc participants are shown in table 1 .
each swcc collaboratively identified a noble purpose goal ( table 2 ) to work toward .
childhood obesity emerged as a primary concern for most of the groups , although each swcc tackled this issue with a different focus .
two communities worked on the food environment , one worked on the physical environment , and four were focused on both .
many swccs also identified the need for better education and support for total community health .
each community was asked to assess its own capacity and resources as part of the benchmark development process .
benchmarks ranged from conducting strategic reviews of available local physical activity resources to changing the food menus at local community events .
some swccs identified the need for education as paramount , while others focused on creating new or improving existing infrastructure .
all communities established at least three benchmarks , and some communities established as many as six .
quantification of benchmark achievement was assessed during the follow - up interviews , and results are shown in table 3 .
several of the key themes discussed in the follow - up interviews with swcc leaders were related to the process in which each group employed to accomplish benchmarks and noble purposes .
for example , community and/or stakeholder involvement was crucial to implementation of all swcc projects .
multiple swccs needed to collaborate with school officials to work toward their noble purpose , which often involved changing school nutrition policies.i'm working with the food service director , the wellness coordinator , a p.e . teacher and the health department .
( wisconsin leader ) i 'm working with the food service director , the wellness coordinator , a p.e .
( wisconsin leader ) leaders talked about the benefits of working together to accomplish goals they would not have attempted or achieved individually.our strongwomen change club was excited and enthusiastic and wanted to be part of the change in the community . and they feel good about what they 've done .
( pennsylvania leader ) our strongwomen change club was excited and enthusiastic and wanted to be part of the change in the community .
( pennsylvania leader ) leaders also mentioned the waning commitment of swcc members regarding potentially working on the project beyond the year they had committed as well as having participation gaps in the process .
we pulled these people from the current strongwomen program and took them a step further in their commitment to wanting to improve the nutrition in the community .
they 've stuck with us , but i also think that we did not intend to carry this project on for an extended period . you know , we told them that we were doing this for a year .
( pennsylvania leader ) we pulled these people from the current strongwomen program and took them a step further in their commitment to wanting to improve the nutrition in the community .
they 've stuck with us , but i also think that we did not intend to carry this project on for an extended period . you know , we told them that we were doing this for a year .
( pennsylvania leader ) swccs were interested in making the community aware of their project .
some swcc enjoyed the support of local media in increasing community awareness of their swcc 's activities and goals . in the first six months ,
seven out of eight communities self - reported that they had successfully accomplished their first benchmark ; some also reported partial success on additional benchmarks . at the 12-month assessment ,
all communities reported success on their first benchmark ; all but one community reported at least partial success on additional benchmarks ( see table 2 ) .
a strong theme in the post - interview was that in addition to their progress in achieving benchmarks , clubs had realized success in building awareness of the food and physical activity environments within communities.it had all those problems , but yet all those people came together because of the change club - bringing it to their attention we feel of anything that we 've done all year long that is our greatest accomplishment .
( alaska leader ) it had all those problems , but yet all those people came together because of the change club - bringing it to their attention we feel of anything that we 've done all year long that is our greatest accomplishment .
( alaska leader ) swcc members , teachers , and children were mentioned specifically as changing or thinking about changing behaviors after becoming aware of swcc activities and problems with the local food and physical activity environment.multiple teachers have come up to me about active classrooms , about concessions , about the school wellness policy , about research on recess , so i think there 's a lot more open communication and you can kind of tell the teachers are really gon na be engaged and i think i did n't have access to that before . (
wisconsin leader ) multiple teachers have come up to me about active classrooms , about concessions , about the school wellness policy , about research on recess , so i think there 's a lot more open communication and you can kind of tell the teachers are really gon na be engaged and i think i did n't have access to that before .
( wisconsin leader ) leaders indicated that being viewed by the community as a group that was accomplishing change helped spur further change.the change club is viewed aspeople who can get things done .
( alaska leader)networking and that professional connection - making is huge and it takes a long time to lay that groundwork but it 's hugely beneficial once you 're no longer sort of just this side group , but this group that has gotten something accomplished and that has been wonderful to work with and i think that 's not to be underestimated .
( alaska leader ) the change club is viewed aspeople who can get things done .
( alaska leader ) networking and that professional connection - making is huge and it takes a long time to lay that groundwork but it 's hugely beneficial once you 're no longer sort of just this side group , but this group that has gotten something accomplished and that has been wonderful to work with and i think that 's not to be underestimated .
( alaska leader ) difficulties included lack of buy - in , limited capacity , slow pace of community change , and waning interest of club members .
buy - in of key people in institutions or from the community in general may be necessary for successful attainment of swcc goals .
change clubs experienced difficulty when there was a lack of support , commitment , or interest from people or groups necessary to implement new
projects.we had a hard time working with the food director within the school district because she was very resistant to change .
( kansas leader)i've been in city council about three times and had asked to be on a committee to discuss it and the person in charge of that said that they had bigger fish to fry .
( pennsylvania leader ) we had a hard time working with the food director within the school district because she was very resistant to change .
( kansas leader ) i 've been in city council about three times and had asked to be on a committee to discuss it and the person in charge of that said that they had bigger fish to fry .
( pennsylvania leader ) when there was a personnel change in key positions , new relationships had to be established before proceeding with project objectives.there is a new director for the program this year
so , we 've kind of had to not start totally fresh , but in a sense we have .
just to kind of start our relationship with the new person and get a feel for what she would like us to do and what she does not want us to do .
so , we 've kind of had to not start totally fresh , but in a sense we have .
just to kind of start our relationship with the new person and get a feel for what she would like us to do and what she does not want us to do .
( kansas leader ) another barrier to successful achievement of benchmarks was the limited capacity of teachers and swcc leaders to devote time to new projects due to existing job duties or other commitments.we can not educate the teachers anyways because they 're already overwhelmed .
( wisconsin leader ) we can not educate the teachers anyways because they 're already overwhelmed .
( wisconsin leader ) some change club leaders mentioned program implementation taking longer than they initially anticipated.it was like an overwhelming thing to do at first , but it 's exciting and we haven't let our sight wane at all . in the next two to five years , you will be buying a plane ticket to come back ( to see the project completed ) .
( missouri leader ) it was like an overwhelming thing to do at first , but it 's exciting and we haven't let our sight wane at all . in the next two to five years , you will be buying a plane ticket to come back ( to see the project completed ) .
the centers for disease control , world health organization ( who ) , and institute of medicine all recognize and recommend environmental change as a key factor in making the healthy choice the easy choice or the default choice [ 3 , 13 , 14 ] .
the who recommends community participation , empowering communities to set priorities , and implements strategies to achieve better health , noting that community participation can draw on the energy and enthusiasm that exist around specific community needs . the swcc project implemented a novel approach to community - based health interventions by employing the change club model in seven rural communities across the united states .
local cooperative extension leaders were successful in creating groups of 815 women to participate in a local change project .
all groups experienced a degree of benchmark success and many leaders expressed increased awareness and feelings of empowerment .
however , it should be noted that during each of the initial swcc workshops , it was clear to the research team that some groups appeared to be further along in terms of readiness to implement change . as this was not a formal component of the evaluation , no correlation between that assessment and benchmark achievement was examined , but that would be important for future studies .
leaders that identified their benchmarks as successful or partially successful indicated that community / stakeholder engagement , swcc dynamics , and community outreach were instrumental to success .
engagement in local politics was a recurring , key theme with leaders recognizing that key community leaders , staff , and boards ( e.g. , public works , school administrators ) are often instrumental to implementing true socioecological changes .
but such entities , or their policies and procedures , are not necessarily well - suited to rapid change .
thus , many groups found it difficult to keep members fully engaged beyond a year because of the substantial time and energy required for this work .
also , it may be valuable to establish short - term goals ( e.g. , start a bicycle education program ) and longer - term targets ( install bike route signage and roadway markings ) to maintain member energy and enthusiasm beyond the initial undertaking .
first , in this project we wanted an in - depth understanding of the processes , facilitators , and barriers , and assessments were largely qualitative .
it may be helpful to assess intermediate factors such as psychosocial markers ( e.g. empowerment , self - efficacy ) and whether these can be linked to community factors and related outcomes
. it would be especially valuable to explore specifically how community groups can effectively influence local elected and appointed leaders , staff , and policies .
however , the findings from this study can be used to help inform quantitative assessment in future studies .
in addition , while this study included communities from across the u.s . and thus provides some degree of geographic diversity , the focus was on rural communities and it is a relatively small sample size .
future studies should focus on adapting the change club model for micropolitan and metropolitan areas . with learning from additional outcome assessments , as well as implementation of the change club in more diverse settings
, a subsequent step will be to consider sustainability of change club groups and mechanisms for curriculum dissemination on a broader scale .
overall , these findings are consistent with previous studies that found leveraging strong local leaders to be important to the success of health and wellness projects . the barriers identified by swcc leaders were also consistent with barriers to obesity prevention and reversal highlighted by the socioecological model , specifically social and environmental supports for behavior change [ 17 , 18 ] .
this project illustrates that it is possible for change clubs to implement local health and wellness projects based on a visioning and planning process facilitated by an academic partner . | introduction .
the epidemic of obesity is a multifaceted public health issue .
positive policy and environmental changes are needed to support healthier eating and increased physical activity .
methods .
strongwomen change clubs ( swccs ) were developed through an academic - community research partnership between researchers at cornell university and tufts university and community partners ( cooperative extension educators ) in rural towns in seven u.s .
states .
extension educators served as the local leader and each recruited 1015 residents to undertake a project to improve some aspect of the nutrition or physical activity environment .
most residents had limited ( or no ) experience in civic engagement . at 6 and 12 months after implementation , the research team conducted key informant interviews with swcc leaders to capture their perceptions of program process , benchmark achievement , and self - efficacy . results . at 12 months , each swcc had accomplished one benchmark ; the majority had completed three or more benchmarks .
they described common processes for achieving benchmarks such as building relationships and leveraging stakeholder partnerships .
barriers to benchmark achievement included busy schedules and resistance to and slow pace of change .
conclusion .
findings suggest that community change initiatives that involve stakeholders , build upon existing activities and organizational resources , and establish feasible timelines and goals can successfully catalyze environmental change . |
the human immunodeficiency virus ( hiv ) pandemic continues to be a major public health threat in the world . since its recognition , hiv has claimed > 34 million lives worldwide and ~36.9 million people were living with hiv by the end of 2014.1 sub - saharan africa accounts for 70% of the world hiv / aids infections and deaths .
ethiopia is one of the sub - saharan african countries most affected by the hiv pandemic .
the adult prevalence of hiv was estimated to be 0.2% in 1985 , 3.2% in 1995 , and 4.4% in 2003.24 trend analysis of national hiv prevalence rate reveals a gradual rise until the late 1990s and a decline after 2000.25 in 2014 , the number of people living with hiv was estimated to be 769,600 , with 15,700 new hiv infections and 35,600 aids - related deaths.6 according to this report , annual new hiv infections and aids - related deaths have been declined by 90% and 53% , respectively , between 2000 and 2011 , due to expansion of hiv counseling and testing , care , and treatment services . despite the significant drop of hiv prevalence in the country , studies show that there is remarkable heterogeneity with geographic regions , study populations , and the period of study.711 further , factors that sustain disparities in hiv prevalence and fueling the epidemic such as high - risk sexual behaviors , low education , limited access to health service , the social stigma surrounding hiv , and poverty are still highly prevalent among the population of ethiopia.1214 furthermore , many of hiv - infected people do not know about their status , and a significant number of the population remain uninformed or misinformed about the cause , transmission , and preventive measures.1517 therefore , there is a pressing need for continuous monitoring and analysis of existing data to generate pragmatic evidence to explain the basis of the prevalence and variations .
such strategies are useful for identification of more vulnerable population groups and to design interventions focusing on those who are at more risk of acquiring hiv infection .
hiv counseling and testing is the key entry point to prevention , care , treatment , and support services , where people learn their hiv status and its implications to make informed decisions about their health.8,12,15 voluntary counseling and testing ( vct ) , provider - initiated testing and counseling , and mandatory hiv screening are the three types of hiv tests being used in ethiopia.18 vct continues to be a cornerstone for hiv prevention and care services in the country .
it increases clients perception of their vulnerability to hiv , promotes behavioral changes , facilitates early referral for care and support , and assists in reducing social stigma .
furthermore , data from vct clients have been used as important sources of information for estimating hiv prevalence and determinants of the epidemic.18,19 although information on hiv prevalence among different segments of population is available,812 data on prevalence of this infection among vct clients from the gondar area are scarce .
thus , we sought to fill this gap by updating the prevalence of hiv infection among vct clients who attended the university of gondar teaching hospital in 3 consecutive years and to identify the sociodemographic determinants for hiv seropositivity .
a retrospective study was conducted at the university of gondar teaching hospital , gondar , northwest ethiopia , to determine the prevalence of hiv infection and the associated risk factors among vct clients between september 2007 and august 2010 .
gondar is located at 737 km from the country s capital addis ababa in north gondar zone of the amhara regional state .
the town has latitude and longitude of 120361n and 370281e , respectively , and an elevation of 2,133 m above sea level .
gondar is a capital of north gondar zone with urban population of ~227,100 in 2007.20 the university of gondar hospital is a tertiary - level teaching and referral hospital with 450 beds for inpatients and provides referral health services for > 5 million inhabitants in northwest ethiopia .
the hospital also has a vct clinic , where clients from gondar town and surrounding areas receive vct for hiv free of any charges .
hiv infection was detected by rapid hiv test kits as per the national algorithm for hiv testing .
briefly , sera prepared from venous blood / whole blood were screened for hiv using a khb diagnostic kit ( shanghai kehua bio - engineering co. ltd , shanghai , people s republic of china ) .
reactive samples were confirmed by stat - pack ( chembio diagnostics , medford , ny , usa ) and uni - gold kits ( trinity biotech , wicklow , ireland ) according to the national algorithm for hiv testing .
data were collected from the vct registration book . data with missing values with respect to the study variables were omitted .
sociodemographic variables such as age , sex , residence , education status , and clinical characteristics such as hiv serostatus were collected .
data analysis was performed using spss version 20 statistical package ( ibm corporation , armonk , ny , usa ) .
data were summarized using frequencies and proportions to describe the study population in relation to relevant variables .
the degree of association between independent and dependent variables was assessed using odds ratio ( or ) with 95% confidence interval ( ci ) .
ethical clearance for this study was obtained from the school of biomedical and laboratory sciences ( sbmls ) , university of gondar .
a permission letter was also obtained from the hospital laboratory head prior to data collection .
the ethics review committee of sbmls did not require written informed consent to be obtained from all patients , as this was retrospective study , and all data was anonymous and handled confidentially .
a retrospective study was conducted at the university of gondar teaching hospital , gondar , northwest ethiopia , to determine the prevalence of hiv infection and the associated risk factors among vct clients between september 2007 and august 2010 .
gondar is located at 737 km from the country s capital addis ababa in north gondar zone of the amhara regional state .
the town has latitude and longitude of 120361n and 370281e , respectively , and an elevation of 2,133 m above sea level .
gondar is a capital of north gondar zone with urban population of ~227,100 in 2007.20 the university of gondar hospital is a tertiary - level teaching and referral hospital with 450 beds for inpatients and provides referral health services for > 5 million inhabitants in northwest ethiopia .
the hospital also has a vct clinic , where clients from gondar town and surrounding areas receive vct for hiv free of any charges .
hiv infection was detected by rapid hiv test kits as per the national algorithm for hiv testing .
briefly , sera prepared from venous blood / whole blood were screened for hiv using a khb diagnostic kit ( shanghai kehua bio - engineering co. ltd , shanghai , people s republic of china ) .
reactive samples were confirmed by stat - pack ( chembio diagnostics , medford , ny , usa ) and uni - gold kits ( trinity biotech , wicklow , ireland ) according to the national algorithm for hiv testing .
data were collected from the vct registration book . data with missing values with respect to the study variables were omitted .
sociodemographic variables such as age , sex , residence , education status , and clinical characteristics such as hiv serostatus were collected .
data analysis was performed using spss version 20 statistical package ( ibm corporation , armonk , ny , usa ) .
data were summarized using frequencies and proportions to describe the study population in relation to relevant variables .
the degree of association between independent and dependent variables was assessed using odds ratio ( or ) with 95% confidence interval ( ci ) .
ethical clearance for this study was obtained from the school of biomedical and laboratory sciences ( sbmls ) , university of gondar .
a permission letter was also obtained from the hospital laboratory head prior to data collection .
the ethics review committee of sbmls did not require written informed consent to be obtained from all patients , as this was retrospective study , and all data was anonymous and handled confidentially .
a total of 2,120 clients data were retrieved from vct registries of the university of gondar teaching hospital . of the 2,120 vct clients , 1,093 ( 51.6% ) were males and 1,027 ( 48.4% ) were females , giving a male - to - female ratio of 1.06:1 .
the median age of the study subjects was 23.0 years ( interquartile range 10 ) .
1,188 ( 56% ) of the clients were from urban areas ; and > 80% of the participants had elementary education and above . in terms of their marital status , 1,334 ( 62.9% )
were never married , followed by married ( 21.2% ) , divorced ( 13.4% ) , and widowed clients ( 2.4% ) . a little over one - third of the vct clients ( 36.7% ) were students , 316 ( 14.9% ) were daily laborers , and 282 ( 13.3% ) were government employees ( table 1 ) .
overall , 17.1% ( 363/2,120 ) of the study participants were found to be hiv positive ( table 2 ) .
higher rates of hiv positivity were observed among female clients ( 20.4% ) than males ( 14.0% ) ( or 1.58 , 95% ci 1.261.98 , p=0.00 ) .
the prevalence of hiv was higher among the participants in the age group of 3549 years ( 35.5% ) , followed by > 50 years ( 35.3% ) and 2024 years ( 22.7% ) . in terms of their marital status ,
higher rates of hiv positivity were observed among widowed ( 64.7% ) and married participants ( 30.2% ) .
the prevalence of hiv among vct clients showed an inverse relationship with their education level , with a higher hiv rate among illiterates ( 28.0% ) than among educated ones .
with respect to occupations of the clients , higher hiv rates of positivity were detected among merchants ( 27.4% ) , daily laborers ( 26.3% ) , and housewives ( 25.7% ) compared to that among other occupations .
year - specific hiv - positive rates were 25.2% , 15.5% , and 14.9% in the years 2007/2008 , 2008/2009 , and 2009/2010 , respectively .
bivariate analysis revealed that widowed ( 95% ci 10.4234.92 , p=0.00 ) , married ( 95% ci 3.425.94 , p=0.00 ) , divorced ( 95% ci 2.795.32 , p=0.00 ) , and illiterate clients ( 95% ci 2.335.47 , p=0.00 ) were associated with hiv infection , with the odds ratios of 19.07 , 4.51 , 3.85 , and 3.57 , respectively .
clients within the age category of 3549 years ( or 5.03 , 95% ci 3.567.12 , p=0.00 ) and over the age of 50 years ( or 4.99 , 95% ci 2.679.34 , p=0.00 ) were more likely to be infected with hiv .
a total of 2,120 clients data were retrieved from vct registries of the university of gondar teaching hospital . of the 2,120 vct clients , 1,093 ( 51.6% ) were males and 1,027 ( 48.4% ) were females , giving a male - to - female ratio of 1.06:1 .
the median age of the study subjects was 23.0 years ( interquartile range 10 ) .
1,188 ( 56% ) of the clients were from urban areas ; and > 80% of the participants had elementary education and above . in terms of their marital status , 1,334 ( 62.9% )
were never married , followed by married ( 21.2% ) , divorced ( 13.4% ) , and widowed clients ( 2.4% ) . a little over one - third of the vct clients ( 36.7% ) were students , 316 ( 14.9% ) were daily laborers , and 282 ( 13.3% ) were government employees ( table 1 ) .
overall , 17.1% ( 363/2,120 ) of the study participants were found to be hiv positive ( table 2 ) .
higher rates of hiv positivity were observed among female clients ( 20.4% ) than males ( 14.0% ) ( or 1.58 , 95% ci 1.261.98 , p=0.00 ) .
the prevalence of hiv was higher among the participants in the age group of 3549 years ( 35.5% ) , followed by > 50 years ( 35.3% ) and 2024 years ( 22.7% ) . in terms of their marital status ,
higher rates of hiv positivity were observed among widowed ( 64.7% ) and married participants ( 30.2% ) .
the prevalence of hiv among vct clients showed an inverse relationship with their education level , with a higher hiv rate among illiterates ( 28.0% ) than among educated ones .
with respect to occupations of the clients , higher hiv rates of positivity were detected among merchants ( 27.4% ) , daily laborers ( 26.3% ) , and housewives ( 25.7% ) compared to that among other occupations .
year - specific hiv - positive rates were 25.2% , 15.5% , and 14.9% in the years 2007/2008 , 2008/2009 , and 2009/2010 , respectively .
bivariate analysis revealed that widowed ( 95% ci 10.4234.92 , p=0.00 ) , married ( 95% ci 3.425.94 , p=0.00 ) , divorced ( 95% ci 2.795.32 , p=0.00 ) , and illiterate clients ( 95% ci 2.335.47 , p=0.00 ) were associated with hiv infection , with the odds ratios of 19.07 , 4.51 , 3.85 , and 3.57 , respectively .
clients within the age category of 3549 years ( or 5.03 , 95% ci 3.567.12 , p=0.00 ) and over the age of 50 years ( or 4.99 , 95% ci 2.679.34 , p=0.00 ) were more likely to be infected with hiv .
hiv / aids continues to be a major public health challenge in the world , despite the major efforts and resources allocated to combat the epidemic .
interventions such as hiv counseling and testing service has been shown to reduce hiv prevalence by promoting safer sexual behavior and serving as a key entry point for hiv treatment and care .
ethiopia has shown an encouraging improvement in the expansion of vct centers and antiretroviral therapy programs over the past decade that has reduced hiv prevalence at the national level.25,10 nevertheless , hiv epidemiology in the country varies markedly in terms of prevalence and risk factors from place to place . therefore , accurate and timely data on the local epidemiology of hiv are a necessity to identify at - risk groups and to tailor intervention strategies accordingly . to this end ,
our study sought to update the prevalence and associated risk factors for hiv seropositivity among vct clients at the university of gondar teaching hospital .
this was higher than the 13.7% vct - based national prevalence rate21 and 13.4% rate in burie , ethiopia.22 yet , it was lower than the previously reported 25.4% rate from gondar,8 24.5% rate in addis ababa , ethiopia,23 and 24.6% in tanzania.24 the variability in hiv prevalence across the studies and our finding could be explained by differences in sociodemographics of the study participants and prevailing hiv risk factors in the study areas .
hiv prevalence rates were declining over the study period , albeit the increasing trend in vct uptake .
this was in agreement with the declining trends of hiv prevalence among the general population of ethiopia and different segments of populations in the country.7,8,11 however , it is not clear whether this decline could be related to the general declining trend of hiv in the country as this is not a community - based study .
this observation was in line with a number of previous studies including the national demographic and health survey that reported higher hiv positivity rate among females.7,8,10 this could be ascribed to biological , cultural , sexual violence , and economic disadvantages related to their sex .
similarly , widowed and married clients were more likely to be infected with hiv than unmarried ones .
this might be partly attributed to the prevalent extramarital sex or successive marriages in the area .
another possible reason that explains the high hiv prevalence among this group is that married / widowed clients often seek vct service upon developing symptoms that suggest hiv infection .
however , this issue remains to be a subject of further investigations to understand the contextual influences of couples relationship and behaviors on their health and to design tailored intervention methods .
this study found that clients above the age of 35 years are at about five times at higher risk of being infected with hiv than the younger ones .
this finding was in agreement with previous studies.23,25 presumably , younger people may be less likely to acquire hiv infection due to the significant reduction in new hiv infections as a result of expanded interventions and hiv testing and care services in the country.6 however , the reason behind the high hiv rate among older age group is not clear .
it appears that older age groups are more likely to be married , divorced , or widowed ( which are found to be risk factors for hiv in this study ) , which could explain the higher prevalence in these groups .
this study also identified higher likelihood of hiv infection among illiterates than educated clients , which was consistent with the report of ethiopian health and demographic survey.7 this suggests that an insufficient awareness regarding hiv transmission and prevention could be the drivers for the seropositivity among this group of the population .
the results of this study were solely derived from records of vct clients ; thus , our inferences are subject to some limitations .
key variables such as clients socioeconomic status , risk behavior , number of sexual partners , and awareness of hiv transmission and prevention methods were missing .
nevertheless , our data provide useful information regarding hiv prevalence and risk factors among vct clients in the gondar area .
hiv is still the major public health concern in the gondar area as evidenced by our data .
being female , widowed , married , illiterate , and of older age were found to be associated with higher likelihood of hiv infection .
thus , concerted efforts and intervention strategies that target these at - risk populations are recommended . | backgroundthe epidemiology of human immunodeficiency virus ( hiv ) infection in ethiopia varies with regions , study population , and time .
thus , timely information on hiv epidemiology is critical for the combat of the epidemic . in this study
, we aim to update hiv prevalence and risk factors among voluntary counseling and testing ( vct ) clients at the university of gondar teaching hospital , northwest ethiopia.methodsa total of 2,120 vct clients records from september 2007 to august 2010 were analyzed retrospectively .
bivariate logistic regression analysis was used to identify significant predictors .
odds ratio ( or ) and 95% confidence intervals ( cis ) were calculated .
statistical significance was set at p - value < 0.05.resultsof 2,120 vct clients , 363 ( 17.1% ) were seropositive for hiv
. a higher rate of hiv positivity was observed among female clients ( 20.4% ) than that in male clients ( 14.0% ) ( or 1.58 , 95% ci 1.261.98 , p=0.00 ) .
widowed ( 95% ci 10.4234.92 , p=0.00 ) , married ( 95% ci 3.425.94 , p=0.00 ) , divorced ( 95% ci 2.795.32 , p=0.00 ) , and illiterate ( 95% ci 2.335.47 , p=0.00 ) clients were associated with hiv infection with the odds ratios of 19.07 , 4.51 , 3.85 , and 3.57 , respectively .
clients within the age category of 3549 years ( or 5.03 , 95% ci 3.567.12 , p=0.00 ) and above the age of 50 years ( or 4.99 , 95% ci 2.679.34 , p=0.00 ) were more likely to be infected with hiv.conclusionhiv is still the major concern of public health in the gondar area as evidenced by our data .
being female , widowed , married , illiterate , and older age were the identified risk factors for hiv infection .
thus , consideration of these factors in future intervention and clinical practice is recommended . |
all the materials used in the experiment were analytical grade . zinc acetate , zinc nitrate hexahydrate and ammonium hydroxide were purchased from sigma aldrich , uk .
zno nanowires were grown on glass substrates using the hydrothermal synthesis method ( 18 ) .
10 mm zinc acetate was drop casted on the glass substrates and annealed on a hotplate for 1 min at 100c for better adhesion .
zno nanowires were grown by immersing the seeded substrates in the mixture of 125 ml 20 mm zinc nitrate and 7 ml 2830% ammonium hydroxide , and annealed on a hotplate at 95c for 5 h. thereafter , substrates were removed from the growth solution and rinsed with de - ionized water several times .
surface analysis of the synthesized zno nanowire films has been carried out using a scanning electron microscope from zeiss ( evo ls10 ) .
surface wettability has been characterized using the contact angle measurement system from the first ten angstrom ( fta 200 ) at room temperature ( rh8% ) .
absorption spectrum of the zno nanowires has been collected using the lambda 950 uv / vis spectrometer from perkin elmer .
all the materials used in the experiment were analytical grade . zinc acetate , zinc nitrate hexahydrate and ammonium hydroxide were purchased from sigma aldrich , uk .
zno nanowires were grown on glass substrates using the hydrothermal synthesis method ( 18 ) .
10 mm zinc acetate was drop casted on the glass substrates and annealed on a hotplate for 1 min at 100c for better adhesion .
zno nanowires were grown by immersing the seeded substrates in the mixture of 125 ml 20 mm zinc nitrate and 7 ml 2830% ammonium hydroxide , and annealed on a hotplate at 95c for 5 h. thereafter , substrates were removed from the growth solution and rinsed with de - ionized water several times .
surface analysis of the synthesized zno nanowire films has been carried out using a scanning electron microscope from zeiss ( evo ls10 ) .
surface wettability has been characterized using the contact angle measurement system from the first ten angstrom ( fta 200 ) at room temperature ( rh8% ) .
absorption spectrum of the zno nanowires has been collected using the lambda 950 uv / vis spectrometer from perkin elmer .
a schematic view of the experimental setup for irradiating the samples with leds is shown in fig .
leds were purchased from nichia corporation ( p=365 nm and 385 nm ) and avago technologies ( p=454 nm and 519 nm ) .
full spectral widths at the half maximum of the leds ( p=365 nm , 385 nm , 454 nm and 519 nm ) were 15 nm , 15 nm , 25 nm and 30 nm , respectively . led emission was coupled through a bi - convex lens and irradiated over the sample placed on a sample stage . a neutral - density filter and aperture
a dichroic mirror was used in the path to reflect a fraction of the light for monitoring the power level with a coherent ( fieldmaster gs ) power meter and/or led spectra with a stellernet ( epp2000c ) spectrometer .
the surface wettability of the developed zno nanowires has been studied using the contact angle measurement system , by measuring the water contact angle ( wca ) before and after the light irradiation for different time durations .
all contact angle measurements were carried out using 2-l water drop under ambient conditions . a schematic view of the experimental set - up .
figure 2 shows a scanning electron microscopy ( sem ) image of zno nanowires at different magnifications . from the sem image
, it can be seen that the synthesized zno nanowires are preferentially oriented toward the c - axis perpendicular to the glass substrate .
the hexagonal structure of the zno nanowires can be seen in the magnified sem image .
also , the magnified view of the nanowire tip reveals the growth of layered hexagonal nanostructures on the tip .
this nanoscale roughness on the tip reduces the contact area between the solid and the droplet , which allows the entrapping of a large amount of air below the droplet and , therefore , enhances the hydrophobicity of the surface ( 13 , 14 ) .
the length and diameter of the synthesized zno nanowires were about 1112 m and 400500 nm , respectively .
the wca was measured immediately after the synthesis of zno nanowires and found to be about 0. this super - hydrophilic nature , shown by the as - synthesized zno nanowires using the hydrothermal method , can be attributed to the hydroxyl groups on the surface .
thereafter , substrates were left to dry under ambient conditions for 72 h. during this process , adsorbed hydroxyl groups on the surface were replaced by atmospheric oxygen , leading to the formation of a hydrophobic surface .
therefore , we measured the wca of zno nanowires after drying the substrate , and it was around 125. zno is a large - bandgap semiconducting material ; therefore , light with energy higher than the bandgap energy is absorbed by the zno .
this absorption of light ( photons ) generates electron hole pairs on the zno surface .
hole pairs will further create the surface defects and composition change that lead to different surface energy and , hence , surface wettability . the process which leads to the surface composition change
can be expressed as ( 13):zno+2hv2e-+2h+zn2++e-zns+(surface - trapped electron)o2-+h+o-(surface - trapped hole)o-+h+1/2o2+vo(oxygen vacancy )
the surface - trapped electrons ( zns+ ) tend to react with oxygen molecules adsorbed on the surface : zns++o2zns2++o2-
at the same time , water molecules may adsorb at the oxygen vacancy ( vo ) site .
these defect sites are kinetically more favorable for hydroxyl group ( oh ) adsorption than oxygen adsorption , and hence promote increased water adsorption at the uv light irradiated areas
. however , the adsorption of oh on the defective sites creates distortion in the electronic structure of the surface and makes it energetically unfavorable . therefore , after the uv illumination stops , the surface tries to recover to its original hydrophobic state by replacing the adsorbed oh with the atmospheric oxygen . during this process ,
therefore , an oxygen - rich environment can promote the recovery rate ( 19 ) .
as discussed , the whole process of change in wettability depends on the absorption of light by zno nanowires . therefore , to understand the spectral effect on the wettability
a normalized absorption spectrum of zno nanowires along with the led s normalized emission spectra are shown in figure 3 .
better spectral overlap indicates that zno nanowires absorb more light at 365 nm than other wavelengths .
led normalized emission spectra (
p=365 nm , 385 nm , 454 nm , 519 nm ) , along with a zno nanowire normalized absorption spectrum in green . to see the effect of light absorption by zno nanowires on their surface wettability
, a static wca has been measured on the zno nanowire sample before and after the light irradiation using uv led ( emission wavelength , p=365 nm ) .
figure 4 shows the measured wca for the 030 min light illumination period , and the inset pictures show the captured images of the water droplet on the sample before ( hydrophobic ) and after ( hydrophilic ) light irradiation . within 5 min ,
this rapid transition rate can be attributed to the nanoscale structures on the tip ( 13 ) .
gradual change in wca continues for a further 5 min , and thereafter it slows down .
the reason behind this observed change in wettability by light irradiation on the zno nanowires can be explained as follows : measured wca change over time on zno nanowires irradiated with 365 nm led .
inset pictures are captured images of water droplet on the zno nanowire sample , before ( hydrophobic ) and after ( hydrophilic ) the light illumination .
surface wettability of the synthesized zno nanowires has been analyzed by measuring the static contact angle .
a young contact angle , y , on the smooth surface can be defined in terms of surface energy as ( 22):cosy=(sv-sl)/lv
where sv , sl , and lv are the surface tension of solid vapor , solid liquid and liquid vapor , respectively .
considering the surface roughness , the wenzel model defines the apparent contact angle , , as : cos=r cosy
where r is a surface roughness factor .
this suggests the effect of change in surface roughness on the hydrophobicity ( contact angle ) .
but the wenzel model assumes that the liquid penetrates the rough surface , which is the case after irradiation of uv light . under unexposed conditions ,
zno nanowires tend to show a hydrophobic surface where liquid does not penetrate inside the rough surface ( nanowire groves )
. therefore , this condition refers to the cassie baxter state ( 20):cos=f(cosy+1)-1
where f is the area fraction of the actual solid surface area to the projected solid surface area .
therefore , upon uv light exposure , zno nanowires follow the wettability transition from the cassie baxter state to the wenzel state . to further understand the effect of wavelength ,
light irradiation has been carried out on the zno nanowires using leds with a peak emission wavelength p=365 nm , 385 nm , 454 nm and 519 nm .
wca has been measured on each sample before and after the light irradiation , and referred to as wca0 and wca , respectively .
figure 5 shows the relative change in wca / wca0 measured with irradiation time for different illumination wavelengths .
rapid change in the wetting angle observed for the led with p=365 nm can be attributed to the higher rate of change in surface energy . a small change observed in the wetting angle under 454 nm and 519
nm illuminations can be attributed to the small fraction of light absorbed by zno nanowires due to the native defects .
although the spectral width of leds emitting at 454 nm and 519 nm is slightly higher than that of the other leds used in the experiment , a negligible effect has been anticipated due to very low absorption by zno nanowires in that range .
trend lines in the graph indicate that the change in wetting angle slows down gradually with increasing wavelength .
in addition , results for the illumination wavelengths of 454 nm and 519 nm reveal that there was no clear hydrophobic - to - hydrophilic transition observed within 30 min of irradiation .
these results indicate that the wettability of the zno nanowires can be controlled by tuning the wavelength of irradiating light .
measured relative contact angle change with irradiation time for different led emissions at
p=365 nm , 385 nm,454 nm , and 519 nm .
the effect of irradiation wavelength and time on tuning the wettability of zno nanowires has been studied for the first time , using different leds .
results have provided significant insight into the role of wavelength on tuning the wettability of zno nanowires .
results show that the spectral overlap between zno absorption spectra and led emission wavelength controls the hydrophobic - to - hydrophilic transition rate .
various controllable parameters of leds can provide advanced control in tuning the desired surface wettability of the zno nanowires .
also , leds can be integrated on the biosensor chip , which allows miniaturizing the sensor and microfluidic devices .
there is no conflict of interest in the present study for any of the authors . | backgroundwettability is an important property of solid materials which can be controlled by surface energy .
dynamic control over the surface wettability is of great importance for biosensing applications .
zinc oxide ( zno ) is a biocompatible material suitable for biosensors and microfluidic devices .
nanowires of zno tend to show a hydrophobic nature which decelerates the adhesion or adsorption of biomolecules on the surface and , therefore , limits their application.methodssurface wettability of the zno nanowires can be tuned using light irradiation . however , the control over wettability using light - emitting diodes ( leds ) and the role of wavelength in controlling the wettability of zno nanowires are unclear .
this is the first report on led - based wettability control of nanowires , and it includes investigations on tuning the desired wettability of zno nanowires using leds as a controlling tool.resultsthe investigations on spectral properties of the led emission on zno nanowires wettability have shown strong dependency on the spectral overlap of led emission on zno absorption spectra .
results indicate that leds offer an advanced control on dynamically tuning the wettability of zno nanowires.conclusionthe spectral investigations have provided significant insight into the role of irradiating wavelength of light and irradiation time on the surface wettability of zno nanowires .
this process is suitable to realize on chip based integrated sensors and has huge potential for eco - friendly biosensing and environmental sensing applications . |
rapid maxillary expansion ( rme ) is a widely used treatment method to widen the maxillary arch .
it operates through moving the right and left maxillary segments apart by opening the mid - palatal suture as well as by buccal tilting of the premolars and molars .
many studies have evaluated the effects of rme , with banded and bonded appliances , on craniofacial structures , sutures , and dentoalveolar regions .
however , they mainly evaluated skeletal changes , dentoalveolar symmetry , dental tipping rates , and root resorption amounts .
clinical and radiographic evaluations are not intended to be adequate to evaluate the results of orthodontic treatment , and thus , the functional aspects of the therapy should also be taken into consideration .
the stomatognathic system includes the functional and physiologic entity of the teeth , the occlusal relationships , the periodontal tissues , the temporomandibular articulations , the craniofacial bones , and the neuromuscular system , whose biology and physiopathology are interdependent .
the results of these studies showed that rme provided an increase in the nasopharyngeal airway dimensions that facilitate nasal respiration .
there are few studies that have analyzed the electromyographic ( emg ) activities of the masseter and temporalis muscles in children treated using rme with a bonded appliance .
de rossi et al . indicated that their results do not suggest that rme harms the muscles , but there is a strong change in muscle activity after removing the disjunctive appliance .
this is mainly attributed to occlusal instability and lack of musculature adaptation to this new condition of the stomatognathic system .
in addition , arat et al . evaluated the condylar response to rme using bilateral temporomandibular joint ( tmj ) magnetic resonance images ( mris ) , and observed a condylar response to rme .
there were increased signal intensities , shown as bright areas on the mris , indicating the red bone marrow edema that is a sign of condylar remodeling .
they concluded that the extensive orthopedic and the functional occlusal forces associated with rme also play roles in condylar and ramal responses .
thus , rme treatment promotes increases in maxillary width and dental arch perimeters , and the new position of the maxilla and the dentition affects both muscles and occlusion .
children with posterior cross - bites are thought to have irregular , complex chewing cycles , notably different from the pattern of patients with normal occlusion , and similar to those with tmj dysfunctions . in previous studies
, it has been reported that after treatment , chewing cycles generally became more regular and symmetrical , similar to those of normocclusive patients . to our knowledge
, there are no previous studies considering the effect of bonded rme on occlusal forces , and thus , our aim in this preliminary study was to evaluate these effects on occlusal force distribution .
we hypothesized that correction of the maxillary cross - bite with a modified bonded rme device would affect the occlusal forces while changing the dentoalveolar structure .
this preliminary study included 12 patients ( 7 girls and 5 boys ) between ages 11 and 15 years ( mean age : 13.10 years ) consecutively admitted to the orthodontic clinic at gazi university faculty of dentistry .
patients were in the permanent dentition stage , excluding third molars , and showed bilateral posterior cross - bite of both premolars and molars , with an indication for rme . in all patients , the maxillary buccal cusps occluded in the central fossa of the mandibular teeth .
additional selection criteria were angle class i ( anb angle : 04 ) or class ii ( anb angle : > 4 ) ; malocclusion with optimal growth pattern ( mandibular plane angle : 2632 ) ; minimum / mild crowding ; no periodontal pathology ; missing teeth ; craniofacial deformation ; crowns and great restorations ; muscular disease ; parafunctional habits ( e.g. , bruxism ) ; and tmj dysfunction .
written informed consent was obtained from each subject following a detailed explanation of the objectives and protocol of the study .
the study was conducted in accordance with the ethical principles stated in the declaration of helsinki and approved by the ethics committee of gazi university ( 25901600/7867 ) .
patients were treated with a modified bonded rme appliance that covered the occlusal surface of the posterior teeth with an acrylic splint [ figure 1 ] .
when the palatal cusps of the maxillary posterior teeth were occluding with the buccal cusps of the mandibular posterior teeth , the screw was fixated using wire .
the average activation time was 14 days , which approximately corresponded to 7 mm of opening of the screw .
modified bonded rapid maxillary device we used the t - scan iii 5.2 device ( tekscan , inc . , south boston , ma , usa ) to analyze the occlusal force distribution [ figure 2 ] .
the instrument was directly interfaced with a computer that presented the data on a screen during the examination and recorded it for further analysis [ figure 3 ] .
the t - scan iii handpiece digital images illustrating maximum intercuspidation using the t - scan iii : ( a ) a 3-dimensional representation of the occlusion and the occlusal force .
( b ) bite force data expressed in percentages during all examinations ; the appliance was removed .
patients were instructed to sit upright on a chair , with their heads unsupported , the frankfurt horizontal plane parallel to the floor , both feet on the floor , hands resting on the lap , and looking forward .
after that , the central incisors widths were measured using a digital caliper to determine the dental arch dimension .
we initiated occlusal evaluation after determining the dental arch dimension and the intra - oral sensor calibration .
the sensor was placed in patients mouths whereas the central line of the sensor 's support was aligned with the upper incisors midline .
patients were instructed to clench the sensor as hard as possible at maximum intercuspidation . when patients bit the sensor ,
the resultant changes in electric resistance were converted into images on the screen , and we analyzed the value of the maximal voltage obtained in the registration of maximal clenching .
the t - scan iii software automatically calculated the distribution of occlusal forces ; the bite force data were expressed in percentages .
a single new sensor was used for each child , and all recordings were performed by the same operator ( sa ) .
recordings were repeated twice , and the averages were calculated to use as the final values .
records were taken at the pretreatment ( t1 ) , the postreatment ( t2 ) , and the postretention ( t3 ) periods .
we evaluated the percentage of occlusal force distribution of ( 1 ) the anterior teeth ( incisors , laterals and canines ) ; ( 2 ) the premolars ( first and second premolars ) ; ( 3 ) the molars ( first and second molars ) ; ( 4 ) the posterior teeth ( both premolars and molars ) ; and ( 5 ) all teeth ( anteriors , premolars , and molars ) .
the third molars are not present in the arch model created by the t - scan , and thus their contacts were not taken into account .
there were no differences in occlusal force distribution between the right and the left sides in all time periods .
a significant decrease was found in the percentage of total occlusal force distribution between the pretreatment and the posttreatment period ( t1t2 ) ( 96.8% and 94.8% , respectively ) ( p < 0.05 ) .
however , during the retention , the percentage of force distribution increased ( 95.9% ) , and no significant differences were found between the pretreatment and the postretention period ( t1t3 ) [ table 1 ] .
percentage force distributions on the right and left sides of the teeth at the pre - treatment ( t1 ) , post - treatment ( t2 ) , and post - retention ( t3 ) periods ( n=12 ) the highest percentage of force was observed in the molar region ( 71.3% ) .
the central and lateral incisors were very frequently without contact , so the percentage of force was low .
there was no statistical difference in the occlusal forces of the premolars in all time periods .
the dental , skeletal , and muscular effects of rme have been well documented in the literature . however , to our knowledge , there have been no studies considering the effect of this therapy on occlusal force distribution .
it was the aim of this study to analyze the effect of modified bonded rme appliances on occlusal force distribution using a t - scan computer - assisted dental occlusion analyzer .
occlusal force can be affected by age , gender , dentition stage , and facial structure , among other factors .
however , differences in bite force between males and females are not found in children , probably because body structure and muscle strength are very similar in this population .
similarly , owais et al . found no significant gender differences in the early primary and permanent dentition stages . in the light of their findings
the relationship between the bite force and the angle classification has been evaluated by sonnesen and bakke .
the authors concluded that bite force did not vary significantly between the angle malocclusion types in children aged 713 years .
contrary to their findings , another study found that the masticatory performance was reduced in subjects with malocclusions , especially in those with class ii and iii , when compared to those with normal occlusion . in this study , we only included patients with class i and ii malocclusions .
a significant positive relationship between the maximum occlusal bite force ( mobf ) and the dentition stage were reported .
this was found to be related to the development of the masticatory system throughout the growth cycle and was explained by the increase in the number of occlusal contacts during the transition to different dentition stages .
correlations between maximum bite force and facial morphology have also been found in pediatric populations .
it has been reported that the maximum molar bite force is nearly twice that of dolichofacial subjects in individuals of normal facial types ; further , that the brachiofacial types showed greater values . this result has also been confirmed by abu alhaija et al . , who reported that subjects with short faces had the highest mobf whereas those with long faces had the lowest .
in this study , all subjects were in a limited age range ( 1115 years ) and at the same dentition stage ( permanent dentition ) , an optimal growth pattern providing sample homogeneity .
the scope of this study was not to compare subjects with posterior cross - bite to those with normal occlusion but to verify the occlusal force changes associated with modified bonded rme .
occlusal bite force values can be directly influenced by the accuracy of the measuring apparatus . in this study
we preferred the t - scan system as a quantitative occlusal analysis technique , rather than the qualitative conventional methods , such as articulating paper , waxes , and silicone impression .
these methods are not ideal occlusal analyzers due to their static nature , subjective interpretation , and limiting factors .
some researchers have defined the t - scan system as providing nonsubjective , precise , and reliable occlusal analysis that offers improved information to clinicians .
it has been claimed that the t - scan iii system measures the relative occlusal force of the subjects instead of the numerical values of absolute occlusal force .
however , some studies of this system found that it produced more comprehensive and evidence - based results ; thus , it has been suggested for use as a measurement tool in clinical experiments .
studies have been performed to determine the relationship between mobf and masticatory efficiency , which concluded that mobf is one indicator of the functional state of the masticatory system that results from the action of jaw elevator muscles modified by the craniofacial biomechanics .
the occlusal contact patterns of the teeth were found to exert a significant influence on the activity of masticatory muscles .
antonarakis et al . have performed the only study that evaluated maximum bite force changes during orthodontic treatment .
they reported decreased maximal molar bite force with functional appliance treatment of class ii patients .
the authors explained that this decrease with the mild muscular atrophy was caused by the decreased functional activity of masticatory muscles , and was related to occlusal instability .
similarly , bonded rme treatment can also cause occlusal contact changes and occlusion instabilities . in this study , we were not surprised to find a significant decrease in total occlusal force distribution during treatment ( t1t2 ) .
in addition , there were decreases in the percentages of force distribution of the anterior and the posterior teeth at the end of the modified bonded rme treatment ( t1t2 ) , although we observed no statistically significant difference . from another point of view
, the decrease might be due to the sensitivity of the periodontal ligament as related to the orthodontic tooth movement during maxillary expansion .
as orthodontic tooth movements alter the periodontal ligament 's vascularity and blood flow and cause inflammatory reactions in the periodontium and dental pulp , they will stimulate the release of various biochemical mediators and cause pain .
the nonsignificant increase in the force distribution percentage at t3 could be related to the masticatory system .
decreased functional activity showed a certain amount of recovery after the interruption of appliances , and the percentage of total force distribution at t3 ( 95.9 0.77 ) seemed to return to the initial values at t1 ( 96.8 0.69 ) .
this increase in occlusal forces was in accordance with the findings of antonarakis et al . , who found that increases in maximal molar bite forces were likely associated with a general increase in muscle force .
similarly , other researchers argued that the jaw - closing muscles might have benefited from a training effect , resulting in stronger muscles .
in support of these findings , two studies that evaluated the emg activity of the masseter and temporalis muscles of children treated with rme using a bonded appliance reported increased activity in anterior temporal and masseter muscles activity after the appliance was removed .
although both the dental and skeletal effects of rme have been evaluated , the effects of this therapy on occlusal force distribution have not been considered , thus limiting direct comparison of our results with other studies .
it would be beneficial to also evaluate the bite forces with temporal and masseter muscle activations .
thus , further studies are needed to verify the influence of occlusal changes on all the muscle activations during modified bonded rme . further
, this study was based on a small sample size;future studies with larger sample sizes are needed .
finally , studies comparing the effects of the banded and the bonded types of rme on occlusal forces and muscle activity might be beneficial in determining the most effective , harmless method to provide more stable results for maxillary expansion .
in this preliminary study , we concluded that , considering the sample and the methodology used , modified bonded rme treatment decreased the percentage of total occlusal force distribution during the treatment period ; however , it returned to its initial value after the postretention period .
| objective : to evaluate the effects of modified bonded rapid maxillary expansion ( rme ) on occlusal force distribution.materials and methods : the sample included 12 patients ( 7 girls and 5 boys ; mean age : 13.1 years ) at the permanent dentition stage with bilateral posterior cross - bite .
the patients were treated with a modified bonded rme appliance , activated twice a day .
the study was terminated when the palatal cusps of the maxillary posterior teeth were occluding with the buccal cusps of the mandibular posterior teeth .
the postretention period was 3 months .
the t - scan iii device was used to analyze the percentages of occlusal force distribution , and records were taken at the pretreatment ( t1 ) , the postreatment ( t2 ) , and the postretention ( t3 ) periods .
wilcoxon signed rank test was used for statistical analyses.results:incisors were most frequently without contact , followed by canines .
the highest forces were seen in the second and first molar regions . a significant decrease was seen in total occlusal force during treatment ( t1t2 ) ; however , during retention , the force returned to its initial value , and no significant differences were found ( t1t3 ) .
no differences were found between right and left sides and in occlusal forces of the teeth in all time periods.conclusion:the use of modified bonded rme decreases the total occlusal forces during the treatment period , but it does returns to its initial value after the postretention period . |
haemolytic uraemic syndrome ( hus ) is a common cause of acute renal failure and microangiopathic haemolytic anaemia ( maha ) .
it is associated with infections ( shiga toxin - producing bacteria in diarrhoea - positive hus and streptococcus pneumoniae ) .
less commonly , it is caused by defects in complement regulation , termed atypical hus ( ahus ) , and early diagnosis is vital for the initiation of plasma exchange ( pex ) , which can lead to improved renal outcomes [ 13 ] .
more recently , eculizumab , a monoclonal antibody which binds complement c5 and blocks terminal complement activation , has been developed .
we describe a case of ahus secondary to complement factor h ( cfh ) mutation , a key regulator of the alternative complement cascade .
a previously healthy 7-month - old girl presented with paleness and easy bruising , preceded by croup .
initial investigations revealed low haemoglobin ( hb ) and platelets ( plts ) , but normal kidney function .
after 6 weeks of follow - up , she developed dark urine , oliguria , fluid overload and hypertension .
hb was 4.5 g / dl , plts were 72 10/l and blood film showed maha with elevated urea and creatinine .
there was no diarrhoea or vomiting and a clinical diagnosis of ahus was made . following transfer to our nephrology centre
a plasma infusion was given on the second day of admission and pex started on the fourth day .
pex consisted of one volume exchange with 5% albumin replacement initially and 10 ml / kg of virion - inactivated plasma ( octaplas ) at the end .
she received nine pex sessions in the first 2 weeks but then developed hypertensive encephalopathy requiring ventilation , continuous veno - venous haemofiltration and sodium nitroprusside .
genetic testing revealed a previously undescribed sequence variant in exon 23 of cfh ( c.3568t > c ; p.tyr1190his ) , substituting a highly conserved amino acid .
other complement tests were normal , including c3 , c4 , classical and alternative pathway activity , factor h and factor i levels .
adamsts13 activity was slightly low at 25% ( normal range 55166 ) but not consistent with deficiency .
other investigations for ahus were negative , including homocysteine , methylmalonic acid and autoimmune profile .
she required nine packed red blood cell transfusions in 4 months and blood priming for pex sessions .
each thin arrow denotes a plasma exchange session ( 1 volume exchange with 5% albumin and 10 ml / kg virion inactivated plasma ) .
horizontal dotted lines represent normal ranges for haemoglobin ( bottom ) and platelets ( top ) respectively .
time on haemodialysis ( hd ) and peritoneal dialysis ( pd ) represented by double arrowed lines . in an attempt to modify the disease process ,
eculizumab dosing was in accordance with manufacturer recommendations of 300 mg weekly for the first two doses and every 3 weeks subsequently .
following the first dose of eculizumab , there was recovery of haematological parameters enabling cessation of pex .
renal function also improved , and dialysis was stopped 1 month after the first dose , with ongoing fall in creatinine .
at last follow - up aged 18 months , she has an estimated glomerular filtration rate of 42 ml / min/1.73 m ( schwartz formula ) , with excellent neurodevelopmental progress .
eculizumab is a humanized monoclonal antibody , which blocks the cleavage of complement c5 to c5a ( a potent chemotactant ) and c5b .
this inhibits the progression of c5b to the terminal complement complex ( tcc ) c5b-9 .
levels of tcc are high in ahus , consistent with increased complement activation , and the improvement post - eculizumab supports this concept of disease pathogenesis
. there are four case reports describing eculizumab treatment of ahus relapses in native kidneys [ 58 ] . in all cases
, there was effective cessation of haemolysis and haematological recovery within 710 days . in three cases , there was also recovery in renal function and these patients avoided dialysis .
eculizumab was used again in a subsequent relapse but there was progression to anuria and eculizumab was stopped after haemodialysis was started .
open - label trials in adolescents and adults have finished recruiting and a multicentre paediatric trial is ongoing though patients receiving chronic dialysis are excluded ( clinicaltrials.gov nct01193348 ) .
cost considerations aside , the long - term side effects of tcc blockade in children are not known , though experiences in paroxysmal nocturnal haemoglobinuria have not highlighted any serious complications .
the main risk is infection from encapsulated organisms and patients require tetravalent meningococcal vaccination beforehand .
however , neisseria meningitidis serogroup b is currently not covered and prophylactic penicillin is strongly advisable .
an alternative strategy would be liver ( with or without kidney ) transplantation , which would restore production of functionally normal cfh .
haller et al . successfully performed an isolated liver transplant using an established protocol with pre - transplant pex and intratransplant plasma infusion and anticoagulation .
liver and renal function remain stable 2 years post - transplant with no ahus relapses though the authors caution that the benefits of this treatment have to be carefully balanced against the risks of major surgery and long - term immunosuppression including calcineurin nephrotoxicity . in our patient ,
not only did eculizumab allow immediate cessation of three times weekly pex , it also resulted in renal recovery and allowed discontinuation of dialysis despite being dialysis dependant for 4 months before eculizumab therapy .
there was a significant improvement in both the clinical condition of the child and the quality of life for the family .
although eculizumab is an expensive drug , the cost is significantly less than that of dialysis and regular pex . in summary ,
eculizumab therapy should be considered in patients with ahus . in those with an underlying complement defect , reversal of the haematological manifestations
is expected , and in addition , some dialysis - dependant patients may be able to stop dialysis . | disorders in complement regulation are a major cause of atypical haemolytic uraemic syndrome ( ahus ) .
eculizumab , a monoclonal antibody targeting complement c5 and blocking the terminal complement cascade , should theoretically be useful in this disease , particularly when associated with specific complement pathway anomalies such as factor h deficiency .
eculizumab is emerging as an effective treatment for post - transplant ahus recurrence and may have a role in treating de novo ahus , halting the haemolytic process . in this case report
, we describe the fourth case of ahus treated with eculizumab . in our patient , with a known complement factor h mutation , not only has the disease process become quiescent but also this therapy has led to significantly improved renal function so that dialysis is no longer necessary . |
in the era of minimally invasive technologies , laparoscopic transperitoneal left adrenalectomy ( ltla ) has become the standard treatment for adrenal masses < 6 cm .
however , masses > 6 cm can be successfully treated using the laparoscopic approach as well , replicating principles of open surgical oncology .
splenorenal and splenoparietal ligaments are dissected during ltla with the lateral approach , and if necessary , the left colic flexure is mobilized .
the middle adrenal vein is identified behind the pancreas so that , after its ligature and dissection , the adrenal gland can be removed .
the dissection of splenic ligaments replicates their congenital absence or weakening , present in cases of wandering spleen ( ws ) .
other possible approaches are laparoscopic transperitoneal anterior adrenalectomy ( during which the patient is supine ) and posterior retroperitoneal laparoscopic adrenalectomy ; neither involves the dissection of splenic ligaments .
ws , also known as splenoptosis or ectopic , free - floating , or aberrant spleen , is the migration of the spleen from the left upper quadrant to a more caudal location in the abdomen . a unique case of ws after ltla was described by corcione et al , who reported the case of a 57-year - old woman who underwent ltla because of a 4-cm nonfunctional mass .
three months later , she was admitted to the surgical emergency department with acute abdominal pain , nausea , and vomiting .
she underwent emergency laparotomy , which revealed an organoaxial gastric volvulus caused by the hypermobile spleen .
another case report , by le et al , showed ws as a rare complication of splenic torsion after laparoscopic nissen fundoplication .
ws , with an incidence of < 0.5% among splenectomies , is rare and is often due to the congenital absence or weakening of splenic ligaments .
the spleen develops from a mesenchymal condensation in the dorsal mesogastrium of the lesser sac in the fourth week of gestation . during fetal development , rotation of the intestinal tract and growth of the dorsal mesentery carry the spleen to the left side of the abdominal cavity , where it is anchored to the left kidney and stomach by gastrosplenic and splenorenal ligaments , respectively .
ws , as well as intestinal malrotation , congenital short bowel , and other abnormalities , has been associated with mutations of filamin a in male patients affected by x - linked periventricular nodular heterotopia .
failure of fusion of the mesogastrium to the posterior body wall leads to incomplete formation of splenocolic , splenorenal , and splenophrenic ligaments , and this is considered the mechanism by which ws , defined as the spleen 's lying outside the left upper quadrant suspended only by hilar vessels , occurs .
consequently , ws has a long mobile mesenteric pedicle that is prone to torsion and ischemia .
this mechanism can also be responsible for gastric rotation ( volvulus ) , rarely associated with ws .
when present in adults , ws may be the delayed onset of congenital conditions or an acquired disease from abdominal wall laxity due to the hormonal effects of pregnancy .
some authors have associated it with splenomegaly , although there is no higher incidence of ws in areas of endemic splenomegaly . in adults
, it usually occurs as a mobile abdominal mass , whereas in children , it presents as acute or chronic abdominal pain and can be associated with a history of congenital diaphragmatic hernia or prune belly syndrome .
the clinical presentation is vague , with symptoms including nausea , vomiting , early satiety , and acute , cramplike abdominal pain due to splenic ischemia and infarction .
bowel obstruction may occur because of the compression of abdominal organs ; enuresis and hydronephrosis may be other consequences , as well as gastric fundal varices , mesenteric varices , and acute pancreatitis .
the progressive splenic enlargement can lead to rare splenic ruptures and to possible spontaneous massive hemoperitoneum .
ultrasonography is considered efficacious in diagnosing ws , as it is a noninvasive , inexpensive test that can confirm both the ectopic location of the spleen and blood flow pattern .
both grayscale and color doppler sonography can easily show the migratory nature and perfusion status of a ws in real time , in the right decubitus position .
sometimes it is necessary to examine patients with computed tomography ( ct ) , which provides information on the location , vascular integrity , and size of the spleen as well as the surrounding organs . with ws ,
ct may show elongated or tortuous splenic vessels and may also reveal multiple collaterals of the hilum of the spleen .
with torsion of the splenic pedicle , a whirled appearance of the splenic vessels may be noticed on ct . despite the advantages offered by ultrasonography and ct ,
the aim of this prospective study was to determine whether the mobilization of the spleen during ltla , replicating the congenital condition of absence or weakening of the splenic ligaments , could be recognized as a cause of postoperative ws .
between january 2006 and may 2011 , 59 patients underwent laparoscopic lateral adrenalectomy ; 30 right adrenalectomies and 29 left adrenalectomies were performed .
we took into consideration the 29 patients who underwent ltla : 9 men ( 31% ) and 20 women ( 69% ) .
twenty - four patients ( 83% ) were included in our ultrasonographic follow - up .
five patients were not included , 3 with terminal cancer and the remaining 2 who had undergone multiple upper gastrointestinal surgical procedures .
therefore , we analyzed 24 patients , 8 men ( 33% ) and 16 women ( 67% ) , with a mean age of 59 13 years ( range , 3784 years ) . among these 24 patients ,
13 were affected by nonfunctional adenoma ( 54% ) , 4 by pheochromocytoma ( 17% ) , 3 by metastasis ( 12% ) , 3 by adrenocorticotropic hormone dependent hyperplasia with cushing 's disease ( 12% ) , and 1 by conn 's adenoma ( 4% ) ( table 1 ) .
demographic and clinical data of 24 patients undergoing ltla acth , adrenocorticotropic hormone ; ltla , laparoscopic transperitoneal left adrenalectomy .
each patient underwent preoperative abdominal ultrasonography , ct , or nuclear magnetic resonance , as well as hormonal laboratory exams .
all ltlas were performed using the same laparoscopic technique : right lateral decubitus pneumoperitoneum induced by a veress needle ; 3 trocars , placed on the midclavicular line ( t3 , 5 mm for the forceps and divaricators ) , midaxillary line ( t2 , 1012 mm for the radiofrequency dissector , scissors , irrigation - aspiration cannula , clips , etc ) , and anterior axillary line in the middle of the line joining the umbilicus to the subcostal margin ( t1 , 1012 mm for the optic ) ( figure 1 ) ; dissection of the splenoparietal and splenorenal ligaments ; en bloc mobilization and medialization of the spleen and pancreas ( figure 2 ) ; sectioning using clips or a radiofrequency dissector of the middle adrenal vein ; and removal of the adrenal gland into an endobag ( covidien , dublin , ireland ) .
mobilization of the left colic flexure was performed only when absolutely necessary , and exposure of the adrenal lodge was limited to the field of view .
furthermore , care was taken in repositioning the spleen and pancreas to their correct seats without using fibrin glue or other devices .
the 24 selected patients were studied postoperatively using abdominal ultrasonography after 2 and 4 months ( 4 patients [ 17% ] ) and after 1 year ( 20 patients [ 83% ] ) , with a 3.5-mhz convex probe , always by the same radiologist , experienced in ultrasonography , who evaluated the following parameters : morphologic and dynamic aspects of the spleen in the supine position , in the right lateral position , and in orthostatism ; potential mobilization of the spleen related to the diaphragmatic dome ; and flow direction and resistive index at the origin of the splenic artery and at the splenic hilum .
the average operation length was 89 35 minutes ( range , 45180 minutes ) ; the average diameter of the lesions was 4 0 cm ( range , 38 cm ) ; and the average postoperative stay was 2 1 days ( range , 15 days ) .
postoperative courses were free of complications in 22 patients . in the remaining 2 patients ( overall morbidity , 8% ) , 1 patient
had left pneumothorax treated with thoracic drainage , and 1 patient had right pleural effusion with hematoma in the left renal region , conservatively treated .
clinical and ultrasonographic follow - up showed an absence of symptoms attributable to ws in each patient , confirming normal position of the spleen with normal blood flow in the supine position , in the right lateral position , and in orthostatism ; an absence of spleen mobilization related to the diaphragmatic dome ; and an absence of free or loculated collections .
the average longitudinal spleen diameter was 9.9 1.3 cm ( range , 7.812.2 cm ) , and the average anteroposterior spleen diameter was 4.8 0.6 cm ( range , 3.85.8 cm ) .
the average resistive index at the origin of the splenic artery was 0.6 0.1 ( range , 0.40.7 ) , and the average resistive index at the splenic hilum was 0.6 0.1 ( range , 0.50.7 ) .
hepatic steatosis was seen in 3 patients ( 12% ) , and ptosis of the right kidney was seen in 1 patient ( 4% ) .
to evaluate whether ws can be considered a frequent postoperative complication of ltla , we considered 29 patients who underwent this surgical procedure , but 5 patients were not included in the study . for the remaining 24 patients , postoperative ultrasonographic follow - up was performed to evaluate the morphologic and dynamic aspects of the spleen , its potential mobilization , and the flow direction and resistive index at the origin of the splenic artery and at the splenic hilum . ws was completely absent , despite the dissection of ligaments and the female majority .
although there are surgical techniques that do not involve dissection of the splenic ligaments , ltla seems to be free of complications related to the supposed hypermobile spleen . given the rarity of ws , we are aware that the number of patients in our study is too small to reach any definitive conclusions , and our results should be regarded as purely exploratory .
ws should not be considered a common complication of ltla , especially in patients with small adrenal masses , which generally do not require extensive dissection ; in our patients , the spleen was repositioned in its correct seat without the necessity of glue or splenopexy , mobilization of the left colic flexure was performed only when absolutely necessary , and exposure of the adrenal lodge was limited to the field of view .
in addition , we have no data regarding postoperative adhesion formation , but we suppose that the healing process of dissected structures may be sufficient to fix the spleen in its natural place .
ws may occur if the dissection is extensive ; for this reason , in these cases we recommend the use of glue or other methods to reposition the spleen in its correct seat .
clinical and ultrasonographic follow - up of 24 patients who underwent ltla showed no symptoms or signs of migration of the spleen .
ltla remains widely recognized as a safe approach , at least as effective as other techniques , such as laparoscopic transperitoneal anterior adrenalectomy and posterior retroperitoneal laparoscopic adrenalectomy . in any case
, dissection should be limited and should not exceed too much in the mobilization of the left colic flexure .
furthermore , it is necessary to pay attention when repositioning the spleen and pancreas in their correct seats .
ws should be therefore considered a very rare complication of ltla that , however , can not be ignored .
it would be useful for other authors to signal this complication , should it occur , so that different approaches and consequent results may be compared . | background and objectives : laparoscopic transperitoneal left adrenalectomy ( ltla ) has become the standard treatment for adrenal masses < 6 cm .
ltla involves the dissection of splenic suspensory ligaments , which replicates their congenital absence or weakening , present in cases of wandering spleen ( ws ) .
ws is a rare condition in which the spleen migrates from the left upper quadrant to a more caudal location in the abdomen . a unique case of ws after ltla was described by corcione et al . in this prospective study , we investigated the possibility of ws as a consequence of ltla.methods:twenty-four patients , 8 men and 16 women , who underwent ltla with the dissection of splenoparietal and splenorenal ligaments were selected.results:clinical and ultrasonographic follow - up showed no evidence of postoperative ws.conclusions:in the literature , ws is not commonly reported as a postoperative complication of ltla . in effect , especially in the case of small adrenal masses ,
the spleen 's repositioning in its seat is autonomous .
however , the alarming possibility of ws should not be ignored , especially in the case of extensive dissection of the left colic flexure . it would be useful for other authors to signal this complication , so that different approaches and consequent results may be compared . |
early repair is important because of risks of incarceration and strangulation of abdominal contents along with respiratory and cardiovascular compromise .
minimally invasive techniques have been useful for diagnosis and treatment of diaphragmatic hernias in both blunt and penetrating trauma .
we present the case of a 54-year - old victim of a motor vehicle crash who presented with a delayed diagnosis of a right - sided traumatic diaphragmatic hernia . by using a 4-port technique and intracorporeal suturing ,
this case highlights the difficulties associated with diagnosing diaphragmatic hernias and the role of minimally invasive techniques to repair them .
minimally invasive surgical techniques are being increasingly used to both diagnose and repair traumatic diaphragmatic injuries with excellent results .
patients are often asymptomatic or comatose , and imaging performed may not clearly depict the dh .
therefore , further studies may not be done , leading to incorrect diagnoses and treatments .
late - onset dh can present as a variety of clinical symptoms , such as cardiorespiratory or gastrointestinal complaints .
right - sided herniation may even be more difficult to detect than left - sided herniation .
thoracoscopy and laparoscopy are 2 of the approaches to minimally invasive repair of these defects .
a 54-year - old woman was brought to the emergency department by ems as a level ii trauma status after a motor vehicle crash .
she was the driver of a vehicle that was t - boned by a deer on the passenger side of the car , and she subsequently lost control of the vehicle .
she was unresponsive at the scene , and an attempt to intubate her in the field failed . she was ventilated with a bag - valve mask and later intubated in the emergency department . on arrival to the er , she was hemodynamically stable with 100% saturation .
her glasgow coma scale was 6 t . a plain chest film performed on arrival revealed elevation of the right hemidiaphragm with linear atelectasis at the right base ( figure 1 ) .
a computed tomographic ( ct ) scan of the chest and abdomen revealed right lower and middle lobe collapse and elevation of the right hemidiaphragm .
there was left lung base atelectasis , and a grade 3 liver laceration , but the great vessels were intact .
the patient also had a closed head injury with subarachnoid hemorrhage , diffuse axonal injury , and a frontal scalp hematoma .
her other injuries included fractures of her right clavicle , multiple bilateral ribs , right wrist , stable pelvic fracture , sacrum , and unstable fracture of c2 .
the patient was admitted to the icu , and her course was complicated by respiratory failure and pulmonary sepsis , which resolved with a tracheostomy and a prolonged course of antibiotics .
the patient was transferred to the surgical floor 20 days later , and after another 5 weeks of hospitalization she was discharged to a long - term rehabilitation facility .
initial chest x - ray reveals elevation of the right hemidiaphragm with linear atelectasis at the right base .
two days later , she was readmitted with shortness of breath , wheezing , and fever .
a chest x - ray showed a moderate - to - large right pleural effusion and opacity at the right lung base ( figure 2 ) . a ct scan of the chest showed right diaphragmatic hernia with herniation of bowel alongside the liver , right lower lobe atelectasis , and mediastinal shift to the left ( figure 3 ) .
bronchoscopy was performed and showed extrinsic compression and complete closure of the right lower lobe orifice and bronchi .
the patient was given a course of antibiotics , and after resolution of her pneumonia , surgery was performed .
chest x - ray on readmission shows a moderate right pleural effusion with opacity at the right lung base .
computed tomographic scan of the chest and abdomen showing right diaphragmatic hernia with stable right pleural effusion , right lower lobe atelectasis , mediastinal shift to the left , and herniation of bowel alongside the liver into the thorax . in the operating room
laparoscopy revealed a large diaphragmatic defect with herniation of the right lobe of the liver into the thoracic cavity ( figure 4 ) .
the liver was pulled back into the abdomen revealing a collapsed right lower lobe , some pleural fluid , and the edges of a large diaphragmatic defect ( figure 5 ) .
adhesions of the lower edge of the diaphragm were taken down , and the defect closed using interrupted figure - of - eight surgidac sutures with ptfe pledgets ( figure 6 ) .
herniation of the right lobe of the liver though the defect in the right diaphragm .
retraction of the liver reveals a collapsed right lung , pleural fluid , and a large defect .
a chest tube was inserted for drainage of the thorax , and the port sites were closed . the postoperative chest x - ray compared with the preoperative chest x - ray showed marked improvement in the borders of the right hemidiaphragm ( figure 7 )
. the patient 's postoperative course was complicated by a flare - up of respiratory infection , but she responded to antibiotics and was discharged to a long - term rehabilitation facility 2 weeks after the surgery .
diaphragmatic hernias ( dh ) can be categorized as occurring in the left or right diaphragm , being congenital or acquired , or having an early or late onset .
congenital diaphragmatic hernias ( cdh ) are well described and occur in 1/2000 to 1/5000 live births , with 30% to 50% diagnosed in utero .
left - sided cdhs are more common than are right - sided and bilateral ones ( 85% vs. 8% to 21% vs 2% ) , with a 1:1.25 male to female ratio .
left - sided cdh may cause severe respiratory distress , gastrointestinal symptoms , or both , in the newborn , whereas right - sided cdh may cause fewer symptoms .
traumatic dhs are mostly left - sided ( 70% to 80% ) vs. right - sided ( 15% to 24% ) vs. bilateral ( 8% ) penetrating injury results in a 5% incidence of diaphragmatic injury , which is usually diagnosed and treated by laparotomy .
laparoscopy is a useful diagnostic tool in low - velocity penetrating injuries to assess violation of peritoneum and to survey the diaphragms .
if not diagnosed promptly , even a small diaphragmatic injury may result in significant morbidity and require repair due to migration of organs aided by the combined push - pull mechanism of positive intraabdominal pressure and negative intrathoracic pressure .
in blunt injury , which includes road accidents and falls , 15% of hospitalized patients have dh .
the first one is immediate herniation of abdominal contents through a large rent in the diaphragm produced by transient but very significant elevation of intraabdominal pressure .
the second , more insidious pattern is a diaphragmatic defect without immediate herniation of intraabdominal contents .
this pattern is often initially overlooked in the acute setting and is only correctly diagnosed in 30% to 40% of cases .
the current practice of nonoperative treatment of solid organ injuries decreases the number of laparotomies in the acute trauma setting , and imaging is notoriously inaccurate in the diagnosis of diaphragmatic injuries .
the time period before onset of symptoms can be variable , sometimes measuring in years .
delayed dh of - ten presents as cardiorespiratory or gastrointestinal complaints , such as dyspnea , coughing , chest pain , abdominal pain , or vomiting .
late presentation of symptoms makes diagnosis even more difficult , and the misdiagnosis rate increases .
when a diaphragmatic hernia presents symptomatically , a 20% to 66% morbidity rate exists due to gastrointestinal obstruction , perforation , incarceration , or respiratory compromise .
other less common causes of dh can be violent coughing , obesity , heavy lifting , multiparity , and iatrogenic causes like esophagogastric surgery . our patient
presented with fever , tachycardia , and respiratory symptoms during her second admission to the hospital , due to the diaphragmatic hernia with simultaneous pneumonia . the inability to communicate with our patient added to the delay in diagnosis and treatment of her dh .
misinterpretation of radiological imaging can miss the diagnosis of dh . in cases of late - presenting cdh ,
baglaj et al report that pneumothorax and pleural effusion were the most common initial findings .
eren et al reviewed imaging of the diaphragm after trauma and suggest that plain radiography as a first - line modality , followed by ct scans , which are feasible in the trauma setting .
mri would be very helpful in visualizing the defect and should be used in patients with a high suspicion of dh .
plain chest film and ct scan of our patient on initial presentation after the motor vehicle crash , only revealed elevation of the right hemidiaphragm and right lower lung collapse ( figures 1 , 2 ) .
this reading perhaps may have been the correct diagnosis at that time , with gradual herniation later on .
interestingly , mris focusing on her spinal injuries were ordered later during hospitalization . in the literature regarding right - sided dh ,
there is controversy about whether right - sided cdh has increased mortality or any influence in the morbidity of the neonate .
the diagnosis of right - sided cdh is often delayed due to milder symptoms compared with symptoms accompanying left - sided cdh .
right - sided cdh can also be associated with a number of congenital defects and complications , and can present with other anomalies.therefore , it is important to recognize that such anomalies exist and perhaps can be present in longstanding right dh .
our patient did not have any anomalies between the lung and liver . however , takedown of adhesions within the diaphragm itself was necessary to free the crura . if this dh had been left unrepaired , it is possible that adhesions between lung , liver , bowel , and diaphragm may have formed .
close proximity of the abdominal contents to the infected lung put our patient at risk for spread of infection .
a recent review of the literature discussing cdh reports that mixed use of thoracoscopy and laparoscopy occurs .
both thoracoscopic and laparoscopic approaches to repair dh have advantages over open surgery in that they result in minimal trauma , promote early recovery , and decrease hospital stay .
thoracoscopy has been successful for repair of dh and may be easier due to good exposure in the thoracic cavity and has decreased the difficulty in reduction of herniated contents .
thoracoscopic repair of cdh in children and in select newborns could be the treatment of choice due to its safety and efficacy .
laparoscopy is a superior early diagnostic tool compared with thoracoscopy , because survey of the entire abdomen can be performed and subsequent repairs made , especially in the acute trauma setting .
laparoscopy provides better orientation and surgical exposure of both the abdominal and thoracic cavity ( through the defect ) , permitting survey for other injuries , whereas in thoracoscopy , only one hemidiaphragm can be inspected at a time .
moreover , laparoscopy allows for the repositioning of herniated contents more easily in the abdominal cavity .
another advantage of laparoscopy is that the patient is not required to undergo single - lung ventilation as needed in thoracoscopy .
a laparoscopic approach is best indicated in anteriorly or centrally located acute or chronic traumatic diaphragmatic hernia without concomitant severe abdominal or thoracic injury .
safety of laparoscopic repair of dh has been proven in many case reports but can be technically challenging .
therefore , surgical experience and meticulous technique are key to the success of laparoscopic repair .
use of a thoracoscopic or laparoscopic approach must be individualized for the patient 's clinical condition , presence or absence of other injuries , type and duration of hernia , and the surgeon 's experience with minimally invasive surgery .
given our patient 's history of blunt trauma , a laparoscopic approach to diaphragmatic repair was preferred over thoracoscopy . using laparoscopy , it was feasible to survey the rest of the abdomen , place the liver and bowel back into the abdominal cavity , and repair the diaphragmatic defect .
diagnosis of traumatic diaphragmatic hernias can be difficult in the acute setting and can present later with variable symptoms and radiological findings .
though right - sided diaphragmatic hernias are less common than left - sided ones , they occur more frequently than was once thought . plain films , ct imaging , and mri can help establish the diagnosis .
both thoracoscopy and laparoscopy are options to minimally invasive surgical approaches to repair , and the approach must be individualized by the clinical condition , presence of other injuries , type of hernia , and the skill level of the surgeon .
laparoscopy is a safe and successful approach to dh repair in the trauma patient , because further assessment and treatment of abdominal and thoracic injury can be facilitated . | background : traumatic diaphragmatic hernias are a diagnostic and therapeutic challenge due to variable presentations .
early repair is important because of risks of incarceration and strangulation of abdominal contents along with respiratory and cardiovascular compromise .
minimally invasive techniques have been useful for diagnosis and treatment of diaphragmatic hernias in both blunt and penetrating trauma.method:we present the case of a 54-year - old victim of a motor vehicle crash who presented with a delayed diagnosis of a right - sided traumatic diaphragmatic hernia . by using a 4-port technique and intracorporeal suturing ,
the hernia was repaired .
this case highlights the difficulties associated with diagnosing diaphragmatic hernias and the role of minimally invasive techniques to repair them.conclusion:minimally invasive surgical techniques are being increasingly used to both diagnose and repair traumatic diaphragmatic injuries with excellent results . |
all - trans - retinoic acid ( atra ) is a family of signalling molecules that are chemically related to vitamin a ( retinol ) .
atra has potent in vitro effects on a number of processes involved in vascular injury and repair , such as modulating smooth muscle cell ( smc ) proliferation and inducing smc differentiation [ 14 ] .
recently , atra was shown to limit restenosis after balloon angioplasty in a rabbit model , as well as favourable effects in reducing neointimal mass and eliciting geometric remodelling in injured rat carotid artery . however , to date there are no reports concerning its role in high - fat diet- ( hfd- ) induced atherosclerosis ( as ) . as is a widespread and one of the most dangerous cardiovascular diseases which cause considerable threat to human health worldwide .
the formation and development of as is a long - term process [ 79 ] which usually lasts for decades and results from complicated environmental factors .
the accumulation of low - density lipoproteins ( ldls ) in the walls of arteries is thought to be an initiator of as , which in turn induces the migration of mononuclear cells and development of inflammation .
eventually , the extracellular matrix interacts with migrating cells to form plaques , which constrict the arteries making them less flexible and more likely to impede blood flow [ 10 , 11 ] .
recently , the activation of platelets and p - selectin are thought to play a central role in homeostasis and thrombosis , which also contribute to the formation of as , as they have been shown to actively promote atherosclerotic lesion development [ 12 , 13 ] .
it has been shown that p - selectin is expressed in mononuclear phagocytes , endothelial cells , and smcs from atheroma [ 11 , 12 ] .
intracellular p - selectin also has the potential to translocate rapidly to the cell surface following stimulation by adenosine diphosphate ( adp ) , thrombin , collagen , and other inflammatory stimuli [ 14 , 15 ] . on the other hand
, fibrinogen has been implicated in the development of inflammation , thrombosis , and as and has been shown to promote p - selectin expression on the surface of platelets [ 16 , 17 ] .
the present study was undertaken to investigate the possible therapeutic effects of atra on hfd - induced as , with a special focus on its potential to inhibit platelet activation , prevent inflammation , and lower blood lipids and reduce aortic lesions , while rosuvastatin was used as a positive control .
new zealand male rabbits ( ordinary class ) were purchased from an experimental animal facility in nanjing , china .
the rabbits ( 2.0 0.2 kg ) were maintained in individual cages under moderate temperature and changing light ( 12-hour light , 12-hour dark ) conditions with access to food and clean water .
the control group ( n = 6 ) was given a basal diet , while another group was given hfd comprising 5% lard and 1% cholesterol .
four weeks later , animals in hfd group were divided into three groups , and received either rosuvastatin calcium ( 3 mg / day ) , atra ( 5 mg / kg / day ) , or the same volume of vehicle by intragastric administration for 8 weeks ( n = 6/group ) .
the rabbits were fasted for 8 hours prior to anaesthesia ( 3% pentobarbital w / v administered at 1 ml / kg ) .
the rabbits were sacrificed , and the aortas were carefully removed and cut open . the tissue was observed and then placed in 10% ( w / v ) neutral formalin for 24 hours .
animal experimental procedures were conducted in accordance with the internal animal care and use committee of the anhui medical university and complied with the guide for the care and use of laboratory .
blood samples were collected in tubes containing 3.8% sodium citrate ( 1/9 v / v ) for biochemical measurements and flow cytometry .
blood sample was centrifuged at 1000 g for 10 minutes to collect the plasma . platelet - rich plasma ( prp ) was obtained by centrifugation at 300 g for 10 minutes .
platelets were then isolated from the prp using pbs to wash twice as previously described [ 13 , 17 ] . resting platelets and platelets activated by thrombin ( 1 u / ml ) or adp ( final concentration is 20 um )
were incubated with fitc - conjugated anti - rabbit cd62p or fibrinogen for 30 minutes at room temperature in the dark .
pbs ( 0.5 ml ) was added to the sample immediately before acquisition of data .
all samples were analyzed by flow cytometry by fc500 ( beckman coulter ) , using 10,000 events per sample with light scatter and fluorescence channels set at a logarithmic gain .
plasma fibrinogen , il-6 , and tnf- were measured using a quantikine rabbit fibrinogen , il-6 , and tnf- enzyme - linked immunosorbent assay ( elisa ) kit ( r&d systems ) .
the root of the aorta from sacrificed animals was longitudinally cut and fixed in 4% paraformaldehyde overnight .
then , the tissue specimens were cut at 5 m thickness for subsequent hematoxylin - eosin ( he ) staining or immunohistochemistry analysis .
the method for he staining of aortic tissues was conducted according to the previous report .
briefly , sections of aortic tissues were deparaffinised , rehydrated , and fixed with methanol-0.3% h2o2 solution for 30 min at room temperature .
then , the sections of aortic tissues were incubated with biotinylated antibody ( anti - rat igg ) and horseradish streptavidin for 30 min at 37c , respectively .
finally , the samples were incubated with diaminobenzidine ( dab ) for coloration and counterstained with hematoxylin for 2 min to stain the nuclei bluish .
the positively stained cells showed brown color ; photographs were taken using the image - pro plus 5.1 image operation system .
the aortic intima - media thickness and vascular wall thickness were measured by jd-801 pathological image analysis system with total 3 slices , each slice was randomly selected five view , and then took a mean value ( m ) .
all the statistical analyses were undertaken using the spss program , version 13.0 , for windows .
comparisons between groups were carried out using one - way analysis of variance ( anova ) and student - newman - keuls ( snk ) method .
compared with control group , hfd - induced significant increases in plasma lipids , including total cholesterol ( total - c ) , low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , and triglycerides ( tg ) .
oral administration of rosuvastatin or atra both significantly reduced total - c and ldl - c , and both significantly increased hdl - c under hfd feeding ( table 1 ) .
these findings suggest that atra intervention may help to restore the lipid imbalance induced by hfd .
the characteristics of arterial lesions were examined by pathological section he staining using light microscopy . in control group ,
no smcs were seen underneath the endothelium ( figure 1(a ) ) . in hfd group ,
there was evidence of foam cells and necrotic substances , where cholesterol crystals and a few inflammatory cells were observed .
the inner elastic plates were broken , and a large proportion of smcs with spindle - shape cores were arranged in disorder ( figure 1(b ) ) .
treatment with rosuvastatin and atra resulted in more even blood vessels , smoother intima , fewer foam cells and inflammatory cells , and less necrotic substances ( figures 1(c ) and 1(d ) ) .
moreover , we measured the aortic intimal thickness and maximal vessel wall thickness in this study .
we observed that hfd feeding resulted in transparent increases in either aortic intimal thickness and maximal vessel wall thickness , which were significantly decreased in either atra or rosuvastatin - treated groups ( figures 1(e ) and 1(f ) ) .
platelet activation was investigated by checking platelet cd62-p expression and fibrinogen binding by flow cytometry . as shown in figures 2 and 3 , the percentage and mfi of fibrinogen and cd62-p
were significantly lower in resting platelets from all groups compared with findings in thrombin and adp - stimulated platelets .
platelets from the control group ( in either resting or activated states ) had lower fibrinogen and cd62-p expression compared with that seen in the hfd , rosuvastatin , and atra groups
. however , when compared to hfd group , platelets from rosuvastatin and atra groups had significantly lower expression of fibrinogen and cd62-p on platelet surfaces , suggesting that atra suppressed platelet activation .
as shown in figures 4(a ) and 5(a ) , respectively , a very low level of fibrinogen deposition and cd62-p expression was found in the intima from the control group , in comparison with that in the hfd group ( figures 4(b ) and 5(b ) ) .
but rosuvastatin or atra treatment resulted in improvements in fibrinogen deposition and cd62-p expression in the intima of arteries ( figures 4(c ) and 4(d ) ) and ( figures 5(c ) and 5(d ) ) .
lesions from hfd group contained extensive foam cell accumulation which was not obvious in atra treatment group .
the presence of significantly larger lesions in hfd group than control group suggested that fibrinogen and cd62-p may play a role in atherosclerotic lesion growth . beside il-6 and tnf- ,
levels of fibrinogen , tnf- , and il-6 were significantly higher in the hfd group than in the control group ( table 2 ) , while they were significantly lowered by both rosuvastatin and atra treatment .
atra is an active metabolite of vitamin a that inhibits cell migration , regulates extrinsic coagulation , and promotes cell differentiation [ 13 , 21 , 22 ] .
although earlier studies had focused on its antitumor effects [ 2123 ] , it has been shown that atra limits restenosis after balloon angioplasty in a rabbit model , suggesting its potential role in other as models . in current study
, we found that oral administration of atra improved characteristics of as and lowered expressions of p - selectin and fibrinogen and concentrations of inflammatory cytokines in as rabbits induced by hfd .
rosuvastatin , a classical lipid - lowering drug of statins in clinic [ 10 , 24 ] , was used as a positive control , and comparable effect was found between atra - and rosuvastatin- the treated rabbits in current study .
the initial stage of as involves the adherence of monocytes to the surface of the injured endothelium , a process facilitated by adhesion molecules .
then , activated platelets cause the weibel - palade body release leading to cd62-p mediated leukocyte rolling , suggesting that platelet cd62-p promotes development of inflammation and deterioration of as [ 2527 ] . on the other hand ,
fibrinogen deposition stimulates chemokine secretion and facilitates a neutrophil - endothelial interaction in septic shock [ 16 , 28 ] .
we have previously shown that fibrinogen enhances platelet intracellular cd62-p levels and affects cd62-p expression on the surface of platelets [ 13 , 17 , 27 , 29 ] .
these findings support a role of fibrinogen in as and suggest that cd62-p may contribute to the formation of as .
although both rosuvastatin and atra treatments resulted in effective amelioration of as in rabbits , much more improvement could be found in the atra group than rosuvastatin - treated rabbits , including less aortic plaques , greater inhibition on fibrinogen deposition , and expression of p - selectin on platelets and intima of aorta .
moreover , we found that the lipids - lowering effect between rosuvastatin and atra showed a little difference , for example the extents of lowering of ldl - c and increasing of hdl - c were greater in the rosuvastatin group than atra .
these findings suggest that atra might reduce as formation through a different mechanism to rosuvastatin .
one possibility is that atra might inhibit pdgf - induced cell proliferation and induce apoptosis in aortic smcs [ 13 ] .
secondly , a more important possibility is that atra might prevent the development of as by greater inhibition on the expression of cd62-p and secretion of fibrinogen , in addition to its lipids - lowering effect , because p - selectin has been demonstrated to be a therapeutic target for as .
clinical and experimental studies support the idea that chronic inflammation plays a major role in the development of as . in our study rosuvastatin and atra
were both shown to counteract the increases in inflammatory cytokines such as il-6 and tnf- resulting from hfd .
these findings are in agreement with previous studies [ 31 , 32 ] which highlight the role of anti - inflammatory properties of rosuvastatin in preventing the development of as , including inhibition of endothelial cell adhesion and monocyte adherence , reduction in tissue plasminogen activation , and suppression of tnf- and mcp-1 in the vessel wall [ 3335 ] .
fibrinogen is not only implicated in development of cardiovascular disease but is also an inflammatory marker .
the inhibition of fibrinogen and inflammatory cytokines by atra underscores the anti - inflammatory effect by atra in as model .
however , it has also been proposed that atra may increase foam cell formation and atherosclerotic lesion via upregulation of cd36 and mcp-1 in vitro , suggesting that the biological functions of atra in as are inclusive which need further investigations .
taken together , the results of present study warrant the further investigation of atra as a potential agent for preventing and treating as and restenosis .
since the underlying signal transduction and mechanisms that result in inhibition of fibrinogen and cd62-p expression by atra remain to be elucidated , further studies should be carried out to investigate the underlying mechanisms of the inhibition of fibrinogen and cd62-p expression by atra , as well as the optimal dosage and route of administration , so that its protective effects can be maximized and side effects be reduced . | background .
all - trans - retinoic acid ( atra ) is effective for many proliferative diseases .
we investigated the protective effects of atra against atherosclerosis . methods .
rabbits were randomly allocated to receive basal diet or an hfd for 4 weeks .
hfd group then received rosuvastatin ( 3 mg / day ) , atra ( 5 mg / kg / day ) , or the same volume of vehicle , respectively , for next 8 weeks .
results .
hfd group showed increases in plasma lipids and aortic plaque formation .
p - selectin expression and fibrinogen binding on platelets or deposition on the intima of the aorta also increased significantly as did the levels of tnf- , il-6 , and fibrinogen in plasma .
after 8 weeks of treatment with atra , there was a significant decrease in plasma lipids and improvement in aortic lesions .
atra also inhibited the expression of p - selectin and fibrinogen binding on platelets and deposition on the intima of the aorta .
conclusion .
atra can ameliorate hfd - induced as in rabbits by inhibiting platelet activation and inflammation . |
driven in part by the human genome1,2 and the hapmap project,3,4 it is now possible to genotype over one million single nucleotide polymorphisms ( snps ) across the human genome in a single assay .
these technologies have made it possible to genotype thousands of cases and controls for any disease and laid the foundation for the genome - wide association study ( gwas ) ( fig.1a ) . with these tools readily available , over 1000 gwass have been published linking nearly 4000 statistically significant loci to over 500 human traits and diseases.5 crucially , the gwas has been very effective in revealing that many immune - related diseases are genetically complex , including multiple sclerosis ( ms ) , ankylosing spondylitis , psoriasis , rheumatoid arthritis ( ra ) , and crohn 's disease.5 one of the strengths of gwas is its unbiased whole genome nature , giving gwas the ability to identify novel genes and pathways linked to common and complex conditions .
surprisingly , many phenotypes of previously unappreciated aetiologies have been linked to immunological genes / pathways by gwass .
representative results from ( a ) a genome - wide association study ( gwas ) and ( b ) a phenome - wide association study ( phewas ) .
( a ) illustration of the phenotype - to - genotype strategy used by gwas along with a representative gwas depicted in the manhattan plot graphing log10(p - values ) across the genome . ( b ) illustration of the genotype - to - phenotype strategy used by phewas including a representative phewas for single nucleotide polymorphism ( snp ) rs1061170 , a snp known to be associated with age - related macular degeneration ( amd).5 the amd icd9 codes are highlighted on the phewas manhattan plot .
due to the burden of multiple comparisons testing , reaching the threshold of statistical significance by gwas can be a challenge . to be considered gwas significant , only those associations with a p < 50e-8 are considered statistically significant.6,7 even if gwas - significant snps are identified , gwass often fail to identify clinically relevant predictive associations and have difficulty explaining a significant portion of the predicted phenotypic heritability .
causative / functional polymorphisms , known or unknown , may be in partial linkage disequilibrium with the genotyped tag snps , resulting in observed weak effects .
addressing these challenges using alternative / complementary strategies is of great importance . without such strategies , our grasp of
one alternative / complementary approach to gwas is the phenome - wide association study ( phewas ) ; this is the same as a gwas , but from a reverse perspective .
whereas a gwas uses a phenotype - to - genotype approach , beginning with a specific phenotype that is associated with genetic variants across the genome , phewas reverses this paradigm by using a genotype - to - phenotype strategy .
phewas can start with a genotype to test for associations over a wide spectrum of human phenotypes the phenome ( fig.1b ) . in 2010 ,
this study demonstrated that a phewas strategy , compared with a gwas strategy , could be applied to identify significant gene disease associations .
for example , it verified that rs3135388 and rs6457620 , two snps in the hla region , were associated with ms and ra , respectively.8 since the first phewas , five additional phewass have focused on genetic targets.913 importantly , four additional phewass have investigated non - genetic targets [ e.g. white blood cell ( wbc ) count],1417 while another phewas assessed both genetic and non - genetic targets.18 a commonality for most phewass is the use of an electronic medical record ( emr ) to define the phenome .
an emr provides an efficient data source for phenotype extraction , as it generally contains longitudinal health histories including prescription records , family histories , laboratory and imaging test results , physician notes , procedure codes , and importantly , the international classification of disease ( icd ) codes .
icd codes are a standardized , internationally recognized , coding system used to define disease status .
the icd codes were initially developed by dr jaxques bertilon in 1893 to classify deaths caused by general diseases affecting specific anatomical sites .
they are now managed by the world health organization ( who ) , although different countries , including the usa , have their own icd adaptations .
in 1975 , who adopted icd version 9 ( icd9).19 in the usa , the implementation of icd9 coding coincided with the digital revolution and has been implemented in emr systems for billing purposes.20 importantly , there are nearly 17 000 possible codes available in the icd9 coding system,21 offering a wide spectrum of phenotypic information at various levels of phenotypic resolution .
for example , icd9 code 714.33 defines monarticular juvenile ra , while 714.32 is pauciarticular juvenile ra .
the highly specific 714.32 , 714.33 , and other related icd9 codes can be consolidated into the 714.3 code , which represents the larger category defining juvenile chronic polyarthritis .
* codes can be combined into the 714 code defining ra and other inflammatory polyarthropathies .
icd9 code 714 is just one of many icd9 codes that fall between icd9 001 ( cholera ) and icd9 999 ( complications of medical care not elsewhere classified ) ( fig.2 ) .
not surprisingly , icd9 codes , with their wide spectrum of phenotypic information , have been employed to define the phenome in most phewass .
this review will discuss such studies and compare general methodologies , describe the limitations and advantages of the phewas design , and provide insight into the potential direction phewas may lead .
international classification of disease version 9 ( icd9 ) spectrum used to define the phenome in phenome - wide association studies ( phewass )
. underlined are icd9 codes represented at varying levels of phenotypic resolution including rheumatoid arthritis ( ra ) and other inflammatory polyarthropathies ( icd9 714 ) , juvenile chronic polyarthritis ( icd9 714.3 ) , and monarticular juvenile ra ( icd9 714.3 ) .
the first phewas was published in 2010 in bioinformatics as proof - of - principle.8 this study associated five genetic targets across a curated phenome built on the backbone of icd9 codes and refined with clinical expertise . in general , high level icd9 codes , including four and five digit icd9 codes ( e.g. 714.3 and 714.33 , respectively ) , were condensed into a common three - digit code ( e.g. 714 ) . under some scenarios , related three - digit codes
were also condensed . under other scenarios , where phenotypes shared very similar four - digit and five - digit icd9 coding , but were deemed to be distinct diseases ( e.g. type i and type ii diabetes mellitus ) , higher order coding was maintained .
icd9 codes with unlikely genetic aetiologies were removed ( e.g. contamination with foreign objects ) . at the simplest level ,
the advantage of using a curated phenome is that the complexity of icd9 coding is reduced .
as such , the power to detect an association may be increased by the increased number of cases and fewer possible phenotypes . by removing selected icd9 codes that appear to have a strong environmental component ,
biases are further introduced when icd9 codes thought to be unrelated to genetics are not analysed .
regardless , a curated phenome can be applied in phewas to validate expected gwas results and to identify potential novel associations .
for example , snp rs6457620 , an hla snp known to be associated with ra,22 was associated with the icd9 code for ra ( icd9 714 ) .
in addition to rs6457620 , another hla snp was genotyped specifically , rs3135388 .
rs3135388 tags for hla - drb1 * 1501 and is known to be strongly associated with ms.5 when hla - drb1 * 1501 ( rs3135388 ) was genotyped , the icd9 code that defines ms ( icd9 340 ) was highly associated with the snp genotype .
novel associations were also reported , including an association between the hla - drb1 * 1501 genotype and erythematous conditions ( icd9 695).8 this example demonstrates how a phewas can complement previously reported gwass and provide novel insights into diseases with unappreciated genetic aetiologies .
another study that used a similar curated phenome was conducted within the electronic medical records and genomics ( emerge ) network.23 unique to this study , gwas was used to inform phewas within the same cohort .
gwas results demonstrated that a common snp near foxe1 ( rs965513 ) was significantly associated with risk for hypothyroidism .
foxe1 , a gene also known as thyroid transcription factor 2 , has been implicated in a variety of thyroid diseases , including a rare form of syndromic congenital hypothyroidism.24 following gwas analysis , rs965513 was assessed by phewas in the same cohort that was used to derive the hypothyroidism cases and controls for the gwas . as expected ,
icd9 codes that define hypothyroidism were significantly associated with the rs965513 genotype by phewas , but other thyroid - related conditions were also associated including non - toxic nodular / multinodular goitre and nutritional deficiency anaemia.9 recently , this gwas - informed phewas approach has also been applied to the study of platelet phenotypes . using a similar emerge population
as described above , this study identified 81 gwas - significant snps including 56 snps associated with platelet count , 29 snps associated with platelet volume , and four snps associated with both .
many of these snps validate previously published gwas results.5 each of the 81 snps was then individually associated with the phenome . for example , rs3819299 , an intronic variant in the hla - b gene , was associated with platelet count as expected.5,25 phewas results for this snp showed that the hla - b genotype is also a risk factor for inflammatory / ankylosing spondylopathies and uveitis .
the role of the hla - b genotype in spondylopathies and uveitis has been previously described.26,27 importantly , a novel association was reported between the hla - b genotype and mastoiditis.13 like the foxe1 example described previously,9 this study demonstrates that when gwas is used to inform phewas in the same population , an expanded understanding of the biological , and potentially clinical , significance for a snp can be achieved .
this gwas - informed phewas approach has also been applied to the study of arrhythmia risk.12 as an alternative approach to a curated phenome , others have applied a holistic method by testing all icd9 codes at multiple levels of phenotypic resolution .
for example , patients coded for monoarticular juvenile ra ( icd9 714.33 ) define a unique case group whereas those coded for pauciarticular juvenile ra ( icd9 714.32 ) define another unique case group . to address the possibility that genetic aetiologies are shared between similar codes
, all 714.3 * codes can be combined into a case group defined by 714.3 and then further combined with like codes to form a separate 714 case group ( fig.2 ) . depending on sample size and frequency restraints , this methodology can generate nearly 17 000 phenotypes.21 the advantage of this method is that it does not make assumptions regarding the genetic or environmental contributions to any one disease .
the disadvantages of using a more holistic phenome include the potential for a reduction in power to detect an association because there are many more phenotypes with small case sizes
regardless , investigators have applied this simplified method to define the phenome , or variations thereof , with success during phewas .
for example , a marshfield clinic patient cohort was genotyped for hla - drb1 * 1501 as a follow up to the first phewas described previously.8 as expected , the hla - drb1 * 1501 genotype was associated with the icd9 code for ms ( icd9 340 ) .
importantly , hla - drb1 * 1501 was also associated with the icd9 code for erythematous conditions ( icd9 695 ) .
this is the first example where a novel phewas finding was independently validated . by applying a holistic approach to define the phenome , and taking advantage of the higher order phenotypes
, it was revealed that the icd9 code for rosacea ( icd9 695.3 ) may be driving the association results of the broader icd9 code defining erythematous conditions .
in addition , this study characterized a novel association between the hla - drb1 * 1501 genotype and the icd9 code that defines alcohol - induced cirrhosis of the liver ( icd9 571.2),10 a phenotype that may have been disregarded in the original phewas due to the potential for a strong environmental component . in support of this novel
phewas finding , previous gwass have demonstrated that hla - drb1 * 1501 is associated with drug - induced liver damage.28,29 another example where an unbiased phenome has been applied in phewas was reported by warner et al.15 this study is unique in that it is the first phewas to use a non - genetic target , specifically wbc count .
the goal of this study was to identify context - dependent associations between wbc count and icd9 codes from patients in an intensive care unit ( icu ) .
expected associations were observed between elevated wbc count and icd9 codes that define leukaemia , including chronic lymphoid ( icd9 204.10 ) , acute myeloid ( icd9 205.00 ) , and chronic myeloid ( icd9 205.10 ) leukaemia .
the wbc count was also associated with diagnosis of clostridium difficile infection , and these patients were at an increased risk for adverse effects because of observed delays in effective treatment and increased length of hospital stay.15 this phewas result may help to alter the current standard of care and to reduce potential adverse effects for icu patients with elevated wbc count and at a high risk for c. difficile infections .
icd9 coding is useful when describing a spectrum of phenotypes , and as a result of its standardized structure and usage , phewas results can be combined or compared across institutions . alternatively , other data types can be applied when defining the phenome .
for example , the population architecture using genomics and epidemiology ( page ) network describes how diverse phenotypes collected from a wide variety of sources , including surveys and medical records , can be applied in phewas across multiple institutions .
first described in 2011,30 and further defined in early 2013,11 the page network focused on 83 previously reported gwas snps that had been genotyped in at least two of the five page network groups .
for example , 3363 phenotypes were described in the women 's health initiative , while 63 were described in the multiethnic cohort study . all phenotypes with p
< 001 were manually grouped into 105 broadly defined standardized phenotypic classes ( e.g. vitamin e levels ) and compared across study groups to identify overlapping significant associations .
this method demonstrated that 48% of expected genotype phenotype associations could be directly validated by phewas , and another 23% represented associations closely related to previously reported genotype phenotype associations .
, this phewas characterized a novel association between the il6r ( rs2228145 ) genotype with numbers of neutrophils and lymphocytes;11 rs2228145 has been previously shown to be associated with c - reactive protein levels.31 regardless of the methodology used to define the phenome , the phewas design has challenges .
conversely , the phewas has unique advantages that make this approach a powerful complementary method for understanding the complexities of human disease .
like gwas , the phewas is a hypothesis - generating approach that is challenged by multiple comparison testing .
for example , if 17 000 phenotypes , the limit of icd9 coding,21 are tested for association with one snp , an association with a p < 29e-6 would be considered statistically significant if a bonferroni correction and an experimental wide of 005 is applied .
conversely , a bonferroni correction may not be an appropriate multiple comparison adjustment because of the lack of independence across many phenotypes , especially in phenomes that use multiple levels of phenotypic resolution when defining individual case groups ( e.g. icd9 codes 714 , 714.3 , 714.33 ) .
the inter - relationship between icd9 codes is depicted in the manhattan plot of fig.1(b ) .
data come from an unpublished phewas for rs1061170 using an unbiased , holistic phenome , as described previously.10 rs1061170 is a non - synonymous snp in the cfh gene and is known to be associated with age - related macular degeneration.5 the most statistically significant icd9 codes for this phewas include 362.52 ( p = 41e-10 ) , 36251 ( p = 33e-8 ) , and 36250 ( p = 40e-7 ) representing exudative , non - exudative and unspecified senile macular degeneration , respectively .
since icd9 362.5 is a composite of all 362.5 * codes , it is intuitive that this code is also significantly associated with the snp genotype ( p = 21e-5 ) .
even though similar codes may co - segregate , divergent icd9 codes may also be correlated for clinical and biological reasons .
for example , it is conceivable that a patient with an icd9 code for hypercholesterolaemia ( icd9 272.0 ) may also have codes that define atherosclerosis ( icd9 440 ) , acute myocardial infarction ( icd9 410 ) , and/or other related comorbidities . the challenge of multiple comparison testing may be further complicated by the ever - increasing granularity of phenotypes , such as the anticipated use of icd10 coding in the usa.21 whereas the icd9 system allows for nearly 17 000 possible codes , the icd10 system allows for more than 155 000 different codes.20 multiple comparison methods that consider inter - relationships between phenotypes ( e.g. permutation testing ) may be more appropriate when measuring statistical significance by phewas .
an advantage of using icd9 codes to define the phenome is that it allows an investigator to comprehensively assemble a wide spectrum of phenotypes in an efficient and cost - effective manner .
each icd9 code is highly variable in frequency and in its positive and negative predictive values.32 it is impractical to manually assess the validity of all phenotypes coded for all patients .
it is also unrealistic to develop sophisticated logic rules to describe every phenotype . to address this challenge , simplistic rules to define cases and controls
can be applied , including the use of the rule - of - two. the rule - of - two states that a patient must be coded two or more times for any icd9 code to be considered a case .
it is intuitive that the rule - of - two increases the positive predictive value , but may result in a reduction in case numbers.32 as phenomes become ever more granulated , average case numbers will undoubtedly be reduced even further .
more advanced high throughput methods , using data beyond icd codes , may be helpful in the future when defining the phenome .
once a phenome is constructed , association testing is often simplified to basic statistical approaches uniformly applied across the phenome .
small numbers of cases for many phenotypes restrict analyses to contingency table tests of independence ( e.g. fisher 's exact test ) precluding the use of covariates . but even if case groups are large enough for regression analysis , standard variables such as age and sex may be inappropriate to implement within a regression model
follow - up phenotype - specific analyses , which may or may not include covariates , may be warranted after an initial phewas screen . like gwas , it is important to validate any phewas association .
, either specific candidate snps can be validated in an independent sample set , or an independent gwas can be conducted . in phewas
, a specific phenotype can be validated in an independent study ( e.g. case control study ) , or an independent phewas can be applied . a specific case
conversely , with imperfect case definitions and small case group sizes in an initial phewas screen , an independent validation phewas may help to identify true associations that may not have been top candidates in the initial phewas . like gwas , and
perhaps even more so for phewas , differences across populations may affect the ability to validate findings .
at the snp level , it would not be unexpected to see very different gwas results from a population with european ancestry compared with a population with african ancestry as a result of significant differences in the linkage disequilibrium structure and allele frequencies between the two populations . in a genetically driven phewas ,
if the snp genotyped is not the functional variant , and/or observed in multiple populations , replicating phewas results may be difficult .
there may be significant differences in clinical care , and the use of icd9 codes , that is observed in different ethnicities , between different physicians , and across different healthcare providers .
even with the challenges of phewas , the utility of phewas has been demonstrated and its application may have significant advantages .
so far , the phewass focusing on genetic targets have concentrated on snps that were already identified by gwas.813,18 even with the complex challenges described above , phewas has demonstrated its capacity to identify expected associations when going in the opposite direction compared with gwas .
the selection of a phenotype for gwas is important for the success of any gwas study .
the selection of a marker , genetic or otherwise , for phewas is also very important . by focusing on variants that have known function and/or clinical significance , phewas has the advantage of simplifying the process by starting with the biology . if there is significant background information regarding a genetic variant 's function and/or clinical significance , that information can be applied directly when interpreting novel phewas results .
this is important because phewas has the distinct advantage of characterizing pleiotropic genetic variants . as of july 2013 ,
1038 gwass had identified 4870 significant associations ( 4018 unique snps ) across the human genome ( p < 50e-8 ) for 547 human traits and diseases .
nearly 13% of these gwas - significant snps ( 519 snps ) are associated with two or more phenotypes , although some phenotypes are similar ( e.g. triglyceride levels and risk for hypertriglyceridaemia).5 rs1260326 on chromosome 2 is an example where multiple phenotypes , some related and some not , are associated with snp genotype at the gwas level . a non - synonymous snp in gckr , rs1260326 was significantly associated with 12 phenotypes in 17 gwass , including triglyceride phenotypes , metabolic networks , urate levels , c - reactive protein levels , total cholesterol , amino acid levels , serum albumin levels , non - albumin protein levels , chronic kidney disease , liver enzyme levels , 2-hr glucose challenge and platelet count.5 this demonstrates that gwass have the capacity to identify pleiotropic variants , but requires that multiple gwass be performed .
phewas has the potential to measure a genetic variant 's pleotropic property in a significantly more comprehensive and efficient manner than gwas .
this point is emphasized by the two phewass described previously where hla - drb1 * 1501 was not only associated with ms , but also with erythematous conditions,8 including rosacea.10 furthermore , other hla variants , such as those described previously in the hla - b gene , can be associated with platelet count , spondylitis , uveitis , and mastoiditis.13 these examples further emphasize the capacity of phewas to study clinically significant diseases that may not otherwise be studied at the genetic level .
understanding the shared genetic aetiologies of multiple diseases by phewas , such as ms and rosacea , is a significant advantage and may provide significant insight into the pathophysiology of numerous conditions
insights that may lead to new treatment strategies while minimizing research costs . for example , medications effective for the treatment of rosacea may be effective for the treatment of ms .
minocycline is an antibiotic with anti - inflammatory properties that is commonly used to treat rosacea .
interestingly , small clinical trials have been conducted to assess the use of minocycline to treat ms and have produced generally positive outcomes.3336 it is the culmination of these advantages and challenges that will dictate how future phewass are conducted .
phewas has the potential to induce new bioinformatic methodologies , result in new disease research opportunities , expand the use of bio - repositories and genetic data , and will hopefully result in clinically significant breakthroughs . in the short term , the number and type of genetic variants assessed by phewas will undoubtedly grow .
approximately 200 snps have been analysed by phewas with all being rooted to previously reported gwas results.913,18 the largest number of snps assessed in a single phewas ( 83 snps ) comes from the page network,11 which falls short of the 4018 unique snps that have been associated with 547 human phenotypes to date.5 although it has been difficult to identify clinically significant associations by gwas , continued focus on gwas snps using higher - resolution phenotypes within a phenome may help to refine initial phenotype
snp associations identified by gwas . as such , phewas may be a complementary strategy to gwas when searching for predictive genetic biomarkers in a clinical setting .
gwas snps are logical starting points for phewas because of the availability of association data , but gwas snps are predominantly tag snps , reside primarily in intergenic regions , and often have no known function . an alternative phewas approach could be to focus on functional variants ( e.g. loss of function variants ) .
the use of gwas snps in phewas exploits known association / phenotypic data while functional variants would exploit known biological insights .
for example , loss of function variants are more likely to be associated with a disease with a stronger effect size compared with other classes of variation.37 in general , mendelian diseases are primarily caused by loss of function mutations,38 and loss of function variants of unknown clinical significance have similar evolutionary selective pressures as known disease - causing mutations.39 focusing on these functional variants in phewas would be analogous to a mouse knockout experiment in a human population without the need to artificially manipulate the human genome .
disease associations while expediting the process of understanding the biology when genetic function is presumed .
as mentioned previously , nearly 13% of all statistically significant gwas catalogue snps are associated with two or more phenotypes.5 many of the pleotropic variants identified to date map to a region on chromosome 6 containing hla genes , suggesting that immunological pathways may play a very important role in many disease aetiologies .
of the 547 phenotypes curated in the gwas catalogue , 5% have at least one gwas significant marker that maps to a 5 mb region containing hla genes .
this is significant because this region makes up less than 1% of the human genome .
very divergent phenotypes map to this region including drug - induced liver injury , ms , stevens johnson syndrome , chronic obstructive pulmonary disease , and narcolepsy.5 as demonstrated by the two hla - drb1 phewas examples mentioned previously,8,10 this region , and variants within , would be a logical target to identify novel association with pleiotropic effects that may have immunological origins . the number of potential phewas targets is only limited by the number of known genetic variants .
an alternative to a focused candidate snp or candidate pathway approach would be a phewas for every snp across the genome .
this strategy would be unbiased to both the phenotypes and genotypes , but would also have a tremendous multiple comparison burden .
the threshold for statistical significance would be orders of magnitude below a gwas significant p - value ( p < 50e-8 ) .
conversely , a phewas - by - gwas approach could be very powerful when developing disease
similar studies have been conducted using combined gwas data,40 but are limited in their ability to address direct disease
network analysis from a single phewas - by - gwas in a large cohort may address this challenge and further describe complex interactions across the phenome and genome .
while a great deal of genetic data can be captured by focused and/or array - based snp genotyping platforms , the future of human genetics will rely heavily on next - generation sequencing ( ngs ) . using
ngs data for phewas - by - gwas analysis , a whole order of complexity will be introduced .
this may include the incorporation of indels , complex rearrangements , and copy number variants in the context of phewas .
furthermore , ngs will result in the identification of many rare variants , many of which may or may not have obvious functions within their respective genes .
incorporating rare variants in the context of phewas ( e.g. gene - specific burden testing ) should be possible .
this reality is quickly coming to fruition as ngs is incorporated into standard medical practices.41,42 although genetic variables served as the focus of the initial phewass , the future of phewas is not limited to genetics .
liao et al.18 associated autoantibody levels to the phenome in patients with or without a diagnosis of ra .
this was done with four different autoantibodies , including anti - citrullinated protein , anti - nuclear , anti - tissue transglutaminase , and anti - thyroid peroxidase antibodies .
anti - thyroid peroxidase antibody levels were associated with hypothyroidism , as expected . moreover , novel statistically significant associations were observed when anti - nuclear antibody levels were associated with the phenomes of ra and non - ra patients .
anti - nuclear antibodies are known to be associated with genetic risk factors linked to systemic lupus erythematosus.43 in ra patients , high - titre antinuclear antibodies were associated with sjgren's / sicca syndrome . in non - ra patients ,
this antibody was associated with chronic non - alcoholic liver diseases.18 whereas liao et al .
assessed autoantibodies in a phewas , warner et al16 conducted a phewas by comparing the frequency of diagnostic codes between multiple myeloma patients in the icu and a larger icu patient cohort .
this study characterized treatment - related and disease - related complications of multiple myeloma over time .
these and other examples14,15,17 demonstrate how phewas can be applied to identify potentially novel / clinically significant associations using non - genetic phewas candidates .
using pre - existing de - identified clinical data may allow investigators to circumvent the difficulties in patient recruitment and avoid the significant resources needed for genotyping .
furthermore , lack of dependence on genetics may allow for exploitation of significantly larger patient cohorts .
this may address some of the challenges described above , most notably , small sample size for many phenotypes within a phenome .
regardless of approach , phewas will always be limited by how well the phenome can be defined .
efforts to reliably define phenotypes using emr data have been limited to specific phenotypes.23 although these disease - specific methods can reliably discriminate between cases and controls , they do not provide a high throughput mechanism to define the thousands of phenotypes within a phenome .
automated medical informatic tools capable of reliably defining the phenotypes within a phenome will be required .
this may include machine learning methods that are able to incorporate various types of data beyond simple icd9 coding , including laboratory values , procedure codes , physician notes , and/or prescription records .
as the number of phenotypes increases and the phenotypic resolution becomes ever more accurate and specific , sample sizes for cases will shrink without expanded cohorts .
this will require multi - institutional collaborative networks working together to develop , implement and apply phewas .
as the use of an emr becomes standard practice , bio - repositories continue to grow , and genomic medicine becomes readily applied in a clinical setting ( e.g. ngs ) , phewas has the potential to unlock novel discoveries that would not be possible otherwise .
| over the last decade , significant technological breakthroughs have revolutionized human genomic research in the form of genome - wide association studies ( gwass ) .
gwass have identified thousands of statistically significant genetic variants associated with hundreds of human conditions including many with immunological aetiologies ( e.g. multiple sclerosis , ankylosing spondylitis and rheumatoid arthritis ) .
unfortunately , most gwass fail to identify clinically significant associations . identifying biologically significant variants by gwas
also presents a challenge .
the gwas is a phenotype - to - genotype approach . as a complementary / alternative approach to the gwas
, investigators have begun to exploit extensive electronic medical record systems to conduct a genotype - to - phenotype approach when studying human disease specifically , the phenome - wide association study ( phewas ) .
although the phewas approach is in its infancy , this method has already demonstrated its capacity to rediscover important genetic associations related to immunological diseases / conditions .
furthermore , phewas has the advantage of identifying genetic variants with pleiotropic properties .
this is particularly relevant for hla variants .
for example , phewas results have demonstrated that the hla - drb1 variant associated with multiple sclerosis may also be associated with erythematous conditions including rosacea .
likewise , phewas has demonstrated that the hla - b genotype is not only associated with spondylopathies , uveitis , and variability in platelet count , but may also play an important role in other conditions , such as mastoiditis .
this review will discuss and compare general phewas methodologies , describe both the challenges and advantages of the phewas , and provide insight into the potential directions in which phewas may lead . |
with reference to the findings of contemporary medicine , diabetes mellitus is a common metabolic disorder .
this complication which is resulted from insulin insufficiency or dysfunction may cover over 5.4% of population or 57.2 million people by the year 2025 .
although the pathophysiology of diabetes is not yet well understood , evidences have suggested the impact of free radicals in the pathogenesis and development of diabetes .
most common cited symptoms of diabetes mellitus are increased serum glucose , unusual thirst , frequent urination , blurred vision , hyperphagia , nausea and vomiting as well as loss of weight . on the treatment pathway , a number of medicaments are administered in addition to the insulin therapy .
other than the current pharmacotherapy of diabetes , interventions concerned to the complementary and alternative medicine are also considerable .
accordingly , extensive information would be obtained from the beliefs of folk medicine and practices by local healers as well as remained manuscripts of traditional medical systems . with respect to the findings of integrative medical systems ,
it is remarkable that traditional persian medicine ( tpm ) plays a considerable role in the development of treatment approaches during the medieval era . during the 8 to 12 century ad
, persian physicians and scholars such as rhazes and avicenna gathered the medical information of traditional remedies from china , egypt , greece and india and also supplemented it by their own findings and experiences . in this regard , remained medical and pharmaceutical manuscripts authored by early persian practitioners encompass large and remarkable information on various categories of ailment . among those , considering the nutritional aspects and pharmacological of diabetes and related complications
accordingly , current study has been carried out to gather the most cited medieval persian information on diabetes as well as those management approaches .
medieval reports encompassing the profile of definition and terminology , classification and etiology , as well as sign and symptoms of diabetes were collected and analyzed from selected medical textbooks of tpm .
the concerned collection was based on the analysis of remaining manuscripts of medieval persia from 9 to 18 centuries ad involving medical and pharmaceutical textbooks of this period . in this
regard , manuscripts namely canon of medicine ( 11 century ) , al- aghraz al- tebbieh va al- mabahes al - alayieh ( 12 century ) , kholasat - ol - tajarob ( 16 century ) , tebb - e - akbari ( 18 century ) and eksir - e - aazam ( 19 century ) were considered for the medical points . on the other hand , pharmacological treatment aspects of diabetes in medieval period were also gathered from main persian pharmaceutical manuscripts containing the liber continents by rhazes ( 9 and 10 centuries ) , the canon of medicine by avicenna ( 11 centuries ) , alabnie an haghaegh - ol - advieh by aboo mansour heravi ( 10 century ) , ikhtiyarat - e - badiyee by zein al - din attar ansari shirazi ( 14 century ) , tohfat ol moemenin by mohammad tonkaboni ( 17 century ) and makhzan ol advieh by aghili - shirazi ( 18 century ) .
these texts are considered as most important sources among medical and pharmaceutical manuscripts of persian medicine .
other textbooks such as matching the old medicinal plant names with scientific terminology,dictionary of medicinal plants , dictionary of iranian plant names , popular medicinal plants of iran and indian medicinal plants were also used to check and reconfirm the plants scientific names .
medieval manuscripts which were reviewed for the survey it is also considerable , that an extensive search on most popular databases as pubmed , sciencedirect , scopus and google scholar was performed to reconfirm the anti diabetic activities of reported herbs as well as concerned pharmacological actions .
as a metabolic disorder , diabetes is characterized in that of unexpected serum glucose elevation .
general manifestation of diabetes is known as polyuria , polydipsia and polyphagia as well as weight loss .
type 1 is resulted from loss of insulin secretion and the most prevalent of type 2 is reported as obesity and unusual weight gain .
in addition to these main groups , gestational diabetes mellitus ( gdm ) is also present as the commonest metabolic disorder during pregnancy .
genetic defects of beta cell function , insulin genetic dysfunction , exocrine pancreatic disorders and endocrinopathies such as hyperthyroidism and glucagonoma may cause diabetes . also a kind of diabetes namely diabetes insipidus is reported in medical textbooks which is related to impaired angiotensin - vasopressin secretion .
main clinical approaches in diabetes mellitus include life style modification , drug therapy to control glycemia and prevention / management of associated complication .
the term , ziabites , or doolab ( water wheel ) in persian , was used by early persian scholars to define and describe what is currently spoken as diabetes . since
, ziabites is a greek word ; it was mentioned by greek physicians well before the entrance to persian manuscripts .
concerning the definition , it was remarked that the disorder of ziabites , which in that patient suffers from excessive thirst , is highly associated with kidney function .
it was said that retentive force of kidney is impaired and hence can be distinguished by excessive urine output .
furthermore , the diffusion of fluid from other organs such as liver and intestines into the kidneys was noted as another etiological symptom that persian scholars believed in .
similar to the fundamental mechanisms of humoral medicine , the disorder was said that may be resulted from an imbalance in the kidney temperament as well as whole body .
hence , treatment was based on the modification of temperament and humors to reach to an optimum or balanced state . regarding the mentioned dystemperament ,
the disorder was categorized into two main types as hot ( ziabites - e - har ) and cold ( ziabites - e - barid ) .
the hot type was reported to be accompanied by unusual and excessive thirst , unusual colored urine and frequent urination as well as excessive augmentation of libido and weight loss or emaciation . on the other hand , in cold type , thirst but less than the above type , bright colored urine , loss or reduction in libido , loss of appetite and also weight loss and slimming . according to the medieval reports , the hot type of ziabites was more prevalent . in a resulted version of ziabites
, persian scholars remarked that ants and other insects may be attracted to the patient 's urine .
also it was mentioned by avicenna that by standing the urine of a diabetic patient under surrounding air , a residue is leaved with sticky and sweet tastes as honey .
ziabites , in this case was named as bole - e - shirin or sweet urine .
therefore , all medications and management strategies which were considered for the impaired kidneys should also be applied for this disorder , as it was mentioned by early persian practitioners .
also a brief overview and comparison between modern aspects of diabetes and medieval description of ziabites is represented in table 3 .
medieval classification of ziabites ziabites and diabetes , the comparison concerning the management strategies as well as preventive approaches for ziabites , there are lots of recommendations in reviewed manuscripts .
the six essential schemes for health maintenance which were called setteh - e - zarurieah ( involving observation and optimization of six main parameters as weather , food and beverage , retention and release , repose and movement , sleep and wakefulness as well as sensual and mental states ) were considered as main preventive approaches .
these parameters were being observed prior medication and are considered as life styles in current medicine .
pharmaceutical manuscripts of persian medicine offer plenty of natural remedies for the management of ziabites .
natural medicines were applied solely ( mono - ingredient ) or in combination to other medicaments ( multi - ingredients ) .
first approach was based on nutritional therapy ( tadbir ba ghaza ) and life style modification . in this condition ,
natural agent with opposite temperament compared to the type of ziabites was administered . in the other word ,
foods or beverages such as beer , milk , barely soap , plum and courgette khoresht , yogurt and verjuice which possess cold temperament were applied for hot type of ziabites . on the other hand ,
medicinal herbs in form of simple or compound dosage forms were administered to manage the disorder .
most cited natural medicaments concerned to either hot or cold ziabites are shown in table 4 .
other than the natural pharmacotherapy , special physical manipulations ( a'mal - e - yadavi ) have also being applied by early persian physicians .
these approaches were usually involved venesection ( fasd ) , cupping ( hijamat ) and massaging ( dalk ) . regarding the type of ziabites and also patient 's dystemperament , these interventions were applied before or after herbal therapy .
in addition to physical manipulation , other approaches were also considered . as an example , sitting in water was recommended by rhazes .
as a metabolic disorder , diabetes is characterized in that of unexpected serum glucose elevation .
general manifestation of diabetes is known as polyuria , polydipsia and polyphagia as well as weight loss .
type 1 is resulted from loss of insulin secretion and the most prevalent of type 2 is reported as obesity and unusual weight gain .
in addition to these main groups , gestational diabetes mellitus ( gdm ) is also present as the commonest metabolic disorder during pregnancy .
genetic defects of beta cell function , insulin genetic dysfunction , exocrine pancreatic disorders and endocrinopathies such as hyperthyroidism and glucagonoma may cause diabetes . also a kind of diabetes namely diabetes insipidus is reported in medical textbooks which is related to impaired angiotensin - vasopressin secretion .
main clinical approaches in diabetes mellitus include life style modification , drug therapy to control glycemia and prevention / management of associated complication .
the term , ziabites , or doolab ( water wheel ) in persian , was used by early persian scholars to define and describe what is currently spoken as diabetes . since
, ziabites is a greek word ; it was mentioned by greek physicians well before the entrance to persian manuscripts . concerning the definition ,
it was remarked that the disorder of ziabites , which in that patient suffers from excessive thirst , is highly associated with kidney function .
it was said that retentive force of kidney is impaired and hence can be distinguished by excessive urine output .
furthermore , the diffusion of fluid from other organs such as liver and intestines into the kidneys was noted as another etiological symptom that persian scholars believed in .
similar to the fundamental mechanisms of humoral medicine , the disorder was said that may be resulted from an imbalance in the kidney temperament as well as whole body .
hence , treatment was based on the modification of temperament and humors to reach to an optimum or balanced state . regarding the mentioned dystemperament ,
the disorder was categorized into two main types as hot ( ziabites - e - har ) and cold ( ziabites - e - barid ) .
the hot type was reported to be accompanied by unusual and excessive thirst , unusual colored urine and frequent urination as well as excessive augmentation of libido and weight loss or emaciation .
on the other hand , in cold type , thirst but less than the above type , bright colored urine , loss or reduction in libido , loss of appetite and also weight loss and slimming .
according to the medieval reports , the hot type of ziabites was more prevalent . in a resulted version of ziabites , persian scholars remarked that ants and other insects may be attracted to the patient 's urine .
also it was mentioned by avicenna that by standing the urine of a diabetic patient under surrounding air , a residue is leaved with sticky and sweet tastes as honey .
ziabites , in this case was named as bole - e - shirin or sweet urine .
therefore , all medications and management strategies which were considered for the impaired kidneys should also be applied for this disorder , as it was mentioned by early persian practitioners .
also a brief overview and comparison between modern aspects of diabetes and medieval description of ziabites is represented in table 3 .
concerning the management strategies as well as preventive approaches for ziabites , there are lots of recommendations in reviewed manuscripts .
the six essential schemes for health maintenance which were called setteh - e - zarurieah ( involving observation and optimization of six main parameters as weather , food and beverage , retention and release , repose and movement , sleep and wakefulness as well as sensual and mental states ) were considered as main preventive approaches .
these parameters were being observed prior medication and are considered as life styles in current medicine .
pharmaceutical manuscripts of persian medicine offer plenty of natural remedies for the management of ziabites .
natural medicines were applied solely ( mono - ingredient ) or in combination to other medicaments ( multi - ingredients ) .
first approach was based on nutritional therapy ( tadbir ba ghaza ) and life style modification . in this condition ,
natural agent with opposite temperament compared to the type of ziabites was administered . in the other word ,
foods or beverages such as beer , milk , barely soap , plum and courgette khoresht , yogurt and verjuice which possess cold temperament were applied for hot type of ziabites . on the other hand ,
medicinal herbs in form of simple or compound dosage forms were administered to manage the disorder .
most cited natural medicaments concerned to either hot or cold ziabites are shown in table 4 .
other than the natural pharmacotherapy , special physical manipulations ( a'mal - e - yadavi ) have also being applied by early persian physicians .
these approaches were usually involved venesection ( fasd ) , cupping ( hijamat ) and massaging ( dalk ) . regarding the type of ziabites and also patient 's dystemperament , these interventions were applied before or after herbal therapy .
in addition to physical manipulation , other approaches were also considered . as an example , sitting in water was recommended by rhazes .
before mid of the nineteenth century , it was believed that diabetes is a disease related to kidney function . however , in 1922 , the isolation of insulin from animal pancreas confirmed the diabetes as an endocrine disorder .
findings from main manuscripts of persian medicine revealed that there are some similarities between the disorder of ziabites and what is currently accepted as diabetes mellitus .
unusual thirst , polyuria and weight loss are some of the main similarities . on the other hand , ziabites may also be closed to diabetes insipidus .
but it should be noted that polyuria would not be stopped in this type of diabetes even if no fluid is ingested . in the type ii of diabetes ,
a large group of affected patients may have no notification about their disorder and serum glucose evaluation should be carried out to confirm the disorder .
it seems that in medieval time , also a large number of patient with this type of diabetes were left uncured in due to lack of paraclinical examinations . with reference to the management of ziabites ,
considering the six essential parameters ( setteh - e - zarurieah ) were highly emphasized by early persian practitioners . similar to these rules , current medicine has also rendered considerable recommendation .
avoidance of environmental pollution , scheduled and adequate exercise and sport , appropriate sleep and awareness ( sleep less than six hours is mentioned as a main predisposing factor for diabetes ) , eluding stress and psychological tensions as well as proper nutritional regimen and rich fiber foods have high impact on diabetes . concerning the treatment of different types of ziabites , medicinal plants having specific temperament which was related to either hot or cold ziabites were used for the treatment .
totally 46 different medicinal herbs were found as cure for hot or cold types of ziabites .
a search through considered databases revealed that almost 70% of reported medicaments were active for anti diabetic effects .
most considered mechanisms for medieval herbs were as reduction in serum glucose level or fasting blood sugar , increasing serum insulin level or stimulating the insulin release and inhibition of intestinal glucose absorption [ table 2 ] .
it was believed that there are differences between various types of temperaments in different patients . in this
regard , persian practitioners differentiated ziabites regarding the patient 's temperament and used the relevant medication .
however , differences in temperament are also evaluated regarding the endocrine and immune system . therefore , according to the temperament , administration of similar medication for patients suffering from a disease should be avoided . as ziabites
is composed of hot and cold types in tpm , different treatments have been recommended for each individual types of the disease and patients .
current research was a survey to clarify the knowledge of medieval persian scientists on disorder of diabetes and related pharmacological intervention strategies . reported remedies are based on centuries of experience and thus might be of beneficial for further studies to the management of diabetes and related unwanted effects . | diabetes mellitus is a common metabolic disorder presenting increased amounts of serum glucose and will cover 5.4% of population by year 2025 . accordingly , this review was performed to gather and discuss the stand points on diagnosis , pathophysiology , non - pharmacological therapy and drug management of diabetes this disorder as described in medieval persian medicine . to this , reports on diabetes were collected and analyzed from selected medical and pharmaceutical textbooks of traditional persian medicine .
a search on databases as pubmed , sciencedirect , scopus and google scholar was also performed to reconfirm the anti diabetic activities of reported herbs .
the term , ziabites , was used to describe what is now spoken as diabetes .
it was reported that ziabites , is highly associated with kidney function .
etiologically , ziabites was characterized as kidney hot or cold dystemperament as well as diffusion of fluid from other organs such as liver and intestines into the kidneys .
this disorder was categorized into main types as hot ( ziabites - e - har ) and cold ( ziabites - e - barid ) as well as sweet urine ( bole - e - shirin ) .
most medieval cite signs of ziabites were remarked as unusual and excessive thirst , frequent urination and polydipsia . on the management , life style modification and observing the essential rules of prevention in persian medicine as well as herbal therapy and special simple manipulations were recommended .
current investigation was done to clarify the knowledge of medieval scientists on diabetes and related interventions . reported remedies which are based on centuries of experience might be of beneficial for- further studies to the management of diabetes . |
it represents a key animal model , with vertebrate - like characteristics as a dorsal nerve cord , a notochord , segmental muscles , pharyngeal gill slits and a post - anal tail , but it is by far less complex than vertebrates .
the genetic programs patterning the amphioxus embryo are also similar to those of vertebrates ' embryo , although its genome did not undergo to extensive gene duplication events that characterize vertebrates ' genomes .
this gives to researchers the opportunity to look at highly conserved embryonic pathways , supported by much simpler genetic information .
amphioxus was at first thought to be a mollusc by the german zoologist peter simon pallas in 1774 1 , until 1834 when the italian naturalist gabriel costa first described its similarity to vertebrates 2 , reconsidering it as a primitive chordate and naming it branchiostoma lubricum .
two years later , in 1836 , william yarrell named it amphioxus ( amphioxus lanceolatus ) and described its notochord 3 .
much later , for its peculiar composition of muscle cells , the amphioxus notochord was defined as an example of evolutionary novelty 4 .
they exists at least 28 different species of the genus branchiostoma described and distributed over the world , but three of them are the mainly studied : the european b. lanceolatum , the floridian - caribbean b. floridae and the east asian b. belcheri .
amphioxus has been successfully used for developmental studies even if it is not easy to obtain embryos in laboratory conditions , due to the unpredictable nature of spawning events .
presently , a full sequencing genome project of the specie b. floridae is nearly to be completed at the joint genome institute ( us department of energy ) , witch will open the door to a new era for the amphioxus community researchers , and it will strongly enhance comparative studies with other vertebrates and invertebrates animal models .
although many studies have been conducted on amphioxus , most of them related to morphological and bio - molecular analyses to elucidate developmental - gene pathways , as far we know investigations concerning the determination of free amino acids have not been carried out in this emerging animal model .
concentrations of free amino acids in animal tissues , particularly in the nervous system , are of great significance from a biochemical and physiological point of view .
indeed , one of the principal roles of free amino acids is to act as substrates for key enzymatic reaction as well as to regulate the balance of nitrogen compounds .
moreover , alterations in the concentration of some amino acids lead to important metabolic disorders . for examples , in humans high concentrations of cystine in the urine
indicate cystinuria , a congenital defect in the amino acid transport system , which controls the transit of small molecular mass substances in and out of cells across their membranes , ultimately resulting in cystine precipitation in the renal pelvis , forming cystine stones ( calculi ) .
an excess of phenylalanine in the blood can cause an illness called phenylketonuria , a mental retardation characterized by the absence of l - phenylalanine hydroxylase , which transforms l - phenylalanine into tyrosine .
high levels of tyrosine in the blood are a symptom of the tyrosinosis , a disease characterized by hepatic cirrhosis and renal damage .
nevertheless , mental retardation can result from high concentration of any of the following amino acids : glycine , valine , lysine , glycine , homocystine and hydroxiproline 5 .
in addition , some amino acids are involved in neurotransmission ; for example , l - glu and l - asp are known to be important neurotransmitters , whereas glycine and gaba as neuroinhibitors 6 .
another amino acid , namely d - aspartic acid ( d - asp ) , has been recently found to be involved in neurotransmission .
evidence has been obtained demonstrating that d - asp plays an important role in hormones release from the neuroendocrine system 7 and acts as a novel neurotransmitter 6 .
d - asp is present at high concentration in the nervous tissues of opistobranchia 8 , molluscs , tunicates , crustaceans , amphibians , reptiles , fishes , birds and mammals 9 .
in addition , d - asp has also been detected as a nuclear component of cells in the mammalian hypothalamus - neurohypophyseal system 10 and in the retina of sepia officinalis , where it is implicated in vision 9 .
therefore , in the present preliminary study , in addition to determining the concentration of free l - amino acids in the neural tube of amphioxus , we determined the concentration of d - asp in order to try to elucidate whether this amino acid could also be involved in neurotransmission .
european specie ) were collected in the bay of argels - sur - mer ( southern coast of france ) , at a depth of approximately 10 mt between october and november 2005 .
animals were kept for a week in tanks in the laboratory before dissection , at a water temperature of 15 c and a 10/14 day - night light cycle .
filtered seawater was changed daily and antibiotics ( streptomycin 100 mg / l and penicillin 10
specimens were dissected under a stereoscopic microscope using a micro - fine knife with a 4 mm cutting edge ( f.s.t . ) , on ice .
no anaesthetic was used to immobilize the animals , in order to not alter hplc measurements .
each group , containing a pool of 10 neural tubes weighting about 3 mg total ( approximately 0.3 mg each ) , was homogenized in 0.1 m tca ( trichloroacetic acid ) at a ratio of 1:20 , and centrifuged at 15,000 g for 15 minutes .
20 l of the tca supernatant obtained from homogenisation of each pool of neural tubes was neutralized with 20 l of 0.1 m naoh and mixed with 100 l of 0.5 m pyrophosphate buffer ( ph 9.5 ) and 20 l of opa - nac reagent , which was obtained by mixing 20 mg opa ( o - phthaldialdehyde ) with 10 mg nac ( n - acetyl - l - cysteine ) in 2.0 ml of methanol .
after 2 min , distilled water was added for a final volume of 1.0 ml , and 100 l of this mixture was injected into a supelcosil c-18 hplc column ( 0.45 x 25 cm in length and 5 m beads ; supelco , bellefonte , pa , usa ) .
amino acids were eluted using two buffers : a ( 5% acetonitrile in 30 mm citrate phosphate buffer , ph 5.6 ) and b ( 90% acetonitrile in double - distilled water ) , using the following gradient : 0 to 5% b over 15 min , 5 - 35% b over 25 min , 30 - 100% b over 5 min , staying at 100% b for 3 min , and then returning to 100% a for 1 min .
amino acids were detected fluorometrically at an excitation wavelength of 325 nm and an emission wavelength of 415 nm .
d - asp eluates with a peak at 6.4 min , followed by l - asp 0.7 - 0.8 min later and remaining well separated from other amino acids ( fig .
in order to confirm that the peak eluting at 6.5 min was d - asp , 20 l of the tca supernatant was neutralized with 20 l of 0.1 m naoh and mixed with 100 l of 0.5 m pyrophosphate buffer ( ph 8.2 ) and 2.0 l of bovine d - aspartate oxidase were added to the reaction ( d - aspo , 1 - 2 mg / ml produced by over - expression in e. coli ) 7 and incubated at 37 c for 20 - 30 min .
subsequently , 200 l of 0.1 m sodium pyrophosphate ( ph 10.0 ) was added and the hplc analysis was repeated as above
. the near complete reduction of the peak at 6.4 min confirmed the presence of d - asp ( data not shown ) . in order to know the exact concentration of d - asp and l - amino acids , a standard curve was made with known amounts of amino acids purchased from sigma chemical co ( st .
louis , mo ) as follows : 10 l of a mixture of 19 different l - amino acids , each at a concentration of 0.1 mol / ml , and d - asp at a concentration of 0.01 mol / ml that was mixed with 100 l of 0.5 m pyrophosphate buffer ( ph 9.5 ) and 20 l of opa - nac .
after 2 min , distilled water was added to obtain a final volume of 1.0 ml , and 100 l of it were injected into the hplc column .
100 l of injected mixture contained 0.01 nmoles of d - asp and 0.1 nmoles of each of the other amino acids .
european specie ) were collected in the bay of argels - sur - mer ( southern coast of france ) , at a depth of approximately 10 mt between october and november 2005 .
animals were kept for a week in tanks in the laboratory before dissection , at a water temperature of 15 c and a 10/14 day - night light cycle .
filtered seawater was changed daily and antibiotics ( streptomycin 100 mg / l and penicillin 10
specimens were dissected under a stereoscopic microscope using a micro - fine knife with a 4 mm cutting edge ( f.s.t . ) , on ice .
no anaesthetic was used to immobilize the animals , in order to not alter hplc measurements .
each group , containing a pool of 10 neural tubes weighting about 3 mg total ( approximately 0.3 mg each ) , was homogenized in 0.1 m tca ( trichloroacetic acid ) at a ratio of 1:20 , and centrifuged at 15,000 g for 15 minutes .
20 l of the tca supernatant obtained from homogenisation of each pool of neural tubes was neutralized with 20 l of 0.1 m naoh and mixed with 100 l of 0.5 m pyrophosphate buffer ( ph 9.5 ) and 20 l of opa - nac reagent , which was obtained by mixing 20 mg opa ( o - phthaldialdehyde ) with 10 mg nac ( n - acetyl - l - cysteine ) in 2.0 ml of methanol .
after 2 min , distilled water was added for a final volume of 1.0 ml , and 100 l of this mixture was injected into a supelcosil c-18 hplc column ( 0.45 x 25 cm in length and 5 m beads ; supelco , bellefonte , pa , usa ) .
amino acids were eluted using two buffers : a ( 5% acetonitrile in 30 mm citrate phosphate buffer , ph 5.6 ) and b ( 90% acetonitrile in double - distilled water ) , using the following gradient : 0 to 5% b over 15 min , 5 - 35% b over 25 min , 30 - 100% b over 5 min , staying at 100% b for 3 min , and then returning to 100% a for 1 min .
amino acids were detected fluorometrically at an excitation wavelength of 325 nm and an emission wavelength of 415 nm .
d - asp eluates with a peak at 6.4 min , followed by l - asp 0.7 - 0.8 min later and remaining well separated from other amino acids ( fig . 2a , 2b ) .
in order to confirm that the peak eluting at 6.5 min was d - asp , 20 l of the tca supernatant was neutralized with 20 l of 0.1 m naoh and mixed with 100 l of 0.5 m pyrophosphate buffer ( ph 8.2 ) and 2.0 l of bovine d - aspartate oxidase were added to the reaction ( d - aspo , 1 - 2 mg / ml produced by over - expression in e. coli ) 7 and incubated at 37 c for 20 - 30 min .
subsequently , 200 l of 0.1 m sodium pyrophosphate ( ph 10.0 ) was added and the hplc analysis was repeated as above
. the near complete reduction of the peak at 6.4 min confirmed the presence of d - asp ( data not shown ) . in order to know the exact concentration of d - asp and l - amino acids , a standard curve was made with known amounts of amino acids purchased from sigma chemical co ( st .
louis , mo ) as follows : 10 l of a mixture of 19 different l - amino acids , each at a concentration of 0.1 mol / ml , and d - asp at a concentration of 0.01 mol / ml that was mixed with 100 l of 0.5 m pyrophosphate buffer ( ph 9.5 ) and 20 l of opa - nac .
after 2 min , distilled water was added to obtain a final volume of 1.0 ml , and 100 l of it were injected into the hplc column .
100 l of injected mixture contained 0.01 nmoles of d - asp and 0.1 nmoles of each of the other amino acids .
to our knowledge , analysis of free amino acids in amphioxus tissues has never been carried out before . in the present study , using a sensitive hplc method , we have determined the concentration of 19 free l - amino acids and d - aspartate in the central nervous system of amphioxus b. lanceolatum that mainly consists in an anterior brain vesicle and a neural tube ( or spinal cord ) ( fig .
1a , 1b , 1c ) . in order to detect fluorimetrically the concentration of each amino acid and of d - asp
, the dissected tissues were reacted with a mixture consisting of o - phthaldialdehyde and n - acetylcysteine , and then injected into the hplc column . using this method
we were able to obtain a clean separation of d - asp from l - asp as well as from other amino acid .
figure 2 shows a typical chromatographic example of a separation and quantification of each individual amino acid at hplc from an extract of amphioxus neural tube ( fig .
2a ) , and a standard mixture of amino acids consisting of 19 different l - amino acids and d - asp ( fig .
in table 1 the composition of each amino acid ( expressed in mol / g tissue standard deviation ) found in amphioxus nervous system is reported , and it is compared to that found in the brain of the cephalopod octopus vulgaris 12 , taken as representative of mollusc phyla , and of human brain as representative of mammals 13 , 14 . as shown in table 1 , in amphioxus nervous system five free amino acids occur at relative concentrations between 1.10 and 4.50 mol / g of tissue , whereas all other amino acids are between 0.11 to 0.45 mol / g of tissue . however , among the higher concentrated amino acids , l - glutamic acid is the compound at highest concentration with 4.50 0.60 mol / g of tissue followed by l - aspartic acid ( 3.90 0.30 ) , l - alanine ( 2.50 0.40 ) , l - glycine ( 2.35 0.35 ) , and l - serine ( 1.10 0.25 ) ( fig . 2a and table1 ) . as in molluscs and mammals , l - glutamic acid ( and its amide derivative l - glutamine ) and l - aspartic acid are present at high concentration in amphioxus neural samples compared to other free amino acids
. these two compounds are well known to be excitatory amino acids and neurotransmitters of the nervous system .
thus , also in amphioxus these amino acids , since they are present at high concentration , are probably involved in neurotransmission . in molluscs ,
interestingly in addition to l - glu and l - asp other two amino acids are found at very high concentration : taurine and alanine , but their role in nervous system is still not completely understood . among other neuroactive substances , we found that the amino acid derivative gaba , the main inhibitory neurotransmitter of vertebrates , is present in low concentration compared to the other two animal 's specie analyzed . about this topic a comprehensive study on gabaergic neuronal network in amphioxus have been carried out , demonstrating that gaba is a widely used neurotransmitter in both its central and peripheral nervous system 15 .
the results obtained in the present study mimic the evolution of the nervous system from invertebrates to vertebrates .
in fact , in molluscs and in mammals l - aspartic acid and especially l - glutamic acid are always found at the highest concentration in the nervous system compared to other amino acids .
our data confirm this statement since also in cephalochordates , which from an evolutionistic point of view are between molluscs and mammals , we found high value of these two excitatory amino acids .
in addition , as shown in table 1 , the nervous system of the mollusc o. vulgaris possess other two non - excitatory neutral amino acids : taurine and alanine .
they are know to be involved in nervous system as neuroinhibitory transmitter , but considering its extremely abundance in molluscs brain it is supposed that they are needed for specific functions in molluscs , since neither the amphioxus nor mammals possess such high concentrations of these two compounds .
the present study thus strongly suggests that there is a relationship between the evolution of the nervous systems and its composition in free amino acids .
the cephalochordates are phylogenetically closer to mammals than molluscs and this largely agree with our biochemical analysis .
still , it is unresolved why amphioxus and mammals , conversely to molluscs , do not possess such a high quantity of taurine and alanine .
another interesting data regards the occurrence of d - aspartic acid in the nervous system of the amphioxus .
in fact , as shown in table 1 , this amino acid is present in amphioxus neural tube at a very high concentration ( considering the d / l ratio percentage ) , as well as in molluscs , while it is present at relatively low concentration in mammals ( table 1 ) , and in another animal phyla 9 , 16 , 17 .
it has been demonstrated that during the last embryonic life in rat and chicken a transient increase of d - asp occurs in the brain ( 200 - 300 nmol / g tissues ) and then it returns to lower levels ( 20 - 30 nmol / g ) , and remains at that concentration for the rest of the life span of the animal 18 , 19 .
in addition , it has been also shown that d - asp is involved in the function of the endocrine system , where it is capable to induce the release of hypothalamic gnrh ( gonadotropin releasing hormone ) , pituitary hormones ( lh , luteinizing hormone and gh , growth hormone ) 7 and testosterone 20 . recently ,
d - asp has also been found in the nervous ganglia of the opistobranchia aplysia limacina , where it plays an active role in neurotransmission processes 8 .
all the above data thus lead us to hypothesize that in amphioxus d - aspartic acid may also have a neuroendocrine role .
it is most likely that the concentration of d - asp ( 0.28 0.04 mol / g tissue ) and of other amino acids reflects in a way the evolution of the nervous system under a biochemical point of view since their amount in amphioxus has been found at concentration between that of molluscs ( invertebrate ) and mammals ( vertebrate ) .
draft of the anterior part of an adult amphioxus ( a ) ( adapted from r. fox ) and the correspondent real picture ( b ) .
c corresponds to the amplification of the red square in b. acronyms : nc , notochord ; nt , neural tube .
scale bar in b = 1 mm ; in c = 250 m ( a ) hplc separation of l - amino acids and of d - asp from amphioxus nervous system . the area of the peaks corresponds to amino acid concentrations coming from 0.1 mg of tissue .
( b ) hplc separation of a standard amino acids mixture containing 10 pmoles of d - asp and 100 pmoles of l - amino acids .
major free l - amino acids and d - aspartate in the nervous system of the amphioxus .
results are expressed in mol / g tissue ( mean sd ) . from ref .
13 ; from ref . 14 . for more details , see in the text . | the cephalochordate amphioxus is the closest invertebrate relative to vertebrates . in this study , using hplc technique , free l - amino acids ( l - aas ) and d - aspartic acid ( d - asp ) have been detected in the nervous system of the amphioxus branchiostoma lanceolatum . among other amino acids
glutamate , aspartate , glycine , alanine and serine are the amino acids found at the greatest concentrations .
as it occurs in the nervous system of other animal phyla , glutamate ( l - glu ) and aspartate ( l - asp ) are present at very high concentrations in the amphioxus nervous system compared to other amino acids , whereas the concentration of taurine and -aminobutyric acid ( gaba ) is very low .
interestingly , as it is the case in vertebrates , d - aspartic acid is present as an endogenous compound in amphioxus nervous tissues . the physiological function of excitatory amino acids , and d - aspartate in particular ,
are discussed in terms of evolution of the nervous system under an evo - fun ( evolution of function ) perspective . |
in a study in the previous issue of critical care , thooft and colleagues investigated the impact of increasing mean arterial pressure ( map ) by norepinephrine ( ne ) on systemic hemodynamics and organ perfusion in a series of patients with septic shock .
the increase in map from 65 to 85 mm hg was associated with increased cardiac output and mixed venous blood oxygen saturation ( svo2 ) and a decreased blood lactate level .
the increase in map was also associated with improvement of the microcirculation state as assessed by near - infrared spectroscopy ( nirs ) at the thenar eminence level in 13 patients and by sidestream dark field ( sdf ) imaging at the sublingual level in 6 of them .
nirs technology allows investigators to evaluate the functionality of the thenar eminence microcirculation through the measurement of the muscle tissue oxygen saturation ( sto2 ) and its changes in response to a vascular occlusion test ( vot ) .
sdf videomicroscopy allows investigators to assess the sublingual microvascular blood flow and capillary density by using several indices .
the presence of marked microvascular abnormalities detected by each of these techniques has been associated with increased mortality in septic shock . before being enrolled ,
the patients of thooft and colleagues had already been stabilized at an map of 65 mm hg with fluid therapy and ne administration and their svo2 was above 65% , in accordance with surviving sepsis campaign guidelines .
although these goals appeared to have been achieved , severe derangements of organ perfusion were detected by both the nirs and sdf techniques .
this agrees with the concept that , in septic shock , microcirculatory abnormalities can persist despite apparent correction of macrocirculatory abnormalities .
however , it is still unclear whether the persistence of microcirculatory abnormalities at the early phase is related only to intrinsic septic organ / tissue injury or is a consequence of insufficient macro - hemodynamic resuscitation and , in particular , achievement of a still suboptimal organ perfusion pressure .
current recommendations suggest an map of at least 65 mm hg given that , in patients with a history of hypertension or other vascular comorbidities or in those with increased abdominal pressure , a higher level of map may be required .
however , increasing the doses of ne carries the theoretical double risk of ( a ) decreasing cardiac output and oxygen delivery through an increased afterload effect and ( b ) worsening tissue perfusion through excessive peripheral vasoconstriction .
recent studies have evaluated the effects on cardiac output and organ perfusion markers of using ne to increase map from 60 ( or 65 ) mm hg to 85 ( or 90 ) mm hg in patients with septic shock [ 8 - 10 ] .
all of the studies supported the idea that , in spite of the increase in cardiac output , achieving the upper map target is not always associated with a remarkable improvement in organ perfusion .
in contrast to previous studies , the study by thooft and colleagues demonstrated that an map target of 85 mm hg might be more appropriate than 65 mm hg for the microcirculation .
one of the strengths of the study is that these results were found by using two different techniques : nirs and sdf imaging .
the higher sto2 reperfusion slope ( during a vot ) at the highest level of map suggests that some degree of microvascular recruitment occurred at this level , confirming results found in patients with severely hypotensive sepsis .
in addition , increasing map from 65 to 85 mm hg significantly improved two of the sdf - derived indices : the perfused vessel density and the microvascular flow index .
this suggests that both microvessel recruitment and microcirculatory blood flow increased at the map of 85 mm hg .
these findings can be the result either of a proper perfusion pressure effect in some pressure - dependent areas or of the increase in systemic blood flow observed at the highest level of map .
the association of increases in cardiac output with ne - induced increases in map is a common finding that has been reported previously [ 8 - 13 ] . clearly , the results of thooft and colleagues argue against the idea that , owing to excessive vasoconstriction , ne could exert deleterious effects on the microcirculation when an map of 85 mm hg is targeted .
it is important to note that 85 mm hg is still low compared with map values of the healthy population , especially in the age group of the study by thooft and colleagues . because the sdf analysis was conducted on only 6 of the 13 patients in the study by thooft and colleagues , the interpretation of their data must be cautious .
it must be underlined that , using study designs similar to that of thooft and colleagues in the early phase of septic shock , jhanji and colleagues and dubin and colleagues reported no overall change in sublingual sdf - derived variables with ne - induced increases in map ( above 60 mm hg in 16 patients and 65 mm hg in 20 patients , respectively ) . nevertheless , dubin and colleagues reported an inverse correlation between the baseline level of the sublingual microcirculation state and the microcirculatory response to the ne - induced increase in map from 65 to 85 mm hg .
thus , septic patients with normalized microcirculation ( after initial hemodynamic resuscitation ) might not benefit from any additional increase in map above 65 mm hg .
in contrast , septic patients with persistent altered microcirculation , though surviving sepsis campaign recommended endpoints are achieved , might benefit from a further increase in map beyond the ' magic ' value of 65 mm hg .
the results of thooft and colleagues are in agreement with this sensible concept , which clearly needs to be implemented in clinical practice .
this paper , like other recent ones , has the merit of bringing to light the idea that , at least during the early phase of sepsis , the microcirculation is not dissociated from the macrocirculation , despite what is claimed by pioneers of the microcirculation evaluation .
more importantly , the paper suggests that bedside assessment of microcirculation might help clinicians to define the optimal level of macrocirculatory resuscitation targets ( for example , map and svo2 ) in each patient .
map : mean arterial pressure ; ne : norepinephrine ; nirs : near - infrared spectroscopy ; sdf : sidestream dark field ; sto2 : muscle tissue oxygen saturation ; svo2 : mixed venous blood oxygen saturation ; vot : vascular occlusion test .
| the surviving sepsis campaign guidelines suggest targeting a mean arterial pressure of at least 65 mm hg to maintain organ perfusion pressure during septic shock . however , the optimal mean arterial pressure can be higher in patients with a history of hypertension or other vascular comorbidities or in those with increased abdominal pressure . in a given individual , the adequate mean arterial pressure target can be difficult to define with the routine hemodynamic parameters ( for example , cardiac output , central or mixed venous blood oxygen saturation , and urine output ) . near - infrared spectroscopy and sidestream dark field imaging have emerged as promising technologies for monitoring the microcirculation at the bedside .
these new methods could provide additional clues to help define the adequate blood pressure to target during the resuscitation phase of septic shock . |
the present study aims to evaluate the epidemiological profile of the 277 patients who suffered facial fractures and were treated at the hospital do trabalhador in curitiba in the state of parana , in 2010 , with an emphasis on variables such as gender , age , cause , and anatomical sites of fractures , and compared with clinical findings of other studies in the literature .
maxillofacial trauma can be considered as a devastating assault found in trauma centers because of the emotional consequences and the possibility of deformity , in addition to its economic impact on a healthcare system1 .
a maxillofacial injury involves not only the soft tissues and bones but also , by extension , can affect the brain , eyes , sinuses , and teeth .
therefore , it is a trauma of a multidisciplinary approach , involving mainly the specialties of otolaryngology , ophthalmology , plastic surgery , maxillofacial , and neurosurgery6 .
severe facial injuries may , in addition to psychological disorders , result in decreased productivity due to visual loss and damage during swallowing and phonation , increasing the costs arising from the trauma .
the group most affected , men of working age , commonly found in many studies , partly explains the impact on productivity2 . in the last 4 decades ,
the incidence of facial trauma has increased , mainly due to increased traffic accidents and urban violence , especially in young individuals21 .
they are very common in emergency rooms around the world and assume a prominent role in the care to polytrauma patients .
the maxillofacial region is very prone to injury because of its prominence and little protection in the region .
today the association of alcohol , drugs , car driving , and increased urban violence are increasingly present as causal factors of facial trauma , and what is worse , increasing its complexity7 .
however , the epidemiology of craniofacial fractures can vary in type , frequency , severity , and cause , depending on the medical center studied and the period considered3 , because many factors influence the cause of maxillofacial injuries around the world , for example , cultural , economic , social , religious and geographical factors4 .
therefore , epidemiological studies focused on facial injuries are of great interest as to the knowledge of the occurrence and quantity and severity of presentation , allowing the adoption of preventive measures for its control and management of patients .
table 1 shows the summary of several epidemiological studies of facial fractures in the literature in several cities .
the table was divided according to the author , publication year , study site , number of patients suffering from facial fractures , male prevalence , age more acomentida , main causes of fractures and other broken facial bones .
we conducted a non - randomized retrospective study , of 277 patients with confirmed facial fractures diagnosed on radiographs and/or computed tomography , from the otolaryngology service at the hospital do trabalhador ( ht ) located in the city of curitiba in paran during the year 2010 .
the study included all patients treated at the emergency room of ht , attended by the department of otolaryngology , who were victims of facial trauma in the period from january 1 to december 31 .
we prepared a protocol for data collection performed by analysis of records and records of emergency in the sector of the medical file of the ht .
this protocol included the following variables : medical record number , patient name , age , sex , origin , birth date , location of facial fracture , and cause .
the cause of the fractures was studied according to : motor vehicle crashes ( combinations of collisions with cars , motorcycles , trucks / buses , and others ) , fall / bicycle accident , interpersonal violence with or without a firearm , drops own height or level falls , fractures arising from sports practices and impact / collisions with objects from accidental causes .
the locations of the fractures were classified into facial : fractures of the nasal bone , mandible , orbital , maxillary , frontal , zygomatic bone fractures , and le fort i , ii , or iii .
of the 277 patients treated in 2010 evaluated in this study , 207 were male ( 74.72% ) and 70 female ( 25.27% ) ; the male to female ration was 3:1 ( figure 1 ) .
the patients ' ages ranged from 1 to 79 years with a mean of 33.57 years .
distribution of patients with facial fractures according to sex in percentage ( n = 277 ) .
the most affected age group was 20 to 39 years with 44.03% ( figure 2 ) .
the extremes of age , younger than 10 years and older than 70 years , accounted for respectively 13 ( 4.69% ) and 8 ( 2.88% ) patients .
distribution of patients with facial fractures according to age in percent ( n = 277 ) .
the merits , 88.44% were residents of the city of curitiba , 10.83% of the metropolitan region of curitiba , and 0.72% from the interior the state of paran .
the number of patients treated per month ranged from 16 to 27 , average of 23 , september being the month with the highest number of visits ( table 2 ) . regarding occupation
, 55.23% had a professional activity , 16.96% were students , 5.77% were dependents , 5.41% were retirees , and 16.60% were unemployed or had indeterminate profession . regarding the cause of facial fractures , our chart review showed that the cause of fractures was interpersonal violence without firearm in 84 ( 30.32% ) cases , interpersonal violence with a firearm in 17 ( 6.31% ) , motor vehicle accidents ( car , truck , bus , motorcycle ) in 48 ( 17.32% ) , injuries while walking in 11 ( 3.97% ) , from falls from a height in 48 ( 17.32% ) , due to drop in level in 16 ( 5.77% ) , due to impact / collisions with objects in 35 ( 12.63% ) , fall / bicycle accidents in 10 ( 3.61% ) , and sports injuries in 8 ( 2.88% ) ( table 3 ) .
the etiologic distribution by age showed a prevalence of violence without firearm in all age groups from 10 to 59 , with a peak incidence in the age group 3039 years with 41% .
falls from height predominated in the age groups 010 and 6079 , representing 57.9% of the causes of facial fractures in the latter .
the motor vehicle crashes were the second most common cause in the age group 2039 years with 22.13% of cases ( table 3 ) .
the motorcycles were involved in 47% of automobile accidents . regarding the cause and gender ,
falls from height were the main cause of facial fractures in women , corresponding to 23 ( 32.85% ) cases in females followed by violence without firearm in 15 ( 21.42% ) cases , and third , vehicle accidents with 12 ( 17.14% ) cases . in the males , the main cause was violence without firearm in 69 ( 33.33% ) cases , followed by motor vehicle accidents in 36 ( 17.39% ) , and third , by impact / collision with objects in 26 ( 12.56% ) cases ( table 4 and figure 3 ) .
distribution of patients with facial fractures by cause and gender ( n = 277 ) .
patients had a total of 391 facial fractures , which were isolated in 205 ( 74% ) cases and associated with 2 or more locations in 72 ( 26% ) .
mandibular fracture was the second most common , found in 56 ( 14.32 ) cases , followed by the orbit with 50 ( 12.78% ) .
fracture of the maxilla was the fourth most frequent with 47 ( 12.02% ) , followed by the zygomatic bone with 39 ( 9.97 ) .
the more complex fractures le fort i , ii , and iii corresponded to 11 ( 2.81% ) cases ( figure 4 ) .
the face is susceptible to a variety of possible traumas , and it is important to note that aggression between facial injuries , especially fractures , plays a major role in emergency care worldwide .
our study revealed a predominance of male patients with facial fractures corresponding to 74.72% , a 3:1 ratio , compatible with the literature including palma et al , 78% , falcon et al10 , 84% , and macedo et al , 72.8% .
this higher incidence in males may be linked to cultural and social factors , considering that the males represent most of the economically active population , exhibit more abuse of alcohol and drugs , practice more contact sports , are involved in the majority in traffic , and thus are more exposed to the factors responsible for facial injuries .
however , the incidence of trauma among women has increased in recent years due to the increased participation of women in the commercial workforce16
17
18 .
the patients ' ages ranged from 1 to 79 years with a mean of 33.57 years .
the most affected age group was 20 to 29 years , with 23.82% of cases .
the age group also is in agreement with the findings of other authors such as silva et al13 .
it is understandable that violence occurs more among young people by their restlessness and risk taking behaviors , including traffic risks influenced by extremely fast behavioral and moral changes16 . at the extremes of age
, patients younger than 10 years and older than 70 years accounted for respectively 13 ( 4.69% ) and 8 ( 2.88% ) patients , consistent with the literature findings6 - 12 there are studies that show low incidence of facial trauma in children and the elderly due to the attention of family , stay at home , and care of children , as well as the characteristics of aging such as lessened social activity and sport , leaving little infrequently , and usually accompanied when they do.14
15 . with regard to professional activity , our study showed that 55.23% had jobs and 16.96% were students , similar to studies by macedo et al12 jcm junior et al in 20106 , and brazil et al 200616 , the latter , which showed 60.5% of patients and 16.9% economically active students .
regarding the cause of facial fractures , our study showed the main causative agent of facial fractures was interpersonal violence without firearm with 84 ( 30.32% ) , and the second leading cause motor vehicle accidents ( car , truck , bus , motorcycle ) with 48 ( 17.32% ) cases , as well as falls from height with the same values .
these data are compatible with the current literature , which shows a growing share of violence as a cause of facial fractures , surpassing automobile accidents .
this confirms the tendency of most recent national studies to show an increased incidence of interpersonal violence and suggest that this is the main cause in facial trauma6
8
12
18 .
this is mainly due to an increase in urban violence and a drop in severity of motor vehicle accidents due to public policies aimed at greater control speeding on the roads and encouraging the use of seat belts .
moreover , the ban on drunk driving and the introduction of air bags and side protection bars have decreased the incidence of facial fractures , as well as the complexity17
20 .
however , it calls attention to the involvement of 47% of motorcycles in traffic accidents , owing to the fact that unsafe vehicle speed abuse is practiced on the streets and that these vehicles are increasingly used because they are a means of low - cost transport .
injurt from falls dominated as the cause in people aged under 10 and over 60 , as these are extremes of age , because locomotion and balance are directly proportional to age .
the consciousness of appearance of the face and its social importance increases with age ( during a fall , older children and adults consider to protect the face)14
15 . in the case of elderly physiological mechanisms such as altered proprioception , weakness , tremor , and
infeco urinary tract and lung , and alcohol are also referred to18 . with the number and location of the fracture , a total of 391 facial fractures , being isolated in 205 ( 74% ) cases , and 2 or more locations associated in 72 ( 26% )
mandibular fracture was the second most commonly found in 14.32% cases , followed by 12.78% of the fractures in the orbit ; maxilla fracture was the fourth most frequent with 12.02% followed by the zygomatic bone with 9.97% .
the more complex fractures le fort i , ii , and iii accounted 2.81% of cases .
these data differ widely in the literature , which show that in many cases , the jaw is the main bone fractured because it is the only mobile bone of the face and thus is more vulnerable to strong impact and fracture10
16 . on the other hand , studies like those by silva et al.13 and leite et al.19 corroborate our data demonstrating the nasal bone as the main fractured bones due to its prominent position , location on the mid - face , as well as the thin structures of the bones that constitute it .
the most serious fractures such as le fort occurred in 2.81% of cases . of the total number of le fort fractures ,
45% were due to interpersonal violence without firearm ; trauma and morbidity are high in this type of fractures , and the cause is easily avoidable .
the study of the epidemiology of facial trauma is important for the studying the cause and effects of facial trauma , assist in the initial care of these patients , and publishing preventive policies .
our study showed that males are more affected and the main causes of facial fractures are assaults , motor vehicle accidents , and falls .
they are more common in young patients aged 2029 years followed by those aged 3039 years . in the vast majority
are isolated fractures and the most affected bone is the nose , followed by the lower jaw , orbit , maxillary , and zygomatic . for the prevention of facial fractures , we must bear in mind the respect for traffic laws and routinely use seatbelts and helmets .
furthermore , the incidence of facial fractures can be reduced by strategies for dealing with falls in children and the elderly , avoiding hostile situations , and creating stricter laws and public policies to reduce traffic accidents and reduce interpersonal violence . | summary
introduction : epidemiological studies that focus on facial injuries are of great interest for the knowledge of occurrence and severity of presentation .
aim : to study the epidemiological profile of 277 patients who suffered facial fractures at the hospital do trabalhador ( ht ) , with an emphasis on variables such as sex , age , cause , and anatomical sites of fractures , comparing the clinical findings with other studies .
method : retrospective nonrandomized chart review of 277 patients who were treated at ht by the ent service during the full year 2010 , victims of facial fractures .
results : of 277 patients , 74.72% were male and 25.27% female ( ratio 3:1 ) . according to age ,
the fractures were distributed as follows : 09 years : 4.69% , 1019 years : 17.32% , 2029 years : 23.82% , 3039 years : 20.21% , 4049 years : 16.24% , 5059 years : 10.83% , 6069 years : 3.97% , and 6079 years : 2.88% .
the cause of trauma was most frequently interpersonal violence , 36.45% , followed by falls , 23.09% , and motor vehicle crashes with 17.32% .
regarding location , nasal fracture was the most common , with 44.75% , followed by the mandible , 14.32% , orbit , 12.78% , maxillary , 12.02% , zygomatic , 9.97% , 3.32% and front le fort 2.88% .
conclusion : the patients were mostly males , aged 2130 years , victims of aggression with the most commonly fractured bone being the nose . the adoption of personal and public strategies and measures may prevent facial fractures . |
well into its third decade , the hiv / aids epidemic continues to pose a major public health challenge . currently , unaids estimates that 40 million persons are living with the disease worldwide and that there are more than 20 million related deaths .
the caribbean region hosts the highest incidence rates for hiv / aids in the americas and the second highest prevalence rates in the world among adults age 1549 years .
it is estimated that between 350,000 and 560,000 adults in the region are living with hiv , resulting in a prevalence rate of 2.3% . in jamaica , as of december 2003 , 8097 cases of aids have been reported with more than 1000 new cases identified in 2003 ; an estimated 1.6% of the adult population is infected with hiv . even in light of a number of education campaigns and interventions ,
hiv / aids and other sexually transmitted infections ( stis ) remain the second leading cause of death among males and females age 3034 years in jamaica .
prevention programs have been promoting hiv testing as one possible way to combat the spread of the disease .
this strategy is based on the premise that an awareness of the risk and severity of hiv will lead to hiv testing and protective behavior adoption .
however , research examining the relationship between hiv testing and subsequent protective behavior has found mixed results .
although some studies did find a significant relationship between awareness of one 's hiv serostatus and protective sexual behaviors , others found no such relationship.[7 - 10 ] when looking specifically at hiv testing behaviors among university students , a limited number of studies were identified .
one study revealed that students seeking hiv testing were more likely to report previous unsafe vaginal and oral sex compared with non - test seekers .
however , those engaging in unprotected sex did not perceive themselves to be at risk of contracting hiv .
another study found similar results , with 40% describing sexual activities that placed them at some risk for hiv infection and other stis .
with respect to predictors of hiv testing , one study found that age was significantly associated with testing ; older students were more likely to seek testing than were younger students .
another study focused on the psychosocial differences between those seeking a test and those who had never had a test .
those seeking testing perceived more benefits and fewer barriers and were ranked higher in consideration of future consequences .
they were also at higher risk of becoming infected with hiv according to reports of risky sexual behavior as compared with those not seeking hiv testing .
very few caribbean - based studies were identified that specifically examined predictors of testing or subsequent behaviors associated with testing .
a study of puerto rican youth in drug treatment found that 66% of those enrolled in ambulatory drug treatment centers agreed to take an hiv test ; volunteers , when compared with nonvolunteers , were more likely to be males who reported fewer years of education and engaged in risky sex and drug behaviors .
with respect to posttest behaviors , a study of adult drug users in puerto rico found that after receiving a positive test result , persons were significantly less likely to continue to engage in unprotected vaginal sex .
another study of at - risk persons from trinidad found that persons who received hiv testing and subsequent counseling decreased their high - risk sexual behaviors with nonprimary partners , but not with primary or steady partners . in an effort to identify studies that specifically targeted university students in the caribbean for the purpose of measuring hiv - related attitudes and behaviors ,
survey data from 7000 university students in puerto rico revealed that students held attitudes that are associated with high - risk sexual behavior ; furthermore , a number of students were engaging in risky sexual activities .
therefore , in an attempt to address the apparent gap in hiv testing research among university students in the caribbean , we drew from research with other populations to develop an explanatory model to examine hiv testing . a hierarchical model
was developed , using submodels that reflect the various factors hypothesized to be related to hiv testing .
the following hypotheses were developed : submodel 1 : sociodemographic characteristics ( age , sex , marital status , hiv education , hiv awareness ) are directly related to perceived risk of hiv .
perceived risk has been found in previous studies to be associated with hiv - testing behaviors.[21 - 24 ]
submodels 3a3c : hiv testing is directly related to sexual behaviors such as condom use and number of sex partners .
previous research has found associations between hiv testing and a number of hiv - related behaviors.[7 - 10 ] therefore , the present study seeks to identify the predictors of hiv testing ( sociodemographics , perceived risk of hiv ) and the role of hiv testing on sex - related behaviors ( condom use , number of sex partners ) .
data for these analyses were taken from the university of the west indies hiv / aids knowledge , attitudes and behaviors study 2001/2002 , a collaborative research effort between the university of the west indies and the ministry of health , kingston , jamaica . a 193-item questionnaire was developed related to hiv / aids education and prevention .
the questionnaire was based upon social - psychological theories of behavior change , including the health belief model , theory of reasoned action , and social cognitive theory .
in addition , instruments from other universities conducting hiv research with students , the us centers for disease control and prevention ( cdc ) , and the ministry of health , jamaica , were used to facilitate the development and inclusion of standard questions that have been found to employ reliable and valid measures of hiv - related attitudes and behaviors across various samples.[27 - 30 ] our survey instrument was reviewed and approved by the research and ethics committee , ministry of health , and included items addressing knowledge of transmission , knowledge of risks associated with specific sexual behaviors , attitudes toward persons living with hiv / aids , hiv testing behaviors , sexual history , attitudes toward condoms and safer sex , sexual behaviors by steady and nonsteady sex partners , and drug and alcohol use during sexual activity .
the instrument was piloted with a sample of 15 students in order to assess the ease of completing the instrument , to determine whether the questions were easily understood , and to ensure that the instrument could be completed in a timely fashion . on the basis of the first piloting phase
, revisions were made and the instrument was piloted again with 10 additional students . following the results of the second piloting phase , minor revisions were made and the instrument was finalized . due to the nature of the questions and the possible perceived threat of addressing issues of a sexual nature , the instrument was self - administered with no identifiers , providing anonymity to the respondents .
data were gathered between june 2001 and february 2002 from 1252 students in various classes across the university faculties , representing 11% of the total student population for the enrollment period .
it is important to note that although the study employed nonrandom sampling , statistical testing indicated no significant differences between the study sample and the university population for both age and sex distributions .
because sexual transmission of hiv is the predominant mode of transmission in jamaica ( less than 2% of reported cases among adults are attributed to injecting drug use ) , sexually inexperienced persons would not be considered at risk of hiv transmission nor an important group to target for hiv testing .
therefore , only data for 961 students ( 77% ) who reported being sexually experienced were included in the present study .
the following operationalizations were used : hiv testing : students were asked if they had ever had an hiv test with responses being categorized as yes ( 1 ) and no ( 0 ) . consistent condom use : frequency of condom use was measured separately with both steady and nonsteady sex partners .
a steady sex partner was defined as " someone with whom you have sexual intercourse on a regular or consistent basis , like a husband / wife or boy / girlfriend . "
a nonsteady sex partner was defined as " someone with whom you have sexual intercourse but only occasionally or even just once . " for both partner types , students who reported always using condoms during the 3 months prior to the survey were coded as consistent condom users ( 1 ) .
remaining students who reported using condoms usually , about half the time , seldom , or never were coded as inconsistent condom users ( 0 ) .
those reporting no sexual partner during the 3 months prior to the survey were excluded from the regression analyses .
multiple sex partners : students reporting 2 or more sex partners in the 12 months prior to the survey were coded as having multiple sex partners ( 1 ) , while those with 1 partner were coded as not having multiple sex partners ( 0 ) . due to issues of reliability associated with long recall periods , those reporting no sex partner during this time period
perceived risk of hiv : students were asked to report their perceived risk of becoming infected with hiv .
response categories included great risk , some risk , not much risk , and no risk .
responses were dichotomized into categories of great / some risk ( 1 ) and little / no risk ( 0 ) .
hiv education : students were asked if they had attended a lecture , course , or community forum about hiv / aids in the 12 months before the survey .
those who reported attending such an activity were coded as receiving hiv / aids education ( 1 ) , while remaining students were coded as not receiving such education ( 0 ) .
hiv awareness : students were asked if they knew someone who was infected with hiv or had died from aids .
those responding yes were coded as having a personal awareness of hiv ( 1 ) , while those reporting knowing no such person were coded as having no personal awareness of hiv ( 0 ) .
marital status : students were asked to report their current relationship status with categories including legally married , common - law , visiting partner ( steady sex partner ) , boy / girlfriend , or no relationship .
students who reported being legally married or involved in a common - law relationship were coded as being married ( 1 ) , while remaining students were coded as not being married ( 0 ) .
age : students were asked to report their age , in years , on their last birthday .
those reporting being under the age of 25 were coded as youth ( 1 ) while those 25 years of age and older were coded as adults ( 0 ) .
this categorization was based on the world health organization 's ( who ) definition of youth .
sex : students were asked to report if they were male ( 1 ) or female ( 0 ) . both bivariate ( chi - square ) and multivariate ( hierarchical logistic regression ) analyses were employed .
chi - square analyses were used to examine the differences between persons who reported previous hiv testing and those with no previous hiv testing .
in addition , in order to understand the relationship among all the model variables with respect to the dependent variables of interest , hierarchical logistic regression modeling was used .
this type of regression analysis takes an iterative form ; an initial simple model is followed by more complex models in which the dependent variable from the immediately preceding model becomes a predictor along with the previous predictors .
all model variables have been dichotomized to facilitate the logistic regression analyses with the comparison group for each variable coded as ( 1 ) as described in the above section .
data for these analyses were taken from the university of the west indies hiv / aids knowledge , attitudes and behaviors study 2001/2002 , a collaborative research effort between the university of the west indies and the ministry of health , kingston , jamaica . a 193-item questionnaire was developed related to hiv / aids education and prevention .
the questionnaire was based upon social - psychological theories of behavior change , including the health belief model , theory of reasoned action , and social cognitive theory .
in addition , instruments from other universities conducting hiv research with students , the us centers for disease control and prevention ( cdc ) , and the ministry of health , jamaica , were used to facilitate the development and inclusion of standard questions that have been found to employ reliable and valid measures of hiv - related attitudes and behaviors across various samples.[27 - 30 ] our survey instrument was reviewed and approved by the research and ethics committee , ministry of health , and included items addressing knowledge of transmission , knowledge of risks associated with specific sexual behaviors , attitudes toward persons living with hiv / aids , hiv testing behaviors , sexual history , attitudes toward condoms and safer sex , sexual behaviors by steady and nonsteady sex partners , and drug and alcohol use during sexual activity .
the instrument was piloted with a sample of 15 students in order to assess the ease of completing the instrument , to determine whether the questions were easily understood , and to ensure that the instrument could be completed in a timely fashion . on the basis of the first piloting phase
, revisions were made and the instrument was piloted again with 10 additional students . following the results of the second piloting phase , minor revisions were made and the instrument was finalized . due to the nature of the questions and the possible perceived threat of addressing issues of a sexual nature , the instrument was self - administered with no identifiers , providing anonymity to the respondents .
data were gathered between june 2001 and february 2002 from 1252 students in various classes across the university faculties , representing 11% of the total student population for the enrollment period .
it is important to note that although the study employed nonrandom sampling , statistical testing indicated no significant differences between the study sample and the university population for both age and sex distributions .
because sexual transmission of hiv is the predominant mode of transmission in jamaica ( less than 2% of reported cases among adults are attributed to injecting drug use ) , sexually inexperienced persons would not be considered at risk of hiv transmission nor an important group to target for hiv testing .
therefore , only data for 961 students ( 77% ) who reported being sexually experienced were included in the present study .
the following operationalizations were used : hiv testing : students were asked if they had ever had an hiv test with responses being categorized as yes ( 1 ) and no ( 0 ) . consistent condom use : frequency of condom use was measured separately with both steady and nonsteady sex partners .
a steady sex partner was defined as " someone with whom you have sexual intercourse on a regular or consistent basis , like a husband / wife or boy / girlfriend . "
a nonsteady sex partner was defined as " someone with whom you have sexual intercourse but only occasionally or even just once . " for both partner types , students who reported always using condoms during the 3 months prior to the survey were coded as consistent condom users ( 1 ) .
remaining students who reported using condoms usually , about half the time , seldom , or never were coded as inconsistent condom users ( 0 ) .
those reporting no sexual partner during the 3 months prior to the survey were excluded from the regression analyses .
multiple sex partners : students reporting 2 or more sex partners in the 12 months prior to the survey were coded as having multiple sex partners ( 1 ) , while those with 1 partner were coded as not having multiple sex partners ( 0 ) . due to issues of reliability associated with long recall periods , those reporting no sex partner during this time period
perceived risk of hiv : students were asked to report their perceived risk of becoming infected with hiv .
response categories included great risk , some risk , not much risk , and no risk .
responses were dichotomized into categories of great / some risk ( 1 ) and little / no risk ( 0 ) .
hiv education : students were asked if they had attended a lecture , course , or community forum about hiv / aids in the 12 months before the survey .
those who reported attending such an activity were coded as receiving hiv / aids education ( 1 ) , while remaining students were coded as not receiving such education ( 0 ) .
hiv awareness : students were asked if they knew someone who was infected with hiv or had died from aids .
those responding yes were coded as having a personal awareness of hiv ( 1 ) , while those reporting knowing no such person were coded as having no personal awareness of hiv ( 0 ) .
marital status : students were asked to report their current relationship status with categories including legally married , common - law , visiting partner ( steady sex partner ) , boy / girlfriend , or no relationship .
students who reported being legally married or involved in a common - law relationship were coded as being married ( 1 ) , while remaining students were coded as not being married ( 0 ) .
age : students were asked to report their age , in years , on their last birthday .
those reporting being under the age of 25 were coded as youth ( 1 ) while those 25 years of age and older were coded as adults ( 0 ) .
this categorization was based on the world health organization 's ( who ) definition of youth .
sex : students were asked to report if they were male ( 1 ) or female ( 0 ) . both bivariate ( chi - square ) and multivariate ( hierarchical logistic regression ) analyses were employed .
chi - square analyses were used to examine the differences between persons who reported previous hiv testing and those with no previous hiv testing .
in addition , in order to understand the relationship among all the model variables with respect to the dependent variables of interest , hierarchical logistic regression modeling was used .
this type of regression analysis takes an iterative form ; an initial simple model is followed by more complex models in which the dependent variable from the immediately preceding model becomes a predictor along with the previous predictors .
all model variables have been dichotomized to facilitate the logistic regression analyses with the comparison group for each variable coded as ( 1 ) as described in the above section .
both bivariate ( chi - square ) and multivariate ( hierarchical logistic regression ) analyses were employed .
chi - square analyses were used to examine the differences between persons who reported previous hiv testing and those with no previous hiv testing .
in addition , in order to understand the relationship among all the model variables with respect to the dependent variables of interest , hierarchical logistic regression modeling was used .
this type of regression analysis takes an iterative form ; an initial simple model is followed by more complex models in which the dependent variable from the immediately preceding model becomes a predictor along with the previous predictors .
all model variables have been dichotomized to facilitate the logistic regression analyses with the comparison group for each variable coded as ( 1 ) as described in the above section .
the sample was predominantly female ( 67.8% ) with a mean age of 28.2 years ( sx = 9.05 ) .
almost one third were married or involved in a common - law relationship ( 31.9% ) with slightly less than one quarter reporting being in no relationship at the time of the survey ( 23.1% ) .
the majority of students ( 77.0% ) had not attended any type of hiv education forum or lecture in the previous 12 months .
however , approximately half ( 51.0% ) did report knowing someone who was infected with hiv or had died from aids . with respect to behaviors ,
less than half of students ( 41.8% ) reported having been previously tested for hiv .
consistent condom use was low , with only one quarter ( 25.8% ) of those engaging in sex with a steady partner in the previous 3 months reporting always using condoms . among those who engaged in sex with a nonsteady partner during the same time , a slight majority ( 58.4% ) reported always using condoms . among those reporting at least 1 sex partner in the 12 months prior to the survey , less than one third ( 30.3% ) reported having 2 or more partners during this time .
table 1 presents the results of the bivariate analysis of hiv testing and selected variables .
overall , persons who reported previous hiv testing , compared with those with no history of testing , were more likely to be older , married or in a common - law relationship , and female .
they were also more likely to have attended an hiv education forum or lecture and know someone who is or has been infected with hiv , and were less likely to report condom use with steady partners .
bivariate results for selected sociodemographic , attitudinal , and behavioral variables by hiv testing ( n = 961 ) * valid percentages presented based on number of respondents providing data for each measure .
table 2 presents the results of the hierarchical logistic regression analyses and consists of 5 models .
the model chi - square test assesses the extent to which the model independent variables , as a whole , are related to the log odds of the dependent variable for a given regression analysis .
the model chi - square results indicated that 4 of the 5 submodels ( submodels 1 , 2 , 3a , and 3c ) in the hierarchical model were statistically significant , p < .001 .
hierarchical logistic regression results * for submodel 1 , in which perceived risk of hiv is the dependent variable , 2 of the 5 independent variables emerged as significant .
persons who were married or had a common - law partner were less likely to perceive some level of hiv risk ( odds ratio [ or ] = 0.61 ; 95% confidence interval [ ci ] = 0.420.89 ) than were unmarried persons . also , persons who reported knowing someone who was hiv - positive or had died from aids were more likely to report some level of hiv risk ( or = 1.46 ; 95% ci = 1.091.95 ) than were those who knew no such person . for submodel 2 ,
in which hiv testing is the dependent variable , 4 of the 6 independent variables were significant .
youth , compared with adults , were much less likely to report ever having been tested for hiv ( or = 0.32 ; 95% ci = 0.220.46 ) , while married persons , compared with those not married , were more likely to report previous hiv testing ( or = 1.51 ; 95% ci = 1.042.19 ) .
hiv education and awareness were both associated with hiv testing ; persons who reported attending an hiv education forum and those who reported knowing someone infected with hiv were more likely to report a previous hiv test ( or = 1.53 ; 95% ci = 1.072.19 ; and or = 1.39 ; 95% ci = 1.021.90 , respectively ) than were those with no previous hiv education or personal awareness . for submodel 3a ,
in which consistent condom use with most recent steady sex partner is the dependent variable , only 2 of the 7 independent variables were significant .
persons who were married or in common - law relationships were much less likely to report consistent condom use ( or = 0.34 ; 95% ci = 0.190.59 ) than were persons in no such relationships .
also , persons who perceived themselves to be at some risk for hiv were less likely to report consistent condom use than were persons who perceived little or no hiv risk ( or = 0.52 ; 95% ci = 0.350.79 ) .
for submodel 3b , in which consistent condom use with most recent nonsteady sex partner is the dependent variable , the overall model was not significant ( p = .13 ) .
persons who were married or in common - law relationships were much less likely to report consistent condom use with their most recent nonsteady sex partner ( or = 0.28 ; 95% ci = 0.090.84 ) than were persons in no such relationship . for submodel 3c ,
in which having multiple sex partners in the previous 12 months was the dependent variable , 4 of the 7 independent variables were significant .
males were far more likely than females to report having multiple sex partners during this time ( or = 3.53 ; 95% ci = 2.495.01 ) .
youth , when compared with adults , were more likely to report having multiple sex partners ( or = 1.56 ; 95% ci = 1.022.37 ) .
persons married or in common - law relationships were less likely than those in no such relationship to report multiple partners ( or = 0.32 ; 95% ci = 0.190.53 ) .
lastly , persons reporting being at some or great risk of hiv were more likely to report multiple partners than were those who perceived no such risk ( or = 1.77 ; 95% ci = 1.262.50 ) .
the sample was predominantly female ( 67.8% ) with a mean age of 28.2 years ( sx = 9.05 ) .
almost one third were married or involved in a common - law relationship ( 31.9% ) with slightly less than one quarter reporting being in no relationship at the time of the survey ( 23.1% ) .
the majority of students ( 77.0% ) had not attended any type of hiv education forum or lecture in the previous 12 months .
however , approximately half ( 51.0% ) did report knowing someone who was infected with hiv or had died from aids . with respect to behaviors ,
less than half of students ( 41.8% ) reported having been previously tested for hiv .
consistent condom use was low , with only one quarter ( 25.8% ) of those engaging in sex with a steady partner in the previous 3 months reporting always using condoms . among those who engaged in sex with a nonsteady partner during the same time , a slight majority ( 58.4% ) reported always using condoms . among those reporting at least 1 sex partner in the 12 months prior to the survey , less than one third ( 30.3% ) reported having 2 or more partners during this time .
table 1 presents the results of the bivariate analysis of hiv testing and selected variables .
overall , persons who reported previous hiv testing , compared with those with no history of testing , were more likely to be older , married or in a common - law relationship , and female .
they were also more likely to have attended an hiv education forum or lecture and know someone who is or has been infected with hiv , and were less likely to report condom use with steady partners .
bivariate results for selected sociodemographic , attitudinal , and behavioral variables by hiv testing ( n = 961 ) * valid percentages presented based on number of respondents providing data for each measure .
table 2 presents the results of the hierarchical logistic regression analyses and consists of 5 models .
the model chi - square test assesses the extent to which the model independent variables , as a whole , are related to the log odds of the dependent variable for a given regression analysis .
the model chi - square results indicated that 4 of the 5 submodels ( submodels 1 , 2 , 3a , and 3c ) in the hierarchical model were statistically significant , p < .001 .
hierarchical logistic regression results * for submodel 1 , in which perceived risk of hiv is the dependent variable , 2 of the 5 independent variables emerged as significant .
persons who were married or had a common - law partner were less likely to perceive some level of hiv risk ( odds ratio [ or ] = 0.61 ; 95% confidence interval [ ci ] = 0.420.89 ) than were unmarried persons .
also , persons who reported knowing someone who was hiv - positive or had died from aids were more likely to report some level of hiv risk ( or = 1.46 ; 95% ci = 1.091.95 ) than were those who knew no such person .
for submodel 2 , in which hiv testing is the dependent variable , 4 of the 6 independent variables were significant .
youth , compared with adults , were much less likely to report ever having been tested for hiv ( or = 0.32 ; 95% ci = 0.220.46 ) , while married persons , compared with those not married , were more likely to report previous hiv testing ( or = 1.51 ; 95% ci = 1.042.19 ) .
hiv education and awareness were both associated with hiv testing ; persons who reported attending an hiv education forum and those who reported knowing someone infected with hiv were more likely to report a previous hiv test ( or = 1.53 ; 95% ci = 1.072.19 ; and or = 1.39 ; 95% ci = 1.021.90 , respectively ) than were those with no previous hiv education or personal awareness . for submodel 3a ,
in which consistent condom use with most recent steady sex partner is the dependent variable , only 2 of the 7 independent variables were significant .
persons who were married or in common - law relationships were much less likely to report consistent condom use ( or = 0.34 ; 95% ci = 0.190.59 ) than were persons in no such relationships . also , persons who perceived themselves to be at some risk for hiv were less likely to report consistent condom use than were persons who perceived little or no hiv risk ( or = 0.52 ; 95% ci = 0.350.79 ) .
for submodel 3b , in which consistent condom use with most recent nonsteady sex partner is the dependent variable , the overall model was not significant ( p = .13 ) .
persons who were married or in common - law relationships were much less likely to report consistent condom use with their most recent nonsteady sex partner ( or = 0.28 ; 95% ci = 0.090.84 ) than were persons in no such relationship .
for submodel 3c , in which having multiple sex partners in the previous 12 months was the dependent variable , 4 of the 7 independent variables were significant .
males were far more likely than females to report having multiple sex partners during this time ( or = 3.53 ; 95% ci = 2.495.01 ) .
youth , when compared with adults , were more likely to report having multiple sex partners ( or = 1.56 ; 95% ci = 1.022.37 ) .
persons married or in common - law relationships were less likely than those in no such relationship to report multiple partners ( or = 0.32 ; 95% ci = 0.190.53 ) .
lastly , persons reporting being at some or great risk of hiv were more likely to report multiple partners than were those who perceived no such risk ( or = 1.77 ; 95% ci = 1.262.50 ) .
first , it is important to note that although the proportion of university students reporting hiv testing was higher than in the sexually experienced jamaican population , the reported levels of testing were still too low to serve as an effective hiv prevention tool .
unfortunately , the stigma , discrimination , and violence faced by jamaicans living with hiv / aids contribute to the avoidance of hiv testing by most people in the country .
concerns regarding violations of confidentiality and test privacy , which may be compounded by negative social conditions , can serve as major barriers to testing .
these issues must be addressed by prevention programs if progress is to be made in promoting universal testing in jamaica . recognizing this need , a number of organizations in jamaica have recently been awarded grants to address this barrier in hopes of improving the social climate and , as such , increasing persons ' willingness to seek hiv testing and counseling .
in addition to fear , students in the present study reported not having an hiv test because they believed they were not at risk of contracting hiv nor infected with the virus .
unfortunately , among those who reported being at no risk , a number of them were engaging in high - risk sexual behaviors . in order for programs promoting hiv testing to be effective
previous research indicates that encouraging individuals to be tested if they engage in at - risk activities will not be appropriate or effective for individuals who have no perception of risk .
the identification of the barriers to hiv testing can help guide the development of appropriate interventions to promote universal testing among persons who may have placed themselves at risk , knowingly or unknowingly , of contracting or transmitting hiv .
as noted earlier , hierarchical modeling was employed to examine the correlates of hiv testing .
the statistical results of the hierarchical modeling provide insight into hiv testing among university students in jamaica . in the first submodel ,
married persons perceived less risk of hiv than did persons with no legal or common - law partner .
this finding supports previous research that has found that persons in stable relationships , due to issues of perceived trust and commitment , perceive less hiv risk than those in unstable relationships . also , knowing someone who is hiv - infected or had died from aids was positively associated with perceived risk of hiv .
it is possible that as persons become more aware of hiv / aids , they are more likely to internalize the disease as a personal danger and recognize the consequences of the disease as well as the importance of protective measures .
it is important for programs to develop strategies that enable persons to accurately assess their risk of contracting hiv .
including persons living with hiv / aids as part of the intervention may be beneficial in achieving this goal . in the second submodel ,
hiv testing was the dependent variable , and perceived risk of hiv became an independent variable along with the sociodemographic variables .
a number of variables were associated with hiv testing , including both age and marital status , a differential that has been documented in previous research . within the jamaican context
, this finding may be more reflective of the fact that older and married persons in the sample were more likely to be employed , and in jamaica , many employers enforce mandatory hiv testing .
therefore , persons who are less likely to perceive themselves at risk for contracting hiv , such as married persons , may be more likely to actually be tested for hiv
. it will be critical for programs to specifically target those at - risk persons who are not employed or are not required to take an hiv test and encourage voluntary hiv testing .
what may be more interesting and informative for developing and implementing programs promoting voluntary hiv testing is the finding that persons who had attended an hiv education forum or lecture and those who reported knowing someone who was living or had lived with hiv / aids were more likely to have had an hiv test .
these findings are not surprising ; previous research has found that persons who have higher levels of hiv knowledge and awareness are more likely to seek hiv testing .
also , knowing someone with hiv / aids may result in more positive attitudes towards hiv testing.[45 - 49 ] increased knowledge and more positive attitudes , as well as a personal awareness of the consequences of the disease , may help persons to see the benefits of hiv testing and decrease some of the perceived barriers .
these findings suggest that bringing those living with hiv / aids into contact with untested persons may have positive outcomes , such as increased knowledge and awareness , improved attitudes , and willingness to seek hiv testing . in the last 3 submodels , when examining the relationship between hiv testing and sexual behaviors , we found no significant relationship between testing and condom use or number of sex partners . although the bivariate analyses indicated a significant difference between those with previous testing and those with no testing , with respect to consistent condom use with most recent steady sex partner , once the variable was entered into the multivariate model , the effect disappeared , indicating that hiv testing , alone , was not an important correlate of condom use .
as noted earlier , a review of hiv testing research has found mixed results with respect to the adoption of behavior change after testing .
it is possible that the adoption of protective sexual behavior is more a function of a test result and not the test itself . when attempting to study behavioral effects of hiv testing , it will be important to gather data on testing results .
when persons in the present study who reported previous hiv testing were specifically asked what behavioral changes , if any , had they made after testing , the majority reported no behavior change .
this may be reflective of an attitude that if an optimal test result was received then it is not necessary to change or adopt protective behavior .
however , a significant proportion of tested persons reported engaging in behaviors associated with increased risk of hiv transmission , including inconsistent condom use and having multiple sex partners .
the risks associated with unprotected sex and multiple partnerships must be elucidated to sexually active persons and the importance of protective sexual behavior , even if a negative test result is received .
it is critical that persons understand that a negative test result does not equate to an absence of hiv risk .
although the present study has provided insight into some of the factors associated with hiv testing among university students in jamaica , it is important to note the limitations of the study that may affect the validity of the findings .
first , the sample was a nonrandom sample , consisting of persons who volunteered to participate in the study .
although the study sample was not statistically different from the university population in terms of age and sex , the generalizability of the results to the university population in jamaica may , nonetheless , be limited .
also , the use of self - reported data may have contributed to threats of internal validity .
as with all surveys of sensitive issues , such data are likely to contain some bias .
intentional misreporting , incomplete recall , and misunderstanding of survey questions can reduce both the reliability and internal validity of the data .
lastly , data on hiv serostatus would have been important for explaining the relationship between hiv testing and sexual risk behaviors .
however , considering the social climate in jamaica with respect to persons living with hiv / aids , we felt that students would not be likely to provide valid information regarding hiv test results , and as such , we did not include this measure in the survey instrument .
irrespective of the study finding that previous hiv testing was not significantly associated with risky sexual behaviors , this should not negate the importance of receiving an hiv test .
one of the first steps in self - protection from hiv is to be informed of one 's hiv status and make appropriate and responsible sexual decisions .
although previous research has found mixed results with respect to hiv testing and subsequent behaviors , most studies have found hiv testing and knowledge of serostatus to be important , to some degree , in the adoption of safer sex behaviors .
effective messages and prevention programs need to be developed and implemented to promote universal hiv testing and counseling , especially among younger , sexually experienced students , so that these persons may make informed sexual decisions with respect to protective sexual behaviors , including having discussions about safer sex , inquiring about a potential partner 's hiv status , and the adoption of condom use with all sex partners , especially where hiv status is unknown . considering the actual and estimated number of hiv / aids cases in jamaica ,
it is clear that effective strategies are urgently needed to prevent both primary and secondary hiv transmission .
programs that can increase persons ' knowledge of hiv , including risks associated with various behaviors , as well as personal awareness , may be more efficacious than current efforts in increasing hiv testing among sexually experienced persons in jamaica .
yitades gebre , md , mph , msc , has disclosed no significant financial interests or relationships . | backgroundprevention programs often promote hiv testing as one possible strategy of combating the spread of the disease.objectiveto examine levels of hiv testing practices among a large sample of university students and the relationship among hiv testing , sociodemographic variables , and hiv - related behaviors.methodsa total of 1252 students were surveyed between june 2001 and february 2002 using a 193-item questionnaire measuring a variety of hiv - related knowledge and attitudinal and behavioral items.resultshierarchical logistic regression analyses revealed that youths , married persons , persons who had attended an hiv education forum , and those who knew someone with hiv / aids were more likely to report a previous hiv test .
however , hiv testing was not associated with condom use or number of sex partners.conclusionthe lack of significant findings between testing and risky sexual behaviors should not negate the importance of hiv testing .
being informed regarding personal hiv serostatus is one of the first steps in self - protection .
effective messages and programs need to be developed and implemented in jamaica to promote hiv testing and help persons to adequately assess their level of risk with respect to contracting hiv . |
seroprevalence among participants of the german health interview and examination survey for adults ( degs ) .
this nationwide cross - sectional survey assessed the health status of 18- to 79-year - old persons in germany during 20082011 ( 10 ) .
survey weights based on age , sex , residence in western or eastern germany , and nationality ( german vs. non - german ) were calculated to correct for deviations from the german population statistics ( december 31 , 2010 ; http://www.destatis.de ) and used throughout the analyses .
the study was approved by the ethical review board of the medical school charit , berlin , germany .
as recommended for serologic confirmation of clinical cases , serum samples were tested for the presence of borrelia
burgdorferi s.l .
, we used an elisa based on b. afzelii extract antigen enriched with recombinant vlse ( an outer - surface protein ) from b. burgdorferi s.s .
elisa - positive results were confirmed by line blot testing , which included purified antigens ospc , dbpa , and p83 from b. afzelii ; recombinant vlse from b. burgdorferi s.s . and b. garinii ; and bmpa and dbpa from b. garinii , b. bavariensis , and b. spielmanii .
all tests were performed / interpreted according to the manufacturer s recommendations . to categorize samples by test results , we applied the rules shown in figure 1 .
categorization , according to elisa and line blot test results , of serum samples tested for borrelia burgdorferi sensu lato igg , germany , 20082011 .
we used sampling weights for all statistical analyses and accounted for the 2-stage sampling structure .
age - related prevalence was graphed and the values were smoothed by using the lowess procedure of stata 12.1 ( statacorp lp , college station , tx , usa ) .
we assessed differences between group prevalences ( explanatory variables ) by using the wald test ( univariable logistic regression ) with 2-sided p values .
a total of 6,945 adults , representing 97.6% of the survey population with available blood samples , were included in the analysis .
the overall weighted seroprevalence for b. burgdorferi s.l . was 9.4% ( 95% ci 8.4%10.0% ) ; seroprevalence was significantly higher among men ( 13.0% ) than women ( 5.8% ) ( p<0.01 ) .
seroprevalence among both sexes increased by age ( figure 2 ) ; the increase was low among 18- to 50-year - old participants , most pronounced among participants > 59 years of age , and higher among women than men > 59 years of age .
seropositivity reached 20.0% ( 95% ci 16.9%23.6% ) in 70- to 79-year - old participants .
estimated seroprevalence of borrelia burgdorferi sensu lato igg among the male and female population , germany , 20082011 . for comparison , results of dehnert et al .
( 9 ) , a previous study among children / adolescents < 18 years of age , were added to the graph . among participants
> 18 years of age , more than twice as many men than women were seropositive for b. burgdorferi s.l .
independent risk factors for seropositivity were residence in a rural area ( p<0.001 ) and in southern germany ( p = 0.032 ) .
non - german citizenship was negatively associated ( p = 0.004 ) with seropositivity ; having a dog / cat in the house was not associated with a higher risk for seropositivity .
to facilitate comparison of our data with data from serosurveys lacking confirmatory testing , we have made our elisa results available online ( technical appendix table ) . * or , odds ratio ; ref , reference ; , not included in the final model .
western states : baden - wrttemberg , bavaria , bremen , hamburg , hesse , lower saxony , northrhine - westfalia , rhineland - palatinate , saarland , schleswig - holstein .
northern states : schleswig - holstein , hamburg , lower saxony , bremen , berlin , brandenburg , mecklenburg - west pomerania .
# middle states : nordrhine - westfalia , hesse , saxony , saxony - anhalt , thuringia .
* * southern states : rhineland - palatinate , baden - wrttemberg , bavaria , saarland .
b. burgdorferi s.l . infections are common in germany ; lyme borreliosis is endemic in all regions , but case numbers are highest in southern germany .
seropositivity in children ( male sex and living in rural areas , small - sized towns , or southern germany ) were identified as risk factors for seropositivity among adults in our study . holding a cat
was previously shown to be a risk factor for children / adolescents ( 9 ) , but was not a risk factor in our study .
seroprevalence among the oldest age group indicates that at least one fifth of the german population becomes infected with b. burgdorferi s.l . during their lifetime .
igg seroprevalence among blood donors in italy ( 4.9% ; n = 365 ) ( 11 ) and romania ( 4.3% ; n = 1,598 ) ( 12 ) was lower than the seroprevalence in our study .
prevalences higher than those in our study have been shown in serosurveys in areas of high disease endemicity in southwestern germany ( 16.9% ; n = 1,228 ) ( 13 ) and finland ( 19.3% ; n = 3,248 ) ( 14 )
. in serosurveys of persons with high exposure to ticks ( e.g. , forestry and agricultural workers ) , similar or higher seroprevalence rates have been described .
seroprevalence rates among men in our study were strikingly higher than rates among women , indicating that tick contact / spirochete transmission is more frequent among men .
prospective studies in germany and sweden and surveillance data from germany show no differences in clinical cases ( except only a slight preponderance among women ) that would point to substantial sex - specific differences in the development of clinical disease ( 57 ) . the age distribution for seropositivity reflects the population s cumulative exposure to b. burgdorferi s.l .. an increased risk for infection among children and persons > 59 years of age suggests that leisure activities / behaviors rather than occupational exposure are the main risk factor for infection . alternatively , these findings might be explained by a birth - cohort effect , in which the force of infection was lower during 19501990 .
seropositivity , suggesting that exposure to infected ticks is higher in rural areas . however , urban populations are also at substantial risk for infection . seropositivity is not equivalent to clinical disease ; thus , seropositivity rates among the different population groups may not necessarily reflect the true effect of infection on disease burden .
furthermore , a us study showed that persons can be consecutively infected by different b. burgdorferi strains and experience clinical manifestations with each infection ( 15 ) .
our seroprevalence estimates can be used , within the context of clinical diagnoses , to assess the likelihood of lyme borreliosis in persons with test results positive for b. burgdorferi s.l .
igg . to reduce the incidence and disease burden of lyme borreliosis , enhanced public health interventions are needed , including education campaigns targeted to parents , children , and the elderly about potential risk factors and preventive measures for lyme borreliosis .
stratified seroprevalence of borrelia burgdorferi sensu lato igg detected by elisa in adults and results of weighted bivariate logistic regression analysis of potential risk factors for seropositivity , germany , 20082011 . | to assess borrelia burgdorferi sensu lato ( the cause of lyme borreliosis ) seropositivity in germany , we tested serum samples from health survey ( 20082011 ) participants .
seroprevalence was 5.8% among women and 13.0% among men ; infection risk was highest among persons > 60 years of age .
public health interventions , including education about risk factors and preventive measures , are needed . |
eighteen nine - week - old sprague - dawley rats were purchased from charles river , inc ( yokohama , japan ) , and were used in the present study .
rats were kept in isolators at a temperature of 23 2c , and a relative humidity of 55 10% , on a 12/12 h dark / light cycle ( 06:0018:00 ) with air exchanged 12 times or more per hour .
rats were fed mf ( oriental yeast co , ltd , tokyo , japan ) , and were allowed free access to water .
all experiments were approved by the institutional review board of azabu university and conducted in accordance with the institute 's animal experimentation guidelines ( japanese association for laboratory animal science , jalas , 1987 ) .
turpentine oil ( wako pure chemical industries , ltd , osaka , japan ) was injected intramuscularly into rats at 0.05 , 0.2 or 0.4 ml / rat .
acute - phase protein levels are known to increase after intramuscular injection of turpentine oil , which has been used to induce acute inflammation in several studies , primarily because turpentine oil is able to induce constant acute inflammation , as compared with infection by microorganisms or other chemical compounds .
blood ( 0.3 ml ) was collected before injection and at 6 , 12 , 24 , 48 , 72 and 96 h after administration .
blood was collected from the venae cervicalis superficialis using a syringe under mild anaesthesia with pentobarbital ( kyoritsu seiyaku corporation , tokyo , japan ) .
pentobarbital was intravenously administered at small doses ( 4 mg / kg ) in order to ensure only short - term effects .
sera were obtained by centrifugation at 1600 g for 15 min , and were stored at 80c until analysis .
serum levels of 2 m were measured by enzyme - linked immunosorbent assay ( elisa ) according to the procedure of honjo et al .
serum levels of aag were measured by single radial immunodiffusion using a commercial kit ( institute for metabolic ecosystem co , ltd , miyagi , japan ) .
serum levels of interleukin-6 ( il-6 ) and cytokine - induced neutrophil chemoattractant ( cinc-1 ) were measured by elisa using commercial kits .
commercial elisa kits were purchased from biosource international , inc ( camarillo , ca , usa ) for il-6 and from panapharm laboratories co , ltd ( kumamoto , japan ) for cinc-1 .
eighteen nine - week - old sprague - dawley rats were purchased from charles river , inc ( yokohama , japan ) , and were used in the present study .
rats were kept in isolators at a temperature of 23 2c , and a relative humidity of 55 10% , on a 12/12 h dark / light cycle ( 06:0018:00 ) with air exchanged 12 times or more per hour .
rats were fed mf ( oriental yeast co , ltd , tokyo , japan ) , and were allowed free access to water .
all experiments were approved by the institutional review board of azabu university and conducted in accordance with the institute 's animal experimentation guidelines ( japanese association for laboratory animal science , jalas , 1987 ) .
rats were divided into three groups ( n = 6 for each group ) . turpentine oil ( wako pure chemical industries , ltd , osaka , japan )
acute - phase protein levels are known to increase after intramuscular injection of turpentine oil , which has been used to induce acute inflammation in several studies , primarily because turpentine oil is able to induce constant acute inflammation , as compared with infection by microorganisms or other chemical compounds .
blood ( 0.3 ml ) was collected before injection and at 6 , 12 , 24 , 48 , 72 and 96 h after administration .
blood was collected from the venae cervicalis superficialis using a syringe under mild anaesthesia with pentobarbital ( kyoritsu seiyaku corporation , tokyo , japan ) .
pentobarbital was intravenously administered at small doses ( 4 mg / kg ) in order to ensure only short - term effects .
sera were obtained by centrifugation at 1600 g for 15 min , and were stored at 80c until analysis .
serum levels of 2 m were measured by enzyme - linked immunosorbent assay ( elisa ) according to the procedure of honjo et al .
serum levels of aag were measured by single radial immunodiffusion using a commercial kit ( institute for metabolic ecosystem co , ltd , miyagi , japan ) .
serum levels of interleukin-6 ( il-6 ) and cytokine - induced neutrophil chemoattractant ( cinc-1 ) were measured by elisa using commercial kits .
commercial elisa kits were purchased from biosource international , inc ( camarillo , ca , usa ) for il-6 and from panapharm laboratories co , ltd ( kumamoto , japan ) for cinc-1 .
changes in serum levels of 2 m , aag , il-6 and cinc-1 are shown in figure 1 .
peak levels of 2 m were observed at 48 h after injection of 0.05 , 0.2 or 0.4 ml / rat .
peak 2 m levels were 1071.2 324.4 g / ml in the 0.05 ml / rat injection group , 1667.4 478.7 g / ml in the 0.2 ml / rat injection group and 1808.0 563.5 g / ml in the 0.4 ml / rat injection group .
peak levels in the 0.05 ml / rat injection group were significantly lower than those in the 0.2 and 0.4 ml / rat injection groups .
however , no significant differences were observed between the 0.2 and 0.4 ml / rat injection groups .
changes in serum levels of acute - phase proteins and cytokines in rats injected with turpentine oil at 0.05 , 0.2 or 0.4 ml / rat .
( a ) 2-macroglobulin ( 2 m ) , ( b ) 1-acid glycoprotein ( aag ) , ( c ) interleukin-6 ( il-6 ) and ( d ) cytokine - induced neutrophil chemoattractant-1 ( cinc-1 ) .
data are expressed as means sd ( n = 6 ) . * p < 0.05 , significantly different versus 0.2 or 0.4 ml / rat injection group , as analysed by student 's t - test . * * p < 0.05 , significantly different versus 0.4 ml / rat injection group , as analysed by student 's t - test serum levels of aag also peaked at 48 h after injection in all groups .
peaks for aag were 1554.8 302.8 g / ml in the 0.05 ml / rat injection group , 2667.6 451.1 g / ml in the 0.2 ml / rat injection group and 2962.8 285.0 g / ml in the 0.4 ml / rat injection group .
peak levels of aag in the 0.05 ml / rat injection group were significantly lower than those in the 0.2 and 0.4 ml / rat injection groups .
peak serum levels of il-6 and cinc-1 were observed at 12 h after injection in all injection groups .
peak levels of il-6 were 120.5 36.0 pg / ml in the 0.05 ml / rat injection group , 361.0 192.9
peak levels in the 0.05 ml / rat injection group were significantly lower than in the 0.2 and 0.4 ml / rat injection groups .
no significant differences were observed between the 0.2 and the 0.4 ml / rat injection groups , except at 24 h after injection .
peaks for cinc-1 were 2812.3 755.8 pg / ml in the 0.05 ml / rat injection group , 6734.8 2898.2
pg / ml in the 0.2 ml / rat injection group and 7449.8 2698.5 pg / ml in the 0.4 ml / rat injection group .
peaks levels in the 0.05 ml / rat injection group were significantly lower than those in the 0.2 and 0.4 ml / rat injection groups .
no significant differences were observed between the 0.2 and 0.4 ml / rat injection groups .
serum levels of the typical acute - phase proteins 2 m and aag in rats after inflammatory stimulation with various doses of turpentine oil were examined in an effort to clarify the differences in production during various degrees of inflammatory stimulation .
serum levels of 2 m and aag in rats injected with 0.05 ml / rat were significantly lower than those injected with 0.2 and 0.4 ml / rat ; however , no significant differences were observed between the 0.2 and 0.4 ml / rat injection groups .
these results suggest that production of 2 m and aag increases in proportion with turpentine oil dose .
production of 2 m and aag was similar above 0.2 ml / rat ; however , limited production was observed .
thus , it was necessary to carefully evaluate the degree of inflammation based on the levels of these acute - phase proteins .
il-6 is known to regulate the synthesis of acute - phase proteins , while il-8 regulates the production of acute - phase proteins in humans .
however , il-8 has not been observed in rats , and cinc-1 is considered to be the counterpart of human il-8 .
on the other hand , serum levels of seven cytokines ( il-1 , il-2 , il-4 , il-6 , cinc-1 , il-10 and interferon- ) in rats injected with turpentine oil have been investigated , and only serum levels of il-6 and cinc-1 were elevated prior to increases in 2 m .
in contrast , levels of the cytokines il-1 , il-2 , il-4 , il-10 and interferon- did not change . il-6 and cinc-1 also increased prior to aag in rats injected with turpentine oil . furthermore , the changes in serum levels of 2 m and aag after repeated injection of turpentine oil at doses of 0.05 and 0.4 ml / rat showed a similar pattern as with single stimulation , and peak serum levels in rats injected with 0.05 and 0.4 ml / rat were largely comparable . this phenomenon was thought to be caused by differences in the production of il-6 and cinc-1 in the two groups .
thus , il-6 and cinc-1 apparently contribute to the production of 2 m and aag in rats . in the present study ,
levels of il-6 and cinc-1 in rats injected with 0.05 ml were also significantly lower than those injected with 0.2 and 0.4 ml / rat .
the levels of both cytokines did not differ significantly between the 0.2 and the 0.4 ml / rat injection groups .
the significant differences in production of 2 m and aag independent of the turpentine oil dosage are considered to be attributable to significant differences in the production of il-6 and cinc-1 .
these results thus suggest that il-6 and cinc-1 regulate the production of 2 m and aag . in summary
, the production of 2 m and aag has an upper limit , irrespective of the magnitude of stimulation .
production of 2 m and aag showed similar trends to those of il-6 and cinc-1 ; thus , the kinetics of these cytokines influenced the production of 2 m and aag . | the relationship between intensity of inflammatory stimulation and production of 2-macroglobulin ( 2 m ) and 1-acid glycoprotein ( aag ) in rats was investigated .
sprague - dawley rats were injected with turpentine oil at doses of 0.05 , 0.2 or 0.4 ml / rat .
serum levels of 2 m , interleukin ( il)-6 and cytokine - induced neutrophil chemoattractant-1 ( cinc-1 ) were measured by enzyme - linked immunosorbent assay , and aag was measured by single radial immunodiffusion .
peak serum levels of 2 m and aag in rats injected at 0.05 ml / rat were significantly lower than those at 0.2 or 0.4 ml / rat .
however , no significant differences were observed for peak serum levels of these acute - phase proteins between 0.2 and 0.4 ml / rat .
furthermore , peak serum levels of il-6 and cinc-1 in rats injected at 0.05 ml / rat were significantly lower than those at 0.2 or 0.4 ml / rat .
thus , the production of these acute - phase proteins has upper limits , even under increased strength of inflammatory stimulation in rats injected with turpentine oil . |
takotsubo cardiomyopathy is a syndrome with transient systolic dysfunction of the left ventricle in the absence of significant coronary artery obstruction .
the contractile function of the mid and apical segments of the left ventricle is classically depressed and there is hyperkinesis of the basal segments .
takotsubo cardiomyopathy is much more common in women than men and is frequently triggered by emotional or physical stress .
patients present most commonly with chest pain , electrocardiographic abnormalities such as st - segment elevation or t - wave inversion and elevated cardiac biomarkers .
complications include acute heart failure , tachyarrhythmias , bradyarrhythmias , mitral regurgitation , cardiogenic shock , transient left ventricular outflow tract ( lvot ) obstruction , formation of a left ventricular thrombus , cardioembolic events , left ventricular free - wall rupture and death [ 15 ] .
a 76-year - old female presented to the coronary care unit with chest pain and an ecg suggestive for an inferolateral myocardial infarction ( fig . 1 ) .
she had never smoked and had two brothers with coronary artery disease before the age of 60 .
it started at the moment she was worrying about loss of eyesight due to macular degeneration.fig .
2left ventricular angiogram a diastolic b systolic left ventricular angiogram a diastolic b systolic a beta blocker , ace inhibitor and oral anticoagulation , to prevent apical thrombus formation , were started . in the following days the patient developed atrial fibrillation and she had recurrent chest and back pain .
echocardiography revealed a pericardial effusion of up to 16 mm , with diastolic collapse of the right ventricle and > 25 % transmitral flow variation with respiration which is indicative for cardiac tamponade ( fig .
the oral anticoagulation was stopped and the inr of 2.6 was antagonised with vitamin k. pericardiocentesis was performed . after draining 120 cc bloody fluid , there was an immediate improvement of the haemodynamics and diuresis.fig . 3echocardiography
a parasternal long axis b transmitral flow c subcostal view echocardiography a parasternal long axis b transmitral flow c subcostal view
catecholamine excess , coronary artery spasm , microvascular dysfunction , dynamic mid - cavity or lvot obstruction , inflammation and regional myocarditis may play a role [ 3 , 6 , 7 ] .
an mri study showed that pericardial effusion is common ( 62 % ) in takotsubo cardiomyopathy .
cardiac tamponade in takotsubo cardiomyopathy , however is very rare . in 2010 , one case was described by yeh et al . in circulation .
so far this complication has only been described in a total number of three cases .
treatment with anticoagulants is recommended although there are no clear guidelines whether or not to start anticoagulation in takotsubo cardiomyopathy .
the incidence of left ventricular thrombus formation and cardioembolic events in takotsubo cardiomyopathy is 5 % and 1.6 % respectively . of the cardioembolic events ,
not all patients with cardioembolic events show a left ventricular thrombus on their first echocardiography . an ischaemic brain event may cause irreversible damage while pericardial effusion can be drained .
treatment with anticoagulants should therefore be considered at the moment of presentation to the hospital , irrespective of the presence of a left ventricular thrombus .
the use of anticoagulants must of course be weighed against a patient s individual bleeding risk .
although takotsubo cardiomyopathy complicated by cardiac tamponade is very rare , echocardiography should be performed in case of haemodynamic deterioration to exclude pericardial effusion . | we describe a 76-year - old patient with takotsubo cardiomyopathy complicated by cardiac tamponade .
pericardial effusion in takotsubo cardiomyopthy is common but a cardiac tamponade is very rare . the use of anticoagulants may increase the risk of pericardial effusion and should be considered with care . |
chilblain lupus erythematosus is a chronic form of lupus erythematosus seen especially in women , affecting the fingertips , rims of ears , calves , and heels .
chle lesions may be clinically indistinguishable from simple chilblains or perniosis , which is seen in healthy individuals . hence , having an understanding of this clinical entity is essential for the clinicians to make a correct diagnosis . here
, we present a case of chle in an adolescent girl with few unique features not previously reported .
a 13-year - old girl presented with reddish raised asymptomatic lesions over both hands and feet for the past five months . the lesions appeared spontaneously as lentil - sized raised lesions over the right index finger and gradually increased in size and number and coalesced to form larger plaques with appearance of similar lesions over other fingers as well as soles .
no personal or family history of oral ulcers , joint pains , photosensitivity , frothing of urine or any systemic complaints in the form of chest pain , abdominal pain , or breathlessness was found .
examination of hands and feet revealed multiple papular lesions , which were erythematous and edematous .
they were discoid erythematous scaly atrophic papules to plaques over the ventral and lateral aspect of the fingers of hand and feet as well [ figure 1 ] .
bilateral concha showed violaceous discoid plaques of size 1 1 cm [ figure 2 ] .
chilblain lupus erythematosus lesions over the ventral aspect of fingers and palms typical discoid lupus erythematosus lesions over the concha hematological investigations were within normal limits except anemia ( hb-10.9g / dl ) and raised erythrocyte sedimentation rate ( 30 mm in first hour ) .
biochemical tests for hepatic and renal functions , serum proteins , urine examination and chest radiograph were normal .
anti - nuclear antibody test was positive with a speckled pattern of immunofluorescence , and the titer being 1:160 .
these antibodies were identified as anti - dsdna , anti - sm , anti - ro52 , anti - histone and anti - nucleosomal antibodies .
histopathology from the lesion over the finger showed focal basal cell vacuolization of the epidermis and moderate perivascular and periadnexal lymphocytic infiltrate in the dermis [ figure 3 ] .
direct immunofluorescence ( dif ) of lesional skin showed igm and c3 deposition along the basement membrane zone .
based on the clinical and laboratory findings a diagnosis of chle was made and treatment was initiated with potent topical steroids twice daily until complete remission .
histopathology showing vacuolization of the basal cell layer and a perivascular and periadnexal lymphocytic infiltrate ( h and e , 10 )
chilblain lupus erythematosus is a rare manifestation of chronic cutaneous lupus erythematosus , first reported by hutchinson in 1888 . according to a review published in 2008 ,
about 70 cases have been reported in the literature , with only few in the adolescent age group .
the two forms of chle are described - familial and sporadic . familial form of chilblain lupus manifests in early childhood and is caused by a heterozygous mutation in the trex1 gene that encodes a 3-5 dna exonuclease .
the pathogenesis of sporadic chle remains unknown , though vasoconstriction or microvascular injury secondary to cold and possible hyperviscosity and stasis due to immunological abnormalities are usually implicated .
an association of chle with anorexia and intestinal lymphoma has also been reported but not conclusively proven .
chle is clinically characterized by symmetrically distributed circumscribed , occasionally infiltrated erythematous to violaceous pruriginous or painful plaques over dorsal and lateral aspects of hands and feet , appearing during cold , damp weather conditions .
chle may be associated with lesions of discoid lupus erythematosus ( dle ) or other forms of cutaneous lupus erythematosus and may progress to systemic lupus erythematosus ( sle ) in up to 18% of patients .
it can clinically simulate lupus pernio , however , it is essential to make a correct diagnosis ; although clinically the two entities are similar and have in common their association with systemic disease ( lupus erythematosus or sarcoidosis ) , the prognosis and treatment differ considerably .
verrucous form of chilblain lupus has also been described in literature . in most patients , immunological abnormalities
are frequently observed in the form of polyclonal hypergammaglobulinemia , raised rheumatoid factor , antinuclear antibodies .
anti - ro / ssa and antiphospholipid antibodies have also been found to be positive in a few studies .
su et al . , proposed the mayo clinic diagnostic criteria for a confirmative diagnosis of chle .
major criteria include skin lesions in acral locations induced by exposure to cold or a drop in temperature , and evidence of lupus erythematosus in the skin lesions by histopathologic examination or dif .
minor criteria are the coexistence of sle or other skin lesions of dle ; response to anti - lupus erythematosus therapy ; and negative results of testing for cryoglobulin and cold agglutinins .
diagnosis of chle is confirmed if patient fulfills both the major and any one of the minor criteria .
our patient presented with erythematous , scaly lesions over both the hands and feet that started during the cold month of december .
the lesions initially appeared to be simple perniosis , but on detailed cutaneous examination , typical dle lesions were observed over bilateral concha though the patient or her family members had not noticed them .
the diagnosis of chle was further confirmed by positive antinuclear antibodies as well as by skin biopsy and direct immunofluorescence .
thus , our patient fulfilled both major as well as two minor criteria as suggested by su et al .
the treatment includes protection from cold by physical measures and topical or oral antibiotics , if the lesions are infected .
topical steroids and calcium channel blockers have been found to be effective . in recalcitrant cases ,
local immunosuppressive agents such as tacrolimus , pimecrolimus or systemic agents such as systemic steroids and mycophenolate mofetil may be tried .
antimalarial drugs ( chloroquine or hydroxychloroquine ) have shown to have good effects on sle typical symptoms but not on chilblain themselves .
the patient was advised protection from cold and application of potent topical steroids twice daily over dle as well as chilblain lesions .
we propose to start her on calcium channel blockers in winters if the lesions become symptomatic during that period .
our patient showed unique features in the form of early age of onset , concentration of chilblain lesions over the ventral aspect compared to the dorsal aspect of the hands and feet and the presence of anti - ro 52 , anti - histone , anti - smith , ds dna and nucleosomal antibodies .
a few case reports have reported the presence of anti ro / ss - a in chle but none has reported the presence of multiple subtypes of antinuclear antibodies .
although rare , it is an important entity that the physician must have an understanding of , as it can be confused with perniosis .
the risk of developing sle in such cases of chle is estimated to be about 18% , hence we plan to follow up the patient for clinical / laboratory evidence of progression to sle . to conclude
, chle lesions may be clinically indistinguishable from simple chilblains or perniosis seen in healthy individuals , hence detailed examination as well as investigations must be done in suspicious cases . | chilblain lupus erythematosus ( chle ) is a rare form of cutaneous lupus erythematosus ( le ) , more frequently seen in middle aged females .
it is characterized by erythematous to violaceous plaques over the acral areas induced by exposure to cold or drop in temperature unlike lesions of lupus erythematosus that worsen with sun exposure . here ,
we present a case of chilblain lupus erythematosus in an adolescent girl with few unique features not previously reported . |
the corpus cavernosum tissue consists of endothelial - lined sinusoidal structures surrounded by a specialized type of smooth muscle cells called corporal smooth muscle .
normal erectile function involves a coordinated relaxation of the cavernous arteries , cavernous sinusoidal endothelium , and the sinusoidal smooth muscle , which results in expansion of the cavernous sinusoids and an increase in intracavernous pressure .
when penile erection is initiated , the cavernous smooth muscle cells relax and allow massive influx of blood into the sinusoidal spaces , which in turn causes rigid penile erection .
the importance of smooth muscle relaxation in penile erection has been demonstrated in both animal and human studies .
the crucial role of the cavernous smooth muscle cells in penile erection is underscored by the fact that more than 60 to 80% of patients with erectile dysfunction ( ed ) can be treated by oral phosphodiesterase ( pde)-5 inhibitors that induce smooth muscle relaxation of the penis .
ed following radical prostatectomy ( rp ) results from inadvertent damage to the cavernous nerves that run close to the prostate capsule .
the mechanisms behind the development of post - rp ed include cavernosal fibrosis and cavernosal smooth muscle apoptosis , resulting from cavernous nerve injury or neurapraxia during surgery . in the hyperlipidemic state , lower numbers of neuronal nitric oxide synthase - positive nerves and a diminished endothelium can negatively affect erectile function because of the reduced bioavailability of nitric oxide .
the diminished endothelium also causes the cavernous smooth muscle to lose contractility as the cells become synthetic .
together , these structural changes may explain why men with cavernous nerve injury and hyperlipidemia are at higher risk of having ed .
although animal models for ed have played a critical role in expanding our understanding of the pathophysiologic mechanisms involved in ed , it is necessary to establish a primary cavernous smooth muscle cell culture system from the rat corpus cavernosum tissue for the further delineation of the cellular and molecular mechanisms involved in smooth muscle cell dysfunction and ed .
however , several technical difficulties remain in obtaining pure primary smooth muscle cells from corpus cavernosum tissue .
impurity and dedifferentiation of isolated cells complicate the interpretation of results from cell culture - based experiments .
matrigel is a gelatinous protein mixture secreted by engelbreth - holm - swarm mouse sarcoma cells .
this mixture resembles the complex extracellular environment found in many tissues and is used by cell biologists as a substrate for cell culture .
successfully isolated endothelial cells from mouse corpus cavernosum tissue by use of a matrigel - based sprouting endothelial cell culture system . in the present study
, we developed a novel technique for the isolation of rat cavernous smooth muscle cells .
we applied a two - stage tissue culture method with the use of a matrigel - based sprouting culture system to facilitate stromal cell sprouting and an adherent culture system using d - valine to eliminate contamination of the smooth muscle cells with fibroblasts .
eight - week - old adult male sprague - dawley rats ( 180 to 190 g , n=7 ) were purchased from daehanbiolink ( chungju , korea ) .
rats were housed under constant optimal temperature and humidity conditions with a normal 12-hour light - dark cycle .
the investigation conformed to the guide for the care and use of laboratory animals published by the us national institutes of health ( nih publication no .
85 - 23 , revised 1996 ) and was approved by the animal subjects committee of the konkuk university school of medicine , korea .
all experiments and animal care were performed in accordance with the institutional guidelines of konkuk university .
muscle strips from aortas were prepared in physiological salt solution ( in mm ; nacl 136.9 , kcl 5.4 , cacl2 1.5 , mgcl2 1.0 , nahco3 23.8 , ethylenediaminetetraacetic acid 0.01 ) , as reported previously .
ex vivo migration of rat cavernous smooth muscle cells was measured by using matrigel with the following modifications .
the corpus cavernosum tissue was harvested with the rats under anesthesia induced by intramuscular injections of ketamine ( 80 mg / kg ) and xylazine ( 8 mg / kg ) .
briefly , the skin overlying the penis was incised and bilateral penile crura were exposed by removing part of the ischiocavernous muscle and fascia .
after dissection and removal of the urethra , the corpus cavernosum tissues were obtained excluding the glans , cartilaginous portion of the penis , and the part of overlying ischiocavernous muscle .
the corpus cavernosum tissue was washed in phosphate - buffered saline ( pbs ) several times .
the samples ( n=7 ) were enzymatically digested with collagenase ( 315 u / ml ) and elastase ( 0.3 u / ml ) for 25 minutes at 37 and were cut into fragments .
the tissue fragments were then placed and embedded in 48-well plates coated with matrigel supplemented with platelet - derived growth factor - bb and incubated for 5 days ( fig .
the sprouting cells were subcultured in dulbecco 's modified eagle medium ( dmem ) containing 920 mg / l d - valine , 10% fetal bovine serum ( fbs ) , 100 u / ml penicillin , 100 g / ml streptomycin , and 200 mm glutamine . for all experiments , cultured rat penile smooth muscle cells ( rpsmcs ) were grown to 70 to 80% confluence and were starved in dmem without fbs for 24 hours .
after treatment with stimulants , cells were lysed with cold extraction buffer ( 20 mm hepes , ph 7.5 , 1% nonidet p-40 , 150 mm nacl , 10% glycerol , 10 mm naf , 1 mm na3vo4 , 2.5 mm 4-nitrophenylphosphate , 0.5 mm phenylmethylsulfonyl fluoride , and 1 tablet of complete proteinase inhibitor cocktail ; roche , indianapolis , in , usa ) .
we also isolated rat aortic smooth muscle cells ( rasmcs ; passages 5 to10 ) from 6 rats and cultured the cells in dmem containing 10% fbs , 100 u / ml penicillin , 100 g / ml streptomycin , and 200 mm glutamine .
rpsmcs and rasmcs lysates were centrifuged ( 13,000g , 15 minutes , 4 ) , and the supernatants were collected as protein samples .
the protein homogenates were diluted 1:1 ( v / v ) with sodium dodecyl sulfate ( sds ) sample buffer containing 40 mm tris - hcl ( ph 6.8 ) , 8 mm ethylene glycol tetraacetic acid , 4% 2-mercaptoethanol , 40% glycerol , 0.01% bromophenol blue , and 4% sds , and then boiled for 5 min .
proteins ( 30 to 50 g per lane ) were separated by using 8 to 12% polyacrylamide sds gels and were then transferred electrophoretically to a polyvinylidene fluoride membrane ( millipore , bedford , ma , usa ) .
the membrane was blocked for 2 hours at room temperature with pbs containing 0.05% tween-20 and 5% fat - free dried milk .
the membranes were incubated with primary antibody against -smooth muscle actin ( -sma ) and platelet / endothelial cell adhesion molecule ( pecam)-1 ( diluted 1:1000 - 2000 ) overnight at 4. immune complexes were detected with horseradish peroxidase - conjugated antibodies ( amersham - pharmacia , piscataway , nj , usa ) diluted 1:1000 and incubated for 1 hour at room temperature .
after application of the secondary antibody , blots were incubated in enhanced chemiluminescence kits ( amersham - pharmacia ) and exposed to las-3000 .
band intensity was measured by computer analysis by using quantitation software ( bio - rad laboratories inc .
the expression of -sma and pecam-1 in the rpsmcs was determined by the standard immunostaining method .
briefly , the cells were fixed with 10% formaldehyde at room temperature for 15 minutes .
after washing twice with pbs containing 0.05% tween 20 and permeabilizing with 0.1% triton x-100 for 5 minutes , the sprouts were treated with 1% bovine serum albumin in pbs for 2 hours and incubated with alexa(594)-conjugated anti--sma and alexa(488)-conjugated anti - pecam antibody ( 1:500 ; sigma - aldrich co. , st .
louis , mo , usa ) overnight at 4. the immunostained sprouts were observed by use of fluorescence microscopy ( axio observer a , zeiss , jena , germany ) .
rna was extracted by using trizol reagent ( invitrogen , carlsbad , ca , usa ) according to the manufacturer 's recommendations .
total rna ( 1 g ) was reverse - transcribed into cdna by using the superscript iii first - strand synthesis system for reverse transcription polymerase chain reaction ( invitrogen ) .
real - time rt - pcr was performed by using iqtmsybr green supermix ( bio - rad laboratory inc . ) and icycleriq real - time pcr detection system ( bio - rad laboratory inc . ) .
the relative mrna expression level was determined by calculating the values of cycle threshold ( ct ) by normalizing the average ct value compared with its endogenous control ( gapdh ) and then calculating 2-ct .
statistical evaluation of the data was performed by using student 's t - tests for comparisons between pairs of groups and analysis of variance for multiple comparisons by use of graphpad prism version 4.0 ( graphpad software inc . , la jolla , ca , usa ) .
eight - week - old adult male sprague - dawley rats ( 180 to 190 g , n=7 ) were purchased from daehanbiolink ( chungju , korea ) .
rats were housed under constant optimal temperature and humidity conditions with a normal 12-hour light - dark cycle .
the investigation conformed to the guide for the care and use of laboratory animals published by the us national institutes of health ( nih publication no .
85 - 23 , revised 1996 ) and was approved by the animal subjects committee of the konkuk university school of medicine , korea .
all experiments and animal care were performed in accordance with the institutional guidelines of konkuk university .
muscle strips from aortas were prepared in physiological salt solution ( in mm ; nacl 136.9 , kcl 5.4 , cacl2 1.5 , mgcl2 1.0 , nahco3 23.8 , ethylenediaminetetraacetic acid 0.01 ) , as reported previously .
ex vivo migration of rat cavernous smooth muscle cells was measured by using matrigel with the following modifications .
the corpus cavernosum tissue was harvested with the rats under anesthesia induced by intramuscular injections of ketamine ( 80 mg / kg ) and xylazine ( 8 mg / kg ) .
briefly , the skin overlying the penis was incised and bilateral penile crura were exposed by removing part of the ischiocavernous muscle and fascia .
after dissection and removal of the urethra , the corpus cavernosum tissues were obtained excluding the glans , cartilaginous portion of the penis , and the part of overlying ischiocavernous muscle .
the corpus cavernosum tissue was washed in phosphate - buffered saline ( pbs ) several times .
the samples ( n=7 ) were enzymatically digested with collagenase ( 315 u / ml ) and elastase ( 0.3 u / ml ) for 25 minutes at 37 and were cut into fragments .
the tissue fragments were then placed and embedded in 48-well plates coated with matrigel supplemented with platelet - derived growth factor - bb and incubated for 5 days ( fig .
the sprouting cells were subcultured in dulbecco 's modified eagle medium ( dmem ) containing 920 mg / l d - valine , 10% fetal bovine serum ( fbs ) , 100 u / ml penicillin , 100 g / ml streptomycin , and 200 mm glutamine .
for all experiments , cultured rat penile smooth muscle cells ( rpsmcs ) were grown to 70 to 80% confluence and were starved in dmem without fbs for 24 hours .
after treatment with stimulants , cells were lysed with cold extraction buffer ( 20 mm hepes , ph 7.5 , 1% nonidet p-40 , 150 mm nacl , 10% glycerol , 10 mm naf , 1 mm na3vo4 , 2.5 mm 4-nitrophenylphosphate , 0.5 mm phenylmethylsulfonyl fluoride , and 1 tablet of complete proteinase inhibitor cocktail ; roche , indianapolis , in , usa ) .
we also isolated rat aortic smooth muscle cells ( rasmcs ; passages 5 to10 ) from 6 rats and cultured the cells in dmem containing 10% fbs , 100 u / ml penicillin , 100 g / ml streptomycin , and 200 mm glutamine .
rpsmcs and rasmcs lysates were centrifuged ( 13,000g , 15 minutes , 4 ) , and the supernatants were collected as protein samples .
the protein homogenates were diluted 1:1 ( v / v ) with sodium dodecyl sulfate ( sds ) sample buffer containing 40 mm tris - hcl ( ph 6.8 ) , 8 mm ethylene glycol tetraacetic acid , 4% 2-mercaptoethanol , 40% glycerol , 0.01% bromophenol blue , and 4% sds , and then boiled for 5 min .
proteins ( 30 to 50 g per lane ) were separated by using 8 to 12% polyacrylamide sds gels and were then transferred electrophoretically to a polyvinylidene fluoride membrane ( millipore , bedford , ma , usa ) .
the membrane was blocked for 2 hours at room temperature with pbs containing 0.05% tween-20 and 5% fat - free dried milk .
the membranes were incubated with primary antibody against -smooth muscle actin ( -sma ) and platelet / endothelial cell adhesion molecule ( pecam)-1 ( diluted 1:1000 - 2000 ) overnight at 4. immune complexes were detected with horseradish peroxidase - conjugated antibodies ( amersham - pharmacia , piscataway , nj , usa ) diluted 1:1000 and incubated for 1 hour at room temperature .
after application of the secondary antibody , blots were incubated in enhanced chemiluminescence kits ( amersham - pharmacia ) and exposed to las-3000 .
band intensity was measured by computer analysis by using quantitation software ( bio - rad laboratories inc .
the expression of -sma and pecam-1 in the rpsmcs was determined by the standard immunostaining method .
briefly , the cells were fixed with 10% formaldehyde at room temperature for 15 minutes .
after washing twice with pbs containing 0.05% tween 20 and permeabilizing with 0.1% triton x-100 for 5 minutes , the sprouts were treated with 1% bovine serum albumin in pbs for 2 hours and incubated with alexa(594)-conjugated anti--sma and alexa(488)-conjugated anti - pecam antibody ( 1:500 ; sigma - aldrich co. , st .
louis , mo , usa ) overnight at 4. the immunostained sprouts were observed by use of fluorescence microscopy ( axio observer a , zeiss , jena , germany ) .
rna was extracted by using trizol reagent ( invitrogen , carlsbad , ca , usa ) according to the manufacturer 's recommendations . total rna ( 1 g )
was reverse - transcribed into cdna by using the superscript iii first - strand synthesis system for reverse transcription polymerase chain reaction ( invitrogen ) .
real - time rt - pcr was performed by using iqtmsybr green supermix ( bio - rad laboratory inc . ) and icycleriq real - time pcr detection system ( bio - rad laboratory inc . ) .
the relative mrna expression level was determined by calculating the values of cycle threshold ( ct ) by normalizing the average ct value compared with its endogenous control ( gapdh ) and then calculating 2-ct .
data are expressed as the meanstandard error . statistical evaluation of the data was performed by using student 's t - tests for comparisons between pairs of groups and analysis of variance for multiple comparisons by use of graphpad prism version 4.0 ( graphpad software inc . , la jolla , ca , usa ) .
to characterize primary cultured cells , we performed immunocytochemical staining for smooth muscle cell marker ( -sma ) and endothelial cell marker ( pecam-1 ) .
the majority of the cells showed positive staining for -sma but did not show positive fluorescence for pecam-1 staining ( fig .
we also performed western blot analysis in rpcmcs and ramscs to determine the protein expression of -sma and pecam-1 .
similar to the result from the immunocytochemical staining , both rpsmcs and rasmcs expressed -sma but did not express pecam-1 ( fig .
3 ) , which indicated that the primary cultured cells were smooth muscle cells . to determine whether the expression patterns of pde family mrna differed between rasmcs and rpsmcs , we performed quantitative real - time pcr .
the levels of calcium / calmodulin - dependent 3',5'-cyclic nucleotide pde1a , pde1b , and pde1c ; cgmp - dependent 3',5'-cyclic nucleotide pde2a ; cgmp - inhibited cyclic nucleotide pde3a ; camp - specific 3',5'-cyclic nucleotide pde4a , pde4b , pde4c , and pde4d ; and cgmp - specific nucleotide pde5a mrna in the rpsmcs were greater by about 3.2- , 4.4- , 3.4- , 29.0- , 3.5- , 2.8- , 2.9- , 6.1- , 45.0- , and 6.0-fold , respectively , than in rasmcs ( fig .
to characterize primary cultured cells , we performed immunocytochemical staining for smooth muscle cell marker ( -sma ) and endothelial cell marker ( pecam-1 ) .
the majority of the cells showed positive staining for -sma but did not show positive fluorescence for pecam-1 staining ( fig .
we also performed western blot analysis in rpcmcs and ramscs to determine the protein expression of -sma and pecam-1 .
similar to the result from the immunocytochemical staining , both rpsmcs and rasmcs expressed -sma but did not express pecam-1 ( fig .
to determine whether the expression patterns of pde family mrna differed between rasmcs and rpsmcs , we performed quantitative real - time pcr .
the levels of calcium / calmodulin - dependent 3',5'-cyclic nucleotide pde1a , pde1b , and pde1c ; cgmp - dependent 3',5'-cyclic nucleotide pde2a ; cgmp - inhibited cyclic nucleotide pde3a ; camp - specific 3',5'-cyclic nucleotide pde4a , pde4b , pde4c , and pde4d ; and cgmp - specific nucleotide pde5a mrna in the rpsmcs were greater by about 3.2- , 4.4- , 3.4- , 29.0- , 3.5- , 2.8- , 2.9- , 6.1- , 45.0- , and 6.0-fold , respectively , than in rasmcs ( fig .
the development of various in vitro model systems from isolated corpus cavernosum tissues has tremendously enhanced our understanding of the biochemical and physiological mechanisms associated with erectile function .
the main function of the corpus cavernosum tissues is to enhance blood flow into the penis through coordinated functions of the cavernous endothelial and smooth muscle cells , which are crucial for penile erection [ 17 - 19 ] .
the corpus cavernosum tissue consists mainly of endothelial cells , smooth muscle cells , and fibroblasts .
whereas intact corporal tissue consists of a heterogeneous mixture of the cells , the isolation of pure primary cell components from erectile tissue may offer the possibility of defining the molecular and functional characteristics of particular cell types .
however , previous studies aimed at the isolation of cavernous smooth muscle cells often lacked detailed isolation protocols or a precise characterization of the culture composition to exclude contamination from other cells , particularly fibroblasts .
the discrepancy in the purity of primary cultured smooth muscle cells in different reports might reflect laboratory and tissue source factors , a lack of precise exclusion of fibroblasts in other studies , and changes in properties of smooth muscle during the subcultivation .
-sma , an isoform typical of smcs and present in high amounts in vascular smcs , was shown in the cytoplasm of pericytes of various rat and human organs by immunocytochemical staining .
on the other hand , pecam-1 is constitutively expressed on all vascular endothelial cells and has been regarded as a standard marker for endothelial cells in a variety of blood vessels , particularly in the setting of angiogenesis . in the present study ,
the fluorescent immunocytochemistry of rpsmcs and rasmcs expressed -sma but did not express pecam-1 ( figs . 2 , 3 ) .
these results suggest that the isolated cells from penile tissues were not endothelial cells but were smooth muscle cells .
research in tissue engineering , stem cells , or cell - based gene therapy primarily involves cultures of the cells on flat plastic dishes , and this technique is known as 2-dimensional cell culture .
the majority of the current cell culture techniques are based on rigid and 2-dimensional substrates .
recently , the use of 3-dimensional cell culture systems has increased in research fields , including drug discovery , cancer biology , regenerative medicine , and basic life science research .
a variety of platforms are used to facilitate cell growth in a 3-dimensional cell culture system , such as nanoparticle - facilitated magnetic levitation , gel matrix scaffolds , and hanging drop plates . in this study
, we applied unique two - stage cell culture methods by use of a matrigel - based sprouting cell culture system and adherent cultures with d - valine .
first , the corpus cavernosum tissue was implanted in matrigel to facilitate stromal cell ( smooth muscle cells and fibroblasts ) sprouting .
second , the sprouting cells were subcultivated in dmem containing 920 mg / l d - valine , 10% fbs , 100 u / ml penicillin , 100 g / ml streptomycin , and 200 mm glutamine .
the cells were cultured in nutrient medium containing l - valine with d - valine added in an attempt to control fibroblast overgrowth .
contamination of rpsmcs cultures with fibroblasts was prevented in cultures maintained in d - valine but not in control cultures containing l - valine .
our results showed that d - valine prevented fibroblast overgrowth without impairment of smooth muscle cell morphology or function .
the physiological importance of pde5 in the regulation of smooth muscle tone has been demonstrated most clearly by clinical use of its specific inhibitors in the treatment of ed .
pde5 inhibitor retards enzymatic hydrolysis of cgmp in the human corpus cavernosum , leading to dilation of the arteries that bring blood into the penis and inducing penile erection .
because pdes form a biochemically and structurally diverse family of proteins , there may be more than one pde isozyme or isogene that could serve as a potential drug target in the treatment of ed .
the pde families include pde1-ca / calmodulin - dependent , pde2-cgmp - stimulated , pde3-cgmp - inhibited , pde4-camp - specific , pde5-cgmp - specific , pde6-photoreceptor cgmp - specific , pde7-camp - specific rolipram - insensitive , pde8-camp - specific 3'isobutyl'1'methylxanthine - insensitive , pde9-cgmp - specific , and pde10-camp - inhibited family members .
different isogenes within a particular family are distinguishable by letters , for example pde1a , pde1b , and pde1c .
most mammalian tissues express several members of pde families and many express more than 1 subtype of an individual family [ 24 - 26 ] .
the presence of mrna specific for 10 different pde isozymes and isoforms in rpsmcs and rasmcs was shown by rt - pcr .
we detected rpsmc and rasmc expression of genes encoding for camp and cgmp - hydrolyzing pdes , including isogenes of pde1 ; pde2a ( which hydrolyzes camp and cgmp ) ; pde3a ; isogenes of pde4 ( the specific camp - hydrolyzing pdes ) ; and pde5a ( the specific cgmp - hydrolyzing pde ) .
it was reported that pde3 and pde4 are highly expressed in the major cardiovascular and arterial system , whereas the expression of pde5 is low .
however , our study showed a higher presence of mrna of 10 pde isogenes in rpsmcs compared with rasmcs ( fig .
this disparity may result from the differences in the preparation of specimens : our study used relatively pure rpsmcs or rasmcs , whereas the previous study used organs , not smooth muscle cells .
the presence of a variety of pde isozymes in the primary cultured rpsmcs further supports the reliability of our protocol for the isolation of cavernous smooth muscle cells .
in this study , we developed a novel method for the isolation of rpsmcs by using a matrigel - based sprouting culture system and subcultivation in dmem containing d - valine .
the in vitro model of primary cultured cavernous smooth muscle cells for the study of erectile function will give us important insights into the physiologic mechanisms of penile erection . | purposeprimary culture of the cavernous smooth muscle cells from corpus cavernous tissues is known to be difficult , mainly because of contamination with fibroblasts .
we applied a new method for better isolation of rat penile smooth muscle cells ( rpsmcs ) from rat corpus cavernosum tissue for reliable ex vivo research on erectile dysfunction.materials and methodswith the use of 8-week - old adult male sprague - dawley rats , ex vivo migrations of rat cavernous tissue were measured by penis and aortic ring assay by use of a matrigel - based d - valine - modified culture method .
the expression of -smooth muscle actin ( -sma ) and platelet / endothelial cell adhesion molecule ( pecam)-1 in the rpsmcs was determined by standard immunofluorescent staining and immunoblotting .
the expression patterns of phosphodiesterase ( pde ) family mrna in rpsmcs were compared with patterns in rat aortic smooth muscle cells ( rasmcs ) by use of quantitative real - time reverse transcription polymerase chain reaction.resultsimmunocytochemical staining showed greater -sma - positive and pcam-1-negative fluorescence . moreover , whereas the expression of -sma was detected in the rpsmcs , that of pecam-1 was not .
the levels of pde1a , pde1b , pde1c , pde2a , pde3a , pde4a , pde4b , pde4c , pde4d , and pde5a mrna in the rpsmcs were about 3.2- , 4.4- , 3.4- , 29.0- , 3.5- , 2.8- , 2.9- , 6.1- , 45.0- , and 6.0-fold the corresponding expression in rasmcs.conclusionswe developed a two - stage tissue culture method utilizing a matrigel - based sprouting culture system to facilitate stromal cell sprouting and an adherent culture system using d - valine to eliminate the contamination of fibroblasts into the smooth muscle cells . |
zizyphus lotus ( z. lotus ) , also known as jujube , belongs to the angiosperm rhamnaceae family .
this family includes about 135170 species of zizyphus . as a tropical and subtropical plant ,
z. lotus grows generally in arid and semiarid countries and is widely distributed in china , iran , africa , south korea , and europe like cyprus , spain , greece , and sicily [ 24 ] . in africa , z. lotus is widely distributed in mediterranean region , like algeria , morocco , tunisia , and libya .
this plant is employed in nutrition , health , and cosmetics in several forms , for example , honey , tea , jam , juice , oil , loaf , and cake .
in addition , in traditional medicine , both in north africa and middle east , several parts of z. lotus are given as antiurinary troubles agents , antidiabetes , skin infections , antifever , antidiarrhea , insomnia agents , sedative , bronchitis , and hypoglycemic activities [ 69 ] .
on the other hand , this plant offers a delicious read fruit ( jujube ) that was consumed fresh , dried , and processed as food by local populations in substantial amounts . in recent years , several scientific reports
have been carried out about the presence of many biologically active molecules from z. lotus , which may have high potential benefit in human nutrition , health , and disease [ 11 , 12 ] . in herbal medicine
, the properties of bioactive compounds from plants depend on the part of the plant concerned ( root , leaf stalk , pulp , or fruit ) and the type of extract used .
z lotus is known for its high content in polyphenols exhibiting antioxidant and antimicrobial , immunomodulatory properties [ 13 , 14 ] .
importantly , others biologically active molecules , particularly cyclopeptide alkaloids , termed lotusines [ 1517 ] , dammarane saponins , and various flavonoids have been isolated from this shrub , along with polyunsaturated fatty acids ( oleic acid and linoleic acid ) , high carbohydrate , and fibers which are abundant in seed extracts and endowed with antiulcerogenic and antioxidants effects [ 11 , 19 ] .
this review is devoted to the most recent findings on biological effects of the major compounds isolated from different parts of z. lotus and to the different usages of this plant in human foods , health promoting , and disease prevention .
z. lotus fruit contains substantial amounts of glutamic acid , mineral matter , sterols , vitamins , tocopherols , fibers , amino acids , triacylglycerol , fatty acid , carbohydrate , and antioxidant compounds ( phenols , flavonoids , etc . ) which have been supposed to be responsible for most of its health benefits such as hypoglycemic , gastroprotective , immunomodulatory , and antioxidant properties [ 14 , 21 , 22 ] . in this respect ,
the fruit of z. lotus is a valuable source of nutrients as well as antioxidant [ 4 , 21 , 23 , 24 ] , antimicrobial , and antifungal [ 13 , 25 ] , immunosuppressive , anti - inflammatory , and antiulcerogenic [ 21 , 27 ] compounds . z.
lotus leaves contain different carbohydrates and dammarane saponins notably jujuboside b , three jujubogenin glycosides , and jujubasaponine iv .
z. lotus seeds are used to prepare lotus oil enriched in essential fatty acids , liposoluble antioxidants , and many sterols .
z. lotus root contains four dammarane saponins , large quantity of polyphenol , essential fatty acids , vitamin c , and several cyclopeptide alkaloids , termed lotusines which have a wide range of pharmacological activities including antioxidant , antiproliferative , and antidiabetic activities [ 12 , 1518 , 22 , 24 , 28 ] .
the pulp of z. lotus contains a significant amount of carbohydrate , phenols , flavonoids , and tannins , which exhibit high antimicrobial activity [ 19 , 25 ] .
as a source of polyphenols , fatty acids , vitamins , and other natural compounds , z. lotus seems to be a potential candidate for human nutrition , health promoting , and disease preventing .
plant - derived polyphenols are a family of organic molecules . during the last decade , there has been a growing interest in the role of polyphenols , in several human pathologies . they have been shown to possess cardioprotective , anticancer , antiviral , antiallergenic , and antispasmodic properties [ 31 , 32 ] . given their chemical structure characterized by the presence of many phenolic groups , polyphenols are also able to scavenge reactive radical species and prevent peroxidative reactions .
numerous studies showed their ability to prevent damage of lipids , proteins , and nucleic acids by reactive oxygen and nitrogen species [ 3436 ] and modulate transcription factors [ 37 , 38 ] and protein tyrosine kinases activation [ 39 , 40 ] .
all parts of z lotus are rich in polyphenol family members such as flavonoids , phenolic acids , and other natural compounds ( table 1 ) . in the fruit ,
total phenols are the major compound , amounting from 297 to 4078.2 mg/100 g of dry matter ; in addition , flavonoids and tannins are present in moderate quantities , 122 and 33 mg/100 g , respectively [ 13 , 23 ] . in the leaf ,
total phenol content is 664 mg/100 g , along with flavonoids ranging from 130 to 199 mg/100 g [ 13 , 18 ] , high content of saponins ( 340 mg/100 g ) , and large amount of carbohydrates ( 8720 mg/100 g ) , and other molecules are found in small quantities under 10 mg/100 g ( see table 1 ) . interestingly ,
z. lotus seeds contain a very high amount of several compounds such as fats ( 29.73 g/100 g ) , fibers ( 16.57 g/100 g ) , and protein ( 14.22 g/100 g ) , along with carbohydrates ( 4720 mg/100 g ) and small amounts of polyphenol ( 14.68 mg/100 g ) . in z. lotus root bark
, polyphenol content is 2009 mg/100 g , along with a high content of saponins 219 mg/100 g , high content of flavonoids ( 120 mg/100 g ) , and large amount of proanthocyanidins ( 156 mg/100 g ) compared to other molecules such as cyclopeptide alkaloids , amounting from 1.4 to 23.95 mg/100 g [ 1517 ] ( table 1 ) .
z. lotus pulp contains high amounts of soluble sugars ( 10.55 g/100 g ) , fibers ( 4.84 g/100 g ) , mineral matter ( 3.2 g/100 g ) , and protein ( 1.18 g/100 g ) , along with tannins ( 922 mg/100 g ) and moderate amounts of polyphenol ( 325 mg/100 g ) . in summary , aerial parts ( leaves and fruits ) of z. lotus are the most important source of polyphenols and flavonoids ( 36308144 mg/100 g ) , while the seeds are rich in fats .
these variations in z. lotus biomolecules content might be due to the environment , soil type , climate , or age of the plant .
it should be noted that the biological activities of z. lotus are allocated to the different classes of pharmacologically active compounds such as flavonoids , several saponins , and alkaloids ( table 1 ) .
it has been reported that z. lotus alkaloids exerted significant antifungal and antibacterial properties [ 12 , 17 ] .
currently , seven alkaloids ( called lotusines , named from a to g ) and nine saponins ( seven jujubogenins and two lotogenins ) ( figure 1 and table 1 ) have been isolated from this plant , and the main chemical compounds including lotusine a , lotusine b , lotusine c , jujuboside a , lotoside i , and 3-o--l - rhamnopyranosyl-(1 - 2)-[(4-sulfo)--dglucopyranosyl-(1 - 3)]--l - arabino - pyranosyl - jujubogenin are presented in figure 2 .
the analysis of lipid composition showed that z. lotus pulp ( table 1 ) was rich in palmitic acid ( c16:0 ) , oleic acid ( c18:1 ) , and linoleic acid ( c18:2 ) , amounting to 27.59% , 24.52% , and 36.63% of total fatty acid content , respectively ( table 2 ) .
its content in z. lotus pulp ( 36.87% ) is thus close to the amount found in olive oil ( 1.1% ) and argan oil ( 31.3% ) but lower to the percentage found in soybean oil ( 50.1% ) and corn oil ( 56% ) ( table 2 ) .
numerous studies reported that all parts of z. lotus particularly , seeds , pulp , fruits , leaves , almond , root , and stem , were rich in palmitic , stearic , linoleic , and oleic acid [ 11 , 13 , 19 , 28 ] .
oleic acid was the most important fatty acid of z. lotus fruits , seeds , and almond at 88.12% , 61.93% , and 49.88% , respectively .
in vivo studies in rabbit ldl model provided evidence that oleic acid is responsible of the potent antioxidant properties attributed to many edible oils rich in this fatty acid .
moreover , it has been reported that oleic acid upregulated the expression of breast cancer resistance protein and thereby modulates intestinal retention of several food toxicants .
this fatty acid is the precursor of arachidonic acid , which has inhibitory effect of colon cancer .
other fatty acids were also present in this plant like linolenic acid ( 9.15% ) particularly in z lotus leaves .
linolenic acid is the precursor of docosahexaenoic acid , known to have potential benefit for health and for other diseases like cardiovascular diseases .
high - performance liquid chromatography ( hplc ) analyses of triacylglycerol ( tag ) composition show that z lotus seed oil contains several tag ( table 3 ) .
the glycerol - trioleate was the most compound , amounting to 26.48 g/100 g , along with glycerol - palmitate - dioleate with 18.78 g/100 g ( table 3 ) .
it has been shown that many types of tg like glycerol - trioleate , glycerol - palmitate - dioleate , glycerol - dioleate - linoleate , and glycerol - palmitate - oleate - linoleate stabilized oil oxidation [ 56 , 57 ] .
the pulp of z. lotus is rich in vitamin c in amounts up to 190.65 mg/100 g , followed by z. lotus seeds , leaves , root , and stem , containing 170.84 , 63.40 , 47.20 , and 24.65 mg/100 g , respectively ( table 4 ) .
z. lotus leaves content is high in vitamin e with 155.71 mg/100 g , while z. lotus 's seeds are enriched in -tocopherols with 130.47 mg/100 g .
a little amount of carotenoids ( 1.47 mg/100 ) was found only in z. lotus fruits .
vitamins b1 and b2 were present in z. lotus seeds with 0.03 and 0.08 mg/100 g. several parts of z. lotus are rich in vitamin a , ranging from 3.8 to 71.63 mg/100 g. collectively , these data provide evidence that z. lotus might be considered as a source of many vitamins for human food .
the sterol analysis of z. lotus seed oil showed that seven compounds have been identified .
-campesterol was the major compound with 147.82 mg/100 g ( 51.86% of total sterol ) , along with -sitosterol and campesterol with 82.10 and 31.89 mg/100 g , respectively ( table 5 ) .
other sterols notably stigmasterol , -avenasterol , , 24-stigmatadienol , and cholesterol are present in small quantities .
total sterols content in z. lotus seed oil was 285.03 mg/100 g. compared to other vegetable oils , this content is better than z. jujuba oil ( 18.56 mg/100 g ) and virgin oil ( 150 mg/100 g ) but lower than those measured in z. zizyphus ( 291.82 mg/100 g ) and soy oil ( 350 mg/100 g ) .
it is important to indicate that there is no available data on the sterol content in the other parts of z. lotus ; this issue remains to be determined .
the mineral analysis of z. lotus fruit showed that calcium , magnesium , and potassium were the predominance compounds with 490.84 , 397.91 , and 134.99 mg/100 g , respectively , ( table 6 ) .
similar amounts for magnesium and calcium were found in z. lotus pulp , while higher contents of these three minerals are present in z. lotus seeds , with amounts ranging from 92.41 to 1349.06 mg/100 g [ 11 , 19 ] .
amino acids composition of z. lotus seeds shows that threonine is the major amino acid in this part with 26.73% of total amino acid content , followed by glutamic acid ( 17.28% ) , leucine ( 13.11% ) , arginine ( 9.47% ) , aspartic acid ( 7.76% ) , and alanine ( 4.56% ) ( table 7 ) .
in z. lotus seed , total proteins represent 14.22% higher than z. lotus pulp with 1.18% .
several parts of z. lotus have been used in traditional medicine for the treatment of bronchitis , diarrhea , and abscess .
in addition , the powder of dried leaves and fruit mixed with water or milk is used for the treatment of boils and the root bark for the treatment of diabetes .
the juice from z. lotus root would be efficient in the treatment of eye leucomas .
the fruits and the leaves of z. lotus are used as emollient and in the treatment of diarrhea and intestinal diseases .
the fruits are dried and processed into flour to make pancakes with very pleasant flavor .
the nutritional virtue of z. lotus is mainly based on its composition rich in vitamin e , vitamin c , fibers , fatty acids , amino acids , calcium , magnesium , and considerable amounts of sugars as mentioned above .
consistent with this , it has been reported that z. lotus oil is of high quality , because of its content in unsaturated fatty acids and other bioactive compounds .
traditional uses of z. lotus have reported several benefits of this plant and its bioactive compounds .
meantime , there has been a growing scientific data to support these beneficial properties of z. lotus through several experimental models devoted to the assessment of z. lotus natural molecules to cure numerous diseases .
this plant is rich in polyphenols , flavonoids , tannins , alkaloids , and saponins which have several healthy properties like antidiabetic , hypoglycemic , and gastroprotective actions [ 21 , 22 ] .
as mentioned above , lotusine b , lotusine c , jujuboside a , and jujuboside c are the main active constituents of z. lotus root bark ( table 1 ) and might exert antibacterial and antifungal activity [ 65 , 66 ] .
therapeutic benefits of z. lotus compounds or extracts have been highlighted by several experimental models ( cell and animal ) through in vivo and in vitro studies .
several studies report that the extracts of z. lotus exhibit anti - inflammatory and antioxidant properties . as shown in table 1 , z. lotus
is rich in many antioxidant compounds such as phenolic acids , flavonoids , alkaloids , and saponins .
these components have been shown to prevent oxidative stress and inflammation by reducing reactive oxygen species ( ros ) .
interestingly , numerous in vitro studies have demonstrated the capacity of the different parts of z. lotus for scavenging free radicals , for instance , in lipid peroxidation , resulting in cell damage prevention [ 4 , 13 , 21 , 23 , 24 , 26 ] .
moreover , in diabetic rats , the aqueous extract of z. lotus roots and leaves strongly increases the rate of haemolysis and glutathione reductase and decreases catalase activity , glutathione peroxidase , and the status of antioxidant , suggesting that this plant corrected diabetes - induced antioxidant status . besides , the involvement of glutathione in protein and dna synthesis , cellular detoxification , and inflammation has been reported .
for this reason , z. lotus extract might have potential benefit for cellular protection . in vitro data on human t cells
suggest that z. lotus fruits have higher antioxidant activities compared to other parts of this plant , followed by leaves , root , and stem .
furthermore , the secondary metabolites of z. lotus administrated orally in carrageenan - induced rat paw edema presented anti - inflammatory effects in dose - dependent manner by inhibiting paw edema and the production of nitrite in lipopolysaccharide - activated raw 264.7 macrophages without cytotoxicity .
these studies sustained that z. lotus biomolecules might have beneficial effects for human health , for example , to reduce or prevent inflammation and oxidative damage .
in vitro studies
have elucidated the effects of z. lotus extracts on the growth of several bacteria and fungi species ( see table 8) .
they demonstrated that the extracts of z. lotus fruits under etheric and methanolic solvents presented the most bactericidal effects to induce growth inhibition [ 13 , 25 ] .
these antimicrobial activities of z. lotus fruits seem to be mediated by phenolic compounds content in this part of z. lotus as shown elsewhere .
altogether , these reports provided evidence that z. lotus with antibacterial effects might be considered as source of natural biomolecules for producing synthetic bactericides and fungicides . in a wistar rat model of streptozotocin - induced hyperglycemia ,
hypoglycemic effects of z. lotus indicate that the aqueous extracts of roots presented the most efficient activities compared to z. lotus leaves .
this beneficial effect might be correlated with the high quantities of vitamin a observed in leaves and roots of z. lotus .
indeed , it has been reported that insulin sensitivity was improved by vitamin a through activation of insulin receptor and protein tyrosine phosphatase 1b . moreover
gastric ulcer is part of gastrointestinal disorder involving inflammation and default of defense mechanism . in many in vivo studies ,
protective effects of aqueous extracts of z. lotus ( root bark , leaves , and fruit ) administered orally were observed in the lesions of several ulcerogenic induced models in wistar rat [ 21 , 27 ] .
these reports suggest that the extracts of this plant act as antiulcer agent by reducing gastric acidity and juice secretion .
helicobacter pylori is the most common bacterium that can survive in the highly acidic environment of the human stomach involving different digestive diseases such as peptic ulcer , dyspepsia ( heartburn , acid indigestion , and nausea ) [ 75 , 76 ] , the stomach cancer ( adenocarcinoma ) [ 77 , 78 ] , and malt lymphoma .
interestingly , the effect of methanol extract of z. lotus ( fruits ) has been studied in vitro on 22 clinical strains of helicobacter pylori , indicating that this plant has bactericidal effects on these clinical strains . in swiss mice , analgesic effects of aqueous extract of z. lotus root barks
were observed in a dose - dependent manner . in acetic acid - induced algesia in mice ,
analgesic activities were also reported by flavonoid and saponin extracts from z. lotus leaves and root bark in vivo , while in vitro , this effect is modulated by nitrite production in raw 264.7 macrophages .
in addition , ex vivo studies on isolated rat duodenum show that aqueous extract of z. lotus leaves and root bark exerts antispasmodic activities by modulating ca signaling via cholinergic receptors .
beneficial effects of z. lotus polyphenols on health might be generated by their antioxidant and radical scavenging properties .
interestingly , our previous studies demonstrated that z. lotus polyphenols also modulate human immune cell signaling and exert immunosuppressive effects .
as shown in figure 3 , in human t cells , z. lotus polyphenols ( zlp ) upregulate thapsigargin- ( tg- , inhibitor of ca - atpase ) mediated calcium signaling at endoplasmic reticulum level , modulate plasma membrane , and , thus , block the entry of ions , decrease erk1 and erk2 activation , diminish cell proliferation and il-2 expression by arresting s cell cycle , and increase intracellular acidification in dose - dependent manner .
zlp alone do not induce elevation of intracellular calcium concentration , [ ca]i , in these cells .
consistent with this , z. lotus might have a potential benefit in human autoimmune diseases .
collectively , this review provides updated comprehensive information on z. lotus as a source of several bioactive compounds which hold therapeutic potentialities for human nutrition , health promoting , and disease preventing . as mentioned in table 8 ,
several scientific papers have clearly reported many biological properties of the different parts of this plant and its constituents through in vitro and in vivo studies .
the potent antioxidant , antimicrobial , and anti - inflammatory effects of z. lotus have been distinctly elucidated . on another side , z. lotus extracts present beneficial effects on metabolic disorders via antidiabetic and hypoglycemic actions . in vivo studies
showed that z. lotus supplementation might be used to treat gastrointestinal disorders . on the nutritional level , this plant is rich in many nutriments which may be used in various fields such as food , cosmetics , and pharmaceutics .
although several studies reported the benefit effects of z. lotus in many facets of human nutrition , health , and disease , the exact mechanisms by which z. lotus bioactive compounds exert their biological and pharmacological activities are not yet entirely elucidated .
therefore , further studies are required to elucidate the effects of z. lotus extracts and active compounds in some unexplored domains such as cancer , metabolic disorders , inflammation , and age - linked diseases as well as their mechanisms of actions . | zizyphus lotus , belonging to the rhamnaceae family , is a deciduous shrub which generally grows in arid and semiarid regions of the globe . in traditional medicine ,
z. lotus is used as antidiabetes , sedative , bronchitis , and antidiarrhea by local populations . recently ,
several scientific reports for health benefit and nutritional potential of bioactive compounds from this jujube have been reported .
this plant is rich in polyphenols , cyclopeptide alkaloids , dammarane saponins , vitamins , minerals , amino acids , and polyunsaturated fatty acids .
these identified compounds were supposed to be responsible for most of z. lotus biologically relevant activities including antimicrobial , anti - inflammatory , hypoglycemic , antioxidant , and immunomodulatory effects .
the aim of the present review was to give particular emphasis on the most recent findings on biological effects of the major groups of zizyphus lotus components and their medical interest , notably for human nutrition , health benefit , and therapeutic impacts . |
liver metastases are being diagnosed at earlier stages because of high - quality imaging techniques and regular follow - ups . among colorectal cancer cases , liver is the common site of metastasis .
colorectal liver metastasis ( clm ) is diagnosed in 20% of cases simultaneously with initial cancer because of their asymptomatic nature and transient symptoms .
extended liver resection in primary intrahepatic tumors or clm is an inevitable surgical approach for the affected patients . associating liver partition and portal vein ligation for staged hepatectomy ( alpps )
has been evolved recently as a salvage therapy for traditionally non - resectable liver tumors .
it has opened a window to the patients with right hepatic lobe tumor with insufficient future liver remnant ( flr ) .
rapid and extensive regeneration of hepatic tissue provided by allps could decrease mortality of patients due to tumor progression .
in 2010 , alpps was applied widespread regardless of its indication among hepatobiliary surgeons and lead to high morbidity and mortality . in some cases with damaged hepatic parenchyma although required regeneration of hepatic tissue occured but the hypertrophied tissue did not show satisfactory function .
further clinical investigation is crucial to define precise indications and also find out whether the patients benefit from the alpps procedure despite high cost of mortality and complications .
till now , limited available experiments have been conducted on technical feasibility , reliability , and safety of the alpps procedure .
this article presents the method in details with precautions need to take , candidates for alpps , their postoperative complications and outcome during near3 years follow up .
during june 2013 to march 2014 , nine patients were scheduled to undergo alpps procedure .
patients enrolled in our study were all candidates for hepatectomy because of primary or metastatic liver tumors .
expected flr to total liver volume ratio for alpps is considered to be 20%30% in normal hepatic tissue and 40% to 45% in patients with an underlying liver disease .
alpps procedure proceeded to the 2 step in patients who achieved adequate hypertrophy in 79 days after the initial step .
patients were followed every 3 months by ultrasound and every 6 months by computed tomography ( ct ) scan . in colorectal cancer with liver metastasis , carcinoembryonic antigen level was checked every 3 months .
a reverse l laparotomy incision is applied based on the location of the primary tumor , abdomen 's anatomy , and resection site .
first , the abdominal cavity is explored through a midline incision 2 cm above the umbilicus .
then , the incision is extended to the right horizontally if it is not contraindicated ( e.g. , multiple small hepatic metastases or peritoneal seeding which was not obvious on ct imaging ) .
if the primary tumor is located in sigmoid or rectosigmoid junction , the incision goes 24 cm below umbilicus [ figure 1 ] .
after identification of the right , left , and middle hepatic arteries , the right hepatic artery is encircled by a vessel loop .
then , cholecystectomy is performed as usual , and the cystic duct is saved for further checking of bile leakage . by reaching hilar plate after common bile duct identification , portal vein is dissected distally up to the bifurcation .
patient d had separate perfusion pathways for anterior and posterior sectors [ figure 2 ] .
additional organ bag covers visceral organs in the first step of procedure anterior and posterior branches of portal vein drain to portal vein separately based on the tumor location , the partition line is defined by electrocautery . in situ splitting
partitioning of the superior part of the liver is done 1 cm distal to hilum . to prevent probable portal triad injury , caudate splitting is postponed to the second step of the procedure . after checking biliary leakage , the right lobe , containing lesions ,
normally , these organ bags are used in transporting the transplant organs . although deserosations had been repaired in patient d , adhesions developed between the incision site and small intestine and caused enterocutaneous fistula . since then
, exposed surface of visceral organs has been covered by another organ bag to prevent developing adhesion bands and facilitate the 2 step of laparotomy [ figure 1 ] .
using organ bags provide greater exposure and better handling , because the loops of intestine bind together . at the end of the first step ,
then , the patient is admitted to intensive care unit , and antibiotics covering gram - positive and negative microorganisms are administered . in the 6 postoperative day ,
flr hypertrophy is assessed by multiple detector ct scan which has been demonstrated in literature . if flr hypertrophy is adequate , the patient will undergo the second step on the 7 postoperative day .
if not , it will be postponed to 3 days later , again after imaging evaluation . during the second step of the procedure , the right hepatic artery is identified and marked by a vascular clamp .
later on , it is preferred to ligate the artery at anterior and posterior sectors to make sufficient perfusion to the biliary tract . while there is not enough space to rotate liver to the right due to the left lobe hypertrophy , it is recommended to extend the incision transversely to the right and cut the rectus muscle .
dissection at hilum plate is continued , till arterial branches of anterior and posterior sectors are identified and ligated .
moreover , it is followed by excision of caudate lobe and removal of the sample .
biliary ducts of the sectors are identified and sutured by polydioxanone ( pds ; ethicon 6 - 0 ) , while biliary leakage is checked by injection into the preserved cystic duct .
if the primary tumor of colon has not been resected previously , colectomy is performed in this stage .
a reverse l laparotomy incision is applied based on the location of the primary tumor , abdomen 's anatomy , and resection site .
first , the abdominal cavity is explored through a midline incision 2 cm above the umbilicus .
then , the incision is extended to the right horizontally if it is not contraindicated ( e.g. , multiple small hepatic metastases or peritoneal seeding which was not obvious on ct imaging ) .
if the primary tumor is located in sigmoid or rectosigmoid junction , the incision goes 24 cm below umbilicus [ figure 1 ] .
after identification of the right , left , and middle hepatic arteries , the right hepatic artery is encircled by a vessel loop .
then , cholecystectomy is performed as usual , and the cystic duct is saved for further checking of bile leakage . by reaching hilar plate after common bile duct identification , portal vein is dissected distally up to the bifurcation .
for instance , patient d had separate perfusion pathways for anterior and posterior sectors [ figure 2 ] .
additional organ bag covers visceral organs in the first step of procedure anterior and posterior branches of portal vein drain to portal vein separately based on the tumor location , the partition line is defined by electrocautery . in situ splitting
partitioning of the superior part of the liver is done 1 cm distal to hilum . to prevent probable portal triad injury , caudate splitting is postponed to the second step of the procedure .
after checking biliary leakage , the right lobe , containing lesions , is wrapped in a sterile organ bag . normally , these organ bags are used in transporting the transplant organs .
although deserosations had been repaired in patient d , adhesions developed between the incision site and small intestine and caused enterocutaneous fistula .
since then , exposed surface of visceral organs has been covered by another organ bag to prevent developing adhesion bands and facilitate the 2 step of laparotomy [ figure 1 ] .
using organ bags provide greater exposure and better handling , because the loops of intestine bind together .
at the end of the first step , a jackson - pratt drain is placed near liver and fascia is sutured .
then , the patient is admitted to intensive care unit , and antibiotics covering gram - positive and negative microorganisms are administered . in the 6 postoperative day ,
flr hypertrophy is assessed by multiple detector ct scan which has been demonstrated in literature . if flr hypertrophy is adequate , the patient will undergo the second step on the 7 postoperative day .
if not , it will be postponed to 3 days later , again after imaging evaluation . during the second step of the procedure , the right hepatic artery is identified and marked by a vascular clamp .
later on , it is preferred to ligate the artery at anterior and posterior sectors to make sufficient perfusion to the biliary tract . while there is not enough space to rotate liver to the right due to the left lobe hypertrophy , it is recommended to extend the incision transversely to the right and cut the rectus muscle .
dissection at hilum plate is continued , till arterial branches of anterior and posterior sectors are identified and ligated .
moreover , it is followed by excision of caudate lobe and removal of the sample .
biliary ducts of the sectors are identified and sutured by polydioxanone ( pds ; ethicon 6 - 0 ) , while biliary leakage is checked by injection into the preserved cystic duct .
if the primary tumor of colon has not been resected previously , colectomy is performed in this stage .
finally , a jackson - pratt drain is placed and fascia is repaired . the incision is sutured through tertiary wound management .
demographic and operative data of the alpps candidates are demonstrated in tables 1 and 2 .
outcome and follow - up of patients underwent successful associating liver partition and portal vein ligation for staged hepatectomy patients who failed to proceed the associating liver partition and portal vein ligation for staged hepatectomy procedure regarding outcome and follow - up , patient b , was a bronchial carcinoid tumor case treated with lobectomy surgery 6 years ago but he came back with presentation of carcinoid syndrome , again few years after .
patient c died because of massive pulmonary thromboembolism ( pte ) 3 days after the second step .
patient d suffered from intestinal fistula leading to sepsis and child class b hepatic failure 7 days after the first step of operation .
furthermore , right - sided pleural effusion developed and was treated by inserting a chest tube .
after 45 days of hospitalization , patient d has made a satisfactory recovery from enterocutaneous fistula and discharged with total bilirubin amount of 5 mg / dl .
patient e developed hepatic failure child class a and right - sided pleural effusion which was drained .
no evidence of recurrence and/or metastasis was detected in the follow - up visits of these two patients . in patient b , the ct after 1 year showed a 2 cm lesion at the cut surface of the liver .
patient a was referred again with few metastatic focuses in segments ii and iii after 11 months and underwent multiple radiofrequency ablations and chemotherapy .
it has been observed 25% recurrence rate among our patients during the follow - up period [ table 1 ] .
four candidates of alpps with diagnosis of pancreatic neuroendocrine tumor ( pnet ) , colon adenocarcinoma , pnet with unknown origin and rectosigmoid tumor had their surgeries canceled intraoperatively [ table 2 ] . in two cases ( g and i ) , fatty firm liver have been detected during primary abdominal exploration .
since hypertrophy of about 23-fold is mandatory and damaged hepatic tissue is unable to produce the required volume , hepatectomy was canceled to prevent postoperative hepatic failure .
resection of the primary tumor was performed in case i who was affected with clm to relieve partial obstruction symptoms .
peritoneal seeding and several small metastases were observed during primary exploration in cases f and h , respectively [ table 2 ] .
alpps is a novel resolution giving hope to some patients with hepatic mass who definitely wo nt survive the disease .
the procedure is consisted of two - staged hepatectomy by which rapid significant hypertrophy of the remnant is induced by transecting the parenchyma while there is not enough time for the development of collateral formation .
the technique is relied on portal vein ligation and in situ splitting through parenchyma so that the two parts are just connected through hilar structures in the first step .
partitioning will be completed after an interval of 7 days if flr hypertrophy is satisfactory .
but the reason that the technique is not approved yet is lack of being evaluated technically in early development phase till now .
this study consists of all the candidates of alpps procedure , either patients for whom operation was done successfully or patients who met the criteria for alpps in initial assessment but failed to complete the procedure due to some reasons .
although there is no oncologic verification of alpps , we only used this method in patients for whom there was no alternative treatment .
however many authors emphasized on necessity of having a common criteria , it is not determined yet .
early experiences of alpps reported postoperative complications which may be due to misusing the technique extensively . then , some restrictions on patient selection were applied in response to high morbidity and mortality including age limitation to under 70 , and exclusion of patients with hilar cholangiocarcinomas , or severe portal hypertension .
previous studies indicated that most of the patients underwent the first step of procedure would complete the second step as well , considering sufficient flr hypertrophy between two steps . in our study , sufficient hypertrophy was developed after the first step in all patients indicating 100% feasibility .
patient d computed tomography scan of hepatocellular carcinoma as mentioned above , one of the limiting factors of this method is high morbidity of about 43%60% due to hepatic failure , bile leakage , and perioperative infections .
despite one postoperative death because of massive pte , hepatic failure rate among our patients was similar to other reports .
unlike other studies , bile fistula and leakage were not found among any of our patients .
according to kawarda 's classification , bile duct of segment iv drains the left hepatic area near the confluence in 35% of cases . based on this fact bile leakage
is due to missed open end of the segment iv a / b ductules which is injured or cut during liver partitioning .
hence , if the transect line is not alongside the falciform ligament , reviewing the biliary tree anatomy of this segment is advised .
as a matter of fact , regenerated hepatic tissue does not have function in proportion to the volume .
to mention one reason , chemotherapy or underlying disease may cause malfunction of liver parenchyma . to proceed to the next step , flr of 20%30% is acceptable in normal hepatic tissue and the flr of 40%50% in damaged hepatic tissue such as chemotherapy - treated parenchyma .
steatosis and fibrosis was seen in 2 of our cases who had diabetes and hypertension and were pretreated by chemotherapy .
since damaged hepatic tissue is unable to produce the required volume , hepatectomy was canceled to prevent postoperative hepatic failure .
therefore , we suggest taking liver biopsies in suspected cases of fatty liver due to an underlying disease and patients underwent chemotherapy .
patient selection will be more precise while mandatory amount of flr hypertrophy is evaluated by liver biopsy . in two scheduled cases ,
the operation was not performed because of small peritoneal seeding and multiple small liver metastases . regarding these cases , although no contraindication for alpps exists before surgery , intraoperative findings caused cancellation of hepatectomy .
the development of inflammatory adhesions following the first step makes the second step more challenging .
here we mentioned a patient with the complication of intestinal fistula after the second step of procedure .
we advised to cover the cut surface of liver and the visceral organs with an extra organ bag to prevent visceral contact with the incision site .
one of the difficulties is that the intestinal loops attach together and could not fill the empty cavity generated after hepatectomy .
the solution to this problem is releasing omentum from stomach and transverse colon and placing it below the right diaphragm .
importantly , the surgical incision type has to be considered , especially among clm cases . based on our experience ,
it is preferred to have a midline incision and decide on where to put the transverse incision according to the primary tumor location .
we used reverse l incision and an extra organ bag for our candidates . furthermore , diagnostic laparoscopy and biopsy before main surgery is suggested .
| background : associating liver partition and portal vein ligation for staged hepatectomy ( alpps ) is a novel surgical technique liver resection in traditionally nonresectable primary intrahepatic tumors or colorectal liver metastases.materials and methods : from june 2013 to march 2014 , patients with primary tumor of liver or colorectal tumors with liver metastasis were selected to evaluate whether they met the initial criteria for alpps procedure.results:nine patients enrolled in the study with primary diagnoses of colon and rectosigmoid cancer , carcinoid tumor , gastrointestinal stromal tumor of small intestine , hepatocellular carcinoma , and pancreatic neuroendocrine tumor ( pnet ) .
four candidates excluded from the study prior or during the first step operation due to fatty liver , hepatic fibrosis , peritoneal seeding , and multiple small intestine metastases .
five patients underwent two stages of alpps with the interval of about 1 week .
liver hypertrophy was 100% among our candidates after the initial step .
one postoperative death happened because of massive pulmonary thromboembolism recurrence of liver metastasis was seen in one patient .
hepatic failure class b and a were observed in two patients which did not progress during follow - up period .
one patient developed an enterocutaneous fistula.discussion:we recommend to use 2 organ bags , one for wrapping right lobe and the other one for covering visceral organs and also do liver biopsy in suspicious cases of damaged liver parenchyma and laparoscopic exploration of abdomen for seeding and multiple metastases prior to laparotomy.conclusion:as the procedure has not been well established and verified by oncologists yet , further studies are required to define the exact indications of alpps . |
age - related macular degeneration ( amd ) is the main cause of legal blindness ( visual acuity lower than 20/200 ) in the western world in people aged over 55 years .
it is a degenerative and progressive macular disease that results in loss of central vision with significant functional impairment .
the most severe vision loss occurs in the neovascular form of amd ( nvamd ) , involving choroidal neovascularization associated with retinal edema . even when amd does not lead to blindness
worldwide , 25 million to 30 million people have severe visual loss due to amd .
the prevalence of amd in spain is estimated between 1.3% ( 65 to 74 years ) and 5.7% ( aged 75 years ) of population , accounting for approximately 485.000 potential patients .
since nowadays life expectancy is growing the prevalence of amd is likely to increase , hence its diagnosis and treatment might represent an important challenge .
therapies currently approved for nvamd include laser photocoagulation , photodynamic therapy , and the new drugs capable of inhibiting vascular growth . the vascular endothelial growth factor ( vegf ) inhibitors demonstrated improved visual outcomes compared with other therapies , becoming the first - line therapy for nvamd .
the phase iii marina and anchor trials with ranibizumab were designed with fixed monthly injections over a period of two years [ 7 , 8 ] . however , as this treatment strategy is difficult to undertake in the regular clinical practice , other alternative approaches have been proposed by some retina specialist societies from several european countries , like the spanish society of retina and vitreous ( serv ) , recommending an initial loading dose followed by maintenance injections on an as - needed basis ( prn : per re nata ) according to visual acuity and optical coherence tomography ( oct ) changes . likewise , three german ophthalmologic societies ( der retinologischen gesellschaft , der deutschen ophthalmologischen gesellschaft und des berufsverbands der augenrzte deutschlands e.v . ) and the prescription guidelines in france recommend an initial 3-month upload phase of monthly injections and retreatment to prevent disease progression if there is evidence of disease activity or vision deterioration .
monitoring visits are recommended at monthly intervals following the upload phase in germany and in spain .
although some recent clinical practice studies have assessed the compliance with these guidelines [ 1115 ] , nothing has been published regarding the spanish situation . in order to face the future needs of nvamd patients ,
therefore , the aim of the present study was to survey the diagnosis and management of patients with nvamd treated with antiangiogenic intravitreal therapy in terms of clinical practice in spanish public health centers .
an observational retrospective study was conducted to assess the diagnostic management , therapeutic approach , and use of resources in spanish public health centers for patients with nvamd treated with ranibizumab or bevacizumab as first line .
the inclusion criteria considered were ( 1 ) patients older than 18 years ; ( 2 ) patients diagnosed with nvamd on the first or second eye between february and june 2009 ; and ( 3 ) patients followed up since the diagnosis of nvamd in the same center .
twelve ophthalmologists specialized in nvamd from different tertiary care public spanish hospitals participated in this study .
patients were selected consecutively to avoid selection bias and each one was observed for a maximum period of two years . the selected eye for
whenever both eyes were diagnosed at the same visit , the studied eye was the one with better corrected visual acuity ( va ) . if va was the same , the right eye was then selected .
the study was approved by the spanish agency of medicines and medical devices ( agencia espaola de medicamentos y productos sanitarios ) and the ethical review board of each participating center .
clinical variables concerning the study eye were collected at baseline and at every follow - up visit , which could be treatment - related or not treatment - related .
baseline visits recorded the number of times that a diagnostic technique ( biomicroscopy , fluorescein angiography ( fa ) , and oct ) was used .
the aim of each follow - up visit was to perform a clinical assessment of the patient : symptoms , lesion size ( number of disc areas : < 1 , 1 - 2 , > 2 ) , foveal thickness ( measured in microns ) , macular thickness ( measured in microns ) , and va ( measured with snellen or etdrs charts ) .
the type of treatment and dose administered for nvamd were also recorded , as well as the number of annual injections .
the administration of an initial loading dose was considered as the injection of 3 doses in a period of maximum 60 15 days between the first and third doses of ranibizumab or bevacizumab . to assess
the evolution of clinical variables several temporal frames were defined : 3 , 6 , 12 , and 24 months after the diagnosis of the study eye .
the nearest follow - up visits to each temporal frame ( 1 month ) were included in the analysis of that frame .
results were expressed as mean and standard deviation ( sd ) for the continuous variables and as the number and percentage of patients per category for categorical variables .
the evolution of clinical variables was assessed by t - student or cochran's - q tests , depending on the variables characteristics ( continuous or categorical , resp . ) .
early treatment diabetic retinopathy study ( etdrs ) scores were converted to snellen scores using a transformation table by patel et al . .
thereafter , snellen fractions were approximately converted to letter count according to an equivalence table .
data analysis was made using spss ( version 19 ) and sas software ( version 9.2 ) .
a total of 221 patients fulfilled the inclusion criteria and 208 were finally included in the analysis .
a summary of the sociodemographic characteristics of the study population and the study eye is provided in table 1 .
of the 208 patients studied , 133 ( 63.9% ) had nvamd in only one eye and 75 ( 36.1% ) in both . smoking history was observed in 19.7% of patients : current smoker ( 5.8% ) and former smoker ( 13.9% ) .
the referral of patients was to general ophthalmologists or other retina specialists in 68.3% of cases , emergency services in 23.6% of cases and general practitioners or optometrists for the rest of the patients .
a total of 95.7% , 76.4% , and 93.8% of patients underwent biomicroscopy , fa and oct , respectively . of note , 67.8% of cases underwent all the three techniques to establish the diagnosis of nvamd .
the mean time between the appearance of symptoms and the diagnosis of the study eye was 1.9 2.3 months .
the administration of first - line treatment took place 14.9 29.6 days after diagnosis in average .
the decrease of foveal thickness was in average 87.80 85.88 m , 85.29 97.74 m , 82.24 104.01 m , and 83.73 126.52 m at 3 , 6 , 12 , and 24 months after diagnosis .
the decrease of macular thickness was in average 34.81 48.72 m , 37.16 66.50 m , 35.26 61.73 m , and 27.92 56.44 m at 3 , 6 , 12 , and 24 months after diagnosis .
thus , at baseline , the mean of foveal and macular thickness was 368.55 125.14 m and 304.59 63.41 m , respectively . and
at the end of the study , mean foveal and macular thicknesses were 267.12 104.18 m and 265.71 30.70 m , respectively .
mean va gains of + 6.45 12.93 , + 3.84 15.17 , + 2.41 16.59 , and + 3.13 19.64 letters were observed at 3 , 6 , 12 , and 24 months of follow - up compared with baseline , respectively ( figure 1 ) .
thereafter , the response decreased until one year of follow - up , when stabilized above the baseline values until the end of the study .
mean va observed at baseline was of 47.93 18.57 letters ( 0.25 or 20/80 approximately ) , and at the end of the study it was of 52.99 22.24 letters ( 0.35 or 20/63 approximately ) .
patients who were treated early after the diagnosis ( less than 7 days ) gained va ( + 6.37 letters in average ) , whereas patients treated later than 14 days after the diagnosis lost va ( 4.41 letters in average ) ( p < 0.001 ) .
a total of 2802 visits were recorded , of which 1230 were treatment - related . at the end of the study , patients had received on average 6.1 intravitreal injections of antiangiogenic drugs : 4.5 1.8 during the first year and 1.6 2.1 during the second year .
overall , 14.7% of the visits were performed following the recommendation of monthly visits established by the serv . during the first and second years of the study a mean of 66.0 57.6 and 82.5 53.1 days between consecutive follow - up visits
a mean of 5.4 2.8 and 3.6 2.2 follow - up visits were performed per patient during the first and second year , respectively .
most patients received ranibizumab ( 96.2% , n = 200 ) as antiangiogenic treatment , although an off - label use of bevacizumab was also reported ( 5.3% , n = 11 ) . during the second year of follow - up about half of the patients ( 51.4% ) received antiangiogenic treatment ( 50% , n = 104 with ranibizumab ; 3.8% , n = 8 with bevacizumab ) for nvamd .
a total of 105 patients ( 50.5% ) did not receive a loading dose . those patients with a loading dose ( 49.5% , n = 103 ) showed better results regarding final va and decrease in foveal and macular thickness over the follow - up period . in particular , mean va gains at 3 , 6 , 12 , and 24 months of follow - up among the group of patients who received a loading dose were + 9.06 12.92 , + 6.69 15.65 , + 5.75 17.86 , and + 4.93 20.31 letters , respectively ; whereas the group without an initial loading dose gained in average + 4.58 13.30 , 0.65 15.54 , + 2.64 16.22 , and + 4.14 17.84 letters at 3 , 6 , 12 , and 24 months of follow - up , respectively .
thus , at the end of the study both groups showed similar va improvements ( figure 2 ) .
the difference reported at 6 months of follow - up between both groups was statistically significant ( p = 0.0038 ) .
total injections received per patient were similar in both groups : a mean of 4.3 2.1 and 1.6 2.1 injections were recorded in the group without an initial loading dose during the first and second years , respectively , and a mean of 4.7 1.8 and 1.6 2.1 injections were recorded in the other group during the first and second years , respectively .
one particular participating center stood out among the others due to a more proactive treatment regimen , consisting of more treatment - related visits instead of follow - up visits . over the study period this center performed a mean of 10.8 treatment - related visits per patient against 5.3 for the rest of the centers .
patients recruited ( n = 33 ) in this hospital obtained substantially greater va gains compared with patients of the rest of the centers .
this observational retrospective study , performed in several public health centers in spain , identified that the management of patients with nvamd treated with vegf inhibitors in routine clinical practice was variable and far away from that recommended by the european specialist guidelines in terms of treatment - related and not treatment - related visits .
for example , the serv guideline and three german ophthalmologic societies recommend a loading treatment initiation followed by monthly follow - up visits to detect early recurrences and perform prompt retreatments to avoid as much as possible the permanent anatomic and functional damage [ 9 , 10 ] .
the results previously described showed that , at the end of the first year , patients gained in average only 2.4 letters from baseline and slightly more than 3 letters at the end of the study period .
these gains are lower than those observed in the pivotal trials with fixed monthly regimen or strict prn schemes with monthly visits [ 18 , 19 ] . these differences can be explained by the wide variability found , which was related to time between first symptoms and diagnosis , time between diagnosis and initiation of treatment , and large follow - up visits intervals .
the delay in the nvamd diagnosis is likely to be caused by a lack of knowledge about the disease , but the treatment delay could be due to the different regional health systems and the accessibility to them .
the large follow - up visit intervals and the fall of the use of antiangiogenic treatment observed during the second year of the study might be due to the big clinical burden in the ophthalmology departments of the public health system in spain that makes a proper monitoring of the increasing nvamd population more complicated .
the initial va gain observed at the first 3 months was mainly attributable to patients who received an initial loading dose .
nevertheless , these patients gradually lost this greater gain and ended the follow - up period close to the gain achieved by the group of patients who did not receive the loading dose .
this can be explained with the fact that , at the end of the study , both groups received a similar number of injections . as reported by the catt research group
, the effect of initial monthly doses disappears once the treatment regimen is not strict and proactive .
the maximum improvement observed in va at 3 months of follow - up is in accordance with other recent clinical practice studies in sweden , germany , denmark , and france , where a peak of va gain occurred after 3 months of treatment followed by a decrease of the response .
few treatment administrations have been recorded in this study , and this fact could be explained by the low number of follow - up visits performed . as it has been reported in the catt study , a prn treatment approach for nvamd patients could offer similar results to fixed monthly injections during the first year of treatment .
unfortunately , a monthly fixed regimen or monthly follow - up visits are hardly feasible in the current clinical practice due to the economic costs and/or the clinical burden in ophthalmology departments of the public health system in spain . therefore , further studies assessing the benefits and the costs associated to a stricter treatment regimen would be of interest .
it is worthy to mention the case of the public hospital with the highest rate of treatment - related visits .
this center performs a more proactive treatment approach than the rest of the centers as more treating than monitoring visits are performed . in addition , va outcomes obtained in this center are substantially better than the overall gains obtained in this study .
although one center out of 12 is not representative , the results found in this hospital support the findings reported in other studies , such us the marina and anchor clinical trials that demonstrated efficacy of ranibizumab with monthly injections , and some recent studies , which demonstrated a higher response as the number of injections increases [ 20 , 21 ] .
in addition , this approach consisting in lower number of follow - up visits but more treatment visits could be affordable to obtain good va results in overloaded health systems where monthly monitoring is not possible and the patient is exposed to large intervals between retreatment with vegf inhibitor drugs , thus increasing the chance of disease reactivation and irreversible damage accumulation .
postponing retreatment until there is evidence of ongoing active disease might be leaving it too late .
design limitations are those typically seen in retrospective studies , such as the lack of data in some medical records or the fact that the information is not directly collected from the patient . however , a retrospective design was the most appropriate in order to achieve the objectives of the present study .
another consideration is concerning the two scoring systems ( snellen and etdrs charts ) , which are known to show differences , particularly in patients with nvamd and poor va . nevertheless , as values are shown in terms of changes in the scores , there is no risk of bias in the results . in conclusion ,
our results show the need for a more standardized approach in the management and diagnosis of nvamd patients treated with vegf inhibitors in spain .
other clinical practice studies [ 12 , 13 , 15 ] achieved the same conclusion in other european countries .
an early diagnosis , a proactive approach , and close follow - up monitoring might be the key to successfully manage this degenerative disease .
earlier treatment including an initial loading dose followed by a stricter reinjection schedule could longer maintain va gains in better values .
an effort should be made to increase the adherence to the retina specialist guidelines in order to offer the best va outcomes to nvamd patients , while new treatments or nvamd management are explored and validated . avoiding reactivation of the disease
should be considered as an aim of the nvamd treatment instead of retreating disease reactivation , since a permanent damage is usually accumulated and never recovered , especially if the monitoring surveillance is not as often as it should be . due to treatment cost and the significant impact that vision loss can have on quality of life , economic evaluations comparing current clinical practice and stricter reinjection regimens could bring valuable information in order to help the decision making process and to standardize a more appropriate treatment schedules . | purpose . to survey the management of patients with neovascular age - related macular degeneration ( nvamd ) in spain
. methods . an observational retrospective multicenter study was conducted .
the variables analyzed were sociodemographic characteristics , foveal and macular thickness , visual acuity ( va ) , type of treatment , number of injections , and the initial administration of a loading dose of an antiangiogenic drug .
results .
208 patients were followed up during 23.4 months in average . during the first and second years , patients received a mean of 4.5 1.8 and 1.6 2.1 injections of antiangiogenic drugs , and 5.4 2.8 and 3.6 2.2 follow - up visits were performed , respectively .
the highest improvement in va was observed at 3 months of follow - up , followed by a decrease in the response that stabilized above baseline values until the end of the study .
patients who received an initial loading dose presented greater va gains than those without . conclusions .
our results suggest the need for a more standardized approach in the management and diagnosis of nvamd receiving vegf inhibitors . to achieve the visual outcomes reported in pivotal trials , an early diagnosis , proactive approach ( more treating than follow - up visits ) , and
a close monitoring might be the key to successfully manage nvamd . |
over the last twenty years a number of scholars have been calling for a bioethics that is culturally sensitive .
many have been critical of traditional bioethics , arguing that it is dominated by the principles and methods of anglo - american philosophy which are too abstract and insensitive to social and cultural realities ( 1 ) . at the same time
, there has been a trend to make bioethics more relevant to real life cases by incorporating the methods of the social sciences ( 2 ) .
my argument is that although these trends in bioethics have their own merits , if overemphasized and not properly conceptualized , they can easily undermine the normativity of bioethics by reducing it to a social science .
bioethicists must endeavor to provide judgments of how things ought to be and not simply describe how things are. it is useful to note that the issues discussed in this paper are part of a wider contro - versy concerning the universality of human rights .
indeed , like in the field of bioethics , similar calls have been made for human rights to be culturally relevant and sensitive to context ( 3 ) .
as i have already pointed out , over the past twenty years there have been incessant calls for bioethics to be more sensitive to culture and social context .
what has motivated these calls is the realization that all individuals see the world through the filtered eyes of their own culture .
consequently , any attempt to impose moral principles based on anglo - american philosophy to people with different cultural perceptions is unjust and amounts to cultural imperialism .
the main criticism leveled against traditional bioethics is that it ignores the role of social and cultural factors in the ethical - decision making process .
a number of scholars , especially those from the developing countries , see the globalization of bioethics as a form of neocolonialism and an attempt by the developed world agencies to advance their biomedical agenda on resource poor nations .
these critics have gone on to call for a truly global bioethics that acknowledges the existence of alternative ethical frameworks ( 4 ) .
he coined the term ethnoethics to refer to the examination of ethical issues in biomedicine in non - western cultures .
he described ethnoethics in the following way :
this would include moral norms and issues in health care as understood and responded to by members of these societies .
ethnoethics should be informative not only about cross - cultural variation in ethical principles of medicine , but also about variations in issues which in different societies become defined as morally relevant or problematic .
ethnoethical information should contribute to the discourse of medical ethics , not only by illuminating culturally distinctive moral views and problems , but also by helping to provide a more realistic and knowledgeable basis for the exploration of cross - cultural ethical similarities ( 5 ) .
this would include moral norms and issues in health care as understood and responded to by members of these societies .
ethnoethics should be informative not only about cross - cultural variation in ethical principles of medicine , but also about variations in issues which in different societies become defined as morally relevant or problematic .
ethnoethical information should contribute to the discourse of medical ethics , not only by illuminating culturally distinctive moral views and problems , but also by helping to provide a more realistic and knowledgeable basis for the exploration of cross - cultural ethical similarities ( 5 ) .
murove ( 6 ) and ogundiran ( 7 ) have separately complained about the western domination of contemporary bioethics .
they have gone on to call for the evolution of an authentic african bioethics , one that benefits from other cultural influences yet not overshadowed by them .
expressing similar sentiments , tai and lin ( 8) have called for a truly asian bioethics , which is based on the traditions and culture of the asian people . taking the principle of informed consent as an example , the two authors have argued that applying this principle to research involving human subjects in the asian context without consulting the family would be inappropriate because of the high value that asians place on community shared decisions .
but de castro ( 9 ) has cautioned that any attempt to assert asian bioethics must recognize that , even within asia , different bioethical perspectives exist and it would therefore be wrong to lump all asian people together as if asia is a homogeneous society .
an understanding of the cultural beliefs of others and how they are influenced by them is especially important in clinical practice . learning how different cultures define and understand health , illness , pain and even death
can go a long way in helping resolve the many ethical dilemmas that healthcare providers routinely encounter .
such an understanding will also translate into improved clinical management ( 10 , 11 , 12 ) . from the foregoing
it is clear that there is an urgent need to recognize that different cultures have different practices and values and we should take this into account when evaluating them .
however , as we shall see later , this does not mean that we can not make important judgments about particular cultural practices .
calls for a culturally sensitive bioethics have coincided with what is now described as the empirical turn in bioethics . as with the calls for a culturally sensitive bioethics ,
the empirical turn in bioethics has come about as a result the traditional bioethics preoccupation with conceptual analysis , which many critics claim has led to a disconnect between theory and practice . while this turn has its own merits , when accompanied by calls to take cross - cultural moral differences seriously , it can undermine our confidence in the normative analytic methods of bioethics . a quantitative analysis of peer reviewed medical ethics journals in the field of bioethics in the period 19902003 showed that the proportion of empirical research in bioethics rose in these journals from 5.4% in 1990 to 15.4% in 2003 ( 2 ) . a more recent study carried out in turkey showed a sharp increase in empirical studies in turkish medical ethics literature during the period 19942009 ( 13 ) .
this turn in bioethics has come as a response to the social science critique of traditional bioethics , with some commentators dismissing it as too abstract and nave . as john irves
forcefully puts it philosophers must get out of the platonic ivory tower , and acknowledge that ethics is about people , not just good arguments .
it is about encounter with experience and using those encounters to inform one s philosophy ( 14 ) the wellcome trust of the united kingdom has described this approach to bioethics as an abstract exercise carried on over sherry in the tutorial rooms of ivory towers ( 15 ) .
it is noteworthy that this trust has been at the fore - front of funding empirical studies in bioethics .
this turn has been well received because it has enriched the field of bioethics in a number of ways
. mildred solomon has identified three different ways in which empirical research can be used in bioethics .
firstly , empirical studies can help facilitate the move from ethical analysis to ethically justifiable behavior ; secondly empirical data can be used to enhance ethical analysis and justification ( this involves testing consequentialist claims ) , and thirdly it can be used to identify and document new moral dilemmas ( 16 ) . however , it must be emphasized that empirical data per se does not determine what is right or wrong although it might be relevant in making such a determination .
but it could be argued that this approach to bioethics is not entirely new . according to sociologist adam hedgecoe those who are interested in incorporating the empirical element into their work
such a discipline already exists in the form of medical sociology . as he forcefully puts it : if medical ethicists are interested in the lived experience of the social world of modern medicine , an obvious solution would be to read some medical sociology rather than look towards developing a new discipline ( 17 ) .
sociology and anthropology have a history of investigating bioethical subjects which are not always recognized by mainstream bioethics ( see for example fox ( 18 ) and edel ( 19 ) . however , the two disciplines i.e. sociology and anthropology do not have ethics as their primary focus .
although both cultural sensitivity and empirical research can enrich bioethical debate , they can be harmful to the extent that they can be used to shield certain harmful but well entrenched cultural practices from external criticism .
indeed the greatest challenge facing bioethicists today is how to produce a bioethics that is both sensitive to culture and lived experience and yet critically normative .
we should be worried about cultural relativism because in the past some people have equated it with ethical relativism , which is the claim that morality is relative to one s culture .
those who argue in this way are accused of deriving an ought from an is or deriving normative claims from factual claims .
the naturalistic fallacy , as this error in reasoning is called , was first pointed out by david hume ( 20 ) in the 18 century and elaborated by george edward moore ( 21 ) in the 20th century . moreover , ethical relativism and its prescription for tolerance conjure images of a world where anything goes .
just because cultures differ in their ethical judgments does not mean that they are justified in holding them or that we should tolerate those beliefs .
the major advocates of ethical relativism were anthropologists who were heavily engaged in the study of indigenous cultures . however , one of the earliest defenders of moral relativism was the greek philosopher protagoras .
he went on to suggest that morality is a matter of social convention and in not found in nature .
for example , brandt ( 22 ) defends the view that there are conflicting moral claims that are equally valid , while hartman ( 23 ) defends what he calls normative moral relativism , which is the claim that different people are subject to different moral demands .
wong ( 24 ) rejects the notion that there is one true morality and defends the view that there is a plurality of true moralities . in the area of anthropology
the main defenders of ethical relativism are benedict ( 25 ) and herskovits ( 26 ) . after studying the cultural practices of different human communities they concluded that what is considered morally normal is culturally bound and historically defined and the western standards of morality should not be considered universal .
a major criticism of moral relativism is that even if different cultures practice different moral behaviors , they may nevertheless share the same underlying moral values .
furthermore , even if we accept that different societies have different moral beliefs , this does not warrant the conclusion that all moral codes are equally valid ( 27 ) .
the field of bioethics can not survive without the belief that there are moral universals .
the truth of the matter is that despite the existence of significant cultural differences , there exist some core moral values that are shared by inhabitants of most human communities .
unfortunately , most people have tended to over - emphasize cultural differences at the expense of the similarities .
many authors have claimed that this concept is absent in both african and asian cultures whose moral theories are communal in nature .
but this view has been challenged by agulanna ( 28 ) . according to him , the idea that africans value collective agreement over individual choice is grossly exaggerated . along the same lines
metz ( 29 , 30 ) has conclusively argued that just like utilitarianism and kantianism , african ethics entails a right to autonomy and informed consent .
but if the later beliefs are better than the earlier ones , it must be because they are closer to what is objectively right .
this concept , which is today very highly valued in western culture , came about as a counter response to the paternalistic hippocratic medicine , which had dominated western medicine since the time of hippocrates .
today cultural relativism has come to mean nothing more than the idea of tolerance . on the face value
this might be considered good but as nafisi rightly pointed out such an attitude neutralizes action instead of galvanizing it ( 31 ) .
indeed , it is possible to practice cultural relativism as a social scientist and the same time maintain a commitment to moral objectivity . by reasoning and argument
further - more , as velasquez ( 32 ) has noted , moral disagreements may be an indication that some people are more morally enlightened than others and we should not assume that if ethical truth exists , then everyone must know it .
as they use empirical studies to enhance bioethical discourse , bioethicists must ensure that bioethics does not become a chapter of sociology or anthropology .
this in essence means that the cross - fertilization between ethics and the social sciences must be done with utmost care .
most bioethicists do nt have a very good grounding in academic moral philosophy . and as david benatar points out , the field of bioethics today suffers from serious quality control problems. the reason for this is that although strictly speaking bioethics is a sub - branch of ethics ( as its name suggests ) which is in turn a branch of philosophy , many of the practitioners in this field are either social scientist , lawyers , health economists , theologians and medical practitioners who are not well grounded in academic moral philosophy .
benatar s further notes :
there has been a proliferation of courses , diplomas and degrees in bioethics . as these courses are often aimed at those without philosophical training and lack the rigor and often duration of other courses of study , there are more and more people with formal and poor bioethics education .
there is a whole enterprise of bioethics education that is creating experts if not instantly then certainly very quickly . in some cases , a brief course or a diploma
is thought sufficient to transform a novice into a so called ethicist , bioethicist or , worse still , bioethics educator ( 33 ) .
there has been a proliferation of courses , diplomas and degrees in bioethics . as these courses are often aimed at those without philosophical training and lack the rigor and often duration of other courses of study , there are more and more people with formal and poor bioethics education .
there is a whole enterprise of bioethics education that is creating experts if not instantly then certainly very quickly . in some cases , a brief course or a diploma
is thought sufficient to transform a novice into a so called ethicist , bioethicist or , worse still , bioethics educator ( 33 ) .
given that they do not have a good grounding in academic moral philosophy , they can easily be tempted to move too quickly from empirical findings to normative conclusions . but this is not to suggest that bioethics should be the exclusive domain of professional philosophers or that empirical research has no role to play in bioethics . on the contrary ,
non - philosophers and especially social scientists have a big role to play in bioethics because empirical findings are often used as premises in ethical arguments .
indeed , good studies in bioethics must be grounded on good empirical data and the philosopher has no choice but to either collaborate with the social scientist or be acquainted with social science methodologies so that she can at least read and interpret empirical data if not carry out the empirical research herself .
bioethicists will also need to know what counts as useful and relevant empirical data and where to find it .
this is because the most useful and original empirical studies , as james dubois recently pointed out , are not published in traditional bioethics journals .
empirical data in bioethics are not data that determine what is right or wrong , but rather are relevant to that determination ( 34 ) .
but as i pointed out previously , in order to avoid the compartmentalization of the empirical and the ethical , collaboration between philosophers and social scientists should be encouraged .
what i am calling for is a moderate form of naturalism that does not threaten to undermine the normativity of bioethics .
the worries i have raised regarding the relationship between facts and values ; ethical theory and empirical data should not be viewed negatively by social scientists . as chris herrera has pointed out this tension
it is a reminder that bioethics is rooted in philosophy , which is a self - reflective discipline that questions its own methods .
in this paper i have argued that although both the cultural and empirical turns in bioethics have enriched the field of bioethics , if not properly conceptualized and integrated with ethical theory , they can easily undermine bioethics normative mandate .
this concern can not be wished away or dismissed with a wave of the hand as it touches on the very essence of bioethics as a philosophical discipline .
as they use empirical data and acknowledge that different cultures have different moral codes , bioethicists must be guided by the belief that objective moral values that transcend culture exist .
this is the only way that bioethics as a discursive discipline can grow and flourish .
bioethicists should debate and , if need be , reject those moral practices that defy rational justification . | in the last two decades , there have been numerous calls for a culturally sensitive bioethics . at the same time , bioethicists have become increasingly involved in empirical research , which is a sign of dissatisfaction with the analytic methods of traditional bioethics . in this article
, i will argue that although these developments have broadened and enriched the field of bioethics , they can easily be construed to be an endorsement of ethical relativism , especially by those not well grounded in academic moral philosophy .
i maintain that bioethicists must resist the temptation of moving too quickly from cultural relativism to ethical relativism and from empirical findings to normative conclusions . indeed , anyone who reasons in this way is guilty of the naturalistic fallacy .
i conclude by saying that properly conceptualized , empirical research and sensitivity to cultural diversity should give rise to objective rational discourse and criticism and not indiscriminate tolerance of every possible moral practice .
bioethics must remain a normative discipline that is characterized by rigorous argumentation . |
primary salivary gland carcinomas with neuroendocrine differentiation are rare accounting for 3.5% of all malignant tumors and less than 1% of all carcinomas of parotid gland .
nicod reported the first case of carcinoid tumor of the parotid gland in a 51 year old lady . following this
there have been occasional reports of round cell tumors of the parotid gland and minor salivary glands with very few reports of primary neuroendocrine tumor [ 14 ] . amongst the various primary tumors showing neuroendocrine differentiation ,
a 48 year old lady presented to the ear nose and throat outpatient department with a small , painless , right sided infra - auricular swelling for 1 year which had suddenly increased in size in the past 1 month ( fig . 1 ) .
local examination revealed a round , well defined , firm , non - pulsatile swelling fixed to the underlying structures measuring 3 3 cms located in tail of parotid .
1right infra - auricular , painless swelling 4.5 3.5 cms right infra - auricular , painless swelling 4.5 3.5 cms contrast enhanced ct ( cect ) scan revealed a well defined , homogenously enhancing soft tissue mass , measuring 3.5 3 2.5 cms , in the superficial lobe of the right parotid gland causing medial displacement of the retromandibular vein .
the deep lobe of the right parotid , left parotid gland and bilateral carotid sheath structure did not reveal any abnormality ( fig . 2 ) .
2cect scan showing a well defined , homogenously enhancing soft tissue mass , measuring 3.5 3 2.5 cms , in the superficial lobe of the right parotid gland with displacement of the retromandibular vein cect scan showing a well defined , homogenously enhancing soft tissue mass , measuring 3.5 3 2.5 cms , in the superficial lobe of the right parotid gland with displacement of the retromandibular vein on follow - up visit the swelling increased in size with prominent cystic change .
patient was taken up for right superficial parotidectomy by modified blair s incision . during surgery
a cystic swelling was found to be involving the superficial lobe of parotid which was excised with a margin of normal surrounding glandular tissue .
on gross examination , the specimen measured 3.8 3.2 2.5 cms in size with focal areas of congestion . on cut section
a well defined nodule was identified measuring 3.3 2.9 2.2 cms , which was grey white with focal areas of hemorrhage ( fig .
also seen was a small , already opened up , part of a cystic structure measuring 1 1 0.7 cms filled with multiple , small , friable , whitish material .
3cut section showing a well defined nodule ( 3.3 2.9 2.2 cms ) , which is grey white to dark in color with areas of congestion and surrounding thin rim of normal salivary gland cut section showing a well defined nodule ( 3.3 2.9 2.2 cms ) , which is grey white to dark in color with areas of congestion and surrounding thin rim of normal salivary gland microscopically , a highly cellular tumor was seen , comprised of small and large cells arranged in prominent organoid pattern , nests , trabeculae and sheets .
the nests of cells were separated by thin to thick , hyalinised fibrous septae ( fig .
the individual cell had a centrally placed , round nucleus with coarsely stippled nuclear chromatin .
the cells displayed moderate to abundant amount of deeply eosinophilic and finely granular cytoplasm with well defined cytoplasmic borders .
few areas showed presence of microcystic spaces filled with pale , eosinophilic secretions lined by the tumor cells ( fig .
prominent thin walled , elongated blood vessels were also seen along with multiple foci of hemorrhage .
both the small and large cell neuroendocrine pattern was seen in the same tumor ( fig .
further sections from the mass revealed variable sized clusters of cells arranged in sheets , with centrally placed nucleus and abundant cytoplasm studded with numerous fine , basophilic granules suggestive of acc ( fig .
6 ) , which was confirmed by pas stain with and without diastase pre - treatment .
the tumor cells strongly expressed s-100 , nse , chromogranin a , and synaptophysin ( fig .
based on the histomorphological and immunohistochemical features a diagnosis of acc with extensive neuroendocrine differentiation was made .
the tumor was seen to focally involve the deep resection plane and one of the lateral margins of resection.fig .
4tumor cells arranged in organoid and nesting pattern , separated by thin and think hyalanised fibrous septae .
both small and large cell neuroendocrine pattern can be identified . ( h&e , 100)fig .
. moderate amount of finely granular and eosinophilic cytoplasm can be seen with well defined cytoplasmic membranes .
b small microcystic areas seen amidst tumor cells filled with pale eosinophilic material ( h&e , 250)fig .
6tumor at the periphery shows cells in clusters and sheets with centrally placed small nuclei and abundant cytoplasm studded with fine basophilic granules suggestive of acinic cell carcinoma ( h&e , 250)fig .
d strong cytoplasmic positivity for synaptophysin ( dab chromogen , 400 ) tumor cells arranged in organoid and nesting pattern , separated by thin and think hyalanised fibrous septae .
( h&e , 100 ) a tumor cells show large round , uniform nuclei with stippled nuclear chromatin
. moderate amount of finely granular and eosinophilic cytoplasm can be seen with well defined cytoplasmic membranes .
b small microcystic areas seen amidst tumor cells filled with pale eosinophilic material ( h&e , 250 ) tumor at the periphery shows cells in clusters and sheets with centrally placed small nuclei and abundant cytoplasm studded with fine basophilic granules suggestive of acinic cell carcinoma ( h&e , 250 ) a strong cytoplasmic positivity for s-100 ( dab chromogen , 400 ) .
d strong cytoplasmic positivity for synaptophysin ( dab chromogen , 400 ) patient was followed up subsequently after 3 months with no clinical evidence of recurrence of the tumor and a good healing of the surgical wound .
since the deep plane and lateral resected plane were microscopically involved , the patient was referred to the department of radiotherapy for further evaluation and management of possible residual tumor . on further follow - up after 6 months from the initiation of radiotherapy
primary carcinoma of salivary gland with neuroendocrine differentiation is rare accounting for only 1% of all parotid gland tumors .
neuroendocrine tumors of the parotid gland that have been reported previously were most commonly metastatic or seen in association with co - existing carcinoid tumor elsewhere [ 14 ] . the various primary salivary gland carcinomas that have been reported to show positivity for neuroendocrine markers include a large majority of small cell carcinoma , occasional cases of adenocarcinoma , warthin s tumor , adenoid cystic carcinoma , and acc [ 58 ] .
accs occur usually in the 3rd and 4th decade with a mean age of 44 years and shows predilection to occur in females [ 9 , 10 ] .
most commonly the parotid gland is affected ( 84% ) as was in our case , however it is also known to occur in submandibular and minor salivary glands .
clinically , it presents as a slowly growing painless or painful mass with infrequent facial nerve involvement ( 510% ) and pursues a protracted clinical course .
our patient had a gradually increasing painless swelling with rapid increase in size of recent onset without clinical and intraoperative evidence of facial nerve involvement .
accs are usually rounded and partially or completely encapsulated with predominant solid , brown cut surface with or without cystic areas [ 9 , 10 ] . in our case
microscopically , the tumor is comprised of variably sized lobules of neoplastic cells with broad fronts of invasion .
the cells have a small central to eccentrically placed nuclei with a finely granular to clear cytoplasm features suggestive of differentiation towards the ductal and acinar cells of normal salivary gland .
the granules are fine , numerous , basophilic , and pas positive [ 9 , 10 ] .
immunohistochemically , the tumor cells express cytokeratin ( especially low molecular weight ) , carcinoembryonic antigen and amylase . in our case
although not yet accepted as a distinct clinicopathological entity , some studies have reported neuroendocrine differentiation in acc based on immunohistochemistry and ultra structural studies [ 5 , 7 ] .
performed a study of expression of neuropeptides in various parotid gland tumors which included 11 cases of accs along with other benign and malignant tumors .
they reported the expression of vasoactive intestinal polypeptide ( vip ) in all the 11 cases of accs however was not seen in any other parotid gland tumor .
the cells of the accs stained with grimelius impregnation and six of these cases on ultra structural study demonstrated presence of large dense core granules along with smaller numerous secretory granules and well developed golgi apparatus and rough endoplasmic reticulum . in another study by hayashi et al . in 1990
, they studied the expression of neuroendocrine markers nse and leu-7 in various parotid gland tumors .
included in this study were 12 cases of acc of which seven expressed only leu-7 only . other tumors of the salivary gland expressed either or both nse and leu-7 .
ito et al . in 1990 reported a case of acc with neuroendocrine differentiation in the parotid gland .
staining for grimelius impregnation and ultra structural evidence of dense core granules were suggestive of neuroendocrine features in the cells of the tumor .
in all the above studies , the evidence of neuroendocrine differentiation was evident only on special staining , immunoexpression of neuropeptides and ultra structural morphology .
the studies conducted by hayashi et al . [ 5 , 6 ] , screened for neuropeptide expression in various tumors of the parotid gland .
none of the cases of acc in their studies showed histomorphological features of neuroendocrine tumor . similarly the reported case by ito et al . had a histomorphology ,
preoperatively there were varied diagnosis of pleomorphic adenoma radiologically and oncocytic tumor versus warthin s tumor on cytopathology .
even the initial histological sections were suggestive of a very rare diagnosis of primary neuroendocrine tumor .
however , after further sectioning of the tumor mass in search of other commoner primary tumors , small areas of acc was documented . such extensive degree of neuroendocrine differentiation in an acc , morphologically and immunohistochemically , to the extent as to mimic a primary neuroendocrine tumor of the parotid gland , has not been reported previously to the best of our knowledge .
a possibility of peripheral entrapped acinic cell with primary neuroendocrine tumor was also ruled out ; since both the morphological areas of the tumor were in gradual continuity with each other and neither did the acinic cells appear to be compressed at the periphery .
hayashi et al . used leu-7 , nse , and vip to demonstrate the neuroendocrine differentiation [ 5 , 6 ] . in our case confirmation of neuroendocrine differentiation
de - differentiated acc is an aggressive form of the tumor requiring adjuvant treatment , carrying a poor prognosis .
it is histologically characterized as a composite tumor with acc along with a poorly or undifferentiated carcinoma .
. suggested that a tumor arising from a common stem cell can lead to divergent differentiation into both acc and neuroendocrine tumor in the same mass .
however , in our case , there were no areas showing poor differentiation or markedly anaplastic morphology .
also after 6 months of follow - up the patient had no evidence of tumor recurrence .
the concept of divergent differentiation could be a possible explanation for the phenomenon , however requiring further studies on these tumors .
grading of acc has been attempted based on histo - morphological features , segregating them into well , moderately and poorly differentiated tumor [ 11 , 12 ] , showing good prognostic correlation .
however , it does not account into presence of neuroendocrine features as affecting the grading of the tumor . also the presented tumor is very rare , making it difficult to assess the impact on grading and prognosis .
the neuroendocrine nature of the cells was proven predominantly on the basis of immunohistochemistry and electron microscopy [ 1315 ] .
some authors have reported that the presence of neuroendocrine differentiation in pancreatic acinar cell carcinoma carries a better prognosis that the tumor without it .
primary neuroendocrine tumors of the parotid gland are of rare occurrence . hence when a salivary gland tumor shows only neuroendocrine histology ,
the possibility of primary salivary gland tumor with neuroendocrine differentiation should be kept in mind and tumor extensively sampled to hunt for such a tumor .
one should also be aware of the fact that accs can show extensive neuroendocrine differentiation so much so as to mimic a primary neuroendocrine tumor of the parotid gland .
since there are very few cases of neuroendocrine differentiation reported in acinic cell tumor , a definite comment on prognostic outcome is not possible .
further studies with long follow - up of these patients need to be done to understand the biological behavior of this special variant of acc . | primary salivary gland carcinoma with neuroendocrine differentiation is of rare occurrence , especially so in the parotid gland . amongst the various reported primary tumors with neuroendocrine differentiation , acinic cell carcinoma ( acc ) one such tumor .
a 48 year old lady presented with a gradually increasing right infra - auricular swelling for a period of 1 year which enlarged suddenly in a short period .
contrast enhanced computed tomography ( cect ) suggested diagnosis of pleomorphic adenoma .
fine needle aspiration cytology ( fanc ) yielded a cystic fluid suggesting a possibility of warthin s tumor or oncocytic lesion .
intraoperative findings
were suggestive of a warthin s tumor . initial histopathological examination of the tumor was suggestive of neuroendocrine carcinoma . however , extensive sectioning revealed peripheral islands of acc .
immunoexpression of s-100 , neuron specific enolase ( nse ) , chromogranin a and synaptophysin confirmed the diagnosis .
the possibility of neuroendocrine differentiation in a primary salivary gland tumor should be kept in mind whenever a salivary gland tumor shows only neuroendocrine histology . |
orthotopic liver transplant remains the treatment of choice for fulminant or acute liver failure and end - stage chronic liver deficiency .
etiologies of these liver failures are numerous , ranging from metabolic liver diseases , infectious causes , nonlife compatible large hepatectomy to alcohol hepatic pathology and others .
unfortunately , the increased need for organ transplantation is met by a lack of organ donors . some surgical techniques such as living donor procedure or cadaveric donor liver split sharing between two recipients ; as well as bioartificial hepatic support can partly alleviate this shortage .
cell transplantation could not only replace solid organ transplantation for the medical indications listed above but also it could , in addition , be proposed for gene therapy applications , in which organ transplant is a procedure too heavy to be ethically accepted such as congenital deficiency of a liver metabolic pathway , that does not impair liver function .
we first focused on the evaluation of adult hepatocyte transplantation for treatment of liver deficiency by cell therapy [ 4 , 5 ] , since it presented many attractive aspects when compared to organ transplant : better availability of cells , less invasive surgery , redo possibility , and lesser cost .
it could also become a bridge between fulminant liver deficiency and organ transplant , to gain time before getting an organ suitable for transplantation [ 6 , 7 ] .
more recently , liver stem cells appeared to also be good candidates for transplantation , with the advantage of being maintained longer in culture than mature hepatocytes which lose their functions after a few days and being easily harvested from various human sources ( adult , embryonic and fetal liver , and nonhepatic mesenchymal tissue , as well as induced pluripotent stem cells ( ips ) ) [ 5 , 8 , 9 ] .
to optimize understanding of cell transplantation mechanisms , small animal models were created to allow the study of cell engraftment conditions and mechanisms of liver regeneration . on the other hand ,
the major species differences in the metabolism of drugs require metabolism and toxicity studies using models as close as possible to humans .
in addition to the current gold standard in vitro approach of cultured adult human hepatocytes for the prediction of in vivo hepatic metabolism , pharmacokinetic and toxicity in humans [ 10 , 11 ] , the use of in vivo approach of animals with humanized livers have been explored [ 1214 ] .
furthermore , viral pathobiological studies , that is , hepatitis b / c viral infections and treatment that are not possible on primary human hepatocyte in culture since the cells become nonpermissive for hb / cv after plating , have been made possible with the development of animal models with humanized livers [ 1517 ] .
for these two research area applications , immunosuppressed animal models , that would bear xenogenic cell transplantation without immunosuppressive drug , needed to be developed . in order to allow
the evaluation of treatment of liver deficiency , an additional request of animal models was that they could be submitted to treatments mimicking human clinical liver pathologies requiring cell transplant therapy .
the vast majority of research began with murine models , such as immunodeficient mice , with normal liver , or undergoing liver failure ( by toxic liver injury , partial hepatectomy , or transgenic modification ) and was tested for xenogenic hepatocyte transplantation , at first murine - murine models secondarily extended to human - murine transplantation ( table 1 ) .
for the evaluation of liver deficiency treatment via cell transplantation , the most commonly used models are the scid mouse , lacking functional t and b cells , and rag2 mouse ( transgenic mouse with recombinant activation gene-2 ( rag-2)s ) , which lack mature t and b lymphocytes .
literature reports less numerous studies with nude mice . yet , this athymic mouse with high deficit of mature t cells due to a fox gene family mutation ( foxn1 [ 34 , 35 ] ) is a more robust model than scid or rag2 mouse , and it can be bred in less immunoprotective environment [ 36 , 37 ] .
nude mice have been used since 1968 to study xenogenic tumor development [ 3842 ] , imaging testing , or gonadal grafts .
in murine models , different liver injuries can be performed , to induce hepatic deficiency comparable to human 's one : ( 1 ) surgical , by extensive partial hepatectomy ; ( 2 ) toxic by administration of carbon tetrachloride ( ccl4 ) ( that can also act on transplanted hepatocyte because of its long half- life ) [ 4547 ] , by d - galactosamine that generates at low dose an apoptosis and at higher dose a necrosis and fulminant hepatitis , by retrorsine [ 4951 ] that suppresses hepatocyte proliferation , or by fas - ligand agonist that will trigger cell apoptosis on mouse hepatocyte , such as jo2 antibody treatment [ 5257 ] ; ( 3 ) radiotherapy was also used to block liver regeneration [ 6 , 58 ] .
but , the most growth selective advantage model used is ( 4 ) transgenic mice , with natural liver injury by hepatotoxicity of metabolism disorders .
there are two main models : upa ( uroplasminogen activator ) mouse [ 59 , 60 ] and fah ( fumarylacetoacetate hydrolase ) mouse .
interestingly , we have found that most of these liver injuries can be applied to nude mice . in our experience , and in contrast to the more resistant wild - type balb / c mice , nude mice did not survive 70% partial hepatectomy performed according to the technique of higgins and anderson and died within 24 h following the surgery .
our finding is not in accordance with the report of strom et al . referring to a high survival rate of nude mice submitted to two third partial hepatectomy .
forty percent partial hepatectomy was successful and consisted , after median laparotomy , in the resection of the left lateral liver lobe of nude mice , after ligation using 3 to 5 titanium 3-mm clips for stapling ( ligacliprm , ethicon , france ) .
hemostasis was performed by dabbing the bloody cut surface with caution using a hemostatic cellulose gauze ( surgicelrm , johnson & johnson , france ) , with removal of this gauze , to decrease postoperative mortality .
after partial hepatectomy , animals were warmed up for at least 20 min under heating lamp to restore their preoperative body temperature and were supplemented for 24 h with 20% glucose in drinking water .
serum alt increased ( 2 compared with the day 0 value ) on day 1 following surgery and returned to basal levels within 3 days .
histological features of liver regeneration , ki67 and caspase 3 expression , proved that both mechanisms of apoptosis and cell proliferation coexist without any necrotic stage during liver regeneration .
various doses were tested on nude mice , with a 20%-mortality at day 10 following an injection of 1700 mg / kg and 100%-mortality at 48 hours with a 2000 mg / kg ta dose .
serum alt values reached 20 times the day 0 values 24 hours after a 1700 mg / kg ta injection to nude mice ; then a progressive return to basal values was observed , which was complete on day 7 .
this correlated with a massive liver necrosis ( up to one - third of liver ) , maximal at 24 hours and then decreasing with normal histology at day 10 .
jo2 antibody is a fas - ligand agonist and acts by stimulating fas antigen and cell apoptosis .
jo2 is mouse specific ; thus , it does not trigger apoptosis of other species , and it acts mainly on hepatocyte .
heart , lung , bone marrow , and kidney express fas , but are not sensitive to fas apoptosis .
the specific anti - mouse fas monoclonal antibody jo2 was injected at 125 , 250 , 375 , or 500 g / kg ip dose to nude mice , with higher sensitivity and mortality compared to balb / c mice .
this anti - fas antibody jo2 was proven to induce fulminant hepatic failure in mice after a single injection [ 6870 ] , characterized by an elevation of serological alt for the first three days .
histologically , massive necrosis ( with less than 5% of healthy parenchyma ) was observed in nude mice at 500 g / kg jo2 .
severe panlobular and panacinar necrosis already occurred at 375 g / kg jo2 on day 1 after treatment ( figure 1 ) .
liver deficiency injury was a dose - dependent effect , and it was reversible within 10 days .
the regenerative capacity of the liver was thus retained after jo2 injection , making its use attractive for studying the efficacy of hepatocyte transplantation .
nowak et al . described a single intraperitoneal administration of 0.7 g / kg body weight d - galactosamine , eventually followed 36 hours later by cell transplantation .
the mortality rate was 62% in absence of transplantation , occurring in the first 72 hours .
ccl4 was also used to induce acute liver deficiency in nude mouse [ 23 , 24 ] by intraperitoneal injection of 100 l/20 g body weight of 10% ccl4 in olive oil .
we succeeded in inducing cirrhosis in nude mice , by repeated thioacetamide injections at 200 mg / kg of body weight , three times a week for 14 weeks .
macroscopically , the liver of nude mice presented a slightly rough surface after 58 weeks of treatment .
nude mice exhibited a spectacular fibrosis and onset of cirrhosis from 5 weeks of treatment .
histological features showed accentuated lobulation , with some nearly complete rings of connective tissue surrounding lobules ( figure 2 ) .
ki67-positive cells were more numerous after 14 weeks of treatment compared with 5 weeks of treatment , reflecting intense proliferation of hepatocytes .
induction of cirrhosis in nude mice was found to reverse to fibrosis within 5 months after cessation of the treatment .
upa - nude mice were used by rhim et al . by crossing alb - upa transgenic mice with athymic nu / nu mice .
this upa mouse model presents an activation of the transgene that expresses uroplasminogen activator under the control of an albumin promoter .
this overexpression of upa causes liver injury with accumulation of hepatotoxic substrate and progressive depletion of hepatocytes , neonatal bleeding , associated to kidney disease .
the homozygous mice die from liver deficiency , unless they received safe non - upa hepatocyte transplantation
. in heterozygous mice , population of non - upa hepatocyte can spontaneously develop and repopulate the liver in about 8 weeks [ 59 , 60 ] .
it has been reported that hepatocytes without upa expression , when transplanted , have a growth advantage over the hepatocytes with transgenic upa leading to cell death [ 59 , 60 , 71 , 72 ] .
the transgenic mouse model fah is a mutant mouse deficient in tyrosine catabolic enzyme fumarylacetoacetate hydrolase , corresponding to clinical model of human type 1 tyrosinemia .
this liver injury can be prevented by a drug : 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione ( ntbc ) , usually put in animal drinking water .
breeding is easier than for upa mice ; there is no renal deficiency , and transplant can be performed at any time .
so , nude mouse is a suitable small animal model in which it is possible to induce different types of hepatic deficiencies , from acute fulminant hepatitis to chronic cirrhosis , including surgical partial hepatectomy , and inborn inherited metabolic disorders .
it also presents the advantage of an inborn immunosuppressed status , which could allow for xenogenic organ or cell transplantation .
thus , it was developed with different hepatic mature and progenitor cell transplantation , isolated from rat and human livers .
before human clinical application of hepatocyte transplantation , it is of high importance to evaluate efficacy of cell transplantation on small animal models , in clinical situation .
as reported in literature [ 49 , 73 ] , engraftment of hepatocyte into recipient livers is largely favored if a selective advantage ( i.e. , existence of growth and proliferation difference ) between donor and recipient hepatocytes does exist .
this was confirmed in nude mouse model transplanted with rat hepatocytes : on healthy liver , mature hepatocyte could engraft but with a very limited proliferative activity , and engraftment after 40% hepatectomy in nude mice liver parenchyma did not differ from non - operated control nude mice .
these results could be due to the time difference between transplanted rat hepatocyte and mouse hepatocyte proliferation , the peak of dna synthesis being observed 24 hours after partial hepatectomy in the rat and 48 hours after partial hepatectomy in the mouse . neither a single dose of jo2 pretransplantation treatment nor ta - liver injury improved rat hepatocyte engraftment . thus , we interpreted this lack of repopulation as a deficit of selective advantage . in literature
, several studies used a growth selective advantage by repeating recipient hepatocyte apoptosis with jo2 while transplanting jo2 resistant cells : for example , bcl - xl overexpressing mouse hepatocytes ( i.e. , resistant to jo2 ) transplanted in a cba mouse resulted in a 4% mean implantation rate ( range from 2 to 6% ) , and bcl2 over - expressing mouse hepatocytes , more resistant to jo2 than bcl - xl cells , resulted in a mean 30% of repopulation ( ranging from 1 to 30% ) .
we hypothesized that because rat hepatocytes were resistant to jo2 drug they would get this selective advantage , on a jo2 treatment repeated model .
we observed that the effects of each weekly jo2 challenge were equivalent during a 3-week experiment : after each 250 g / kg body weight dose of jo2 , liver injury could be proven by the increases in serum alt levels following 24 h. routine histology showed panlobular necrosis 24 h after the last jo2 administration , identical to the necrosis seen 24 h after the first jo2 administration , meaning liver remained sensitive to anti - fas antibody .
three weeks after transplantation , engraftment rate was determined by immunodetection of the transplanted rat hepatocytes using an anti - rat mhc type i antibody ( figure 3 ) .
engraftment of xenogeneic rat hepatocytes , when transplanted to nude mice presenting an acute liver failure induced by a single sublethal injection of this anti - fas antibody , could be improved when mice were further submitted to a weekly repeated jo2 apoptosis - inducing treatment . in the latter case , engraftment was increased about sevenfold ( about 2.4% of repopulation ) .
on genomic analysis comparing jo2 single dose versus weekly repeated jo2 treatment in nude mice receiving rat hepatocytes , the altered pathways suggested a blockade of cell cycle and proliferation ( upregulation of cell cycle regulation and downregulation of circadian transcripts ) , activation of interferon- pathways , activation of antigen - presenting cells that probably reflects the immune system activation secondary to hepatocyte necrosis and liver injury , and metabolic pathway inhibition confirming liver injury .
this overall transcriptome profile might correspond to a selective advantage model where cell cycle blockade occurs in mouse hepatocytes submitted to weekly jo2 treatment , while natural jo2 resistance of rat hepatocytes allows them to proliferate . apart from toxic liver injury ,
rhim et al . transplanted rat hepatocytes into a transgenic upa - nude mice , with high rate ( near 100% ) of repopulation achieved in 6 to 14 weeks .
presence of rat hepatocytes was confirmed by histology , immunostaining , and rat transferrine measurement .
weglarz and sandgren used the same model to determine that hepatocyte entry into dna synthesis depends on each species and is not influenced by nature of animal recipient and engraftment . for rat hepatocyte transplantation model
, nude mouse seems to be efficient , with complete repopulation in transgenic upa model , and less important engraftment in other hepatic injury models , comparable to results obtained in similar allogenic models .
we recently demonstrated that human hepatocyte transplantation into recipient nude mice submitted to sublethal and lethal repeated jo2 liver injury could present a hepatoprotective effect , despite a very poor engraftment rate , lasting up to 7 weeks after transplant .
genomic analysis correlated this lack of engraftment to an absence of selective advantage , cumulated with a paradoxical increased survival rate linked to stimulation of host cell proliferation .
these genomic results are in contradiction with those observed with the same treatment protocol and rat hepatocyte transplantation .
this hepatoprotective effect of mature hepatocyte transplantation was also described by banas et al . .
they generated in vitro human adipose - derived stem cells- ( asc- ) derived hepatocytes , and they transplanted these hepatocyte - like cells into female nude mouse 24 hours after an intraperitoneal injection of 10 l/20 g of ccl4 .
transplanted hepatocyte - like cells derived from human embryonic stem cell ( ips ) to nude mice and found an hepatoprotective effect , not only on engrafted nodules but also on places far from them , which could be linked to delivered trophic factors ( same effect observed after injection of secreted proteins alone ) .
commenting on the very poor engraftment in the repeated jo2 nude mouse model , it is of common knowledge that even in more immunosuppressed and transgenic models , an important rate of failure of chimerism has been observed .
for example , dandri et al . described a 70%-success rate to raise minimal human hepatocyte engraftment in a upa / rag2 mouse liver , with an all - or - none response for human hepatocyte engraftment .
it means that in one - third of their mice , human cells did not engraft at all .
they correlated this 30%- failure result with time of prolonged warm ischemia and with poor viability of transplanted cells .
these results were confirmed in a model of upa / scid mouse where engraftment was successful in a median of 22% ( 087% ) of animals , linked with donor age and warm ischemia .
human fetal liver progenitor cells are able to engraft in nude mouse model , after retrorsine injection followed three weeks later by 30%-partial hepatectomy challenging , 50%-partial hepatectomy alone , or galactosamine - induced liver deficiency .
repopulation yield could be triggered by cotransplantation with fetal liver mesenchymal cells or repeated liver biopsies .
interestingly , in these nude mouse models , human fetal liver progenitor cells not only demonstrate proliferation but also differentiation into both mature hepatocyte and cholangiocyte pathways .
four to six weeks after transplantation , clusters of cells have developed , that have lost their progenitor markers and display morphological and immunohistological markers of mature cells [ 2022 ] .
we can compare these results to those obtained by transplanting heparg naturally immortalized human liver cell line into scid / beige mice ( scid - nk cell deficient mice ) that were thereafter ( first injection began at day one posttransplantation ) treated with 0.2 mg / kg jo2 once a week for 10 weeks .
heparg cells are bipotent progenitor and in vitro express biologic functions at the same level as primary human mature hepatocytes .
they engrafted in jo2-treated mice liver with a 1520% repopulation of recipient liver , which is comparable to results obtained with fetal hepatoblast transplanted into nude mice .
the results altogether suggest that it is of most importance to pay attention to chronology ( to avoid transplantation preceding liver injury ) , to be the closest to human clinical scene .
most of maximal human cell engraftment was described on two transgenic models with highly immunosuppressed mouse models , upa mice and fah mice .
this engraftment could become sufficient to talk of humanized liver , with human hepatic functions and human pharmacological proprieties [ 1214 , 80 ] .
, there was no publication of human liver cell transplanted in a nude mouse upa model .
the second transgenic fah mouse model was used by azuma et al . in fah nude mice model to create humanized liver , but they did not obtain sufficient human cell engraftment in this model and had to use a more immunosuppressed transgenic model ( fah / rag2-/il2rg- mice ) reinforced by upa - adenovirus administration intravenously 2448 hours before human hepatocyte transplantation , associated to ntbc withdrawal over five days after transplantation . to our knowledge , no success in humanized livers in nude mice has been reported , most likely because of an insufficient immunosuppression of this mouse . in other upa immunosupressed mouse models , such as rag2 mice ,
human hepatocytes can engraft , although at lower rate than murine hepatocytes [ 79 , 81 ] .
in such a model , dandri et al . reported 8 weeks after human hepatocyte transplantation a 2 to 10% of repopulation by human cells . in the fah mouse model ,
human hepatocytes could repopulate fah / rag2/il2rg mouse liver with a range from 5 to 34% , 12 weeks after transplant .
but these two models have specific disadvantages , directly linked with their concept ; upa mice , because of their inborn metabolic abnormally , have a poor breeding efficiency , a quite narrow window in time to perform transplantation ( on neonatal age , between1 to 3 weeks of age , before they die of severe bleeding ) , and a renal disease that still exists despite hepatocyte transplantation [ 19 , 27 , 59 , 60 ] .
moreover , there is a continuous and progressive hepatic parenchyma injury , possibly via activation of plasminogen and modified activity of matrix metalloproteinase : thus , this modified metabolism can interfere with liver cell growth and distort a physiopathologic model .
fah deficient mice also have inconveniences : their metabolic pathway leads to development of liver hepatocellularcarcinomas , requiring treatment by ntbc - diet repeated cycles to prevent tumor formation and maintain long - term survival .
this diet cancels the natural selective advantage that triggers xenogenic cells proliferation and can give possible bias in results interpretation . to increase human hepatocyte repopulation efficiency ,
some teams use a transfection of upa gene , which adds the same issues as encountered in upa model . another strategy
is represented by liver suicide model : it consists to transfect recipient liver hepatocytes with an apoptotic gene ; this gene being under the control of herpes virus type 1 thymidine kinase ( hsv tk ) : it can be activated by administration of gancyclovir , and thus could destroy specifically cells targeted with this suicide gene .
hasegawa et al . obtained an nog ( nod / scid / il2rg ) mouse expressing hsv tk transgene in their liver . by inducing apoptosis of liver recipient cells five days before transplantation of human hepatocyte
, they observed a high index of repopulation ( average up to 43% ) , correlated with elevated human albumin in plasma , and functional human hepatocyte .
who used upa - scid model optimized it to achieve total replacement of upa hepatocytes by human hepatocytes , by associating mouse liver suicide to upa - scid model , so that ganciclovir treatment could induce conditional selective murine hepatocyte death in humanized scid - upa mouse liver . but
unexpected , mice survival was not increased by humanized liver , whatever the repopulation index ( 32 to 87% ) .
as illustrated by the results of douglas et al , the limits of the humanized liver reside intrinsically in the principle of xenogenic transplantation : whatever the models , even in the best repopulation performance such as upa or fah transgenic mouse , xenogenic murine models gave best engraftment results compared to human cells transplantation .
interestingly , it does not seem possible to avoid a percentage of nearly complete failure of human cells engraftment in mouse liver , for each experiment , even in the studies reporting very high repopulation index [ 28 , 79 ] .
this disparity between animals of the same study could be due to imperfect immunosuppression or inadequation between murine and human metabolism [ 74 , 81 ] .
in favor of the first hypothesis , tateno et al . showed that when human hepatocyte engraftment in upa - scid mouse results in more than 50% repopulation , this high repopulation index leads to death of recipient .
this mortality can be corrected by a treatment that blocks human complement factor activity . in favor of the second hypothesis
of a failure to induce chimerism in a fah / rag2/il2rg mouse transplanted with human hepatocyte , in 6 over 14 mice despite an immunosuppressive drug ( fk506 ) .
also in favour of this hypothesis is the observation that mouse survival was not increased by humanized liver , whatever the repopulation index ( 32 to 87% ) , in the upa - scid model of liver failure challenged with liver suicide gene activation .
there seems to be an incompatibility directly linked to animal species differences : although human and murine cells can create narrow cellular junctions , morphologically and architecturally subnormal links confirming integration of xenogenic cells but human cells will develop unexplained glycogen storage or steatosis anomalies [ 81 , 86 ] . therefore , the hypothesis that humanized liver would allow performing experiments not feasible on humans thus helping to predict pharmacotoxicological and pathobiological effects in humans needs additional demonstration .
but , even in this case , transgenic models would mimic only limited aspects of human clinical liver failure , and one should be aware that humanized liver may not be transposable to real human physiology . as a last comment
, the use of an immunodeficient animal model could also be a bias in these liver cell transplantation studies .
their absence in immunodeficient animals could modify the liver response to acute injury ( demonstrated by strick - marchand et al . ) and to chronic injury , as well as engraftment of hepatic cells .
in the other hand , the use of immunosuppressive treatment for transplanted nonimmunodeficient animal models could also interact with liver physiology [ 91 , 92 ] and become another bias .
this raises the question of the bias of using xenogenic models , with respect to the risk taken by xenogenic animal models not reflecting human physiology .
as a conclusion , nude mice can sustain various liver injuries and are good recipients for xenogenic rodent hepatocyte transplantation .
they remain an acceptable model for human hepatocyte engraftment and for human hepatic progenitor cell transplantation , by exploring the beneficial environment allowing the differentiation of the latter into mature hepatocyte as well as biliary cells .
the low immunosuppressive background of nude mice is both an advantage , with easy breeding conditions , and disadvantage , with difficulties to raise humanized liver .
they are certainly an interesting model to study liver regeneration mechanisms in a context of human clinical liver deficiency situations . | we aimed at reviewing the various uses of nude mouse for the development of liver deficiency models and evaluation of efficacy of hepatic cell xenotransplantation . the first part records the large range of liver deficiency models that can be developed in nude mice : surgical partial hepatectomy , acute toxic liver deficiency , chronic cirrhosis , and transgenic liver injury .
the second part tackles the outcome of rat hepatocyte as well as human cell transplantation , both mature hepatocyte and hepatic progenitor , into nude mouse submitted to liver injury .
results are discussed and compared to other available immunodeficient mouse models .
the issue of humanized liver creation is also addressed .
altogether , these results show that nude mouse appears to be a suitable small animal model to expand our insight into liver cell engraftment and regeneration . |
perception of healthy body size and composition differs considerably across the globe , ethnic groups , cultures , and even inside medical community . during lifetime ,
these clearly represent a major health problem worldwide with over a billion persons , mostly women and children , suffering from hunger .
more persons die from hunger each year than aids , malaria , and tuberculosis combined .
however , in the developed world and much of the developing world disease - related weight loss , which is assuming epidemic proportions , is frequently ignored .
body wasting and cachexia , however , can develop in a variety of acute and chronic conditions to be associated with grim outcome [ 1 , 2 ] .
these include infectious diseases , such as hiv / aids , malaria , and tuberculosis , as well as many chronic conditions like cancer , chronic heart failure ( chf ) , chronic obstructive pulmonary disease ( copd ) , chronic kidney disease ( ckd ) , cystic fibrosis , liver cirrhosis , crohn s disease , rheumatoid arthritis , stroke , and neurological degenerative disease [ 35 ] .
cachexia has also been noted in patients after extensive traumatic injury and sepsis [ 1 , 2 ] .
the prevalence of an illness or condition is defined as the number of individuals who have the condition at any moment .
table 1 summarizes the prevalence of various conditions from world health organization ( who ) regions .
several relevant diseases that can lead to cachexia are not included in this report thus worldwide estimation of cachexia burden remains inconclusive [ 1 , 7 ] .
we therefore often have to rely on fragmented data from individual countries [ 8 , 9 ] or on several studies focusing on various conditions ( table 2 ) .
unfortunately , the estimate is only as good as the available data . across the existing literature ,
several definitions and different populations have been evaluated . with recent cachexia definition from the cachexia consensus working group at hand ( fig . 1 ) , more reliable data about cachexia should be available in the next years . with new data at hand , past estimates may need adjustment and prevalence of cachexia is likely to increase .table 1prevalence ( in millions ) of selected conditions by world health organization region , 2004condition ( millions)worldeuropeafricaamericassouth - east asiawestern pacificeastern mediterraneantuberculosis13.90.63.00.55.03.81.1hiv infection31.42.021.72.83.31.00.5stroke survivors30.79.61.64.84.59.11.1copd63.611.31.513.213.920.23.3rheumatoid arthritis23.76.21.24.64.46.01.3modified from hiv human immunodeficiency virus , copd chronic obstructive pulmonary diseasetable 2prevalence ( in percent ) of cachexia in some diseasesdiseaseprevalence ( % ) cancer2857chronic heart failure1642chronic kidney disease3060copd2735rheumatoid arthritis1867hiv / aids1035modified from [ 1 , 8]copd chronic obstructive pulmonary disease , hiv / aids human immunodeficiency virus / acquired immunodeficiency syndromefig .
1cachexia definition from the cachexia consensus working group . modified from prevalence ( in millions ) of selected conditions by world health organization region , 2004 hiv human immunodeficiency virus , copd chronic obstructive pulmonary disease prevalence ( in percent ) of cachexia in some diseases
copd chronic obstructive pulmonary disease , hiv / aids human immunodeficiency virus / acquired immunodeficiency syndrome cachexia definition from the cachexia consensus working group .
misconceptions about cachexia , even in medical and scientific community , are many . terms as cachexia , malnutrition ,
anorexia , and sarcopenia are frequently used as synonyms although they relate to completely different conditions . with regard to topic of this article ,
it is frequent around the world and represents a major issue for developing countries and community as a whole .
however , it can be reversed by simple refeeding whereas such a strategy alone is ineffective in cachexia . on the other hand , in developing countries malnutrition
is , if present sufficiently long and particularly in combination with rheumatic heart disease or chagas disease , a major contributor to chf and increased risk to develop cachexia .
a recent estimate suggests that approximately nine million patients have cachexia of chronic disease , with the highest prevalence ( currently about 1 % ) being recorded in north america , europe , and japan .
given the population size in asia , it is very likely that the worldwide burden of cachexia will continue to escalate .
the data for africa and south america are scarce , although reports on cardiovascular disease [ 1214 ] suggest that epidemiological transition has broadened the spectrum of cachexia aetiology in these regions .
different figures have been reported for prevalence of cachexia in individual conditions ; yet , common belief is that patients with advanced conditions , particularly cancer , are more prone to develop cachexia ( table 2 ) . in terms of absolute numbers , conditions with high population burden like copd and
cachexia increases mortality in chronic disease and is present in about 30 % of patients that die .
annual mortality rates are disease dependent : copd , 1015 % and chf or ckd , 2030 % .
an exponential increase to 80 % in cancer translates to two million cancer - related cachexia deaths , i.e. , about every second patient with malignant disease will die with some degree of cachexia . from this point of view , cachexia is not merely a complex syndrome that occurs in advanced stages of various chronic diseases but one of the major killers worldwide .
the ultimate goal of public health has always been and remains the prevention of disease and the promotion of health in communities .
in contrast to clinical medicine , public health addresses population groups rather than single individuals and acts through comprehensive and multisectoral interventions . whereas target diseases and risk factors are largely shared with clinical medicine , public health aims to prevent development of clinically relevant or even full - blown disease to reduce the burden for population and healthcare system .
although no mandatory definition of public health problem is accepted , some general guidance is adopted in the scientific community . for a health condition to be considered as a public health problem ,
firstly , the condition burden is high in terms of morbidity , mortality , quality of life , and costs .
secondly , the problem is distributed unfairly , affecting disadvantaged population groups to a greater extent .
thirdly , there must be some evidence that public health strategies could substantially reduce the burden of the condition .
lastly , such preventive strategies are not yet fully in place [ 17 , 18 ]
. next to the epidemiological and causation background , maybe the most important requirement should be the feasibility of relatively simple and cost - effective population - wide strategies to reverse the natural course of the condition .fig .
2characteristics of a public health problem characteristics of a public health problem although many people still believe that chronic diseases are problems of developed communities who have acquired them primarily through indulging in four preventable behaviors ( insufficient physical activity , tobacco use , unhealthy diet , and the harmful use of alcohol ) , the situation around the globe has already started to change [ 12 , 20 , 22 , 23 ] . based on the latest who global status report on non - communicable diseases
, it is estimated that out of 57 million global deaths in 2008 , 36 million ( 63 % ) were due to chronic diseases , principally cardiovascular diseases , diabetes , cancers , and chronic respiratory diseases .
nearly 80 % ( 29 million ) of chronic disease deaths occur in the developing world : about 80 % of cardiovascular and diabetes deaths , 90 % of copd deaths , and about two thirds of cancer deaths .
importantly , in low- and middle - income countries deaths from chronic diseases occur at younger age that hampers social and economic development [ 20 , 24 ] .
as chronic diseases have already started to dominate global public health , low- and middle - income countries are facing a double burden of disease .
while they continue to deal with the problems of infectious diseases , they are experiencing a rapid upsurge in chronic disease risk factors and deaths , especially in urban settings . over last decades
, we have witnessed dramatic changes in eating , drinking , and moving habits around the globe .
generally , these now clash with our biology and physiology to cause major shifts in body size and composition .
the body mass index had a constant trend of increase by 0.4 kg / m per decade thus doubling the worldwide obesity and overweight prevalence from 1980 to 2008 [ 27 , 28 ] .
importantly , this increase occurred in an accelerated fashion with half of the rise occurring in 1980 to 2000 and half in only 8 years from 2000 to 2008 .
there is some variation in levels and trends [ 2729 ] but generally speaking , the increase is more pronounced in low and middle income countries .
similarly , the prevalence of hypertension is increasing in developing countries with the current prevalence of 1637 % , and increases of up to 30 % in last decades .
this eventually translates into double burden of disease as these countries are facing an epidemic of chronic diseases through increasing prevalence of risk factors .
indeed , the risk factor associated population attributable fractions of fatal coronary heart disease and stroke indicate further increase in double burden with the growing potential for wasting and cachexia associated with chronic disease . taking different projections of global mortality and disease burden into account , the world will experience further shift in the distribution of deaths from younger to older age groups and from infectious to chronic diseases during next decades .
large declines in mortality are projected to occur between 2002 and 2030 for all of the principle communicable , maternal , perinatal , and nutritional causes , with the exception of hiv / aids .
the four leading causes of death globally in 2030 will be ischemic heart disease , stroke , copd and hiv / aids . based on epidemiological data and public health problem characteristics ,
this should in our opinion increase the awareness about cachexia and help the community to comprehend its complexity and magnitude .
cardiovascular disease , cancer , diabetes , and chronic respiratory diseases are both most prevalent and largely preventable conditions .
population - wide interventions to prevent chronic disease are feasible at low - cost which gives ample opportunities even in countries with low income levels . bringing this to a higher level , who considers some interventions as
best buys when immediate actions should be undertaken to produce accelerated results in terms of lives saved , diseases prevented , and heavy costs avoided ( table 3 ) . for those at high risk or with chronic disease , an alternate approach through individual healthcare interventions is necessary.table 3examples of best buys according to world health organizationpopulation - wide interventionsindividual healthcare interventionsbans : on smoking in public places and on tobacco and alcohol advertisingaspirin , statin , and blood pressure - lowering agents for people at high cardiovascular riskraising taxes : on tobacco and alcoholadministration of aspirin to people who develop a myocardial infarctionraising public awareness : about diet and physical activityblood pressure , glycemic control , and foot care for patients with diabetesvaccination : against hepatitis b and human papilloma virusmodified from examples of best buys according to world health organization evidence from high - income countries shows that a focus on both prevention and improved treatment following cardiovascular events led to dramatic declines in mortality rates .
similarly , progress in cancer treatment combined with early detection and screening have improved survival rates for many cancers . as these approaches address risk factors and progression of disease ,
effective preventive strategies to tackle socio - economic inequalities , malnutrition , infectious disease , and modern community - related conditions will merely shift the burden towards refractory chronic disease stage with significant potential to increase cachexia burden .
future management , provided that preventive strategies are implemented and successful , will therefore critically depend upon potent remedy against cachexia ( not available yet ) and supportive measures to meet needs of dependent patients ( currently not affordable by any community ) . in other words
, we may experience a transition of cachexia aetiology rather than a story of success .
therefore , a joined perspective of public health practitioners and clinicians about issues dealing with cachexia should be initiated . meanwhile , intense efforts to find treatment for cachexia should be maintained . to identify suitable patients , a reliable and routine marker reflecting therapeutic target ( muscle mass in this case ) would be preferred , and creatinine appears as an attractive option .
public and medical community awareness about cachexia should be a primary target for future interventions .
this could enhance detection of individuals at risk of or those with body wasting and pre - cachexia . around the globe
socio - economic inequalities like poverty , level of education , housing and type of occupation are in an independent manner associated with risk factors , disease progression and outcome . as a key and
maybe even a rate limiting step in disease s natural course , they require multisectoral and comprehensive measures , which to a great extent are beyond the scope of medicine alone .
accessibility to healthcare system and basic level of drug availability remain an issue at large .
unmanaged chronic conditions can therefore easily and more quickly develop into full - blown disease and eventually cachexia .
after several trials with pharmacological interventions in chronic disease , including the polypill in cardiovascular disease , such an approach is promoted over standard public health measures .
the jury about this disputed topic , however , is still out and additional research is warranted before accepting such an approach as first line management .
identifying condition as a public health problem often serves implicit normative or political purposes . yet , in case of cachexia this is true reflection of epidemiological and clinical circumstances , which has not been acknowledged neither by the community nor by the medical care professionals .
we have to perceive the dynamic course of body weight through lifespan where weight loss can have more deleterious effects than weight gain [ 37 , 38 ] .
this should in our opinion increase the awareness about cachexia and help the community to comprehend its complexity and magnitude .
action to prevent and control chronic diseases requires a comprehensive approach that targets a population as a whole and includes both prevention and treatment interventions .
it has to be multisectorial with collaboration efforts from government , international organizations , academic institutions , civil society and the private sector . around the globe
, a shift in current public health priorities to highlight the magnitude of the problem and areas of interventions is necessary .
simultaneously , scientific efforts should provide us with more reliable estimates of body wasting and cachexia as well as pathophysiology of cachexia - associated death . as a certain proportion of patients will , irrespective of preventive measures , eventually develop cachexia , a quest for effective remedies remains vital . | perception of healthy body size and composition differs considerably across the globe , ethnic groups , cultures , and even inside medical community .
although the concept of ideal body weight has evolved over the past decades , the observation that weight loss can have more deleterious effects within a short - term period than weight gain has remained rather consistent .
weight loss , as a prelude to cachexia , occurs frequently in a variety of disease states and meets the requirements of a global public health problem .
consequently , interventions to prevent and control chronic diseases require a comprehensive approach that targets a population as a whole and includes both prevention and treatment strategies . around the globe , cachexia awareness campaigns and expanding the current public health priorities to highlight the cachexia magnitude and areas of interventions is necessary .
simultaneously , scientific efforts should provide us with more reliable estimates of body wasting and cachexia as well as pathophysiology of cachexia - associated death .
as certain proportion of patients will , irrespective of preventive measures , eventually develop cachexia , a quest for effective remedy remains vital . |
the quest for ideal fillers for soft tissue augmentation has resulted in the development of many diverse products with divergent augmentation strategies and mechanisms of action .
this is partially a testament to the fact that no one product will ideally suit the desires of all patients and all clinicians .
moreover , the formidable biological and aesthetic criteria of the ideal filler have driven researchers to take a variety of development paths .
this has led to the development of multiple categories of soft tissue fillers that are shown in tables 1 and 2 .
the primary actions of these products are to occupy physical space , to induce collagen formation , and/or increase volume .
convenience drives the use of more durable ( semi - permanent and permanent ) soft tissue fillers or implants , because these products require fewer or no follow - up treatments .
however , because the face continues to age following procedures , temporary or semi - permanent effects may be preferred , so the face can be periodically augmented to accommodate ongoing morphological changes .
some clinicians have also voiced concerns about the potential for long - term complications that may arise after treating with permanent soft tissue fillers . in general ,
soft tissue augmentation techniques have evolved from an early focus on the treatment of lines to , more recently , focusing on the treatment of facial areas .
in addition , earlier methods focused on injection techniques that treated the superficial levels of the skin .
another important development in the science of soft tissue augmentation is the growing acknowledgment of the fundamental morphological changes in the aging face that cause the outward appearance of aging .
the culprit , generally implicated for the appearance of the aging face , is gravity .
rather , one of the primary causes of the aging face is the redistribution of fat ( donofrio 2000 ) .
the other causes of the aging face include environmental factors , dermal thinning , bone resorption ( vleggaar and bauer 2004 ) , and the loss of elasticity and collagen in the skin .
rhytids and wrinkles , caused by facial expressions , also contribute to the aging face .
however , we now know that lines , caused by the underlying facial muscle contractions , respond best to botulinum toxin a , as opposed to using soft tissue fillers to fill in the lines and creases .
, superficial and deep fat is distributed evenly , creating a homogenous topographical appearance with smooth primary arcs and convexities . with intrinsic aging
fat atrophy and hypertrophy cause hills and valleys to develop , and produce demarcations between the cosmetic units .
fat atrophy begins to become clinically apparent in the temple and cheek and is followed by fat loss around the chin and mandibular areas .
features become concave , characterized by flat lips , sunken temple and cheek , scalloped mandible , and increased shadowing among the resulting hills and valleys .
with aging , the most significant change in appearance is the sagging of excess skin caused by the conversion of primary arcs to straight lines .
when evaluating the tendencies of facial skin vectors , the skin drapes inferiorly and diagonally from the temporal area towards the perioral region , thus creating a central focus on the perioral and mandibular regions .
intrinsic aging is characterized by fat atrophy in the periorbital , forehead , buccal , temporal , and perioral areas .
fat atrophy results from the collective effects of decreased fat cell size , diminished fat cell function , impaired fat cell differentiation , and redistribution of fat cells .
fat hypertrophy occurs submentally , in the jowl , lateral nasolabial fold , lateral labiomental crease , and lateral malar areas .
in contrast , the loss of fat leaves excessive skin in proportion to the diminished volume , causing the excess skin to sag ( alster et al 2004 ; donofrio 2004 ) . reversing the effects of fat atrophy / hypertrophy
can not be adequately accomplished through conservative techniques . a comprehensive approach that considers the entire face and all the underlying causes of aging is necessary .
volume must be restored in regions experiencing fat atrophy and excess fat must be removed from regions where fat has accumulated .
correcting the distribution of volume throughout the face can help restore homogenous topography , eliminate demarcations between cosmetic units , and restore the primary arcs ( donofrio 2004 ) .
the atrophy / hypertrophy model for aging represents a shift from focusing on wrinkles and gravitational sagging to a more comprehensive approach that treats the aging face from the inside out .
as stated earlier , acquiring the ideal soft tissue filler is hopeless , since preferences of patients and clinicians are not universal .
however , the goal of soft tissue augmentation should involve a universal acknowledgment of the fundamental causes of the aging face .
chronological age is among a variety of factors that may influence the choice of soft tissue filler(s ) . in general , older patients are likely to require soft tissue fillers that primarily produce volume and correct the results of fat atrophy by re - creating the foundation and underlying structure of the face .
younger patients are more likely to require soft tissue fillers that smooth surfaces , such as creases and folds . in younger patients ,
the typical goal of lip augmentation is to make lips appear bigger and fuller , whereas in older patients the goal is to restore volume to lips that has been lost through fat atrophy .
ethnicity is another significant determinant used to match soft tissue fillers with the specific needs of the patient .
based on the particular ethnic skin - type , some soft tissue fillers will achieve better results than others will .
for example , volumizers are generally more effective in ethnic skin , due to the presence of sagging skin .
histologically , there is less thinning of collagen bundles and elastic tissue in this skin - type .
the treatment of ethnic skin does not involve correcting the typical photodamage that occurs in caucasian skin , such as creases and surface lines .
for soft tissue augmentation , a variety of products exists , and new products are continually entering the market .
these soft tissue fillers provide correction by two basic mechanisms : ( 1 ) the filler material occupies a void space , or ( 2 ) the filler material stimulates processes that produce volume .
an overview of historical developments in the science of soft tissue augmentation is presented here , including a discussion of current products , patient selection , and injection techniques .
in the young face , superficial and deep fat is distributed evenly , creating a homogenous topographical appearance with smooth primary arcs and convexities . with intrinsic aging
fat atrophy and hypertrophy cause hills and valleys to develop , and produce demarcations between the cosmetic units .
fat atrophy begins to become clinically apparent in the temple and cheek and is followed by fat loss around the chin and mandibular areas .
features become concave , characterized by flat lips , sunken temple and cheek , scalloped mandible , and increased shadowing among the resulting hills and valleys .
with aging , the most significant change in appearance is the sagging of excess skin caused by the conversion of primary arcs to straight lines .
when evaluating the tendencies of facial skin vectors , the skin drapes inferiorly and diagonally from the temporal area towards the perioral region , thus creating a central focus on the perioral and mandibular regions .
intrinsic aging is characterized by fat atrophy in the periorbital , forehead , buccal , temporal , and perioral areas .
fat atrophy results from the collective effects of decreased fat cell size , diminished fat cell function , impaired fat cell differentiation , and redistribution of fat cells .
fat hypertrophy occurs submentally , in the jowl , lateral nasolabial fold , lateral labiomental crease , and lateral malar areas .
in contrast , the loss of fat leaves excessive skin in proportion to the diminished volume , causing the excess skin to sag ( alster et al 2004 ; donofrio 2004 ) . reversing the effects of fat atrophy / hypertrophy
a comprehensive approach that considers the entire face and all the underlying causes of aging is necessary .
volume must be restored in regions experiencing fat atrophy and excess fat must be removed from regions where fat has accumulated . correcting the distribution of volume throughout the face can help restore homogenous topography , eliminate demarcations between cosmetic units , and restore the primary arcs ( donofrio 2004 ) .
the atrophy / hypertrophy model for aging represents a shift from focusing on wrinkles and gravitational sagging to a more comprehensive approach that treats the aging face from the inside out . as stated earlier , acquiring the ideal soft tissue filler is hopeless , since preferences of patients and clinicians are not universal .
however , the goal of soft tissue augmentation should involve a universal acknowledgment of the fundamental causes of the aging face .
chronological age is among a variety of factors that may influence the choice of soft tissue filler(s ) . in general , older patients are likely to require soft tissue fillers that primarily produce volume and correct the results of fat atrophy by re - creating the foundation and underlying structure of the face .
younger patients are more likely to require soft tissue fillers that smooth surfaces , such as creases and folds . in younger patients ,
the typical goal of lip augmentation is to make lips appear bigger and fuller , whereas in older patients the goal is to restore volume to lips that has been lost through fat atrophy .
ethnicity is another significant determinant used to match soft tissue fillers with the specific needs of the patient . based on the particular ethnic skin - type , some soft tissue fillers will achieve better results than others will .
for example , volumizers are generally more effective in ethnic skin , due to the presence of sagging skin .
histologically , there is less thinning of collagen bundles and elastic tissue in this skin - type .
the treatment of ethnic skin does not involve correcting the typical photodamage that occurs in caucasian skin , such as creases and surface lines . for soft tissue augmentation ,
these soft tissue fillers provide correction by two basic mechanisms : ( 1 ) the filler material occupies a void space , or ( 2 ) the filler material stimulates processes that produce volume . an overview of historical developments in the science of soft tissue augmentation is presented here , including a discussion of current products , patient selection , and injection techniques .
groundbreaking work in the science of soft tissue fillers began in the late 1800s after neuber reported using blocks of free fat , harvested from the arms to reconstruct depressed facial defects .
only a few years later , gersuny became the first to use the injection technique for cosmetic correction ; the injected substance was paraffin . in 1911 ,
however , advances in the use of autologous fat were tempered by the limited efficacy of fat injection , since transplants lost 50% of their volume after only one year ( klein and elson 2000 ) .
, autologous fat transfer remains a popular procedure because there is no risk of rejection and no cost for materials .
recent advances in fat transfer include suspending the autologous fat in the patient s plasma , which increases the longevity of the procedure .
the treatment lasts from several months to several years , depending on technique and transplant preparation ( burgess 2005 ) .
collagen is a fibrous protein found in both humans and animals . in soft tissue augmentation ,
development of bovine collagen for soft tissue augmentation began in the 1950s with the work of gross and kirk , who were the first to successfully prepare a bovine collagen gel .
however , in the 1960s , the use of bovine collagen received a significant boost from the development of a process that reduced the antigenicity by removing the nonhelical amino and carboxy terminal telopeptides .
this led the way to the development of a clinically useful preparation of bovine collagen . in 1981 , zyderm i collagen implant ( inamed aesthetics , ca , usa ) received approval from the food and drug administration ( fda ) and quickly became a popular filler for soft tissue augmentation ( klein and elson 2000 ) .
these fillers are injected intradermally into the superficial papillary dermis of the nasolabial folds ( klein 1989 ) .
they usually require overcorrection due to resorption of water from the preparation , which occurs within 24 h ( cheng et al 2002 ) .
further developments led to a more durable bovine collagen filler that retains its integrity and inherent water content to a greater degree because of cross - linking with glutaraldehyde .
cross - linking strengthens the collagen and makes it more resistant to proteolytic degradation ( elson 1999 ) .
bovine collagen cross - linked glutaraldehyde is injected into the mid - reticular or deep reticular dermis at the dermal subcutaneous interface ( cheng et al 2002 ) . unlike collagen fillers with low concentrations of cross - linking
human - based collagen is available in two forms : ( 1 ) isolated from human fibroblasts cell cultures , and ( 2 ) cross - linked with glutaraldehyde .
cosmoderm ( inamed aesthetics , ca , usa ) is an example of highly purified human - based collagen , available in two different concentrations .
cosmoplast ( inamed aesthetics , ca , usa ) is an example of human - based collagen cross - linked with glutaraldehyde .
cosmoderm is used more superficially ( bisaccia and scarborough 1992 ) , whereas cosmoplast is reserved for deeper lines and is used off - label for the lips .
corrections using collagen - based fillers last from several weeks to several months ( glogau et al 2004 ) .
hyaluronic acid is a naturally occurring polysaccharide , a glycosaminoglycan that is a component of the ground substance or extracellular matrix of the dermis ( lupton and alster 2000 ) .
hyaluronic acid provides volume to the skin , shape to the eyes , and is chemically identical across all species and all tissue types ( larsen et al 1993 ) .
it has natural hydrating functions that form the basis of its role in soft tissue augmentation ( lupton and alster 2000 ) .
fillers containing hyaluronic acid are true fillers , in which the mechanism of augmentation is simply to fill space .
there are two sources of hyaluronic acid used for soft tissue augmentation : ( 1 ) nonanimal stabilized hyaluronic acid ( nasha ) produced from a bacterial biofermentation process ( eg , restylane , medicis aesthetics , az , usa ; and juvederm , inamed aesthetics , ca , usa ) , and ( 2 ) animal - derived hyaluronic acid purified from rooster combs ( eg , hylaform , inamed aesthetics , ca , usa ) ( burgess 2005 ) .
hyaluronic acid is readily broken down in the dermis and eliminated via the lymphatics and the liver ( klein and elson 2000 ) . however , cross - linking with ester and ether linkages stabilizes the molecule for greater durability in dermal processes ( burgess 2005 ) .
depending on the concentration , it can be injected into either the ( 1 ) reticular dermis and/or subcutaneous - dermal plane , or ( 2 ) papillary dermis .
various injection techniques apply and should not be overcorrected ( lowe et al 2001 ) .
viscoelastic nonanimal hyaluronic acid gel ( eg , juvederm ) is available in three different concentrations .
it is intended for injection , based on concentration , into either : ( 1 ) the superficial dermis , ( 2 ) the mid dermis , or ( 3 ) the mid to deep dermis .
hyaluronic acid derived from rooster combs ( eg , hylaform ) is available in a 5.5 mg / ml concentration with a particle size of 500 m , and 20% cross - linking as a result of glutaraldehyde and vinyl sulfone stabilization ( burgess 2005 ) .
it can be used in younger patients for shaping and volumizing in addition to restoring lost lip structure in older patients .
all hyaluronic acid fillers can be used in the body of the lips to reshape or enlarge the lips .
fillers containing hyaluronic acid are generally considered to last twice as long as fillers containing collagen .
both collagen and hyaluronic acid are indicated for the treatment of more superficial corrections , such as smoothing surfaces , as opposed to fillers used in deeper regions to create more supportive structure and foundation .
theoretically , hyaluronic acid can be used to volumize . however , this application is considered cost - prohibitive .
poly - l - lactic acid ( pla ) ( sculptra , sanofi - aventis , nj , usa ) is a biodegradable bioabsorbable synthetic polymer that has been used for several decades in resorbable medical devices , such as sutures , surgical sealant meshes , screws , plates , and membranes for guided tissue regeneration ( vleggaar and bauer 2004 ; burgess and quiroga 2005 ) . in soft tissue augmentation
, pla is used exclusively to produce volume in areas of sinking and sagging skin .
it is not intended for the focused treatment of specific wrinkles , as is the case with most traditional soft tissue fillers .
although other volumizers can be used to treat facial areas , this can be quite costly .
in many patients , pla can also be used in combination with other soft tissue fillers for a variety of applications in soft tissue augmentation .
poly - l - lactic acid was recently approved in the us for the treatment of facial lipoatrophy in patients with human immunodeficiency virus ( hiv ) .
moreover , it has been used since 1999 in more than 30 other countries to treat a variety of facial volume and contour deficiencies ( vleggaar and bauer 2004 ) .
facial lipoatrophy may also occur as a result of surgical procedures to cause extensive weight loss .
it can also be manifested by specific disease states , either inherited or acquired , in which lipodystrophy results in metabolic disturbances that lead to abnormalities in adipose tissue .
in addition to disease states , facial lipoatrophy also results from the treatment of certain diseases , such as the use of anti - retroviral therapy to treat hiv .
in particular , facial lipoatrophy has been associated to a large degree with the use of protease inhibitors , and to a lesser degree with the use of nucleoside reverse transcriptase inhibitors , as part of highly active anti - retroviral therapy for treatment of hiv ( ascher et al 2006 ) .
facial lipoatrophy has been treated using silicone oil , bovine collagen , particulate human fascia lata , and autologous fat .
safety and efficacy of pla for treatment of facial lipoatrophy has been demonstrated in several studies with immunocompromised patients ( valantin et al 2003 ; moyle et al 2004 ; onesti et al 2004 ; mest 2005 ) .
for example , burgess and quiroga ( 2005 ) used pla to treat lipoatrophy in 61 hiv - infected patients over a 5-month period .
the amount of pla varied from 14 vials , depending on the degree of lipoatrophy severity and areas of involvement .
mild cases required 12 treatment sessions ; moderate cases , approximately 4 treatment sessions ; and severe lipoatrophy required 4 or more treatment sessions .
all patients experienced significant clinical improvement , defined as smoothing of the skin with decreased concavities or depressions , and improved overall appearance .
the effect was long lasting , remaining for up to 30 months in patients , depending on initiation of treatment and length of follow - up ( burgess and quiroga 2005 ) . at the time of writing
in addition to treating hiv - infected patients , significant experience has also accumulated in the treatment of immunocompetent patients for cosmetic reasons ( vleggaar and bauer 2004 ) .
poly - l - lactic acid is distinct from other soft tissue fillers , due to its mechanism of action , treatment plan , preparation of injection material , and injection technique ( burgess and lowe 2006 ) .
the application of pla in soft tissue augmentation exploits a mechanism of action not seen in any other soft tissue filler .
although the injection of pla causes an immediate effect by physically occupying space , this initial response is temporary and only lasts one week or less ( mest 2005 ) . once the carrier solution is absorbed , a delayed but progressive volumizing effect begins .
the process of hydration , loss of cohesion and molecular weight , and solubilization and phagocytosis of pla by the host s macrophages , slowly degrades pla into lactic acid microspheres and eliminates co2 by way of respiratory excretion .
this inflammatory reaction elicits resorption and the formation of fibrous connective tissue about the foreign body , causing dermal fibroplasia that leads to the desired cosmetic effect ( burgess and quiroga 2004 ) .
poly - l - lactic acid is injected into deeper regions for creating supportive structure and foundation .
effects of volume restoration can persist for as long as 1824 months , but are not permanent ( burgess and quiroga 2005 ; mest 2005 ) .
beljaards and colleagues ( 2005 ) has reported the late onset of granulomatous skin reactions in three immunocompetent patients receiving pla treatment .
the report underscores necessity for dermatologists to recognize and acknowledge the distinct differences between pla and other soft tissue fillers .
the characteristics that distinguish pla from traditional fillers are : ( 1 ) the mechanism of action , ( 2 ) the treatment plan , ( 3 ) the preparation of the injection material , and ( 4 ) the injection technique ( burgess and lowe 2006 ) .
poly - l - lactic acid causes a gradual volume restoration that may take 36 months to develop .
in addition , the rate of dermal thickening increases from the first to last injection . since
immediate correction is not the primary goal , as is the case in most traditional fillers , the use of pla requires a markedly different approach and treatment plan .
although manufacturers recommend diluting in 35 ml of sterile water , 57 ml of sterile water is the customary reconstitution volume in the us .
what is most critical for successful outcomes is that physicians receive sufficient training before attempting to inject pla .
for example , as opposed to treating a specific line , the strategy of pla treatment is to return volume to a facial area . in addition
, pla is injected into the dermal subcutaneous plane , unlike collagen or hyaluronic acids .
finally , it is important that pla injection be accompanied by massage of the treatment area and continued by the patient post - treatment ( werschler 2005 ) . at the center for dermatology and dermatologic surgery ( washington d.c . , us ) , pla has been successfully used to treat lipoatrophy of the hands , feet , face , and buttocks in both immunocompetent and immunocompromised patients .
in addition , pla has been used effectively in both the younger and older patient . however , in the older or aging patient , the thickness of the dermis plane is diminished , leading to a greater likelihood for bruising that results from increased fragility of blood vessels . in these patients , it is recommended that clinicians allow 8 weeks between treatment sessions , as opposed to the regular 46 week waiting period . for clinicians ,
the learning curve is longer in these patients , due to the concerns associated with treating patients with a thin dermis plane ( burgess 2006 ) .
groundbreaking work in the science of soft tissue fillers began in the late 1800s after neuber reported using blocks of free fat , harvested from the arms to reconstruct depressed facial defects .
only a few years later , gersuny became the first to use the injection technique for cosmetic correction ; the injected substance was paraffin . in 1911 ,
however , advances in the use of autologous fat were tempered by the limited efficacy of fat injection , since transplants lost 50% of their volume after only one year ( klein and elson 2000 ) .
, autologous fat transfer remains a popular procedure because there is no risk of rejection and no cost for materials .
recent advances in fat transfer include suspending the autologous fat in the patient s plasma , which increases the longevity of the procedure .
the treatment lasts from several months to several years , depending on technique and transplant preparation ( burgess 2005 ) .
collagen is a fibrous protein found in both humans and animals . in soft tissue augmentation ,
development of bovine collagen for soft tissue augmentation began in the 1950s with the work of gross and kirk , who were the first to successfully prepare a bovine collagen gel .
however , in the 1960s , the use of bovine collagen received a significant boost from the development of a process that reduced the antigenicity by removing the nonhelical amino and carboxy terminal telopeptides .
this led the way to the development of a clinically useful preparation of bovine collagen . in 1981 , zyderm i collagen implant ( inamed aesthetics , ca , usa ) received approval from the food and drug administration ( fda ) and quickly became a popular filler for soft tissue augmentation ( klein and elson 2000 ) .
these fillers are injected intradermally into the superficial papillary dermis of the nasolabial folds ( klein 1989 ) .
they usually require overcorrection due to resorption of water from the preparation , which occurs within 24 h ( cheng et al 2002 ) .
further developments led to a more durable bovine collagen filler that retains its integrity and inherent water content to a greater degree because of cross - linking with glutaraldehyde .
cross - linking strengthens the collagen and makes it more resistant to proteolytic degradation ( elson 1999 ) .
bovine collagen cross - linked glutaraldehyde is injected into the mid - reticular or deep reticular dermis at the dermal subcutaneous interface ( cheng et al 2002 ) . unlike collagen fillers with low concentrations of cross - linking
human - based collagen is available in two forms : ( 1 ) isolated from human fibroblasts cell cultures , and ( 2 ) cross - linked with glutaraldehyde .
cosmoderm ( inamed aesthetics , ca , usa ) is an example of highly purified human - based collagen , available in two different concentrations .
cosmoplast ( inamed aesthetics , ca , usa ) is an example of human - based collagen cross - linked with glutaraldehyde .
cosmoderm is used more superficially ( bisaccia and scarborough 1992 ) , whereas cosmoplast is reserved for deeper lines and is used off - label for the lips .
corrections using collagen - based fillers last from several weeks to several months ( glogau et al 2004 ) .
hyaluronic acid is a naturally occurring polysaccharide , a glycosaminoglycan that is a component of the ground substance or extracellular matrix of the dermis ( lupton and alster 2000 ) .
hyaluronic acid provides volume to the skin , shape to the eyes , and is chemically identical across all species and all tissue types ( larsen et al 1993 ) .
it has natural hydrating functions that form the basis of its role in soft tissue augmentation ( lupton and alster 2000 ) .
fillers containing hyaluronic acid are true fillers , in which the mechanism of augmentation is simply to fill space .
there are two sources of hyaluronic acid used for soft tissue augmentation : ( 1 ) nonanimal stabilized hyaluronic acid ( nasha ) produced from a bacterial biofermentation process ( eg , restylane , medicis aesthetics , az , usa ; and juvederm , inamed aesthetics , ca , usa ) , and ( 2 ) animal - derived hyaluronic acid purified from rooster combs ( eg , hylaform , inamed aesthetics , ca , usa ) ( burgess 2005 ) .
hyaluronic acid is readily broken down in the dermis and eliminated via the lymphatics and the liver ( klein and elson 2000 ) . however , cross - linking with ester and ether linkages stabilizes the molecule for greater durability in dermal processes ( burgess 2005 ) .
depending on the concentration , it can be injected into either the ( 1 ) reticular dermis and/or subcutaneous - dermal plane , or ( 2 ) papillary dermis .
various injection techniques apply and should not be overcorrected ( lowe et al 2001 ) .
viscoelastic nonanimal hyaluronic acid gel ( eg , juvederm ) is available in three different concentrations . it is intended for injection , based on concentration , into either : ( 1 ) the superficial dermis , ( 2 ) the mid dermis , or ( 3 ) the mid to deep dermis .
hyaluronic acid derived from rooster combs ( eg , hylaform ) is available in a 5.5 mg / ml concentration with a particle size of 500 m , and 20% cross - linking as a result of glutaraldehyde and vinyl sulfone stabilization ( burgess 2005 ) .
it can be used in younger patients for shaping and volumizing in addition to restoring lost lip structure in older patients .
all hyaluronic acid fillers can be used in the body of the lips to reshape or enlarge the lips .
fillers containing hyaluronic acid are generally considered to last twice as long as fillers containing collagen .
both collagen and hyaluronic acid are indicated for the treatment of more superficial corrections , such as smoothing surfaces , as opposed to fillers used in deeper regions to create more supportive structure and foundation .
poly - l - lactic acid ( pla ) ( sculptra , sanofi - aventis , nj , usa ) is a biodegradable bioabsorbable synthetic polymer that has been used for several decades in resorbable medical devices , such as sutures , surgical sealant meshes , screws , plates , and membranes for guided tissue regeneration ( vleggaar and bauer 2004 ; burgess and quiroga 2005 ) . in soft tissue augmentation
, pla is used exclusively to produce volume in areas of sinking and sagging skin .
it is not intended for the focused treatment of specific wrinkles , as is the case with most traditional soft tissue fillers .
although other volumizers can be used to treat facial areas , this can be quite costly . in many patients , pla can also be used in combination with other soft tissue fillers for a variety of applications in soft tissue augmentation .
poly - l - lactic acid was recently approved in the us for the treatment of facial lipoatrophy in patients with human immunodeficiency virus ( hiv ) .
moreover , it has been used since 1999 in more than 30 other countries to treat a variety of facial volume and contour deficiencies ( vleggaar and bauer 2004 ) .
facial lipoatrophy may also occur as a result of surgical procedures to cause extensive weight loss .
it can also be manifested by specific disease states , either inherited or acquired , in which lipodystrophy results in metabolic disturbances that lead to abnormalities in adipose tissue .
in addition to disease states , facial lipoatrophy also results from the treatment of certain diseases , such as the use of anti - retroviral therapy to treat hiv .
in particular , facial lipoatrophy has been associated to a large degree with the use of protease inhibitors , and to a lesser degree with the use of nucleoside reverse transcriptase inhibitors , as part of highly active anti - retroviral therapy for treatment of hiv ( ascher et al 2006 ) .
facial lipoatrophy has been treated using silicone oil , bovine collagen , particulate human fascia lata , and autologous fat .
( james et al 2002 ) . safety and efficacy of pla for treatment of facial lipoatrophy has been demonstrated in several studies with immunocompromised patients ( valantin et al 2003 ; moyle et al 2004 ; onesti et al 2004 ; mest 2005 ) .
for example , burgess and quiroga ( 2005 ) used pla to treat lipoatrophy in 61 hiv - infected patients over a 5-month period .
the amount of pla varied from 14 vials , depending on the degree of lipoatrophy severity and areas of involvement .
mild cases required 12 treatment sessions ; moderate cases , approximately 4 treatment sessions ; and severe lipoatrophy required 4 or more treatment sessions .
all patients experienced significant clinical improvement , defined as smoothing of the skin with decreased concavities or depressions , and improved overall appearance .
the effect was long lasting , remaining for up to 30 months in patients , depending on initiation of treatment and length of follow - up ( burgess and quiroga 2005 ) . at the time of writing
in addition to treating hiv - infected patients , significant experience has also accumulated in the treatment of immunocompetent patients for cosmetic reasons ( vleggaar and bauer 2004 ) .
poly - l - lactic acid is distinct from other soft tissue fillers , due to its mechanism of action , treatment plan , preparation of injection material , and injection technique ( burgess and lowe 2006 ) .
the application of pla in soft tissue augmentation exploits a mechanism of action not seen in any other soft tissue filler .
although the injection of pla causes an immediate effect by physically occupying space , this initial response is temporary and only lasts one week or less ( mest 2005 ) .
the process of hydration , loss of cohesion and molecular weight , and solubilization and phagocytosis of pla by the host s macrophages , slowly degrades pla into lactic acid microspheres and eliminates co2 by way of respiratory excretion . crystals are left behind to stimulate collagen and a granulomatous reaction .
this inflammatory reaction elicits resorption and the formation of fibrous connective tissue about the foreign body , causing dermal fibroplasia that leads to the desired cosmetic effect ( burgess and quiroga 2004 ) .
poly - l - lactic acid is injected into deeper regions for creating supportive structure and foundation .
effects of volume restoration can persist for as long as 1824 months , but are not permanent ( burgess and quiroga 2005 ; mest 2005 ) .
beljaards and colleagues ( 2005 ) has reported the late onset of granulomatous skin reactions in three immunocompetent patients receiving pla treatment .
the report underscores necessity for dermatologists to recognize and acknowledge the distinct differences between pla and other soft tissue fillers .
the characteristics that distinguish pla from traditional fillers are : ( 1 ) the mechanism of action , ( 2 ) the treatment plan , ( 3 ) the preparation of the injection material , and ( 4 ) the injection technique ( burgess and lowe 2006 ) .
poly - l - lactic acid causes a gradual volume restoration that may take 36 months to develop .
in addition , the rate of dermal thickening increases from the first to last injection . since immediate correction is not the primary goal , as is the case in most traditional fillers , the use of pla requires a markedly different approach and treatment plan .
physicians should treat , wait , assess , and then decide how to proceed . unlike other cosmetic injectables , pla requires reconstitution before use . although manufacturers recommend diluting in 35 ml of sterile water , 57 ml of sterile water is the customary reconstitution volume in the us .
what is most critical for successful outcomes is that physicians receive sufficient training before attempting to inject pla .
for example , as opposed to treating a specific line , the strategy of pla treatment is to return volume to a facial area . in addition
, pla is injected into the dermal subcutaneous plane , unlike collagen or hyaluronic acids .
finally , it is important that pla injection be accompanied by massage of the treatment area and continued by the patient post - treatment ( werschler 2005 ) . at the center for dermatology and dermatologic surgery ( washington d.c . , us ) , pla has been successfully used to treat lipoatrophy of the hands , feet , face , and buttocks in both immunocompetent and immunocompromised patients .
in addition , pla has been used effectively in both the younger and older patient . however , in the older or aging patient , the thickness of the dermis plane is diminished , leading to a greater likelihood for bruising that results from increased fragility of blood vessels . in these patients , it is recommended that clinicians allow 8 weeks between treatment sessions , as opposed to the regular 46 week waiting period . for clinicians ,
the learning curve is longer in these patients , due to the concerns associated with treating patients with a thin dermis plane ( burgess 2006 ) .
the growing marketplace of available soft tissue fillers is a testament to the necessity for individualized treatment to suit the individual desires of the patient and clinician .
the fact that fillers provide augmentation through a variety of divergent mechanisms of action is also reflective of the great challenges of developing an effective filler .
the attributes and use of one particular filler are not exclusive to the use of another filler .
however , not all fillers are appropriate for various indications and successful treatment depends on tailoring appropriate fillers to the desired outcome . for example
, pla can not be injected into the lips , whereas collagen and hyaluronic acid are well suited for injection into the lips . to accommodate the patient s individual needs , soft tissue fillers can be used either individually or combined , depending on treatment goals .
soft tissue fillers may be more effective when used within a comprehensive strategy that addresses the underlying morphological causes that produce facial signs of aging .
although the aging face is a result of a number of processes , the primary cause of the aging face is the redistribution of fat and facial muscular activity .
fat atrophy and fat hypertrophy cause the development of hills and valleys , demarcation of cosmetic units , and disruptions of the primary arcs in the face .
therefore , strategies to restore the youthful appearance of the face should address these changes in a comprehensive manner .
this represents a significant shift from merely treating lines to treating entire areas of the face .
this can be accomplished by using volumizers that are interjected more deeply , alone , or in combination with products that are injected more superficially . however , as the marketplace of soft tissue fillers continues to grow , techniques become more divergent , and therefore , requirements for adequate training on specific products becomes increasingly crucial .
the potential of soft tissue augmentation continues to grow in step with increasing demand . today
, new clinical applications are gaining interest , such as the use of soft tissue fillers in the treatment of nonfacial areas .
moreover , an increasing number of clinical trials are underway to assess the effectiveness of these new clinical applications . for the dermatologist and cosmetic surgeon , this represents an exciting , but challenging era . | in the quest for the ideal soft tissue filler , many diverse products have been developed .
the expanding market of available fillers is a testament that no one product will ideally suit all patients or clinicians .
in addition , the challenge of satisfying the criteria of an ideal filler has driven researchers to take a variety of development paths .
this has resulted in multiple categories to characterize soft tissue fillers .
these fillers are categorized according to : ( 1 ) filler material , eg , autologous , natural , synthetic ; ( 2 ) mechanism of action , eg , void filler , neocollagenesis , fibroblast stimulation ; ( 3 ) patient type and profile , eg , younger versus older patient , rhytids versus sinking and sagging skin ; or ( 4 ) durability of treatment effects , eg , temporary , semi - permanent , or permanent .
although strategies for soft tissue augmentation may be quite diverse , strategies should share a universal goal to address fat redistribution ( atrophy and hypertrophy ) , the primary underlying morphological cause of facial aging . to accomplish this ,
volumizers are now available that are injected more deeply , resulting in the restoration of supportive structure and foundation .
these can be used in combination with other products that are used more superficially for smoothing skin surfaces . as numerous soft tissue fillers enter the market , mechanisms and injection techniques become more divergent , and therefore require that the dermatologist and cosmetic surgeon receive adequate training to use products safely and effectively .
this manuscript provides an overview of soft tissue fillers and their proper use . |
the morphology of the root canal systems of mandibular molars may vary according to ethnic differences and origin , age , gender , and study design .
the knowledge of morphological characteristics and variations of root canal plays an important role in the success of endodontic diagnosis and treatment .
the quality of root canal fillings is associated with ideal biomechanical instrumentation and followed by homogeneity obturation of the root canal structure . on the other hand , the type of roots and the morphology characteristics of mandibular molar teeth present clinical complications that often jeopardize the endodontic therapy .
therefore , clinicians must be able to identify the root canal structure before or during a root canal treatment .
it is known that the presence of additional canals or deviations of the main root canals can cause endodontic flare - ups and failures .
successful treatment of endodontic complications is associated with diagnostic imaging techniques that provide information about the teeth and their surrounding structures .
these techniques include panoramic , full - mouth periapical radiograph , and cone - beam computed tomography ( cbct ) .
conventional radiograph images are widely used in endodontic treatment , but they are not very beneficial in the assessment of complex root canal anatomies where anatomic structures may be confused with periapical pathosis . recently , the use of three - dimensional ( 3d ) imaging techniques such as cbct provides much more detail about the root canal morphology and is more sensitive to detection of supplemental canals than are radiographic images .
studies suggest that cbct potentially provides the clinician a more accurate assessment of the outcome of root canal treatments .
recently , some studies have shown that cbct scanning is able to evaluate the morphology of root canals .
a review of the literature on root and canal morphology using the pubmed database identified several reports on the root and canal morphology with different techniques in a turkish population .
however , there is one study that evaluated root and canal morphology of the mandibular molars in a turkish population by using cbct .
the aim of this retrospective study was to analyze the root and canal morphology of mandibular molar teeth in a turkish population from the south - eastern region of turkey by using cbct images .
cbct images of mandibular molars were obtained from patients who visited the faculty of dentistry , dicle university , diyarbakir , turkey , for various purposes between may 2009 and april 2012 .
all records were selected from a turkish population from the south - eastern region of turkey .
cbct images of 421 women and 429 men between the ages of 14 and 70 years were examined . in total , 2131 teeth ( 966 first mandibular molars and 1165 second mandibular molars ) were evaluated in this study .
this study was based on the retrospective evaluation of cbct , and approved by the medical ethics committee of sifa university ( protocol # 33 - 2013 ) .
the following were the inclusion criteria for selecting the teeth :
permanent molars with no periapical lesionsno root canals with open apices , resorption , or calcificationfully erupted teethno root canal fillings , posts , and crown restorationscbct images of good quality .
permanent molars with no periapical lesions no root canals with open apices , resorption , or calcification no root canal fillings , posts , and crown restorations cbct images of good quality .
the exclusion criteria included the following :
image deformity on cbctincomplete root formationpermanent mandibular third molars that had shifted mesially to the position of early lost second molars were identified by tooth morphology and tilting and were excluded .
image deformity on cbct incomplete root formation permanent mandibular third molars that had shifted mesially to the position of early lost second molars were identified by tooth morphology and tilting and were excluded .
the cbct images were obtained using a cbct scanner ( i - cat vision tm ; imaging science international , hatfield , pa , usa , 2008 ) at 120 kvp , 18.54 ma with an exposure time of 8 - 9 s. the voxel size of the images was 0.3 mm . an experienced radiologist acquired the images according to the manufacturer 's instructions
. intra - examiner calibration of the cbct images was first performed to evaluate the reliability of the assessment .
all the images were assessed separately twice by two examiners ( one endodontist and one maxillofacial radiologist ) with a 2-week interval between the assessments .
the specimens were investigated radiographically by cbct and the following were observed :
the number of rootsthe number of canals per rootthe canal configuration in each root using vertucci 's classificationthe frequency of additional rootsdifferences in the teeth of males and females .
the number of canals per root the canal configuration in each root using vertucci 's classification the frequency of additional roots differences in the teeth of males and females .
statistically significant differences were evaluated using the chi - square test with spss ( version 17.0 ; spss inc . , chicago , il , usa ) software , and p < 0.05 were considered as statistically significant .
the results of this study are summarized in tables 1 and 2 , and variants in the root canal morphology of the mandibular first and second permanent molars are shown in figure 1 . the frequency and distribution of root canals in mandibular first and second permanent molar teeth the frequency distribution and percentage of root canal configurations in mandibular permanent molar teeth ( a ) mandibular bilateral first molars with two roots and four canals .
( b ) mandibular left first molar with two roots , right first molar with three roots .
( e ) mandibular right first molars with three roots and four canals , mandibular right second molar with three roots and three canals , and left second molar with one root and one canal .
( f ) mandibular right first molars with two roots and two canals and left first molars with three roots and three canals the majority of first molars ( 99.2% ) had one mesial and one distal root .
only five patients ( three females and two males ) were detected as having three - rooted ( 0.5% ) molars and three patients ( all females ) were detected as having one - rooted ( 0.3% ) molars .
the three - rooted molars had distolingual or mesiolingual roots and all additional roots had a type i configuration .
there was no statistical difference related to gender ( p > 0.05 ) in the number of roots of mandibular first molars .
the frequency and distribution of all root canals are listed in table 1 . in total ,
96.8% of the mesial roots had two canals , 3% had one canal , and 0.2% had three canals . in the distal root ,
49.8% had two canals , 49.8% had one canal , and 0.4% had three canals .
the distribution and percentages of the six categories of variants in the root canal anatomy of the mandibular first molars are listed in table 2 .
type iv canal configuration was the most prevalent in the mesial roots , whereas type i canal configuration was the most prevalent in the distal roots .
additionally , type ii , type iii , type v , and type viii canal configurations were observed in mesial and distal roots .
moreover , the incidences of varying root canal configurations did not statistically differ between females and males ( p > 0.05 ) .
most of the second molars ( 90% ) had one mesial and one distal root , whereas 10% had one root .
no statistical gender - related difference ( p > 0.05 ) was detected in the incidence of root numbers of mandibular second molars .
the majority of distal roots had one canal ( 97% ) and 3% had two canals .
additionally , three patients ( 0.01% ) had additional ( distolingual or mesiolingual ) canals .
the frequency distribution of the number of root canals did not differ between females and males ( p > 0.05 ) .
the distribution and percentages of the five categories of variants in the root canal anatomy of the mandibular second molars are listed in table 2 .
type iv canal configuration was the most prevalent in the mesial roots , whereas type i canal configuration was the most prevalent in the distal roots .
additionally , type ii , type iii , and type v canal configurations were also observed in mesial and distal roots , and the incidences of varying root canal configurations did not statistically differ between females and males ( p > 0.05 ) , with the exception being the mesial canal of the left mandibular second molars ( p < 0.05 ) .
the majority of first molars ( 99.2% ) had one mesial and one distal root .
only five patients ( three females and two males ) were detected as having three - rooted ( 0.5% ) molars and three patients ( all females ) were detected as having one - rooted ( 0.3% ) molars .
the three - rooted molars had distolingual or mesiolingual roots and all additional roots had a type i configuration .
there was no statistical difference related to gender ( p > 0.05 ) in the number of roots of mandibular first molars .
the frequency and distribution of all root canals are listed in table 1 . in total ,
96.8% of the mesial roots had two canals , 3% had one canal , and 0.2% had three canals . in the distal root , 49.8% had two canals , 49.8% had one canal , and
the distribution and percentages of the six categories of variants in the root canal anatomy of the mandibular first molars are listed in table 2 .
type iv canal configuration was the most prevalent in the mesial roots , whereas type i canal configuration was the most prevalent in the distal roots .
additionally , type ii , type iii , type v , and type viii canal configurations were observed in mesial and distal roots .
moreover , the incidences of varying root canal configurations did not statistically differ between females and males ( p > 0.05 ) .
most of the second molars ( 90% ) had one mesial and one distal root , whereas 10% had one root .
no statistical gender - related difference ( p > 0.05 ) was detected in the incidence of root numbers of mandibular second molars .
the majority of distal roots had one canal ( 97% ) and 3% had two canals .
additionally , three patients ( 0.01% ) had additional ( distolingual or mesiolingual ) canals .
the frequency distribution of the number of root canals did not differ between females and males ( p > 0.05 ) .
the distribution and percentages of the five categories of variants in the root canal anatomy of the mandibular second molars are listed in table 2 .
type iv canal configuration was the most prevalent in the mesial roots , whereas type i canal configuration was the most prevalent in the distal roots .
additionally , type ii , type iii , and type v canal configurations were also observed in mesial and distal roots , and the incidences of varying root canal configurations did not statistically differ between females and males ( p > 0.05 ) , with the exception being the mesial canal of the left mandibular second molars ( p < 0.05 ) .
it is essential to have a thorough knowledge of root canal morphology and configuration for successful endodontic treatment .
the failure to determine additional canals and incomplete instrumentation are the most likely causes of endodontic flare - ups and failures .
many studies have examined root and canal morphology using various methods such as canal staining and clearing technique , cross - sectioning technique , contrast medium - enhanced radiography , modified canal staining and clearing , radiographic examination , and computed tomography scanning
. however , canal staining and clearing technique and cross - sectioning technique are invasive and result in irreversible damage to samples .
cbct is a non - invasive method compared with cross - sections and the clearing technique , and can be used directly to evaluate patients .
conventional intraoral periapical radiographs provide only two - dimensional images , and in some cases , anatomic structures can be superimposed in these images ; therefore , they are not beneficial in the evaluation of complex root canal anatomies due to their natural limitations . cbct has been widely used to evaluate the endodontic applications by clinicians in the past few years , and provides clinicians with three - dimensional information about the external and internal morphology of the root and canal systems . in the present study
, cbct provides a comprehensive report on the root canal morphology of mandibular first and second molars in a turkish population .
it has usually been accepted that mandibular first molars have two roots located mesially and distally and three canals ( one root canal in the distal root and two root canals located in the mesial root ) ; but in populations with mongoloid traits , the additional root in a mandibular first molar is considered to be a normal morphologic version and may be defined as a mongolian trait or an asian trait .
it has been described that the mongoloid population exhibits significantly more mandibular first molars with three roots ( 3:1 ) than the other populations , and this version could be considered a genetically determined characteristic . in this study , we found that 0.5% of mandibular first molars and
this frequency is less than that reported in a previous study of turkish population ( 2.06% ) , but is very less than that reported in japanese ( 22.7% ) , koreans ( 22.3% ) , hong kong population ( 15.0% ) , and by zhang et al .
( 29% ) who reported a high prevalence of three roots in an asian population .
furthermore , this variant has a frequency of lower than 5% in european people ( british , dutch , german , finnish ) , african ( bushmen , bantu , senegalese ) and indian populations . in the case of mandibular second molars , the majority ( 90% ; 1051 teeth )
had two roots located mesially and distally and 114 teeth ( 10% ) had one root .
the incidence of two separate roots is similar to that reported in a previous study of turkish population by demirbuga et al .
( 85.4% ) ; however , it is higher than that reported in burmese ( 58.2% ) and thai ( 54% ) populations . in the present study ,
the frequency of two canals in the mesial and distal roots of the first molar tooth was 96.8% and 49.8% , respectively , whereas the second molar tooth had two canals in 90.2% and 3.15% of the mesial and distal roots , respectively .
this incidence is similar to those reported in the previous studies of turkish and western chinese populations by using cbct , except for the distal roots of the first molar tooth . in the present study ,
type iv configuration was the most prevalent ( 89% in females and 93% in males ) in mesial roots .
and al - nazhan reported type ii being the most prevalent followed by type iv .
only one patient ( 0.2% of females ) had a type viii configuration in the mesial root , which is in agreement with the study by chen et al .
the most common canal configuration of distal roots was type i , with a reported frequency between 54% and 72% .
likewise , in our study , type i configuration was the most prevalent in females ( 60% ) and males ( 59% ) in the distal roots .
furthermore , in this study , type ii , type iv , and type v configurations were found to be higher than that reported by demirbuga et al .
examined 20 extracted three - rooted mandibular first molars in a micro - computed tomography study of chinese patients .
they found that all the additional roots contained a type i ( 100% ) canal configuration .
in the present study , all the additional roots contained a type i canal configuration the results of the present investigation are in agreement with previous studies .
the most common root canal configuration of second molar teeth in the present study was type iv ( 76% for females and
84% for males ) in the mesial roots and type i ( 95% for females and
vertucci and williams , and pineda and kuttler who observed type i as the most frequent canal configuration in the mesial root of the second molar tooth in a caucasian population .
several possible reasons may account for the differences , such as ethnic population , study design ( the samples in the other studies were extracted teeth ) , and the sample size .
the root number and morphology of 850 turkish mandibular molars were examined using cbct . in the present study ,
type iv and type i canal configurations were the most prevalent in the mesial and distal roots , respectively , of both the mandibular first and second molar teeth .
there was a low prevalence of three - rooted mandibular molars in this turkish population .
cbct scanning provides supplemental information about the root canal configurations of mandibular molars in a turkish population , and this study may help clinicians in the root canal treatment of mandibular molars . | objective : the aim of this retrospective study was to determine the root and canal morphology of the mandibular first and second permanent molars in a turkish population using cone - beam computed tomography ( cbct).materials and methods : cbct images of mandibular first ( n = 966 ) and second molar ( n = 1165 ) teeth from 850 turkish patients were evaluated .
the root canal configurations were classified according to the method of vertucci .
the data were analyzed by pearson 's chi - square test.results:the majority of mandibular molars were two rooted with three canals ; however , three roots were identified in 0.05% of the first molars and 0.01% of the second molars , and 100% of the additional root canals were of type i configuration .
mesial roots had more complex canal systems with more than one canal , whereas most distal roots had a type i configuration.conclusions:within the limitations of this study , it can be concluded that cbct scanning provides supplemental information about the root canal configurations of mandibular molars in a turkish population .
this study may help clinicians in the root canal treatment of mandibular molars . |
portal hypertension ( ph ) is a common clinical syndrome defined as the elevation of hepatic venous pressure gradient ( hvpg ) above 5 mm hg .
ph is caused by a combination of two simultaneous occurring hemodynamic processes : ( 1 ) increased intrahepatic resistance to passage of blood flow through the liver due to cirrhosis and ( 2 ) increased splanchnic blood flow secondary to vasodilatation within the splanchnic vascular bed .
ph can be due to many different causes at prehepatic , intrahepatic , and posthepatic sites ( table 1 ) .
cirrhosis of the liver accounts for approximately 90% of cases of ph in western countries .
the importance of ph is defined by the frequency and severity of its complications including variceal bleeding , spontaneous bacterial peritonitis , and hepatorenal syndrome , which represent the leading causes of death and of liver transplantation in patients with cirrhosis .
ph is considered to be clinically significant when hvpg exceeds 10 to 12 mm hg , since this is the threshold for the clinical complications of ph to appear .
proper diagnosis and management of these complications are vital to improving quality of life and patients ' survival .
approximately 515% of cirrhotics per year develop varices , and it is estimated that the majority of patients with cirrhosis will develop ge varices over their lifetime .
the presence of ge varices correlates with the severity of liver disease ; while only 40% of child a patients have varices , they are present in 85% of child c patients ( table 2 ) .
the resistance in the portal vessels is normally lower than in the collateral circulation , and so blood flows from the systemic bed into the portal bed .
however , when ph develops , the portal pressure is higher than systemic venous pressure , and this leads to reversal of flow in these collaterals .
in addition , the collateral circulatory bed also develops through angiogenesis and the development of new blood vessels in an attempt to decompress the portal circulation .
the areas where major collaterals occur between the portal and systemic venous system are shown in table 3 .
unfortunately these collaterals are insufficient to decompress the ph , leading to complications including variceal bleeding .
esophageal varices ( ev ) form when the hvpg exceeds 10 mm hg . in the lower 2 to 3 cm of the esophagus ,
in addition , these varices do not communicate with the periesophageal veins and therefore can not easily be decompressed .
these are the reasons why ev bleeds only at this site . over the last decade
, most practice guidelines recommend to screen known cirrhotics with endoscopy to look for ge varices .
varices should be suspected in all patients with stigmata of chronic liver disease such as spider nevi , jaundice , palmar erythema , splenomegaly , ascites , encephalopathy , and caput medusae .
ev are graded as small ( < 5 mm ) and large ( > 5 mm ) , where 5 mm is roughly the size of an open biopsy forceps .
decompensated cirrhosis ( child b or c ) , presence of red wale marks ( defined as longitudinal dilated venules resembling whip marks on the variceal surface ) , and alcoholic cirrhosis at the time of baseline endoscopy are the main factors associated with the progression from small to large ev .
the predictors of first bleeding include the size of varices , severity of cirrhosis ( child b or c ) , variceal pressure ( > 12 mm hg ) , and the endoscopic presence of red wale marks [ 7 , 8 ] . although ev bleeding stops spontaneously in up to 40% of patients , and despite improvements in therapy over the last decade , the 6 weeks mortality rate is still 20% .
gastroesophageal varices ( gov ) are an extension of ev and are categorized based on sarin 's classification into 2 types ( figure 1 ) .
the most common are type 1 ( gov1 ) varices , which extend along the lesser curvature .
isolated gastric varices ( igv ) occur in the absence of ev and are also classified into 2 types .
type 1 ( igv1 ) are located in the fundus and tend to be tortuous and complex , and type 2 ( ivg2 ) are located in the body , antrum , or around the pylorus .
gv are less common than ev and are present in 5%30% of patients with ph with a reported incidence of bleeding of about 25% in 2 years , with a higher bleeding incidence for fundal varices .
predictors of gv bleeding include the size of fundal varices ( large ( > 10 mm ) > medium ( 510 mm ) > small ( < 5 mm ) ) , severity of cirrhosis ( child class c > b > a ) , and endoscopic presence of variceal red spots ( defined as localized reddish mucosal area or spots on the mucosal surface of a varix ) .
ecv are best defined as large portosystemic venous collaterals occurring anywhere in the abdomen except for the ge region .
they are an unusual cause of gi bleeding , but account for up to 5% of all variceal bleeding . compared to ge varices ,
ecv are difficult to locate , occur at distal sites , and when identified , the choice of therapy is unclear , therefore representing a clinical challenge .
furthermore , bleeding ecv may be associated with poor prognosis , with one study quoting mortality reaching 40% .
different areas of ecv are the duodenum , jejunum , ileum , colon , rectum , peristomal , biliary tree , gallbladder , peritoneum , umbilicus , bare area of the liver , ovary , vagina , and testis [ 15 , 16 ] .
the prevalence of ecv varies in the literature and seems to be related to the etiology of the ph and the diagnostic modalities used . in patients with ph due to cirrhosis , duodenal
results of a survey for ecv conducted over 5 years in japan identified 57 cases of duodenal varices ; they were located in the duodenal bulb in 3.5% , the descending part in 82.5% , and the transverse part in 14.0% .
in contrast to duodenal varices , it appears that most cases of varices in other portions of the small bowel and colonic varices are seen in patients with cirrhosis who have previously undergone abdominal surgery . using advanced endoscopic technologies ,
particularly capsule endoscopy and enteroscopy , the prevalence of small bowel varices is estimated to be approximately 69% in patients with ph .
the prevalence of colonic varices and rectal varices has been found to be 34% to 46% [ 20 , 21 ] and 10% to 40% , respectively , in patients with cirrhosis undergoing colonoscopy .
it is important to differentiate rectal varices from hemorrhoids ; rectal varices extend more than 4 cm above the anal verge , are dark blue in color , collapse with digital pressure , and do not prolapse into the proctoscope on examination , whereas hemorrhoids do not extend proximal to the dentate line , are purple in color , do not collapse with digital pressure , and often prolapse into the proctoscope [ 22 , 23 ] .
stomal varices are a particularly common cause of ecv and can occur in patients with cirrhosis secondary to primary sclerosing cholangitis ( psc ) . in the west ,
because the prevalence of noncirrhotic ph is low , most bleeding ecv is usually associated with cirrhotic ph ( 6,8 ) .
although ecv can occur at several sites , bleeding ecv are most commonly found in the duodenum and at sites of previous bowel surgery including stomas . in a review of 169 cases of bleeding ecv , 17% occurred in the duodenum , 17% in the jejunum or ileum , 14% in the colon , 8% in the rectum , and 9% in the peritoneum . in the review ,
26% bled from stomal varices and a few from infrequent sites such as the ovary and vagina .
portal biliopathy , which includes abnormalities ( stricture and dilatation ) of both extra and intrahepatic bile ducts and varices of the gallbladder , is associated with ph , particularly extrahepatic portal vein obstruction [ 25 , 26 ] .
they are also seen associated with cirrhosis , non - cirrhotic portal fibrosis , and congenital hepatic fibrosis . while a majority of these patients are asymptomatic , some present with a raised alkaline phosphatase level , abdominal pain , fever , and cholangitis .
choledocholithiasis may develop as a complication and manifest as obstructive jaundice with or without cholangitis .
on cholangiography , bile - duct varices may be visualized as multiple , smooth , mural - filling defects with narrowing and irregularity resulting from compression of the portal vein and collateral vessels .
mucosal changes in the stomach in patients with ph include portal hypertensive gastropathy ( phg ) and gastric vascular ectasia .
phg describes the endoscopicappearance of gastric mucosa with a characteristic mosaic , or snake - skin - like appearance with or without red spots .
the prevalence of phg parallels the severity of ph and it is considered mild when only a mosaic - like pattern is present and severe when superimposed discrete red spots are also seen . bleeding ( acute or chronic ) from these lesions is relatively uncommon , and rarely severe .
patients with chronic bleeding usually present with chroniciron deficiency anemia . in gastric vascular ectasia ,
collection of ectatic vessels can be seen on endoscopy as red spots without a mosaic - like pattern . when the aggregates are confined to the antrum of the stomach
, the term gastric antral vascular ectasia ( gave ) is used , and if aggregates in the antrum are linear , the term watermelon stomach is used to describe the lesion .
the prevalence of gave syndrome in cirrhosis is low and can be endoscopically difficult to differentiate from severe phg .
therefore , gastric biopsy may be required to differentiate them as histologically gave lesions are completely distinct from phg ( table 4 ) .
small bowel might also show mucosal changes related to ph , which is called portal hypertensive enteropathy ( phe ) .
the diagnosis of phe has been limited in the past due to the difficult access to the small bowel . with advanced endoscopic techniques such as capsule endoscopy and enteroscopy ,
phe is now thought to be a frequent finding in patients with cirrhosis , perhaps as common as phg , and may cause occult gi blood loss [ 34 , 35 ] .
portal hypertensive colopathy ( phc ) refers to mucosal edema , erythema , granularity , friability , and vascular lesions of the colon in ph .
although they are found in up to 70% of patients with ph and are more common in patients with ev and phg , they rarely cause bleeding [ 38 , 39 ] .
cirrhotic ph is the most common cause of ascites , which accounts for approximately 75% patients with ascites .
the development of ascites is an important event in cirrhosis as the mortality is approximately 50% at 2 years without a liver transplantation .
the formation of ascites in cirrhosis is due to a combination of abnormalities in both renal function and portal and splanchnic circulation .
the main clinical symptom of patients with ascites is an increase in abdominal girth , often accompanied by lower - limb edema . in some cases ,
the accumulation of fluid is so severe that respiratory function and physical activity is impaired . in most cases ,
patients must have approximately 1500 ml of fluid for ascites to be detected reliably by physical examination .
dyspnea in these patients can occur as a consequence of increasing abdominal distension and/or accompanying pleural effusions .
increased intra - abdominal pressure might favour the development of abdominal hernias ( mainly umbilical ) in patients with cirrhosis and longstanding ascites .
the current classification of ascites , as defined by the international ascites club , divides patients in three groups ( table 5 ) .
patients with refractory ascites are those that do not respond to sodium restriction and high doses of diuretics or develop diuretic - induced side effects that preclude their use .
ascites may not be clinically detectable when present in small volumes . in larger volumes ,
the classic findings of ascites are adistended abdomen with a fluid thrill or shifting dullness .
ascites must be differentiated from abdominal distension due to other causes such as obesity , pregnancy , gaseous distension of bowel , bladder distension , cysts , and tumours .
paracentesis with analysis of ascitic fluid is the most rapid and cost - effective method of diagnosis .
it should be done in patients with ascites of recent onset , cirrhotic patients with ascites admitted to hospital , or those with clinical deterioration .
the most important analyses are cell count , fluid culture , and calculation of the serum : ascites albumin gradient ( saag ) , which reflects differences in oncotic pressures and correlates with portal venous pressure .
it saag is greater or equal to 1.1 g / dl ( or 11
patients with cirrhosis and ascites are also at risk of developing infections , particularly spontaneous bacterial peritonitis ( sbp ) . sbp occurs in approximately 10% of hospitalized cirrhotic patients , with an associated mortality of 2040% if untreated .
many patients are asymptomatic , but clinical signs can include abdominal pain , fever , and diarrhea . the diagnosis of sbp is based on neutrophil count > 250 cells / mm in the ascitic fluid .
hepatorenal syndrome ( hrs ) is a common complication seen in patients with advanced cirrhosis and ph .
hrs can also be seen in other types of severe chronic liver disease , alcoholic hepatitis , or in acute liver failure .
hrs has been defined in the literature as a reversible functional renal impairment in the absence of other causes of renal failure , tubular dysfunction , proteinuria , or morphological alterations in histological studies .
precise and accurate diagnostic criteria have been established in order to clearly define this syndrome ( table 6 ) .
the reported incidence of hrs is approximately 10% among hospitalized patients with cirrhosis and ascites .
the probability of occurrence of hrs in patients with cirrhosis is around 20% after 1 year and 40% after 5 years .
the pathogenesis of hrs is not completely understood , but is likely the result of an extreme underfilling of the peripheral arterial circulation secondary to arterial vasodilatation in the splanchnic circulation . in addition
, recent data indicates that a reduction in cardiac output also plays a significant role .
hrs - associated renal failure is seen in late stages of cirrhosis and is marked by severe oliguria , increased sodium and water retention , volume overload , hyperkalemia , and spontaneous dilutional hyponatremia .
type 1 hrs is a rapidly progressive renal failure that is defined by doubling of serum creatinine > 2.5 mg / dl ( > 221 mol / l ) or a decrease of 50% in creatinine clearance ( < 20 ml / min ) in less than 2 weeks .
this form of hrs is usually precipitated by gastrointestinal bleeds , large volume paracenthesis , acute alcoholic hepatitis and sbp .
in addition to renal failure , patients with type 1 hrs present deterioration in the function of other organs , including the heart , brain , liver , and adrenal glands . the median survival of these patients without treatment is < 2 weeks , and almost all of them die within 10 weeks after onset of hrs .
type 2 hrs is a moderate and stable renal failure with a serum creatinine of > 1.5 mg / dl ( > 133
mol / l ) that remains stable over a longer period and is characterized by diuretics resistant ascites [ 49 , 54 ] .
hepatic encephalopathy ( he ) is defined as neurologic and psychiatric dysfunction in a patient with chronic liver disease . the exact mechanism leading to this dysfunction is still poorly understood , but multiple factors appear to play a role in its genesis .
the liver normally metabolizes ammonia , produced by enteric bacteria and enterocytes [ 57 , 58 ] . in a patient with ph
, ammonia bypasses the liver through portosystemic shunt and reaches the astrocytes in the brain . within the astrocyte
, ammonia is metabolized into glutamine , which acts as an osmole to attract water , thus causing cerebral edema .
in addition , direct ammonia toxicity triggers nitrosative and oxidative stress , which lead to astrocyte mitochondrial dysfunction [ 59 , 60 ] .
another important factor is the enhancement of gamma - aminobutyric acid ( gaba - a ) receptors through neuroinhibitory steroids ( i.e. , allopregnanolone ) and benzodiazepine .
finally , tryptophane byproducts indole and oxindole , manganese , inflammation , hyponatremia , and reduced acetylcholine through acetylcholinesterase activity also contribute to cerebral dysfunction .
minimal hepatic encephalopathy ( grade 0 ) ( table 7 ) can present with impaired driving ability , minimally impaired psychometric tests , decreased global functioning , and increased risk of falls . in overt hepatic encephalopathy , diurnal sleep pattern changes will often precede neurologic symptoms . to add to the complexity , he can be intermittent or persistent .
the severity of presentation is usually classified using the west haven criteria ( table 7 ) .
grade 1 hepatic encephalopathy represents lack of awareness , anxiety or euphoria , and short attention span .
change of personality , lethargy , and inappropriate behavior can be seen in grade 2 encephalopathy .
more advanced features include disorientation , stupor , confusion ( grade 3 ) , and can even reach coma ( grade 4 ) .
physical examination may be normal , but typical signs include bradykinesia , asterixis , hyperactive deep tendon reflexes and even decerebrate posturing .
hepatopulmonary syndrome ( hps ) is a triad of liver disease , pulmonary vascular ectasia and impaired oxygenation .
hps is defined in the literature as a widened alveolar - arterial oxygen difference ( a - a gradient ) in room air ( > 15 mm hg or > 20 mm hg in patients > 64 years of age ) with or without hypoxemia due to intrapulmonary vasodilatation in the presence of hepatic dysfunction [ 70 , 71 ] .
this syndrome occurs mostly in those with ph ( with or without cirrhosis ) and indicates poor prognosis and higher mortality .
estimates of the prevalence of hps among patients with chronic liver disease range from 4 to 47% , depending upon the diagnostic criteria and methods used [ 7173 ] .
hps results in hypoxemia through pulmonary microvascular vasodilatation and intrapulmonary arteriovenous shunting resulting in ventilation - perfusion mismatch , and can occur even with mild liver disease .
clinically , patients with hps complain of progressive dyspnea on exertion , at rest , or both .
severe hypoxemia ( pao2 < 60 mm hg ) is often seen and strongly suggests hps [ 70 , 71 ] .
a classical finding in hps is orthodeoxia defined as a decreased arterial oxygen tension by more than 4 mm hg or arterial oxyhemoglobin desaturation by more than 5% with changing position from supine to standing .
it is associated with platypnea defined as dyspnea worsened by upright position [ 70 , 71 ] .
platypnea - orthodeoxia is caused by the worsening of diffusion - perfusion matching and increased shunting at the lung bases in the upright position .
there are no hallmark signs on physical exam ; however , cyanosis , clubbing , and cutaneous telangiectasia ( spider nevi ) are commonly noted .
furthermore , systemic arterioembolisation may cause stroke , cerebral hemorrhage , or brain abscess , and can present with neurological deficits .
portopulmonary hypertension ( pph ) , a well - recognized complication of chronic liver disease , refers to pulmonary arterial hypertension ( pah ) associated with ph when no alternative causes exist .
it is defined by the presence of elevated pulmonary arterial pressure ( mean pressure > 25 mm hg at rest and 30 mm hg on exertion ) elevated pulmonary vascular resistance ( > 240 dyne s cm ) in the presence of a pulmonary capillary wedge pressure < 15 mm hg .
the prevalence of pph depends on the patient population studies and severity of the liver disease , 0.72% and 3.516.1% in cirrhotics and patients undergoing liver transplantation , respectively .
the development of pph is independent of the cause of ph , and it is often seen in cirrhosis .
it is however , also described in those with ph due to nonhepatic pathologies such as portal venous thrombosis [ 71 , 77 ] .
the pathogenesis of pph is not completely understood ; however , several theories have been offered .
the most widely accepted theory is that a humoral vasoactive substances ( e.g. , serotonin , endothelin-1 , interleukin-1 , thromboxane b2 , and secretin ) , normally metabolized by the liver , is able to reach the pulmonary circulation via portosystemic shunts , resulting in pph [ 71 , 78 , 79 ] .
the most common presenting symptom is progressive dyspnea on exertion and less frequently fatigue , palpitations , syncope , hemoptysis , orthopnea , and chest pain . on physical exam ,
classical features include edema , an accentuated p2 and a systolic murmur , indicating tricuspid regurgitation [ 71 , 77 , 80 ] . in severe cases , signs and symptoms of right - heart failure can be noted .
it is defined as a pleural effusion greater than 500 ml in patients with cirrhosis in absence of primary cardiac , pulmonary , or pleural disease .
patients with cirrhosis and ph have abnormal extracellular fluid volume regulation resulting in passage of ascites from the peritoneal space to the pleural cavity via diaphragmatic defects generally in the tendinous portion of the diaphragm .
negative intrathoracic pressure during inspiration pulls the fluid from the intra - abdominal cavity into the pleural cavity .
hydrothorax develops when the pleural absorptive capacity is surpassed , leading to accumulation of fluid in the pleural space .
multiple studies have shown the passage of fluid from the intra - abdominal space to the pleural space via 99mtc - human albumin or 99mtc - sulphur colloid .
clinical manifestations of hepatic hydrothorax include shortness of breath , cough , hypoxemia , and chest discomfort .
hepatic hydrothorax should always be suspected in patients with cirrhosis or ph and undiagnosed pleural effusion , regardless of the presence of ascites .
serious complications include acute tension hydrothorax with dyspnoea and hypotension and spontaneous bacterial empyema .
it is characterized by impaired contractile response and/or altered diastolic relaxation in the absence of other cardiac diseases .
the pathophysiology of this condition is complex , and seemingly related to ph and cirrhosis . in advanced liver disease ,
the arterial vessel wall thickness and tone decreases , leading to reduced arterial compliance [ 87 , 88 ] .
ultimately , these factors lead to systolic and diastolic dysfunction . symptoms associated with cirrhotic cardiomyopathy include dyspnea with exertion , impaired exercise capacity , paroxysmal nocturnal dyspnea , peripheral edema , and orthopnea .
less - frequent presentations include long qt on electrocardiography , arrhythmia , and sudden death .
hepatic osteodystrophy is defined as bone disease ( osteomalacia , osteoporosis , and osteopenia ) associated with liver disease .
osteomalacia and osteoporosis are frequently seen in cirrhotic patients and can predispose to pathologic fractures .
the leading hypothesis suggests that it is related to the uncoupling of osteoblastic and osteoclastic activity .
osteoclastogenic proinflammatory cytokines ( interleukin 1(il-1 ) and tumor necrosis factor ( tnf ) ) are increased in hepatic fibrosis .
decreased osteoblastic activity has also been linked with insulin - like growth factor 1 in a rat model ( igf-1 ) .
increasing igf-1 levels are associated with liver disease severity . finally , vitamin k mediates the carboxylation of glutamyl residues on osteocalcin , stimulating osteoclastic activity .
. they may present with pain following a nontraumatic fracture of the axial skeleton or bone deformity , including pronounced cervical kyphosis .
hypersplenism is a common complication of massive congestive splenomegaly in patients with cirrhosis and ph . in this condition ,
splenomegaly is associated with thrombocytopenia , leucopenia , or anemia or a combination of any the three [ 95 , 96 ] .
severe hypersplenism is present in about 1/3 of patients with cirrhosis being assessed for liver transplantation .
most patients have no symptoms related to hypersplenism , however severe thrombocytopenia may increase the risk of bleeding , especially after invasive procedures .
once a patient develops such complications , they are considered to have decompensated disease with the high morbidity and mortality .
the quality of life and survival of patients with cirrhosis can be improved by the prevention and treatment of these complications . | the portal hypertension is responsible for many of the manifestations of liver cirrhosis .
some of these complications are the direct consequences of portal hypertension , such as gastrointestinal bleeding from ruptured gastroesophageal varices and from portal hypertensive gastropathy and colopathy , ascites and hepatorenal syndrome , and hypersplenism .
in other complications , portal hypertension plays a key role , although it is not the only pathophysiological factor in their development .
these include spontaneous bacterial peritonitis , hepatic encephalopathy , cirrhotic cardiomyopathy , hepatopulmonary syndrome , and portopulmonary hypertension . |
the posterior parietal cortex ( ppc ) of primates has been shown to be important for multisensory space representation and the control of goal directed action ( culham et al . ,
traditionally , ppc has been considered a key node of the dorsal visual stream involved in the vision for action neural processing ( goodale and milner , 1992 ) .
the ppc comprises the superior parietal lobule ( spl ) and the inferior parietal lobule ( ipl ) , separated by the intraparietal sulcus ( ips ) .
these regions include v6 and v6a in the anterior bank of the parieto - occipital sulcus ( galletti et al . , 1999 )
, the medial intraparietal area ( mip ) in the medial bank of the ips , [ mip , together with the most lateral part of v6a is included within the functionally defined parietal reach region ( prr , snyder et al . , 1997 ) ] , areas pe ( area 5 ) and pe caudal ( pec ) , and pgm in the medial part of spl ( pandya and seltzer , 1982 ; shipp et al . , 1998 ;
the ventral intraparietal area ( vip ) , located at the fundus of ips between spl and ipl , can also be grouped with the spl regions due to its functional properties ( colby et al . , 1993 ) .
( a ) location and extent of the areas that form the superior parietal cortex of the macaque brain .
the occipital pole has been partially removed and inferior parietal lobule of the right hemisphere has been cut away at the level of the fundus of the intraparietal sulcus to show the cortex of the medial bank of this sulcus .
the occipital lobe of the same hemisphere has been cut away at the level of the fundus of the parieto - occipital and lunate sulci to show the cortex of the anterior bank of the parieto - occipital sulcus .
the medial surface of the left hemisphere is drawn to show the location on it of all areas that extend medially .
pos , parieto - occipital sulcus ; cal , calcarine sulcus ; cin , cingulate sulcus ; ips , intraparietal sulcus ; ios , inferior - occipital sulcus ; ots , occipitotemporal sulcus ; sts , superior temporal sulcus ; lf , lateral fissure ; cs , central sulcus ; sas , superior arcuate sulcus ; ias , inferior arcuate sulcus ; ps , principal sulcus ; v6 , area v6 ; v3 , area v3 ; v2 , area v2 ; v1 , area v1 ; pec , area caudal pe ; pe , area pe ; peip , intraparietal area pe ; mip , medial intraparietal area , pgm , area medial pg ; vip , ventral intraparietal area ; lip , lateral intraparietal area ; aip , anterior intraparietal area ; mt , middle temporal area ; mst , medial superior temporal area ; brodmann 's areas 23 , 31 , 46 , and dorsal premotor areas f2 and f7 in are also shown .
( b ) flow chart of the connections of v6a modified from passarelli et al .
rostral / caudal brain areas are shown at the top / bottom part of the figure .
areas in the ventral part of the parieto - occipital sulcus ( v6 , ventral v6a ) are dominated by visual input , whereas as one proceeds toward the dorsal part of v6a sensory association and visuomotor / premotor connections prevail .
the above areas form together an important network that performs the integration of visual and somatic spatial information necessary for the control of arm movements in space ( snyder et al .
, 1997 ; buneo et al . , 2002 ; galletti et al . , 2003
figure 1b summarizes the flow of information in spl by illustrating the major connections of area v6a .
this brain region receives visual information from the extrastriate areas v2 , v3 , v4 , v6 , and mst ( gamberini et al . , 2009 ; passarelli et al . , 2011 ) .
moreover , it receives input from areas mip and vip , where neurons with both visual and somatosensory sensitivity have been found ( colby and duhamel , 1991 ) .
v6a also shows strong reciprocal connections with lip and area pg of the ipl , involved in encoding spatial parameters for ocular and manual actions ( barash et al . , 1991 ; heider et al . ,
v6a , together with mip and pec , send a strong projection to the arm region of area f2 in the premotor cortex ( godschalk et al . , 1995 ; matelli et al . , 1998 ;
spl areas through their reciprocal interactions , collectively process visual , somatosensory and motor information to program and control reaching movements .
the anatomical and neurophysiological evidence from monkeys is in line with data from neurological patients .
lesions in human spl have been reported to produce deficits in the perception of the spatial relationship between objects and the subjects ' own body that is manifested with inaccurate reaching movements ( critchley , 1953 ; perenin and vighetto , 1988 ; wolpert et al . ,
furthermore , damages to spl were shown to compromise more severely the depth component of visually guided arm movements ( baylis and baylis , 2001 ; danckert et al . ,
the encoding by spl of the visually guided behavior in depth has been addressed by relatively few studies ( lacquaniti et al . , 1995 ; bhattacharyya et al . , 2009 ;
a strong influence of vergence angle on the activity of neurons involved in the planning of reaches has been demonstrated ( bhattacharyya et al . , 2009 ) , whereas in area pe signals related to the position of the hand prevailed ( ferraina et al . , 2009 ) .
eye position signals are critical in the visuomotor transformations performed by the ppc , as they are used to compute the position of visual targets with respect to the body ( andersen and mountcastle , 1983 ; andersen et al . , 1990 ; bremmer et al . , 1997 )
the computation of target location in an egocentric frame of reference is realized through the modulation of a visual response by gaze position in what has been referred to as a gain field
gain fields have been demonstrated in many areas of both the dorsal and ventral stream , in the primary visual cortex and in subcortical structures as well ( salinas and sejnowski , 2001 ) . regarding the encoding of target location in depth , a model of disparity - selective neurons
gain modulated by the vergence angle has been proposed ( pouget and sejnowski , 1994 ) and subsequent studies in prr and lateral intraparietal area ( lip ) provided experimental support for it ( genovesio and ferraina , 2004 ; bhattacharyya et al . , 2009 ) .
apart from the spatial localization of targets , gain modulations of activity by eye position have been linked with systematic biases in space representation that serve behaviorally important perceptual and motor actions .
for example , in the primary visual cortex the majority of neurons that represent the peripheral visual field increase their firing as the eyes attain eccentric fixation , thus facilitating the processing of targets presented at straight - ahead directions ( durand et al . , 2010 ) .
similarly , in areas v2 and v4 most of the neurons with distance tuning for near space preferred also downward gaze positions ( rosenbluth and allman , 2002 ) and this could reflect the fact that it is more usual to look down when we fixate near targets .
these associations between eye position signals and behavioral context could be the result of learning and adaptation processes . in the rest of this review we focus on the multiple functions of 3d eye position signals in spl , in particular in area v6a .
we review recent neurophysiological studies performed by our group to address whether signals related to vergence angle and to the encoding of the peripersonal space are processed in area v6a .
we also present the results of studies in putative homologue regions in human spl that also addressed these two issues .
furthermore , we will review evidence showing the influence of eye position activity on the spatial tuning of reaching discharges . in addition
, we will review recent findings from human studies that support the involvement of spl in non - spatial cognitive processes .
to estimate the position of a foveated target in 3d space , information about the direction of gaze ( version ) and the depth of fixation ( vergence ) is needed .
electrophysiological studies in various ppc areas found that neuronal activity was modulated by version signals ( andersen et al .
1995 ; bremmer et al . , 1997 ; nakamura et al . , 1999 ) .
similarly , ppc neurons affected by vergence signals were reported ( genovesio and ferraina , 2004 ) .
in addition , vergence is a valid cue for distance estimation within the space that can be reached by the hands ( viguier et al . , 2001 ) .
a combined encoding of the direction and depth of gaze in single cells has been only demonstrated in a small number of cells in area 7a of ipl ( sakata et al . , 1980 ) .
in our study ( breveglieri et al . , 2012 ) , we set out to investigate the effect of vergence angle and its interaction with version signals in v6a .
two monkeys were trained to fixate targets located within the peripersonal space at different distances and directions from the eyes ( figure 2a ) . in total ,
74% of the cells were affected by the vergence and/or the version angle , with the majority of the modulated cells being affected by both gaze variables .
figure 2b shows a neuron displaying its maximum discharge when the gaze was directed to the near and contralateral space .
in addition , this neuron showed a linear increase of activity as the gaze shifted from far to near and from ipsilateral to contralateral positions .
a multilinear regression analysis showed that in the majority of modulated neurons ( ~85% ) vergence and version had a linear effect on the neural activity .
this finding suggests that intermediate depths and straight - ahead gaze positions did not activate maximally most of v6a cells .
when the average activity of all cells modulated by vergence ( figure 2c , left ) and version ( figure 2c , right ) was plotted as a population spike density function , the central space evoked significantly ( permutation test , p < 0.05 ) lower activity compared to contralateral and ipsilateral space ( figure 2c , right ) . at the same time , no difference was found in the average activity for near , intermediate and far positions ( figure 2c , left ) . while the underrepresentation of central gaze positions confirms studies of eye position encoding in two dimensional space ( galletti et al .
, 1995 ; nakamura et al . , 1999 ) , the monotonic tuning of most v6a cells by vergence is a new finding that is in agreement with evidence from area lip ( genovesio and ferraina , 2004 ) . at the population level
, monotonic cells could equally increase their activity toward near and far peripersonal space ( n = 38 , 44% near , n = 49 , 56% far ) .
this result was in contrast with the study of genovesio and ferraina ( 2004 ) , where the majority of cells increased their activity with increasing vergence .
this suggests that there is a different encoding of depth in spl compared to ipl . in neurons showing a linear effect of version
there was a trend for representing contralateral space locations that was not statistically significant ( test , p > 0.05 ) . in summary ,
our study revealed a strong effect of signals related to vergence angle in area v6a .
furthermore , these signals were often integrated with version information in single neurons thus allowing the encoding of locations in 3d space . ( a ) scheme of the experimental setup set up used for the fixation - in - depth and reaching - in - depth tasks .
exact distances and angles of the targets are indicated in the lateral view ( left ) and top view ( right ) , respectively .
( b ) example of a v6a neuron modulated by both version and vergence during fixation .
the discharge to the nine leds arranged from near ( bottom ) to far ( top ) was aligned twice ( at the start of the fixation and at the led change ; dashed line : point where trials were cut because of double alignment ) . from left to right , the behavioral events are : led onset , saccade offset ( first alignment marker ) , led change ( second alignment marker ) .
bin size for spike histograms : 20 ms ; scale for version and vergence traces is 100 and 20 , respectively .
population average spike density functions ( sdf ) were constructed by ranking the response for each tested row of fixation targets .
the thick lines indicate average normalized sdf ; the light lines indicate variability bands ( sem ) .
the peak of the sdf of the row showing the maximum activity was set to 1 ( 100% ) and was used to normalize the sdf curves of the other rows .
the black rectangles below each plot indicate the periods where the permutation test was run . in the left plot ,
no statistical difference was observed between the curves ( permutation test , p > 0.05 ) , whereas in the right , the central row is statistically different from the other two ( permutation test , p < 0.05 ) .
scale on abscissa : 100 ms / division ; vertical scale : 70% of normalized activity ( 10% per division ) .
the putative human homologue of monkey v6a is presumably located in the dorsal part of the parieto - occipital sulcus ( pos ) , anterior to the human v6 ( pitzalis et al . , 2006 ; cavina - pratesi et al . ,
2010 ) . in that region , modulations of activity by the direction of gaze ( law et al . , 1998 ; williams and smith , 2010 ) , but also by eyes ' vergence have been reported ( quinlan and culham , 2007 ) . in the latter study ,
the authors found higher activity in the dorsal pos while the subjects were viewing moving or stationary stimuli located in near space , compared to the intermediate or far space .
it should be noted that the intermediate spatial location was within the limits of reachable space , whereas the far location was outside of reachable space .
fixating small leds placed at the same range of distances ( near , intermediate and far ) resulted in the same pattern of modulation in dpos .
given the consistency of their results , quinlan and culham ( 2007 ) attributed them to the vergence of the eyes .
their findings are in line with our neurophysiological data from monkey v6a and argue strongly for the encoding of 3d space by early visuomotor areas in pos , in both human and non - human primates .
a subsequent question we examined was the influence of depth information on the reaching activity .
to this end , monkeys were trained to fixate first and then reach the targets of the same experimental set up where the fixation task was performed ( figure 2a ) . figure 3
this neuron displayed a modulation by depth that started from movement planning and was present also during movement execution and holding of the target epochs .
in addition , during movement and target holding the cell showed a preference for central and ipsilateral targets . in most of the neurons recorded
, there was an effect of both depth and direction signals in at least one motor epoch , a finding similar to the combined influence of vergence and version signals found in the fixation task ( breveglieri et al . , 2012 ) .
these findings suggest the presence of depth information in v6a not only for the purpose of target localization , but also for movement control ( hadjidimitrakis et al . , 2011a ) .
example of a v6a neuron modulated by depth and direction signals during several epochs of a reaching - in - depth task .
spike histograms and eye traces were aligned twice , at the fixation onset and at the start of movement .
this cell prefers far space during movement planning , execution and holding of the target epochs . in the last two epochs
bin size for spike histograms ; 20 ms ; scale for version and vergence traces is 100 and 20 , respectively .
few studies have so far documented the effect of eye vergence signals in the cortex of non - human primates .
sakata et al . ( 1980 ) found in the ipl area pg - pfg cells that were modulated by both gaze direction and depth of fixation , while fewer cells were influenced by only one of these factors .
rosenbluth and allman ( 2002 ) found that a significant number of neurons ( 3050% ) in visual areas v1 , v2 , v4 were influenced by either gaze direction , depth of fixation , or their interaction , though they did not report the number of cells affected by both signals .
these areas , directly connected to v6a ( see also figure 1b ) may be sources of vergence information to area v6a , which receives also input from the posterior parietal areas lip and pg ( gamberini et al . , 2009 ) , where vergence angle has been reported to have an effect on presaccadic and fixation activities , respectively ( sakata et al . , 1980 ; genovesio and ferraina , 2004 ) .
other possible sources of vergence input are the medial superior temporal area ( mst ) and the frontal eye fields ( fef ) .
, 1998 ; gamlin and yoon , 2000 ; akao et al . , 2005 ) and send efferent inputs to v6a ( gamberini et al . , 2009 ; passarelli et al . ,
in addition , modulations of neural activity by vergence have been found in the vip ( colby et al . , 1993 ) and in visual areas v2 and v4 ( dobbins et al . , 1998 ;
rosenbluth and allman , 2002 ) , all of them directly connected with v6a , as also summarized in figure 1b . the two extrastriate areas , in particular , send strong inputs to the ventral part of v6a ( passarelli et al . , 2011 )
the space within arm 's reach is also termed peripersonal space ( previc , 1998 ) .
several lines of evidence from monkey neurophysiology seem to support the view that the encoding of peripersonal space is being processed in ppc , and more in particular in spl .
for instance , neurons with multimodal ( e.g. , visual - tactile ) receptive fields ( rfs ) have been reported in several ppc areas ( colby et al . , 1993 ; avillac et al . ,
overrepresentation of body parts ( e.g. , face or hand ) ( colby et al . , 1993 ; breveglieri et al . , 2002 ; graziano and cooke , 2006 ) that are behaviorally important has been found in several spl areas and these representations show adaptation after training or are modified depending on the task performed ( graziano et al . , 2000 ) .
in addition , representational biases in the encoding of visual space could be attributed to the natural position of the effectors in space . in this context , areas of the dorsal visual pathway
show a preferential encoding of the lower visual field where the hands are usually located ( previc , 1998 ) .
area v6a belongs to the dorsal pathway and the vast majority of v6a neurons have their visual rfs in the lower contralateral visual field ( galletti et al .
in addition , many v6a neurons have somatosensory rfs centered on the arm ( breveglieri et al . , 2002 ) and even more show spatially tuned responses during reaches in 3d space ( fattori et al . ,
recently , the orientation of the hand and the type of hand grip were found to affect the activity of v6a cells during reach - to - grasp movements ( fattori et al . , 2009 , 2010 ) .
the hypothesis that spl could process specific information related to the peripersonal space prompted us to study ( hadjidimitrakis et al .
, 2011b ) whether there is a preferential encoding of that part of space compared to the extrapersonal space in v6a .
to test this , we studied the oculomotor and fixation responses of v6a neurons to targets presented at near and far parts of space .
, 2009 , 2011 ) with small led targets arranged in a horizontal board below eye level , in order to simulate the natural perimanual space .
the monkeys were trained to direct their gaze to the led targets located in different positions in the 3d space without performing any arm movement ( figure 4a ) .
ten targets were divided into two rows , one located centrally , in the mid - sagittal plane , and the other located contralateral to the recording site , in a parasagittal plane ( figure 4a ) . in several neurons where isolation was held constant for longer periods ,
the horizontal board was translated so the targets were distributed between the central row and a parasagittal row in the ipsilateral space . even though no reaches were performed by the animals
, we checked that the two nearest led targets in each row were within reaching distance ( < 30 cm ) .
( a ) scheme of the set up used for the fixation - in - depth task below eye level .
the leds are depicted with different colors according to their distance from a frontal plane passing from monkey 's eyes .
( b ) example of a v6a neuron modulated by vergence during fixation below eye level .
top / middle / bottom : neural responses ( peristimulus time histograms ) and eye traces to the five leds of the contralateral / central / ipsilateral space arranged from near ( left ) to far ( right ) , aligned at the end of the saccade .
( c ) frequency histogram of the positions that neurons preferred in perisaccadic ( n = 91 ) and fixation ( n = 167 ) epochs .
( d ) population activity for each target position , illustrated with a different color , of v6a neurons with activity modulated during perisaccadic ( left ) and fixation ( right ) epochs .
neural activity was quantified in a time epoch around the saccadic eye movement ( perisaccadic activity ) and during the subsequent fixation period ( fixation activity ) .
a one - way anova test performed independently in each row showed , on average , a significant effect ( p < 0.05 ) of the depth of fixation in perisaccadic and fixation epochs in about 30 and 40% of v6a cells , respectively . about 15% of the cells were modulated by the depth of fixation in both epochs .
figure 4b shows the firing activity of a neuron with a tonic pattern during fixation .
the greatest activity was evoked while the eyes were fixed on the nearest target , irrespectively of gaze angle ( figure 4b ) .
in addition , at constant fixation depth the responses showed an increasing trend from contralateral to ipsilateral space ( figure 4b ) .
thus , the neuron was significantly modulated by the depth of fixation and displayed monotonic tuning for near and ipsilateral space ( anova , p < 0.05 ) .
we applied a bonferroni post hoc test on the anova significant cells to define the preferred position in depth in each part of space .
perisaccadic activity ( figure 4c , left ) , was typically higher for saccades to the two nearest targets : 6075% of the cells preferred one of these leds in the different rows .
we compared the incidence of preference for the two nearest ( reachable ) targets with the three that were outside the peripersonal , reachable space .
the preference for reachable locations was highly significant ( test , two nearest leds against the three farthest ones , p < 0.0001 ) . in neurons with modulation during fixation , there was also a strong preference for the peripersonal space ( figure 4c , right ) .
their percentage ranged from 65 to 74% depending on the different parts of space tested : 74% ( 45/61 ) for straight - ahead , 73% ( 56/77 ) for contralateral and 65% ( 19/29 ) for ipsilateral locations .
the preference for fixating targets placed within the reachable space with respect to those positioned outside was again highly significant ( test , two nearest leds against the three farthest ones , p < 0.0001 ) .
it was also demonstrated that the fixation distance of 45 cm ( third led in each row ) , which was outside the monkey 's reachable space , was the least represented in our cell population ( figure 4c , right ) .
this gap zone of fixation preference , between the reachable and unreachable space , could serve to signal targets that are definitely within or beyond reach .
overall , at the population level perisaccadic and fixation activity of v6a neurons showed a strong bias for representing the 3d space within reaching distance ( figure 4d ) .
our study showed many similarities with a human functional imaging study performed by gallivan et al .
they used an experimental setup similar to ours with objects being placed in the perimanual space , below eye level .
the authors reported that passive viewing of reachable objects evoked higher activity compared to objects located in non - reachable space in the superior part of parieto - occipital cortex ( spoc ) .
the spoc , which was also activated during arm reaching movements ( cavina - pratesi et al . , 2010 ) , includes the cortex anterior to the human homologue of v6 ( pitzalis et al . , 2006 ) ,
objects within reachable space evoked stronger activation in spoc , and it was suggested that this modulation could be related to the objects ' reachability .
the authors proposed that a reachability signal could be extracted in spoc by the combination of gaze and object depth signals .
given the response properties of cells modulated by depth described in our study ( hadjidimitrakis et al .
, 2011b ) , it is likely that both human spoc and macaque v6a encode the difference in depth between current fixation and location of objects to be grasped . in this context , the abundance of strong tuning for near space observed could be a result of an adaptive cognitive mechanism that satisfies two conditions : the natural tendency for fixating far and the necessity to respond to behaviorally relevant stimuli appearing in the near space .
( 2009 ) suggest that in v6a visual , eye position and motor related information is integrated to encode more cognitive variables such as motor affordance and/or potential actions ( andersen and cui , 2009 ; cisek and kalaska , 2010 ; macaluso and maravita , 2010 ) . in line with this
( 2011 ) that activation in spoc region was found to be correlated with the hand dominance , with the later being crucial in defining the subjects ' typical reachable space .
as described in the previous section , neural discharges during the presentation and ocular fixation of targets can be an important cue to estimate whether they are reachable or not .
but what happens when an arm reaching movement is actually performed ? does the activity during arm movement show the same spatial tuning with that of the visual fixation - related discharges ? it has been suggested that ppc neurons integrate spatially consistent retinal , eye and hand information into a global tuning field , gtf ( mascaro et al . , 2003 ) .
this type of common tuning of different effectors could be advantageous for the control of eye - hand coordination . according to caminiti and colleagues
, optic ataxia could be the result of the breakdown of gtf in spl ( battaglia - mayer and caminiti , 2002 ) .
evidence for neurons with consistent spatial tuning between eye position , arm movement and position signals has been reported in the spl ( battaglia - mayer et al . , 2001 ) .
in that study , in a center - out reaching task about 60% of the neurons showed gtf in several epochs and across various oculomanual tasks .
the same group found in area 7a a much lower incidence ( ~25% ) of cells with gtf ( battaglia - mayer et al . , 2005 ) .
we addressed this issue in another study of our group ( fattori et al . , 2005 ) , where monkeys were trained to perform 3d reaching movements toward foveated led targets arranged on a plane ( figure 5a ) .
the monkeys were required to fixate the targets and wait for a go cue signal ( led color change ) to perform a body - out reach toward the led and hold it for a variable time , till another cue ( switching off of the led ) signaled the monkey to return to the initial hand position .
many neurons in v6a were spatially tuned ( one - way anova , p < 0.05 ) , both during the outward movement execution ( figure 5b , raw m1 ) and in the holding of target period ( figure 5b , raw hold ) .
modulations in these two periods often coincided and in many cases they were spatially consistent .
figure 5c shows an example of this pattern of activity in a neuron that fired strongly not only when the hand moved , but also when it held targets located on the right .
in addition , this neuron showed a preference for right space in the fixation period , i.e. , it displayed a pattern of activity similar to the cells with gtf . to test whether the tuning of arm - related activity could be explained by gain modulation by eye position ,
an anova ( p < 0.05 ) then was performed to detect significant effects of target position on the residual firing activity .
the substraction of fixation activity abolished the spatial tuning in about one - third of v6a neurons in both movement and holding periods ( figure 5b , proper m1 , proper hold ) , whereas about 40% of the v6a maintained their spatial tuning . in line with our data
are those obtained in area 7a in ipl ( heider et al . , 2010 ) .
the authors , using a body - out reaching task very similar to ours , found that about 60% of the neurons changed their directional tuning from eye fixation till preparation and movement execution ( heider et al . , 2010 ) .
both their results and ours suggest that global tuning is probably implemented in ppc , but in a much more limited extent with respect to the one originally reported ( battaglia - mayer et al . , 2001 ) .
( a ) experimental set - up and time course of a frontal reaching task .
reaching movements were performed in darkness , from a home - button ( black rectangle ) toward one out of nine targets ( open circles ) located on a panel in front of the animal .
columns indicate the percentages of spatially modulated v6a cells during outward reaching movements ( m1 ) , and static position of the arm in the peripersonal space ( hold ) .
neural activity and eye - traces have been aligned twice in each inset , with the onsets of outward ( 1st ) and inward ( 2nd ) reach movements .
the mean duration of epochs fix , m1 , hold , m2 is indicated in the bottom left inset .
behavioral markers on rasters from left to right : led color change , outward movement onset , outward movement end , led offset , inward movement onset , inward movement end , end of trial .
bin size in peri - event time histograms : 15 ms ; eye traces : scale bar , 60. other details as in figure 2 .
regardless of its incidence in the ppc , the concept of gtf has some interesting cognitive aspects . in everyday life and under certain conditions ,
when we want to reach and/or grasp an object , the eyes fixate on the target first and then the hand starts to move ( land et al .
, the common spatial preference for eye and arm could be the result of a learned association between eye position and arm movement signals .
learning could be the result of many repetitions of combinations of eye and arm positions in space .
the spatial coincidence would reinforce the response of neurons that are tuned ( ahissar et al .
, 1992 ; rosenbluth and allman , 2002 ) . as a result , in these neurons , whenever the eyes move to a certain location , a plan that would carry the arm in the same location is formed . in this way
, eye position signals can predict the sensory consequences of future motor actions ( land and furneaux , 1997 ) .
apart from its crucial role in spatial perception and visually guided motor action , experimental evidence accumulated over the past decade suggests that ppc is also involved in non - spatial , higher order cognitive functions .
such functions include attention , categorization , reward , working memory , encoding of task rules , and behavioral context ( for references see culham and kanwisher , 2001 ; husain and nachev , 2007 ; gottlieb and snyder , 2010 ) .
most studies of cognitive processes in monkeys and humans have found the neural substrate of these functions to be located in areas of the ipl and this created a view of a gradient of increasing non - spatial , cognitive processing going from spl to ipl ( husain and nachev , 2007 ) . here , without claiming the opposite to this view , we focus on evidence that relates the spl activity to cognitive processing . yantis and colleagues identified a region in the medial spl of humans that was transiently activated during shifts of spatial attention ( yantis et al . , 2002 ) . in that study , subjects were presented with two stimulus streams of numerical cues at opposite peripheral locations ( left - right ) . their task was to attend one of the two streams for the presentation of a cue that signaled them , either to shift their attention to the unattended stream , or to maintain it on the attended one .
the authors found that a region in the medial spl showed a transient activation during the shift of attention , irrespectively of the direction of the shift .
this area is likely homologous to macaque v6a , where modulations during covert shifts of attention have been reported ( galletti et al . , 2010 ) .
furthermore , in subsequent studies , the same human medial spl region was found to exhibit transient activity in a variety of conditions : during non - spatial shifts of attention between objects , features and working memory representations , and during shifts between visual features that represented different categorization rules ( chiu and yantis , 2009 ; esterman et al . , 2009 ) .
these findings suggest that spl activity is related to the encoding of the change in task demands . in line with this assumption
( 2010 ) observed that the perceptual switch rate between bistable structure - from - motion stimuli in humans was correlated with macroscopic anatomical features of an spl area that mostly corresponded with the region identified by yantis and colleagues .
( 2009 ) found that humans with lesions in spl displayed deficits in working memory tests that required the manipulation and rearrangement of working memory , but not the simple retrieval of it .
the authors found that the manipulation in the working memory was impaired not only for visuospatial stimuli , but also for verbal - auditory ones .
furthermore , their findings are consistent with a meta - analysis of several neuroimaging studies that highlighted the involvement of spl in the updating of the working memory ( wager and smith , 2003 ) .
finally , activity in spl has been linked with mental navigation ( ino et al . , 2002 ) and visuomotor learning ( inoue et al . , 2000 ) . in summary
, the above studies strongly suggest that beyond its role in goal directed motor behavior , the cortex of spl is also involved in processing cognitive information to enable the perception of a continuously changing environment .
in this review we have presented evidence that the cortex of the spl , in particular area v6a processes signals related to eye position in depth .
we showed that these signals can be used in simple spatial computations like the encoding of the location of visual targets in 3d space , but also in more cognitive ones , like the encoding of peripersonal space in terms of reachability by the arm .
in addition , we reviewed studies in humans that suggest the involvement of spl in other cognitive processes . overall , these findings rather than challenging the classic view that spl play a role as an interface between sensory information and motor action , highlight the fact that this interface shows a remarkable flexibility and is influenced by attention , learning , and other cognitive factors .
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 .
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 . | research conducted over the last decades has established that the medial part of posterior parietal cortex ( ppc ) is crucial for controlling visually guided actions in human and non - human primates . within this cortical sector
there is area v6a , a crucial node of the parietofrontal network involved in arm movement control in both monkeys and humans .
however , the encoding of action - in - depth by v6a cells had been not studied till recently .
recent neurophysiological studies show the existence in v6a neurons of signals related to the distance of targets from the eyes .
these signals are integrated , often at the level of single cells , with information about the direction of gaze , thus encoding spatial location in 3d space .
moreover , 3d eye position signals seem to be further exploited at two additional levels of neural processing : ( a ) in determining whether targets are located in the peripersonal space or not , and ( b ) in shaping the spatial tuning of arm movement related activity toward reachable targets .
these findings are in line with studies in putative homolog regions in humans and together point to a role of medial ppc in encoding both the vergence angle of the eyes and peripersonal space .
besides its role in spatial encoding also in depth , several findings demonstrate the involvement of this cortical sector in non - spatial processes . |
the function of the metalloenzyme sod1 is to convert superoxide , a toxic by - product of mitochondrial oxidative phosphorylation , to water or hydrogen peroxide .
however , alteration in wild type sod1 expression or mutations in the gene have been held responsible for the activation of catabolic pathways associated with degenerative diseases , including amyotrophic lateral sclerosis ( als ) .
als is a disorder involving the degeneration of motor neurons , muscle atrophy , and paralysis . in few familiar forms of als , mutations in sod1 gene
initially it has been suggested that mutation in sod1 gene led to a decrease in the protein enzymatic activity ( loss of function hypothesis ) .
however , subsequent studies have clarified that mutant sod1 possesses a neurotoxic property ( gain of function hypothesis ) responsible for the pathogenic mechanism of the disease . indeed , the finding that overexpression of mutant sod1 in transgenic mice recapitulates several clinical features of als disease even in the presence of endogenous mouse sod1 has led to the conclusion that the disease results from a toxic gain of function .
mutations in sod1 that impair its functions may lead to increased oxidative damage , promoting the activation of apoptotic pathways .
the effects of the reaction oxidative species ( ros ) are dose - dependent , and low ros concentration is necessary to guarantee cellular homeostasis while high ros dose exerts toxic effects on the cells and may contribute to cellular dysfunction . indeed , oxidative stress is a hallmark of aging and several chronic diseases such as alzheimer 's disease , duchenne dystrophy , and als .
how such an oxidative insult plays a direct role in the disease - related decrease of muscle performance and mass remains largely unknown .
in addition , the discrepancy among different studies has further complicated the achievement of a conclusive link between altered balance of ros generation and altered homeostasis - associated diseases . in a previous work we demonstrated that muscle specific expression of the mutant isoform of sod1 gene ( sod1 ) induces muscle
atrophy associated with a significant reduction in muscle strength and alterations in the contractile apparatus .
we provided evidences that muscle - restricted expression of sod1 gene is sufficient to increase oxidative stress and to induce a reduction in protein synthesis and the activation of proteolytic pathway .
it has been demonstrated that lactate - induced oxidative stress delays c2c12 differentiation while treatment of the same cell line with resveratrol , that confers resistance against oxidative stress , promotes myogenesis and hypertrophy .
interestingly , high glucose - induced oxidative stress has been correlated with lipid deposition in muscle derived stem cells leading to their adipogenic differentiation . in this study , we address the role of the toxic effect of mutant sod1 gene ( sod1 ) on in vitro myogenic program and we demonstrate that sod1 expression prevents myoblasts differentiation and retains c2c12 cells in an undifferentiated state that show features common to fibro / adipogenic cells .
c2c12 cells were stably transfected with ppuro and pmexmlc / sod1 plasmids ( ratio 1 : 10 ) by using superfect transfection reagent ( qiagen ) according to the manufacturer 's instructions , as control c2c12 cells were also transfected with ppuro and pmex empty vector . after 1 day from transfection
the medium was replaced with fresh medium containing puromycin 3 g / ml ( sigma aldrich ) .
after 48 hours the cells were split 1 : 10 into the selective medium .
c2c12 and c2c12 mlc / sod1 cells were maintained in dulbecco 's modified eagle 's medium ( dmem ) with 4.5 g / l glucose , l - glutamine ( sigma ) , supplemented with 15% fetal bovine serum ( gibco ) , 100 u / ml penicillin ( sigma aldrich ) , 100 g / ml streptomycin ( sigma aldrich ) , and 2,5 g / ml of puromycin ( sigma aldrich ) for c2c12 mlc / sod1 cells . to induce differentiation ,
cells were shifted to differentiation medium ( dm ) , dmem with 2% horse serum ( gibco ) .
cells were harvested at d0 , d2 ( 48 h after shift in dm ) , and d5 ( 120 h after shift in dm ) .
c2c12 and c2c12 mlc / sod1 cells grown in 60 mm culture dishes were washed twice with cold phosphate - buffered saline , pelleted , resuspended in 100 ul of modified lysis buffer ( tris - hcl , ph 7.5/20 mm , edta/2 mm , egta/2 mm , sucrose/250 mm , dtt/5 mm , triton - x/0.1% , pmsf/1 mm , naf/10 mm , sov4/0.2 mm , and cocktail protease inhibitors/1x ( sigma aldrich ) ) , and processed for western blot analysis .
filters were blotted with antibodies against hsod1 ( santa cruz ) , gp91phox ( bd ) anti - perilipin 2 ( lifespan biosciences ) , phospho - p42/p44 mapk ( millipore ) , p42/p44 mapk ( cell signaling ) phospho - akt ( thr 308 ) ( sigma aldrich ) , akt ( cell signaling ) , phospho - p70 ( thr389 ) ( cell signaling ) , p70 ( cell signaling ) , and hdac4 ( cell signaling ) .
signals were acquired by chemidoc - it imaging system ( uvp , llc ) and the analysis was performed using visionworks ls image acquisition analysis software .
total rna extraction was performed using trizol reagent method ( sigma aldrich ) as described by the manufacturers .
microrna ( mirna ) was reverse - transcribed using the taqman microrna reverse transcription kit ( life technologies ) , and mrna was reverse - transcribed using quantitect reverse transcription kit ( qiagen ) .
quantitative pcr was performed on an abi prism 7500 sds ( life technologies ) , using premade 6-carboxyfluorescein- ( fam- ) labeled taqman assays for beta actin , pax7 , pax3 myod , myogenin , mrf4 , and smarcd3 ( life technologies ) .
fam - labeled taqman microrna assays for mir1 , mir133a , mir206 , and u6 snrna ( applied biosystems , usa ) were performed as described .
quantitative rt - pcr sample values were normalized to the expression of beta - actin or u6 snrna for mrna and microrna , respectively .
the relative level for each gene and mirna was calculated using the 2-ddct method and reported as mean fold change in gene expression .
cells were fixed in 4% paraformaldehyde for 1 h. after being washed with ddh2o , cells were treated with 100% propylene glycol for 5 min and stained with a filtered oil red o solution ( 0.5% oil red o in propylene glycol ) for 8 min at 60c .
the cells were treated with 85% propylene glycol solution for 5 min , washed twice with ddh2o , and mounted with glycerol .
samples were visualized using an inverted microscope ( axioskop 2 plus ; carl zeiss microimaging inc . ) .
c2c12 and c2c12 mlc / sod1 cells were exposed to 100 nm ( sigma aldrich ) trichostatin a ( tsa ) in gm ( growth medium ) for 24 h. tsa was removed and the cells were analyzed for myosin expression ( at day 5 in dm ) or by cytofluorimetric profile ( at day 2 in dm ) .
cells were fixed in 4% paraformaldehyde and incubated overnight at 4c with primary antibody against mhc ( mf-20 hybridoma bank ) ; nuclei were visualized using hoechst staining .
samples were viewed under an inverted microscope ( axioskop 2 plus ; carl zeiss microimaging inc . ) . to quantify the differentiation and fusion of control and tsa treated we calculated the differentiation index as the percentage of mhc - positive cells above total nuclei and the fusion index as the average number of nuclei in mhc - positive cells with at least three nuclei above total number of nuclei , respectively .
c2c12 mlc / sod1 were detached from culture with 0.25% trypsin , 2 mm edta ( sigma aldrich ) at indicated times .
the isolated cells were then filtered through a 40 m cell strainer ( falcon ) and incubated with the following antibodies ( 10 ng / ml ) : cd31-pecy7 , cd45-efluor 450 ( ebioscience ) , sca-1-fitc ( macs ) , and 7 integrin - pe ( r&d systems inc . ) . a subsequent incubation with 7-aminoactinomycin d ( 1/1000 ; sigma )
, data were collected from 10,000 cells and analyzed on cyan adp ( dako ) flow cytometer using the summit 4.3 software ( dako ) .
groups were compared using nonparametric tests ( mann whitney rank sum test ) and student 's t - test . a p value of < 0.05 was considered statistically significant .
to investigate the role of mutant sod1 gene in myoblast differentiation we stably transfected the c2c12 cells line with the mlc / sod1 expression cassette ( figure 1(a ) ) that allows the expression of the mutated isoform of sod1 gene under the control of the myosin light chain promoter .
as expected , sod1 transgene expression was accumulated in c2c12 mlc / sod1 cells induced to differentiate ( figure 1(b ) ) .
the postmitotic expression of sod1 transgene induced the accumulation of gp91 protein , a marker of mitochondria oxidative damage ( figure 1(c ) ) that might mediate the toxic properties of mutant sod1 on muscle differentiation and homeostasis . to explore if sod1 transgene expression directly interferes with muscle differentiation , we stimulated differentiation by shifting the cells from growth to the differentiation medium ( dm ) ( figure 2(a ) ) .
morphological and immunofluorescence analysis revealed a dramatic inhibition of muscle differentiation in c2c12 mlc / sod1 cells , with significant reduction in the number and size of the myosin positive cells , compared with control c2c12 myotubes ( figures 2(a)-2(b ) ) .
morphometric analysis revealed a complete inhibition of fusion index , quantified as the percentage of hoechst - stained nuclei located within multinucleated cells , positive to sarcomeric myosin ( figure 2(b ) ) .
the altered differentiated muscle phenotype was also confirmed by western blot analysis , revealing a drastic downmodulation of the sarcomeric myosin heavy chain expression , a specific marker of myogenic differentiation ( figure 2(c ) ) . of note
, c2c12 cells stably transfected with wild type sod1 cassette ( mlc / sod1 ) did not show any morphological differences compared to control c2c12 cell lines ( data not shown ) . a key myogenic factor that triggers myoblast differentiation is myod [ 12 , 13 ] , which resulted in significant downregulation throughout the time course of differentiation in c2c12 mlc / sod1 cells ( figure 3(a ) ) .
myogenin is the myogenic factor that functions downstream of myod and plays a critical role in triggering terminal differentiation process of myoblasts [ 12 , 14 ] .
myogenin expression resulted in significant downregulation in c2c12 mlc / sod1 cells during differentiation ( figure 3(b ) ) .
the final stage of skeletal muscle differentiation and maturation program is also dependent on the concerted action of another myogenic factors , namely , mrf4 , which promotes the activation of myosin heavy chain expression .
real time pcr analysis revealed a significant reduction of mrf4 transcripts in c2c12 mlc / sod1 cells during differentiation , compared to control c2c12 myotubes ( figure 3(c ) ) .
recent works have shown that among genes which are important for proper muscle differentiation and function , micrornas ( mirnas ) play a crucial role [ 1517 ] . among them mir133 , mir206 , and mir1 are abundantly expressed in muscle tissue and specifically induced during myogenesis and c2c12 differentiation .
it has been reported that mir1 and mir133 are involved in a complex molecular mechanism by which mir1 induces the downmodulation of an inhibitor of muscle differentiation , namely , histone deacetylase ( hdac ) 4 and mir133 , which is clustered on the same chromosomal loci of mir1 and enhances myoblast proliferation inhibiting the serum response factor ( srf ) .
in addition , mir206 facilitates satellite cell differentiation [ 20 , 21 ] by restricting its proliferative potential through the repression of pax-7 expression .
these findings implicate these myomirnas in a complex regulatory loop to control cell proliferation , commitment , and differentiation .
real time pcr analysis revealed a significant downmodulation of mir133 , mir206 , and mir1 during the differentiation time course of c2c12 mlc / sod1 cells compared to control c2c12 cells ( figures 3(d ) , 3(e ) , and 3(f ) ) .
the activation of a specific developmental program requires the integration of multiple extrinsic signals from the cell membrane that culminate in changes of nuclear gene expression patterns . among the known signal transduction intermediates in muscle cells ,
the serine / threonine kinase akt and the mitogen - activated protein kinases ( mapk ) have been shown to modulate myogenic differentiation . in a previous study
, we have demonstrated that muscle specific expression of sod1 gene in transgenic animals promotes a reduction of the phosphatidylinositol 3-kinase ( pi3k)/akt pathway and leads to muscle atrophy . in this study
, we explored whether the inhibitory effects of mutant sod1 gene on myogenic program perturb the relevant signaling pathways of the myogenic program .
figure 4 shows that the absolute ratio of pakt / akt was negatively regulated ( figure 4(a ) ) and pp70/p70 , the downstream effector of akt , was significantly reduced in c2c12 mlc / sod1 cultures compared with control c2c12 cells ( figure 4(b ) ) .
in addition we observed a significant downmodulation of the phosphorylated active form of a factor associated with mapk differentiation cascade , namely , erk1/2 ( figure 4(c ) ) .
overall these results demonstrate that the postmitotic expression of sod1 mutant gene prevents c2c12 differentiation , affecting the activation of the muscle regulatory factors , muscle mirna , and the signal transduction cascades responsible for myogenic differentiation , and might impinge the maintenance of the muscle phenotype . to validate this hypothesis
, we analyzed the expression of pax-7 , which is a key factor that triggers the specification of uncommitted skeletal muscle progenitors to myogenic cells .
of note , we observed a complete inhibition of pax-7 expression in c2c12 mlc / sod1 cells compared to c2c12 myoblasts , suggesting that sod1 expression not only inhibits muscle differentiation but also confers to transfected cells an immature state ( figure 5(a ) ) .
recently , it has been demonstrated that satellite cells in adult muscle are bipotential stem cells that can give rise to brown adipogenic as well as myogenic progenitors .
the lineage switch between myogenic and brown adipogenic commitment is controlled by the muscle specific mir133a , which is highly expressed in satellite cells and can repress the expression of adipogenic markers to enforce myogenic commitment in satellite cells .
further , recent evidences revealed that pax-3 transcription factor , whose ectopic expression in c2c12 myoblasts efficiently inhibits myogenic specification , plays a pivotal role during differentiation into adipocytes cells of human - induced pluripotent stem cells .
in addition , the exclusive expression of pax-3 or myod gene in stem cells allows a clear and distinct choice between myogenic and fibro / adipogenic potential cell lineage .
based on these evidences , we performed real time pcr and histochemical analysis in both c2c12 and c2c12 mlc / sod1 cells to evaluate the potential adipogenic features in c2c12 mlc / sod1 transfected cell lines .
as shown in figure 5(b ) the levels of pax-3 transcript were significantly upregulated in c2c12 mlc / sod1 during all stages in culture , compared to c2c12 .
moreover oil red o staining revealed the accumulation of intracellular lipid droplets in c2c12 mlc / sod1 cells ( figure 5(c ) ) .
these data were corroborated by western blot analysis for perilipin 2 ( plin2 ) , a marker of fatty acids uptake and storage .
western blot analysis revealed that plin2 protein was accumulated in c2c12 mlc / sod1 during the differentiation process compared to control cell line ( figure 5(d ) ) .
all these results together with the data of mir133a and mrfs downmodulation in c2c12 mlc / sod1 ( figure 3 ) suggest that muscle specific expression of sod1 mutant gene inhibits myoblasts differentiation and promotes adipogenic features in c2c12 cells through a mir133a and pax-3 dependent mechanism .
fibro - adipogenic progenitors ( faps ) are multipotent mesenchymal cells residing in skeletal muscle interstitium [ 3032 ] .
these cells are negative for cd31 , cd45 , and 7 integrin surface antigens and are characterized by the expression of the stem cell antigen 1 ( sca1 ) .
faps convert environmental cues into signals that modulate muscle regeneration or turn themselves into fibro - adipocytes , inducing fat deposition and fibrosis under pathologic conditions , such as dystrophic muscles . to verify whether postmitotic expression of sod1 mutant gene promotes a faps phenotype in c2c12 cells , we performed facs analysis ( figure 6(a ) ) for sca1 , cd31 , cd45 , and 7 integrin .
c2c12 and c2c12 mlc / sod1 cells were negative for cd31 and cd45 antigens ( data not shown ) .
c2c12 were mainly 7 integrin and a low percentage of them was sca1 and 7 integrin ( figures 6(a)6(c ) ) .
in contrast , c2c12 mlc / sod1 were mainly sca1 and 7 integrin , with a significant reduction in the number of 7 integrin cells ( figures 6(a)6(c ) ) .
these results clearly evidenced the presence of fibro / adipogenic features in c2c12 mlc / sod1 . the molecular mechanism that , in concert with environmental cues , controls
the identity and activity of fap cells involves a hdac - regulated network ; this network consists of muscle specific mirnas that target two alternative variants of the swi / snf chromatin remodeling complex , baf60a and baf60b , and favor the formation of a baf60c - based swi / snf complex able to confer on myod the ability to activate the myogenic program .
we analyzed the protein and transcript levels , respectively , of hdac4 and baf60c in both c2c12 and c2c12 mlc / sod1 cells .
we observed that hdac4 was significantly upregulated in cultures of c2c12 mlc / sod1 cells compared to control differentiated ( day 5 in dm ) c2c12 cells ( figure 7(a ) ) .
in contrast , baf60c was significantly downregulated in c2c12 mlc / sod1 cells ( figure 7(b ) ) .
to better correlate the induction of faps phenotype and hdac activity with sod1 expression and toxic properties , we treated c2c12 mlc / sod1 cells with the hdac class ii inhibitor trichostatin ( tsa ) and analyzed the signature profile of both faps and myogenic cells .
cytofluorimetric profile revealed that tsa treatment induced a significant downmodulation of sca1 and 7 integrin cells ( figure 7(c ) ) and an increased percentage of double positive sca1 and 7 integrin cells ( figure 7(d ) ) .
of note we did not observe any significant difference in the number of 7 integrin sca1 cells after treatment ( data not shown ) suggesting that tsa treatment is able to counteract the establishment of a fap phenotype but it only partially rescued the muscle phenotype . to support this evidence , we induced muscle differentiation in c2c12 mlc / sod1 cells treated with tsa and we revealed that inhibition of hdac activity partially rescued myogenic differentiation , promoting myoblast fusion and differentiation ( figures 7(e)-7(f ) ) .
overall these data suggest that the postmitotic expression sod1 mutant gene promotes a faps phenotype in c2c12 cells , by upregulating hdac4 protein and preventing the baf60c - swi / snf complex myogenic commitment .
in this work we defined the specific toxic effects of postmitotic expression of mutant sod1 gene on the myogenic program , demonstrating that mutant sod1 alters the identity of muscle cells , affects cell homeostasis , and inhibits muscle differentiation .
the physiological activity of the metalloenzyme sod1 is to detoxify the cells from the accumulation of free radicals , converting superoxide , a toxic by - product of mitochondrial oxidative phosphorylation , to water or hydrogen peroxide .
in contrast , the mutant sod1 possesses a toxic property that is responsible for the pathogenic mechanism of als , a neurodegenerative disease associated with the degeneration of motor neurons , muscle atrophy , and paralysis .
different studies also support the evidence that skeletal muscle is a primary target of mutant sod1 toxicity in mice [ 5 , 35 ] , indicating that dysfunctions of affected muscle cells are not only a marginal consequence of denervation associated with motor neurons loss , but a direct consequence of cell muscle toxicity of mutant sod1 .
the understanding of the mechanisms involved in mutant sod1 toxicity in muscle may facilitate the design of treatments directed toward this specific tissue to treat als or at least to delay disease progression .
the aim of our work was to define the responses of myogenic cells to the toxic effects of sod1 and the signaling pathways that mediated the toxic properties of mutant sod1 product .
to this purpose , we generated a stable transfected c2c12 cell line , overexpressing the mutated isoform of the sod1 gene under the control of the mlc promoter [ 11 , 37 ] .
postmitotic expression of sod1 gene induced an excess of oxidative stress , as evidenced by the increased expression of gp91 protein , and impaired muscle differentiation and fusion of c2c12 cells , inducing a significant downregulation in the expression of molecular markers of myogenic differentiation , like myod , myogenin , mrf4 , and sarcomeric myosin heavy chain .
epigenetic factors , including mirnas expression , play important role in muscle homeostasis and represent good molecular markers to define the stage of myogenic program and to monitor the effects of toxic factors on the modulation of muscle phenotype .
mir206 , mir133a , and mir1 [ 18 , 20 , 38 ] contribute to the proper development of the myogenic program , and their alteration can impair the myogenic differentiation .
based on this evidence , we investigated their expression levels in c2c12 mlc / sod1 cells and demonstrated that transfected mlc / sod1 induced a significant downmodulation of all muscle micrornas analyzed .
here we have demonstrated that the inhibitory effect of the toxic sod1 protein on myogenic differentiation acts negatively on two major signaling pathways involved in muscle differentiation , such as the akt / p70 and mapk pathways .
these results suggest that muscle expression of sod1 impinges muscle differentiation and might alter the identity of muscle cells in line with previous studies that demonstrate the impairment of myofiber - associated skeletal muscle satellite cells function in sod1-g93a mice and the altered expression of myogenic regulatory factors in the mouse model of amyotrophic lateral sclerosis .
aguiari and colleagues have shown that high glucose growth medium induces an increase in ros production and promotes the adipocyte differentiation of muscle derived stem cells . since c2c12 mlc / sod1 cells show higher levels of oxidative stress and impaired myogenic differentiation process , modulating relevant genes and myomirna of the maintenance of the muscle phenotype , we supposed a link between toxic properties of sod1 and activation of adipogenic differentiation .
in particular , we demonstrated that transfected mlc / sod1 cells show low levels of mir133a , pax-7 , and myod expression and high levels of pax-3 and perilipin 2 , a marker of lipid droplets .
our data support the evidences by yin and colleagues who showed that mir133a controls satellite cells commitment to the adipocyte lineage ; in this work the authors demonstrated that mir133a inhibition promotes proadipogenic differentiation of satellite cells and that the new - formed preadipocytes completely lose the expression of both pax-7 and myod .
moreover , mohsen - kanson et al . demonstrated that pax-3 plays a pivotal role during stem cell differentiation into adipocytes and that the exclusive expression of pax-3 or myod allows cells to choose between myogenic and fibro / adipogenic cell lineage .
our data indicate that muscle expression of sod1 induced deregulation of myogenic process , led to the impairment of myoblast differentiation , and promoted adipogenic commitment . the fibro - adipogenic precursors or faps cells are bipotent cells positive for sca1 and negative for 7 integrin , cd31 , and cd45 , markers of satellite cells , endothelial cells , and hematopoietic cells , respectively .
faps are mesenchymal cells residing in skeletal muscle interstitium and quiescent in physiological conditions and are efficiently activated to proliferate after muscle injury .
it has been shown that faps may exacerbate the dystrophic phenotype turning into fibro - adipocytes , mediating fat deposition and fibrosis and thereby disrupting the environment conducive for muscle regeneration .
interestingly , it has been demonstrated that , during ischemia - induced regeneration , oxidative stress negatively modulates myogenic differentiation and it is well known that oxidative stress is a hallmark of chronic disease including the duchenne muscle dystrophy where faps have been deeply studied . here
we demonstrate , by histochemical assay and facs analysis , the presence of lipid droplets in c2c12 mlc / sod1 cells and their significant and exclusive positivity for sca1 , demonstrating that c2c12 mlc / sod1 cells share common features with faps .
a recent study has revealed a link between hdacs , myomirs , and chromatin remodeling that underlies faps commitment to the promyogenic phenotype .
the authors demonstrated that the signaling pathway that mediates the transition from myogenic to fibro / adipogenic phenotype requires the action of mir1 , mir206 , and mir133a , which favor the incorporation of specific core - protein , baf60c , into the chromatin remodeling swi / snf complex .
moreover , the authors also demonstrated that in a murine model of duchenne dystrophy treatments with the hdacs inhibitor ( tsa ) address faps to a proper myogenic fate , blocking the fibro - adipogenic one . since hdac4 regulates faps fate and since our transfected cell line shares common features with faps , including higher levels of hdac4 protein , lower levels of baf60c , and deregulation of myomirna expression
, we hypothesized that hdac4 is responsible for c2c12 mlc / sod1 adipogenic choice . to prove our hypothesis we treated c2c12 mlc / sod1 cells with tsa and interestingly
this suggests that inhibition of hdac activity interferes with the establishment of a fap phenotype and partially rescues muscle differentiation .
three main conclusions can be drawn from the present work ; the postmitotic expression of sod1 mutant gene ( 1 ) induces the impairment of the myogenic differentiation process of c2c12 cells ; ( 2 ) triggers the myoblast towards an adipogenic phenotype ; ( 3 ) promotes faps features in c2c12 cells by epigenetic changes that involve hdacs proteins .
further studies will clarify the different molecular mechanisms that are modulated by multiple toxic effects of mutant sod1 protein in skeletal muscle and whether oxidative stress can represent a determinant for myoblasts choice toward a fibro / adipogenic fate . | to determine the role of mutant sod1 gene ( sod1g93a ) on muscle cell differentiation , we derived c2c12 muscle cell lines carrying a stably transfected sod1g93a gene under the control of a myosin light chain ( mlc ) promoter - enhancer cassette .
expression of mlc / sod1g93a in c2c12 cells resulted in dramatic inhibition of myoblast differentiation . transfected sod1g93a gene expression in postmitotic skeletal myocytes downregulated the expression of relevant markers of committed and differentiated myoblasts such as myod , myogenin , mrf4 , and the muscle specific mirna expression .
the inhibitory effects of sod1g93a gene on myogenic program perturbed akt / p70 and mapk signaling pathways which promote differentiation cascade .
of note ,
the inhibition of the myogenic program , by transfected sod1g93a gene expression , impinged also the identity of myogenic cells .
expression of mlc / sod1g93a in c2c12 myogenic cells promoted a fibro - adipogenic progenitors ( faps ) phenotype , upregulating hdac4 protein and preventing the myogenic commitment complex baf60c - swi / snf .
we thus identified potential molecular mediators of the inhibitory effects of sod1g93a on myogenic program and disclosed potential signaling , activated by sod1g93a , that affect the identity of the myogenic cell population . |
the implantation of polyether - polyurethane synthetic matrix has been used as a framework to induce fibrovascular tissue growth that has similarities with tissue formed in the physiological and pathological processes where angiogenesis , inflammation , and fibrogenesis are important concurrent processes .
this model provides a chronically inflamed environment that mimics the ones that occur after mechanical tissue injuries such as in balloon angioplasty insertion , as well as during wound healing , inflammatory diseases , and foreign body reaction .
indeed , at present , there is considerable interest in implantation techniques and scaffolds for tissue engineering and , for safety and biocompatibility reasons , inflammation , angiogenesis , and fibrosis need to be determined , as the success of the procedure is conditioned by an adequate inflammatory angiogenesis response to the device as well as by its degree of encapsulation [ 1 , 2 ] .
one of the main inflammatory mediators involved in both inflammation and angiogenesis is the nitric oxide ( no ) .
no can be synthesized by three different isoforms of no synthase : neuronal ( nnos ) , endothelial ( enos ) , and inducible ( inos ) synthases .
no production due to cytokine - induced expression of inducible nitric oxide synthase ( inos ) is largely involved in the pathophysiology of inflammation [ 46 ] .
studies conducted in inos - deficient mice and/or under inos inhibition have provided evidence that inducible no governs a broad spectrum of processes , such as recruitment and adhesion of leukocyte [ 7 , 8 ] , inflammatory diseases [ 9 , 10 ] , wound healing [ 11 , 12 ] , ischemia [ 1315 ] , and tumor - induced angiogenesis .
in addition , no - releasing compounds have proved effective in attenuating foreign body reaction to subcutaneous implant [ 17 , 18 ] .
while these studies have been decisive in demonstrating the potential activity of no in minimizing the adverse foreign body reaction , we found no study that investigated the role of endogenous inos in the regulation of the foreign body reaction induced by subcutaneous implantation of synthetic matrix in mice .
therefore , our aim was to study the effects of inos deletion on different features of the foreign body response induced by polyether - polyurethane implants in terms of inflammation , neovascularization , and fibrogenesis in the synthetic matrix .
we report that the deletion of inos was able to modulate critical features of inflammation , neovascularization , and collagen deposition on the fibrovascular tissue induced by sponge implants in mice as the absence of this enzyme leads to reduced angiogenesis and exacerbated inflammation and fibrosis in the synthetic implants .
all animal care and experimental procedures complied with the guidelines established by our local institutional animal welfare committee .
male c57bl/6 mice aged 7 - 8 weeks ( 2025 g body weight ) with genetic deletion of inos ( inos ) and wild - type ( wt ) mice were provided by dr .
leda qurcia vieira ( department of biochemistry and immunology at the institute of biological sciences , federal university of minas gerais , brazil ) .
the light / dark cycle was 12:12 h with lights on at 7:00 am and lights off at 7:00 pm .
manchester , uk ) was used as the implanted material , as described previously [ 2 , 19 , 20 ] . the implants were in the shape of discs , 5 mm thick 8 mm diameter .
they were soaked overnight in 70% v / v ethanol and sterilized by boiling in distilled water for 15 minutes before implantation .
the animals were anaesthetized with a mixture of ketamine 100 mg / kg and xylazine 10 mg / kg and the dorsal hair was shaved and the exposed skin wiped with 70% ethanol .
the sponge discs were aseptically implanted into a subcutaneous pouch , which had been made with curved artery forceps through a 1-cm long dorsal midline incision .
postoperatively , the animals were monitored for any signs of infection at the surgical site , discomfort , or distress ; any animals showing such signs were promptly euthanized with anesthetic excess .
the implants were evaluated 14 days after implantation to assess vascularization ( hemoglobin content , cytokines levels , and histological analysis ) , inflammatory markers ( mpo and nag activities and cytokines levels ) , and collagen deposition ( picrosirius - red staining ) .
the extent of vascularization of the sponge implants was assessed by the amount of hemoglobin ( hb ) detected in the tissue using the drabkin method [ 19 , 20 ] . at 14 days after implantation
, the animals were euthanized with an excess of anesthetic and the sponge implants were carefully removed , dissected , cleared of any adherent tissue , and weighed .
each implant was homogenized ( tekmar tr-10 , oh ) in 2 ml of drabkin reagent ( labtest , so paulo , brazil ) and centrifuged at 12000 g for 20 min .
the hemoglobin concentration in the samples was determined spectrophotometrically by measuring absorbance at 540 nm using an elisa plate reader and comparing it against a standard hemoglobin curve .
hemoglobin content in the implant was expressed as g of hb per mg wet tissue .
the supernatants from centrifugation of sponge homogenates ( see hemoglobin measurement method ) were used to examine the levels of vegf , cxcl1/kc , ccl2/mcp-1 , tnf- , il-10 , and ifn- produced in sponge implants by elisa .
the assays were performed using kits from r&d systems and according to the manufacturer 's instructions .
standards were 0.5-log10 dilutions of recombinant murine cytokines from 7.5 pg ml to 1000 pg ml .
the threshold of sensitivity for each cytokine / chemokine was 7.5 pg / ml .
pellets from centrifugation of sponge homogenates ( see hemoglobin measurement method ) were divided into two portions and suspended with different buffers specific for measurement of myeloperoxidase ( mpo ) or n - acetyl--d - glucosaminidase ( nag ) activities used as neutrophil and macrophage accumulation indexes , respectively , as described previously .
the sponge implants from a separate group of mice were carefully excised , dissected free of adherent tissue , and fixed in formalin ( 10% w / v in isotonic saline ) .
sections ( 5 m ) were stained with hematoxylin and eosin ( h&e ) or picrosirius - red and processed for light - microscopic studies . to perform a morphometric analysis of blood vessels , cross
section images obtained from 15 sequential fields ( 8533 m ) from each implant were captured with a plan apochromatic objective ( 40x ) in light microscopy ( final magnification = 400x ) .
a countable vessel was defined as a structure with a lumen whether or not it contained red blood cell . for collagen analysis
, images were obtained from 10 fields ( area = 343,592 m ) at 20x ( final magnification = 200x ) under polarized light ( olympus ) .
the images were digitized through an olympus bx43 with an olympus q - color 5 microcamera and morphometric analyses were performed on digital images using imageproplus 7.0 software .
additionally , to standardize the image analysis , the fields were photographed on the same day to avoid any variability associated with the light source .
statistical comparisons between two groups of mice were carried out using student 's t - test for unpaired data .
the sponge matrix was well tolerated by animals of both wild - type and knockout groups .
no signs of infection or rejection were observed in the implant location during the 14-day period of the experiment .
angiogenesis was assessed by evaluating implants hemoglobin content and blood vessels counting in h&e - stained tissue sections , besides vegf levels by elisa .
we observed reduced hemoglobin content ( figure 1(a ) ) and blood vessels count ( figures 1(b ) and 1(d ) ) in the fibrovascular tissue at day 14 after sponge implantation in inos mice when compared to the wt counterparts . in wt mice ,
the hemoglobin content ( g / mg wet tissue ) was 2.52 0.12 and , in inos mice , it fell to 1.88 0.13 ( p < 0.01 ) .
corroborating these data , the measurement of the proangiogenic cytokine vegf was also decreased in sponges from inos mice , as shown in figure 1(c ) .
altogether , these data suggest that sponge - induced angiogenesis is impaired in the absence of inos activity .
the inflammatory component was determined by estimating the neutrophil and macrophage accumulation into the implants through assaying mpo and nag enzyme activities , respectively , as well as measuring levels of cytokines ( cxcl1 , ccl2 , tnf- , il-10 , and ifn- ) in the implants .
oppositely to the reduced formation of new blood vessels in the newly formed fibrovascular tissue , we observed an enhancement in the leukocyte accumulation into implants of inos mice when compared to wt , as assayed by mpo ( figure 2(a ) , p < 0.05 ) and nag activities ( figure 2(b ) , p < 0.05 ) , suggesting increased infiltration of neutrophils and macrophages , respectively . the histopathology confirmed the increased leukocyte infiltration in implants from inos mice compared to wt mice ( figure 2(c ) ) .
interestingly , levels of the proinflammatory cytokine tnf- ( figure 3(a ) , p < 0.05 ) were decreased , while levels of the anti - inflammatory cytokine il-10 ( figure 3(b ) , p < 0.001 ) were increased in sponges from inos mice when compared with wt mice .
however , no differences were observed in cxcl1 ( figure 3(c ) ) , ccl2 ( figure 3(d ) ) , or ifn- ( figure 3(e ) ) levels between both groups . another important component of the fibrovascular tissue is the collagen deposition .
here , we estimated the content of collagen present in the implants by calculating the total area occupied by collagen ( m ) in picrosirius - red - stained histological sections evaluated under polarized light ( figure 4(a ) ) .
as shown in figure 4(b ) , there was a significant increase in collagen deposition in implants from inos mice when compared with wt mice ( p < 0.001 ) , suggesting inos activity plays a role in adjusting collagen deposition during the inflammatory angiogenesis process induced by an implanted device .
nitric oxide ( no ) is a diffusible gas involved in a vast number of biological processes that cover the cardiovascular , immune , and neural systems .
its production can be achieved via three known different no synthase ( nos ) enzymes isoforms : nnos ( neuronal ; nos1 ) , inos ( inducible ; nos2 ) , and enos ( endothelial ; nos3 ) .
different from neuronal and endothelial isoforms , inos is regulated mainly on transcriptional level and is independent of intracellular calcium concentration . besides its important physiological roles in cardiovascular functions , especially in endothelium - dependent vasorelaxation , antiplatelet aggregation , and leukocyte adhesion ,
no is a key mediator of inflammation ensuring antimicrobial and immunoregulatory functions as well as modulating by leukocyte recruitment .
the expression of inos , specifically , is involved in many inflammatory and neoplastic conditions .
more recently , the role of inos - induced no during ischemia was also demonstrated .
all these physiopathological situations depend on new blood vessel formation to perpetuate or even cease .
in addition , considering the interest in implantation techniques and scaffolds for tissue engineering , it is worth mentioning that , as demonstrated by our group , levels of no in polyether - polyurethane synthetic sponge implants are modulated by diverse angiogenesis - modifying treatments without , however , a definite correlation with inflammatory cell accumulation [ 23 , 24 ] .
moreover , inos activity seems to be crucial for adequate response to polypropylene implant integration in the peritoneum , although not necessary for the collagen deposition in synthetic pva ( polyvinyl - alcohol ) sponges . nevertheless , the contribution of the inos isoform to the formation of a fibrovascular tissue in the polyether - polyurethane sponge implants was never investigated . here
, we studied the role of inos in different features of the foreign body response induced by that matrix implantation into the subcutaneous compartment of mice .
such implants are known to be infiltrated by a number of cells such as inflammatory cells , endothelial cells , and fibroblasts .
we demonstrated that inos expression may modulate the major concurrent components of the newly formed fibrovascular tissue ( angiogenesis , inflammation , and collagen deposition ) induced by sponge implantation . in the first set of results
, we observed that angiogenesis and vegf levels are reduced in the implants from inos mice when compared to wt animals .
those data are in agreement with various reports in different in vitro and in vivo systems that demonstrated inhibition of inos results in decreased vegf levels and consequently impaired angiogenesis [ 2729 ] .
indeed , the no production by inos may , for instance , contribute to the release of vegf , a growth factor known to be important to stimulate endothelial cell migration and the angiogenic process in vivo [ 31 , 32 ] .
vegf , in turn , may stimulate enos activity resulting in further release of no [ 26 , 33 , 34 ] .
therefore , our results suggest that impaired angiogenesis in the sponge implants from inos mice may be attributed , at least partially , to the reduced no - induced vegf levels .
angiogenesis and inflammation are interconnected processes in many pathophysiological conditions as well as in response to synthetic devices . here
, we measured several markers of inflammation ( mpo and nag activities and tnf- , cxcl1/kc , ccl2/mcp-1 , ifn- , and il-10 levels ) in 14-day - old implants from wt and inos animals .
we observed that inos deficiency resulted in a prolonged inflammatory cell infiltration into the implants with both neutrophils and macrophages content being increased in sponges from inos when compared to wt mice .
however , no differences were observed in the levels of the respective chemoattractants cxcl1 or ccl2 . in effect , there is a vast literature demonstrating that no is a vascular protective molecule that prevents leukocyte adhesion to the endothelium and trafficking .
likewise , there is evidence that inos - derived no may modulate leukocyte and platelet functions that are involved in leukocyte recruitment to inflammatory site [ 37 , 38 ] .
hickey et al . , using a lps in vitro model , showed that inos - derived nitric oxide was able to reduce the adhesion of leukocytes under hydrodynamic flow conditions , providing a plausible explanation for the enhanced leucocyte recruitment observed in vivo in inos mice .
therefore , it is reasonable to speculate that the signaling pathways of the inflammatory chemokines cxcl1 and ccl2 are preserved at some extension in the implants from inos mice and that the increased accumulation of inflammatory cells in the sponge from these animals may be attributed to the lack of inos - derived no production , but not increase in the chemoattractant production .
intriguingly , we found reduced levels of the proinflammatory cytokine tnf- in the implants from inos mice but increased levels of the anti - inflammatory cytokine il-10 , when compared with wt mice . in accordance , decreased production of proinflammatory cytokines in inos mice has been shown [ 40 , 41 ] .
in addition , a compensatory overproduction of il-10 may be observed in mice lacking the gene for the enzyme inos [ 42 , 43 ] . of note , il-10 is associated with an antiangiogenic effect by its ability to decrease vegf levels which , in turn , could partially help to explain the reduced levels of vegf in the implants .
the collagen deposition during tissue remodeling is regulated by complex interactions of pro- and antifibrogenic proteins within the inflammatory tissue [ 4446 ] .
there are some pieces of evidence in the literature regarding the no regulation of collagen deposition as , for example , increased collagen deposition observed in excisional wounds in inos mice .
in addition , neutrophils and macrophages are well known to be involved in inflammation - induced fibrosis .
therefore , the accumulation of inflammatory cells into the implants of inos - deficient mice could be one possible explanation of why there is an exaggerated fibrotic response in the implants from those animals .
in fact , in the absence of inos activity , macrophages may acquire a nonclassical activated phenotype that may have profibrotic characteristics [ 48 , 49 ] and then may account for the increased collagen deposition in the implants from inos - deficient mice .
in addition , it is worth mentioning that although il-10 is understood as an antifibrotic cytokine , it has been suggested that il-10 cooperates with th1 cytokines , such as ifn- ( and even tnf- ) , to suppress collagen deposition [ 50 , 51 ] .
interestingly , here , there were no changes in ifn- levels , and tnf- levels were even decreased , indicating a possible lack of the regulatory pathway for collagen deposition and , consequently , a supportive microenvironment for increasing fibrogenesis in the implants is endorsed in the absence of inos activity . altogether
, our data suggest that inos expression helps in controlling fibrosis in the sponge implants .
overall , the absence of inos leads to increased foreign body reaction to implants in mice , observed as reduced angiogenesis and exacerbated inflammation and fibrosis in the synthetic implants .
therefore , the present work suggests a potential role for inos as a regulator of inflammatory angiogenesis during the fibrovascular tissue formation in sponge implants . these observations may provide relevant information for future insights in therapeutic strategies for conditions where
inflammation and angiogenesis are associated as well as for the development of scaffolds for tissue engineering . | there is considerable interest in implantation techniques and scaffolds for tissue engineering and , for safety and biocompatibility reasons , inflammation , angiogenesis , and fibrosis need to be determined .
the contribution of inducible nitric oxide synthase ( inos ) in the regulation of the foreign body reaction induced by subcutaneous implantation of a synthetic matrix was never investigated . here
, we examined the role of inos in angiogenesis , inflammation , and collagen deposition induced by polyether - polyurethane synthetic implants , using mice with targeted disruption of the inos gene ( inos/ ) and wild - type ( wt ) mice .
the hemoglobin content and number of vessels were decreased in the implants of inos/ mice compared to wt mice 14 days after implantation .
vegf levels were also reduced in the implants of inos/ mice .
in contrast , the inos/ implants exhibited an increased neutrophil and macrophage infiltration . however , no alterations were observed in levels of cxcl1 and ccl2 , chemokines related to neutrophil and macrophage migration , respectively .
furthermore , the implants of inos/ mice showed boosted collagen deposition .
these data suggest that inos activity controls inflammation , angiogenesis , and fibrogenesis in polyether - polyurethane synthetic implants and that lack of inos expression increases foreign body reaction to implants in mice . |
expression profiles were generated using affymetrix u133 plusv2 ( mrna human ) and 430v2 ( mrna mouse ) arrays and agilent mirna arrays ( human ) .
expression profiles of 53 human medulloblastomas and 76 glioblastomas were obtained from previously published studies29,30 .
cnas were validated by real - time pcr ( see supplemental table 4 ) and/or fish as appropriate7 .
mrna and mirna expression profiles and dna cnas were analyzed , validated and integrated using established and novel bioinformatic and statistical approaches ( see supplemental methods ) .
egfp cells were isolated from blbp - egfp mouse brains and spines and cultured in neural stem cell medium as described7 .
nscs were transduced with rfp - retroviruses and egfp / rfp cells sorted using a becton dickinson aria ii cell sorter .
clonal neurosphere formation and differentiation capacities were assessed in serial dilutions of single - cell suspensions7 .
nscs were implanted under stereotactic control into the forebrain of immunocompromised mice and animals were observed daily for signs of tumor development7 .
protein expression was detected in formalin fixed tissue sections and differentiated nscs using standard immunohistochemical and immunofluorescence techniques .
electron microscopy was performed using standard tissue preparations and a fei tecnai 20 200kv feg electron microscope .
western blot analysis was used to detect protein expression in fresh tissue and cells ( for antibodies see supplemental methods ) .
expression profiles were generated using affymetrix u133 plusv2 ( mrna human ) and 430v2 ( mrna mouse ) arrays and agilent mirna arrays ( human ) .
expression profiles of 53 human medulloblastomas and 76 glioblastomas were obtained from previously published studies29,30 .
cnas were validated by real - time pcr ( see supplemental table 4 ) and/or fish as appropriate7 .
mrna and mirna expression profiles and dna cnas were analyzed , validated and integrated using established and novel bioinformatic and statistical approaches ( see supplemental methods ) .
egfp cells were isolated from blbp - egfp mouse brains and spines and cultured in neural stem cell medium as described7 .
nscs were transduced with rfp - retroviruses and egfp / rfp cells sorted using a becton dickinson aria ii cell sorter .
clonal neurosphere formation and differentiation capacities were assessed in serial dilutions of single - cell suspensions7 .
nscs were implanted under stereotactic control into the forebrain of immunocompromised mice and animals were observed daily for signs of tumor development7 .
sections of human and mouse ependymomas were prepared from formalin paraffin embedded tissues . protein expression was detected in formalin fixed tissue sections and differentiated nscs using standard immunohistochemical and immunofluorescence techniques .
electron microscopy was performed using standard tissue preparations and a fei tecnai 20 200kv feg electron microscope .
western blot analysis was used to detect protein expression in fresh tissue and cells ( for antibodies see supplemental methods ) .
| understanding the biology that underlies histologically similar but molecularly distinct subgroups of cancer has proven difficult since their defining genetic alterations are often numerous , and the cellular origins of most cancers remain unknown13 . we sought to decipher this heterogeneity by integrating matched genetic alterations and candidate cells of origin to generate accurate disease models .
first , we identified subgroups of human ependymoma , a form of neural tumor that arises throughout the central nervous system ( cns ) .
subgroup specific alterations included amplifications and homozygous deletions of genes not yet implicated in ependymoma . to select cellular compartments most likely to give rise to subgroups of ependymoma
, we matched the transcriptomes of human tumors to those of mouse neural stem cells ( nscs ) , isolated from different regions of the cns at different developmental stages , with an intact or deleted ink4a / arf locus .
the transcriptome of human cerebral ependymomas with amplified ephb2 and deleted ink4a / arf matched only that of embryonic cerebral ink4a / arf/ nscs .
remarkably , activation of ephb2 signaling in these , but not other nscs , generated the first mouse model of ependymoma , which is highly penetrant and accurately models the histology and transcriptome of one subgroup of human cerebral tumor .
further comparative analysis of matched mouse and human tumors revealed selective deregulation in the expression and copy number of genes that control synaptogenesis , pinpointing disruption of this pathway as a critical event in the production of this ependymoma subgroup .
our data demonstrate the power of cross - species genomics to meticulously match subgroup specific driver mutations with cellular compartments to model and interrogate cancer subgroups . |
we discuss the management of a patient after its recurrence , involving the upper respiratory tract i.e. nasopharynx and oropharynx , which compromised the airway .
the patient was referred for anesthesia on three different occasions with different presentations owing to the recurrence of symptoms .
the presence of an oropharyngeal membrane with a small opening made airway management a challenge .
rhinoscleroma is a chronic , granulomatous infection , caused by klebsiella rhinosleromatis , affecting the nasopharynx , oro - pharynx , larynx , trachea and bronchi .
it is common in the second and third decade of life , with females more frequently affected .
treatment consists of prolonged antibiotic treatment , surgery to remove the obstruction and laser therapy to treat airway compromise and tissue deformity .
this progressive infection , involving the upper respiratory tract , may pose a challenge in airway management .
however , he had no long - term relief and the symptoms progressed . in the last six months , he developed difficulty in breathing and swallowing .
oral examination revealed a membrane at the level of uvula covering the oropharynx with a central opening of approximately 8 mm diameter through which he managed swallowing [ figure 1 ] .
ct scan and mri scan showed a concentric narrowing at the level of the oropharynx [ figures 2 and 3 ] with sparing of the lower respiratory tract .
flexible fiber - optic bronchoscopy ( with a 6 mm scope ) under la revealed uninvolved vocal cords , but patient had discomfort and was unable to hold his breath during the procedure .
oro - pharyngeal membrane visible at the level of uvula covering the oropharynx with a central opening of approximately 8 mm diameter mri of the airway sagittal section showing the membrane in the oropharynx and no involvement of the airway below mri coronal view showing narrowing of the oropharynx due to the membrane ( shown by arrow ) premedication with 20 mg omeprazole and glycopyrrolate 0.2 mg intramuscularly was given .
after preoxygenation , anesthesia was induced with 120 mcg fentanyl , propofol 1mg / kg and sevoflurane in titrated doses . after ensuring adequate mask ventilation , succinylcholine 1.5mg / kg was given .
although an ideal laryngoscopy could not be performed , moving the tongue gently with the laryngoscope helped visualize the membrane .
a 15 fr bougie was inserted via the hole in the membrane and tracheal rings were well felt .
a non - laser shielded 5 mm i d endotracheal tube ( ett ) was negotiated , over the bougie , through the opening .
membrane was resected with diode contact laser and the tissue was sent for histopathology evaluation .
the induction was done as in the previous instance but we were unable to negotiate the bougie , and two attempts at blind intubation with 5 mm i d ett and an attempt with a smaller ett were unsuccessful .
post operatively , the tracheostomy was kept and patient discharged on fourth postoperative day with tracheostomy tube in situ .
rhinoscleroma leads to fibrosis of the airways with consequent deformity , atresia or complete obstruction of the airway .
diagnosis is made on the basis of clinical presentation , histopathology , bacteriological culture and serological investigations .
the extent of oropharyngeal and tracheobronchial involvement can be accurately assessed with ct or mri scans , although very few studies have described imaging features of rhinoscleroma.[79 ] the nasal cavity is the most often affected ( 95 - 100% ) , but the lesions may also involve larynx ( 15 - 40% ) , nasopharynx ( 18 - 43% ) , oral cavity , paranasal sinuses ( 26% ) , soft tissues of lips , nose , trachea ( 12% ) and bronchi ( 2 - 7% ) .
sharing the airway with the surgeon is of utmost concern to an anesthesiologist . in the case reported , a trial at oral intubation guided by a gum elastic bougie was done as the patency of the pharynx distal to the membrane on mri and ct scan was confirmed [ figures 24 ] .
we avoided blind nasal intubation since the nares seemed blocked and the ct image showed some synechiae in the nasal passage .
the feel of the tracheal rings by the bougie guided us the insertion of the ett .
a fiberoptic bronchoscope would have helped in visualizing the membrane , as it done in the ent clinic , but we only had a 6 mm scope , which would have permitted only a larger ett over it .
trauma to the membrane or bleeding had to be avoided . a supraglottic airway could not be used .
jet ventilation would have made the patient prone to barotrauma as airway pressures can not be controlled with its use .
retrograde intubation would have been tedious as passing the guide wire through the small opening would have been very difficult .
as the pharyngoplasty took only fifteen minutes , ett was not changed to a bigger size later .
ct scan showing narrowing of the oropharynx due the membrane ( shown by arrow ) fatal airway obstruction has been reported after evading tracheostomy in a patient who seemed to be improving following antibiotic treatment .
pharyngoplasty is often a day care procedure performed using laser , and is associated with minimal blood loss , edema , and quick recovery .
routinely , it does not need the patient to have a tracheostomy , as lubricated airways , post - surgery , maintain the airway patency well . in our patient , at the second instance , the opening in the oropharyngeal membrane was smaller .
we were however reluctant to do a tracheostomy knowing that the symptoms would be relieved on resection of the membrane . despite three attempts with smaller sized ett
we did not expect recurrence , as the patient had undergone laser and electrical cautery resection , but there was recurrence .
the third time , the patient presented with respiratory distress that warranted an immediate tracheostomy .
he had not responded to prolonged antibiotics , anti - inflammatory drugs , steroids or even surgery and finally needed a permanent tracheostomy .
close observation for prolonged periods of time is essential to recognize reactivation of quiescent lesions .
trauma caused by excision and laser can incite more fibrosis and lead to recurrence of the primary problem with a vengeance .
the recurrence rate of rhinoscleroma has been cited to be up to 25% of the treated cases in 10 years . in patients with an oropharyngeal membrane with a solitary opening ,
the size of the opening , pathology and involvement of the oropharynx , nasopharynx and the tracheobronchial tree must be evaluated .
it is important to consider the site of airway obstruction and ensure availability of equipment to secure the airway .
fiberoptic bronchoscope is an important tool in assessing the airway patency but can precipitate respiratory distress and a bougie may be helpful .
tracheostomy should be performed if the opening is small or if the airway is affected at multiple levels . | rhinoscleroma is a rare entity encountered in anesthesia practice .
we discuss the management of a patient after its recurrence , involving the upper respiratory tract i.e. nasopharynx and oropharynx , which compromised the airway .
the pateint was referred for anesthesia on three different occasions with different presentations owing to the recurrence of symptoms.the presence of an oropharyngeal membrane with a small opening made airway management a challenge .
the patient was successfully managed on all three occasions .
imaging facilitated assessment and subsequent airway management . |
closure of wounds to achieve an esthetically pleasing result has always been a challenge . since time immemorial
the introduction of tissue adhesives heralded the era of suture free closures which led to better results .
the aim of this study is to compare the efficacy of octyl- 2-cyanoacrylate with that of the conventional sutures , in facial skin closure .
the use of octyl-2-cyanoacrylate offers many advantages such as rapidity and ease of application and superior results .
wound closure techniques have evolved from the earliest development of suturing materials to resources that include synthetic absorbable sutures , staples , tapes , and adhesive compounds . the creation of natural glues , surgical staples and tapes to substitute sutures has supplemented the armamentarium of wound closure techniques .
the use of tissue adhesives has long appealed to surgeons and they have been extensively studied for nearly four decades for diverse applications including tissue adhesion , wound closure , hemostasis , closure of cerebrospinal fluid ( csf ) leaks , vascular embolization and application of skin grafts .
the ideal method of laceration and incision closure should be simple , safe , rapid , inexpensive , painless , bactericidal , and result in optimal cosmetic appearance of the scar .
the cyanoacrylates are supplied as monomers in a liquid form . on contact with tissue anions , they polymerize forming a strong bond that holds the apposed wound edges together .
the cyanoacrylate adhesives usually slough off with wound re - epithelialization within 510 days and do not require removal .
the aim of this study is to compare the efficacy of octyl-2-cyanoacrylate with that of the conventional sutures , in closure of facial skin .
twenty patients were enrolled in the study and they were randomly divided into two groups . in group
i , octyl-2-cyanoacrylate ( dermabond , ethicon inc , johnson and johnson , somerville , new jersey , usa . ) [ figure 1 ] , was used for skin closure [ figure 2 ] . in group
patients were eligible for inclusion in the protocol if they were of generally good health without significant systemic abnormalities , agreed to return for 10th - day and second month follow - up assessment , and provided written informed consent .
closure with dermabond specific exclusion criteria were patients with multiple trauma , peripheral vascular disease , insulin - dependent diabetes mellitus , known bleeding diathesis , known personal or family history of keloid formation or scar hypertrophy , or a known allergy to cyanoacrylate compounds or formaldehyde .
in addition to inclusion and exclusion criteria for patients , the study also had specific criteria based on laceration etiology , degree of wound contamination , and location .
eligible wounds were those that required 3 - 0 or smaller sutures for skin closure .
although the functional tensile strength of 2-octylcyanoacrylate is comparable to that of 5 - 0 sutures , as we commonly use 3 - 0 braided black silk sutures for skin closure , we designed the study with the same .
wounds as a result of animal or human bites , punctures , ulcers , or crush injuries were excluded .
wounds with visual evidence of active local or systemic infection , gangrene , contaminated or devitalized tissue , or within active rashes were also excluded . in addition , wounds located at the vermilion border of the lip , the mucosa , or in areas covered by natural hair ( precluding an assessment of cosmetic outcome at 2 months ) were excluded .
dermabond is supplied in a single use sterile plastic vial containing 0.5 ml of octyl-2-cyanoacrylate adhesive within an inner glass ampoule . just before application , the outer plastic vial is gently crushed between index finger and thumb to break the inner glass ampoule and the adhesive is expressed through the tip of applicator . as the adhesive moves through the applicator tip , it mixes with an initiator and begins the chemical change from monomer to polymer .
moisture on the surface of the skin adds the final catalyst to create the strong polymer bond that bridges the wound edges .
care is taken to avoid introducing the adhesive between the wound edges since it would impede healing .
the adhesive is then carefully expressed through the tip of the applicator and gently brushed over the wound surface in a steady continuous motion .
it is made sure that the adhesive covers the entire wound and an area covering 510 mm on either side of the wound edges .
initial layer was allowed to polymerize for approximately 1530 seconds , two additional layers of adhesive are similarly brushed onto the surface of the wound , with a waiting period of 510 seconds between successive layers .
third postoperative evaluation was done at the end of second month for cosmesis . out of the 20 patients 14 were males and 6 were females . on 10 day ,
19 patients reported for follow up in which , 9 belonged to group i and 10 belonged to the group ii .
statistical analysis was performed using chi - square test for parametric variables and the p ( probability ) and z values were calculated .
one patient in group i had immediate postoperative bleeding after application of adhesive , which had resolved itself within 23 minutes .
the incidence of bleeding may be attributed to incomplete hemostasis prior to closure . on the 10 day
follow up , one patient in group i had wound dehiscence in the chin region .
the wound finally healed uneventfully . although it was in 10% of the total cases , the difference was statistically insignificant .
one case in each group had the presence of infection for which the patients were treated by standard protocol .
statistically , the overall difference between results in both the groups on 10 postoperative day was insignificant .
similar studies by jim quinn et al toriumi , and singer et al have reported results in compliance with the present study .
next postoperative evaluation was done at the end of second month [ figures 4 and 5 ] for cosmesis .
scar was evaluated for patient 's and surgeon 's satisfaction on a 110 point visual analog scale ( vas ) , where 1 denotes worst possible outcome and 10 is the best possible outcome .
earlier study by quinn has demonstrated vas as a valid scale to measure the cosmetic outcome .
the mean of the total patient satisfaction score and the surgeon 's satisfaction score was calculated with standard deviation .
patient satisfaction score in group i was higher as compared to group ii , but this difference was statistically insignificant ( z = 1.405 , p = 0.500 ) .
postoperative view at the end of 2 months ( dermabond ) postoperative view at the end of 2 months ( sutures ) the surgeon 's satisfaction score was also higher in group i [ graph 1 ] as compared to group ii [ graph 2 ] , although this difference was statistically insignificant ( z = 1.50 , p = 0.773 ) .
similar result was found in several other prior studies by quinn , toriumi , and singer et al .
however , a study by bernard laurie et al showed a statistically significant difference on vas scale in favor of sutures .
surgeon 's satisfaction score patient 's satisfaction score the cost effectiveness in both the groups were also measured and it was found that although cost of the material in the case of group i was higher , total effective cost including transportation cost for follow up , loss of wages , cost of dressing and local antibacterial medicaments was higher in group ii . thus , overall effective cost was almost equal in both groups .
martin et al did an economic comparison between adhesives and sutures and found tissue adhesives to be more efficient economically . the time required for closure with dermabond was one - third of the time required for suture closure .
early , uncomplicated wound healing has been a subject of intensive research over the ages .
the complexities involved in wound healing , such as involvement of more than one type of tissue , various degrees of wound strength during the process of healing , exposure of the biomaterials to body fluids and a variety of wounds , each with its own healing problems , call for different types of wound closure materials in general , wound closure biomaterials are divided into three major categories : suture materials , staples and tissue adhesives .
suturing has been the most widely used method for wound closure because of high reliability of suture materials .
however , alternative techniques have long been sought , since suturing technique requires skill and experience , a relatively longer time and the need for its removal . due to these reasons
several studies regarding the use of the tissue adhesives in closure of facial wounds have been conducted to compare their efficacy against the conventional sutures.[1014 ] although most facial wounds heal without complications , owing to the abundant blood supply of the region , mismanagement may result in infection , wound dehiscence , and unsightly and dysfunctional scar . historically , the autologous and homologous fibrin tissue adhesives have been extensively used due to their safety and reliability . however , fibrin tissue adhesives carry the risk of viral transmission . epoxy resins ( polyurethanes ) also have been considered for surgical applications .
the polyurethanes have high bond strengths as compared to adhesive systems , but apparently form bonds at a rate too low for practical applications .
also , it has been seen that they exhibit adverse bonding behavior in the presence of moisture , making them unreliable .
cyanoacrylate - based adhesive systems are most recent tissue adhesives . the rapid setting time and desirable effect of moisture on polymerization
early attempts at developing a cyanoacrylate - based tissue adhesive have been fraught with handling problems and associated histotoxicity .
further studies demonstrated that the histotoxicity of cyanoacrylate tissue adhesives can be attributed to the by - products of cyanoacrylate polymer degradation , i.e. , cyanoacetate and formaldehyde .
this rate of degradation is affected by the length of the alkyl group of the cyanoacrylate derivative .
shorter chain derivatives such as methyl and ethyl cyanoacrylates degrade quickly and therefore have more toxicity than longer chain derivatives .
octyl-2-cyanoacrylate ( dermabond , ethicon , inc . ) is a recent cyanoacrylate derivative with eight alkyl constituents off the carboxyl group , which slows down the degradation and by - product release into the surrounding tissues .
additionally , plasticizers have been added which make the adhesive bond stronger and more durable but allow flexion of the skin .
cyanoacrylates have a number of advantages over conventional sutures like their fast and painless application , rapid setting which reduces the total operating time , their antibacterial properties .
cyanoacrylate itself acts as a water proof dressing and helps in reduction in the number of follow - up visits . as they do not require any needles ,
accidental needle stick injuries are prevented . however , there are certain disadvantages of cyanoacrylates like their less tensile strength and chances of adhesive seepage if edges are not properly approximated .
multiple studies have shown equivalence of octyl cyanoacrylate to 5 - 0 skin sutures in esthetic facial surgery and repair of traumatic facial wounds . however , it is important to remember that dermal suture support is still needed ( in wounds that traverse the full thickness ) and skin must be held together as the adhesive is applied to prevent the deposition of the cyanoacrylate polymer into the wound , potentially delaying or preventing the healing .
the popularity of dermabond for closure of elective surgical incisions , repair of traumatic facial lacerations and in esthetic facial surgery is limited in india , primarily due to cost considerations and a dearth of studies conducted on indian population .
although the cost of dermabond tissue adhesive is more compared to conventional sutures , the total cost effectiveness of using the material is equivalent or even better compared to conventional sutures .
we may conclude that the use of octyl-2-cyanoacrylate is better than sutures in the closure of facial wounds . however , further studies with a larger sample size are necessary on indian population for octyl cyanoacrylate to replace sutures as a primary method for repair of facial wounds . | introduction : closure of wounds to achieve an esthetically pleasing result has always been a challenge . since time immemorial
, surgeons have strived to produce invisible scars .
this , however , has always been elusive .
the introduction of tissue adhesives heralded the era of suture free closures which led to better results.aims and objectives : the aim of this study is to compare the efficacy of octyl- 2-cyanoacrylate with that of the conventional sutures , in facial skin closure.results and conclusion : the use of octyl-2-cyanoacrylate offers many advantages such as rapidity and ease of application and superior results . |
mesothelial cells ( mc ) , which form the innermost monolayer of the peritoneal cavity , are critical in morphological and functional alterations of the peritoneal membrane in patients who undergo peritoneal dialysis ( pd ) . they are a major source of intraperitoneal monocyte chemoattractant protein-1 ( mcp-1 ) which is a chemokine that has been reported to play a key role in the recruitment of monocytes toward the peritoneal cavity .
monocytes in turn contribute to peritoneal fibrosis by producing various cytokines and growth factors that induce cell proliferation and extracellular matrix production in mesothelial cells and fibroblasts [ 3 , 4 ] .
thiazolidinediones ( tzd ) are a novel group of antidiabetic agents that act via the activation of peroxisome proliferator - activated receptor - gamma ( ppar- ) , a nuclear hormone receptor .
therefore , tzd like rosiglitazone are not only capable of increasing the insulin sensitivity in peripheral organs ( as e.g. , adipose tissue ) and thus lowering blood glucose levels in diabetic patients ; they also possess anti - inflammatory capacities in certain circumstances as they decrease the expression of inflammatory proteins like inos and mmp9 in macrophages [ 6 , 7 ] .
a reduced expression of mcp-1 due to tzd treatment has been shown for diverse cell types as , for example , lung epithelial , endothelial , and mesangial cells . in this context , the present study was designed to investigate the presence of ppar- on human peritoneal mc and to characterize the effect of ppar- activation by tzd on mesothelial mcp-1 mrna transcription and release .
medium m199 and newborn calf serum were obtained from gibco brl ( eggenstein , germany ) ; tissue - culture plates came from costar ( cambridge , massachusetts , usa ) .
human serum was prepared from freshly collected blood of healthy donors and stored at 20c .
fibronectin from human serum , trypsin , and tnf were purchased from boehringer ( mannheim , germany ) , collagenase type ii from worthington ( freehold , ny , usa ) .
monoclonal antibodies against cytokeratins 8 and 18 as well as vimentin were a gift from dr .
antibody against ppar- was from santa cruz biotechnology ( santa cruz , california , usa ) .
mtt was from sigma - aldrich ( st . louis , mo , usa ) .
rosiglitazone was from molekula ( nienburg / weser , germany ) , and gw-9662 was purchased from cayman chemical ( ann arbor , michigan , usa ) .
mc were isolated from the omental tissue of consenting patients undergoing elective surgery , as described previously .
cells were grown in fibronectin - coated dishes in m199 medium supplemented with 25 mmol / l hepes ( ph 7.3 ) , 2 mmol / l glutamine , 10% ( v / v ) human serum and 10% ( v / v ) newborn calf serum ( heat inactivated ) , penicillin ( 100 iu / ml ) , and streptomycin ( 100 g / ml ) at 37c under 5% co/95% air atmosphere .
subcultures were obtained by trypsin / edta treatment at a split ratio of 1 : 3 .
the cells were purely mc , as assessed by their uniform cobblestone appearance at confluence , by the absence of von willebrand factor , and by their uniform positive staining for cytokeratins 8 and 18 as well as for vimentin . for the experiments ,
confluent cultures were used at the second or third passage , and cells were refed with 2% human serum 24 hours before the experiment .
conditioned media were obtained by incubating cells in 2 cm dishes at 37c with 0.5 ml serum - free m199 containing the appropriate concentration of the test compound .
serum - free medium m199 served as a control . in coincubation experiments with tnf ,
cells were preincubated for 24 hours with rosiglitazone , and then tnf was added . in experiments using a ppar- blocker ,
supernatants were centrifuged 5 minutes at 2000 g to remove cells and cellular debris , and the samples were frozen at 20c until use .
all experiments were done with cells from 3 to 6 individual donors and were measured in triplicate .
cultured human mesothelial cells were harvested with lysis buffer ( 50 mm tris - hcl ph7.4 , 1% nonidet p-40 , 0.25% sodium deoxycholate , 150 mm nacl , 1 mm egta , and 1 mm na3vo4 , complete protease inhibitor cocktail ( roche , mannheim , germany ) ) .
extracted proteins were boiled in loading buffer for 30 min , resolved by 8% sds - page under reducing conditions , and transferred to an immobilon - p membrane ( millipore , eschborn , germany ) .
the membrane was blocked with 3% skim milk , incubated in a 1 : 1000 dilution of a rabbit polyclonal igg - antibody against human ppar-1 ( sc-7196 , santa cruz biotechnology , heidelberg , germany ) over night , and rinsed with pbs containing 0.1% tween 20 .
immune complexes were visualized using enhanced chemoluminescence ( ecl , amersham biosciences , freiburg , germany ) .
human mesothelial cells ( 30 10/100 l medium ) were cultured in 96-well microtiter plates for 24 h under standard conditions to yield firmly attached and stably growing cells . after discarding supernatants , 50 l of medium m199 containing rosiglitazone in concentrations of 1 and 10 g / l or medium m199 as a control was added to the cells and incubated for 48 h. then 50 l of a 1 mg / ml solution of ( 3,5-dimethylthiazol-2-yl]-2,5-diphenyl - tetrazolium bromide ) mtt ( sigma - aldrich , taufkirchen , germany no .
after 3 h incubation at 37c , formazan crystals were dissolved by addition of 100 l isopropanol and 0.04 n hcl .
absorbance was then measured at 590 nm using a genios plus tecan elisa reader . for each experiment
mcp-1 antigen levels [ pg/10 cells ] were measured by quantikine human mcp-1 immunoassay from r&d systems ( minneapolis , mn , usa ) . diluted aliquots of the cell supernatants were assayed without prior purification .
total rna was extracted from cells using silica gel columns ( rneasy , qiagen , hilden , germany ) .
2 g of isolated total rna underwent random hexamer - primed reverse transcription for one hour at 42c using a modified molony murine leukaemia virus ( mmlv ) reverse transcriptase ( superscript , life technologies , karlsruhe , germany ) . real - time quantitative reverse transcription - polymerase chain reaction ( rt - pcr )
was performed on a taq - man abi 7700 sequence detection system ( pe biosystems , weiterstadt , germany ) using heat - activated taqdna polymerase ( amplitaq gold , applied biosystems , darmstadt , germany ) .
thermal cycler conditions contained holds for two minutes at 50c and ten minutes at 95c , followed by 40 cycles of 15 seconds at 95c and one minute at 60c .
glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) and 18s ribosomal rna ( rrna ) served as the reference housekeeping gene .
controls consisting of h2o or samples that were not reverse transcribed were negative for the target and reference .
sequences with following gene bank accession numbers served for the design of the predeveloped taq man assay reagents ( pdar ) or primers and probe , purchased from applied biosystems ( darmstadt , germany ) : x14768 ( human mcp-1/ccl2 ) m33197 ( human gapdh ) and x03205.1 ( human18s - rrna ) .
statistical analysis was performed using the anova analysis . a p value < 0.05 was considered to indicate statistically differences .
analysis of the extracted total protein of unstimulated mc showed a single band at 67 kda as has been described for ppar-1 and was comparable to the recommended positive control ( protein from human breast carcinoma cells ) ( figure 1 ) .
confluent unstimulated mc were incubated with increasing concentrations of rosiglitazone ( 0.110 mol / l ) for 48 hours .
this resulted in a concentration - dependent decrease of mcp-1 protein levels in the cell culture supernatants .
rosiglitazone treatment reached statistical significancy at each employed concentration : a concentration of 0.1 mol / l reduced the mcp-1 level to 13200 pg/10 cells versus control 18200 pg/10 cells ( p = 0.013 ) whereas a concentration of 1 mol / l reduced mcp-1 levels to 12700 pg/10 cells ( p = 0.001 ) and a concentration of 10 mol / l to 9400 pg/10 cells ( p < 0.001 ) ( figure 2(a ) ) .
rosiglitazone treatment in the shown concentrations did not effect cell viability as assessed in trypan blue staining or cell proliferation as assessed in an mtt assay ( figure 3 ) .
the incubation of mc with 10 mol / l rosiglitazone resulted in a marked decrease of mcp-1 mrna after a 4 hours ' dwell time ( 0.09 versus 1 ) ( figure 2(b ) ) . a longer incubation time ( 8 hours ) and
the use of another housekeeping gene ( gapdh ) resulted in comparable findings ( data not shown ) . in order to investigate whether the effect of tzd on the mesothelial mcp-1 release is dependent on the tzd property to activate ppar- , mc were preincubated with the ppar- inhibitor gw-9662 ( 10 mol / l ) or control media for 24 hours .
mol / l , gw-9662 10 mol / l , or the combination of gw-9662 and rosiglitazone for 8 hours . in accordance with longer incubation times ( figure 2 ) , rosiglitazone treatment led to a significant decrease in mcp-1 protein ( 1700 pg/10 cells versus control : 2660 pg/10 cells ; p = 0.028 ) .
gw-9662 did not have an effect on mcp-1 secretion and resulted in similar mcp-1 protein levels compared to control .
the blocking of ppar- by administration of gw-9662 before a rosiglitazone application could completely prevent the rosiglitazone - induced attenuation in mcp-1 release ( 2870 pg/10 cells versus 1700 pg/10 cells ; p = 0.039 ) ( figure 4(a ) ) .
corresponding results could be found on the transcriptional level : whilst rosiglitazone markedly reduced mcp-1 mrna levels ( 0.28 versus 1 ) , ppar- blockade by gw-9662 resulted in considerably higher mcp-1 mrna levels ( 0.62 ) .
gw-9662 alone had no effect on the mc mcp-1 mrna production , as the administration resulted in a mcp-1 mrna level comparable to the control ( 0.86 ) ( figure 4(b ) ) .
the incubation of mc with tnf ( 100 u / ml ) resulted in a drastic increase in mesothelial mcp-1 secretion ( 67500 pg/10 cells versus control 4000 pg/10 cells ) .
mol / l and subsequent coincubation resulted in a one - third reduction in mesothelial mcp-1 levels ( 43000 pg/10 cells versus 67500 pg/10 cells ) ( figure 5 ) .
mc are supposed to be critical in the pathogenesis of complications following pd treatment . by producing profibrotic [ 13 , 14 ] and neoangiogenetic factors , they contribute to peritoneal fibrosis .
in addition , mc are a major source of intraperitoneal mcp-1 and thus account for the recruitment of monocytes toward the peritoneal cavity . apart from an inflammatory reaction
mcp-1 can be found in markedly elevated concentration in the dialysate of pd patients during and after episodes of peritonitis [ 16 , 17 ] .
il-1 , ifn , and tnf as well as high glucose concentrations ( due to high osmolality and the polyol pathway ) are found to increase the mesothelial mcp-1 synthesis rate [ 18 , 19 ] .
tzd are activators of ppar-. this receptor is a member of the nuclear receptor family that includes 48 human transcription factors regulated by direct binding of steroid and thyroid hormones , vitamins , lipid metabolites , and xenobiotics . by differential promotor usage and splicing two isoforms are generated : ppar-1 which can be found on a variety of cell types and ppar-2 , which has an additional 30 amino acids at its n - terminal end and is expressed specifically in adipocytes .
we could now demonstrate the constitutive expression of ppar-1 on human mc . in pd patients ,
described that rosiglitazone improved glucose metabolism in nondiabetic uremic patients on capd , and wong et al .
found reduced insulin requirement and c - reactive protein levels in type 2 diabetic patients receiving peritoneal dialysis . in the present study
, we could demonstrate that tzd are able to reduce the constitutive mcp-1 release in mc by ppar- stimulation .
furthermore , rosiglitazone was able to attenuate enhanced mcp-1 secretion resulting from a stimulation with the proinflammatory cytokine tnf. peng et al .
found a decrease in high glucose concentration - induced mesothelial production of tgf , collagen i , and fibronection secretion after treatment with troglitazone .
some in vivo animal model studies point toward positive tzd effects on the peritoneal membrane in pd or peritoneal inflammation : yao et al .
found maintained peritoneal morphology and increased ultrafiltration after intraperitoneal administration of rosiglitazone in comparison to commercial pd solution alone .
sandoval et al . described a reduction in the accumulation of ages as well as reduced fibrosis and angiogenesis resulting in an improved peritoneal function . in accordance with our findings , hornung et al .
were able to demonstrate that the intraperitoneal administration of ciglitazone was able to significantly reduce the number of invading peritoneal macrophages following a thioglycollate - induced peritoneal inflammation .
ppar- stimulation in mc may be a promising possibility in the attempt to minimize long - term pd complications
. however , potential negative effects of the tzd ( the only commercially available ppar- activators at present ) such as their ability to cause edema and their negative cardiovascular risk profile should be considered critically .
its activation via rosiglitazone decreases the mesothelial release and mrna expression of mcp-1 and attenuates the tnf-induced enhancement in mcp-1 release in these cells .
therefore , the ppar-1 receptor may be a therapeutic target to ameliorate peritoneal inflammation and long - time survival of the peritoneal membrane in pd
. however , potential negative systemic effects of the tzd have to be considered critically . | human peritoneal mesothelial cells ( mc ) play an important role in inflammatory processes of the peritoneal cavity by producing various cytokines and chemokines , such as monocyte chemoattractant protein-1 ( mcp-1 ) .
the present study was designed to assess the effect of the peroxisome proliferator - activated receptor - gamma- ( ppar- ) activator rosiglitazone on the mesothelial mcp-1 expression and release .
primary cultures of mc were obtained from omental tissue .
mcp-1 antigen concentrations were measured in the cell supernatant by elisa and mcp-1 mrna levels by real - time polymerase chain reaction .
the presence of ppar on mc was assayed in a western blot analysis .
mc constitutively express ppar. activation of this receptor via rosiglitazone ( 0,110
mol / l ) resulted in significantly reduced amounts of mesothelial mcp-1 release as well as mcp-1 mrna .
the use of the ppar inhibitor gw-9662 could completely prevent the rosiglitazone effects .
rosiglitazone was also effective in reducing tnf-induced enhanced secretion of mcp-1 .
our findings indicate that glitazones are effective in reducing constitutive and tnf-stimulated mesothelial mcp-1 mrna expression and release . |
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