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the use of various imaging techniques in post - treatment assessment of head neck cancer is gradually on the rise , though clinical examinations at periodic intervals still remains the mainstay of follow - up . 18f fdg pet / ct is increasingly used in the post - treatment setting to diagnose residual / recurrent disease early so that appropriate treatment can be initiated . the effects of multimodality treatment such as surgery , radiation , and chemotherapy can distort normal anatomical landmarks induce inflammation and infection , which can mask disease . it is important to understand the anatomical and functional effects as well as complications produced by these treatment modalities in order to diagnose the resultant imaging pitfalls on fdg pet / ct . knowledge of the common and unusual patterns of loco - regional recurrent disease and distant metastases on fdg pet / ct is also crucial in the post - treatment assessment of head - neck cancer patients . radiation therapy alone or in combination with chemotherapy is often used to treat head - neck cancer patients . there are several tissue changes , which are a result of early reaction to radiation . these include thickening of the skin and platysma , stranding of the subcutaneous fat , pharyngeal and laryngeal wall thickening and increased vascularity of the major and minor salivary glands . edema , inflammation , and increased vascularity in these structures lead to a pattern of fdg uptake which is low grade , diffuse , symmetrical , and restricted to the radiation field [ figure 1 ] . these findings are seen during the course or within 3 months of radiation therapy and are often reversible . several muscle groups within the radiation field also demonstrate an elevated fdg activity which can be potentially confused with neoplastic uptake . however , absence of a mass lesion on ct and reduction over time differentiate an inflammatory or physiological uptake from disease . axial pet and fused pet / ct images show diffuse symmetrical fdg uptake in the tongue and oropharynx ( arrows in the b and c ) and in the floor of mouth ( arrows in e and f ) . diffuse low intensity fdg uptake is also seen in the neck muscles ( arrowheads in b , c , e , f ) . diffuse uptake is seen within a few days of radiation and can last up to 810 weeks timing of pet / ct after radiation therapy is an important and often debated clinical issue . performing pet / ct as early as possible to detect recurrent disease seems logical as salvage treatment can be initiated early enough to derive the best possible clinical benefit . however , studies performed too early may lead to false positive as well as false negative results . possible mechanism for false negative studies has been attributed to radiation induced vascular damage , which temporarily prevents concentration of radiotracer in the viable tumor cells . imaging after a further few weeks delay in such cases may allow accumulation of tracer in the viable cells leading to a true positive pet result . waiting too long to image might result in a loss of therapeutic window and also a more complicated surgical procedure due to fibrosis setting in . in order to strike a balance between misleading pet results when study is performed early and the clinical drawbacks of imaging late , a time interval of 12 weeks after completion of radiation therapy is generally recommended . absence of fdg uptake at both the primary and nodal sites has a high negative predictive value in ruling out residual disease [ figure 2 ] . studies have shown that a negative pet scan can potentially defer planned neck dissections after chemo - radiation therapy . pretreatment coronal fused pet / ct ct studies show fdg avid right sided hypopharyngeal mass ( arrow in a and b ) . study performed 8 weeks after completion of chemo - radiation therapy show complete metabolic and morphologic response ( arrow in c and d ) which rules out the possibility of residual disease curative resection involves complex surgical procedures , which result in loss of symmetry and anatomical landmarks . various reconstructive techniques are used to close the surgical defects and restore function which produces characteristic well - recognizable imaging findings . procedures such as glossectomy , marginal or segmental mandibulectomy , maxillectomy leave large surgical defects , which are closed by flaps and grafts . pectoralis myocutaneous flap is an example of a composite flap which is often used for reconstruction of large surgical defects in the face , tongue , and skull base . they can be recognized by the well marginated soft tissue / muscle density at the site of the defect and is often accompanied by fat density [ figure 3a , b ] produced by a denervation atrophy and fatty replacement of the muscle . normal symmetric pattern of tracer concentration is disturbed due to absence of physiological fdg uptake at the resected and reconstructed site [ figure 3b and d ] . in patients undergoing total laryngo - pharyngectomy , a neopharynx is reconstructed which can be recognized as a simple tubular structure sans the accompanying complex anatomy of hypopharynx and larynx [ figure 4 ] . the neopharynx is often constructed by using a jejunal free flap , which is recognised recognized by the mesenteric fat along with its native vessels . axial fused pet / ct images show myocutaneous flap with fat density used to reconstruct the defect after hemimandibulectomy ( arrow in a ) and mandibular symphysectomy ( arrow in b ) performed for buccal cancer absence of normal physiological fdg uptake in the reconstructed flap ( arrowheads in a and b ) tissue changes after surgery . axial ct shows a reconstructed neopharynx after a laryngopharyngectomy seen as a tubular structure ( arrow in a ) . diffuse low grade physiological uptake is seen around the neopharynx ( arrow head in b ) during or after the course of treatment pitfalls in image interpretation can occur due to several factors . asymmetric nature of physiological uptake , inflammations / infective processes , and treatment - related complications are some of the common causes of false positive results leading to imaging pitfalls . sometimes , as a consequence of late effects of radiation therapy , there is a reduction of the normal physiological fdg uptake in structures such as the salivary glands and pharyngeal mucosal lining on the side of the therapy , leading to an appearance of asymmetric increased uptake in the normal tissues on the contralateral side [ figure 5 ] . coronal and axial ( pet ) and fused pet / ct images show reduction in physiological uptake ( arrows in a , b , d ) on the left side due to long term effects of radiation therapy . note the relative increase in physiological uptake in the parotid and submandibular glands and the oropharynx on the contralateral normal side which can mimic pathology ( arrowheads in a and d ) absence of physiological fdg uptake in the region of the surgically removed part and persistence of physiological uptake in the normal contralateral side produces an appearance of asymmetric tracer concentration that can mimic disease [ figure 6 ] . occasionally , after partial resection of an organ such as the tongue , the remnant portion retains its physiological uptake , which appears focal in nature mimicking disease [ figure 7 ] . after extensive jaw surgeries and reconstructive procedures , the altered mechanics of mastication can result in physiologically increased focal uptake in the adjacent masticator muscles that can be mistaken for disease [ figure 8 ] . axial ct shows changes of right hemiglossectomy with a myocutaneous flap containing fat ( arrowhead in a ) . axial pet and fusion pet / ct show physiological uptake in the remnant tongue which is focal and asymmetric in nature ( arrows in b and c ) and can potentially mimic disease pitfalls of due asymmetric physiological uptake . coronal pet and fusion pet / ct show physiological uptake along the right side of the oral cavity and the floor of mouth which is asymmetric in nature ( arrows in b and c ) and can potentially mimic disease . note the absence of normal physiological uptake on the left side ( arrowheads in b and c ) . this pattern was seen more than a year after treatment pitfalls of due asymmetric physiological uptake . axial pet and fusion pet / ct show focal asymmetric uptake in the left masseter which is physiological in nature ( arrows in a and b ) and is produced due to altered mechanics of mastication secondary to jaw surgery physiological uptake in the tongue tip and oral cavity arising due to close approximation of tongue and palate , buccal and gingival mucosae can be better resolved by performing certain maneuvers like puffing the cheek and placing a gauzegauze that can help mouth opening . surgical complications occur early and include serous collections , infections , abscess and fistula formation , flap necrosis . benign serous collections are self - limiting and should be distinguished from infected abscesses that might need a drainage procedure . benign serous collections also called seromas usually do not show any fdg avidity unless they get infected . on fdg pet / ct abscesses show a rim of tracer uptake in the periphery with a photopenic center which corresponds to a hypodense collection on ct with air pockets seen occasionally . linear fdg uptake is seen along the fistulous communications associated with abscesses [ figure 9 ] . these complications occur at the site of surgical resection and reconstructed flaps in the head neck region . rarely , one can come across infections / inflammations at the site of the donor flap in the chest or the abdominal wall and focal tracer uptake in this region can potentially mimic metastatic disease [ figure 10 ] . axial fused pet / ct shows an intense focus of fdg uptake in the infratemporal fossa mimicking disease recurrence ( arrow in b ) . corresponding contrast ct image shows a hypodense collection with an air pocket ( arrow in a ) suggestive of an abscess . note the linear fdg uptake along the enhancing fistulous tract opening on the skin surface . ( arrowheads in a and b ) pitfalls due to treatment related complications ( surgery ) . coronal mip image shows an intense focus of fdg uptake in the left hemithorax ( arrow in a ) which appears to be metastatic disease . fused pet / ct shows focal uptake in the anterior chest wall ( arrow in c ) which was the site for the pmmc flap . arrowhead in b ) radiation toxicity can lead to intense inflammatory changes in the mucosal structures and the soft tissues of the neck that can cause intense fdg accumulation [ figure 11 ] . coronal pet ( b ) and fused pet / ct ( c ) shows intense fdg uptake in the naso , oro , and hypopharyngeal structures and the soft tissue of the neck bilaterally , with associated ill - defined fat stranding ( a arrowhead ) . such intense non - infective inflammation is seen on rare occasions after radiation and should not be confused with recurrence radiation induced necrosis can occur in bones ( osteoradionecrosis [ orn ] ) , cartilage ( chondronecrosis / laryngeal necrosis ) and even in irradiated soft tissues . osteoradionecrosis ( orn ) occurs due to devitalization of irradiated bone which gets exposed through the skin and mucosa and remains without healing for at least 3 months . the risk of orn is greatest at 6 - 12 months after radiation therapy and it is uncommon to see orn at radiation doses below 60 gy . lytic destruction , cortical erosion and fragmentation of the mandible with associated fistulae and soft tissue thickening are some of the features seen on ct scan [ figure 12 ] . increased fdg avidity is seen in areas affected by orn [ figure 12 ] , but its specificity in differentiating viable tumor from radionecrosis is not reported to be very high . orn can occur at rare sites such as the hyoid bone and increased fdg avidity may lead to the erroneous diagnosis of viable disease unless the physician is aware of its occurrence [ figure 13 ] . characteristics osseous changes of orn and absence of associated soft tissue lesion make the diagnosis of radiation necrosis more likely axial ct in soft tissue ( a ) and bone window settings ( b ) show soft tissue thickening , erosion and fragmentation of the mandible ( arrows ) . coronal fused pet / ct shows intense fdg uptake restricted to right hemimandible ( arrowhead in c and d ) . findings suggest the diagnosis of orn pitfalls due to treatment related complications - osteoradionecrosis [ orn ] . axial fused pet / ct shows intense fdg uptake along the hyoid bone ( arrowhead in a and b ) . axial ct shows subtle erosion of the hyoid bone along with a small collection and air pockets ( arrows in c and d ) . sometimes , as a consequence of late effects of radiation therapy , there is a reduction of the normal physiological fdg uptake in structures such as the salivary glands and pharyngeal mucosal lining on the side of the therapy , leading to an appearance of asymmetric increased uptake in the normal tissues on the contralateral side [ figure 5 ] . coronal and axial ( pet ) and fused pet / ct images show reduction in physiological uptake ( arrows in a , b , d ) on the left side due to long term effects of radiation therapy . note the relative increase in physiological uptake in the parotid and submandibular glands and the oropharynx on the contralateral normal side which can mimic pathology ( arrowheads in a and d ) absence of physiological fdg uptake in the region of the surgically removed part and persistence of physiological uptake in the normal contralateral side produces an appearance of asymmetric tracer concentration that can mimic disease [ figure 6 ] . occasionally , after partial resection of an organ such as the tongue , the remnant portion retains its physiological uptake , which appears focal in nature mimicking disease [ figure 7 ] . after extensive jaw surgeries and reconstructive procedures , the altered mechanics of mastication can result in physiologically increased focal uptake in the adjacent masticator muscles that can be mistaken for disease [ figure 8 ] . axial ct shows changes of right hemiglossectomy with a myocutaneous flap containing fat ( arrowhead in a ) . axial pet and fusion pet / ct show physiological uptake in the remnant tongue which is focal and asymmetric in nature ( arrows in b and c ) and can potentially mimic disease pitfalls of due asymmetric physiological uptake . coronal pet and fusion pet / ct show physiological uptake along the right side of the oral cavity and the floor of mouth which is asymmetric in nature ( arrows in b and c ) and can potentially mimic disease . note the absence of normal physiological uptake on the left side ( arrowheads in b and c ) . this pattern was seen more than a year after treatment pitfalls of due asymmetric physiological uptake . axial pet and fusion pet / ct show focal asymmetric uptake in the left masseter which is physiological in nature ( arrows in a and b ) and is produced due to altered mechanics of mastication secondary to jaw surgery physiological uptake in the tongue tip and oral cavity arising due to close approximation of tongue and palate , buccal and gingival mucosae can be better resolved by performing certain maneuvers like puffing the cheek and placing a gauzegauze that can help mouth opening . surgical complications occur early and include serous collections , infections , abscess and fistula formation , flap necrosis . benign serous collections are self - limiting and should be distinguished from infected abscesses that might need a drainage procedure . benign serous collections also called seromas usually do not show any fdg avidity unless they get infected . on fdg pet / ct abscesses show a rim of tracer uptake in the periphery with a photopenic center which corresponds to a hypodense collection on ct with air pockets seen occasionally . linear fdg uptake is seen along the fistulous communications associated with abscesses [ figure 9 ] . these complications occur at the site of surgical resection and reconstructed flaps in the head neck region . rarely , one can come across infections / inflammations at the site of the donor flap in the chest or the abdominal wall and focal tracer uptake in this region can potentially mimic metastatic disease [ figure 10 ] . axial fused pet / ct shows an intense focus of fdg uptake in the infratemporal fossa mimicking disease recurrence ( arrow in b ) . corresponding contrast ct image shows a hypodense collection with an air pocket ( arrow in a ) suggestive of an abscess . note the linear fdg uptake along the enhancing fistulous tract opening on the skin surface . ( arrowheads in a and b ) pitfalls due to treatment related complications ( surgery ) . coronal mip image shows an intense focus of fdg uptake in the left hemithorax ( arrow in a ) which appears to be metastatic disease . fused pet / ct shows focal uptake in the anterior chest wall ( arrow in c ) which was the site for the pmmc flap . arrowhead in b ) radiation toxicity can lead to intense inflammatory changes in the mucosal structures and the soft tissues of the neck that can cause intense fdg accumulation [ figure 11 ] . coronal pet ( b ) and fused pet / ct ( c ) shows intense fdg uptake in the naso , oro , and hypopharyngeal structures and the soft tissue of the neck bilaterally , with associated ill - defined fat stranding ( a arrowhead ) . such intense non - infective inflammation is seen on rare occasions after radiation and should not be confused with recurrence radiation induced necrosis can occur in bones ( osteoradionecrosis [ orn ] ) , cartilage ( chondronecrosis / laryngeal necrosis ) and even in irradiated soft tissues . osteoradionecrosis ( orn ) occurs due to devitalization of irradiated bone which gets exposed through the skin and mucosa and remains without healing for at least 3 months . the risk of orn is greatest at 6 - 12 months after radiation therapy and it is uncommon to see orn at radiation doses below 60 gy . lytic destruction , cortical erosion and fragmentation of the mandible with associated fistulae and soft tissue thickening are some of the features seen on ct scan [ figure 12 ] . increased fdg avidity is seen in areas affected by orn [ figure 12 ] , but its specificity in differentiating viable tumor from radionecrosis is not reported to be very high . orn can occur at rare sites such as the hyoid bone and increased fdg avidity may lead to the erroneous diagnosis of viable disease unless the physician is aware of its occurrence [ figure 13 ] . characteristics osseous changes of orn and absence of associated soft tissue lesion make the diagnosis of radiation necrosis more likely ( b ) show soft tissue thickening , erosion and fragmentation of the mandible ( arrows ) . coronal fused pet / ct shows intense fdg uptake restricted to right hemimandible ( arrowhead in c and d ) . findings suggest the diagnosis of orn pitfalls due to treatment related complications - osteoradionecrosis [ orn ] . axial fused pet / ct shows intense fdg uptake along the hyoid bone ( arrowhead in a and b ) . axial ct shows subtle erosion of the hyoid bone along with a small collection and air pockets ( arrows in c and d ) . fdg pet / ct has been used to detect recurrent disease in the head and neck cancer patients . it can confirm the site of clinically suspected local recurrence and determine its true extent . recurrences at the site of the treated primary disease , subtle disease in the reconstructed flaps and nodal / soft tissue recurrence in the irradiated neck , the clinical assessment of which is difficult can be seen on pet / ct as enhancing lesions with intense fdg avidity [ figures 14 and 15 ] . at the same time , it can pick up distant failures with a good accuracy and thus is performed before salvage surgery can potentially change the intended treatment plan . in addition to common sites of distant metastases like the lungs and bones , pet / ct has the potential to unmask second primaries as well as certain unusual and rare metastatic sites such as the subcutaneous tissue , muscles , and feeding stomies [ figures 16 and 17 ] . recurrent disease . operated case of buccal cancer . coronal mip shows intense foci of fdg uptake on the left side of the face and neck ( arrowhead and arrows in a ) . axial fused pet / ct shows fdg avid recurrent nodule in the pmmc flap ( arrowhead in b and c ) and a metastatic cervical lymph node ( arrow in d and e ) recurrent disease . sagittal mip shows foci of fdg uptake in the face ( arrows in a ) . axial fused pet / ct ( arrowhead in b and c ) shows subtle uptake at the cut margin of the mandible due to local recurrence as well as metastatic deposit in the pterygopalatine fossa ( arrows in d and e ) distant metastases . fusion pet / ct images show metastatic deposits in spleen ( b ) , lungs ( c ) , liver ( d ) , bone ( e ) , subcutaneous region ( f ) and muscle ( g ) distant metastases ( unusual sites ) . operated case of base tongue cancer with feeding gastrostomy . sagittal pet and pet / ct images show a fdg avid mass in the anterior abdominal wall at the site of the gastrostomy ( arrows in a - b ) s / o metastatic deposit . local recurrence in the tongue base fdg pet / ct is a useful modality in the post - treatment setting of head and neck cancers . knowledge of certain characteristic imaging appearances and a few commonly encountered treatment related complications is important to avoid pitfalls in post - treatment imaging . understanding the role of fdg pet / ct in detecting local recurrence and distant metastases plays a crucial role in deciding salvage treatment .

majority of patients with head and neck cancer are treated with combined treatment regimes such as surgery , radiation therapy , and chemotherapy . the loss of structural symmetry and imaging landmarks as a result of therapy makes post - treatment imaging a daunting task on conventional modalities like computed tomography ( ct ) scan and magnetic resonance imaging ( mri ) as well as on 18 fluorine - fluorodeoxyglucose positron emission tomography / computed tomography ( 18f fdg pet / ct ) . combined multimodality treatment approach causes various tissue changes that give rise to a spectrum of findings on fdg pet / ct imaging , which are depicted in this atlas along with a few commonly encountered imaging pitfalls . the incremental value of fdg pet / ct in detecting locoregional recurrences in the neck as well as distant failures has also been demonstrated .

the bacterial isolates were collected throughout finland during 19982007 as described ( 10 ) . of bionumerics version 5.1 software ( applied maths , kortrijk , belgium ) was used for sequence assembly . allele numbers , sequence types ( sts ) , and clonal complexes ( ccs ) were assigned by using the pubmlst database ( 5 ) . a serum sensitivity assay was conducted with 73 c. jejuni isolates according to a described protocol ( 8) . the same pool of serum samples from 10 healthy blood donors was used in all experiments . c. jejuni all statistical analyses were performed by using graphpad prism version 4.03 ( graphpad software , san diego , ca , usa ) and pasw statistics version 18 ( spss inc . , chicago , il , usa ) . a total of 72 c. jejuni isolates from blood were successfully typed by mlst ; 1 isolate had a mixed mlst pattern . five isolates were in unassigned sts , and the rest were distributed among 11 ccs ( table ) . genetic relatedness of these isolates was further confirmed by using pulsed - field gel electrophoresis . * mlst , multilocus sequence typing ; st , sequence type ; nd , none detected . however , bacteremia episodes caused by st-677 cc isolates were exclusively diagnosed during the seasonal peak during may august ( figure 1 ) . of c. jejuni blood culture isolates detected during may august , most ( 64% ) were st-677 cc . furthermore , st-677 cc was the most prevalent complex in 4 geographic regions of finland . annual and seasonal distribution of 72 camplyobacter jejuni blood culture isolates belonging either to the st-677 clonal complex ( cc ) or to the other multilocus sequence typing ( mlst ) ccs . one isolate with a mixed multilocus sequence type was not included . c. jejuni bacteremia was diagnosed during may august ( m a ) or during any other month of the year ( o ) . susceptibility to human serum varied between c. jejuni isolates from different ccs ( figure 2 ) . st-677 cc isolates were significantly less susceptible to human serum than all other isolates ( p<0.0001 ) . st-45 cc isolates were significantly more susceptible to human serum than all other isolates ( p<0.0001 ) . percentage of surviving bacteria in human serum for 73 blood culture isolates of campylobacter jejuni ( cj ) , grouped according to major multilocus sequence typing clonal complexes ( ccs ) , and for controls c. jejuni cj11168 and c. fetus . we characterized a unique collection of 73 c. jejuni isolates from blood obtained during a nationwide study in finland over a 10-year period . despite the high population diversity of c. jejuni , nearly half of the isolates from blood showed clustering within the st-677 cc , a rare cc in other countries ( 12,13 ) . thus , invasiveness of blood culture isolates could not be solely explained by their serum resistance , although the predominant isolates of st-677 cc were more serum resistant than other isolates . c. jejuni has high st diversity . as of may 2 , 2013 , a total of 6,564 sts were registered ( 5 ) . in this study , we detected clustering of c. jejuni isolates from blood in an uncommon st-677 cc . further studies are needed to clarify whether bacterial characteristics might explain this finding . in our previous study , which included human fecal c. jejuni isolates obtained in finland from the mid-1990s through 2007 , which is nearly the same period as in the current nationwide study , 11.7% of the isolates belonged to st-677 cc ( 11 ) . the 2 most prevalent ccs in that study , st-45 cc ( 43.6% of fecal isolates ) and st-21 cc ( 19.4% of fecal isolates ) , were detected only among 12 ( 16% ) and 10 ( 14% ) of blood culture isolates , respectively , in the present study . st-45 cc and st-21 cc have been shown to be prevalent in several countries ( 4,13 ) . however , our results suggest that these 2 ccs are not common among c. jejuni isolates from blood in finland , which cluster more in the st-677 cc . on the basis of the present results , we speculate that st-677 cc might have a special invasive capability or has adapted to the environment in finland . in general , complement - mediated killing of serum - susceptible isolates plays a major role in restricting access of pathogens to the bloodstream . however , available information about possible serum sensitivity of c. jejuni isolates from blood is scarce ( 8,9 ) . in our study of nonselected c. jejuni isolates from blood , susceptibility to human serum varied according to mlst cc . in conclusion , in this nationwide study during a 10-year period in finland , we found by mlst analysis that half of the bacteremia isolates of c. jejuni clustered within an otherwise uncommon st-677 cc . whether this finding indicates special adaptation of st-677 cc to finland or to the human bloodstream our findings emphasize the role of using well - defined clinical materials in studies on bacterial pathogenicity and severity of human disease .

campylobacter jejuni bacteria are highly diverse enteropathogens . seventy - three c. jejuni isolates from blood collected in finland were analyzed by multilocus sequence typing and serum resistance . approximately half of the isolates belonged to the otherwise uncommon sequence type 677 clonal complex . isolates of this clonal complex were more resistant than other isolates to human serum .

in particular , osteoimmunology investigates how the immune system impacts on bone turnover in physiological and pathological conditions through the immunoskeletal interface . researchers recently hav - egained better understanding of the dialogue between immune and bone cells and a new reading register of bone remodeling is emerging , in which the various phases of bone formation and resorption , that coexist in a dynamic equilibrium , are under strict immunological control . bone and immune system are functionally integrated through complex homeostatic networks and , in all respects , osteoporosis could be considered a chronic immune mediated inflammatory disease which shares clinical and biological features with many other inflammatory conditions , as well as of other immune mediated diseases . in this context immune and bone systems appear to be integrated and sharing signaling pathways and regulatory mechanisms whose understanding provides a framework for obtaining new insights for the discovery of novel treatments for diseases related to both systems . thus osteoimmunology appears definitely as an interdisciplinary research and clinical field which also allowed new pathogenetic and clinical interpretations of well - known and common diseases , such as osteoporosis . its fields of interest are constantly expanding , thus enriching with an increasing number of translational implications , even in clinical practice and in various branches of medicine . here , the most important concepts in osteoimmunology are addressed in the context of physiopatological states bridging these two organ systems , including osteoporosis , ageing , menopause , inflammatory arthritis , cancer , dysmetabolism and neurological disorders . purpose of this review is to outline a new panorama of osteoimmunology that is not limited to immune mediated bone turnover but also consider , in the light of the latest findings in this field , interesting connections with other systems and regulatory functions over bone remodeling . bone is a dynamic tissue formed by a protein and mineral salt matrix in which are embedded the bone cells , osteocytes ( ocy ) , osteoblasts ( ob ) and osteoclasts ( oc ) . in addition , many other types of cells take part in bone composition , including cartilage , stromal , hematopoietic and mesenchimal stem cells , all linked by a dense network of signals . antagonistic signaling between skeletal stem cell - derived subsets is a key mechanism in skeletal subset lineage commitment . bone tissue undergoes continuous adaptation during lifetime to preserve the structure of the skeleton and to control the mineral homeostasis . bone turnover requires two coordinated processes : bone formation , driven by ob and bone resorption , mediated by oc . ocy , through a complex network of tiny channels , transmit mechanical and microtraumatic signals leading to the activation of repair processes . moreover , ocy synthesize the bone matrix proteins which , along with the mineral component , determine the quality of the bone . ob are the precursors of ocy , i.e , the structural cells in the bone , and interact with oc to drive their differentiation and function . the mesenchymal stem cell ( msc ) from which the ob originate , can also give rise to chondrocytes , marrow stromal cells , and adipocytes . there are multiple subpopulations of perisinusoidal mesenchymal stem / progenitor cells ( mspcs ) , that have specific relationships with the different kinds of niche , i.e. the surrounding microenvironment in which the self - renewal and multilineage stem cells proliferate and differentiate [ 7 - 9 ] . the stem cells that maintain and repair the postnatal skeleton is an osteochondroreticular ( ocr ) stem cell that generate ob , chondrocytes , and reticular marrow stromal cells , but not adipocytes . they are characterized by the expression of the bone morphogenetic protein ( bmp ) antagonist gremlin 1 ( grem 1 ) . the perisinusoidal msc population also contains nes - gfp , leptin receptor ( lepr)-cre and cd146 expressing cells with osteogenic and adipogenic potential . the osteoblast precursor cells ( obp ) after increasing the osteopontin receptor ( cd44 ) and the receptor for stromal cell - derived factor 1 - sdf1 ( cxcr4 ) expression , migrate and become mature ob , attracted by vascular endothelial cells expressing sdf1 along chemotactic gradients into regions of bone formation . oc are multinucleated myeloid cells , specialized to remove mineralized bone matrix through the production of lysosomal enzymes , such as tartrate - resistant acid phosphatase ( trap ) and catepsin k , against which a selective inhibitor ( odanacatib ) has been recently synthesized to be employed in osteoporotic patients . they derive from a bone marrow precursor which gives rise also to professional antigen presenting cells ( apc ) , i.e. dendritic cells and macrophages . ob , ocy and oc continuously communicate with each other to optimize the quality of the bone . for example , ob provide essential signals for the differentiation of the myeloid lineage precursors of oc by producing macrophage colony - stimulating factor ( m - csf ) , receptor activator of nuclear factor - kb ( nf - kb ) ligand ( rankl ) and other co - stimulatory factors . the binding of rank receptor on oc and their precursors by its ligand rankl , expressed by ob and stromal cells , is the main activation signal for bone resorption . the ob derived m - csf links to its receptor c - fms on the surface of osteoclast cell precursors ( ocp ) , enabling the rank / rankl signal . osteoprotegerin ( opg ) inhibits osteoclastogenesis by acting as a decoy receptor of rankl , thus preventing bone resorption . rank receptor on oc , through the adapter protein tumor - necrosis - factor - receptor - associated factor 6 ( tr - af6 ) , bound to its cytoplasmic tail , activates nf - kb and other transcription factors , such as mapks , c - fos , activator protein 1 ( ap1 ) , up to nuclear factor of activated t cells ( nfatc1 ) , the hub of various signaling pathways . simultaneously , the activation of rank induces the phosphorylation of ig - like receptor associated adaptor proteins , such as the immunoreceptor tyrosine - based activation motif ( itam ) and fc - receptor common gamma ( fcr ) subunit . in the nucleus nfatc1 , together with other transcription factors , such as ap1 , pu.1 , microphthalmia - associated transcription factor ( mitf ) and cyclic amp responsive - element - binding protein ( creb ) , induces oc specific genes , including those codifying for calcitonin receptor , cathepsin k and trap , leading to oc differentiation and proliferation . many other receptor pathways interact with rank , some costimulators and amplificators , others inhibitors and modulators , and many of these are shared by immune cells . an inhibitor receptor system for rank signal is ephrin ( eph ) b2/b4 . ephb2 receptor on oc , stimulated by ephb4 ligand on ob , inhibits the oc differentiation blocking c - fos and the nfatc1 transcriptional cascade . a peculiar property of this membrane receptor complex is its capacity to control bone turnover through bidirectional signals : the cell expressing the receptor and the one that expresses the ligand influence each other at the same time . therefore , ephb4 activation on ob , through the induction of osteogenetic regulatory genes , contemporaneously favours the coupling of bone formation and resorption . the canonical wingless ( wnt)/ catenin pathway , involved mainly in the response to mechanical load , promotes differentiation , proliferation and mineralization activity of ob and also inhibits their apoptosis . it encompasses a family of proteins that bind to complex transmembrane receptors , formed by the association of frizzled ( fz ) proteins and low density lipoprotein related receptors ( lrp-5 , lrp-6 ) which stabilize the -catenin substrate that concentrates in the cytosol and migrates into the obp nucleus to regulate the transcription of target genes to induce ob differentiation and bone formation . wnt signaling inhibits msc commitment towards adipogenic and chondrogenic lineages , stimulating the differentiation towards osteoblastogenesis . wnt--catenin signaling also indirectly inhibits osteoclastogenesis and bone resorption by increasing opg secretion in ob and ocy . the bmp pathway acts as a wnt associative stimulator signal in skeletogenesis by expanding primitive mesenchymal cells and thus laying the foundation for subsequent endochondral ossification . at the same time , various natural inhibitors , produced both by ob and ocy , exert a negative feed - back control on wnt system , such as the wnt/-catenin signaling pathway inhibitor dickkopf homolog-1 ( dkk-1 ) , the secreted frizzled related protein ( sfrp ) and the sclerostin , synthesized by the sost gene , which binds to lrp5/6 receptors . the inflammatory cytokine tumor necrosis factor- ( tnf- ) induces ob apoptosis and reduces osteoblastogenesis by stimulating dkk-1 and sost expression . cortison augments dkk-1 and sfrp expression thus suppressing the wnt/-catenin signal on ob inducing osteoporosis . an anti - sclerostin moab has recently been produced as a new osteoanabolic drug useful in the therapy of osteoporosis . other potential osteoanabolic drugs acting on the wnt pathway are also dkk inhibitor molecules , now in the experimental stage . bone is a dynamic tissue formed by a protein and mineral salt matrix in which are embedded the bone cells , osteocytes ( ocy ) , osteoblasts ( ob ) and osteoclasts ( oc ) . in addition , many other types of cells take part in bone composition , including cartilage , stromal , hematopoietic and mesenchimal stem cells , all linked by a dense network of signals . antagonistic signaling between skeletal stem cell - derived subsets is a key mechanism in skeletal subset lineage commitment . bone tissue undergoes continuous adaptation during lifetime to preserve the structure of the skeleton and to control the mineral homeostasis . bone turnover requires two coordinated processes : bone formation , driven by ob and bone resorption , mediated by oc . ocy , through a complex network of tiny channels , transmit mechanical and microtraumatic signals leading to the activation of repair processes . moreover , ocy synthesize the bone matrix proteins which , along with the mineral component , determine the quality of the bone . ob are the precursors of ocy , i.e , the structural cells in the bone , and interact with oc to drive their differentiation and function . the mesenchymal stem cell ( msc ) from which the ob originate , can also give rise to chondrocytes , marrow stromal cells , and adipocytes . there are multiple subpopulations of perisinusoidal mesenchymal stem / progenitor cells ( mspcs ) , that have specific relationships with the different kinds of niche , i.e. the surrounding microenvironment in which the self - renewal and multilineage stem cells proliferate and differentiate [ 7 - 9 ] . the stem cells that maintain and repair the postnatal skeleton is an osteochondroreticular ( ocr ) stem cell that generate ob , chondrocytes , and reticular marrow stromal cells , but not adipocytes . they are characterized by the expression of the bone morphogenetic protein ( bmp ) antagonist gremlin 1 ( grem 1 ) . the perisinusoidal msc population also contains nes - gfp , leptin receptor ( lepr)-cre and cd146 expressing cells with osteogenic and adipogenic potential . the osteoblast precursor cells ( obp ) after increasing the osteopontin receptor ( cd44 ) and the receptor for stromal cell - derived factor 1 - sdf1 ( cxcr4 ) expression , migrate and become mature ob , attracted by vascular endothelial cells expressing sdf1 along chemotactic gradients into regions of bone formation . oc are multinucleated myeloid cells , specialized to remove mineralized bone matrix through the production of lysosomal enzymes , such as tartrate - resistant acid phosphatase ( trap ) and catepsin k , against which a selective inhibitor ( odanacatib ) has been recently synthesized to be employed in osteoporotic patients . they derive from a bone marrow precursor which gives rise also to professional antigen presenting cells ( apc ) , i.e. dendritic cells and macrophages . ob , ocy and oc continuously communicate with each other to optimize the quality of the bone . for example , ob provide essential signals for the differentiation of the myeloid lineage precursors of oc by producing macrophage colony - stimulating factor ( m - csf ) , receptor activator of nuclear factor - kb ( nf - kb ) ligand ( rankl ) and other co - stimulatory factors . the binding of rank receptor on oc and their precursors by its ligand rankl , expressed by ob and stromal cells , is the main activation signal for bone resorption . the ob derived m - csf links to its receptor c - fms on the surface of osteoclast cell precursors ( ocp ) , enabling the rank / rankl signal . osteoprotegerin ( opg ) inhibits osteoclastogenesis by acting as a decoy receptor of rankl , thus preventing bone resorption . rank receptor on oc , through the adapter protein tumor - necrosis - factor - receptor - associated factor 6 ( tr - af6 ) , bound to its cytoplasmic tail , activates nf - kb and other transcription factors , such as mapks , c - fos , activator protein 1 ( ap1 ) , up to nuclear factor of activated t cells ( nfatc1 ) , the hub of various signaling pathways . simultaneously , the activation of rank induces the phosphorylation of ig - like receptor associated adaptor proteins , such as the immunoreceptor tyrosine - based activation motif ( itam ) and fc - receptor common gamma ( fcr ) subunit . in the nucleus nfatc1 , together with other transcription factors , such as ap1 , pu.1 , microphthalmia - associated transcription factor ( mitf ) and cyclic amp responsive - element - binding protein ( creb ) , induces oc specific genes , including those codifying for calcitonin receptor , cathepsin k and trap , leading to oc differentiation and proliferation . many other receptor pathways interact with rank , some costimulators and amplificators , others inhibitors and modulators , and many of these are shared by immune cells . an inhibitor receptor system for rank signal is ephrin ( eph ) b2/b4 . ephb2 receptor on oc , stimulated by ephb4 ligand on ob , inhibits the oc differentiation blocking c - fos and the nfatc1 transcriptional cascade . a peculiar property of this membrane receptor complex is its capacity to control bone turnover through bidirectional signals : the cell expressing the receptor and the one that expresses the ligand influence each other at the same time . therefore , ephb4 activation on ob , through the induction of osteogenetic regulatory genes , contemporaneously favours the coupling of bone formation and resorption . the canonical wingless ( wnt)/ catenin pathway , involved mainly in the response to mechanical load , promotes differentiation , proliferation and mineralization activity of ob and also inhibits their apoptosis . it encompasses a family of proteins that bind to complex transmembrane receptors , formed by the association of frizzled ( fz ) proteins and low density lipoprotein related receptors ( lrp-5 , lrp-6 ) which stabilize the -catenin substrate that concentrates in the cytosol and migrates into the obp nucleus to regulate the transcription of target genes to induce ob differentiation and bone formation . wnt signaling inhibits msc commitment towards adipogenic and chondrogenic lineages , stimulating the differentiation towards osteoblastogenesis . wnt--catenin signaling also indirectly inhibits osteoclastogenesis and bone resorption by increasing opg secretion in ob and ocy . the bmp pathway acts as a wnt associative stimulator signal in skeletogenesis by expanding primitive mesenchymal cells and thus laying the foundation for subsequent endochondral ossification . at the same time , various natural inhibitors , produced both by ob and ocy , exert a negative feed - back control on wnt system , such as the wnt/-catenin signaling pathway inhibitor dickkopf homolog-1 ( dkk-1 ) , the secreted frizzled related protein ( sfrp ) and the sclerostin , synthesized by the sost gene , which binds to lrp5/6 receptors . the inflammatory cytokine tumor necrosis factor- ( tnf- ) induces ob apoptosis and reduces osteoblastogenesis by stimulating dkk-1 and sost expression . cortison augments dkk-1 and sfrp expression thus suppressing the wnt/-catenin signal on ob inducing osteoporosis . an anti - sclerostin moab has recently been produced as a new osteoanabolic drug useful in the therapy of osteoporosis . other potential osteoanabolic drugs acting on the wnt pathway are also dkk inhibitor molecules , now in the experimental stage . both clinical observations and basic research demonstrated that mediators driving inflammatory processes are also closely involved in bone remodeling . inflammation and bone turnover share the same mediators , such as cytokines and transcription factors . various molecules mediating communication between bone cells have been identified and several immunological mediators are involved in this crosstalk . t lymphocytes resident in the bone marrow are the key immune cells that regulate bone remodeling and responsiveness of bone cells to parathyroid hormone ( pth ) , in physiological and pathological conditions . during inflammatory diseases or in conditions characterized by low - grade systemic inflammation , such as menopause and aging , oc bone resorption is driven by inflammatory cytokines produced by activated t lymphocytes . however , bone marrow t cells also support bone homeostasis by inducing bone formation via direct interactions with bone cells . two mechanisms are involved : the binding of t cell costimulatory molecules to their counter receptors on bone cells and their precursors , and the release of cytokines and wnt ligands that activate wnt signaling in osteoblastic cell lineage . the final effect of t lymphocytes on bone depends on their activation state and their specific phenotype . the prevailing bone marrow t cells are activated central memory cd8 + lymphocytes , secreting relatively high levels of effector cytokines , mainy tnf-. these cells are abundant in postmenopausal women with osteoporotic fractures . t helper ( th17 ) cells are capable of stimulating bone resorption and play a pivotal role in the bone loss of inflammatory conditions such as psoriasis , rheumatoid arthritis , periodontal disease , and inflammatory bowel disease . th17 cells induce osteoclastogenesis by secreting interleukin ( il)-17 , rankl , tnf- , il-1 , and il-6 , along with low levels of ifn-. moreover , il-17 stimulates the release of rankl by ob and ocy and upregulate rank expression on oc . treg exert anti - osteo - clastogenic activity by producing suppressor cytokines , including il-4 , il-10 , and tgf- . for example it is interesting to note how even the cells of the immune sytem , mainly activated t and b lymphocytes and dendritic cells , express rankl . moreover , ran - kl , the principal osteoclastogenic mediator , stimulates bone resorption through the nfatc1 , which is also a crucial factor in the immune system regulation . rankl , initially regarded as activator of apc by t lymphocytes , also plays an important role in the generation of treg , which suppress the development of cd8 + lymphocytes into cytotoxic cells . the expression of rankl on t lymphocytes is also central for the differentiation of medullary epithelial cells which are responsible for self - reactive t lymphocyte negative selection in the thymus . thus , depending on the context in which it acts , rankl can stimulate or suppress immune reactions . other examples of shared receptor signals are the immunoglobulin ( ig)-like receptors which amplify the nfatc1 signal . the toll like receptors ( tlr ) , stimulated by pathogen associated molecular patterns ( pamp ) , utilize traf6 in their cascade signaling . tlr are able to activate both the synthesis and release of proinflammatory and osteoclastogenic cytokines from immune cells , leading to bone resorption stimulation . their involvement in the bone remodeling process provides a further key in the comprehension of the osteoporosis of infectious diseases . the osteoclast associated receptor ( oscar ) , which belongs to the ig - like receptor family , mediates interactions between ob and oc and is also involved in the regulation of both the adaptive and innate immunity . it associates with the adaptor molecule fcr subunit , which harbors an immunoreceptor tyrosine - based activation motif ( itam ) critical for calcium signaling activation in the immune system . also , the itam - harboring adaptor dap12 plays a role in oc differentiation and function . therefore , receptors such as dap12 and ig - like receptors associated with fcr , initially characterized in myeloid cells and in natural killer lymphocytes , are also involved in rank induced osteoclastogenesis . tnf induces the expression of oscar and other receptors important for oc differentiation on the surface of monocytoid peripheral blood cells . for example , cd80/cd86 blocks oc generation by binding to ctla4 , an inhibitory molecule of the monocyte induced t lymphocyte costimulation , which is highly expressed on treg surface . cathepsin k is expressed in oc and plays a central role in the degradation of bone matrix components , such as type i collagen . in addition to osteoclastic bone resorption , cathepsin k is also implicated in dendritic cell activation through tlr 9 . moreover , cathepsin k supports the secretion of il-6 and il-23 , inflammatory cytokines involved in the production of th17 cells , which in turn promote osteoclastic bone resorption . eph receptors and their associated ligands , expressed by cells found within the bone marrow microenvironment , including ob and oc , are implicated in the regulation of physiological and pathological bone remodeling , but also are central in many other different cellular processes including , in addition to immune regulation , angiogenesis , neuronal development and neoplastic metastatization . osteoclast semaphorin 4d sustains bone resorption by inhibiting osteoblastogenesis . since sema4d also regulates a variety of immune functions , such as antigen presentation , b lymphocyte activation and chemotaxis of monocytes , it could be regarded as an osteoimmunological mediator . the matrix glycoprotein osteopontin ( opn ) , produced by different types of cells , including immune cells , oc , endothelial and epithelial cells , increases bone resorption by inducing the expression of the osteoclastic immune receptor cd44 , essential for cell migration , and by directly enhancing oc attachment to bone extracellular matrix ( ecm ) , required for ocp activation . as a consequence of bone resorption , more opn is further released from the ecm into the bone microenvironment and into the blood , thus amplifying local and systemic osteoclastogenesis . rankl and cd40l expressed on t cells , apc , stromal cells and ob , activate the cognate receptors rank and cd40 in ocp and ob , respectively . cd40/cd40l signaling promotes macrophage activation and differentiation , antibody isotype switching , and the development of b cell memory . cd40l also increases the commitment of msc to the osteoblastic lineage . through cd80/86 signaling in ocp boh these conditions are mimicked by continuous pth infusion ( cpth ) , whereas daily or intermittent pth injection ( ipth ) , therapeutically employed in several osteoporotic conditions , stimulates bone formation . pth binds its receptors ( ppr ) on stromal cells , ob , and ocy but also on t cells and macrophages . the catabolic effect of cpth is mostly mediated by enhanced production of rankl and decreased production of opg by ob and stromal cells . the pth anabolic effect is mediated by wnt signaling activation : pth increases -catenin levels in ob , promotes lrp6 signaling and decreases the production of sclerostin . cpth stimulates bone cells and immune cells to release growth factors and cytokines , including il-6 and tnf- , which induce th17 cell differentiation and the production of il-17 , that plays a pivotal role in the pth induced bone loss . tnf- in turn stimulates oc formation and activity via multiple mechanisms , including increased rankl production . moreover , tnf- upregulates the expression of cd40 in ob and stromal cells , increasing their sensitivity to cpth and suppressing opg . bone marrow t cells provide cell surface signals and secrete cytokines that direct the differentiation of mesenchimal progenitors towards ob characterized by a high sensitivity to pth . therapy with teriparatide , a form of ipth treatment , increases the bone marrow levels of wnt10. bone marrow cd8 + t cells potentiate the anabolic activity of pth by providing wnt10 . inflammation results in disturbances in the immunoskeletal interface , i.e. the convergence of cells and cytokines that regulate both the immunity that the bone , causing osteoporosis . the inflammatory cytokines tnf- , il-1 , il-6 and il-17 are crucial in acute and chronic inflammation and strong inducers of bone resorption . an excessive or abnormal immune activation can induce osteoporosis , as for example in autoimmune diseases , infections and also in senile and postmenopausal osteoporosis . all these conditions go along with an increased inflammatory background and the presence of rankl producing activated t cells . however , in addition to osteoclastogenic cytokines , there are also cytokines which counteract bone resorption and exert osteoblastogenic properties , resulting in a complex bone remodeling cytokine network . each cytokine has also pleiotropic functions and it is therefore not surprising that same cytokines can exert different and often contrasting effects depending on the specific context in which they act , the maturation stage of target cells and/or the influence of other cytokines . for example , the pleiotropic cytokine ifn- exerts anti - osteoclastogenic effects in physiological bone remodeling , by binding to specific oc receptors and inducing traf6 proteosomal degradation with consequent inhibition of the transduction signal mediated by rankl . however , in postmenopausal osteoporosis , inflammation or infections , the final effect of ifn- is skewed towards bone resorption through t lymphocyte activation and rankl expression . in fact , ifn- is a powerful stimulator of class ii major histocompatibility complex ( mhcii ) antigen expression on apc , with consequent increased stimulation of t cells through their antigen specific receptor ( tcr ) , inducing further immune activation and production of osteoclastogenic proinflammatory cytokines [ 35 - 40 ] . a cascade of cytokines drives ocp homing , differentiation and activation . circulating bone marrow produced ocp function as a tank of progenitor cells for several effector cells , in relation to the different cytokines implicated . the activation of cd8 t cells by oc induces il-2 , il-6 , il-10 and ifn- production . ocp can also enhance the expression of the suppressor of cytokine signaling ( socs ) . treg , whose main marker is the transcription factor foxp3 , balance il-17 induced bone resorption closely interacting with oc and expressing ctla-4 , which in turn inhibits oc activity . endothelial cells , activated by il-1 and tnf- , attract circulating ocp at sites of inflammation where they migrate through high endothelial venules driven by the expression of cell adhesion molecules ( cam ) , such as icam-1 and cd44 . these cd14 + monocytoid cells , under the stimulation of rankl , become activated bone resorbing oc . resident tissue macrophages of bone , termed osteal macrophages , are predominantly located adjacent to ob . osteal macrophages play diverse roles in skeletal homeostasis , their specific functions depending on the macrophage subset considered . a central function of macrophages is their phagocytic ability . in particular , efferocytosis ( phagocytosis of apoptotic cells ) is a critical process in both clearing dead cells and replacement of progenitor cells to maintain bone homeostasis . finally , not only the immune system regulates bone remodeling , but also the bone is able to influence the immune system , actively interacting with immune cells . the same bone cells would then be able to influence or even also perform many immune functions , such as cytokine production and antigen presentation [ 46 - 48 ] . in this sense , the bone would be regarded as a sort of expanded immune system . cytokines secreted by bone cells drive naive t cell differentiation into several lineages , leading to expansion of mature t cell populations that further regulate bone homeostasis . oc selectively recruit and activate cd8 + t cells expressing cd25 and foxp3 ( oc - induced regulatory cd8 t cells ) . in turn , these cd8 + treg cells suppress bone resorption , decrease inflammatory / osteoclastogenic cytokine production , and stimulate bone formation , creating a regulatory loop : oc and rankl induce treg , and then treg blunt osteoclastic bone resorption . osteocalcin , expressed on mature bone cells , regulates the production of thymic - seeding t lymphoid progenitors . they secrete a variety of proinflammatory and immunosuppressive factors . a subset of mesenchimal cells expressing osterix , a marker of bone precursors , regulate the maturation of early b lymphoid precursors by promoting pro - b to pre - b cell transition through insulin - like growth factor 1 ( igf-1 ) production . skeletal stem cells are also able to recruit and activate neutrophils via the release of il-6 and il-8 , ifn- , gm - csf and mif . they inhibit b cell proliferation , differentiation , and antibody production , and can also directly inhibit t cell function , rendering them anergic or shifting their phenotype to that of functional regulatory cells . msc induce macrophages to switch from classically activated proinflammatory ( m1 ) to alternatively activated anti - inflam - matory ( m2 ) phenotype , and inhibit mast cell degranulation attenuating allergic reactions . msc express active tlr , through which they sense bone microenvironment , recognizing exogenous ( bacterial products ) and endogenous ( heat shock proteins , rna ) danger signals . the common tlr signaling feature is the activation of the nf- b transcription factors implicated in controlling the expression of inflammatory cytokines and cell maturation molecules . both clinical observations and basic research demonstrated that mediators driving inflammatory processes are also closely involved in bone remodeling . inflammation and bone turnover share the same mediators , such as cytokines and transcription factors . various molecules mediating communication between bone cells have been identified and several immunological mediators are involved in this crosstalk . t lymphocytes resident in the bone marrow are the key immune cells that regulate bone remodeling and responsiveness of bone cells to parathyroid hormone ( pth ) , in physiological and pathological conditions . during inflammatory diseases or in conditions characterized by low - grade systemic inflammation , such as menopause and aging , oc bone resorption is driven by inflammatory cytokines produced by activated t lymphocytes . however , bone marrow t cells also support bone homeostasis by inducing bone formation via direct interactions with bone cells . two mechanisms are involved : the binding of t cell costimulatory molecules to their counter receptors on bone cells and their precursors , and the release of cytokines and wnt ligands that activate wnt signaling in osteoblastic cell lineage . the final effect of t lymphocytes on bone depends on their activation state and their specific phenotype . the prevailing bone marrow t cells are activated central memory cd8 + lymphocytes , secreting relatively high levels of effector cytokines , mainy tnf-. these cells are abundant in postmenopausal women with osteoporotic fractures . t helper ( th17 ) cells are capable of stimulating bone resorption and play a pivotal role in the bone loss of inflammatory conditions such as psoriasis , rheumatoid arthritis , periodontal disease , and inflammatory bowel disease . th17 cells induce osteoclastogenesis by secreting interleukin ( il)-17 , rankl , tnf- , il-1 , and il-6 , along with low levels of ifn-. moreover , il-17 stimulates the release of rankl by ob and ocy and upregulate rank expression on oc . treg exert anti - osteo - clastogenic activity by producing suppressor cytokines , including il-4 , il-10 , and tgf- . for example it is interesting to note how even the cells of the immune sytem , mainly activated t and b lymphocytes and dendritic cells , express rankl . moreover , ran - kl , the principal osteoclastogenic mediator , stimulates bone resorption through the nfatc1 , which is also a crucial factor in the immune system regulation . rankl , initially regarded as activator of apc by t lymphocytes , also plays an important role in the generation of treg , which suppress the development of cd8 + lymphocytes into cytotoxic cells . the expression of rankl on t lymphocytes is also central for the differentiation of medullary epithelial cells which are responsible for self - reactive t lymphocyte negative selection in the thymus . thus , depending on the context in which it acts , rankl can stimulate or suppress immune reactions . other examples of shared receptor signals are the immunoglobulin ( ig)-like receptors which amplify the nfatc1 signal . the toll like receptors ( tlr ) , stimulated by pathogen associated molecular patterns ( pamp ) , utilize traf6 in their cascade signaling . tlr are able to activate both the synthesis and release of proinflammatory and osteoclastogenic cytokines from immune cells , leading to bone resorption stimulation . their involvement in the bone remodeling process provides a further key in the comprehension of the osteoporosis of infectious diseases . the osteoclast associated receptor ( oscar ) , which belongs to the ig - like receptor family , mediates interactions between ob and oc and is also involved in the regulation of both the adaptive and innate immunity . it associates with the adaptor molecule fcr subunit , which harbors an immunoreceptor tyrosine - based activation motif ( itam ) critical for calcium signaling activation in the immune system . also , the itam - harboring adaptor dap12 plays a role in oc differentiation and function . therefore , receptors such as dap12 and ig - like receptors associated with fcr , initially characterized in myeloid cells and in natural killer lymphocytes , are also involved in rank induced osteoclastogenesis . tnf induces the expression of oscar and other receptors important for oc differentiation on the surface of monocytoid peripheral blood cells . for example , cd80/cd86 blocks oc generation by binding to ctla4 , an inhibitory molecule of the monocyte induced t lymphocyte costimulation , which is highly expressed on treg surface . cathepsin k is expressed in oc and plays a central role in the degradation of bone matrix components , such as type i collagen . in addition to osteoclastic bone resorption , cathepsin k is also implicated in dendritic cell activation through tlr 9 . moreover , cathepsin k supports the secretion of il-6 and il-23 , inflammatory cytokines involved in the production of th17 cells , which in turn promote osteoclastic bone resorption . eph receptors and their associated ligands , expressed by cells found within the bone marrow microenvironment , including ob and oc , are implicated in the regulation of physiological and pathological bone remodeling , but also are central in many other different cellular processes including , in addition to immune regulation , angiogenesis , neuronal development and neoplastic metastatization . osteoclast semaphorin 4d sustains bone resorption by inhibiting osteoblastogenesis . since sema4d also regulates a variety of immune functions , such as antigen presentation , b lymphocyte activation and chemotaxis of monocytes , it could be regarded as an osteoimmunological mediator . the matrix glycoprotein osteopontin ( opn ) , produced by different types of cells , including immune cells , oc , endothelial and epithelial cells , increases bone resorption by inducing the expression of the osteoclastic immune receptor cd44 , essential for cell migration , and by directly enhancing oc attachment to bone extracellular matrix ( ecm ) , required for ocp activation . as a consequence of bone resorption , more opn is further released from the ecm into the bone microenvironment and into the blood , thus amplifying local and systemic osteoclastogenesis . rankl and cd40l expressed on t cells , apc , stromal cells and ob , activate the cognate receptors rank and cd40 in ocp and ob , respectively . cd40/cd40l signaling promotes macrophage activation and differentiation , antibody isotype switching , and the development of b cell memory . cd40l also increases the commitment of msc to the osteoblastic lineage . through cd80/86 signaling in ocp boh these conditions are mimicked by continuous pth infusion ( cpth ) , whereas daily or intermittent pth injection ( ipth ) , therapeutically employed in several osteoporotic conditions , stimulates bone formation . pth binds its receptors ( ppr ) on stromal cells , ob , and ocy but also on t cells and macrophages . the catabolic effect of cpth is mostly mediated by enhanced production of rankl and decreased production of opg by ob and stromal cells . the pth anabolic effect is mediated by wnt signaling activation : pth increases -catenin levels in ob , promotes lrp6 signaling and decreases the production of sclerostin . cpth stimulates bone cells and immune cells to release growth factors and cytokines , including il-6 and tnf- , which induce th17 cell differentiation and the production of il-17 , that plays a pivotal role in the pth induced bone loss . tnf- in turn stimulates oc formation and activity via multiple mechanisms , including increased rankl production . moreover , tnf- upregulates the expression of cd40 in ob and stromal cells , increasing their sensitivity to cpth and suppressing opg . bone marrow t cells provide cell surface signals and secrete cytokines that direct the differentiation of mesenchimal progenitors towards ob characterized by a high sensitivity to pth . therapy with teriparatide , a form of ipth treatment , increases the bone marrow levels of wnt10. bone marrow cd8 + t cells potentiate the anabolic activity of pth by providing wnt10 . inflammation results in disturbances in the immunoskeletal interface , i.e. the convergence of cells and cytokines that regulate both the immunity that the bone , causing osteoporosis . the inflammatory cytokines tnf- , il-1 , il-6 and il-17 are crucial in acute and chronic inflammation and strong inducers of bone resorption . an excessive or abnormal immune activation can induce osteoporosis , as for example in autoimmune diseases , infections and also in senile and postmenopausal osteoporosis . all these conditions go along with an increased inflammatory background and the presence of rankl producing activated t cells . however , in addition to osteoclastogenic cytokines , there are also cytokines which counteract bone resorption and exert osteoblastogenic properties , resulting in a complex bone remodeling cytokine network . each cytokine has also pleiotropic functions and it is therefore not surprising that same cytokines can exert different and often contrasting effects depending on the specific context in which they act , the maturation stage of target cells and/or the influence of other cytokines . for example , the pleiotropic cytokine ifn- exerts anti - osteoclastogenic effects in physiological bone remodeling , by binding to specific oc receptors and inducing traf6 proteosomal degradation with consequent inhibition of the transduction signal mediated by rankl . however , in postmenopausal osteoporosis , inflammation or infections , the final effect of ifn- is skewed towards bone resorption through t lymphocyte activation and rankl expression . in fact , ifn- is a powerful stimulator of class ii major histocompatibility complex ( mhcii ) antigen expression on apc , with consequent increased stimulation of t cells through their antigen specific receptor ( tcr ) , inducing further immune activation and production of osteoclastogenic proinflammatory cytokines [ 35 - 40 ] . a cascade of cytokines drives ocp homing , differentiation and activation . circulating bone marrow produced ocp function as a tank of progenitor cells for several effector cells , in relation to the different cytokines implicated . the activation of cd8 t cells by oc induces il-2 , il-6 , il-10 and ifn- production . ocp can also enhance the expression of the suppressor of cytokine signaling ( socs ) . treg , whose main marker is the transcription factor foxp3 , balance il-17 induced bone resorption closely interacting with oc and expressing ctla-4 , which in turn inhibits oc activity . endothelial cells , activated by il-1 and tnf- , attract circulating ocp at sites of inflammation where they migrate through high endothelial venules driven by the expression of cell adhesion molecules ( cam ) , such as icam-1 and cd44 . these cd14 + monocytoid cells , under the stimulation of rankl , become activated bone resorbing oc . resident tissue macrophages of bone , termed osteal macrophages , are predominantly located adjacent to ob . osteal macrophages play diverse roles in skeletal homeostasis , their specific functions depending on the macrophage subset considered . a central function of macrophages is their phagocytic ability . in particular , efferocytosis ( phagocytosis of apoptotic cells ) is a critical process in both clearing dead cells and replacement of progenitor cells to maintain bone homeostasis . finally , not only the immune system regulates bone remodeling , but also the bone is able to influence the immune system , actively interacting with immune cells . the same bone cells would then be able to influence or even also perform many immune functions , such as cytokine production and antigen presentation [ 46 - 48 ] . in this sense , the bone would be regarded as a sort of expanded immune system . cytokines secreted by bone cells drive naive t cell differentiation into several lineages , leading to expansion of mature t cell populations that further regulate bone homeostasis . oc selectively recruit and activate cd8 + t cells expressing cd25 and foxp3 ( oc - induced regulatory cd8 t cells ) . in turn , these cd8 + treg cells suppress bone resorption , decrease inflammatory / osteoclastogenic cytokine production , and stimulate bone formation , creating a regulatory loop : oc and rankl induce treg , and then treg blunt osteoclastic bone resorption . osteocalcin , expressed on mature bone cells , regulates the production of thymic - seeding t lymphoid progenitors . a subset of mesenchimal cells expressing osterix , a marker of bone precursors , regulate the maturation of early b lymphoid precursors by promoting pro - b to pre - b cell transition through insulin - like growth factor 1 ( igf-1 ) production . skeletal stem cells are also able to recruit and activate neutrophils via the release of il-6 and il-8 , ifn- , gm - csf and mif . they inhibit b cell proliferation , differentiation , and antibody production , and can also directly inhibit t cell function , rendering them anergic or shifting their phenotype to that of functional regulatory cells . msc induce macrophages to switch from classically activated proinflammatory ( m1 ) to alternatively activated anti - inflam - matory ( m2 ) phenotype , and inhibit mast cell degranulation attenuating allergic reactions . msc express active tlr , through which they sense bone microenvironment , recognizing exogenous ( bacterial products ) and endogenous ( heat shock proteins , rna ) danger signals . the common tlr signaling feature is the activation of the nf- b transcription factors implicated in controlling the expression of inflammatory cytokines and cell maturation molecules . osteoporosis is a systemic disease of the skeleton , whose main features are loss of bone mass , bone mineral density ( bmd ) decrease and disruption of bone microarchitecture , so the skeleton becomes fragile , exposing patients to increased risk of fractures . aside from senile and postmenopausal osteoporosis , the first recognized types of primary osteoporosis , many other causes of secondary osteoporosis have been subsequently recognized , for example vitamin d and calcium deficits , lack of sun exposure , immobility , drugs such as cortisone , malabsorption syndromes , endocrine and dismetabolic diseases such as diabetes , disthyroidisms , hypercortisolism , and so on . only later , clinical and experimental findings evidenced a close connection between osteoporosis and immune mediated inflammatory conditions , for example , rheumatoid arthritis ( ra ) and acquired immune deficiency syndrome ( aids ) , and a common inflammatory background has been finally discovered as pathogenetic factor even in conditions of major osteoporotic risk , such as old age and estrogen deficiency [ 57 - 59 ] . more recently , other unpredictable pathological conditions , such as obesity , are coming out as potential osteoporotic risk factors . even in these cases , the main pathogenetic mechanism leading to bone tissue alteration seems to be inflammation . from this point of view osteoporosis could be therefore regarded as an immune mediated disease in which immune activation , through the induction of cytokine production and inflammation , leads to a remodeling of oc and ob activity and dysregulation of bone turnover with consequent increased bone resorption and osteoporosis . paradigmatic examples of the link between inflammation and osteoporosis are inflammatory arthritis , mainly ra ( fig . 1 ) . ra is an autoimmune disease that is characterized by inflammation of the synovial joint , leading to severe structural damage and bone destruction . an increased bone resorption is the main pathogenetic mechanism in both disease progression leading to juxta - articular bone erosions and irreversible joint damage and systemic osteoporosis . bisphosphonates , drugs used for some time in the therapy of osteoporosis , are potent inhibitors of oc activity both in the primitive and secondary osteoporosis , such as that associated with autoimmune diseases . a decreased bmd in the spine and hip and higher prevalence of osteoporosis have been described in ra patients . in early untreated ra , bmd is related to longer symptom duration , the presence of rheumatoid factor ( rf ) and cyclic citrulinated peptide antibodies ( anti - ccp ) , disease activity score , and the presence and progression of joint damage [ 1 , 19 ] . monoclonal antibodies ( moab ) against various proinflammatory cytokines and their receptors , such as tnf- , are useful in preventing and/or reversing bone erosions as well as systemic osteoporosis . autoimmune reactions induce rankl expression and subsequent osteoclastogenesis . that the rank/ rankl / opg pathway is central to the osteoporosis pathogenesis is confirmed by the elevated antiresorptive capacity of denosumab , an anti - rankl moab , utilized in osteoporosis therapy . activated immune cells at sites of inflammation produce a wide spectrum of proinflammatory and osteoclastogenic cytokines , resulting in bone erosions , osteitis , and peri - inflammatory and systemic bone loss . local peri - inflammatory bone loss and osteitis occur early and precede and predict erosive bone destruction in ra . moreover , peri - inflammatory bone formation is impaired , resulting in non - healing of erosions , and this allows a local vicious circle of inflammation between synovitis , osteitis , and local bone loss . rankl is highly expressed in the ra synovium , and inflammation - mediated bone damage is largely attributable to its abnormally high expression . in addition to activated t lymphocytes and macrophages , a pivotal role in inducing bone erosions is also played by rankl - expressing b cells , as highlighted by the observation of the therapeutic effect of anti - cd20 antibody in ra . cd4+t - cells , especially th17 cells , play a prominent role , particularly in the initiation of systemic immune response in ra . the interaction between immune and mesenchymal cells in joints , including synovial fibroblasts , which are characterized by hyperproliferative and hyperactive properties in response to an inflammatory environment , is of paramount importance in rheumatoid inflammation . in fig . thus , in the affected joints , hyperplasia of the synovial membrane is characterized by both hyperproliferation of synovial fibroblasts and massive infiltration of inflammatory immune cells , including cd4 + t cells and innate immune cells . autoimmune diseases , including arthritis , often result from an imbalance between treg cells and th17 cells . the th17 cells derived from foxp3 + t cells in ra comprise a novel th17 cell subset with a distinct pattern of gene expression and arthritogenic properties . the fate of plastic foxp3 + t cells may be a key determinant of the treg / th17 balance that is critically involved in the self - tolerance and autoimmunity . mesenchymal cells are a determinant of the development of ra that links the systemic immune response and the local disorder in the joints . mesenchymal cells contribute to the th17 mediated chronic inflammation by promoting the migration of th17 cells to the inflamed joint and concomitant increase in il-17 production . thus , the interaction of immune and mesenchymal cells plays a key role in both the chronic inflammation and bone destruction in ra . in particular , pathogenetic autoreactive immune cells migrate into joints and activate the mesenchymal cells resident in joint , such as synovial fibroblasts . moreover , since soluble inhibitors of the wnt pathway , such as dkk-1 , produced by synovial fibroblasts , are upregulated by tnf- , antibodies against dkk1 could be able to both promote bone formation and prevent bone erosions in ra . osteoporosis has long been regarded as simply the consequence of the menopausal estrogen decline . os- the progression of articular erosions is clearly driven by proinflammatory and osteoclastogenic cytokines produced by immune cells in the inflamed joint . both activated lymphocytes and macrophages stimulate osteoclast differentiation by producing proinflammatory mediators . activated lymphocytes of the synovial pannus overexpress rankl and tnf- that stimulate bone marrow cd11b+ ocp to proliferate and enter the bloodstream where they themselves produce inflammatory factors amplifying inflammation . activated macrophages in the inflamed joints produce various chemokines which drive the localization of periarticular bone ocp . the stimulation of osteoclastogenesis induced by the high concentrations of rankl and tnf- results in bone resorption . these cells do not produce antiosteoclastogenic cytokines , such as ifn- or il4 , while expressing high levels of rankl and secrete il-17 , that in turn stimulates ob , synoviocytes and fibroblasts to express rankl , and induces macrophages to produce proinflammatory cytokines , such as tnf- and il-6 in the synovium amplifying local inflammation . il-17 also induces the synthesis of enzymes capable of degrading the bone matrix such as matrix metalloproteinases ( mmp ) . these effects are balanced by treg that inhibit bone destruction by suppressing oc formation through both cell - cell contact and the secretion of inhibitory cytokines such as tgf- , il-4 and il-10 . teoimmunology , suggesting that immune cells take part in the bone changes typical of menopause , has led to a shift in the concept of osteoporosis , that is currently considered an inflammatory condition . post - menopausal osteoporosis is a clear example of the mutual influences between immune system , bone and endocrine system . like many other hormones , in addition to specific target organs ( breast and reproductive system ) , estrogens have their receptors also on immune cells and bone , as well as on bone marrow precursors . ( 3 ) shows the effects of estrogen deficiency on cells and molecules involved in bone metabolism . menopausal estrogen decline leads to proliferation and activation of t cells by increasing mhcii expression on monocytes and apc function and by inhibiting t cell apoptosis . these effects leads to the expansion of activated t lymphocytes resulting in hyperproduction of inflammatory cytokines and chronic oc stimulation which is responsible for bone loss and increased fracture risk . maintenance of inflammatory reactions leading to bone resorption and skeletal fragility is also present during senescence and inflammaging ( fig . 4 ) , that is the condition of chronic inflammation characterizing aging , as the result of the immune system s ability to cope with stressors . inflammaging is now considered the background of a broad range of age - related diseases with an inflammatory pathogenesis . many of the biological mechanisms implicated in the aging process , such as cell senescence , proinflammatory immune profile , apoptosis and metabolism imbalance , are also implicated in bone remodeling . also in the absence of overt inflammatory diseases , the heightened catabolic signals induced by inflammation enhance apoptosis of ob and muscle cells , causing both osteoporosis and sarcopenia . during aging , the lifelong exposure to oxidative stress and chronic antigenic load leads to the loss of the regulatory process which counteracts t cell activation induced bone resorption . in aged people , lipid oxidation mediated by ros oxidized polyunsaturated fatty acids induce the association of ppar with -catenin and promote its degradation . oxidized lipids also potentiate oxidative stress , enhance ob apoptosis and inhibit bmp-2 induced ob differentiation . antioxidant agents seem to have some action on bone remodeling : resveratrol decreases nf - k activation induced by rankl and oc differentiation and also promotes osteogenesis in msc via the sirt1/foxo3 axis stimulation . during senescence , besides the impaired treg function , the number of effector memory cells is increased . these are senescent cells with proinflammatory properties , secreting several inflammatory cytokines able to influence bone remodeling . interestingly , this immunological pattern ( accumulation of activated cells and memory / effector lymphocytes secreting proinflammatory cytokines ) characterizing immunosenescence , is also peculiar of other immunological conditions associated with osteoporosis , such as ra , aids , chronic viral infections , etc . . the already complex cross - talk between bone and immune system is further expanding as they emerge new mediators involved . interestingly , the scenario of osteoimmunolgy is increasingly including interconnections with other organs and systems , therefore influencing several homeostatic functions in addition to bone turnover and immunity , such as hemopoiesis , metabolic and neuro - endocrine functions . the crosstalk between skeletal and bone marrow is crucial to hematopoietic stem cell ( hsc ) function and throughout the hemopoiesis . hsc occupy specific locations in the bone marrow microenvironment , commonly referred to as niches , comprising multiple cell types able to regulate hsc proliferation and differentiation . hematopoietic and skeletal stem cell homeostasis are closely related : msc progeny express numerous cytokines necessary for hsc maintenance and hematopoiesis , including kit ligand and stromal - derived factor , but also stem and progenitor cells of the hematopoietic system may reciprocally regulate skeletal progenitors by expressing myriad factors associated with skeletogenesis , whose cognate receptors are highly expressed by skeletal stem cells . interestingly , these latter exhibit receptors to circulating hormones , such as leptin and thyroid - stymulating hormone , suggesting that skeletal stem cells and their progeny may link systemic exocrine regulation to both skeletal and hematopoietic system . primitive mesenchymal progenitors are more critical for hsc function , whereas lineage - restricted mesenchymal cells control more committed hematopoietic progenitors . bone microenvironment is therefore a composite of specialized niches providing distinctive functional units to regulate hematopoiesis . the estrogen deficiency induces a marked increase of inflammatory cytokines and mediators of inflammation , in particular ifn- , m - csf , tnf- , il-1 , il-6 and il-7 , in addition to prostaglandins ( pge ) and reactive oxygen species ( ros ) , which play a driving role in the development of osteoporosis . of particular interest is the expansion in the peripheral blood from postmenopausal women of two particular lymphocyte subsets , cd3 + cd56 + t lymphocytes , major producers of tnf- , and b220 + igm - b lymphocyte precursors , that under certain stimuli can differentiate into ocp and are strong producers of inflammatory cytokines . the enhanced tnf- production by activated t cells has a central role in bone loss due to estrogen decline in menopausal women . the same ifn- , which in some situations could be protective against osteoporosis , during estrogen deficiency has a prevailing osteoclastogenic stimulating action . the estrogen decline also results in the decrease of opg and tgf- that normally contrast the effects of inflammatory mediators on bone . the age - related increase of inflammatory cytokines results from a chronic activation of macrophages and memory / effector t cells , in addition to an impaired treg function . a peculiar finding of immunosenescence is the increased number of senescent memory cells expressing rankl and secreting osteoclastogenic cytokines , mainly tnf- , il-1 , il-6 and il-17 . these cytokines are able to facilitate ocp expansion which amplify the systemic inflammation by producing further proinflammatory factors able to recruit other inflammatory cells and perpetuating the flogistic vicious cycle . both the increased transcriptional activity of nf - kb due to genotoxic , inflammatory , and oxidative stresses in aging , and the chronic p53 activation induced by the age - related progressive loss of telomere length , result in impaired ob proliferation and osteoporosis development . cells of the immune system and bone cells derive from bone marrow precursors and develop in the same bone marrow microenvironment . hematopoietic and immune cells share same signaling pathways with cells of the bone and their precursors , which surprisingly do not take part in the regulation of immunity and hemopoiesis . ob , together with msc , are crucial components of the niche of growth of hsc . ob overexpression of the wnt antagonist dkk1 reduces wnt signaling in hsc and disrupts hsc self - renewal potential . the wnt antagonist secreted frizzled - related protein 1 ( sfrp1 ) is involved in osteoblastic mediated hsc regulation ; through its regulation of ob , pth controls the hematopoietic niche function , involving crosstalk with wnt signaling . the upregulation of notch ligand protein jagged-1 ( jag1 ) expression in ob and the increased notch signaling are involved in increasing hsc numbers ; angiopoietin-1 receptor activation on hsc by ob produced angiopoietin-1 promotes strict adhesion of hsc to the niche , inducing quiescence of these cells . osteopontin , an ob - secreted protein , participates in hsc location and is a negative regulator of their proliferation . also , ocy derived from ob that become embedded within the bone matrix , are involved in the complex regulation of hemopoiesis through the osteoimmune interface : mainly by the production of sclerostin , they appear to have an inhibitory effect on hsc support . even oc , as well as activated endothelial cells , are likely involved in the mobilization of hsc by cytokine induced cam expression . being the only cells capable of bone resorption , in addition to allowing the renewal of the skeleton , they also open the space in the bone marrow for hematopoietic cells . moreover , osteoclastic bone resorption releases calcium , which attracts and retains calcium sensing receptor expressing hsc at the endosteal region . macrophages play diverse roles in the bone and marrow . at the sites of bone remodeling , a subset of hsc is located in close proximity to megakaryocytes in the bone marrow , where ob undergo rapid expansion in response to the secretion of megakaryocyte - derived mesenchymal growth factors , such as platelet derived growth factor ( pdgf)- , to promote hsc proliferation . the bone marrow microenvironment can also act as a niche for the onset and progression of neoplastic diseases , including both hematologic malignancies and metastases of solid tumors , mainly breast and prostatic cancer . in multiple myeloma , an osteolytic hematological malignancy characterized by an important skeletal involvement , neoplastic plasma cells produce a large amount of mediators that induce osteoclastic bone resorption and block the activity of ob . in addition to osteolytic factors , myeloma cells produce dkk1 , which inhibits obp differentiation by binding to the lrp5/6 coreceptors expressed on their surface . on the other hand , the same bone cells produce growth factors and angiogenetic cytokines able to support the development and progression of myeloma , perpetuating a vicious circle of mutual reinforcement . a promising new field of interest in this regard is the osteo - immune - oncology . there is in fact a close relationship between immune regulation of bone turnover and tumor growth . often , cancer cells express rank and in proneoplastic inflammation various cell types express rankl that acts as chemotactic factor favoring the skeletal metastasis . tumor cells in the bone cause activation of osteoclasts that mediate bone resorption and additional growth factor release from the bone matrix , fueling a vicious circle of impaired bone turnover and tumor proliferation ( fig . not always the tumor associated inflammation is the expression of an immune system that successfully opposes the neoplastic growth , as previously believed , but sometimes some patterns of immune activation can be facilitators of the development of tumors . then the block of rankl , in addition to inhibiting bone resorption , also decreases tumor growth , enhances apoptosis of malignant cells and diminishes proneoplastic inflammation . osteal macrophages , as well as macrophages in other tissues , have a role in tumor progression , supporting cancers which preferentially metastasize to the skeleton , in particular breast and prostate cancer . osteal macrophages expressing cd68 , a phagocytic capacity marker of cells infiltrating metastatic lesions , could facilitate tumor establishment and growth . tumor - derived pthrp drives myeloid cell recruitment via ob produced ccl2 , which is high in the bone microenvironment and whose levels are associated with poor prognoses in primary breast tumors . as for bone , adipose tissue is a kind of immune tissue and produces immunoregulatory factors . adipocytes and ob derive from the same msc , as well as visceral adipose tissue macrophages and oc derive from the same hsc . shared transcription factors regulate the shift between the different cell lines and the subsequent differentiation cascade . by action of peroxisome proliferative activated receptor gamma ( ppar ) , the principal regulator of adipogenesis , the msc differentiates into adipocytes rather than into ob . in contrast , the expression of runt - related transcription factor 2 ( runx2 ) , associated with ob differentiation , and osterix , an ob zinc finger mechanical loading promotes ob differentiation and inhibits adipogenesis by down - regulating ppar or by stimulating a durable -catenin signal . not surprisingly , ppar agonists such as thiazolidinediones used to increase insulin sensitivity in type 2 diabetes , also increase the risk of osteoporotic fractures . leptin is a pro - inflammatory adipokine that exerts its actions via central nervous system regulation of feeding behavior , where it promotes satiety and prevents weight gain . leptin can also directly signal through its receptor expressed on immunocytes , where it induces tnf- and il-6 production by monocytes , chemokines by macrophages , and th1 cytokines from polarized cd4 + t cells . adiponectin is an anti - inflammatory adipokine whose plasma levels are strongly correlated with insulin sensitivity and glucose tolerance . it can directly interfere with inflammatory cytokine production in macrophages and can induce expression of the anti - inflammatory cytokine il-10 . tnf- and il-6 inhibit adiponectin production in adipocytes . adipose tissue produced pro - inflammatory cytokines and adipokines further modulate the activity of oc and ob . fat tissue , mainly visceral fat tissue , may increase bone resorption through the production of inflammatory cytokines such as il- 6 and tnf- , which stimulate oc activity through the regulation of the rankl / rank / opg pathway . leptin and adiponectin act on the bone through different signaling pathways with contrasting effects ( table 2 ) . it promotes adipogenesis and inhibits osteogenesis in response to diet and adiposity by activating jak2/stat3 signaling . therefore , leptin receptors on skeletal msc function as a sensor of systemic energy homeostasis . various cell populations within the fat tissue can exacerbate the development of the chronic , low - grade inflammation associated with obesity and metabolic dysfunction . white adipocytes store lipid as triglycerides within unilocular droplets , and are responsive to various stimuli , such as insulin . brown adipocytes store lipid in multilocular droplets that are quickly catabolized for fuel when the tissue is stimulated . beige adipocytes , dispersed within white adipose tissue , can dissipate heat , similarly to classical brown adipocytes , when exposed to cold temperatures or after prolonged highfat diet feeding . visceral fat tissue infiltrating macrophages in the setting of diet - induced obesity , have a pro - inflammatory phenotype which initiates and/or exacerbates the chronic inflammation that contributes to adipocyte dysfunction in obesity . conversely , in lean humans adipose tissue macrophages may promote tissue remodeling and temper inflammation by secreting anti - inflammatory cytokines . notwithstanding epidemiological evidence indicates that an increase in body mass index ( bmi ) is related to increased bone mass , probably due to the effects of the mechanical load of the body weight on the skeleton , not always obesity , and mainly the increase in visceral fat mass , has a positive effect on bone . obesity is associated with increased leptin and decreased adiponectin serum levels . moreover , in the visceral adipose tissue there are activated macrophages producing cytokines . in obese subjects , especially with central obesity , in which the visceral fat is increased , there is a significant increase of several markers of inflammation such as c reactive protein ( crp ) , il-1 , il-6 and tnf- , that can alter the quality of the bone , making it more fragile . therefore , these new clinical and experimental evidences definitively connect obesity and other related pathological conditions , such as metabolic syndrome , nonalcoholic fatty liver disease ( nafld ) and diabetes , to impaired bone health and fragility fractures [ 90 - 93 ] . in conclusion , it is currently emerging that adipose tissue , liver , bone and immune system modulate each other through a complex network of interconnected signals . both adipocytes and ob express opg and rankl and their modulation is influenced by adipokines , sex hormones , redox balance , ppar and liver x receptors ( lxr ) . osteocalcin , an ob secreted bone matrix noncollagen protein , takes part in calcium metabolism and in bone deposition , as well as in energy homeostasis and glucose metabolism . fetuin - a , produced by the liver , is involved in the regulation of bone metabolism and insulin action , vascular calcification , neurodegenerative diseases and cancer cell proliferative signaling . ob lineage cells express receptors for adiponectin , leptin , angiotensin ii , insulin and insulin - like growth factor-1 , able to influence rank / rankl / opg signaling pathway . interestingly , these hormones are implicated in the pathogenesis of type - ii diabetes , obesity and hypertension , all of which are risk factors for metabolic syndrome . the development and progression of diabetes - associated osteoporosis are mediated by the interaction between advanced glycation end products ( age ) and receptor of age ( rage ) . age are the end products of glucose , as well as other sugars , proteins , lipids , and nucleic acids via a non - enzymatic glycosylation reaction , able to bind to multiple membrane receptor proteins , including rage . age / rage interaction is involved in the pathophysiological processes of many inflammatory and dysmetabolic diseases . in particular , age are involved in the development and progression of osteoporosis by inhibiting proliferation and inducing ob apoptosis . the binding of age to organic bone matrix may also increase the fragility of bones . autophagy is a metabolic process by which eukaryotic cells degrade and recover damaged macromolecules and organelles into autophagosomes autophagy is upregulated in stressful conditions . however , excessive autophagy is harmful to cells and leads to damage or massive death of cells . autophagy deficiency increases oxidative stress levels in ob , decreases bone mineralization and induces ran - kl secretion . it is well eatablished that several neuroendocrine pathways modulate both immune responses and bone remodeling . in turn sympathetic nerves produce catecholamines , which are delivered to the bone microenvironment by the blood circulation or by secretion from the nerve endings . hsc express catecholaminergic receptors , suggesting that they are able to directly respond to signals from the sympathetic nervous system . adrenergic signaling reduces cxcl12 expression in the bm . affecting maintenance of hsc and the differential lineage commitment . an association between major depression and osteoporosis has been recognized [ 96 - 99 ] . the prevalence of low bmd is higher in people with depression than the general population . interestingly , patients on antidepressant therapy with selective serotonin reuptake inhibitors develop decreased bmd and increased risk of fracture compared to those treated with tricyclic antidepressants such as amitriptyline or patients with untreated depression , who also have a lower bmd compared with healthy controls [ 100 , 101 ] . neuroendocrine abnormalities of the hypothalamo - pituitary - adrenal ( hpa ) and sympathoadrenal axes are a common finding in depressed patients leading to increased catecholamine turnover and hypersecretion of corticotropin - releasing hormone ( crh ) . leptin is expressed in the hypothalamus and pituitary gland , where it modulates corticotropin - releasing hormone and acth secretion , probably acting in an autocrine - paracrine manner . it inhibits steroid - hormone secretion from the adrenal cortex but enhances catecolamine release from the adrenal medulla , activating the sympathoadrenal axis . the neuromodulator serotonin or 5-hydroxy - tryptamine ( 5ht ) , which is an important vasoactive mediator of allergic reactions , in addition , it has recently been emerged that proinflammatory cytokines can modulate the central nervous system 5ht signaling , resulting in the conceptualization that 5ht participates in an integrated behavioral response to pathogens and inflammatory events . on the other hand , 5ht receptor expression on ocy and their precursors is involved in bone metabolism and its mechanoregulation . moreover , serotonin blocks the proliferation of ocp through the suppression of intracellular transcription factor creb , which regulates many genes involved in circadian rhythms in different tissues ( period 1,2,3 and clock genes ) . there are two anatomically and functionally distinct pools of serotonin : the first one , synthesized through the activity of the enzyme tryptophane - hydroxylase type 2 in the central nervous system , where it functions as neurotransmitter ; the second one is synthesized in the periphery through the activity of the tryptophane - hydroxylase 1 , regulated by the low - density lipoprotein receptor ( ldlr)-related protein-5 ( lrp5 ) . the circulating serotonin does not exceed the blood brain barrier and is for 95% produced by intestinal enterochromaffin cells in the intestine , where it stimulates peristalsis in response to the meal . the peripherally produced serotonin acts as a hormone inhibiting bone formation , whereas serotonin produced in the brain functions as a neurotransmitter , enhancing bone formation and limiting bone resorption . the neurological mechanism of action of serotonin on bone implicates also the interaction with the adipokine leptin , in an integrated homeostatic network with fat tissue and metabolism ( fig . a portion of 5ht is also produced by the mammary gland , where it acts as an autocrine - paracrine regulator of the epithelial homeostasis exerting antiproliferative and proapoptotic effects . in the course of pregnancy , lactation and menopause , the local serotonin production increases , contributing to the increased bone resorption typical of these phases of the woman 's life . small molecules able to specifically inhibit the intestinal tryptophane - hydroxylase and do not cross the blood brain barrier , have recently been proposed for the treatment of osteoporosis . in alzheimer s disease ( ad ) , a neurodegenerative disorder characterized by cortical and cerebrovascular amyloid peptide ( a ) deposits , neurofibrillary tangles , chronic inflammation , and neuronal loss , increased bone fracture rates and reduced bmd are commonly observed , suggesting common denominators between both disorders . amyloid precursor protein ( app ) is transmembrane protein involved in ad pathogenesis : app gene mutations characterize early - onset ad , and many risk factors or genes associated with late - onset ad appear to affect app trafficking and a production . rage , acting as an app / a binding partner , is therefore implicated in the pathogenesis of both ad and osteoporosis . the role of rage in oc maturation and activation is also mediated by its interaction with proinflammatory associated mac-1/2 integrin , the s100 family , and the high mobility group box 1(hmgb1 ) . in particular , hmgb1 is a proin - flammatory cytokine released from activated macro - phages , that promotes rankl - induced oc differentiation in a rage - dependent manner . shared signaling pathways among the complex immunological machineries involved in bone remodeling activate vicious cycles underlying both bone destruction and cancer growth . the release of growth factors in the skeletal microenvironment as a result of osteolysis induced by oc mediated bone resorption enhances metastases and cancer cell proliferation . in addition to the hyperproduction of proinflammatory cytokines , rank /rankl signal alterations are central in the pathogenesis of neoplastic osteolysis . the upregulation of rankl , particularly in myeloma and breast cancer , promotes the growth of neoplastic cells which express rank and protects them from dna damage induced programmed cell death . in this context , bone cells may represent potential therapeutic targets in the treatment of both secondary neoplastic osteoporosis and the underlying neoplasia . for example , bisphosphonate treatment of individuals with multiple myeloma reduces osteolytic events and tumor burden as well . the block of rankl by the monoclonal antibody denosumab , in addition to inhibiting bone resorption , is also useful in reducing tumor growth , in increasing apoptosis of malignant cells and in decreasing the inflammation that supports the neoplasia . its predominant effect on bone is through the central nervous system : by stimulating specific receptors ( lepr ) on both serotonergic brainstem neurons and the hypothalamic ventromedial nucleus , which interact each other , it increases central sympathetic activity . inhibitory signals are transmitted through sympathetic fibers from the hypothalamic ventromedial nucleus to 2-adrenergic receptors ( 2-ar ) expressed on ob , suppressing their differentiation and osteocalcin production . serotonin is synthesized in serotonergic neurons of the central nervous system , exerting various functions in the brain ; it is also synthesized in the gut , mediating different peripheral functions . it acts on bone cells using three different receptors : through 5ht1b receptors , it negatively regulates bone mass , while it enhances bone formation through 5ht2b and 5ht2c receptors . the crosstalk between skeletal and bone marrow is crucial to hematopoietic stem cell ( hsc ) function and throughout the hemopoiesis . hsc occupy specific locations in the bone marrow microenvironment , commonly referred to as niches , comprising multiple cell types able to regulate hsc proliferation and differentiation . hematopoietic and skeletal stem cell homeostasis are closely related : msc progeny express numerous cytokines necessary for hsc maintenance and hematopoiesis , including kit ligand and stromal - derived factor , but also stem and progenitor cells of the hematopoietic system may reciprocally regulate skeletal progenitors by expressing myriad factors associated with skeletogenesis , whose cognate receptors are highly expressed by skeletal stem cells . interestingly , these latter exhibit receptors to circulating hormones , such as leptin and thyroid - stymulating hormone , suggesting that skeletal stem cells and their progeny may link systemic exocrine regulation to both skeletal and hematopoietic system . primitive mesenchymal progenitors are more critical for hsc function , whereas lineage - restricted mesenchymal cells control more committed hematopoietic progenitors . bone microenvironment is therefore a composite of specialized niches providing distinctive functional units to regulate hematopoiesis . the estrogen deficiency induces a marked increase of inflammatory cytokines and mediators of inflammation , in particular ifn- , m - csf , tnf- , il-1 , il-6 and il-7 , in addition to prostaglandins ( pge ) and reactive oxygen species ( ros ) , which play a driving role in the development of osteoporosis . of particular interest is the expansion in the peripheral blood from postmenopausal women of two particular lymphocyte subsets , cd3 + cd56 + t lymphocytes , major producers of tnf- , and b220 + igm - b lymphocyte precursors , that under certain stimuli can differentiate into ocp and are strong producers of inflammatory cytokines . the enhanced tnf- production by activated t cells has a central role in bone loss due to estrogen decline in menopausal women . the same ifn- , which in some situations could be protective against osteoporosis , during estrogen deficiency has a prevailing osteoclastogenic stimulating action . the estrogen decline also results in the decrease of opg and tgf- that normally contrast the effects of inflammatory mediators on bone . the age - related increase of inflammatory cytokines results from a chronic activation of macrophages and memory / effector t cells , in addition to an impaired treg function . a peculiar finding of immunosenescence is the increased number of senescent memory cells expressing rankl and secreting osteoclastogenic cytokines , mainly tnf- , il-1 , il-6 and il-17 . these cytokines are able to facilitate ocp expansion which amplify the systemic inflammation by producing further proinflammatory factors able to recruit other inflammatory cells and perpetuating the flogistic vicious cycle . both the increased transcriptional activity of nf - kb due to genotoxic , inflammatory , and oxidative stresses in aging , and the chronic p53 activation induced by the age - related progressive loss of telomere length , result in impaired ob proliferation and osteoporosis development . cells of the immune system and bone cells derive from bone marrow precursors and develop in the same bone marrow microenvironment . hematopoietic and immune cells share same signaling pathways with cells of the bone and their precursors , which surprisingly do not take part in the regulation of immunity and hemopoiesis . ob , together with msc , are crucial components of the niche of growth of hsc . ob overexpression of the wnt antagonist dkk1 reduces wnt signaling in hsc and disrupts hsc self - renewal potential . the wnt antagonist secreted frizzled - related protein 1 ( sfrp1 ) is involved in osteoblastic mediated hsc regulation ; through its regulation of ob , pth controls the hematopoietic niche function , involving crosstalk with wnt signaling . the upregulation of notch ligand protein jagged-1 ( jag1 ) expression in ob and the increased notch signaling are involved in increasing hsc numbers ; angiopoietin-1 receptor activation on hsc by ob produced angiopoietin-1 promotes strict adhesion of hsc to the niche , inducing quiescence of these cells . osteopontin , an ob - secreted protein , participates in hsc location and is a negative regulator of their proliferation . also , ocy derived from ob that become embedded within the bone matrix , are involved in the complex regulation of hemopoiesis through the osteoimmune interface : mainly by the production of sclerostin , they appear to have an inhibitory effect on hsc support . even oc , as well as activated endothelial cells , are likely involved in the mobilization of hsc by cytokine induced cam expression . being the only cells capable of bone resorption , in addition to allowing the renewal of the skeleton , they also open the space in the bone marrow for hematopoietic cells . moreover , osteoclastic bone resorption releases calcium , which attracts and retains calcium sensing receptor expressing hsc at the endosteal region . macrophages play diverse roles in the bone and marrow . at the sites of bone remodeling , a subset of hsc is located in close proximity to megakaryocytes in the bone marrow , where ob undergo rapid expansion in response to the secretion of megakaryocyte - derived mesenchymal growth factors , such as platelet derived growth factor ( pdgf)- , to promote hsc proliferation . the bone marrow microenvironment can also act as a niche for the onset and progression of neoplastic diseases , including both hematologic malignancies and metastases of solid tumors , mainly breast and prostatic cancer . in multiple myeloma , an osteolytic hematological malignancy characterized by an important skeletal involvement , neoplastic plasma cells produce a large amount of mediators that induce osteoclastic bone resorption and block the activity of ob . in addition to osteolytic factors , myeloma cells produce dkk1 , which inhibits obp differentiation by binding to the lrp5/6 coreceptors expressed on their surface . on the other hand , the same bone cells produce growth factors and angiogenetic cytokines able to support the development and progression of myeloma , perpetuating a vicious circle of mutual reinforcement . a promising new field of interest in this regard is the osteo - immune - oncology . there is in fact a close relationship between immune regulation of bone turnover and tumor growth . often , cancer cells express rank and in proneoplastic inflammation various cell types express rankl that acts as chemotactic factor favoring the skeletal metastasis . tumor cells in the bone cause activation of osteoclasts that mediate bone resorption and additional growth factor release from the bone matrix , fueling a vicious circle of impaired bone turnover and tumor proliferation ( fig . 5 ) . in skeletal metastases , not always the tumor associated inflammation is the expression of an immune system that successfully opposes the neoplastic growth , as previously believed , but sometimes some patterns of immune activation can be facilitators of the development of tumors . then the block of rankl , in addition to inhibiting bone resorption , also decreases tumor growth , enhances apoptosis of malignant cells and diminishes proneoplastic inflammation . osteal macrophages , as well as macrophages in other tissues , have a role in tumor progression , supporting cancers which preferentially metastasize to the skeleton , in particular breast and prostate cancer . osteal macrophages expressing cd68 , a phagocytic capacity marker of cells infiltrating metastatic lesions , could facilitate tumor establishment and growth . tumor - derived pthrp drives myeloid cell recruitment via ob produced ccl2 , which is high in the bone microenvironment and whose levels are associated with poor prognoses in primary breast tumors . as for bone , adipose tissue is a kind of immune tissue and produces immunoregulatory factors . adipocytes and ob derive from the same msc , as well as visceral adipose tissue macrophages and oc derive from the same hsc . shared transcription factors regulate the shift between the different cell lines and the subsequent differentiation cascade . by action of peroxisome proliferative activated receptor gamma ( ppar ) , the principal regulator of adipogenesis , the msc differentiates into adipocytes rather than into ob . in contrast , the expression of runt - related transcription factor 2 ( runx2 ) , associated with ob differentiation , and osterix , an ob zinc finger containing transcription factor , shift the equilibrium towards the osteoblastogenesis . mechanical loading promotes ob differentiation and inhibits adipogenesis by down - regulating ppar or by stimulating a durable -catenin signal . not surprisingly , ppar agonists such as thiazolidinediones used to increase insulin sensitivity in type 2 diabetes , also increase the risk of osteoporotic fractures . leptin is a pro - inflammatory adipokine that exerts its actions via central nervous system regulation of feeding behavior , where it promotes satiety and prevents weight gain . leptin can also directly signal through its receptor expressed on immunocytes , where it induces tnf- and il-6 production by monocytes , chemokines by macrophages , and th1 cytokines from polarized cd4 + t cells . adiponectin is an anti - inflammatory adipokine whose plasma levels are strongly correlated with insulin sensitivity and glucose tolerance . it can directly interfere with inflammatory cytokine production in macrophages and can induce expression of the anti - inflammatory cytokine il-10 . tnf- and il-6 inhibit adiponectin production in adipocytes . adipose tissue produced pro - inflammatory cytokines and adipokines further modulate the activity of oc and ob . fat tissue , mainly visceral fat tissue , may increase bone resorption through the production of inflammatory cytokines such as il- 6 and tnf- , which stimulate oc activity through the regulation of the rankl / rank / opg pathway . leptin and adiponectin act on the bone through different signaling pathways with contrasting effects ( table 2 ) . it promotes adipogenesis and inhibits osteogenesis in response to diet and adiposity by activating jak2/stat3 signaling . therefore , leptin receptors on skeletal msc function as a sensor of systemic energy homeostasis . various cell populations within the fat tissue can exacerbate the development of the chronic , low - grade inflammation associated with obesity and metabolic dysfunction . white adipocytes store lipid as triglycerides within unilocular droplets , and are responsive to various stimuli , such as insulin . brown adipocytes store lipid in multilocular droplets that are quickly catabolized for fuel when the tissue is stimulated . beige adipocytes , dispersed within white adipose tissue , can dissipate heat , similarly to classical brown adipocytes , when exposed to cold temperatures or after prolonged highfat diet feeding . visceral fat tissue infiltrating macrophages in the setting of diet - induced obesity , have a pro - inflammatory phenotype which initiates and/or exacerbates the chronic inflammation that contributes to adipocyte dysfunction in obesity . conversely , in lean humans adipose tissue macrophages may promote tissue remodeling and temper inflammation by secreting anti - inflammatory cytokines . notwithstanding epidemiological evidence indicates that an increase in body mass index ( bmi ) is related to increased bone mass , probably due to the effects of the mechanical load of the body weight on the skeleton , not always obesity , and mainly the increase in visceral fat mass , has a positive effect on bone . obesity is associated with increased leptin and decreased adiponectin serum levels . moreover , in the visceral adipose tissue there are activated macrophages producing cytokines . in obese subjects , especially with central obesity , in which the visceral fat is increased , there is a significant increase of several markers of inflammation such as c reactive protein ( crp ) , il-1 , il-6 and tnf- , that can alter the quality of the bone , making it more fragile . therefore , these new clinical and experimental evidences definitively connect obesity and other related pathological conditions , such as metabolic syndrome , nonalcoholic fatty liver disease ( nafld ) and diabetes , to impaired bone health and fragility fractures [ 90 - 93 ] . in conclusion , it is currently emerging that adipose tissue , liver , bone and immune system modulate each other through a complex network of interconnected signals . both adipocytes and ob express opg and rankl and their modulation is influenced by adipokines , sex hormones , redox balance , ppar and liver x receptors ( lxr ) . osteocalcin , an ob secreted bone matrix noncollagen protein , takes part in calcium metabolism and in bone deposition , as well as in energy homeostasis and glucose metabolism . fetuin - a , produced by the liver , is involved in the regulation of bone metabolism and insulin action , vascular calcification , neurodegenerative diseases and cancer cell proliferative signaling . ob lineage cells express receptors for adiponectin , leptin , angiotensin ii , insulin and insulin - like growth factor-1 , able to influence rank / rankl / opg signaling pathway . interestingly , these hormones are implicated in the pathogenesis of type - ii diabetes , obesity and hypertension , all of which are risk factors for metabolic syndrome . the development and progression of diabetes - associated osteoporosis are mediated by the interaction between advanced glycation end products ( age ) and receptor of age ( rage ) . age are the end products of glucose , as well as other sugars , proteins , lipids , and nucleic acids via a non - enzymatic glycosylation reaction , able to bind to multiple membrane receptor proteins , including rage . age / rage interaction is involved in the pathophysiological processes of many inflammatory and dysmetabolic diseases . in particular , age are involved in the development and progression of osteoporosis by inhibiting proliferation and inducing ob apoptosis . the binding of age to organic bone matrix may also increase the fragility of bones . autophagy is a metabolic process by which eukaryotic cells degrade and recover damaged macromolecules and organelles into autophagosomes autophagy is upregulated in stressful conditions . however , excessive autophagy is harmful to cells and leads to damage or massive death of cells . autophagy deficiency increases oxidative stress levels in ob , decreases bone mineralization and induces ran - kl secretion . it is well eatablished that several neuroendocrine pathways modulate both immune responses and bone remodeling . in turn sympathetic nerves produce catecholamines , which are delivered to the bone microenvironment by the blood circulation or by secretion from the nerve endings . hsc express catecholaminergic receptors , suggesting that they are able to directly respond to signals from the sympathetic nervous system . adrenergic signaling reduces cxcl12 expression in the bm . affecting maintenance of hsc and the differential lineage commitment . an association between major depression and osteoporosis has been recognized [ 96 - 99 ] . the prevalence of low bmd is higher in people with depression than the general population . interestingly , patients on antidepressant therapy with selective serotonin reuptake inhibitors develop decreased bmd and increased risk of fracture compared to those treated with tricyclic antidepressants such as amitriptyline or patients with untreated depression , who also have a lower bmd compared with healthy controls [ 100 , 101 ] . neuroendocrine abnormalities of the hypothalamo - pituitary - adrenal ( hpa ) and sympathoadrenal axes are a common finding in depressed patients leading to increased catecholamine turnover and hypersecretion of corticotropin - releasing hormone ( crh ) . leptin is expressed in the hypothalamus and pituitary gland , where it modulates corticotropin - releasing hormone and acth secretion , probably acting in an autocrine - paracrine manner . it inhibits steroid - hormone secretion from the adrenal cortex but enhances catecolamine release from the adrenal medulla , activating the sympathoadrenal axis . the neuromodulator serotonin or 5-hydroxy - tryptamine ( 5ht ) , which is an important vasoactive mediator of allergic reactions , is also likely involved in interactions between the central nervous and immune systems . in addition , it has recently been emerged that proinflammatory cytokines can modulate the central nervous system 5ht signaling , resulting in the conceptualization that 5ht participates in an integrated behavioral response to pathogens and inflammatory events . on the other hand , 5ht receptor expression on ocy and their precursors is involved in bone metabolism and its mechanoregulation . moreover , serotonin blocks the proliferation of ocp through the suppression of intracellular transcription factor creb , which regulates many genes involved in circadian rhythms in different tissues ( period 1,2,3 and clock genes ) . there are two anatomically and functionally distinct pools of serotonin : the first one , synthesized through the activity of the enzyme tryptophane - hydroxylase type 2 in the central nervous system , where it functions as neurotransmitter ; the second one is synthesized in the periphery through the activity of the tryptophane - hydroxylase 1 , regulated by the low - density lipoprotein receptor ( ldlr)-related protein-5 ( lrp5 ) . the circulating serotonin does not exceed the blood brain barrier and is for 95% produced by intestinal enterochromaffin cells in the intestine , where it stimulates peristalsis in response to the meal . the peripherally produced serotonin acts as a hormone inhibiting bone formation , whereas serotonin produced in the brain functions as a neurotransmitter , enhancing bone formation and limiting bone resorption . the neurological mechanism of action of serotonin on bone implicates also the interaction with the adipokine leptin , in an integrated homeostatic network with fat tissue and metabolism ( fig . a portion of 5ht is also produced by the mammary gland , where it acts as an autocrine - paracrine regulator of the epithelial homeostasis exerting antiproliferative and proapoptotic effects . in the course of pregnancy , lactation and menopause , the local serotonin production increases , contributing to the increased bone resorption typical of these phases of the woman 's life . small molecules able to specifically inhibit the intestinal tryptophane - hydroxylase and do not cross the blood brain barrier , have recently been proposed for the treatment of osteoporosis . in alzheimer s disease ( ad ) , a neurodegenerative disorder characterized by cortical and cerebrovascular amyloid peptide ( a ) deposits , neurofibrillary tangles , chronic inflammation , and neuronal loss , increased bone fracture rates and reduced bmd are commonly observed , suggesting common denominators between both disorders . amyloid precursor protein ( app ) is transmembrane protein involved in ad pathogenesis : app gene mutations characterize early - onset ad , and many risk factors or genes associated with late - onset ad appear to affect app trafficking and a production . rage , acting as an app / a binding partner , is therefore implicated in the pathogenesis of both ad and osteoporosis . the role of rage in oc maturation and activation is also mediated by its interaction with proinflammatory associated mac-1/2 integrin , the s100 family , and the high mobility group box 1(hmgb1 ) . in particular , hmgb1 is a proin - flammatory cytokine released from activated macro - phages , that promotes rankl - induced oc differentiation in a rage - dependent manner . shared signaling pathways among the complex immunological machineries involved in bone remodeling activate vicious cycles underlying both bone destruction and cancer growth . the release of growth factors in the skeletal microenvironment as a result of osteolysis induced by oc mediated bone resorption enhances metastases and cancer cell proliferation . in addition to the hyperproduction of proinflammatory cytokines , rank /rankl signal alterations are central in the pathogenesis of neoplastic osteolysis . the upregulation of rankl , particularly in myeloma and breast cancer , promotes the growth of neoplastic cells which express rank and protects them from dna damage induced programmed cell death . in this context , bone cells may represent potential therapeutic targets in the treatment of both secondary neoplastic osteoporosis and the underlying neoplasia . for example , bisphosphonate treatment of individuals with multiple myeloma reduces osteolytic events and tumor burden as well . the block of rankl by the monoclonal antibody denosumab , in addition to inhibiting bone resorption , is also useful in reducing tumor growth , in increasing apoptosis of malignant cells and in decreasing the inflammation that supports the neoplasia . its predominant effect on bone is through the central nervous system : by stimulating specific receptors ( lepr ) on both serotonergic brainstem neurons and the hypothalamic ventromedial nucleus , which interact each other , it increases central sympathetic activity . inhibitory signals are transmitted through sympathetic fibers from the hypothalamic ventromedial nucleus to 2-adrenergic receptors ( 2-ar ) expressed on ob , suppressing their differentiation and osteocalcin production . serotonin is synthesized in serotonergic neurons of the central nervous system , exerting various functions in the brain ; it is also synthesized in the gut , mediating different peripheral functions . it acts on bone cells using three different receptors : through 5ht1b receptors , it negatively regulates bone mass , while it enhances bone formation through 5ht2b and 5ht2c receptors . immune system and skeletal system interact with each other both in physiological and pathological conditions . considerable progress has been made in clarifying the crosstalk between bone and immune system that occurs in a complex and dynamic bone microenvironment . a central role in this crosstalk osteoimmunology therefore represents a new approach to studying osteoporosis that in the past was not considered an inflammatory condition . moreover , the discoveries of the existence of the immunoskeletal interface has also highlighted interesting repercussions for a wide range of pathological conditions beyond osteoporosis , including infections , autoimmune diseases , and neoplasia , in which same pathogenetic pathways are shared . osteoporosis could be therefore considered as a systemic model of integrated signaling pathways and cytokines working in a cooperative fashion . further important research horizons are opened with the extension of the discoveries of osteoimmunology and the disclosure of the immunoskeletal interface , whose practical implications may provide novel therapies for diseases other than osteoporosis , by targeting specific transcription factors , cytokines and their receptors . the correct understanding and decoding of the complex language through which immune system and bone communicate , although still in its dawn , is the essential requirement for the identification of such potentially useful therapeutic targets for both osteoporosis and other correlated inflammatory conditions , which share same mediators and signaling pathways .

abstract : objectiveosteoimmunology investigates interactions between skeleton and immune system . in the light of recent discoveries in this field , a new reading register of osteoporosis is actually emerging , in which bone and immune cells are strictly interconnected . osteoporosis could therefore be considered a chronic immune mediated disease which shares with other age related disorders a common inflammatory background . here , we highlight these recent discoveries and the new landscape that is emerging.methodextensive literature search in pubmed central.resultswhile the inflammatory nature of osteoporosis has been clearly recognized , other interesting aspects of osteoimmunology are currently emerging . in addition , mounting evidence indicates that the immunoskeletal interface is involved in the regulation of important body functions beyond bone remodeling . bone cells take part with cells of the immune system in various immunological functions , configuring a real expanded immune system , and are therefore variously involved not only as target but also as main actors in various pathological conditions affecting primarily the immune system , such as autoimmunity and immune deficiencies , as well as in aging , menopause and other diseases sharing an inflammatory background.conclusionthe review highlights the complexity of interwoven pathways and shared mechanisms of the crosstalk between the immune and bone systems . more interestingly , the interdisciplinary field of osteoimmunology is now expanding beyond bone and immune cells , defining new homeostatic networks in which other organs and systems are functionally interconnected . therefore , the correct skeletal integrity maintenance may be also relevant to other functions outside its involvement in bone mineral homeostasis , hemopoiesis and immunity .

there appears to have been consensus in the past decades in classifying systemic sclerosis ( ssc ) according to extension of skin involvement as limited and diffuse , using the elbows and knees as limits to distinguish between them . however , although this has been used to dissect two more or less distinct clinical subsets , there are patients who do not fit this classification and several others have been proposed [ 1 , 2 ] . the growing demonstration that autoantibodies specific for ssc such as anticentromere , anti - scl-70 ( anti - topoisomerase 1 ) , and more recently a variety of antinucleolar antibodies , particularly anti - rna polymerase 3 , correlate with these subsets and/or visceral manifestations has led to them being used to predict clinical subsets in early disease and also raise awareness for the possibility of certain organ involvement ( anti - scl-70 and interstitial lung disease [ 3 , 4 ] , anticentromere associated with digital ulcers , debatably pulmonary hypertension , and anti - rna polymerase 3 with renal crisis ) . decades ago , barnett classified ssc as type 1 ( only sclerodactyly ) , type 2 ( acrosclerosis - distal but may reach up to elbows and/or knees plus face ) , and type 3 ( diffuse skin involvement ) ( figure 1 ) . patients with type 2 had an intermediate degree of both skin and organ involvement compared to type 1 ( less ) and type 3 ( more ) [ 810 ] . this classification by barnett was extensively discussed in an editorial by masi many years later . the division into three categories according to extension of skin involvement was not agreed to by others , who could not find real differences between what we could call types 1 and 2 as determined by barnett [ 1 , 2 ] . a similar classification but this time with 4 categories , proposed by giordano et al . simultaneously , as reasonable stress was laid on early disease detection , the extent of cutaneous involvement was perhaps not considered so relevant , particularly as opposed to the autoantibodies as subset identifiers . however , truth is that many patients present with evolving disease , and all of ssc specific antibodies are not widely available in many parts of the world , the best example being anti - rna polymerase . many years ago but with a small number of patients , we noted that the limited subgroup could be , indeed , separated clinically as barnett had suggested according to skin involvement into patients with only fingers , which we proposed to call crest , and those with skin distal to elbows and knees but proximal to mcp or mtp joints , which we proposed to call creat ( a instead of s for acrosclerosis ) . in that report , crest patients had more anticentromere antibodies than those with creat and the presence of anti - scl-70 occurred more frequently in the patients with creat than those with crest . small numbers did not allow for detection of clinical differences . over the past decades , many of the larger series [ 416 ] and particularly the eustar database , one of the largest registries , have not differentiated subsets within the limited form ( scleroderma sine scleroderma excluded ) . very recently however , in a very large series from a single center followed for years , the hopkins group emphasized just this , showing differences between the two limited groups and stressing the higher prevalence of lung disease and anti - scl-70 antibodies in the type 2 ( barnett ) or intermediate form when compared with type 1 , a behavior more similar to the diffuse form , and similar to what we had supported and barnett proposed years ago . their conclusion was that dividing into limited and diffuse as we do now left out an intermediate form with different clinical and serological characteristics . since over the years it was our feeling that this subsetting was clinically useful , we always registered extension of skin involvement in our patients , particularly identifying those with intermediate form within the lssc . we therefore decided to examine the clinical and serologic characteristics of our patients with limited disease seen over the last decade to see if we could find differences within them according to the degree of skin involvement . rheumatology section database , patients with icd 9 code 710.1 ( scleroderma ) in the inpatient hospital database , patients with the item : scleroderma , crest , or other synonyms as registered in the hospital 's electronic clinical history ; and laboratory database : patients with positive anticentromere , antinucleolar , or anti - scl-70 results . all medical records so identified were then manually reviewed to confirm diagnosis and obtain demographic data such as date of onset , clinical characteristics , and serologic profile . only cases fulfilling acr 1980 criteria were included and were classified as diffuse or limited according to leroy 's criteria . this difference in distribution was established considering the involvement of skin at its maximum extension at any point in the disease course . within the limited subset , they were separated into sclerodactyly ( only fingers ) and acrosclerosis ( fingers and up to elbows and/or knees , face ) ( barnett 's types 1 and 2 ) . ssc clinical manifestations were considered to be present if predefined criteria were met during the course of the illness . organ involvement definitions were the following : ( 1 ) upper gastrointestinal tract ( one or more of the following : manometry with esophageal dysmotility , esophagram with gastroesophagic reflux or peristaltic alterations , or upper endoscopy with esophagitis , ( 2 ) pulmonary hypertension ( ph ) : echocardiogram with estimated pulmonary systolic artery pressure equal to or greater than 40 mmhg or right heart catheterization with mean pulmonary artery pressure at rest over 25 mmhg , ( 3 ) interstitial lung disease ( ild ) : pulmonary interstitial disease observed in high resolution computerized tomography ( hrct ) or pulmonary function tests with vital forced capacity ( vfc ) lower than 70% of the expected and/or carbon monoxide lung diffusion ( dlco ) test under 80% of the expected , ( 4 ) echocardiographic alterations ( aside from ph ) : left or right ventricular diastolic dysfunction in absence of arterial hypertension or pulmonary hypertension , respectively , or pericardial effusion , ( 5 ) digital ulcers : active digital ulcers or pitting scars confirmed by a physician , ( 6 ) renal involvement : history of scleroderma renal crisis ( abrupt onset of accelerated arterial hypertension and/or rapidly progressive renal failure ) . laboratory detection methods were indirect immunofluorescence on hep-2 cells ( antinuclear antibodies , antinucleolar , and anticentromere ) . chi - square analysis was used to determine significant differences between sets of categorical data and fisher 's exact test when appropriate . two hundred and thirty four ssc patients ( 216 females ) fulfilled the established criteria . female / male ratio was 12 : 1 ; 24% had diffuse ssc and 76% limited ( 64% sclerodactyly and 12% acrosclerosis ) . ten - year survival rate was 80% for limited and 70% for diffuse variants , respectively ( hr : 0.88 , 95% ci : 0.71.1 ) . anti - scl-70 was present in 16% , anticentromere in 53% , and nucleolar ana in 7% of overall patients . several characteristics in the acrosclerosis ( type 2 ) group were more similar to the diffuse patients than those with type 1 ( sclerodactyly ) . in patients with acrosclerosis , duration of raynaud 's was shorter , and they had significantly more anti - scl-70 and less anticentromere antibodies than those with type 1 . in particular , interstitial lung disease ( ild ) was significantly more prevalent in type 2 group and similar to type 3 . comparisons between patients with acrosclerosis and diffuse disease are presented in table 2 , showing no differences in clinical manifestations . as described elsewhere , autoantibodies were associated with visceral involvement and also with clinical subset . in univariate analysis , anticentromere antibodies were associated with pulmonary hypertension , or of 8.25 ( ci 1.935.7 ) , and inversely correlated with ild ( or 0.18 , ci 0.110.29 ) . they were also associated with limited disease , or 34.4 ( ci 10.2116.6 ) . when performing multivariate analysis adjusting by disease subset , association between anticentromere and pulmonary hypertension was lost . all of the patients with ph had lssc ( 11 with sclerodactyly and 1 with acrosclerosis ) except for one patient with diffuse disease with anticentromere antibodies . in summary , in multivariate analysis , both anti - scl-70 ( or 5.2 , ci 1.517.6 , p = 0.08 ) and diffuse disease ( or 4.1 , ci 1.213.5 , p = 0.021 ) were associated with ild . these findings are very similar to those recently described by cottrell et al . within the limited patients , dissecting between what could be barnett 's type 1 and 2 subsets . our results support the initial description by barnett et al . that there is an intermediate form of ssc between the some of these patients , albeit with limited scleroderma because skin involvement does not exceed elbows and/or knees , have a clinical behavior which can be described as intermediate between the strictly limited to fingers patients and those with diffuse disease . in particular , a sort of gradient from milder to more severe disease was confirmed in our patients , as suggested by barnett many years ago . this was also shown by the hopkins group in a much larger number of patients . indeed , in type 2 as defined by barnett , serology may be more similar to the diffuse type and different from type 1 . the gradient is also reflected by the accompanying antibodies which further define visceral involvement such as pulmonary hypertension and interstitial lung disease . these results appear to confirm that extension of skin involvement within limited ssc may identify two different subsets with clinical and serologic characteristics . autoantibodies , in many cases present years prior to diagnosis and established skin disease , play an essential role here as predictors of clinical subsets and visceral involvement . however , some of these are not widely available . furthermore , many patients present with established disease , although early , and in these a clinical approach contemplating extent of skin involvement coupled with the classic autoantibodies may indeed be useful and support a division within the limited form which may have useful clinical implications . a retrospective review of data in the large registries trying to document skin extension may or may not confirm this . the problem may arise in the fact that since the extension of skin involvement within limited disease was not considered important , this may not have been adequately registered . our smaller series completely agrees with the data shown by the hopkins group with a very large number of patients . we believe identifying these differences within limited ssc is useful and resuscitating the intermediate form or barnett type 2 within limited ssc may contribute to a better clinical assessment of systemic sclerosis .

objectives . to examine the characteristics of our patients with limited systemic sclerosis ( lssc ) for differences between barnett type 1 ( sclerodactyly only ) and type 2 or intermediate ( acrosclerosis - distal but may reach up to elbows and/or knees plus face ) subsets . methods . records of patients between january 1 , 2000 , and december 31 , 2011 , with ssc or those with anti - scl-70 , anticentromere , or antinucleolar antibodies were reviewed . only cases fulfilling acr 1980 criteria were included and classified as diffuse or limited according to leroy 's criteria . limited ssc was separated into sclerodactyly and acrosclerosis ( barnett 's types 1 and 2 ) . results . 234 ssc patients ( 216 females ) fulfilled criteria . female / male ratio was 12 : 1 ; 24% had dssc and 76% lssc ( 64% type 1 and 12% type 2 ) . total follow - up was 688 patient - years . within lssc , the type 2 group had significantly shorter duration of raynaud 's and more anti - scl-70 and less anticentromere antibodies . in particular , interstitial lung disease ( ild ) was significantly more prevalent in type 2 group and similar to type 3 . conclusions . these results appear to confirm that extension of skin involvement within limited ssc may identify two different subsets with clinical and serologic characteristics .

common failure of the root canal obturation process is because of the presence of gaps and porosities at the sealer / dentin interface . several studies have shown that filled root canals can allow the re - colonization of micro - organisms . the presence of micro - organisms and their products in the treated root canal can led to failure of the root canal treatment and the necessity of retreatment . the analysis of the dentin / sealer interface allows the determination of which materials and filling techniques could obturate the root canals with less gaps and voids . several microscopy techniques are currently used to evaluate the sealer / dentin interface , including stereomicroscopy , scanning electron microscopy ( sem ) , transmission electron microscopy , and confocal laser scanning microscopy ( clsm ) . in comparison to conventional sem , clsm has the advantage of providing detailed information about the presence and distribution of sealers or dental adhesives inside dentinal tubules in the total circumference of the root canal walls at relative low magnification as 100x through the use of fluorescent rhodamine marked sealers . placement and activation of a sealer into the root canal system should be done in a manner which is predictable and completely covers the dentin walls . accepted means of sealer placement include the use of endodontic files or reamers , lentulo spirals , gutta - percha cones , ultrasonic files , and counter - clockwise motion of rotary systems . very few studies have been done to evaluate the efficacy of different methods of sealer placement and activation effectively . mta fillapex is a sealer that is composed of mta and has excellent flow , radiopacity , easy handling , working time , and low solubility . because of its suitable physicochemical properties and excellent biocompatibility and bioactivity , hence , this study was planned to evaluate percentage and the depth of dentinal tubule sealer penetration of mta fillapex when agitated with three different techniques and to compare it with ah plus using clsm as the evaluative tool . sixty teeth with single canal and free of cracks , caries , resorption , calcification , previous endodontic treatment , and less than 10- root curvature were selected and stored in distilled water until use . for standardization of the root length ( 10 mm ) , the crowns were resected with a diamond disc in a slow speed straight handpiece under constant water cooling . the working length was determined by inserting # 15 k - file ( dentsply maillefer ) into the canal until it was just seen at the apical foramen and then 0.5 mm was subtracted from this length . the roots were instrumented by using the protaper universal root canal files ( dentsply maillefer ) in a sequential manner from s1 till f5 . canals were irrigated between files with 2 ml of 3% naocl ( sree rayalaseema alkalies and allies chemicals limited , india ) . to eliminate the smear layer 2 ml of 17% ethylenediaminetetraacetic acid ( edta ) ( ph 7.7 ) for 3 min was used , followed by a final rinse of 2 ml distilled water . the roots were randomly divided into three groups on the basis of sealer activation technique utilized : g1 : ultrasonic ( woodpecker dte - d5 ultrasonic scaler , china ) ; g2 : lentulo spiral ( dentsply , maillefer ) ; g3 : counter - clockwise motion ( x smart , densply , maillefer , ballaigues , switzerland ) . each group was further divided into two subgroups on the basis of type of sealer . subgroup a utilized ah plus sealer while subgroup m utilized mta fillapex sealer . ah plus sealer and mta fillapex sealer were mixed according to the manufacturer instructions and to allow analysis under the clsm , each sealer was labelled with rhodamine b ( mayor diagnostics , mumbai , india ) to an approximate concentration of 0.1%(by weight ) . group 1 ( g1 ) : the ultrasonic unit ( woodpecker dte - d5 ultrasonic scaler , china ) was used in endo mode with a satelec ultrasonic endodontic tip k15 sonofile ( dentsply tulsa ) for activation . the file was inserted 2 mm short of the working length inside the canal and was ultrasonically activated for 20 seconds . gutta - percha was compacted 1 mm below the canal orifice and the teeth were sealed with cavit ( 3 m , espe ) . in g1a ah plus sealer and in g1 m , mta fillapex sealer was used . group 2 ( g2 ) : a size 30 lentulo spiral ( dentsply , maillefer ) was selected that would not bind in the prepared canal and that would reach the prepared working length . the lentulo spiral was rotated at speed of 300 rpm keeping the instrument 2 mm from apex for 20 seconds . thereafter , the obturation of canal was conducted as described for g1 . in g2a ah plus sealer and in g2 m , mta fillapex sealer was used . group 3 ( g3 ) : f1 protaper file attached in handpiece of endodontic micromotor ( x - smart , dentsply tulsa ) was kept 2 mm short of apex and rotated at 300 rpm in reverse mode for 20 seconds . thereafter , the obturation of canal was conducted as described for g1 . in g3a ah plus sealer and in g3 m , mta fillapex sealer was used . the specimens were kept in an incubator at 37c and 100% humidity for 2 days . each root was sectioned at 90- to the long axis by using diamond disc removing two , 1 mm sections at 3 mm and 6 mm from the apex . coronal surface of each section was polished with sand paper ( politriz , arotec , cotia , sp , brazil ) . the dentin segments were examined on a confocal microscope ( olympus fluoview fv 1000 ) . the respective absorption and emission wave lengths for the rhodomine b were 540 nm and 590 nm . dentin samples were analyzed using the 10x lens . to calculate the percentage of sealer penetration around the root canal , first each image was imported into the iob software and the circumference of root canal measured using its ruler tool . next , areas along the canal walls in which the sealer penetrated into dentinal tubules were outlined and measured using the same method [ figure 1 ] . subsequently , the percentage of root canal sealer penetration in that section was established . to determine the maximum depth of penetration , the point of deepest penetration was measured from the canal wall to the maximum depth of penetration [ figure 2 ] . the effects of sealer agitation techniques and type of sealer on depth and percentage of sealer penetration were analyzed , by performing nonparametric kruskal - wallis for overall analysis , and a series of mann - whitney u tests for pairwise comparison using statistical package of social sciences ( spss ) statistics 20 software . confocal laser scanning microscopic photograph showing calculation of percentage of penetration of sealer in the dentinal tubules ( original magnification 10 ) confocal laser scanning microscopic photograph showing calculation of depth of penetration of sealer in the dentinal tubules ( original magnification 10 ) sixty teeth with single canal and free of cracks , caries , resorption , calcification , previous endodontic treatment , and less than 10- root curvature were selected and stored in distilled water until use . for standardization of the root length ( 10 mm ) , the crowns were resected with a diamond disc in a slow speed straight handpiece under constant water cooling . the working length was determined by inserting # 15 k - file ( dentsply maillefer ) into the canal until it was just seen at the apical foramen and then 0.5 mm was subtracted from this length . the roots were instrumented by using the protaper universal root canal files ( dentsply maillefer ) in a sequential manner from s1 till f5 . canals were irrigated between files with 2 ml of 3% naocl ( sree rayalaseema alkalies and allies chemicals limited , india ) . to eliminate the smear layer 2 ml of 17% ethylenediaminetetraacetic acid ( edta ) ( ph 7.7 ) for 3 min was used , followed by a final rinse of 2 ml distilled water . the roots were randomly divided into three groups on the basis of sealer activation technique utilized : g1 : ultrasonic ( woodpecker dte - d5 ultrasonic scaler , china ) ; g2 : lentulo spiral ( dentsply , maillefer ) ; g3 : counter - clockwise motion ( x smart , densply , maillefer , ballaigues , switzerland ) . each group was further divided into two subgroups on the basis of type of sealer . ah plus sealer and mta fillapex sealer were mixed according to the manufacturer instructions and to allow analysis under the clsm , each sealer was labelled with rhodamine b ( mayor diagnostics , mumbai , india ) to an approximate concentration of 0.1%(by weight ) . group 1 ( g1 ) : the ultrasonic unit ( woodpecker dte - d5 ultrasonic scaler , china ) was used in endo mode with a satelec ultrasonic endodontic tip k15 sonofile ( dentsply tulsa ) for activation . the file was inserted 2 mm short of the working length inside the canal and was ultrasonically activated for 20 seconds . canal was obturated with protaper f5 gutta - percha cone . gutta - percha was compacted 1 mm below the canal orifice and the teeth were sealed with cavit ( 3 m , espe ) . in g1a ah plus sealer and in g1 m , mta fillapex sealer was used . group 2 ( g2 ) : a size 30 lentulo spiral ( dentsply , maillefer ) was selected that would not bind in the prepared canal and that would reach the prepared working length . the lentulo spiral was rotated at speed of 300 rpm keeping the instrument 2 mm from apex for 20 seconds . thereafter , the obturation of canal was conducted as described for g1 . in g2a ah plus sealer and in g2 m , mta fillapex sealer was used . group 3 ( g3 ) : f1 protaper file attached in handpiece of endodontic micromotor ( x - smart , dentsply tulsa ) was kept 2 mm short of apex and rotated at 300 rpm in reverse mode for 20 seconds . thereafter , the obturation of canal was conducted as described for g1 . in g3a ah plus sealer and in g3 m , mta fillapex sealer was used . the specimens were kept in an incubator at 37c and 100% humidity for 2 days . each root was sectioned at 90- to the long axis by using diamond disc removing two , 1 mm sections at 3 mm and 6 mm from the apex . coronal surface of each section was polished with sand paper ( politriz , arotec , cotia , sp , brazil ) . the dentin segments were examined on a confocal microscope ( olympus fluoview fv 1000 ) . the respective absorption and emission wave lengths for the rhodomine b were 540 nm and 590 nm . dentin samples were analyzed using the 10x lens . to calculate the percentage of sealer penetration around the root canal , first each image was imported into the iob software and the circumference of root canal measured using its ruler tool . next , areas along the canal walls in which the sealer penetrated into dentinal tubules were outlined and measured using the same method [ figure 1 ] . subsequently , the percentage of root canal sealer penetration in that section was established . to determine the maximum depth of penetration , the point of deepest penetration was measured from the canal wall to the maximum depth of penetration [ figure 2 ] . the effects of sealer agitation techniques and type of sealer on depth and percentage of sealer penetration were analyzed , by performing nonparametric kruskal - wallis for overall analysis , and a series of mann - whitney u tests for pairwise comparison using statistical package of social sciences ( spss ) statistics 20 software . confocal laser scanning microscopic photograph showing calculation of percentage of penetration of sealer in the dentinal tubules ( original magnification 10 ) confocal laser scanning microscopic photograph showing calculation of depth of penetration of sealer in the dentinal tubules ( original magnification 10 ) the percentage and depth of sealer penetration of different groups and intergroup comparison are summarized in tables 1 and 2 , respectively . shows mean and standard deviation of depth and percentage of sealer penetration of various groups at 3 and 6 mm levels intergroup comparison of depth and percentage of sealer penetration at 6 and 3 mm levels mean percentage and depth of sealer penetration of mta fillapex and ah plus sealer was greatest when ultrasonics was used for agitation and statistically significant ( p < 0.001 ) than lentulo spiral and counter - clockwise agitation techniques . irrespective of method used for agitation , the percentage and depth of sealer penetration for mta fillapex was significantly greater ( p < 0.001 ) than ah plus . the percentage and depth of sealer penetration for mta fillapex and ah plus were greater and highly significant ( p < 0.001 ) at 6 mm than 3 mm . removal of smear layer and use of sealer to attain and impervious seal between the core material and root canal walls is considered an essential step of root canal treatment . sealers which can penetrate into the dentinal tubules exert bactericidal effect by having a closer contact with the residual bacteria within the tubules . in addition , sealer plugs inside the dentinal tubules provide a mechanical interlocking , thereby improving the retention of the filling material and reducing the microleakage along the root canal walls . thus , both percentage and depth of sealer penetration might influence the outcome or success rate of endodontic therapy . factors influencing sealer depth penetration in dentinal tubules are presence / absence of dentinal permeability ( the number and the diameter of tubules ) , root canal dimension , presence of water , and physical and chemical properties of the sealer . the compound effect of physical properties viz flow , surface tension , solubility , viscosity , chemical composition , and working and setting time may influence penetration . flow which is defined as the ability of a sealer to penetrate in irregularities , lateral canals , or dentinal tubules of the root canal system is one of the main physical factors to influence the tubular penetration . as most endodontic sealers are pseudoplastic , their flow increases with increase of shear rate . on comparison with conventional sealers patel dv et al . , and gharib sr et al . , found better depth of penetration of resin - based sealer in dentinal tubules . mta fillapex has excellent biocompatibility and bioactivity and displayed significantly higher ph up to 7 days period than ah plus . studies based on the physical properties reported significantly greater flow of mta fillapex than ah plus . therefore , this comparative study was conducted to evaluate and compare the quality of sealing of ah plus and mta fillapex . clinically , rapid insertion or activation of a pseudoplastic sealer into the canal would decrease viscosity and increase the flow of sealer . as very few studies have been conducted on the effect of sealer activation / placement and activation on sealing ability of root canal sealers therefore in the present study three activation techniques ( ultrasonics , lentulo spiral , and rotary counter - clockwise motion ) were chosen and the sealer distribution was analyzed . amount of sealer , extent of activating instrument , and time for activation were standardized to minimize the errors . all the analyzed activation techniques failed to show a consistent adaptation of both the sealers to the total circumference of the root canal walls . however , mta fillapex showed significantly better ( p < 0.001 ) percentage and depth of sealer penetration than ah plus with all the techniques of sealer activation . kuc , ia et al . , also reported greater mta fillapex sealer penetration than ah plus . zhou h et al . , and silva ej et al . , found mta fillapex significantly more flowable than ah plus and attributed difference in composition and smaller particle size of the sealer for this property . application of ultrasonics for activation led to significantly more ( p < 0.001 ) percentage and depth of both the sealers and is accordance to guimaraes bm et al . , the explanation for this is that the oscillating files in ultrasonics transmits the acoustic microstreaming energy and cause a greater depth of dentinal sealer penetration and coverage of root canal walls in the same manner as it promotes the penetration of irrigants in an area of anatomic complexities and the dentinal tubules . although activation of both the sealers with lentulo spiral resulted in more percentage and depth of penetration as compare to counter - clockwise rotation however , the difference was statistically insignificant ( p > 0.05 ) . this is one of the pioneer studies evaluating effect of methods of activation on mta fillapex so it was difficult to compare the results obtained with previous studies . lentulo spiral rotates and the spring portion pushes the sealer centrifugally while in counter - clockwise rotation with a file , the sealer tends to pool toward the tip of file but is not forced toward the walls . irrespective of technique of activation and type of sealer , the depth , and percentage of penetration of sealer was significantly better at the 6 mm level than 3 mm level . these findings are similar to other studies and could be because the number and diameter of dentinal tubules decreases on descending apically in the root canal and superior removal of smear layer . in addition , the apical dentin is irregular in direction and density ; even some areas are devoid of dentinal tubules . under the parameters of present study , it can be concluded that mta fillapex sealer exhibited better percentage and depth of penetration in the radicular dentinal tubules than ah plus . ultrasonic activation of pseudoplastic sealers can significantly increase its percentage and depth of penetration and level of root canal can affect both , percentage , and depth of penetration of sealers in the dentinal tubules .

aim : to compare the effect of three root canal sealer activation techniques on percentage and depth of sealer penetration of mta fillapex and ah plus sealers.materials and methods : sixty teeth prepared till f5 protaper size were divided into three equal groups on the basis of sealer activation technique ( g1 : ultrasonics , g2 : lentulo spiral , and g3 : counter - clockwise rotary motion ) . each group was further divided into two equal subgroups on the basis of type of sealer used : ah plus ( denstply , konstanz , germany ) or mta fillapex ( angelus , londrina , pr , brazil ) and obturated with gutta - percha . horizontal sections at 3 and 6 mm from the apex were obtained and the percentage and depth of penetration of sealers into dentinal tubules were measured using confocal laser scanning microscopy ( clsm ) . statistical analysis was performed utilizing kruskal - wallis and mann - whitney u tests with a significance level of 5%.results : g1 showed significantly ( p < 0.001 ) high percentage and depth of sealer penetration than g2 and g3 while the difference was insignificant ( p > 0.05 ) between g2 and g3.conclusion:percentage and depth of sealer penetration are influenced by the type of sealer used sealer activation technique and by the root canal level . ultrasonic method of sealer activation and mta fillapex showed the best results .

vision 2020 is a joint initiative by the world health organization ( who ) and the international agency for the prevention of blindness , that aims to eliminate avoidable blindness by the year 2020 . the vision 2020 strategy depends on the development of district level plans for the prevention of avoidable blindness.[26 ] india was the first country in the world to initiate a public funded program for the prevention of blindness as a national priority health problem . population - based surveys have been the main stay of information regarding the effective implementation and monitoring of such eye care programs . are expensive and time consuming . rapid assessment of avoidable blindness ( raab ) survey is cheap and easy means of getting population - based data on prevalence and causes of blindness in people aged more than 50 years . raab also has the other utility of monitoring programs at the unit / district level.[911 ] kolar district in south karnataka , india , has an estimated population of around 1.5 million , 51% of the population being males and 29% being over 50 years . the aim of the study was to conduct a raab study in persons aged more than 50 years in kolar district in order to estimate the prevalence and causes of blindness . this was also to help the ophthalmology and community medicine departments of the medical institution to develop student capacity as well as expose them to scientific survey methods of generating evidence for planning of eye care services . the survey was carried out by a team consisting of trained personnel from the departments of ophthalmology and community medicine , sri devaraj urs medical college , kolar , karnataka , india . the survey was carried out between march and june 2011 ( 4 months ) , in accordance with the helsinki declaration . written informed consent was obtained from all study participants , after explaining the purpose of the study in their local language . sample size was determined using a prevalence estimate of 4% for blindness ( who definition of presenting vision < 20/400 in better eye ) among those aged over 50 years . the prevalence estimate was assumed to be around 4% considering the recent raab survey conducted across india which gave prevalence of blindness using the same definition to be around 3.6% . using 95% confidence interval , 22.5% precision , design effect of 1.5 and 10% nonresponse rate , sample size was calculated to be 3017 , which would require 61 clusters of 50 people aged over 50 years . using the population data from the last census ( 2001 ) and the growth rate for 9 years , the population was estimated for end of 2010 and used as a sampling frame . a list of wards and villages in urban and rural areas was prepared taluk ( revenue division ) wise . in each , the population size of people aged over 50 years was listed . households within each cluster were selected by compact segment sampling . a map of the selected cluster was drawn and divided into equal segments that would give segments were numbered and one segment was chosen by draw of lots . to be eligible for inclusion an individual had to reside in that household for at least the previous 6 months . in each cluster , the people in the selected cluster were briefed about the survey 23 days in advance by the local health worker along with the public relations officer of the team . if an eligible person was not available during the survey , at least two more attempts were made to assess information . if after repeated visits , examination could not be done , information of his visual status was obtained from his relatives or neighbors . standard raab protocol was used for gathering information and for eye examination . a survey form comprising seven sections the form consisted of general information ; vision and pin hole examination ; lens status ; principal cause of visual impairment ; history if not examined ; and barriers to uptake of cataract surgery and details of cataract surgery if operated . visual acuity ( va ) was measured using a tumbling snellen - e chart using optotype size 20/60 on one side and 20/200 on the other . all measurements were taken in full daylight with available correction . if visual acuity was less than 20/60 in either eye , pin hole vision was tested . if presenting vision was < 20/60 , then pupil was dilated and ophthalmoscopy done to assess the cause of blindness . the interobserver agreement between the two teams was good as observed by the kappa value ( > 0.60 ) . double data entry and analysis was done using the raab software program version 4.02 . to check for errors made during data entry of the survey record forms , the data are entered twice by different data entry clerks in two separate databases and then compared . sample size was determined using a prevalence estimate of 4% for blindness ( who definition of presenting vision < 20/400 in better eye ) among those aged over 50 years . the prevalence estimate was assumed to be around 4% considering the recent raab survey conducted across india which gave prevalence of blindness using the same definition to be around 3.6% . using 95% confidence interval , 22.5% precision , design effect of 1.5 and 10% nonresponse rate , sample size was calculated to be 3017 , which would require 61 clusters of 50 people aged over 50 years . using the population data from the last census ( 2001 ) and the growth rate for 9 years , the population was estimated for end of 2010 and used as a sampling frame . a list of wards and villages in urban and rural areas was prepared taluk ( revenue division ) wise . in each , the population size of people aged over 50 years was listed . households within each cluster were selected by compact segment sampling . a map of the selected cluster was drawn and divided into equal segments that would give segments were numbered and one segment was chosen by draw of lots . to be eligible for inclusion an individual had to reside in that household for at least the previous 6 months . in each cluster , the survey team visited each household accompanied by local health worker to facilitate compliance . the people in the selected cluster were briefed about the survey 23 days in advance by the local health worker along with the public relations officer of the team . if an eligible person was not available during the survey , at least two more attempts were made to assess information . if after repeated visits , examination could not be done , information of his visual status was obtained from his relatives or neighbors . standard raab protocol was used for gathering information and for eye examination . a survey form comprising seven sections vision and pin hole examination ; lens status ; principal cause of visual impairment ; history if not examined ; and barriers to uptake of cataract surgery and details of cataract surgery if operated . visual acuity ( va ) was measured using a tumbling snellen - e chart using optotype size 20/60 on one side and 20/200 on the other . all measurements were taken in full daylight with available correction . if visual acuity was less than 20/60 in either eye , pin hole vision was tested . if presenting vision was < 20/60 , then pupil was dilated and ophthalmoscopy done to assess the cause of blindness . the interobserver agreement between the two teams was good as observed by the kappa value ( > 0.60 ) . double data entry and analysis was done using the raab software program version 4.02 . to check for errors made during data entry of the survey record forms , the data are entered twice by different data entry clerks in two separate databases and then compared . a total of 3050 persons aged over 50 years were included in the study and 2907 ( 95.3% ) of them were examined . of them the sampled population was relatively representative of the district population in terms of age and sex distribution . the overall unadjusted prevalence of blindness from all causes in persons aged over 50 years was 3.9% ( 95% ci 2.75.1 ) . table 1 shows the data regarding prevalence of blindness ( va < 20/400 in better eye with available correction ) ; severe visual impairment ( va < 20/200 20/400 in better eye with available correction ) and moderate visual impairment in persons ( va < 20/60 20/200 in better eye with available correction ) . the age and sex adjusted prevalence of blindness , severe visual impairment , and moderate visual impairment was 3.4% , 3.1% , and 9.7% , respectively . sample prevalence of blindness , severe visual impairment and moderate visual impairment all causes untreated cataract was the primary cause of bilateral blindness ( 75% ) and severe visual impairment ( 73% ) . refractive errors were the most common cause of moderate visual impairment ( 56% ) and second most common cause of severe visual impairment ( 11% ) . avoidable causes accounted for 91% of all cases of blindness and 95% of cases of severe visual impairment [ table 2 ] . causes of blindness ( va < 20/400 ) , severe visual impairment ( va 20/20020/400 ) and moderate visual impairment ( va 20/6020/200 ) the main barriers to uptake of cataract surgery were no one to accompany ( 27% ) ; waiting for maturity ( 27% ) ; do not know how to get surgery done ( 10% ) ; and old age no need ( 7% ) . the cataract surgical coverage ( csc ) in persons with va < 20/400 was high , with 82% of those requiring surgery having received the same . for people with va < 20/200 and va < 20/60 , 72% and 64% , respectively of those needing surgery had received it [ table 3 ] . cataract surgical coverage by persons and eyes sex wise of the 707 eyes , which had received surgery , 641 ( 90.7% ) had an intra - ocular lens ( iol ) implantated . among the patients with iol , 72.7% had va of 20/60 or more with available correction [ table 4 ] . in patients operated less than 5 years back , 86% of patients with iol had best corrected va of more than or equal to 20/60 , when compared with 79% in patients with iol operated more than 5 years back . around 85% of patients were satisfied with the results of cataract surgery . ocular comorbidities , operative complications and long - term complications were the principal reasons for poor outcome in eyes operated less than 3 years back . postoperative visual acuity with available correction low vision ( persons with va < 20/60 in better eye with correction and not due to cataract , refractive error , or uncorrected aphakia ) was seen in 46 persons ( 1.6% ) . in india , eye care planning and monitoring under the national program for control of blindness has been guided by population - based surveys . rapid assessment techniques , which provide reliable estimates have been used for the past 16 years and have been the basis for district level programming . the prevalence of blindness ( va < 20/400 ) was 3.9% in our study , which is almost similar to the raab study conducted across various states in india , which gave a prevalence of 3.6% . the raab india study which covered gulbarga district in karnataka , gave a prevalence of 4.3% for that district . the prevalence of blindness in our study is on the higher side when compared with other studies from kenya , bangladesh , china , palestinian territories and malawi where blindness prevalence ranged from 2.0% to 3.7% . consequently , even though there has been a sharp increase in outreach programs and service delivery , this has been offset by the increasing elderly population as a result of increased life expectancy . in our study , almost 91% of blindness was avoidable . untreated cataract still continues to be the major cause of blindness and severe visual impairment . refractive errors and uncorrected aphakia are the other leading avoidable causes . despite the increased csc , there are an estimated 4700 people [ table 5 ] who are having bilateral cataract in kolar district , extrapolating the survey findings . this cataract burden can only be reduced by proper taluk wise segmentation of the cataract blind and targeting the community outreach programs toward these areas . spectacle provision to the needy at a peripheral level by means of mobile refraction units and spectacle dispensing outfits age and sex adjusted prevalence modified strategies in the form of extensive health awareness and health education campaigns through a decentralized approach involving all major health care providers at the grass root level , down to the remote rural level could be one of the positive steps in reducing this cataract burden . waiting for maturity. this indirectly indicates that patients have been told to wait and sent back , which is an indicator of service delivery deficiency . such people once turned back are unlikely to return back because of reasons such as lack of funds for conveyance to the hospital , loss of daily wages during the visit to the hospital , and relative lack of knowledge about when to return . thus not only increasing the outreach programs but also concentrating on proper case selection , good surgical techniques , proper follow - up care and spectacle provision will definitely reduce the barriers and improve the outcome after cataract surgery . this could be due to the fact that increased percentage of males seeks eye care services as reflected in the increased csc among males . also females are less likely to report a need for sight than males.[1622 ] the strengths of our study was ready availability of taluk wise population data and the survey teams being trained by a certified raab trainer . even though , there is a decline in prevalence of blindness , modified strategies need to be implemented to tackle the burden of untreated cataract . raab done at regular intervals is an effective tool to quantify the problem of blindness and monitor the implementation of eye care programs .

background : more than 90% of visual impairment can either be treated or avoided . rapid assessment of avoidable blindness methodology provides valid estimates in short time to assess magnitude and causes of blindness.aims:to estimate the prevalence and causes of blindness in persons above 50 years in kolar , south india , using the above methodology.materials and methods : sixty one clusters of 50 people aged above 50 years were selected by probability - proportionate to size sampling . participants were evaluated using a standardized survey form . persons with vision < 20/60 were dilated and examined by an ophthalmologist.results:of the 3050 people listed 2907 were examined ( 95.3% ) . prevalence of bilateral blindness in persons was 3.9% ; severe visual impairment 3.5% , and visual impairment 10.4% . untreated cataract was the leading cause of blindness ( 74.6% ) and severe visual impairment ( 73.3% ) . avoidable causes of blindness accounted for 91.2% of all blindness and 95.0% of severe visual impairment . waiting for maturity and no one to accompany were the most common barriers to uptake of cataract surgery.conclusion:untreated cataract continues to be the leading cause of avoidable blindness . modified strategies need to be implemented to tackle the burden of cataract blindness .

they are usually encountered as incidental findings in magnetic resonance imaging ( mri ) , or in arthroscopy . they may originate from both the cruciate ligaments and the menisci , from the popliteus tendon and alar folds , infrapatellar fat pad of hoffa , and subchondral bone cysts . those arising from the hoffa s fat pad , usually present as palpable mass at anterior aspect of the knee joint . we report a case of intraarticular ganglion cyst of knee arising from the infrapatellar fat pad and protruding anterolaterally through retinacular rent into the subcutaneous plane . a 19-year - old young man , presented with a painless gradually increasing swelling at the anterior aspect of left knee of 9 months duration . mri scan revealed a multilobulated , cyst with septations within the anterior aspect of the knee joint , just inferolateral to the patella , with deep extension into the infrapatellar fat pad , and superficial extension into the subcutaneous space across the retinaculum . after diagnostic arthroscopy , we performed an open excision of the cystic mass and confirmed the retinacular rent pre - operatively . arthroscopic resection and debridement is the gold standard treatment in ganglion cyst of the knee . however , a subcutaneous extension may lead to incomplete arthroscopic resection : leaving behind the residual tissue which may cause recurrence . therefore , proper pre - operative evaluation of mr images of these cases is very important . a ganglion is a cystic tumor - like lesion of unknown etiology , surrounded by a dense network of connective tissue , filled with a gelatinous fluid containing hyaluronic acid and other mucopolysaccharides . intra - articular ganglion cysts of the knee joint are uncommon . they are usually encountered as incidental findings in magnetic resonance imaging ( mri ) , or in arthroscopic examination . they may originate from both the cruciate ligaments and the menisci , from the popliteus tendon and alar folds , infrapatellar fat pad of hoffa , and subchondral bone cysts . those arising from the hoffa s fat pad have been reported to present as palpable mass at anterior aspect of knee joint [ 2 - 5 ] ; however , to the best of our knowledge , none of them had subcutaneous communications across the retinaculum . we report a case of intra - articular ganglion cyst of knee arising from the infrapatellar fat pad and protruding anterolaterally through retinacular rent into the subcutaneous plane . a.a.m . , a 19-year - old young man , presented with a painless gradually increasing swelling at the anterior aspect of left knee for last 9 months . there was no history of trauma , local injections , instability , locking episodes , clicking , snapping , or any constitutional symptoms . clinical examination revealed a 2.5 cm x 2.5 cm round , soft , cystic , non - tender , irreducible swelling at anterolateral aspect of left knee 1.5 cm above the lateral joint line just lateral to the patellar tendon with discoloration of the overlying skin . it was partially mobile , not fixed to the extensor mechanism or overlying skin , and was more prominent in knee extension ( fig . 1 ) . he could squat and sit cross - legged without any discomfort with a normal range of movement . there was no effusion , no joint line tenderness , and no evidence of ligamentous laxity . pre - operative clinical pictures showing the ganglion cyst at anterolateral aspect of left knee in various degrees of knee flexion . mri scan revealed a multilobulated , cyst with septations within the anterior aspect of the knee joint , just inferolateral to the patella , with deep extension into the infrapatellar fat pad . ( a ) normal radiograph except increased soft tissue shadow , ( b ) parasagittal and axial mr images showing the ganglion cyst from infrapatellar fat pad extending subcutaneously across the retinaculum . then we went for open excision of the cystic mass through lateral parapatellar approach ( incision included that of the anterolateral arthroscopic portal ) . the cyst was found to be protruding just beneath the skin through a retinacular rent ( fig . however , the entire mass was taken out , but we had to sacrifice the fat pad almost entirely . post - operatively the patient underwent appropriate rehabilitation making an uneventful recovery and returned to his normal activities . he was symptom - free in his last follow - up at 6 months post - operatively . between the first description of an anterior cruciate ligament ( acl ) ganglion by caan in 1924 during a routine autopsy and the early 1990s , only a handful of sporadic cases were described in the literature . the last 30 years have seen a huge increase in the number of these cases being reported owing to the widespread use of both mri and arthroscopy . the prevalence of intra - articular cystic knee masses is 1.3% in mri and 0.6% in arthroscopy . several theories have been suggested : ( a ) synovial tissue herniation , ( b ) mucinous degeneration of connective tissue after trauma , ( c ) synovial ectopia or displacement during embryogenesis , etc . . the histological finding that ganglia are fluid - filled structures without any epithelial lining confirms that they are not true cysts , and thus favors the degenerative theory over others ; and , even though , ganglia may develop in the absence of trauma , it is postulated that repetitive microtrauma from joint and soft tissue motion may play an important role . intra - articular ganglia of knee originates most commonly from the acl accounting for more than 50% cases ; whereas , the location at infrapatellar fat pad is far less common ( approximately 4% ) . however , only 10% of them are symptomatic with most being incidental finding on mri or arthroscopy . the symptoms are usually non - specific , like knee pain , locking , clicking or popping sensation , decreased range of motion , etc . , and depend on the site and size of the cyst . ganglia anterior to the acl tend to limit extension , whereas those posterior to the posterior cruciate ligament often limit knee flexion . mri is the investigation of choice because it is the most sensitive , specific accurate , and noninvasive method for depicting such cystic masses , including their size and location , and to detect associated intra - articular pathologies . for intra - articular ganglion cyst in the infrapatellar fat pad , fat - suppressed contrast - enhanced mri could be useful , because a thin , rim - enhancing feature of intra - articular ganglion cyst allows it to differentiate from synovial hemangioma and synovial sarcoma . among the various treatments available , arthroscopic resection and debridement are currently the preferred procedure ; open surgery may be necessary in particular cases only . after diagnostic arthroscopy , we performed an open excision of the ganglion cyst since it was not clearly visible on arthroscopy ; moreover , we did not have any previous experience of arthroscopic resection of intra - articular ganglion cysts . hence , in view of the high recurrence rate after incomplete removal of ganglion , we preferred open excision over arthroscopic resection . although recurrence is rare after arthroscopic or open surgical treatment of ganglion cyst of knee , the fact that it does occur in other areas even after excision , this patient should be followed up on regular basis . current literature suggests arthroscopic resection and debridement as the gold standard treatment of ganglion cyst of the knee . however , a subcutaneous extension may lead to incomplete arthroscopic resection : leaving behind the residual tissue which may cause recurrence . therefore , proper pre - operative evaluation of mr images of these cases is very important . they are usually encountered as incidental findings in mri , or in arthroscopy . arthroscopic resection and debridement is regarded as the gold standard treatment in ganglion cyst of the knee . however , very rarely , these masses may extend subcutaneously across the retinaculum . this may lead to incomplete arthroscopic resection : leaving behind the residual tissue which may cause recurrences . therefore , proper pre - operative evaluation of mr images of these cases is very important .

introduction : intra - articular ganglion cysts of the knee joint are rare occurrences . they are usually encountered as incidental findings in magnetic resonance imaging ( mri ) , or in arthroscopy . they may originate from both the cruciate ligaments and the menisci , from the popliteus tendon and alar folds , infrapatellar fat pad of hoffa , and subchondral bone cysts . those arising from the hoffa s fat pad , usually present as palpable mass at anterior aspect of the knee joint . we report a case of intraarticular ganglion cyst of knee arising from the infrapatellar fat pad and protruding anterolaterally through retinacular rent into the subcutaneous plane.case report : a 19-year - old young man , presented with a painless gradually increasing swelling at the anterior aspect of left knee of 9 months duration . mri scan revealed a multilobulated , cyst with septations within the anterior aspect of the knee joint , just inferolateral to the patella , with deep extension into the infrapatellar fat pad , and superficial extension into the subcutaneous space across the retinaculum . after diagnostic arthroscopy , we performed an open excision of the cystic mass and confirmed the retinacular rent pre-operatively.conclusion:arthroscopic resection and debridement is the gold standard treatment in ganglion cyst of the knee . however , a subcutaneous extension may lead to incomplete arthroscopic resection : leaving behind the residual tissue which may cause recurrence . therefore , proper pre - operative evaluation of mr images of these cases is very important .

pancreatitis is a major well - known complication of endoscopic retrograde cholangiopancreatography ( ercp ) with reported incidence ranging from 1% to 10% in various series [ 1 - 3 ] . but the most accepted theory is mechanical trauma to papilla or pancreatic sphincter causing transient obstruction to outflow of pancreatic juice . another theory suggests the increased hydrostatic pressure in pancreatic duct caused by injection of contrast or saline cause injury to parenchyma . regardless of mechanism , the cascade of events is initiated resulting in activation of proteolytic enzymes causing autodigestion of pancreas and impaired acinar secretion . this results in activation of inflammatory cascade causing both local inflammation and systemic effects [ 5 , 6 ] . non - steroidal anti - inflammatory drugs ( nsaids ) are potent inhibitor of phospholipase a2 which is thought to play a critical role in early inflammatory cascade . rectal diclofenac is a cheap , widely available agent with easy method of administration and favorable side effect profile makes it an attractive option . there are limited data on efficacy of nsaids in prevention of post - ercp pancreatitis . rectal diclofenac has been evaluated in few trials earlier but most trials included low - risk patients and sample size of these trials was very small . till date , sparse indian data are available which evaluated rectal diclofenac in prevention of post - ercp pancreatitis . we conducted a prospective , single - center , open - labeled , randomized placebo - controlled trial evaluating role of rectal diclofenac in prevention of post - ercp pancreatitis in high - risk patients and whether it has any implications on severity of post - ercp pancreatitis . this study was performed at a tertiary care center between august 2011 and june 2014 . inclusion criteria involved only those patients with high risk of developing post - ercp pancreatitis . patients were considered having high risk of development of post - ercp pancreatitis if they had one or more major risk factors : suspected sphincter of oddi dysfunction ( sod ) , prior history of post - ercp pancreatitis , difficult or failed cannulation ( more than five attempts ) , repeated pancreatic cannulation , pancreatic duct injection with acinarization , pancreatic sphincterotomy , precut sphincterotomy , biliary sphincterotomy for suspected sod , and ampullectomy ; or if they had two or more minor risk factors for development of post - ercp pancreatitis : female gender , young age , history of recurrent acute pancreatitis , normal serum bilirubin , lack of choledocholithiasis , pancreatic brush cytology , and balloon dilatation of intact biliary sphincter . those patients not fitting the high - risk criteria , acute pancreatitis at the time of ercp , contraindications to the use of nsaids ( active peptic ulcer disease or s. creatinine > 1.4 mg / dl ) , and who had ingested nsaids in last 1 week were excluded from the study . one hundred twenty - six patients were excluded from the study depending on the exclusion criteria . one group ( n = 200 ) received rectal diclofenac suppository ( containing 100 mg of diclofenac ) immediately prior to or during the procedure . other group ( n = 200 ) received glycerine suppositories as placebos . injection hyoscine was given for control of bowel motility . during the procedure , an assistant recorded the details of the procedure like timing of procedure , number of pancreatic duct cannulation and injection , difficulty in cannulation , and whether precut , pancreatic sphincterotomy , and balloon sphincteroplasty were done . patients in rectal diclofenac or placebo group received rectal suppository immediately prior to or during the procedure . pancreatic duct stents were placed in those patients in which pancreatic cannulation occurred more than two times or pancreatic injection with contrast or saline during the procedure . patients were subjected to testing of serum amylase at 2 and 32 h post - procedure . those patients who had no abdominal pain , vomiting , back pain and 2 h serum amylase levels less than two times upper limit of normal were started on oral liquids 3 - 4 h after ercp . post - ercp pancreatitis was defined as rise in serum amylase more than three times upper limit of normal 24 h after ercp with associated clinical feature of severe abdominal pain requiring persistent hospitalization . primary endpoint of the study was to detect number of patients developing post - ercp pancreatitis in both groups . patients were subjected to routine biochemical investigations , imaging modalities like ultrasound abdomen and contrast - enhanced computed tomography to detect complications of pancreatitis . the severity of pancreatitis was graded as mild , moderate and severe according to days of hospitalization required and complications of pancreatitis . mild post - ercp pancreatitis was defined as requiring an unplanned admission or prolongation of hospitalization by 2 - 3 days . moderate post - ercp pancreatitis was defined as requiring hospitalization of 4 - 10 days and severe post - ercp pancreatitis was defined as requiring hospitalization of greater than 10 days or requiring intensive care or intervention for local complications of pancreatitis . the secondary endpoint of the study was to assess the severity of post - ercp pancreatitis in both groups . , we used fisher exact test to analyze the difference in proportion of patients with post - ercp pancreatitis in rectal diclofenac and placebo groups with p value < 0.05 indicating significant difference . patients demographic and clinical factors were compared using fisher exact test or x test as appropriate . for the analysis of primary endpoint , we used fisher exact test to analyze the difference in proportion of patients with post - ercp pancreatitis in rectal diclofenac and placebo groups with p value < 0.05 indicating significant difference . patients demographic and clinical factors were compared using fisher exact test or x test as appropriate . a total of 400 patients entered the study ; 200 patients received rectal diclofenac , while 200 patients received glycerine suppository ( control group ) . there were 128 ( 64% ) female patients in rectal diclofenac group while 123 ( 61.5% ) in placebo group . the mean age in rectal diclofenac group was 45.44 years while in placebo group was 47.86 years . the patients in both groups were well matched for the indication of ercp as depicted in table 1 . we compared various risk factors prior to or during the procedure which might increase the risk of post - ercp pancreatitis like pancreatic cannulation , precut sphincterotomy , suspected sod , pancreatic sphincterotomy , pancreatic duct injection , balloon sphincteroplasty in patients with suspected sod , and difficulty of failed cannulation in both groups . the baseline characteristics of the patients in both groups were identical as depicted in table 2 . a total of 23 patients , 12 in placebo group and 11 patients in rectal diclofenac group , received pancreatic stents due to recurrent pancreatic duct cannulation or pancreatic duct injection with acinarization . twenty - nine out of 400 ( 7.2% ) patients developed post - ercp pancreatitis . six out of 200 ( 3% ) patients in rectal diclofenac group developed post - ercp pancreatitis compared to 23 out of 200 ( 11.5% ) patients in placebo group ( fig . the difference was statistically significant with p value < 0.05 . in a subgroup analysis , incidence of post - ercp pancreatitis in patients with suspected sod in rectal diclofenac group was 6% ( 4/66 ) while in placebo group was 18.8% ( 13/69 ) . among patients who received pancreatic stent , only one patient in rectal diclofenac group ( 1/11 ) developed post - ercp pancreatitis compared to two patients in placebo group ( 2/12 ) ; however , the number of patients in which pancreatic stenting was performed was low in our study . the secondary endpoint of the study was to assess the severity of post - ercp pancreatitis in both groups . the severity of post - ercp pancreatitis in rectal diclofenac group was mild in all six patients ( 100% ) , while in placebo group , it was severe in four patients ( 4/23 , 17.3% ) , moderate in five patients ( 5/23 , 21.7% ) and mild in 14 patients ( 14/23 , 60.8% ) ( fig . the difference in severity of pancreatitis in rectal diclofenac and placebo groups was statistically significant . two out of four patients in placebo group who had severe post - ercp pancreatitis developed pancreatic pseudocyst , but were managed conservatively . one out of four patients in placebo group with severe post - ercp pancreatitis developed right - sided pleural effusion requiring therapeutic pleural tapping . one out of four patients in placebo group with severe post - ercp pancreatitis developed subacute intestinal obstruction . two deaths occurred in the placebo group , both with severe post - ercp pancreatitis , one with right - sided pleural effusion and the other with subacute intestinal obstruction . adverse events like bleeding while ercp in sphincteromized patients were noted in 16 patients , six in placebo group and eight patients in rectal diclofenac group but responded to adrenaline injection and coagulation . rectal diclofenac suppository was well tolerated in all the patients with no adverse event noted with use of rectal diclofenac . our study has shown that single dose administration of rectal diclofenac prior to or during the procedure has reduced the incidence of post - ercp pancreatitis . , our study has shown that rectal diclofenac was also effective in this group of patients in reducing post - ercp pancreatitis . rectal diclofenac is a cheap drug , easily available and with a favorable side effect profile . pancreatic stents are of proven benefit in preventing post - ercp pancreatitis [ 8 , 9 ] but difficult to use in routine practice because of difficulty in pancreatic duct cannulation and requirement of operator expertise . but most accepted theory is trauma of thermal injury to papilla causing transient obstruction to pancreatic secretion . another accepted theory is hydrostatic pressure due to contrast or saline injection into pancreatic duct . this injury initiates an inflammatory cascade leading to activation of proteolytic enzymes intraductally causing pancreatic autodigestion and impaired secretion . diclofenac inhibits phospholipase a2 which is an important mediator of various reactions in this inflammatory cascade breaking this cycle [ 5 , 6 ] . peak concentration of rectal diclofenac occurs between 30 and 90 min after insertion with complete bioavailability . the elimination half life is 2 h. rectal diclofenac has been evaluated in different randomized clinical trials . in a study by murray et al , 220 patients with high risk of post - ercp pancreatitis were randomized in two groups , rectal diclofenac vs. placebo . in these patients there was significant reduction in incidence of post - ercp pancreatitis in rectal diclofenac group as compared to placebo which was similar to our study . however , in this study , there was no significant difference in patients with sod in rectal diclofenac vs. placebo group . our study demonstrated that there is significant reduction in incidence of post - ercp pancreatitis even in patients with suspected sod . khoshbaten et al evaluated the use of rectal diclofenac in patients with extrahepatic cholestasis undergoing ercp . this study also reported significant reduction in incidence of post - ercp pancreatitis in rectal diclofenac group similar to our study . however , this study has reported very high incidence of post - ercp pancreatitis ( 26% ) in the control group . oral diclofenac has been evaluated for prevention of post - ercp pancreatitis by cheon et al , but was found to be of no benefit . though rectal diclofenac is such an attractive option in prevention of post - ercp pancreatitis , it is still underutilized . there are very few trials evaluating its use in prevention of post - ercp pancreatitis . this prospective single - center , open - labeled , randomized placebo - controlled trial has shown that use of single dose of rectal diclofenac immediately prior to or during the ercp in high - risk patients is effective in not only reducing the incidence but also severity of post - ercp pancreatitis . the major limitation of this study was severity of pancreatitis was graded according to the consensus guideline depending on days of hospitalization and complications . various scoring systems like ranson s score and apache ii score were not utilized . however , similar guidelines were followed in previous studies . rectal diclofenac prior to or during ercp in high - risk patients reduces the incidence as well as severity of post - ercp pancreatitis compared to placebo . rectal diclofenac prior to or during ercp in high - risk patients reduces the incidence as well as severity of post - ercp pancreatitis compared to placebo .

backgroundthe aim was to study the role of rectal diclofenac in prevention of post - endoscopic retrograde cholangiopancreatography ( ercp ) pancreatitis and its impact on severity of post - ercp pancreatitis.methodswe conducted a single - center , prospective , open - labeled , randomized trial for evaluating the use of rectal diclofenac in prevention of post - ercp pancreatitis in high - risk patients . we assessed 526 patients coming for ercp for different indications . four hundred patients were eligible for the study . those not fitting the high - risk criteria and with acute pancreatitis were excluded . these patients were randomized in two groups : 200 patients received rectal diclofenac prior to or during the procedure , while 200 patients received placebos . serum amylase was measured at 2 and 36 hours . post - ercp pancreatitis was defined as serum amylase > 3 times upper limit of normal with associated severe abdominal pain . severity was graded according to days of hospitalization and complications.resultstwenty-nine out of 400 ( 7.2% ) patients developed post - ercp pancreatitis . six out of 200 ( 3% ) patients in rectal diclofenac group developed post - ercp pancreatitis compared to 23 out of 200 ( 11.5% ) patients in placebo group . the difference was statistically significant ( p = 0.001 ) . all patients ( six ) in rectal diclofenac group developed mild pancreatitis as compared to severe pancreatitis in four and moderate pancreatitis in five patients in the placebo group.conclusionrectal diclofenac prior to or during ercp in high - risk patients reduces the incidence as well as severity of post - ercp pancreatitis compared to placebo .

a 75-year - old male farmer presented to a primary care skin cancer practice in west australia with a 6-month history of an enlarging painless lesion on his left cheek with no history of any possible precursor lesion . there was a past history of treatment of two separate squamous cell carcinomas in - situ , one on the forehead and the other on an ear , in the previous two years . other comorbidities included asthma , coronary heart disease , chronic obstructive pulmonary disease and arthritis . there was no history of nevus sebaceous and there was neither history nor clinical signs suggestive of cowden s syndrome . examination showed a single small non - pigmented nodular lesion located on the left cheek in continuation with a small , pigmented macular lesion ( figure 1 ) . dermatoscopy ( figure 2 ) revealed a raised pink lesion , 3.5 mm in diameter , with a radial arrangement of vessels peripherally , with centered blood vessels in the middle of the lesion , these vessels being centered in skin - colored clods . no keratin structures ( keratin scale , white structureless areas or white circles ) were seen . at one edge of this lesion , in direct contact with it , was a heavily pigmented flat lesion , 1 mm in diameter with a pattern of pigmented lines radial , converging ( figure 2 ) . the lesion was subjected to an excision biopsy with a 4 mm peripheral margin and submitted for dermatopathological evaluation . dermatopathology ( figure 3 ) showed both the trichilemmoma and bcc ( basal cell carcinoma ) components . the trichilemmoma ( figure 3 a1 and a2 ) was a well - circumscribed , sharply demarcated lesion with surface papillomatosis , horn cysts and a degree of central desmoplasia . under high power an eosinophilic mantle of thickened basement membrane , mimicking the outer root sheath of the hair follicle infundibulum , was seen . peripheral palisading was evident adjacent to the basement membrane and extending from that there were clear cells which were banal cytologically , lacked mitosis or individual cell necrosis ( apoptosis ) and contained an abundant amount of glycogen within their cytoplasm . the bcc component ( figure 3 b1 and b2 ) showed superficial bcc at the dermo - epidermal junction . in the high power view melanin pigment trichilemmoma is a benign tumor derived from the external sheath cells of pilosebaceous units and desmoplastic trichilemmoma is a rare variant , which can present clinically with features suggesting invasive malignancy . there has been controversy as to whether trichilemmomas are actually aged warts , this belief being proposed by well - known dermatopathologist bernie ackerman but vigorously opposed by others including marty brownstein . basal cell carcinoma is a malignant tumor , of trichoblast differentiation but uncertain origin , although it is probably of follicular derivation , predilection for non - glabrous skin being consistent with this . trichilemmoma has previously been reported in association with bcc in the context of lesions arising in a preexisting nevus sebaceous . in addition , three cases of desmoplastic trichilemmoma arising in association with bcc without a preexisting nevus sebaceous have been reported . the dermatoscopy of four trichilemmomas has been reported with images being presented for three of them . in the first case keratin masses ; perivascular whitish halos . the other three were reported together , with the first two described as having a hyperkeratotic central area with peripheral erythematous radial lines , the third having a peripheral radiated red area . all three were then presented as depicting what the authors called a red iris like structure . examination of the photographs published in fact displayed a non - pigmented lesion in each case with a radial arrangement of vessels peripherally and structureless white centrally with some centred vessels . in the case we report , the clinician interpreted the lesion as a pigmented lesion and assessed it using the chaos and clues decision - algorithm . the lesion was regarded as chaotic ( asymmetrical ) with the clues of an eccentric structureless ( pink ) area as well as lines radial segmental , which lead to a clear decision to do an excision biopsy . the pathologist , as is often the case with esoteric lesions , was able to deliver the answer to which particular type of malignancy this was . the dermatoscopic features of the trichilemmoma presented here are consistent with those published previously , with a radial pattern of vessels peripherally and centred vessels centrally . association with a bcc is a previously reported , unusual variation , consistent with both lesions having hair - follicle derivation . this case demonstrates the utility of using a decision algorithm in esoteric cases with the final diagnosis to be delivered by the gold standard of dermatopathology .

a case of trichilemmoma in continuity with a pigmented basal cell carcinoma is presented with dermatoscopy and dermatopathology . the distinction between the two lesions was evident dermatoscopically and was confirmed dermatopathologically . while trichilemmoma has been reported in association with basal cell carcinoma and dermatoscopy images of four previous cases of trichilemmoma have been published , no previous dermatoscopy image has been published of trichilemmoma associated with basal cell carcinoma .

today , the treatment of choice is surgical excision of the tongue , but before 1900 , surgery was often temida1 . only in 1673 niels the size and shape of the teeth are directly influenced by the size of tongue3 . the shape of the teeth is determined by forces employed on the teeth , especially the muscles of the tongue , lips and cheek . due to the effects caused by the aesthetic and functional macroglossia the macroglossia is classified as true when there is excessive enlargement of the language and a relative when there is an imbalance between the size of the tongue and oral cavity , resulting in insufficient space for organ4 . the goal is to establish technical bases associated with partial glossectomy orthodontic treatment of dentofacial deformity in patients with macroglossia . three patients underwent orthognathic surgery associated with partial glossectomy from 1995 to 1999 , a multidisciplinary team - doctor and dentist . all patients had macroglossia relative and underwent clinical assessment taking into account the respiratory function , swallowing and speech deficit , as well as changes in dental occlusion and was also performed radiological evaluation . the main problem was manifested by the presence of steep lower dent alveolar , destabilizing the use of dentures causing joint dysfunction with severe pain . she underwent a single surgical intervention in the partial glossectomy and orthognathic surgery for targeting sub apical segment dent alveolar anterior inferior . the main problem of the second patient was the anterior open bite accompanied by difficulty in breathing and articulation of words . after treatment for orthodontic tooth alignment leveling , underwent surgery for a single suspension by corticotomy posterior maxillary le fort i type , reduction of mandibullary prognathism by sagittal technical branches , targeting sub apical posterior - inferior right and partial glossectomy . the third patient had mandibullary prognathism and , during orthodontic treatment in preparation for orthognathic surgery , the surgery was anticipated , since the interposition of the tongue between the back teeth did not allow the evolution of orthodontic treatment . he underwent a partial glossectomy to reduce the transverse diameter of the tongue and mandibullary prognathism by sagittal technical branches . we used rigid skeletal fixation with titanium plates and screws so that patients could stay without intermaxillary block in the immediate postoperative period . to control tongue edema , the technique used consist of segmental resection along the median raphe of the tongue and suture by planes - figure 1 . as a routine the symptoms regressed completely and all skeletal segments remained stable . demarcation and resection of the lingual and final appearance . the classification of macroglossia is not yet consensus in the literature . according to shafer ( 1968)5 , or primary congenital macroglossia is due to the excessive development of the musculature , which may or may not be associated with generalized muscular hypertrophy or unilateral hypertrophy . since the secondary macroglossia may occur as a result of a tumor in the tongue , as a diffuse hemangioma or lymphangioma , neurofibromatosis , and occasionally blocking efferent vessels in cases of malignant neoplasm of the tongue . wolford et al . ( 1996)6 refer to as macroglossia on pseudomacroglossia , separating it from the true macroglossia . ( 1980)7 macroglossia consider the functional as a third classification , occurring when the language does not fit into the oral cavity after a surgical procedure . the true when there are histological abnormalities associated with the increase of the tongue , such as vascular malformation , stretching and tumors . relative macroglossia includes cases of apparent increase in volume without an explanation of the language exam , as in down syndrome . the decision to refer the patient to partial glossectomy should be based on the volume of the language , mobility , position , function , symptoms , speech intelligibility , skeletal anterior open bite , interference in orthodontic treatment , drooling , swallowing and tongue recurrent trauma9 . the language has increased in volume expansive effect on the lower dental arch , being blamed as the cause and maintenance of open bite , bimaxillary protrusion or diastemas10 . being interposed between the arches , is an important etiologic factor for malocclusion listed ( figures 2 and 3 ) . a partial glossectomy performed simultaneously with mandibullary osteotomy for treatment of patients with mandibullary prognathism and anterior open bite is advantageous to prevent recidivas11 . the tongue can cause deformity increased dental - muscle - skeletal , instability in orthodontic treatment and orthognathic surgery , masticatory disability , communication problems and respiratory6 . there are several clinical and radiographic findings , but not all features are always present and their existence is not necessarily path gnomonic for the diagnosis of macroglossia . should be included the clinical , radiographic and functional for the interference with speech , mastication , airway and stability of orthodontic treatment and orthognathic surgery . there are basically three choices in the surgical sequence : ( i ) stage 1 : partial glossectomy , stage 2 : orthognathic surgery ( ii ) stage 1 : orthognathic surgery , stage 2 : partial glossectomy and ( iii ) partial glossectomy and orthognathic surgery in a single stage surgery .

summary introduction : macroglossia is a condition which influences the size and shape of the teeth employed due to the forces on teeth . objective : to establish bases for the indication of partial glossectomy associated with orthodontic treatment and surgical dento - facial deformity in patients without tumors and down syndrome as a cause of macroglossia . case reports : three patients underwent orthognathic surgery associated with partial glossectomy under general anesthesia . all patients had macroglossia relative and underwent clinical assessment taking into account the respiratory function , swallowing and speech deficits and radiological evaluation . the technique used consist of segmental resection along the median raphe of the tongue and suture by planes . we used rigid skeletal fixation with titanium plates and screws so that patients could stay without intermaxillary block in the immediate postoperative period . were followed over five years . the symptoms regressed completely and all skeletal segments remained stable . discussion : the decision to refer the patient to partial glossectomy should be based on the volume of the language , mobility , position , function , symptoms , speech intelligibility , skeletal anterior open bite , interference in orthodontic treatment , drooling , swallowing and tongue trauma applicant .

nevertheless , many people do not have adequate physical activity and sedentary lifestyle has become an important public health issue in all age groups , including adolescents . because of increasing consumption of high - calorie food , use of digital technologies , and adopting a sedentary lifestyle in older age , physical activity promotion has become more important in adolescents . nowadays , about 80% of adolescents have neither severe nor mild physical activity more than 60 min per day . many studies show that self - efficacy ( se ) is one of the major determinants of physical activity . this construct plays an important role as the predictor of exercise , and people who have more confidence about their ability show greater participation in physical activity . likewise , those who engage more in exercise programs also achieve a higher level of general se . according to bandura , se expresses one 's beliefs in his or her ability to successfully carry out a course of action . he posits that cognitive constructs such as se have a great impact on physiologically and/or psychologically demanding behaviors like physical activities . the role of se is strongest during the early stages of participation in physical activities and when this behavior is difficult to do because of some barriers such as fatigue and time constraints . se has indirect effects on behavior too , exerting its influence through goals , ideas , outcome expectations , intentions , and perceived barriers and opportunities . physical activity is a multiple behavior and its performance needs the involvement of a range of motor skills . therefore , se about physical activity reflects a range of capabilities required to achieve a final consequence . some aspects of se that play a role in initiation of physical activity may differ from other aspects of se that relate to maintenance of physical activity . according to bandura , beliefs of se on special behavior include multiple detention set of management of intellectual , emotional , motivational , and functional processes . because attention to these dimensions can be helpful in the achievement of better understanding of the relationship between se and physical activities , it is necessary to identify the relationship between different aspects of se and physical activity . mcauley and mihalko explained task se and regulatory se as the two main types of se . task se is defined as beliefs on ability to perform constituent components of a behavior or a skill . when we consider the type , intensity , duration , and frequency of physical activity , task se should be studied . barrier se is one of the most common and known aspects of regulatory se that is applied in explanation , prediction , and modification of physical activity . other aspects of regulatory se like goal setting efficacy , scheduling efficacy , relapse prevention efficacy , asking efficacy , and environmental change efficacy have already been studied . it seems that different aspects of regulatory se have more importance in promoting participation in physical activity in public health issues . se reflects confidence of a person to management and coordination between a set of abilities and skills , not only in countering the barrier but also in commonplace condition . this shows the importance of se in design and execution of program , besides its ability to overcome the barriers . for this reason , some researchers believe that adoption and maintenance of physical activities require the se beliefs in goal setting , designing , and implementing . maddux found that the effect of se on setting and performing a program is more fundamental than task efficacy . nevertheless , few researchers have considered barrier se and se in designing and implementation of physical activities , simultaneously . nowadays , most studies are performed by considering in addition , researchers who intend to perform cross - sectional or interventional studies on physical activities in iranian population have attempted to design , translate , or re - translate se measurement tools . however , there is no evidence of a specially designed questionnaire for measuring barrier se , program designing se , and program execution se among iranian male adolescents . because the lack of attention to different aspects of se can lead to bias in the results of studies and interventions that deal with the relationship between se and physical activities , this study aims at introducing and evaluating the psychometric properties of the se questionnaire of leisure time physical activity among iranian male adolescents , emphasizing the barrier se , program designing se , and program execution se . this study was cross - sectional in design and was conducted in isfahan in the central region of iran in 2013 . recommended sample size for confirmatory factor analysis ( cfa ) is at least 5 to 20 cases per parameter . comrey and lee recommended the sample sizes of 200 as fair , 300 as good , and 500 as very good for exploratory factor analysis ( efa ) . because cfa and efa were used to evaluate the psychometric properties of the questionnaire , the main sample was divided into two separate samples for each analysis . overall , 750 adolescents aged 1519 years were recruited , of whom 16 participants were excluded while completing the questionnaire . thus , 734 male adolescents aged 1519 who fulfilled the inclusion criteria remained and were randomly divided into sample 1 and 2 . while entering the data , it was found that the 62 questionnaires had to be excluded due to incomplete filling ( ntotal = 672 ) . the data of sample 1 ( n1 = 325 ) were used for item analysis and efa and those of sample 2 ( n2 = 347 ) for cfa . in order to increase generalizability , isfahan was divided into three regions with low , intermediate , and high socioeconomic levels , based on previous studies and expert opinions . then five high schools were selected randomly in each region , as clusters of sampling ( totally 15 high schools ) . after calculating the study sample size ( 750 ) , the allocated sample size to each high school inclusion criteria were parental consent and student assent , lack of health problem that prevented them from performing physical activities , and not being a member of a professional sport teams . data were gathered via a self - administrated questionnaire that consisted of three main parts : some of main characteristics of participants , such as age and familial income ( demographic characteristics)physical activity was measured using the long - version of international physical activity questionnaire ( ipaq ) . this questionnaire was designed in 1998 by a group of italian researchers and suggested as the international measurement of physical activity for the age range of 1569 by the world health organization ( who ) and the centers for disease control and prevention ( cdc ) . ipaq is used to evaluate a subject 's estimated metabolic equivalent ( met ) on five domains of physical activity consisting of occupational , home and domestic , transportation , and leisure time physical activity ( ltpa ) . ipaq divides individuals into three groups of total pa : low activity ( less than 600 met - min / week ) , moderate activity ( between 600 and 3000 met - min / week ) , and severe activity ( more than 3000 met - min / week ) . the amount of ltpa is divided into three levels based on the leisure time that a person spends per week for pa : less than 60 min , 60180 min , and over 180 min assumed as low , medium , and high ltpa , respectively . reliability and validity of persian version of ipaq were verifiedthe questionnaire offered for se consisted of 13 items . content of the items and the process of selection and generation are described in the following sections . these items measured the degree of confidence of participants in their ability in overcoming barriers , goal setting , and implementation of programs about physical activity using a 5-point likert scale ( 1 = absolutely correct , 5 = absolutely incorrect ) . some of main characteristics of participants , such as age and familial income ( demographic characteristics ) physical activity was measured using the long - version of international physical activity questionnaire ( ipaq ) . this questionnaire was designed in 1998 by a group of italian researchers and suggested as the international measurement of physical activity for the age range of 1569 by the world health organization ( who ) and the centers for disease control and prevention ( cdc ) . ipaq is used to evaluate a subject 's estimated metabolic equivalent ( met ) on five domains of physical activity consisting of occupational , home and domestic , transportation , and leisure time physical activity ( ltpa ) . ipaq divides individuals into three groups of total pa : low activity ( less than 600 met - min / week ) , moderate activity ( between 600 and 3000 met - min / week ) , and severe activity ( more than 3000 met - min / week ) . the amount of ltpa is divided into three levels based on the leisure time that a person spends per week for pa : less than 60 min , 60180 min , and over 180 min assumed as low , medium , and high ltpa , respectively . reliability and validity of persian version of ipaq were verified the questionnaire offered for se consisted of 13 items . content of the items and the process of selection and generation are described in the following sections . these items measured the degree of confidence of participants in their ability in overcoming barriers , goal setting , and implementation of programs about physical activity using a 5-point likert scale ( 1 = absolutely correct , 5 = absolutely incorrect ) . generation of the items and evaluation of psychometric properties of the questionnaire were done in a current and logical direction that included the following stages : creating the initial draft of the instrument and selection or generation of items , establishing a jury of experts for completing the qualitative review , and completing the quantitative review based on lawshe , venezizno , and hooper method . in this method , content validity ratio ( cva ) and content validity index ( cvi ) were estimated and then , reliability of the questionnaire was evaluated by efa and cfa . from the very moment of designing and/or item selection , the validity of the questionnaire was tried to be achieved . so , after review of the literature and some current questionnaires , the main aspects of se were extracted according to the characteristics of the target group . then 30 items for the evaluation of se were designed in persian on the three following domains : overcoming barriers , program adjustment , and implementation of programs . so , the process of translation and backward translation of these items from english into persian was performed by two independent health professionals who were fluent in both languages . to evaluate the we requested five independent health professionals to give their opinions about the face validity and cultural adaptation of the initial questionnaire . thirty items were evaluated based on criteria such as simplicity , intelligibility , relevance , and appropriateness to the target group and also absence of ambiguity . at this stage , content validity was primarily evaluated by an expert panel that consisted of 10 health educators . they were asked about some of the qualitative characteristics of items , such as compliance to principles of grammar , wording , item allocation , and scaling . according to lawshe 's method , the minimum acceptable cut - off for cvr was 0.62 . based on related equation , in addition to quantitative evaluation of cvr , propositions for reform were requested from experts for each item that was selected as an unnecessary item . cultural and linguistic characteristics of iranian population are the main criteria for item evaluation . according to lynn 's method , simplicity , clarity , and specificity were considered to calculate cvi . since 0.79 was selected as the criterion for acceptable cvi , all the 13 items had the efficient criterion for remaining in the questionnaire . prior to the pilot study , comprehensibility of the questionnaire was evaluated through the opinions of 35 adolescent boys , none of whom was a member of either sample 1 or sample 2 . they stated their opinions about each item via a likert scale that consisted of the options , quite understandable , the number of selected options of quite understandable and understandable was divided by 35 and comprehensibility coefficient of each item was calculated . acceptable criterion for comprehensibility of each item was equal to or greater than 0.79 and all items met this criterion . finally , for estimation of reliability of the questionnaire , internal consistency and test retest methods were used . in a pilot study , the questionnaires were given to 73 members of the target group who were not from the study population . they completed the questionnaire 2 weeks prior to the study , and then cronbach 's alpha coefficient of the first survey and pearson correlation coefficient between both surveys were calculated . we considered cronbach 's alpha coefficient and correlation coefficient equal to or greater than 0.70 as satisfactory . a total of 62 students participated in the second survey and pearson correlation coefficient between test and retest showed a strong correlation between se score in the first and second steps ( r = 0.73 , n = 62 , p < 0.005 ) . item analysis was performed for the evaluation of reliability of the questionnaire and its items on sample 1 ( n1 = 325 ) . so , inter - item correlation , item total correlation , variance , squared multiple correlation , and cronbach 's alpha if item deleted were evaluated for each item . if the mean score of an item greatly diverged from the total mean score of the questionnaire or its variance was near to zero , that item was deleted . in addition , we used efa to evaluate the construct validity of the questionnaire on sample 1 . since the suggested questionnaire consisted of three types of se , extraction step was performed by pre - assumption of principle component analysis . we also chose direct oblimin rotation because of the possibility of correlation between components . based on pre - assumption of statistical software , the amount of eigenvalue ( variance of the factor ) was determined to be equal to 1 and the number of items of rotation to establish an appropriate rotational factor was determined to be equal to 25 . recent procedure is suitable for the evaluation of some characteristics such as unidimensionality of items . so , cfa was performed on sample 2 ( n2 = 347 ) , for determination of construct validity , performed to test the fit of data to the model that was suggested by efa . in this stage , several alternative models were tested . we used amosgraphic version 20 software ( amos graphic ) with maximum likelihood estimation procedure assumption and evaluated absolute , comparative , and parsimonious fit indices . the model was considered acceptable if cmin / df was between 1 and 5 , cfi was greater than 0.8 , parsimonious comparative fit index ( pcfi ) was less than 0.6 , root mean squared error of approximation ( rmsea ) was less than 0.8 , and pclose was greater than 0.005 . fitness of model was confirmed if p value of cmin was greater than 0.05 , cmin / df was between 2 and 3 , cfi was greater than 0.9 , and pcfi was less than 0.5 . the study was started after it was approved by isfahan university of medical sciences and isfahan education organization . ethical approval was granted by the deputy of research and technology of isfahan university of medical sciences ( i d : 39147 , date : december 30 , 2012 ) . the purpose and procedures of the study were explained to the participants , and researcher emphasized on the confidentiality of the data and voluntary nature of participation . this study was cross - sectional in design and was conducted in isfahan in the central region of iran in 2013 . recommended sample size for confirmatory factor analysis ( cfa ) is at least 5 to 20 cases per parameter . comrey and lee recommended the sample sizes of 200 as fair , 300 as good , and 500 as very good for exploratory factor analysis ( efa ) . because cfa and efa were used to evaluate the psychometric properties of the questionnaire , the main sample was divided into two separate samples for each analysis . overall , 750 adolescents aged 1519 years were recruited , of whom 16 participants were excluded while completing the questionnaire . thus , 734 male adolescents aged 1519 who fulfilled the inclusion criteria remained and were randomly divided into sample 1 and 2 . while entering the data , it was found that the 62 questionnaires had to be excluded due to incomplete filling ( ntotal = 672 ) . the data of sample 1 ( n1 = 325 ) were used for item analysis and efa and those of sample 2 ( n2 = 347 ) for cfa . in order to increase generalizability , isfahan was divided into three regions with low , intermediate , and high socioeconomic levels , based on previous studies and expert opinions . then five high schools were selected randomly in each region , as clusters of sampling ( totally 15 high schools ) . after calculating the study sample size ( 750 ) , the allocated sample size to each high school inclusion criteria were parental consent and student assent , lack of health problem that prevented them from performing physical activities , and not being a member of a professional sport teams . data were gathered via a self - administrated questionnaire that consisted of three main parts : some of main characteristics of participants , such as age and familial income ( demographic characteristics)physical activity was measured using the long - version of international physical activity questionnaire ( ipaq ) . this questionnaire was designed in 1998 by a group of italian researchers and suggested as the international measurement of physical activity for the age range of 1569 by the world health organization ( who ) and the centers for disease control and prevention ( cdc ) . ipaq is used to evaluate a subject 's estimated metabolic equivalent ( met ) on five domains of physical activity consisting of occupational , home and domestic , transportation , and leisure time physical activity ( ltpa ) . ipaq divides individuals into three groups of total pa : low activity ( less than 600 met - min / week ) , moderate activity ( between 600 and 3000 met - min / week ) , and severe activity ( more than 3000 met - min / week ) . the amount of ltpa is divided into three levels based on the leisure time that a person spends per week for pa : less than 60 min , 60180 min , and over 180 min assumed as low , medium , and high ltpa , respectively . reliability and validity of persian version of ipaq were verifiedthe questionnaire offered for se consisted of 13 items . content of the items and the process of selection and generation are described in the following sections . these items measured the degree of confidence of participants in their ability in overcoming barriers , goal setting , and implementation of programs about physical activity using a 5-point likert scale ( 1 = absolutely correct , 5 = absolutely incorrect ) . some of main characteristics of participants , such as age and familial income ( demographic characteristics ) physical activity was measured using the long - version of international physical activity questionnaire ( ipaq ) . this questionnaire was designed in 1998 by a group of italian researchers and suggested as the international measurement of physical activity for the age range of 1569 by the world health organization ( who ) and the centers for disease control and prevention ( cdc ) . ipaq is used to evaluate a subject 's estimated metabolic equivalent ( met ) on five domains of physical activity consisting of occupational , home and domestic , transportation , and leisure time physical activity ( ltpa ) . ipaq divides individuals into three groups of total pa : low activity ( less than 600 met - min / week ) , moderate activity ( between 600 and 3000 met - min / week ) , and severe activity ( more than 3000 met - min / week ) . the amount of ltpa is divided into three levels based on the leisure time that a person spends per week for pa : less than 60 min , 60180 min , and over 180 min assumed as low , medium , and high ltpa , respectively . reliability and validity of persian version of ipaq were verified the questionnaire offered for se consisted of 13 items . content of the items and the process of selection and generation are described in the following sections . these items measured the degree of confidence of participants in their ability in overcoming barriers , goal setting , and implementation of programs about physical activity using a 5-point likert scale ( 1 = absolutely correct , 5 = absolutely incorrect ) . generation of the items and evaluation of psychometric properties of the questionnaire were done in a current and logical direction that included the following stages : creating the initial draft of the instrument and selection or generation of items , establishing a jury of experts for completing the qualitative review , and completing the quantitative review based on lawshe , venezizno , and hooper method . in this method , content validity ratio ( cva ) and content validity index ( cvi ) were estimated and then , reliability of the questionnaire was evaluated by efa and cfa . from the very moment of designing and/or item selection , so , after review of the literature and some current questionnaires , the main aspects of se were extracted according to the characteristics of the target group . then 30 items for the evaluation of se were designed in persian on the three following domains : overcoming barriers , some of these items also had already and similarly been used in previous studies . so , the process of translation and backward translation of these items from english into persian was performed by two independent health professionals who were fluent in both languages . to evaluate the we requested five independent health professionals to give their opinions about the face validity and cultural adaptation of the initial questionnaire . thirty items were evaluated based on criteria such as simplicity , intelligibility , relevance , and appropriateness to the target group and also absence of ambiguity . at this stage , eight items were revised and changed in wordage and 10 items were deleted . content validity was primarily evaluated by an expert panel that consisted of 10 health educators . they were asked about some of the qualitative characteristics of items , such as compliance to principles of grammar , wording , item allocation , and scaling . according to lawshe 's method , the minimum acceptable cut - off for cvr was 0.62 . based on related equation , in addition to quantitative evaluation of cvr , propositions for reform were requested from experts for each item that was selected as an unnecessary item . cultural and linguistic characteristics of iranian population are the main criteria for item evaluation . according to lynn 's method , simplicity , clarity , and specificity were considered to calculate cvi . since 0.79 was selected as the criterion for acceptable cvi , all the 13 items had the efficient criterion for remaining in the questionnaire . prior to the pilot study , comprehensibility of the questionnaire was evaluated through the opinions of 35 adolescent boys , none of whom was a member of either sample 1 or sample 2 . they stated their opinions about each item via a likert scale that consisted of the options , quite understandable , the number of selected options of quite understandable and understandable was divided by 35 and comprehensibility coefficient of each item was calculated . acceptable criterion for comprehensibility of each item was equal to or greater than 0.79 and all items met this criterion . finally , for estimation of reliability of the questionnaire , internal consistency and test retest methods were used . in a pilot study , the questionnaires were given to 73 members of the target group who were not from the study population . they completed the questionnaire 2 weeks prior to the study , and then cronbach 's alpha coefficient of the first survey and pearson correlation coefficient between both surveys were calculated . we considered cronbach 's alpha coefficient and correlation coefficient equal to or greater than 0.70 as satisfactory . a total of 62 students participated in the second survey and pearson correlation coefficient between test and retest showed a strong correlation between se score in the first and second steps ( r = 0.73 , n = 62 , p < 0.005 ) . item analysis was performed for the evaluation of reliability of the questionnaire and its items on sample 1 ( n1 = 325 ) . so , inter - item correlation , item total correlation , variance , squared multiple correlation , and cronbach 's alpha if item deleted were evaluated for each item . if the mean score of an item greatly diverged from the total mean score of the questionnaire or its variance was near to zero , that item was deleted . in addition , we used efa to evaluate the construct validity of the questionnaire on sample 1 . since the suggested questionnaire consisted of three types of se , extraction step was performed by pre - assumption of principle component analysis . we also chose direct oblimin rotation because of the possibility of correlation between components . based on pre - assumption of statistical software , the amount of eigenvalue ( variance of the factor ) was determined to be equal to 1 and the number of items of rotation to establish an appropriate rotational factor was determined to be equal to 25 . all these statistical tests were performed using spss version 20 software . today , use of cfa is common for semantic matching of questions with factors . recent procedure is suitable for the evaluation of some characteristics such as unidimensionality of items . so , cfa was performed on sample 2 ( n2 = 347 ) , for determination of construct validity , performed to test the fit of data to the model that was suggested by efa . in this stage , several alternative models were tested . we used amosgraphic version 20 software ( amos graphic ) with maximum likelihood estimation procedure assumption and evaluated absolute , comparative , and parsimonious fit indices . the model was considered acceptable if cmin / df was between 1 and 5 , cfi was greater than 0.8 , parsimonious comparative fit index ( pcfi ) was less than 0.6 , root mean squared error of approximation ( rmsea ) was less than 0.8 , and pclose was greater than 0.005 . fitness of model was confirmed if p value of cmin was greater than 0.05 , cmin / df was between 2 and 3 , cfi was greater than 0.9 , and pcfi was less than 0.5 . the study was started after it was approved by isfahan university of medical sciences and isfahan education organization . ethical approval was granted by the deputy of research and technology of isfahan university of medical sciences ( i d : 39147 , date : december 30 , 2012 ) . the purpose and procedures of the study were explained to the participants , and researcher emphasized on the confidentiality of the data and voluntary nature of participation . main characteristics of the participants including samples 1 and 2 are shown in table 1 . characteristics of male adolescents who participated in study after deletion of outliers and missing data , the average of physical activity and ltpa , based on met - min / week , was equal to 2421 ( sd = 1543 ) and 902 ( sd = 938 ) , respectively . based on the correlation matrix of sample 1 , each of the suggested items had a correlation coefficient higher than 0.4 at least with one of the other items ( p 0.005 ) . according to the results shown in table 2 , all items were suitable and there was no need to remove any item . statistics of self - efficacy questionnaire abut leisure time physical activity in iranian male adolescents the data of sample 1 were analyzed so as to extract the principal components with direct oblimin rotation . olkin ( kmo ) index was equal to 0.89 and the results of bartlett 's test of sphericity were significant at the confidence interval of 95% ( = 1544 , df = 78 , p 0.00 ) . according to the adequacy of sample volume and proportion of correlation matrix with factor analysis , the data were entered into the efa process . regarding the theoretical framework of the study and as the questionnaire consisted of three aspects of se however , principle component analysis showed two components with eigenvalue up to 1 . observing a point of failure after three components convinced us to examine other probable solutions . in addition , results of parallel analysis that were analyzed by monte carlo physical activity software also supported the two - component solution . based on these results performed with 13 variables , 325 participants , and 100 repeats and match assumption , only two components had eigenvalue higher than the criterion values that were appropriate with randomized data . these two components explain 51.6% of variance of se and the results of rotated pattern matrix are shown in table 3 . rotated component and pattern matrix with pca and direct oblimin rotation for items of self - efficacy questionnaire related to leisure time physical activity in iranian male adolescents in order to verify the construct validity of the questionnaire , fitness of several models , including 13 items , was specified and evaluated with the sample 2 using cfa . the second - order model demonstrated more acceptable indices compared to first - order model . however , only some fit indices supported the acceptable fitness of second - order model with data of sample 2 , so the model needed to be modified ( cmin = 212.50 , cmin / df = 3.32 , cfi = 0.92 , pcfi = 0.76 , rmsea = 0.082 , pclose = 0.000 ) . since all unstandardized estimated parameters were significantly different from zero at the 0.001 level ( two - tailed ) , no items were removable . it was found that addition of two parameters between error variables of items 2 and 4 and items 6 and 12 led to a decrease in chi - square . moreover , theoretical framework of the study supported the correlation between error variables of these items . so , corrected model was designed via two rating reduction of degree of freedom . fitness indices approved the appropriateness of the corrected model totally ( cmin = 165.53 , cmin / df = 2.67 , cfi = 0.95 , pcfi = 0.75 , rmsea = 0.069 , pclose = 0.007 ) . results of descriptive and bivariate correlation test such as main score of se and its subconstructs are shown in table 4 . the mean , standard deviation , and reciprocal correlation between different aspects of self - efficacy and leisure time physical activity in adolescent boys based on the correlation matrix of sample 1 , each of the suggested items had a correlation coefficient higher than 0.4 at least with one of the other items ( p 0.005 ) . according to the results shown in table 2 , all items were suitable and there was no need to remove any item . the data of sample 1 were analyzed so as to extract the principal components with direct oblimin rotation . olkin ( kmo ) index was equal to 0.89 and the results of bartlett 's test of sphericity were significant at the confidence interval of 95% ( = 1544 , df = 78 , p 0.00 ) . according to the adequacy of sample volume and proportion of correlation matrix with factor analysis , the data were entered into the efa process . regarding the theoretical framework of the study and as the questionnaire consisted of three aspects of se however , principle component analysis showed two components with eigenvalue up to 1 . observing a point of failure after three components convinced us to examine other probable solutions . in addition , results of parallel analysis that were analyzed by monte carlo physical activity software also supported the two - component solution . based on these results performed with 13 variables , 325 participants , and 100 repeats and match assumption , only two components had eigenvalue higher than the criterion values that were appropriate with randomized data . these two components explain 51.6% of variance of se and the results of rotated pattern matrix are shown in table 3 . rotated component and pattern matrix with pca and direct oblimin rotation for items of self - efficacy questionnaire related to leisure time physical activity in iranian male adolescents in order to verify the construct validity of the questionnaire , fitness of several models , including 13 items , was specified and evaluated with the sample 2 using cfa . the second - order model demonstrated more acceptable indices compared to first - order model . however , only some fit indices supported the acceptable fitness of second - order model with data of sample 2 , so the model needed to be modified ( cmin = 212.50 , cmin / df = 3.32 , cfi = 0.92 , pcfi = 0.76 , rmsea = 0.082 , pclose = 0.000 ) . since all unstandardized estimated parameters were significantly different from zero at the 0.001 level ( two - tailed ) , no items were removable . it was found that addition of two parameters between error variables of items 2 and 4 and items 6 and 12 led to a decrease in chi - square . adding these covariance parameters had methodological acceptance . moreover , theoretical framework of the study supported the correlation between error variables of these items . so , corrected model was designed via two rating reduction of degree of freedom . fitness indices approved the appropriateness of the corrected model totally ( cmin = 165.53 , cmin / df = 2.67 , cfi = 0.95 , pcfi = 0.75 , rmsea = 0.069 , pclose = 0.007 ) . results of descriptive and bivariate correlation test such as main score of se and its subconstructs are shown in table 4 . the mean , standard deviation , and reciprocal correlation between different aspects of self - efficacy and leisure time physical activity in adolescent boys this article reports the development process and evaluation of psychometric properties of a questionnaire for the determination of se about leisure time physical activity ( selpa ) . selpa was developed to evaluate the main aspects of se among iranian male adolescents via 13 items . after item generation or translation ( first stage ) , comprehensibility of the items was evaluated ( second stage ) . the psychometric properties of selpa were evaluated in a current and logical direction , including face validity ( third stage ) , content validity ( fourth stage ) , item analysis ( fifth stage ) , and construct validity by efa ( sixth stage ) and cfa ( seventh stage ) . brons and gorow believe that review of literature , obtaining comments from experts , and targeted population are the most important steps to achieve content validity of the measurement tools . in the current study , after the literature review , recommendation of 10 health experts from the research group helped us to achieve content validity . cultural and linguistic characteristics of the target group were considered in modifying the remaining items in different aspects of se related to ltpa in all stages . in addition to expert panel , during the primary study , the members of the target group represented their recommendation about comprehensibility of the questionnaire . reliability was evaluated in three steps by internal consistency and test retest method . in each stage , the results supported the reliability of the questionnaire . one of the main strengths of this study is that two different populations were employed for the evaluation and confirmation of construct validity of the questionnaire by efa and cfa . efa showed that two factors with eigenvalues greater than 1.00 loaded on all 13 items significantly and explained 51.6% of the variance of se . the first factor loaded on items 6 , 7 , 8 , 9 , 10 , 11 , 12 , and 13 , as we expected . these items addressed the current impediments and challenges of ltpa in iranian male adolescents that were derived from literature review and expert panel . this factor is called barrier se and its related items consist of obstacles such as fatigue , lack of money , lack of support , time conflict , bad weather , lack of facilities , and present other competitor entertainment . the highest correlation was related to item 12 ( 0.86 ) . this item was considered the participant 's ability in continuing physical activity even in the compacted curriculum . the lowest correlation rate was allocated to item number 11 ( 0.52 ) that is associated with lack of suitable place for physical activity . results of this study about the beliefs of adolescents about overcoming barriers may be different from those of other investigations . reported that the strongest and weakest factor loading belonged to feeling depressed and feeling physical discomfort after exercise , respectively . that investigation was conducted on iranian diabetic women and the difference in characteristics of the target groups justifies such results . the second factor loaded on items 1 , 2 , 3 , 4 , and 5 . this can be attributed to the confidence of a person to his or her ability to design a program for physical activity . all the items on which factor 2 loaded were related to scheduling se . compared to other questionnaires that commonly focus on barrier se , selpa considers an important aspect of regulatory se , named scheduling se about physical activity in leisure time . rodgers et al . noted that considering scheduling se is essential for improving the effectiveness of interventions in physical activity . since using cfa is recommended to confirm the fitness of conceptual model suggested by efa , the research group operated this statistical analysis on a separate population . correction of the model was conducted based on modification indices by adding two covariance to the model , all fit indices shift into good fitness as noroozi 's study . results of descriptive and bivariate correlation test , such as main score of se and its subconstructs , can be considered as the evidence that target group members also perceive moderate confidence to overcome the barriers and design or operate program for physical activity [ table 4 ] . robbins et al . showed a similar result in american adolescents in 2004 and reported the perceived se score of adolescents as 34.262.5 . this result is consistent with previous researches , especially those that have posited the importance of social cognitive constructs on physical activity . for example , sriramatr et al . explained a moderate correlation between coping ( barrier ) ( r = 0.33 ) and scheduling se ( r = 0.35 ) with energy consumption via physical activity in 364 young students in thailand . kim et al . showed a moderate correlation between se and exercise in adolescents too ( r = 0.45 ) . taymoori et al . have reported the highest correlation coefficient ( r = 0.62 ) between barrier se and physical activity in iranian female adolescents . this difference can be justified by population characteristics , idiographic nature of barriers , or instruments specifications . finally , the correlation pattern supports the importance of barrier compared with scheduling se , similar to other investigations . since the availability of a specific questionnaire based on specific characteristics of the target groups is important and as all important subconstructs of se should be taken into consideration , selpa was developed for the assessment of important aspects of self - regulatory efficacy , such as barrier and schedule se , in iranian male adolescents . findings support that barrier and scheduling se about physical activity can be conceptually and statistically distinguished from each other . this study supported the comprehensibility , face validity , content and construct validity , reliability , and internal consistency of selpa . however , further investigations are necessary to evaluate the reliability , concurrent validity , comprehensibility , and applicability of the questionnaire through supplementary descriptive and interventional studies . also , regression analysis is recommended to explore the predictive power of this questionnaire on physical activity . we thank those who helped us in conducting this investigation , including all involved students , teachers , and school managers . in addition , the expert panel is appreciated for revising and estimating the questionnaire 's validity and the research and technology deputy of isfahan university of medical sciences for funding this research .

background : attention to different aspects of self - efficacy leads to actual evaluation of self - efficacy about physical activity . this study was carried out in order to design and determine psychometric characteristics of a questionnaire for evaluation of self - efficacy about leisure time physical activity ( selpa ) among iranian adolescent boys , with an emphasis on regulatory self-efficacy.materials and methods : this descriptive analytic study was conducted in 734 male adolescents aged 1519 years in isfahan . after item generation and item selection based on review of literature and other questionnaires , content validity index ( cvi ) and content validity ratio ( cvr ) were determined and items were modified employing the opinions of expert panel ( n = 10 ) . comprehensibility of the questionnaire was determined by members of target group ( n = 35 ) . exploratory factors analysis ( efa ) was operated on sample 1 ( n1 = 325 ) and confirmatory factors analysis ( cfa ) on sample 2 ( n2 = 347 ) . reliability of selpa was estimated via internal consistency method.results:according to efa , barrier self - efficacy and scheduling self - efficacy are the two main aspects of selpa with the total variance of 65% . the suggested model was confirmed by cfa and all fitness indices of the corrected model were good . cronbach 's alpha was totally estimated as 0.89 and for barrier and scheduling self - efficacy , it was 0.86 and 0.81 , respectively.conclusions:the results provide some evidence for acceptable validity and reliability of selpa in iranian adolescent boys . however , further investigations , especially for evaluation of predictive power of the questionnaire , are necessary .

neurodegenerative conditions , such as dementia , multiple sclerosis ( ms ) , parkinson s disease , and huntington s disease , are often characterized by significant neuro - psychological deficits that interfere significantly with patient s abilities and quality of life.14 cognitive impairment is a common feature in ms affectin43%72% of patients5,6 and is characterized as other signs and symptoms of the disease by variability and heterogeneity . however , dementia is rare , and the more common clinical presentation is one of the specific and subtle cognitive deficits.7 the most frequently affected cognitive functions are attention , speed of information processing , memory , executive functions , and visuospatial abilities,8 although new domains such as theory of mind have also been identified.9 involvement of cognitive functions such as language , praxis , and gnosis is rare and less well studied . the influence of the duration of the illness on cognitive functioning is still a matter of controversy . as regards clinical presentation , the progressive forms of ms ( both secondary progressive ms and primary progressive ms ) are more involved in both degree and altered domains as compared to relapsing - remitting multiple sclerosis ( rrms).10,11 some authors point out that impairments are almost invariably a complication of the later stages of the disease when there is axonal loss with the involvement of large areas of white matter , disconnection between several cortical areas of association , and disconnection between cortical and subcortical areas , but impaired cognitive function appears to be present even in the early stages of the disease . studies with long - term follow - up indicate that cognitive function decreases as the disease progresses.12,13 the degree of neurological impairment is shown as a predictor of cognitive decline in the follow - up studies.14 in the case of fatigue , this can interfere negatively , especially in tasks that require sustained mental effort such as working memory tests or sustained attention.15 neuropsychological dysfunction severely affects the patients lives16 in terms of their ability to keep their jobs,17 and they require greater assistance with daily living activities . cognitively compromised patients are also more likely to have problems with socialization than cognitively intact ms patients.18 cognitive impairment has been associated with nonsomatic symptoms of depression in rrms , and this finding supports the importance of evaluating depressive symptoms when cognitive impairment is suspected in patients with rrms.19 anxiety has been less studied than depression in ms patients , although there is evidence that high levels of anxiety are associated with poor performance in cognitive tasks , especially processing speed , working memory , and visual spatial memory.20,21 this study was conducted in lanzarote , the easternmost landmass of the canary islands , spain ( figure 1 ) . the only study of the prevalence of ms on this island was published in 1980s , which reported a low prevalence of 15/100,000.22 the population of the island at that time was 60,000 inhabitants , and the authors concluded that the rate was higher than expected because of the geographical situation of the island . more recently , a study presented at the lxv annual meeting of the spanish society of neurology identified a total of 70 patients with ms on the island . this figure gives a prevalence of 49/100,000.23 other epidemiological studies conducted in the region have found that the canary islands is a medium - to - high risk area like spain and other mediterranean countries.24,25 thus , the canary islands , despite being closer to the equator , are still a medium risk area . the aim of this study was to describe the extent and pattern of the involvement of cognitive impairment and the psychological status in all patients of a ms cohort . a study of the full ms population , which rules out a possible selection bias , should provide a more comprehensive view of the cognitive impairment spectrum in ms . the study protocol was approved by the ethics committee for clinical research of the university hospital of the canary islands and the medical director of hospital doctor jos molina orosa . the study was conducted in accordance with the declaration of helsinki.26 all patients signed written informed consent . seventy patients with ms or clinically isolated syndrome ( cis ) treated at the doctor jos molina orosa hospital in lanzarote ( the hospital sees all the ms or cis patients on the island ) and 56 healthy controls ( hcs ) matched by sex , age , and schooling were included in the study . the inclusion criteria for the patients were as follows : willing and able to sign written informed consent;ms diagnosed according to the mcdonald criteria 200527 or cis ; andneurologically stable ms ( with no evidence of relapse or steroids treatment in the last 4 weeks preceding the enrollment ) . willing and able to sign written informed consent ; ms diagnosed according to the mcdonald criteria 200527 or cis ; and neurologically stable ms ( with no evidence of relapse or steroids treatment in the last 4 weeks preceding the enrollment ) . the inclusion criterion for the control group ( cg ) was willing and able to sign the written informed consent . the exclusion criteria for the cg were as follows : no history of psychiatric or neurological disorders andno history of drug abuse or any major medical illness . no history of psychiatric or neurological disorders and no history of drug abuse or any major medical illness . six patients did not participate in the study : three patients were not on the island at the time , one patient could not attend due to severe physical disability , and two patients refused to participate in the study . the spanish version of the brief repeatable battery of neuropsychological test ( brb - n)6 is used to assess the cognitive status of all the participants . the brb - n includes the following tests : selective reminding test ( srt)28 as a measure of verbal learning and delayed recall . this test gives three different scores : srt - long - term storage ( srt - lts ) , srt - consistent long - term retrieval ( srt - cltr ) , and srt - delayed ( srt - d).10/36 spatial recall test ( spart ) , an extended version of the original form the 7/24 spart to assess visuospatial learning and delayed recall.29 this test gives two scores : spart - total and spart - delayed ( spart - d).symbol digit modalities test ( sdmt)30 as a measure of complex attention and processing speed.paced auditory serial addition test ( pasat)31 as a measure of sustained attention and working memory.controlled oral word association test32 as a measure of verbal fluency . this test gives three different scores : srt - long - term storage ( srt - lts ) , srt - consistent long - term retrieval ( srt - cltr ) , and srt - delayed ( srt - d ) . 10/36 spatial recall test ( spart ) , an extended version of the original form the 7/24 spart to assess visuospatial learning and delayed recall.29 this test gives two scores : spart - total and spart - delayed ( spart - d ) . symbol digit modalities test ( sdmt)30 as a measure of complex attention and processing speed . paced auditory serial addition test ( pasat)31 as a measure of sustained attention and working memory . the multiple sclerosis neuropsychological questionnaire ( msnq),33 the hospital anxiety and depression scale,34 and the fatigue severity scale35 were administered to the patients . all evaluations were performed by a neuropsychologist who was an expert in ms and familiar with the administration of all tests . these tests were administered in a single session of ~1 hour . a z score ( direct score mean / standard deviation [ sd ] ) was created for each cognitive domain according to standard practice to obtain a global cognitive - performance score . verbal memory = z - lts+z - cltr+z - srt - d3(1 ) visuospatial memory = z - spart total+z - spart - d2(2 ) processing speed = z - total correct responses of sdmt(3 ) working memory = z - total correct responses of pasat3+pasat22(4 ) verbal fluency = z - semantic+z - phonetic total correct responses2(5 ) finally , a global cognitive function score ( z - global ) was obtained by calculating the mean of the z scores from the five cognitive domains . different cognitive impairment criteria were used : < 1.0 sd , < 1.5 sd , and < 2.0 sd ( compared to the cg ) in one , two , or three subtests of the battery , respectively . interviews were also conducted with family members about their level of functionality ( food preparation , medication management , use of money , travel outside home ) in the case of patients with suspected dementia . dementia was diagnosed following the fourth edition of the diagnostic and statistical manual of mental disorders , text revised ( dsm - iv - tr).36 categorical variables were expressed as frequencies and percentages . all the variables analyzed followed a normal distribution except pasat 3 , and the scores were log transformed before analysis of covariance . analysis of covariance was performed , including cognitive domains such as dependent variables and controlling for age and schooling . correlation between the cognitive domains and the patient s clinical variables and questionnaires were assessed using the pearson s correlation test . statistical analysis was performed with spss v20.0 software ( ibm corporation , armonk , ny , usa ) . p - values were corrected for multiple testing problem using the bonferroni correction method ( /k : 0.05/6=0.008 ) , considering p - values < 0.008 as statistically significant . seventy patients with ms or clinically isolated syndrome ( cis ) treated at the doctor jos molina orosa hospital in lanzarote ( the hospital sees all the ms or cis patients on the island ) and 56 healthy controls ( hcs ) matched by sex , age , and schooling were included in the study . the inclusion criteria for the patients were as follows : willing and able to sign written informed consent;ms diagnosed according to the mcdonald criteria 200527 or cis ; andneurologically stable ms ( with no evidence of relapse or steroids treatment in the last 4 weeks preceding the enrollment ) . willing and able to sign written informed consent ; ms diagnosed according to the mcdonald criteria 200527 or cis ; and neurologically stable ms ( with no evidence of relapse or steroids treatment in the last 4 weeks preceding the enrollment ) . the inclusion criterion for the control group ( cg ) was willing and able to sign the written informed consent . the exclusion criteria for the cg were as follows : no history of psychiatric or neurological disorders andno history of drug abuse or any major medical illness . no history of psychiatric or neurological disorders and no history of drug abuse or any major medical illness . six patients did not participate in the study : three patients were not on the island at the time , one patient could not attend due to severe physical disability , and two patients refused to participate in the study . the spanish version of the brief repeatable battery of neuropsychological test ( brb - n)6 is used to assess the cognitive status of all the participants . the brb - n includes the following tests : selective reminding test ( srt)28 as a measure of verbal learning and delayed recall . this test gives three different scores : srt - long - term storage ( srt - lts ) , srt - consistent long - term retrieval ( srt - cltr ) , and srt - delayed ( srt - d).10/36 spatial recall test ( spart ) , an extended version of the original form the 7/24 spart to assess visuospatial learning and delayed recall.29 this test gives two scores : spart - total and spart - delayed ( spart - d).symbol digit modalities test ( sdmt)30 as a measure of complex attention and processing speed.paced auditory serial addition test ( pasat)31 as a measure of sustained attention and working memory.controlled oral word association test32 as a measure of verbal fluency . this test gives three different scores : srt - long - term storage ( srt - lts ) , srt - consistent long - term retrieval ( srt - cltr ) , and srt - delayed ( srt - d ) . 10/36 spatial recall test ( spart ) , an extended version of the original form the 7/24 spart to assess visuospatial learning and delayed recall.29 this test gives two scores : spart - total and spart - delayed ( spart - d ) . symbol digit modalities test ( sdmt)30 as a measure of complex attention and processing speed . paced auditory serial addition test ( pasat)31 as a measure of sustained attention and working memory . the multiple sclerosis neuropsychological questionnaire ( msnq),33 the hospital anxiety and depression scale,34 and the fatigue severity scale35 were administered to the patients . all evaluations were performed by a neuropsychologist who was an expert in ms and familiar with the administration of all tests . these tests were administered in a single session of ~1 hour . a z score ( direct score mean / standard deviation [ sd ] ) was created for each cognitive domain according to standard practice to obtain a global cognitive - performance score . verbal memory = z - lts+z - cltr+z - srt - d3(1 ) visuospatial memory = z - spart total+z - spart - d2(2 ) processing speed = z - total correct responses of sdmt(3 ) working memory = z - total correct responses of pasat3+pasat22(4 ) verbal fluency = z - semantic+z - phonetic total correct responses2(5 ) finally , a global cognitive function score ( z - global ) was obtained by calculating the mean of the z scores from the five cognitive domains . different cognitive impairment criteria were used : < 1.0 sd , < 1.5 sd , and < 2.0 sd ( compared to the cg ) in one , two , or three subtests of the battery , respectively . interviews were also conducted with family members about their level of functionality ( food preparation , medication management , use of money , travel outside home ) in the case of patients with suspected dementia . dementia was diagnosed following the fourth edition of the diagnostic and statistical manual of mental disorders , text revised ( dsm - iv - tr).36 all the variables analyzed followed a normal distribution except pasat 3 , and the scores were log transformed before analysis of covariance . analysis of covariance was performed , including cognitive domains such as dependent variables and controlling for age and schooling . correlation between the cognitive domains and the patient s clinical variables and questionnaires were assessed using the pearson s correlation test . statistical analysis was performed with spss v20.0 software ( ibm corporation , armonk , ny , usa ) . p - values were corrected for multiple testing problem using the bonferroni correction method ( /k : 0.05/6=0.008 ) , considering p - values < 0.008 as statistically significant . table 1 shows the clinical and demographical characteristics of hc ( n=56 ) and patients ( ms ; n=60 ; both groups had similar distributions of sex , age , and schooling ) . the patients scored less than controls , in the controlling for age and schooling , on all variables of the brb - n except visuospatial memory ( table 2 ) . a high prevalence of cognitive impairment ( 75.0% ) was obtained when using a lenient criterion ( < 1.0 sd in one sub - test ) . the prevalence was 35.0% with a moderately stringent criterion ( < 1.5 sd in two subtests ) , and the prevalence was 16.6% when using the most stringent criterion of cognitive impairment ( < 2 sd in three tests ; table 3 ) . the most frequently affected domain was working memory with an impairment in 43.8% of the patients ( 28 patients ) , followed by verbal memory in 21.7% ( 13 patients ) , processing speed in 15.0% ( nine patients ) , verbal fluency in 13.3% ( eight patients ) , and finally , visuospatial memory in 11.7% ( seven patients ) . when clinical variables were analyzed , disease duration had a moderate correlation with visuospatial memory and processing speed ( r=0.400 and r=0.370 , p<0.008 ) . the expanded disability status scale ( edss ) score showed correlations with verbal memory , processing speed , and global cognitive index ( r=0.344 to 0.395 , p<0.008 ; table 4 ) . symptoms of anxiety were present in 18.3% of patients , and depression symptoms were present in 33.3% of patients . msnq score and anxiety symptoms were not associated with performance in any of the cognitive domains . verbal memory correlated with symptoms of depression ( r=0.415 , p=0.001 ) and fatigue ( r=0.383 , p=0.003 ) . msnq correlated with depression ( r=0.405 , p=0.001 ) and anxiety ( r=0.255 , p=0.049 ) . the aim of this study was to describe the extent and pattern of cognitive impairment in all the population affected by ms on a small island located in the southwest of europe . lanzarote is an interesting place for ms study because its geographical location confers a high exposure to uv radiation for most of the year and patients probably have an optimal vitamin d serum level . low vitamin d status is a possible environmental risk factor for ms development and outcome.37,38 severe cognitive impairment , with clear criteria for dementia ( dsm - iv - tr ) , was found in four patients in the study sample ( 6.3% of the total ) . all these four patients had an obvious neuropsychological deficit , and they were unable to perform all the selected tests . interviews with family members revealed significant functional impairment with an impact on activities of daily living . the mean age of the dementia patients was 71 years ( 6078 years ) , and they obtained an edss score between 6.0 and 8.0 as well as had progressive forms of the disease . the remaining nondementia patients formed a population with a low moderate degree of disability ( < 3.0 on the edss ) , and most patients had an rrms . the patient group underperformed the ( demographically equivalent ) cg in most of the battery tests after controlling the results for age and schooling , suggesting an overall performance deficit . the range of variability observed in the prevalence of cognitive impairment is possibly associated with the methodological differences between the studies such as the study design and setting , as well as the neuropsychological tests selected for the study . however , the biggest difference could be due to the consideration of the concept of cognitive impairment . for example , a performance < 1.0 sd in one subtest may reflect the fluctuant nature of the disease and does not necessarily reflect the real pattern of cognitive impairment . the concept of cognitive impairment proposed by amato et al12 as a failure in two or more brb - n subtests with scores at least 1.5 sd below the scores of hc is suggested for use here . using similar figures were found by nogales - gaete et al39 with a sample of chilean patients with rrms , but the finding here differs from other studies conducted in oslo ( norway ) and the uk and from another study in spanish speaking patients in argentina,40 where the prevalence of cognitive impairment was > 45% . the pattern of involvement found suggests that working memory and verbal memory are the most affected areas . physical disability has been associated with cognitive impairment in some studies,6,14 while others found no such association.41 in the present work , a negative correlation between edss score and verbal memory , processing speed , and a global cognitive index was found . these data could indicate that deficit in processing speed might be a good predictor of disease progression and , therefore , constitutes a key deficit in time monitoring . however , the involvement of other domains such as working memory or verbal fluency could start in any stage of the disease and does not evolve in parallel with other symptoms of this disease . msnq - self report ( as opposed to msnq - informant report ) is not considered a sensitive screen for neuropsycho - logical impairment in ms , and indeed , no correlation with this test was found here , suggesting that it is not an effective screening method for detecting cognitive impairment . depressive symptoms were less ( present in 33.3% of the sample ) than those found in the literature,42 and higher scores on the depression scale were correlated with poorer performance in verbal memory . this finding has been associated with the mineralocorticoid expression in the brain that diminishes during depression , especially in the hippocampus and the prefrontal cortex , which are critical brain areas for memory.43 anxiety ( present in 18.3% of patients ) was not associated with cognitive performance . this confirms findings of another study that has linked cognitive functioning with the state of fatigue in patients with ms44 and more specifically with memory.45 as a limitation of the study , the use of a more extensive evaluation protocol could provide more information about the true neuropsychological profile of studied patients . at least 35% of the study population had mild - to - moderate cognitive impairment relative to a control sample . the results of this study confirm the high prevalence of cognitive impairment in an ms cohort . the most affected domains were working memory and verbal memory . working memory , verbal memory , and verbal fluency did not deteriorate as the disease progressed , and therefore could not be used as a means of monitoring it . cognitive decline was frequently related to clinical variables ( disease duration and edss score ) . verbal memory was associated with depression and fatigue , but none of the cognitive domains showed a strong correlation with anxiety and subjective perception of cognitive impairment .

objectivescognitive impairment is a common feature in multiple sclerosis affecting ~43%72% of patients , which involves cognitive functions such as memory , processing speed , attention , and executive function . the aim of this study was to describe the extent and pattern of the involvement of cognitive impairment and psychological status in all patients with multiple sclerosis on a small spanish island.patients and methodsin all , 70 patients and 56 healthy controls were included in the study between february 2013 and may 2013 . all participants were assessed using the brief repeatable battery of neuropsychological test . the patients also completed instruments to evaluate the presence of fatigue , perceived cognitive dysfunction , and symptoms of anxiety and depression . all procedures were performed in a single session.resultscognitive impairment , defined as a score < 1.5 standard deviation on two subtests of the battery , was present in 35% of the participants . the most frequently affected domain was working memory , followed by verbal memory and processing speed . disease duration showed a moderate correlation with visuospatial memory and processing speed . the expanded disability status scale score correlated with verbal and processing speed . verbal memory was correlated with depression symptoms and fatigue.conclusioncognitive impairment was present in 35% of the study population . the most affected domains were working memory and verbal memory . working memory and verbal fluency deficit are independent factors of disease evolution . cognitive decline is related to clinical variables and psychological measures such as fatigue or depression but not to anxiety .

lumbosacral radicular pain is a common and disabling condition in adults with pathology of the bones in the distal spine , and it represents a significant quality - of - life issue.1 lower back pain is not always associated with radicular symptoms . clinical findings of neuropathy may vary significantly , depending on the pathogenesis of the pain symptoms . neuropathic pain is still a dark zone for neuroscientists and treatment remains challenging for clinicians . it has been described as a direct consequence of involvement of the somatosensory system , with a population prevalence between 7 and 10% , thus stressing the importance of a standardized approach for its recognition.2 pharmacological therapies have often proved ineffective , surgery has poorly characterized indications , and the use of interventional neuromodulation ( eg , pulsed radiofrequency and spinal cord stimulation ) is still debated or not always applicable.3 some older studies reported that opioids have poor efficacy for neuropathic pain , although this conclusion has been progressively questioned because of the lack of compelling evidence , and several reports arguing that a substantial proportion of patients with neuropathic pain might significantly respond to opioids.4 some investigators have evaluated the additional efficacy of local anesthetics , stressing evidence for the development of synergistic effects after coadministration with opioids , and found that bupivacaine and morphine together showed better results than either alone , in terms of patient satisfaction , medication intake , and pain clinic admissions.5 different routes of administration have been tested , and epidural and intrathecal drug delivery confers benefits in comparison with other routes . for instance , these routes avoid first - pass metabolism and the blood brain barrier and allow the use of lower doses at comparable efficacy , given that the medications are deposited directly at the spinal cord.68 considering that epidural administration can be maintained for longer periods with lower risks relative to the intrathecal route , it may be possible to utilize other analgesics to obtain adequate analgesia for prolonged periods . to the best of our knowledge , no previous study has assessed how epidural therapy with combined opioids and local anesthetics might provide long - lasting pain modulation . the aim of this study was to investigate the effectiveness of epidural morphine and bupivacaine in patients with chronic neuropathic pain associated with lumbosacral radiculopathy . the research protocol was approved by the institutional review board committee of the santa maria maddalena hospital , occhiobello ( ro ) , italy . informed consent was obtained from each patient , and the patients enrolled in the research were allowed to withdraw from the study at any point if they wished . prior to the enrollment , every patient was evaluated to confirm probable or definite neuropathic involvement.9 the following criteria were adopted as inclusion criteria for the study subjects : severe pain intensity assessed with a numeric rating scale ( nrs ) score of 7/10.definite or probable neuropathic involvement as assessed by clinical examination and grading system.chronic lower extremity radicular pain lasting for > 6 months.clinical examination suggestive of radicular pain with 1 positive provocation tests ( eg , straight leg raise test).poor response to pharmacological therapy , physical therapy , or epidural injection of anti - inflammatories ( ie , pain reduction in nrs 30% during the 6 months before enrollment).magnetic resonance imaging and/or electromyographic test suggestive of radiculopathy . severe pain intensity assessed with a numeric rating scale ( nrs ) score of 7/10 . definite or probable neuropathic involvement as assessed by clinical examination and grading system . clinical examination suggestive of radicular pain with 1 positive provocation tests ( eg , straight leg raise test ) . poor response to pharmacological therapy , physical therapy , or epidural injection of anti - inflammatories ( ie , pain reduction in nrs 30% during the 6 months before enrollment ) . magnetic resonance imaging and/or electromyographic test suggestive of radiculopathy . the following exclusion criteria were applied : possible or unlikely neuropathic pain.positive response to previous treatments.comorbid neurological or psychiatric disorders.significant motor deficits . . significant motor deficits . the procedure was performed in a suitable operating room , with disinfection and sterilization of all devices . a sterile operative field was maintained while each patient was lying on a fluoroscopy table in the prone position for needle introduction . after injection of local anesthetic to numb the operative zone , a 16-g hollow needle was inserted through the paramedian tissues at a 40 angle , with the skin and epidural space located by standard loss of resistance to air techniques . an epidural catheter was inserted at the t12l1 intervertebral space , with the correct placement verified by fluoroscopy . the position of the catheter tip was confirmed by injection of x - ray contrast medium ( iopamiro 300 ; bracco imaging , s.p.a . , the catheter was tunneled for 5 cm subcutaneously and then fixed at 10 cm in the abdominal skin , and the patient was transported to the recovery room ( figure 1 ) . on the following day , each patient received an epidural analgesic injection followed by a 2 ml flush with normal saline . the injection solution was composed of morphine chlorhydrate ( 0.1 mg / ml ) and bupivacaine ( 1 mg / ml ) , and the dose was progressively increased to 3 ml three times a day ( total dosage per day : morphine 0.9 mg and bupivacaine 9 mg ) . a variable ratio of the opioid and local anesthetic agents was used , such that the dosages of the two drugs were adjusted in order to obtain the optimal analgesia with the fewest side effects in comparison to the postoperative pain treatment ( ie , morphine 24 mg / day and bupivacaine 0.125%).10 the medication was administered in the hospital for the first week and self - administered by the patient in the outpatient setting for the following 3 weeks . proper medication intake and catheter functioning were evaluated every week by an expert nurse and a pain physician . on the 30th day , the catheter was removed and an appointment for clinical follow - up in 1 month was scheduled . each patient was evaluated for pain levels by two independent reviewers not involved with catheter placement and drug administration , before catheter placement and at 1 month after removal . pain intensity was assessed with the 010 nrs and by the italian version of the mcgill pain questionnaire ( quid ) , which has 42 descriptors divided into four main pain rating index ranks ( sensory , affective , evaluative , and mixed ) . the total prir ( prir - t ) value , given by the sum of all the rank values , describes and quantifies the pain.11,12 response to treatment was considered successful with a pain reduction in nrs > 30% at follow - up . the wilcoxon s rank sum test was applied to evaluate changes in nrs and prir - t , comparing baseline and follow - up levels . moreover , differences between the four prir coefficients describing sensory ( prirc - s ) , affective ( prirc - a ) , evaluative ( prirc - e ) , and mixed ( prirc - m ) aspects of pain were compared in the responders and nonresponders . the association between pain variation in nrs and quid at the follow - up was calculated using the spearman s test . the kolmogorov the sample size was chosen in order to obtain a pain reduction > 30% at follow - up in patients with a pretreatment nrs pain intensity 7/10 . considering an alpha level of < 5% , a study group of 20 patients allowed achievement of a statistical significance of > 95% with statistical power of > 80% . the research protocol was approved by the institutional review board committee of the santa maria maddalena hospital , occhiobello ( ro ) , italy . informed consent was obtained from each patient , and the patients enrolled in the research were allowed to withdraw from the study at any point if they wished . prior to the enrollment , every patient was evaluated to confirm probable or definite neuropathic involvement.9 the following criteria were adopted as inclusion criteria for the study subjects : severe pain intensity assessed with a numeric rating scale ( nrs ) score of 7/10.definite or probable neuropathic involvement as assessed by clinical examination and grading system.chronic lower extremity radicular pain lasting for > 6 months.clinical examination suggestive of radicular pain with 1 positive provocation tests ( eg , straight leg raise test).poor response to pharmacological therapy , physical therapy , or epidural injection of anti - inflammatories ( ie , pain reduction in nrs 30% during the 6 months before enrollment).magnetic resonance imaging and/or electromyographic test suggestive of radiculopathy . severe pain intensity assessed with a numeric rating scale ( nrs ) score of 7/10 . definite or probable neuropathic involvement as assessed by clinical examination and grading system . clinical examination suggestive of radicular pain with 1 positive provocation tests ( eg , straight leg raise test ) . poor response to pharmacological therapy , physical therapy , or epidural injection of anti - inflammatories ( ie , pain reduction in nrs 30% during the 6 months before enrollment ) . magnetic resonance imaging and/or electromyographic test suggestive of radiculopathy . the following exclusion criteria were applied : possible or unlikely neuropathic pain.positive response to previous treatments.comorbid neurological or psychiatric disorders.significant motor deficits . . significant motor deficits . the procedure was performed in a suitable operating room , with disinfection and sterilization of all devices . a sterile operative field was maintained while each patient was lying on a fluoroscopy table in the prone position for needle introduction . after injection of local anesthetic to numb the operative zone , a 16-g hollow needle was inserted through the paramedian tissues at a 40 angle , with the skin and epidural space located by standard loss of resistance to air techniques . an epidural catheter was inserted at the t12l1 intervertebral space , with the correct placement verified by fluoroscopy . the position of the catheter tip was confirmed by injection of x - ray contrast medium ( iopamiro 300 ; bracco imaging , s.p.a . , the catheter was tunneled for 5 cm subcutaneously and then fixed at 10 cm in the abdominal skin , and the patient was transported to the recovery room ( figure 1 ) . on the following day , each patient received an epidural analgesic injection followed by a 2 ml flush with normal saline . the injection solution was composed of morphine chlorhydrate ( 0.1 mg / ml ) and bupivacaine ( 1 mg / ml ) , and the dose was progressively increased to 3 ml three times a day ( total dosage per day : morphine 0.9 mg and bupivacaine 9 mg ) . a variable ratio of the opioid and local anesthetic agents was used , such that the dosages of the two drugs were adjusted in order to obtain the optimal analgesia with the fewest side effects in comparison to the postoperative pain treatment ( ie , morphine 24 mg / day and bupivacaine 0.125%).10 the medication was administered in the hospital for the first week and self - administered by the patient in the outpatient setting for the following 3 weeks . proper medication intake and catheter functioning were evaluated every week by an expert nurse and a pain physician . on the 30th day , the catheter was removed and an appointment for clinical follow - up in 1 month was scheduled . each patient was evaluated for pain levels by two independent reviewers not involved with catheter placement and drug administration , before catheter placement and at 1 month after removal . pain intensity was assessed with the 010 nrs and by the italian version of the mcgill pain questionnaire ( quid ) , which has 42 descriptors divided into four main pain rating index ranks ( sensory , affective , evaluative , and mixed ) . the total prir ( prir - t ) value , given by the sum of all the rank values , describes and quantifies the pain.11,12 response to treatment was considered successful with a pain reduction in nrs > 30% at follow - up . the wilcoxon s rank sum test was applied to evaluate changes in nrs and prir - t , comparing baseline and follow - up levels . moreover , differences between the four prir coefficients describing sensory ( prirc - s ) , affective ( prirc - a ) , evaluative ( prirc - e ) , and mixed ( prirc - m ) aspects of pain were compared in the responders and nonresponders . the association between pain variation in nrs and quid at the follow - up was calculated using the spearman s test . the sample size was chosen in order to obtain a pain reduction > 30% at follow - up in patients with a pretreatment nrs pain intensity 7/10 . considering an alpha level of < 5% , a study group of 20 patients allowed achievement of a statistical significance of > 95% with statistical power of > 80% . eighty - three consecutive patients , diagnosed with radicular lumbosacral pain between january 2014 and december 2015 , were evaluated in the pain medicine unit of santa maria maddalena hospital . oral medications that were prescribed for therapy before the enrollment included non - steroidal anti - inflammatory drugs , antidepressants , anticonvulsants , and opioids , and the pre - enrollment physical therapy was based on a progressive specific exercise program involving the training of the deep abdominal and lumbar muscles . radicular epidural injection with triamcinolone 40 mg / ml and lidocaine 2% 0.3 ml was performed with ultrasound guidance.13,14 fifty - three patients did not meet inclusion criteria and were excluded from the study . two patients were excluded during the treatment period after malfunction and subsequent removal of the epidural catheter , and six patients reported no improvement in pain level at the fourth week and were removed from follow - up . ultimately , 22 patients remained enrolled in the study , and most ( 73% ) of these patients reached significant pain relief during treatment . the study profile and the epidural treatment effect on pain scores are summarized in figure 2 and table 2 , respectively . nrs and prir - t were significantly reduced at follow - up ( p=0.001 and p=0.03 , respectively ) . these data were confirmed by the parallel evolution in the two scores ( r=0.75 , p<0.001 ) , strengthening marked pain relief lasting up to 1 month after treatment cessation ( figure 3 ) . if the four pain rating coefficients were analyzed , none of the single items was significantly modified compared to the others either in responders or in nonresponders . the oswestry disability index was applied for clinical purposes and showed an overall improvement at follow - up . nevertheless , considering the frequent association with low back pain , disability caused by axial pain therefore , we chose not to include this index in the study due to the complex nature of lumbosacral radicular pain . a successful clinical outcome was observed in eleven of the 22 patients at the follow - up , with 50.4% of effect size and 97.2% of statistical power . in this study , we evaluated pain improvement after the cessation of epidural treatment with morphine and bupivacaine . at follow - up , half of the patients suffering from moderate - to - severe pain at baseline reported a significant improvement , which was confirmed with two validated scores , affecting the sensory and evaluative as much as affective aspects of pain . to our knowledge , this is the first study to report the effectiveness of epidural treatment with combined opioids and anesthetics persisting after catheter removal . if pain improvement had occurred , it was maintained for 1 month after therapy cessation , thereby suggesting a potential role for modifying the natural history of the disease . although the protocol was complex , involving hospital admission , placement of epidural catheter in the operating room , and the administration of medications for 4 weeks , all the patients included in the study had severe chronic pain that was refractory to several standard treatments . opioid infusion has increasingly been used as a treatment in noncancer pain and more recently has been assessed in patients with chronic pain . pain levels improve significantly with higher doses , but these levels can promote the risk of abuse and often burden the patient with adverse effects . one of the main pitfalls in opioid therapy lies in the progressive development of tolerance , which is responsible for decreasing efficacy.15 epidural delivery of anesthetics may be associated with prolonged analgesia and reduced mechanical allodynia in rats with nerve injury , whereas similar effects have also been described in humans with neuropathic pain after lidocaine administration.16 in other trials , the combination of intrathecal bupivacaine and morphine showed good efficacy in most cancer and nonmalignant pain patients with neuropathic or mixed pain . in addition to enhancing analgesia , bupivacaine combinations seem to be responsible for opioid - sparing regimens with diminished adverse effects.17 opioids are usually claimed to target dorsal horn cell receptors and cause pre- and postsynaptic modulation of pain , although peripheral effects have also been taken into account due to the identification of opioid receptors on peripheral sensory neuron processes.18 a previous study has also shown that microinjection of opioids into the brainstem may elicit powerful antinociceptive effect in animals and humans , likely involving the periaqueductal gray ( pag ) and the rostro - ventromedial medulla ( rvm).19 although the cellular mechanisms and sites of action are still debated , many local anesthetics block ion channels at the dorsal entry root zone and antagonize pain transmission via ad and c fibers.20 moreover , bupivacaine may inhibit spinal glutamatergic transmission , and its analgesic effect might also be subsequent to the modulation of n - methyl - d - aspartate receptors in the superficial dorsal horn.21 compelling evidence suggests that ectopic neural discharge may represent an underlying mechanism of neuropathic pain . these abnormal impulses may arise at the site of nerve injury or far from it , in apparently intact axons or structures , driven by abnormal discharges in ab or c fibers.22 in this regard , the role of the dorsal root ganglion ( drg ) has been widely investigated , due to its location as a border area between spinal cord and peripheral nerves . abnormal discharges in the drg tend to be more sensitive to lidocaine injection than those at the site of nerve injury . several studies highlighted that hyperexcitable drg neurons begin to show ongoing activity that lasts many days or weeks after nerve injury . in turn , this pathological activity might induce reversible central sensitization.23 in experimental models , systemic lidocaine silences the drg ectopic discharges without blocking nerve conduction , whereas local anesthetic agents applied intrathecally and to the drg surface in patients with phantom limb pain produce transient improvement of painful and nonpainful sensations in the majority of subjects , strengthening the conclusion that lidocaine can antagonize spontaneous bursting action potential discharge and sensitization.24 despite the distance from the catheter tip , sensitized drg might therefore have responded to low - dose bupivacaine and , because of a lack of efficient neurovascular barriers , it could be considered as a potential target in our study . in addition , ion channels are widely expressed in the central nervous system as much as in the periphery . the number of sodium channels ( eg , nav 1.3 ) has been found to be increased in dorsal horn and thalamic neurons after traumatic spinal cord injury , producing central pain . blocking the expression of such channels may reverse the excitability changes in either the central nervous system or the peripheral nervous system , whereas injection of lidocaine into the rvm blocks tactile and thermal hyperesthesia.25 increasing evidence is also documenting the anti - inflammatory properties of anesthetics , resulting from their modulating action on the immune system . manchikanti et al26 showed comparable long - lasting efficacy of epidural steroids and anesthetics in patients suffering from chronic lumbar disk herniation . this result might explain the improvement in low - back pain with radicular involvement , which often combines mixed features of nociceptive and neuropathic effects . how can we explain the maintenance of pain control efficacy in our results , lasting up to 1 month after the cessation of treatment and catheter removal ? several experiments showed that administration of systemic or local anesthetics may improve symptoms related to neuropathic pain for variable amounts of time after the end of therapy.27 some data stress the important role of bupivacaine in pain control : 1 ) the common tendency to increase the dose of opioids to optimize pain control , whereas in our study , it was maintained at a constant dose , and 2 ) the reported pain improvement after the addition of intrathecal bupivacaine in patients unresponsive to opioid alone.28 bupivacaine may enhance the analgesic effect of opioids through its action on voltage - gated calcium channels , changing opioid pharmacokinetics and receptor conformation.29 moreover , chronic pain has been associated with structural and functional changes in the human cortex and with dysfunctional descending pain modulation , which may be affected by medications and pain resolution , suggesting cortical plasticity processes.30 epidural addition of bupivacaine to opioids might help in restoring physiological antinociceptive mechanisms , therefore preventing or antagonizing chronic neuropathic pain.31 because our patients were suffering from refractory neuropathic pain , we considered a reduction of pain > 30% ( at follow - up ) as utilized in clinical trials , as significant.32 the analysis of quid coefficients failed to disclose significant pain variation among prirc - s , prirc - a , prirc - e , and prirc - m . the treatment improved sensory pain as much as affective quality of pain in responders , whereas none of these items changed compared to the others in poor responders . nevertheless , the use of pain coefficients is not sufficient to rule out placebo effects , which may play an important role in the subjective response to treatments.33 the heterogeneous and small number of patients may be another issue , given that neuropathic pain may develop from different disorders and etiologies , making tighter criteria too restrictive . nevertheless , we believe that the statistical power in this study was strong enough to support reliable results . finally , the short duration of follow - up in this study does not allow us to comment on the potential for long - term pain relief . in animal models , intravenous infusion of lidocaine was able to eliminate mechanical allodynia for at least 3 weeks after treatment , but the maximal duration in humans needs to be elucidated in further studies . the present results showed significant efficacy of combined epidural morphine and bupivacaine in the treatment of radicular neuropathic pain , which was maintained for up to 1 month after the end of administration . as a result of multiple pathophysiological events , central and peripheral neural changes may produce clinical symptoms and lead to chronic pain . maintaining significant pain relief with low doses of epidural bupivacaine / opioid may allow the physician to minimize adverse effects , neurotoxicity , and drug dosages , or to adopt more conservative approaches ( eg , physical therapy ) in pain management . therefore , further studies are needed to evaluate the safety and reliability of this regimen over long periods , to promote the use of epidural treatment in selected cases , or ultimately to develop new implantable pump systems engineered to provide infusions for extended pain relief .

objectivethe aim of this study was to investigate the therapeutic effectiveness of epidural morphine and bupivacaine in patients with chronic lumbosacral radicular neuropathic pain after the cessation of treatment.methodstwenty-two patients with chronic lumbosacral pain with neuropathic features were enrolled . an indwelling catheter was placed into the epidural space , and each patient received an epidural injection of morphine chlorhydrate and bupivacaine up to three times a day . the medication was administered for 4 weeks . the pain intensity score on a 010 numeric rating scale ( nrs ) , the total pain rating index rank ( prir - t ) , and its coefficients were evaluated before treatment and 1 month after catheter removal . p - value < 0.05 was considered statistically significant.resultsnrs and prir - t were significantly reduced at follow - up ( p=0.001 and p=0.03 , respectively ) , whereas the parallel evolution of the two scores ( r=0.75 and p<0.001 , respectively ) confirmed significant pain relief lasting up to 1 month after treatment cessation . none of the four pain rating coefficients was significantly modified compared to the others in either responders or nonresponders . successful clinical outcome ( pain reduction > 30% in nrs ) was reached and maintained in half of the patients at follow-up.conclusioncombined epidural morphine and bupivacaine seems to be effective in the treatment of neuropathic pain .

behet 's disease ( bd ) is an immune - mediated , systemic vasculitis , in which blood vessels of all sizes ( small , medium , and large ) in both the venous and arterial circulation can be affected . clinically it is characterised by recurrent aphthous ulcers of the mouth and/or genitals in combination with other systemic manifestations involving the skin , eyes , joints , vessels , gastrointestinal tract , or central nervous system . it lies at the crossroad between autoimmune and autoinflammatory disorders , may be triggered by infectious agents , and is characterised by a number of immunological aberrations , such as neutrophil hyperactivity ( reviewed in [ 3 , 4 ] ) . bd is most frequently observed in populations around the mediterranean basin , the middle east , and the far east , and the clustering of bd in populations along the ancient silk route suggests that an inherited tendency to bd was spread by travellers along these trading routes . multiple studies , in multiple populations , have confirmed a strong association between hla - b51 and bd , but other genes are also likely to be involved . copy number variation ( cnv ) is departure from the normal diploid number of genes ( n = 2 ) which may arise from gene duplication and deletion events and which may contribute substantially to quantitative variation in expression . an increasing number of cnvs have been characterised in the human genome with implications for both evolution and disease susceptibility . this cluster carries 5 highly homologous genes that encode for low affinity receptors for igg - complexed antigens , which are expressed widely throughout the haematopoietic system . these low affinity fc - gamma receptors are involved in the regulation of a multitude of innate and adaptive immune responses , with implications for both response to infection and susceptibility to autoimmunity . fcgr3b is expressed almost exclusively on neutrophils , and there is a clear correlation between gene copy number and fcgr3b cell surface expression , neutrophil adherence to igg - coated surfaces , and immune complex uptake . further , multiple studies have identified low fcgr3b cn ( i.e. , < 2 copies ) as a risk factor for systemic autoimmune diseases , such as systemic lupus erythematosus [ 810 ] , rheumatoid arthritis [ 11 , 12 ] , and primary sjgren 's syndrome [ 9 , 10 ] . we therefore examined the association between fcgr3b cnv and bd , in a cohort of iranian patients . ethics approval was obtained from the rheumatology research centre 's ethics committee of tehran university of medical sciences . 187 iranian bd patients were recruited from the behet 's clinic at shariati hospital , tehran , from early 2005 until late 2006 . 178 ethnically - matched , unrelated healthy volunteers were also recruited as normal controls between 2005 and 2007 . all the bd patients recruited for this study met the international study group ( isg ) criteria for bd . information was obtained regarding their age , gender , ethnicity , clinical features , and family history . serotyping for hla - b51 status was carried out in selected general immunologic laboratories within iran using routine commercial kits . genomic fcgr3b cn was determined using a quantitative real - time pcr method , as previously described . briefly , a duplex taqman copy number assay was performed , using fcgr3b specific primers ( applied biosystems , hs04211858 , fam - mgb dual labeled probe ) and rnase p ( applied biosystems , product 4403326 , vic - tamra dual - labeled probe ) as the reference assay . the assay was performed according to the manufacturer 's instructions and pcr reactions were run on an applied biosystems 7300 real - time pcr machine . copy number was determined using copy caller software ( v.1.0 , applied biosystems , usa ) , and results were accepted only when calling confidence was > 80% , and cq standard deviation between replicates was < 0.20 . analysis of fcgr3b cn in bd patients compared to controls , and with clinical manifestations within bd patients , was performed using logistic regression . 187 bd patients were included in the study ( mean age 32.7 8.2 , 48% female ) . baseline characteristics are summarised in table 1 . of those who were tested for hla status , 51% ( 89 of 175 ) were hla b5 positive and 51% ( 41 of 80 ) were hla b51 positive . the frequency of fcgr3b cn variants in both bd patients and controls is presented in table 2 . copy numbers ranged from 1 to 4 , and no null genotypes were observed in this cohort . the odds ratio for low copy number ( < 2 cn ) was 0.6 ( 95% ci 0.30 , 1.21 , p = 0.16 ) and the odds ratio for high copy number ( > 2 cn ) was 0.75 ( 96% ci 0.33 , 1.73 , p = 0.50 ) . therefore there was no evidence that either low or high fcgr3b was associated with bd , and in fact , both low cn and high cn were slightly decreased in bd patients relative to controls . further , there was no evidence of associations between fcgr3b cn variants and clinical manifestations within bd patients as shown in table 3 . this is the first study to examine the relationship between fcgr3b cn variants and behet 's disease , and we report no evidence of an association in terms of either disease susceptibility or clinical manifestations in this cohort of patients from iran . however , a previous study of turkish bd patients has reported associations between fcgr2a , fcgr3a , and fcgr3b snps and bd , in terms of both disease susceptibility and clinical manifestations , but this remains unconfirmed . other studies have reported intriguing but conflicting relationships between fcgr3b cn and vasculitis in the context of different diseases . for example , fcgr3b low cn ( < 2 ) is associated with granulomatosis with polyangiitis ( wegener 's granulomatosis ) and microscopic polyangiitis ( antineutrophil cytoplasmic antibody - associated systemic vasculitidies ) in one study , and high fcgr3b cn ( > 2 ) in another , and no association has been observed with vasculitis in conjunction with systemic lupus erythematosus . further , similar to bd , susceptibility to other systemic vasculitidies including giant cell arteritis and kawasaki 's disease has also been linked to snps within the fcgr gene cluster [ 16 , 17 ] . the fcgr gene cluster is a complex genomic region , with both snp and cnv polymorphism . while we were unable to demonstrate an association between fcgr3b cn and bd in this study , there are undoubted links to polymorphism in this region with vasculitic conditions . in future , the challenge will be to integrate cnv and snp data into haplotypes in order to systematically evaluate and contrast associations with different vasculitidies .

behet 's disease ( bd ) is an immune - mediated systemic vasculitis associated with hlab51 . other gene associations are likely and may provide further insight into the pathogenesis of this disease . fc - gamma receptors play an important role in regulating immune function . copy number variation ( cnv ) of the fc - gamma receptor 3b ( fcgr3b ) gene is associated with other inflammatory conditions and may also play a role in bd . the aim of this study was to determine whether cnv of the fcgr3b gene is associated with bd or its clinical features . fcgr3b copy number was determined for 187 iranian patients and 178 ethnicity - matched controls using quantitative real - time pcr . the genotype frequencies were comparable in both bd patients and controls . the odds ratio for low copy number ( < 2cn ) was 0.6 ( p = 0.16 ) and the odds ratio for high copy number ( > 2cn ) was 0.75 ( p = 0.50 ) . there was no association found between high or low cn of the fcgr3b gene and bd or its clinical features in this iranian population . we are the first to report this finding which , when looked at in the context of other genetic studies , gives us further insight into the complex pathogenesis of bd .

epidemiological studies during the past decade have shown a positive correlation between the general consumption of cruciferous vegetables and decreased incidence of some cancers , including lung , stomach , colon , breast,[23 ] bladder,[45 ] prostate,[67 ] and non - hodgkin lymphoma . lung cancer risk is reduced especially by dietary intake of isothiocyanates or cruciferous vegetables in persons with genotypes of glutathione s - transferase mu 1-null and glutathione s - transferase theta 1-null , highlighting an important interplay between genetic susceptibility factors and chemopreventive agents.[1910 ] the cancer chemopreventive effect of cruciferous vegetables is due to glucosinolates . sulforaphane ( sfn ) is formed by enzymatic transformation of a glucosinolate , glucoraphanin , present in cruciferous vegetables , during chewing or through conversion through the gut microflora . sfn acts as a chemopreventive agent by inducing phase - ii enzymes by activating the nuclear factor erythroid-2 related factor 2 ( nrf2 ) pathway.[1213 ] evidence is mounting that sfn acts through other chemopreventive mechanisms such as blocking cancer initiation via inhibition of cytochrome p-450-dependent monoxygenases that convert procarcinogens into active carcinogens and modulating signaling pathways that regulate cell growth and apoptosis to suppress cancer progression.[1415 ] several groups have reported that sfn is effective in preventing chemical carcinogen - induced breast , stomach , pancreas , and colon cancer , as well as decreased polyp formation in apc mice . in the lungs , sfn and its n - acetylcysteine conjugate inhibit malignant progression of adenomas induced by tobacco carcinogens . in addition to its anticarcinogenic effect , sfn treatment inhibits the growth of established prostate,[2123 ] breast , pancreas,[2527 ] and lung cancer xenografts in mice . preclinical study results have also shown that sfn - mediated activation of nrf2 improves bacterial clearance and corticosteroid sensitivity in mice with chronic cigarette smoke exposure.[2930 ] several clinical trials are underway for evaluating sfn for treatment of various diseases ( www.clinicaltrials.gov ) . somatic loss of function mutations in kelch - like ech - associated protein 1 ( keap1 ) and oncogenic gain of function mutations in nrf2 have been reported in lung , prostate , skin , esophagus , gallbladder , and ovarian cancer . more recently , oncogenic activation of k - ras , myc , and b - raf have been shown to activate nrf2 signaling , and this cross talk has been shown to be important for oncogene - induced senescence evasion and tumorigenesis.[3637 ] for future development of sfn as a chemopreventive or therapeutic agent , we have evaluated the risk of long - term sfn administration on lung tumorigenesis by using oncogenic k - ras mouse model and human lung cancer xenograft mouse models . d , l - sulforaphane was purchased from toronto research chemicals , ontario ( cat#s699115 ) and stored at -20c . the sfn was freshly diluted in phosphate - buffered saline ( pbs ) before treatment . a549 and h1975 cells were purchased from american type culture collection ( manassas , virginia , united states ) and cultured under recommended conditions . cells were cultured in dulbecco modified eagle medium ( invitrogen , carlsbad , california , united states ) containing 10% fetal bovine serum and 1% penicillin streptomycin . ad - cmv - cre ( 1 10 pfu / ml ) was obtained from vector biolabs . kras ( strain number 01xj6 ) mice were obtained from a mouse repository ( nci , frederick ) and were bred in our facility . athymic ncr - nu / nu ( strain number 01b74 ) nude mice ( 6 - 8 weeks ) were obtained from nci . mice were fed the 2018sx diet ( harlan teklad ) and had access to water ad libitum ; they were housed under controlled conditions ( 232c ; 14-h light/ 10-h dark cycles ) . all experimental protocols conducted on the mice were performed in accordance with national institutes of health guidelines and were approved by the johns hopkins university animal care and use committee . eight - week - old kras mice were anesthetized with ketamine ( 100 mg / kg of body weight ) and xylazine ( 10 mg / kg of body weight ) . five million adenovirus cre - recombinase particles diluted in 100 l of minimal essential medium were administered by oropharyngeal aspiration ( 50 l , twice at an interval of 5 min ) according to the standard protocol . five days after adenovirus cre treatment , mice were randomly placed into one of the two groups . sfn ( 0.5 mg ) suspended in 50 l of pbs was administered 5 d / wk for 14 weeks by using a nebulizer ( aeroneb lab ) in the sfn group ( n = 9 ) . the lesions were assessed subjectively for the percentage of lesion area to total lung section area . the lesions were categorized as mild hyperplasia , severe hyperplasia , adenoma , or adenocarcinoma . either a549 cells ( 5 10 ) or h1975 cells ( 1 10 ) were injected subcutaneously into the right flank of the anesthetized athymic nude mice . when the volume of the tumor reached 50 20 mm , the mice were separated randomly into different treatment groups , and sfn treatment was started . the mice were weighed and the tumor dimensions were measured with calipers once a week until 1 week before harvesting of the tumor . the tumor volumes were calculated by using the following formula : length ( mm ) width ( mm ) width ( mm ) 0.52 . the subcutaneous sfn injections were administered 0.51 cm adjacent to the left side of the tumor . the control mice in all the experiments received the same volume of pbs as did their respective cohorts . the mice were euthanized by means of cervical dislocation , and the tumors were removed without skin and weighed . design for xenograft experiments total rna was extracted from tissues and cells by using the rneasy kit ( qiagen ) and was quantified by means of ultraviolet absorption spectrophotometry . the reverse transcription reaction was performed by using a high - capacity cdna synthesis kit ( applied biosystems , foster city , california , united states ) and the multiscribe first strand synthesis system ( applied biosystems , foster city , california , united states ) in a final volume of 20 l containing 1 g of total rna , 100 ng of random hexamers , 1 reverse transcription buffer , 1 mm deoxyribonucleotide triphosphate , multiscribe reverse transcriptase , and nuclease - free water . a quantitative real - time polymerase chain reaction analysis of nad(p)h dehydrogenase , quinone 1 ( nqo1 ) , heme oxygenase 1(ho-1 ) , and glutamate - cysteine ligase , catalytic subunit ( gclc ) was performed by using assay - on - demand primers and probe sets from applied biosystems . tumor volume , tumor weight , and body weight were measured and presented as mean plus or minus standard error and compared by means of a t - test or anova . d , l - sulforaphane was purchased from toronto research chemicals , ontario ( cat#s699115 ) and stored at -20c . the sfn was freshly diluted in phosphate - buffered saline ( pbs ) before treatment . a549 and h1975 cells were purchased from american type culture collection ( manassas , virginia , united states ) and cultured under recommended conditions . cells were cultured in dulbecco modified eagle medium ( invitrogen , carlsbad , california , united states ) containing 10% fetal bovine serum and 1% penicillin streptomycin . ad - cmv - cre ( 1 10 pfu / ml ) was obtained from vector biolabs . kras ( strain number 01xj6 ) mice were obtained from a mouse repository ( nci , frederick ) and were bred in our facility . athymic ncr - nu / nu ( strain number 01b74 ) nude mice ( 6 - 8 weeks ) were obtained from nci . mice were fed the 2018sx diet ( harlan teklad ) and had access to water ad libitum ; they were housed under controlled conditions ( 232c ; 14-h light/ 10-h dark cycles ) . all experimental protocols conducted on the mice were performed in accordance with national institutes of health guidelines and were approved by the johns hopkins university animal care and use committee . eight - week - old kras mice were anesthetized with ketamine ( 100 mg / kg of body weight ) and xylazine ( 10 mg / kg of body weight ) . five million adenovirus cre - recombinase particles diluted in 100 l of minimal essential medium were administered by oropharyngeal aspiration ( 50 l , twice at an interval of 5 min ) according to the standard protocol . five days after adenovirus cre treatment , mice were randomly placed into one of the two groups . sfn ( 0.5 mg ) suspended in 50 l of pbs was administered 5 d / wk for 14 weeks by using a nebulizer ( aeroneb lab ) in the sfn group ( n = 9 ) . the lesions were assessed subjectively for the percentage of lesion area to total lung section area . the lesions were categorized as mild hyperplasia , severe hyperplasia , adenoma , or adenocarcinoma . either a549 cells ( 5 10 ) or h1975 cells ( 1 10 ) were injected subcutaneously into the right flank of the anesthetized athymic nude mice . when the volume of the tumor reached 50 20 mm , the mice were separated randomly into different treatment groups , and sfn treatment was started . the mice were weighed and the tumor dimensions were measured with calipers once a week until 1 week before harvesting of the tumor . the tumor volumes were calculated by using the following formula : length ( mm ) width ( mm ) width ( mm ) 0.52 . the subcutaneous sfn injections were administered 0.51 cm adjacent to the left side of the tumor . the control mice in all the experiments received the same volume of pbs as did their respective cohorts . the mice were euthanized by means of cervical dislocation , and the tumors were removed without skin and weighed . total rna was extracted from tissues and cells by using the rneasy kit ( qiagen ) and was quantified by means of ultraviolet absorption spectrophotometry . the reverse transcription reaction was performed by using a high - capacity cdna synthesis kit ( applied biosystems , foster city , california , united states ) and the multiscribe first strand synthesis system ( applied biosystems , foster city , california , united states ) in a final volume of 20 l containing 1 g of total rna , 100 ng of random hexamers , 1 reverse transcription buffer , 1 mm deoxyribonucleotide triphosphate , multiscribe reverse transcriptase , and nuclease - free water . a quantitative real - time polymerase chain reaction analysis of nad(p)h dehydrogenase , quinone 1 ( nqo1 ) , heme oxygenase 1(ho-1 ) , and glutamate - cysteine ligase , catalytic subunit ( gclc ) was performed by using assay - on - demand primers and probe sets from applied biosystems . tumor volume , tumor weight , and body weight were measured and presented as mean plus or minus standard error and compared by means of a t - test or anova . we determined whether chronic sfn administration affects oncogenic k - ras ( k - ras)-driven lung tumorigenesis . the k - ras mutant mouse develops progressively less differentiated lung tumors after the administration of adenoviral cre - recombinase . in this study , we evaluated the effect of sfn on k - ras mediated lung tumorigenesis post oncogenic k - ras activation . one week after administration of a high titer adenoviral cre to induce mutant k - ras expression , 1 cohort of mice was treated with sfn ( 0.5 mg , 5 d / wk ) for 3 months by means of a nebulizer [ figure 1a ] . we have reported that sfn ( 0.5 mg / mice ) administered using nebulizer significantly induces nrf2-dependent gene expression in lungs . fourteen weeks after adenovirus - cre - recombinase administration and mutant k - ras activation , mice were sacrificed , and lung sections were screened for neoplastic foci . histological analyses of the lung sections from the k - ras mutant mice showed the presence of atypical adenomatous hyperplasia and adenomas with infiltration of inflammatory cells . although tumor number , histological subtypes , and grades were not significantly different between the two groups , we noticed a trend toward reduced tumor burden in the sfn - treated cohort of mice [ figure 1b ] . extended exposure to sfn transcriptionally induced the expression of the nrf2 target genes nqo1 , gclc , and ho-1 in the lungs [ figure 1c ] . thus , our data demonstrate that prolonged exposure to sfn via aerosol inhalation does not promote or increase tumorigenesis in the mutant k- ras - driven mouse model of lung tumorigenesis . ( a ) schematic of the experimental design . ( b ) bar graph showing relative number of neoplastic lesions ( hyperplasia , adenoma , adenocarcinoma ) and percentage of the area of the lung covered by the tumor cells . ( c ) real - time rt - pcr data showing induction of nrf2-dependent gene expression in the lungs of the sfn - treated cohort of mice . p - values are calculated using t test to evaluate the effect of prolonged sfn treatment on the growth of established lung cancer xenografts , we selected two cell lines , a549 and h1975 . a549 cells with a mutant k - ras and keap1 allele , and h1975 with a mutant epidermal growth factor receptor ( egfr ) allele , are the two commonly used lung cancer xenograft models . a549 and h1975 cells were injected into the flanks of nude mice , and change in tumor volume was recorded . once the tumor volume reached 50 20 mm , mice were randomly allocated into two groups , and sfn treatment was initiated [ figures 2a and 3a ] . mice were treated with vehicle ( pbs ) or sfn by means of intraperitoneal injection ( 5 d / wk ) , and tumor volume was measured weekly . sfn dose was selected based on previously published results where sfn administration at a dose 250400 mol / kg body weight , has been reported to be well tolerated . we did not notice a significant reduction in the tumor growth rate / weight of a549 xenografts treated with 3 mol dose of sfn , and thus , we increased the sfn dose and repeated the experiment with 3 and 6 mol . again , sfn treatment at a dose of 3 or 6 mol did not inhibit the growth of a549 tumors significantly , although we noticed a trend toward lower tumor volume and weight in the sfn - treated group [ figures 2b and c and 3b and c ) . for h1975 cells expressing mutant egrf allele , we increased the sfn dose further ( 9 mol ) since these tumors proliferate rapidly in vivo . similar to our results with a549 tumors , sfn at a dose 9 mol did not attenuate the growth of h1975 xenografts significantly , although we noticed a trend toward lower tumor volume and weight in the sfn - treated group [ figures 3b and 3c ] . to ascertain whether systemic sfn administration causes weight loss , we recorded body weights of the control and treated mice . average body weights of the control and sfn - treated mice did not differ significantly throughout the treatment protocol [ figures 2d and 3d ] . systemic delivery of sfn does not promote growth of keap1 and k - ras mutant , a549 , lung cancer xenografts . tumor volume is not significantly different between the three cohorts of mice ( anova ) . ( e ) real - time rt - pcr showing expression of the nrf2-dependent gene , nqo1 , in the liver and tumor tissues.*p < 0.05 ( t test ) . systemic delivery of sfn does not promote growth of egfr mutant ( h1975 ) lung cancer xenografts . tumor volume is not significantly different between the two cohorts of mice ( t test ) . ( c ) tumor weights ( day 30 ) are not significantly different between the two groups ( t test ) . ( e ) real - time pcr showing expression of the nrf2-dependent gene , nqo1 , in the liver and tumor tissues.*p < 0.05 ( t test ) to demonstrate that systemic delivery of sfn induced nrf2-dependent target gene expression in mice , we measured nqo1 expression in the liver [ figures 2e and 3e ] . in addition to the liver , we also measured the induction of nrf2 signaling in a549 and h1975 tumors . however , sfn treatment did not upregulate nrf2 signaling in tumors [ figures 2e and 3e ] . these data indicate that systemic sfn treatment induces nrf2 signaling but does not promote growth of lung cancer xenografts . to determine whether localized delivery of sfn that results in higher accumulation of sfn in the tumor and neighboring areas can retard the growth of subcutaneous tumors , we selected a549 cells as the model system . a549 cells were injected into the flanks of nude mice , and change in tumor volume was recorded . once the tumor volumes reached 50 20 mm , mice were randomly allocated into 2 groups , and treatment groups were administered sfn ( 1 mol , 5d / wk ) by means of subcutaneous injection adjacent to the tumor [ figure 4a ] . the average change in tumor volume was significantly different between the vehicle- and sfn - treated tumors ( p < 0.05 ) [ figure 4b ] . tumor weights were significantly higher in the vehicle - treated tumors than in the sfn - treated tumors ( p = 0.05 ) [ figure 4c ] , with no apparent difference in total body weight [ figure 4d ] between the two groups . graph showing relative growth of a549 tumors treated with 1000 nmol of sfn . * p < 0.05 ( t test ) . ( c ) tumor weights were significantly lower in the sfn - treated group . * p < 0.05 ( t test ) . ( e g ) relative tumor growth , average tumor weight , and body weight in the cohort of mice treated with 500 nmol of sfn . ( h j ) relative expression of nrf2-dependent genes in the liver and tumor tissues . * significant inhibition of tumor growth by sfn at a dose of 1 mol prompted us to determine whether sfn exerts a similar effect at a lower dose ( 0.5 mol ) as well . treatment of a549 xenograft tumors with a lower dose of sfn significantly retarded the growth of a549 tumors in nude mice . average tumor volume and weight were significantly lower in the sfn - treated cohort of mice [ figures 4e and 4f ] . there was no adverse effect of localized sfn treatment on animal health or body weight , although we noticed slight thickening of skin in the area surrounding the injection spot [ figure 4 g ] . to demonstrate that localized delivery of sfn induced nrf2-dependent target gene expression , we measured nqo1 , ho-1 , and gclc expression in the skin and liver . in addition to these two tissues , we measured the induction of nrf2 signaling in a549 tumors . sfn treatment significantly induced the expression of ho-1 in the skin with no apparent effect on nrf2-dependent gene expression in the tumors [ figures 4h j ] . since sfn was administered locally , we did not notice significant change in nrf2 dependent gene expression in the liver . these data indicate that localized sfn treatment probably results in greater accumulation of sfn in subcutaneous tumors leading to significant growth inhibition . we determined whether chronic sfn administration affects oncogenic k - ras ( k - ras)-driven lung tumorigenesis . the k - ras mutant mouse develops progressively less differentiated lung tumors after the administration of adenoviral cre - recombinase . in this study , we evaluated the effect of sfn on k - ras mediated lung tumorigenesis post oncogenic k - ras activation . one week after administration of a high titer adenoviral cre to induce mutant k - ras expression , 1 cohort of mice was treated with sfn ( 0.5 mg , 5 d / wk ) for 3 months by means of a nebulizer [ figure 1a ] . we have reported that sfn ( 0.5 mg / mice ) administered using nebulizer significantly induces nrf2-dependent gene expression in lungs . fourteen weeks after adenovirus - cre - recombinase administration and mutant k - ras activation , mice were sacrificed , and lung sections were screened for neoplastic foci . histological analyses of the lung sections from the k - ras mutant mice showed the presence of atypical adenomatous hyperplasia and adenomas with infiltration of inflammatory cells . although tumor number , histological subtypes , and grades were not significantly different between the two groups , we noticed a trend toward reduced tumor burden in the sfn - treated cohort of mice [ figure 1b ] . extended exposure to sfn transcriptionally induced the expression of the nrf2 target genes nqo1 , gclc , and ho-1 in the lungs [ figure 1c ] . thus , our data demonstrate that prolonged exposure to sfn via aerosol inhalation does not promote or increase tumorigenesis in the mutant k- ras - driven mouse model of lung tumorigenesis . ( a ) schematic of the experimental design . ( b ) bar graph showing relative number of neoplastic lesions ( hyperplasia , adenoma , adenocarcinoma ) and percentage of the area of the lung covered by the tumor cells . ( c ) real - time rt - pcr data showing induction of nrf2-dependent gene expression in the lungs of the sfn - treated cohort of mice . to evaluate the effect of prolonged sfn treatment on the growth of established lung cancer xenografts , we selected two cell lines , a549 and h1975 . a549 cells with a mutant k - ras and keap1 allele , and h1975 with a mutant epidermal growth factor receptor ( egfr ) allele , are the two commonly used lung cancer xenograft models . a549 and h1975 cells were injected into the flanks of nude mice , and change in tumor volume was recorded . once the tumor volume reached 50 20 mm , mice were randomly allocated into two groups , and sfn treatment was initiated [ figures 2a and 3a ] . mice were treated with vehicle ( pbs ) or sfn by means of intraperitoneal injection ( 5 d / wk ) , and tumor volume was measured weekly . sfn dose was selected based on previously published results where sfn administration at a dose 250400 mol / kg body weight , has been reported to be well tolerated . we did not notice a significant reduction in the tumor growth rate / weight of a549 xenografts treated with 3 mol dose of sfn , and thus , we increased the sfn dose and repeated the experiment with 3 and 6 mol . again , sfn treatment at a dose of 3 or 6 mol did not inhibit the growth of a549 tumors significantly , although we noticed a trend toward lower tumor volume and weight in the sfn - treated group [ figures 2b and c and 3b and c ) . for h1975 cells expressing mutant egrf allele , we increased the sfn dose further ( 9 mol ) since these tumors proliferate rapidly in vivo . similar to our results with a549 tumors , sfn at a dose 9 mol did not attenuate the growth of h1975 xenografts significantly , although we noticed a trend toward lower tumor volume and weight in the sfn - treated group [ figures 3b and 3c ] . to ascertain whether systemic sfn administration causes weight loss , we recorded body weights of the control and treated mice . average body weights of the control and sfn - treated mice did not differ significantly throughout the treatment protocol [ figures 2d and 3d ] . systemic delivery of sfn does not promote growth of keap1 and k - ras mutant , a549 , lung cancer xenografts . tumor volume is not significantly different between the three cohorts of mice ( anova ) . ( e ) real - time rt - pcr showing expression of the nrf2-dependent gene , nqo1 , in the liver and tumor tissues.*p < 0.05 ( t test ) . systemic delivery of sfn does not promote growth of egfr mutant ( h1975 ) lung cancer xenografts . tumor volume is not significantly different between the two cohorts of mice ( t test ) . ( c ) tumor weights ( day 30 ) are not significantly different between the two groups ( t test ) . ( e ) real - time pcr showing expression of the nrf2-dependent gene , nqo1 , in the liver and tumor tissues.*p < 0.05 ( t test ) to demonstrate that systemic delivery of sfn induced nrf2-dependent target gene expression in mice , we measured nqo1 expression in the liver [ figures 2e and 3e ] . in addition to the liver , we also measured the induction of nrf2 signaling in a549 and h1975 tumors . however , sfn treatment did not upregulate nrf2 signaling in tumors [ figures 2e and 3e ] . these data indicate that systemic sfn treatment induces nrf2 signaling but does not promote growth of lung cancer xenografts . to determine whether localized delivery of sfn that results in higher accumulation of sfn in the tumor and neighboring areas can retard the growth of subcutaneous tumors , we selected a549 cells as the model system . a549 cells were injected into the flanks of nude mice , and change in tumor volume was recorded . once the tumor volumes reached 50 20 mm , mice were randomly allocated into 2 groups , and treatment groups were administered sfn ( 1 mol , 5d / wk ) by means of subcutaneous injection adjacent to the tumor [ figure 4a ] . the average change in tumor volume was significantly different between the vehicle- and sfn - treated tumors ( p < 0.05 ) [ figure 4b ] . tumor weights were significantly higher in the vehicle - treated tumors than in the sfn - treated tumors ( p = 0.05 ) [ figure 4c ] , with no apparent difference in total body weight [ figure 4d ] between the two groups . ( b ) graph showing relative growth of a549 tumors treated with 1000 nmol of sfn . * p < 0.05 ( t test ) . ( c ) tumor weights were significantly lower in the sfn - treated group . * p < 0.05 ( t test ) . ( e g ) relative tumor growth , average tumor weight , and body weight in the cohort of mice treated with 500 nmol of sfn . ( h j ) relative expression of nrf2-dependent genes in the liver and tumor tissues . * p < 0.05 . significant inhibition of tumor growth by sfn at a dose of 1 mol prompted us to determine whether sfn exerts a similar effect at a lower dose ( 0.5 mol ) as well . treatment of a549 xenograft tumors with a lower dose of sfn significantly retarded the growth of a549 tumors in nude mice . average tumor volume and weight were significantly lower in the sfn - treated cohort of mice [ figures 4e and 4f ] . there was no adverse effect of localized sfn treatment on animal health or body weight , although we noticed slight thickening of skin in the area surrounding the injection spot [ figure 4 g ] . to demonstrate that localized delivery of sfn induced nrf2-dependent target gene expression , we measured nqo1 , ho-1 , and gclc expression in the skin and liver . in addition to these two tissues , we measured the induction of nrf2 signaling in a549 tumors . sfn treatment significantly induced the expression of ho-1 in the skin with no apparent effect on nrf2-dependent gene expression in the tumors [ figures 4h j ] . since sfn was administered locally , we did not notice significant change in nrf2 dependent gene expression in the liver . these data indicate that localized sfn treatment probably results in greater accumulation of sfn in subcutaneous tumors leading to significant growth inhibition . since the early 1980s , naturally occurring antioxidants and vitamins have been investigated and promoted as chemopreventive agents . antioxidants have been popular as chemopreventive agents because there is strong scientific evidence supporting reduced incidence of epithelial cancers associated with high dietary intake of fruits and vegetables . because of this evidence , major clinical trials were launched to test -carotene , alone or in combination with vitamin e or vitamin a. the largest trials for chemoprevention of lung cancers in high - risk smoker populations were atbc , the alpha tocopherol ( vitamin e ) , beta - carotene trial in finland , and caret , the beta - carotene and retinol efficacy trial smokers in the united states . the results were devastatingly similar , with excess lung cancer incidence and mortality rates in the active treatment arm , compared with those in the placebo arm of each trial . the unexpected and unwanted outcomes from the caret and atbc trials prompted us to reconsider issues related to both the effectiveness and the safety of micronutrient supplementation and careful evaluation of chemopreventive agents in preclinical cancer models . sfn derived from broccoli sprouts is currently under clinical evaluation in diseases in which oxidative stress and inflammation play important roles in disease pathogenesis , namely , pulmonary diseases , such as chronic obstructive pulmonary disease , asthma , and cystic fibrosis , and cardiovascular disease , and protection against radiation dermatitis . in addition to these , sfn is being evaluated as a potential chemopreventive agent in melanoma and breast cancer and as an anticancer agent in patients with recurrent prostate cancer . at present , there are 17 clinical trials listed at www.clinicaltrials.gov that are using sfn or an enriched broccoli sprout preparation for treatment of a variety of diseases although sfn is known to modulate several signaling pathways , it is a potent inducer of nrf2-keap1 signaling , and the anticarcinogenic and anti - inflammatory activities of sfn are mediated primarily by nrf2-dependent induction of phase - ii detoxification and antioxidant enzymes.[13142930 ] results from recent studies suggest that sfn offers protection against tumor development and progression during the post - initiation phase by modulating diverse cellular processes that inhibit the growth of transformed cells . the ability of sfn to induce reactive oxygen species production , apoptosis , and cell cycle arrest is associated with regulation of many molecules , including the bcl-2 family of proteins , caspases , p21 , cyclins , cyclin - dependent kinases , and histone deacetylases and nuclear factor - kappa - b pathways . sfn also suppresses angiogenesis and metastasis by downregulating hypoxia - inducible factor 1 alpha , matrix metallopeptidase 2 , matrix metallopeptidase 9 , and vascular endothelial growth factor in various preclinical models of cancer however , in the past few years , loss of function mutations in keap1 and activating mutations in nrf2 leading to gain of nrf2 have been reported in lung cancer . in addition to lung cancer , gain of nrf2 function has been reported in other cancers such as prostate , gallbladder , esophagus , breast , skin , and ovarian cancer . in addition to mutations , results from recent studies have demonstrated that the oncogenes k - ras , b - raf , and myc upregulate nrf2 signaling to evade senescence and apoptosis and promote tumorigenesis . a comprehensive profiling of sfn bioavailability and tissue distribution kinetics revealed a dose - dependent increase in tissue concentrations after oral administration of sfn in mice . sfn accumulation peaked at 2 h in lung , liver , kidney , brain , and plasma , but in small intestine , colon , and prostate , the highest concentrations were recorded at 6 h. maximum sfn accumulation was detected in small intestine . furthermore , tissue concentrations of sfn metabolites varied as much as 100-fold between different tissues suggesting that peak plasma concentrations do not always align precisely with target tissues . thus , route of administration may have a critical impact on sfn tissue distribution and accumulation . in this study , we compared three routes of sfn administration ; ( a ) direct delivery to the target organ ( lung ) using nebulizer , ( b ) localized delivery to tumor bearing area ( subcutaneous injection ) , and ( c ) systemic delivery with minimal risk of potential side effects due to systemic distribution ( intraperitoneal injection ) . to evaluate the effect of prolonged sfn treatment on growth of lung cancer xenografts , we selected two models : ( 1 ) a549 cells harboring mutant k - ras oncogene and mutant keap1 gene and ( 2 ) h1975 cells harboring mutant egfr oncogene but wild - type keap1 and nrf2 . prolonged systemic sfn treatment did not promote the growth of these tumors in vivo . on the contrary , localized delivery of sfn significantly abrogated the growth of keap1 mutant a549 tumors in vivo suggesting that the tumor suppression effects of sfn are mediated by non - nrf2-dependent signaling mechanisms . importantly , significant tumor growth inhibition and a trend toward reduction with localized and systemic route of sfn administration , respectively , suggests that localized delivery of sfn probably results in better target tissue distribution and accumulation as compared to the systemic route . next , we investigated whether prolonged sfn administration affects oncogenic k - ras ( k - ras)-driven lung tumorigenesis . a study by denicola et al . showed that oncogenic k - ras signaling upregulates nrf2 expression and its transcriptional network . interestingly , although sfn administration via aerosol inhalation only modestly affected the nrf2 signaling , we still noticed a trend toward reduced tumor burden in sfn treated cohort of mice . this raises a possibility that alternative route of administration resulting in localized accumulation and robust induction of nrf2 signaling may significantly inhibit k - ras mediated lung tumor growth . in summary , sfn treatment however , one limitation of our study is that we only evaluated the effect of sfn on k - ras - mediated lung tumorigenesis post oncogenic k - ras activation and tumor foci formation . it remains to be investigated if sfn administration prior to cre - recombinase mediated oncogenic k - ras activation may impact the overall tumor burden . pharmacological activators of nrf2 ( dithiolethiones , isothiocyanates , and triterpenoids ) including sfn have been evaluated as modifiers of multistage carcinogenesis in animal models and no protumorigenic effects have been reported to date . our data from three preclinical mouse models of lung cancer suggest that prolonged sfn treatment does not promote tumor growth in mice . these findings , along with those from previously published studies on the chemopreventive properties of sfn against chemical carcinogens , support its future clinical development as a drug for chronic obstructive pulmonary disease and other pulmonary diseases . dr . anju singh , department of environmental health sciences , bloomberg school of public health , johns hopkins university , baltimore , maryland dr . shyam biswal , department of environmental health sciences , bloomberg school of public health , johns hopkins university , baltimore , maryland department of oncology , sidney kimmel comprehensive cancer center , school of medicine , johns hopkins university , baltimore , maryland mr . shengbin yang , department of environmental health sciences , bloomberg school of public health , johns hopkins university , baltimore , maryland dr . edward gabrielson , department of oncology , sidney kimmel comprehensive cancer center , school of medicine , johns hopkins university , baltimore , maryland dr . jinfang ma , department of environmental health sciences , bloomberg school of public health , johns hopkins university , baltimore , maryland dr . rajesh k thimmulappa , department of environmental health sciences , bloomberg school of public health , johns hopkins university , baltimore , maryland dr . ponvijay kombairaju , department of environmental health sciences , bloomberg school of public health , johns hopkins university , baltimore , maryland

background : sulforaphane ( sfn ) , an activator of nuclear factor erythroid-2 related factor 2 ( nrf2 ) , is a promising chemopreventive agent which is undergoing clinical trial for several diseases . studies have indicated that there is gain of nrf2 function in lung cancer and other solid tumors because of mutations in the inhibitor kelch - like ech - associated protein 1 ( keap1 ) . more recently , several oncogenes have been shown to activate nrf2 signaling as the main prosurvival pathway mediating ros detoxification , senescence evasion , and neoplastic transformation . thus , it is important to determine if there is any risk of enhanced lung tumorigenesis associated with prolonged administration of sfn using mouse models of cancer.materials and methods : we evaluated the effect of prolonged sfn treatment on oncogenic k - ras ( k - raslsl - g12d)-driven lung tumorigenesis . one week post mutant - k - ras expression , mice were treated with sfn ( 0.5 mg , 5 d / wk ) for 3 months by means of a nebulizer . fourteen weeks after mutant k - ras expression ( k - raslsl - g12d ) , mice were sacrificed , and lung sections were screened for neoplastic foci . expression of nrf2-dependent genes was measured using real time rt - pcr . we also determined the effect of prolonged sfn treatment on the growth of preclinical xenograft models using human a549 ( with mutant k - ras and keap1 allele ) and h1975 [ with mutant epidermal growth factor receptor ( egfr ) allele ] nonsmall cell lung cancer cells.results:systemic sfn administration did not promote the growth of k - raslsl - g12d - induced lung tumors and had no significant effect on the growth of a549 and h1975 established tumor xenografts in nude mice . interestingly , localized delivery of sfn significantly attenuated the growth of a549 tumors in nude mice , suggesting an nrf2-independent antitumorigenic activity of sfn.conclusions:our results demonstrate that prolonged sfn treatment does not promote lung tumorigenesis in various mouse models of lung cancer .

maternal moderate and severe anemia have been considered as conditions that affect adversely maternal and perinatal outcomes . for such reason the identification of these cases is important since appropriate treatment may reduce the risk for adverse outcomes . iron is the most common treatment currently used to control iron deficiency and anemia particularly in low - income countries , where anemia is more frequent . physiologically , hemoglobin concentration drops during gestation , and for such reason the hemoglobin cutoff to define anemia was set at 11 g / dl of hb . maternal hb levels are higher at high altitudes in all trimesters of pregnancy than at sea level . however , the reduction of hemoglobin levels during pregnancy occurs in populations at low and at high altitudes . this is due to plasma volume expansion as a mechanism to improve arterial uterine flow to the placentae . thus , a normal hb value at first booking in the first trimester does not preclude the presence of anemia in a second measurement of hemoglobin as pregnancy advances . a recent study showed a higher risk for anemia in pregnant women with low body mass index ( bmi ) . , anemia , particularly moderate and severe anemia is associated with high risk for low birthweight [ 3 , 9 , 10 ] , it is important to monitor hemoglobin values during pregnancy . the present study has been designed to determine changes in hemoglobin concentration at second measurements after a normal hemoglobin concentration was detected at first booking during pregnancy at low and at high altitudes . we will determine rates of anemia , normal hemoglobin values and erythrocytosis ( hb > 14.5 g / dl ) . in addition , risk factors for moderate / severe anemia will be assessed in a multivariable analysis . this is a secondary analysis of a large data base obtained from the perinatal information system in peru which includes 379,816 pregnant women and their babies obtained from 43 maternity units in peru . data were obtained at the three geographical regions in peru : coast , highlands , and amazon . the study was authorized by the institutional review board at the universidad peruana cayetano heredia . as the study used secondary analysis of data , the study was exempt from formal review . from the total sample size , a selection was made of all women diagnosed as nonanemic and without erythrocytosis at first booking ( hb ranging from 11 to 14.5 g / dl ) and having a second measurement of hemoglobin during the same pregnancy . , 57,231 pregnant women were living at low altitude ( < 2000 m ) , 11,349 women at moderate altitude ( 20003000 m ) , and 20,714 women were living at high altitudes ( > 3000 m ) . data on gestational age at first and second hemoglobin measurement were recorded . with a second hemoglobin value , rates of pregnant women with anemia ( hb < 11 g / dl ) , normal hemoglobin values ( 1114.5 g / dl ) , and erythrocytosis ( hb>14.5 g / dl ) were determined . as demonstrated previously , no correction of hemoglobin cutoff to define anemia at high altitude is needed ; hence , the present study used the definition of anemia as suggested who for populations at sea level [ 1 , 11 ] . the degrees of anemia were classified as mild ( hb 9.010.9 g / dl ) , moderate ( hb 7.0 < 9 g / dl ) , and severe ( hb < 7 g / dl ) . body mass index was calculated from prepregnancy body weight and height . low bmi was defined as < 19.9 kg / m and high bmi as > 25 altitude was defined as low ( 01999 m ) , moderate ( 20002999 m ) , and high ( 3000 m ) . the following perinatal outcomes were assessed : stillbirths , preterm deliveries and small for gestational age . stillbirths were diagnosed when fetal deaths occurred after 20 weeks of gestation or with a weight higher than 500 g. preterm births were defined as deliveries before 37 weeks of gestational age . small for gestational age was defined if birthweight was below the 10th percentile of the williams reference chart . the adverse maternal outcomes assessed were preeclampsia , premature rupture of membranes ( prom ) , and postpartum hemorrhage ( pph ) . preeclampsia was defined as the presence of pregnancy - induced hypertension ( systolic pressure of 140 mm hg and/or diastolic pressure of 90 mm hg ) and proteinuria ( 300 mg/24 h ) after 20 weeks of gestation . pph was defined as women after parturition with blood losses of 500 ml or more . data are assessed according level of altitude ( low , moderate , and high ) and according to degree of anemia ( moderate / severe and mild ) , and erythrocytosis . if data were normally distributed , then a one - way analysis of variance were used ( anova ) . data expressed in proportions were assessed by the chi - square test . the confidence interval ( ci ) the prevalences of women with moderate / severe anemia ( hb < 9 g / dl ) , mild anemia ( hb 910.9 g / dl ) , normal hemoglobin ( hb 1114.5 g / dl ) , and erythrocytosis ( hb > 14.5 g / dl ) were calculated for the total sample , as well as for groups at different altitudes ( low , moderate , and high altitude ) . estimates of crudes and adjusted odds ratio ( or ) with 95% ci were computed as measures of association between the variables . a logistic regression analysis to determine the risk for moderate / severe anemia at second hemoglobin measured was assessed . the estimates were adjusted for gestational age at second hemoglobin , hemoglobin value at first measurement , altitude , maternal age , body mass index , maternal education , marital status , prenatal care visits , parity , the presence of preeclampsia , cardiopathy , and diabetes . concentration of hemoglobin at first booking in nonanemic women increased as altitude increased ( p < 0.05 ) , whereas birthweight decreased as altitude increased . no differences were observed in gestational age at birth at the three altitude groups studied . concentration of hemoglobin at second measurement also was increased as altitude increases , but values were lower than those at first measurement , particularly at low altitude , in which values were reduced to 94% , whereas at moderate altitude . they were reduced to 98.4% and at high altitude to 99.2% . gestational age at first and second hemoglobin was higher as altitude of residence increased ( table 1 ) . most of the studied sample remained with normal hemoglobin values at second hemoglobin measurement ( 75.1 ) . however , 21.4% of women became anemic at the second hb measurement . in all , 2.8% resulted with moderate / severe anemia and 3.5% with erythrocytosis ( figure 1 ) . women with anemia in the second hb measurement showed lower hb measurements in the first hb . conversely , women with erythrocytosis at the second hb measurement showed higher hb levels in the first measurement ( table 2 ) . in all cases hb was higher as altitude increased . the rate of stillbirths was higher with moderate / severe anemia and with erythrocytosis ( p < 0.05 ) , which remained higher after controlling for confounders ( table 3 ) . the rate of preterm births was increased in women with severe anemia ( 9.01% ; or = 1.81 ) . after adjusting for confounders , moderate / severe anemia ( or = 1.73 ) and mild anemia ( or = 1.28 ) were risks for preterm deliveries ( table 4 ) . the rate of sga was significantly higher in mothers with erythrocytosis , almost twice , than in women with normal hemoglobin . lowest rate of sga was observed with a maternal hb level of 910.9 g / dl ( mild anemia ) ( table 5 ) . the rate of preeclampsia was significantly higher in moderate / severe and mild anemia and in mothers with erythrocytosis , which remained after controlling for confounders ( table 6 ) . the highest risk for pph was observed with moderate / severe anemia ( twenty times higher than in woman with normal hb levels ) ( table 8) . an increased risk for moderate / severe anemia was associated with higher gestational age at second measurement of hemoglobin , bmi < 19.9 kg / m , living without partner , prenatal visits care < 5 , first parity , multiparity , and preeclampsia . lower risk for moderate / severe anemia were observed with normal high hb level at first booking , living at moderate and high altitude , and with high bmi ( table 9 ) . the present study has been designed to determine changes in hb levels after a first hb measurement in 89,294 pregnant women detected normality ( hb 1114.5 g / dl ) . according to the study results an increase in the rate of anemia according to pregnancy progress has been previously published in other settlings . moderate / severe anemia has been characterized as the conditions more associated with maternal and perinatal adverse outcomes . our results demonstrated that 2.8% of women classified as nonanemic in the first hb measurement showed moderate / severe anemia in the second measurement . our data showed that moderate / severe anemia were associated with stillbirths , preterm births , preeclampsia , and pph . the strength of this study is that it was done thanks to a large sample of women recorded through a perinatal information system that included 379,816 pregnant women and their newborn babies from different maternity units throughout peru . this database is an important tool for monitoring conditions and trends related to pregnancies and newborns , including service characteristics and women 's risk factors . data revealed that a percentage of women became anemic during the progression of pregnancy despite having a first normal hb measurement . this is an important finding since moderate and severe anemia are harmful for mothers and neonates . risks for moderate / severe anemia are preventable , or can be managed through appropriate management , such as with prenatal care , parity , preeclampsia , and low bmi . increasing the number of prenatal visits will result in better monitoring of hemoglobin values , and can control or reduce the effect of preeclampsia . low bmi is a characteristic of low - income countries and is associated with increased risk of maternal anemia , low birthweight , and preterm births . the present results are in accordance with other studies where low prevalence of anemia was observed in women with highest bmi , and high prevalence of moderate / severe anemia were associated with malnutrition [ 14 , 15 ] . our data suggest that in situation in which a normal hemoglobin value was first observed in a woman with low body mass index , supplementation with iron should be recommended . it is interesting to note that populations living at moderate and high altitudes had lower risk for moderate / severe anemia in a second measurement of hemoglobin during pregnancy . this suggests that women living in these altitudes bearing normal hemoglobin values at first booking should not be treated with iron supplementation . in recent years , it has been demonstrated that anemia defined as less than 11 g / dl of hb is lower at moderate and at high altitudes , since populations at these altitudes are characterized by increased hemoglobin values to compensate for the effect of altitudinal hypoxia [ 3 , 10 ] . however , we assumed that most cases of anemia were related to iron deficiency , which was supported by the finding that low bmi was associated with moderate / severe anemia . however , results of our analysis were similar to those obtained in a study in which bmi was obtained at first prenatal care . it is possible that erythrocytosis found in the second measurement of hb after a normal value of hb at first booking could be due to the effect of iron supplementation , since in peru a ministry of health regulation orders pregnant women be supplemented with iron . however , low adherence may explain the low prevalence of erythrocytosis in a second hb measurement during pregnancy . the results of this study confirm that prevalence of anemia increases as pregnancy progresses and that a normal value at first booking should not be considered sufficient , as further hb values should be indicated and observed during the course of pregnancy .

objective . to determine changes in hemoglobin concentration at second measurements after a normal hemoglobin concentration was detected at first booking during pregnancy at low and at high altitudes . methods . this is a secondary analysis of a large database obtained from the perinatal information system in peru which includes 379,816 pregnant women and their babies from 43 maternity units in peru . results . most women remained with normal hemoglobin values at second measurement ( 75.1% ) . however , 21.4% of women became anemic at the second measurement . in all , 2.8% resulted with moderate / severe anemia and 3.5% with erythrocytosis ( hb>14.5 g / dl ) . in all cases hb was higher as altitude increased . risk for moderate / severe anemia increased associated with higher gestational age at second measurement of hemoglobin , bmi < 19.9 kg / m2 , living without partner , < 5 antenatal care visits , first parity , multiparity , and preeclampsia . lower risk for moderate / severe anemia was observed with normal high hb level at first booking living at moderate and high altitude , and high bmi . conclusion . prevalence of anemia increases as pregnancy progress , and that a normal value at first booking may not be considered sufficient as hb values should be observed throughout pregnancy . bmi was a risk for anemia in a second measurement .

isolated left ventricular noncompaction ( lvnc ) is a congenital cardiomyopathy caused by a defect in endomyocardial morphogenesis.1 ) it is characterized by prominent trabeculation and deep intertrabecular recesses , resulting in thickened myocardium consisting of 2 layers - compacted and noncompacted myocardium.2 ) lvnc is occasionally combined with other congenital cardiac diseases3 - 6 ) and genetic cardiomyopathies.7 ) its clinical manifestations include heart failure , thromboembolism , ventricular tachyarrhythmia , and sudden death.8 ) the risk of embolic complications was mostly associated with impaired systolic function.9 ) there were only a few cases about coexisting left ventricular aneurysm.10 - 12 ) we report a rare case of lvnc associated with left ventricular ( lv ) aneurysm , presenting with recurrent embolism . a 40-year - old woman was admitted to our hospital for abdominal and lower back pain lasting for 10 days . in 1999 , she had an embolic stroke of the right middle cerebral artery territory ; however , after 2007 , she was lost to follow - up and discontinued all medications , including antiplatelet and anticoagulant therapy she had none of the classic cardiovascular risk factors such as diabetes or hypertension nor any family history of cardiomyopathy or heart failure . her chest radiography and laboratory data , including b - type natriuretic peptide and cardiac enzymes levels , were unremarkable . abdominal computed tomography , performed because of her abdominal pain , revealed left renal infarction . to evaluate for an embolic source , transthoracic and transesophageal echocardiography were performed . two - dimensional echocardiography showed prominent trabeculations at the interventricular septum and the lateral wall . color doppler imaging revealed deep intertrabecular recesses filled by blood flowing from the ventricular cavity ( fig . the ratio of maximal thickness of the noncompacted to compacted layers in the lateral wall was greater than 2 at end - systole . the 4-chamber and 2-chamber views defined the lvnc in the mid - septal and inferior walls and apical wall thinning ( fig . because wall motion of the apex was akinetic at end - systole and end - diastole , we confirmed the diagnosis as an apical aneurysm . the lv ejection fraction by modified simpson 's rule was 48% ( lv end - diastolic volume 89 ml and lv end - systolic volume 49 ml ) and the ratio of early diastolic mitral inflow velocity to early diastolic mitral septal annular velocity ( e / e ' ) was 7.62 . we performed coronary computed tomography angiography and cardiac magnetic resonance imaging ( mri ) to differentiate the aneurysm from post - infarction aneurysms . on computed tomography 4 ) . cardiac mri showed two - layered appearance of trabeculated and compacted myocardium , it revealed a thin compacted layer preserving contractility and an apical aneurysm with akinetic motion at end - diastole and end - systole ( fig . we therefore diagnosed her with left renal infarction caused by lvnc , coexisting with an lv aneurysm . lvnc is a rare congenital cardiomyopathy characterized by multiple prominent trabeculations with deep intertrabecular recesses.1 ) an arrest of compaction of the developing myocardium is strongly suggested as the probable mechanism of lvnc.1)9 ) recently , the american heart association classified lvnc as a primary genetic cardiomyopathy.13 ) in contrast , the european society of cardiology considers lvnc to be an " unclassified cardiomyopathy".14 ) multiple diagnostic criteria for lvnc have been proposed on the basis of echocardiography and cardiac mri findings . they are as follows : 1 ) thickened myocardium with a 2-layered structure consisting of a thin compacted epicardial layer [ c ] and a much thicker , noncompacted endocardial layer [ n ] or trabecular meshwork with deep endomyocardial spaces ( n / c ratio > 2.0 at end - systole ) ; 2 ) predominant location of the pathology in the mid - lateral , mid - inferior , and apical areas ; 3 ) color doppler evidence of deep intertrabecular recesses filled with blood from the lv cavity ; and 4 ) absence of coexisting cardiac abnormalities ( in isolated lvnc ) . there have been many reports of coexistent congenital cardiac disorders , including atrial septal defect , ventricular septal defect , pulmonary stenosis , anomalous pulmonary venous connection , ebstein 's anomaly , and a bicuspid aortic valve.3 - 6 ) however , only a few cases of lvnc with lv aneurysm have been reported.10 - 12 ) the mechanism of aneurysm is uncertain . sato et al.10 ) proposed impaired microcirculation of noncompacted and compacted layers as the mechanism of aneurysm formation in lvnc . however , in our patient , the epicardial coronary arteries appeared normal on coronary computed tomography angiography and neither perfusion defects nor delayed enhancement were seen on cardiac mri . we therefore thought that our patient 's aneurysm might be congenital rather than degenerative change of lvnc . the classical triad of complications with lvnc is heart failure , ventricular arrhythmia , and systemic embolic events.8 ) because there are limited data regarding treatment of this condition , it is recommended that clinical complications be managed according to the current guidelines for each clinical complication . the prevalence of systemic embolic events in patients with lvnc varied in reports . based on the high rate of embolic events reported in long - term follow - up data , oechslin et al.8 ) recommended anticoagulant therapy for these patients , independent of ventricular systolic function . however , oechslin and jenni9 ) recently recommended anticoagulation therapy for patients with impaired systolic function ( lv ejection fraction < 40% ) because deep intertrabecular recesses and slow blood flow might increase the risk of thrombus formation . we believed that the apical thrombus was the embolic source of her presentation with renal infarction and that the apical aneurysm with slow blood flow was a risk factor for recurrent embolic events . therefore , we suggest that anticoagulation therapy might be needed in patients with lvnc with coexisting lv aneurysm , even in the absence of systolic dysfunction or atrial fibrillation . in conclusion , we described a rare case of lvnc with lv aneurysm presenting as recurrent thromboembolic events . we believe that careful evaluation of lvnc patients for coexisting heart abnormalities such as aneurysms is essential for making the best clinical decisions for their management .

isolated left ventricular noncompaction ( lvnc ) is a rare disorder caused by embryonic arrest of compaction . lvnc is sometimes associated with other congenital cardiac disorders ; however , there have been few reports of its coexistence with a left ventricular aneurysm . a 40-year - old woman was admitted to our hospital for renal infarction . she had a history of embolic cerebral infarction 10 years ago . transthoracic echocardiography showed prominent trabeculae and deep intertrabecular recesses which are filled with blood from the left ventricular ( lv ) cavity . a thrombus in the akinetic apical wall was confirmed by contrast echocardiography . using cardiac computed tomography and magnetic resonance imaging , we rejected a possible diagnosis of suspicion of coronary artery disease . she was diagnosed lvnc with a thrombus in apical aneurysm . here , we report the first patient in korea known to have lvnc accompanying lv congenital aneurysm presenting with recurrent embolism .

the reverse shoulder arthroplasty was approved by the fda in 2004 and has proven a very effective prosthesis for treatment of cuff tear arthropathy and other shoulder conditions requiring arthroplasty in the setting of a deficient rotator cuff . however , in some series , the overall revision rate for the reverse shoulder is approximately 10% , with instability and infection being the most common precipitating causes.[13 ] studies have also shown that aseptic loosening may contribute to 7% of failures , but as indications expand and long - term follow - up increases , the incidence of aseptic loosening will likely increase . have further shown that pain increases with longer term follow - up of reverse replacements ( 510 years ) . therefore , in the future , surgeons will be faced with difficult choices when revising the reverse prosthesis , and optimal management of the failed reverse replacement is not known . when instability or loosening of a reverse prosthesis does occur , retention of the reverse prosthesis can be performed by ( 1 ) increasing deltoid tension with additional polyethylene or metallic spacers , ( 2 ) increasing constraint with a retentive cup or ( 3 ) revision of the humeral or glenoid components.[514 ] however , due to poor bone stock , infection or refractory instability , removal of a reverse replacement ( explant ) or conversion to hemiarthoplasty can be required . we present a series of failed reverse replacements that were converted back to hemiarthroplasty with either a metallic prosthesis or preformed antibiotic shoulder spacer for infection , instability or glenosphere loosening . to date , there are few reports of reverse replacement conversion to hemiarthroplasty , and none specifically examining the shoulder function in this unique group . we examine a group of patients after conversion of failed or infected reverse shoulder replacement to hemiarthroplasty ; the level of function and pain in this group is important for surgeons and patients considering a revision reverse shoulder replacement . from 2008 to 2010 , the senior author performed 115 primary and revision shoulder arthroplasty procedures . as part of the shoulder registry ( irb#10 - 000859 ) , seven patients were identified that had presented to the senior author 's clinic with a reverse replacement that was dislocated ( 3 ) , infected ( 4 ) or loose ( 3 ) . all of the failed reverse replacements in this study were referred from outside hospitals for tertiary care ; revision surgery was performed an average of 9.2 months after implantation of the reverse prosthesis . patients with known infection were treated with explant of the reverse prosthesis using a slot osteotomy , cement extraction with an oscar device ( orthosonics , chatham , nj , usa ) and conversion mean size of the defect from 273 ( n = 3 ) ( exactech , interspace , gainesville , fl , usa ) . this antibiotic spacer was cemented in place with additional antibiotic - loaded cement ( tobramycin and vancomycin ) . in three cases of gross loosening of the glenosphere these patients were treated with removal of glenosphere only , bone grafting of glenoid with allograft including cancellous bone chips and demineralized bone matrix , and retention of the humeral stem by converting it via the manufacturer 's guidelines to a hemiarthroplasty ( n = 4 ) . at the time of reverse removal in this elderly population , the bone stock of the glenoid was cavitary and uncontained , not deemed of sufficient quality to support immediate replacement of a single - stage revision glenosphere . preoperative computated tomography ( ct ) was also used in four patients to evaluate the glenoid bone stock , but was not clinically very useful due to metal artifact . this group of seven patients status post removal of reverse replacement were contacted for reevaluation with physical exam and outcome scores specifically for this study . one patient who had been converted from a dislocated , infected reverse replacement to preformed antibiotic spacer chose not to participate , but has not had any further surgery . the case details of the remaining six patients who consented for examination and inclusion in this study are contained in table 1 . details of six cases of failed reverse shoulder arthroplasty converted to hemiarthroplasty using either a metallic head and retention of the reverse stem or an explant and implantation of a preformed antibiotic - loaded cement hemiarthroplasty there were three males and three females ( average age 74.5 years ) who had had an average of three previous shoulder surgeries including the index reverse replacement . the reverse replacement had been implanted an average of 9.2 months before failure of the prosthesis required removal . radiographic evidence of technical problems with the index reverse arthroplasty surgery was present in five of six cases , with superior implantation of the glenosphere ( n = 4 ) being the most common . average follow - up after removal of reverse replacement was 26.5 months ( range 1041 months ) . the decision to include patients with less than 2-year follow - up was made to maximize inclusion of patients that have been offered conversion back to reverse replacement . shoulder function was very poor postoperatively ( average forward elevation of 42.5 degrees and average external rotation near zero ) , but the pain level was generally low . sf-12 scores were similar to us averages in the 75 years - plus age group . anterosuperior escape was present in five patients ( vas instability score averaged 4.7 [ range 08 ] ) . in the sixth patient , there was static radiographic anterior subluxation ( case 1 ) . in the four cases where bone graft was used for glenoid deficiency , radiographic follow - up showed good incorporation of graft and reimplantation with a reverse prosthesis seemed feasible , although none of the patients elected to have this performed . a peripherally inserted central catheter ( picc ) line was inserted and culture - specific intravenous antibiotics were administered for 6 weeks postoperatively . there were no reoperations and , to date , no patient has accepted an offer to be converted back to a reverse replacement . a 58-year - old male ( case 2 ) presented 6 months after reverse arthroplasty for failed fracture fixation with anterior dislocation of the prosthesis . notice the superior placement of the glenosphere with slight superior tilt . at the time of revision surgery , no combination of glenosphere or reverse humeral component could result in stability as the prosthesis levered out with adduction past 40 degrees . removal of a well - fixed glenosphere and baseplate was performed and glenoid bone stock was not sufficient for immediate revision of the baseplate to an improved position . ( c and d ) conversion to large head hemiarthroplasty and bone grafting of glenoid defect . cultures were positive for p. acnes at the time of revision a 74-year - old female ( case 4 ) underwent reverse replacement for severe cuff tear arthropathy ( a ) . the glenosphere loosened at 8 months postoperatively with breakage of the inferior screw ( c ) . ( d and e ) ap and axillary views after conversion to hemiarthroplasty an 80-year - old man ( case 5 ) presented with a draining sinus and a dislocated , infected reverse replacement 6 months after primary reverse replacement at an outside institution that was complicated with reoperation the same day after a recovery room dislocation . glenosphere position is acceptable , but the entire humeral head was essentially resected , likely compromising stability . ( c and d ) postoperative radiographs of preformed antibiotic spacer preoperative draining sinus an intraoperative photograph after irrigation and debridement , removal of glenosphere , slot osteotomy and cementation of preformed antibiotic spacer ( exactech interspace , gainesville , fl , usa ) with additional antibiotic - loaded cement . notice how the humeral head is devoid of any soft tissue attachments creating a shoulder with profound anterosuperior escape in farshad and gerber 's review of reverse arthroplasty complications , they state that the optimal management for a failed reverse arthroplasty is not known and that complications that require removal of the prosthesis result in poor shoulder function . once a severe complication in reverse arthroplasty is established , retention of a stable reverse prosthesis is difficult and multiple reoperations are common . gallo et al . reviewed a series of nine cases of reverse replacement requiring revision for instability in the setting of subscapularis deficiency . in these nine patients , three eventually required explant , three remained chronically dislocated and three were concentrically intact . in a retrospective review of describe their experience with dislocation and infection of reverse prosthesis . in a series of 284 reverse replacements , the rate of instability was similar in patients with primary ( 11 of 212 [ 5% ] ) and revision ( six of 72 [ 8% ] ) reverse arthroplasty . the rate of infection was higher in the revision ( five of 72 [ 7% ] ) than in the primary ( three of 212 [ 1% ] ) group . in this series , the authors reported that a stable noninfected prosthesis was present in only 12 of 25 shoulders revised for infection or instability , and multiple reoperations for both persistent infection and recurrent instability were common in this series . in 2008 , norris presented a series of 26 revision reverse replacements for various causes . not all series of revision reverse arthroplasty have reported such a high rate of rerevision and reoperation . if retention of the reverse shoulder prosthesis in the setting of infection , dislocation or loosening is to be performed , the reoperation rate could be as high as 50% , and there is a potential for multiple reoperations and eventual resection arthroplasty . because of poor bone stock , persistent infection or persistent instability , conversion back to reverse replacement is not always possible in the immediate revision setting . in addition , due to the very elderly nature of this population and the often presence of severe medical comorbidities , a surgical strategy that could result in multiple reoperations can be unwise . for this reason , conversion of the reverse replacement to metallic or cement hemiarthroplasty is an attractive option , especially in the multiply operated patient unwilling to harbor further surgical risk . there seems to be less risk of reoperation using this surgical strategy , and the option remains to revise back to reverse replacement at a later date . however , successful single - stage treatment of infected and loose reverse replacements have been reported in the literature . grammont revised one patient who was revised on postoperative day 1 due to an intraoperative glenoid fracture , and this patient maintained only 40 of active elevation and had persistent shoulder pain . frankle reported on two patients who were converted to a hemiarthroplasty because of insufficient bone stock and a deep infection ; these patients rated the outcome of their revision as good and satisfactory . in another large series of reverse replacements , steinmann reported on a subset of four patients who were converted to a hemiarthroplasty as a result of loosening of the glenoid component in three and recurrent instability in the other . three of these patients had moderate pain and one had severe persistent pain , and patient satisfaction was low . this case series can be criticized for the small number of patients included and only very short - term follow - up . while the short - term follow - up may be used to predict future shoulder function , pain has been shown to increase with time . therefore , it is possible that shoulder pain after hemiarthroplasty will become more significant over time , compelling patients to seek further treatment such as reimplantation of a reverse prosthesis . despite these limitations , the literature is very sparse on what comes next ? after reverse replacement . we felt that this is the optimal time to report this series as it provides a snapshot of patients that are now candidates for reimplantation of reverse replacement to restore shoulder function . in addition , we feel that this series does provide the shoulder arthroplasty surgeon with several pieces of useful information : ( 1 ) in five of six patients , technical error was evident on postoperative radiographs with superior placement of the glenosphere often with superior tilt accounting for three cases of glenosphere loosening and one case of dislocation . these technical errors likely resulted in the average failure of the index reverse replacement at 9.2 months . . the reoperation rate of this series will certainly increase if patients choose to have conversion back to reverse replacement . ( 3 ) pain level was acceptable in most patients after conversion of reverse to hemiarthroplasty , with an average vas pain score of 2.4 . ( 4 ) shoulder function was poor , with anterosuperior escape present in five of six patients . this escape is likely accentuated by reversion to a state where the deltoid is again detensioned . failure of reverse replacement will increase in the future , with expansion of indications to younger patients and longer term follow - up . the optimal treatment for a failed reverse replacement is unknown and often very unique to each individual patient and each mode of failure . when counseling a patient with an infected , dislocated or loose reverse replacement , the patient should understand that attempts at retention and salvage of the reverse configuration of their shoulder is not always successful and can be associated with a high rate of reoperation . this small series of patients shows that safe removal of a reverse replacement and conversion to hemicement spacer or hemiarthroplasty can provide the shoulder with a relatively low pain level , but that the shoulder will be nearly devoid of meaningful function postoperatively . every effort should be made to meticulously assure proper initial implant placement and avoid infection in primary reverse replacement . in addition , when complications do occur , retention of the reverse replacement is necessary to maintain shoulder function as conversion to hemiarthroplasty results in poor function . the failed reverse replacement can result in extraordinarily difficult salvage situations and therefore we echo the sentiment published by rockwood that the reverse replacement should be implanted most frequently by shoulder arthroplasty specialists prepared to treat the severe complications encountered too frequently by even the world 's experts . removal of the reverse shoulder replacement and conversion to hemiarthroplasty results in a shoulder with minimal meaningful function but with an acceptably low level of pain . this information can be helpful when discussing preoperatively the relative risk of revision reverse arthroplasty versus conversion to hemiarthroplasty in patients with a failed reverse replacement .

background : the reverse total shoulder replacement has become a popular treatment option for cuff tear arthropathy and other shoulder conditions requiring arthroplasty in the setting of a deficient rotator cuff . despite a revision rate of as much as 10% , to date , there are few reports of reverse replacement conversion to hemiarthroplasty , and none specifically examining shoulder function.materials and methods : six patients with a reverse replacement that was dislocated , infected or loose were revised an average of 9.2 months after the reverse replacement . two of the three patients that were dislocated also had a known deep infection . patients with known infection were treated with explant of the reverse prosthesis and conversion to a preformed antibiotic spacer hemiarthroplasty . in three cases with gross loosening of the glenosphere without infection , treatment was performed with removal of glenosphere only , bone grafting of glenoid with allograft and conversion of humeral stem to hemiarthroplasty . patients were evaluated with outcome scores and physical examination an average of 26.5 months after removal of the reverse prosthesis.results:the average range of motion postoperatively was forward elevation 42.5 degrees and external rotation 1.7 degrees . the vas pain score was 2.42 ( range 06 ) ; simple shoulder test was 3.17 ( range 15 ) ; and ases score was 52.1 8.5 . there were no reoperations to date , and five patients had anterosuperior escape.conclusions:safe removal of a reverse replacement and conversion to hemicement spacer or hemiarthroplasty can provide pain relief in those patients with a dislocated or infected reverse replacement . however , the shoulder will likely have very poor function and anterosuperior escape postoperatively . further studies are needed to determine the optimal treatment for the failed reverse shoulder replacement.level of evidence : therapeutic level iv .

we applied the blastp program ( 5 ) to identify , for each predicted protein sequence in the h37rv genome ( genbank accession no . ae000516 ) . following genbank annotations , we compared 3,972 predicted proteins in h37rv with 4,187 predicted proteins in cdc1551 . we used a strict search criterion ( e = 10 - 50 ) to identify truly orthologous gene pairs . we aligned ( 6 ) the putative orthologous pairs of amino acid sequences ( n = 3,428 ) , then imposed this alignment on the dna sequences . visual inspection of amino acid alignments showed that certain alignments , usually near the n - terminus or c - terminus , had regions of very low sequence identity . examination of the dna sequences of the corresponding genes showed that these regions of low identity were typically caused by a frameshift in one of the two genomes relative to the other . whether these frameshifts are biologically real or result from sequencing error was uncertain ; therefore , we eliminated 119 such gene pairs from our data set . for the remaining gene pairs ( n = 3,309 ) , we computed the proportion of synonymous substitutions per synonymous site ( ps ) and the proportion of nonsynonymous substitutions per site ( pn ) by using nei and gojobori s method ( 7 ) . because values of ps and pn were very low in most cases , we did not correct for multiple hits . because ps values appeared to fall into two groups ( see results ) , we used a simple probabilistic model to separate these two sets of gene pairs . we assumed that the probability of synonymous substitution followed two separate binomial distributions , designated models a and b , with probabilities of success ( i.e. , of a synonymous difference ) designated pa and pb , respectively . using the bayes equation , for each gene pair with a given ps value , we computed the probability that model a applies , given the observed ps : p(a|ps ) = ( psa ) fa /[(psa ) fa + ( psb ) fb ] , where fa is the frequency of cases to which model a applies , fb the frequency of cases to which model b applies , psa is the binomial probability of obtaining the observed ps , given the number of synonymous sites in the gene and a probability of a synonymous difference equal to pa ; and psb is the binomial probability of obtaining the observed ps , given the number of synonymous sites in the gene and a probability of a synonymous difference equal to pb . of 3,309 pairs of putatively homologous protein - coding genes in the h37rv and cdc1551 genomes of m. tuberculosis , 2,662 ( 80.5% ) showed no synonymous or nonsynonymous nucleotide differences between the two genomes , and 3,010 ( 91.0% ) showed no synonymous differences between the two genomes . however , in a small number of gene pairs , the proportion of synonymous differences per synonymous site ( ps ) was surprisingly high . in 13 ( 0.4% ) gene pairs , ps was > 0.01 , and in 3 gene pairs ps exceeded 4% . these extreme ps values seen in a small number of gene pairs are much higher than generally observed between alleles at neutrally evolving loci in eukaryotes ( 8) . thus , the comparison of protein - coding genes between the two m. tuberculosis genomes suggested the existence of two distinct groups of gene pairs : a large group having few or no synonymous differences and a much smaller group with a substantial degree of synonymous divergence . we used a simple probabilistic model ( see methods ) to separate these two sets of gene pairs , designated group a and group b , respectively ( figure ) . the application of this method showed 11 loci with unusually high ps values and probabilities of assignment to group a of < 50% ( figure ) . a plot of p(a|ps ) , the probability of assignment of a locus to group a given the observed ps value , as a function of ps at 3,309 loci compared between the h37rv and cdc1551 genotypes of mycobacterium tuberculosis . the plot shows the bimodal nature of the distribution of ps values , with overall higher values of ps at the 11 loci having p(a|ps ) > 50% . we assumed that group a members are truly orthologous gene pairs that diverged at the time of the common ancestor of the h37rv and cdc1551 genomes . group a included 3,298 pairs , with mean ps for all genes of 0.000328 0.000022 standard error . when ps was estimated for the 3,298 genes concatenated together ( a total of 934,413 synonymous sites ) , an estimate of ps = 0.000348 0.00019 was obtained . the range of ps values in group a was between zero and 0.012 ; a total of 288 loci in group a had ps values other than zero . these results show a substantial level of nucleotide diversity , approximately half the level of nucleotide diversity in humans ( 9 ) . rates of nucleotide substitution per unit time are difficult to estimate in bacteria given the lack of calibration from the fossil record ( 10 ) . to obtain an estimate of the rate of synonymous nucleotide substitution , we used published data on comparisons of escherichia coli and salmonella typhimurium ( 11,12 ) , which are believed to have diverged approximately 100 million years ago ( 13,14 ) ( table 1 ) . this procedure yielded estimates for the last common ancestor of h37rv and cdc1551 in the range of 34,00038,000 years ( table 1 ) . these estimates are approximately twice previous estimates of the age of the common ancestor of worldwide m. tuberculosis ( 2,3 ) . to obtain the observed mean ps value between h37rv and cdc1551 within 15,00020,000 years would require a rate of synonymous substitution approximately twice that observed in enterobacteria . based on synonymous substitutions between escherichia coli and salmonella typhimurium , assumed to have diverged 100 million years ago ( 13,14 ) . estimates are shown standard error , based on standard error of mean ps . group b consisted of 11 gene pairs with mean ps of 0.0286 0.0050 ( table 2 ) . paired sample t - test of the hypothesis that ps = pn , p<0.01 . the quantities ps and pn are the proportion of nucleotide difference per synonymous site and per nonsynonymous site , respectively . in enterobacteria , a negative correlation exists between observed proportions of synonymous difference and codon bias ( 11 ) . in the case of mycobacterium , codon bias results mainly from the very high third position g+c content of most genes ( 15 ) . in our data , however , we observed no correlation between ps and proportion g+c at third codon positions ( r = -0.010 ; not significant ) . a number of additional possibilities may explain the occurrence of gene pairs with higher than expected ps values : 1 ) balanced polymorphism . selectively maintained polymorphisms are expected to be much older than neutral polymorphisms and may even predate speciation events ( 16 ) . in the case of haploid organisms such as bacteria , balancing selection would take the form of frequency - dependent selection rather than overdominant selection . 2 ) differential deletion . in a multi - gene family , if one member of an orthologous pair of genes were deleted in one genotype , the gene pairs would involve paralogous , not orthologous comparisons . 3 ) horizontal gene transfer . a gene obtained by one of the two genotypes from another bacterial species one indication of a balanced polymorphism is a higher rate of nonsynonymous than synonymous substitution ( 8) . there was no strong evidence of such selection in the present case ; ps was greater than pn at 10 of the 11 loci , and pn exceeded ps only slightly at one locus ( table 2 ) . in addition , we compared ps and pn in sliding windows of 30 codons along the length of these genes . no regions were observed in which pn was greater than ps ( data not shown ) . thus , there was no evidence of positive selection acting on specific regions of these genes . on the other hand , differential deletion can probably explain some cases , most notably members of the pe multi - gene family ( 11 ) ( table 2 ) . the remaining gene pairs are possibly cases of horizontal gene transfer ( table 2 ) , for which there is some recent evidence in m. tuberculosis ( 17 ) . our results did not support the hypothesis that the common ancestor of m. tuberculosis was relatively recent ( 2 ) . rather , the pattern of nucleotide substitution at synonymous sites suggested a divergence time for the two available genotypes of this species approximately 35,000 years ago . since h37rv and cdc1551 represent two genotypes sampled from within the species , they are probably not the most divergent genotypes possible . thus , the last common ancestor of m. tuberculosis likely occurred considerably earlier than 35,000 years ago . while the difference between an estimate of 15,00020,000 years and one of 35,000 years is not large on an evolutionary time scale , such a difference is substantial on the scale of human history . for example , the existence of two genotypes in the current population of m. tuberculosis with a common ancestor at 35,000 years is evidence against the hypothesis that m. tuberculosis arose , presumably from m. bovis , at the time of human domestication of cattle ( 18 ) . our result is thus consistent with phylogenetic analyses based on insertion - deletion events , which suggest that the m. tuberculosis lineage was a human pathogen well before the origin of m. bovis ( 19 ) . thus , along with recent evidence of an ancient origin and extensive polymorphism in the malaria parasite plasmodium falciparum ( 20,21 ) , our study provides evidence against the long - held view that virulent pathogens are invariably evolutionarily recent ( 22 ) . our estimate is conservative because the rate of synonymous substitution may actually be lower in mycobacterium than in enterobacteriaceae , given the highly skewed g+c content in the former . furthermore , our estimate of the mean proportion of synonymous difference was conservative because we excluded 119 loci with potential frameshifts between the two genotypes as well as a set of 12 loci with unusually high ps values . in addition to the 12 loci assigned to our group b , certain other loci might also have originated from horizontal gene transfer . however , even if horizontal gene transfer has occurred at other loci besides those in group b , eliminating further loci with relatively high ps values from group a will not affect the results greatly . for example , if we eliminate the 10 loci with highest ps values from group a , mean ps will be reduced only to 0.000299 , and the estimated age of the common ancestor will be barely affected . the degree of polymorphism observed in this study is unlikely to have been substantially influenced by sequencing errors . the error rate for finished sequences from the institute for genomic research ( where cdc1551 was sequenced ) has been independently estimated at < 1 in 88,000 bases ( 23 ) . assuming a similar error rate for both 4.4 mega - bp m. tuberculosis genomes , we would expect to see approximately 100 differences between them due to sequencing errors . approximately 21 such differences would be expected in the 938,778 synonymous sites in group a and group b genes . in fact , 411 synonymous differences were observed at these sites ; thus , even if present , sequencing errors are likely to have made up only a small fraction ( approximately 5% ) of the total synonymous polymorphism . at such a rate , sequencing errors would have little effect on our estimates of nucleotide diversity at synonymous sites or the age of the common ancestor of the two genomes . in addition , the hypothesis that the single nucleotide polymorphisms ( snps ) observed between these genotypes are real received strong support from a recent study that observed a number of the same snps in clinical isolates ( 24 ) . moreover , since sequencing errors are expected to occur at random with respect to the reading frame of coding sequences , the fact that mean ps exceeded mean pn in both group a and group b was strong evidence against the hypothesis that a substantial proportion of the observed polymorphism was due to sequencing error . simple considerations of probability can explain why earlier studies produced relatively low estimates of this species age . if we assume that the per - site probability of a synonymous difference between two m. tuberculosis genomes is equal to the mean ps observed between h37rv and cdc1551 ( 0.000328 ) , then the probability is approximately 95% that no synonymous differences will be seen in a gene with 150 synonymous sites . the probability that no synonymous differences will be seen in 10 such loci chosen at random is approximately 60% , and the probability that no synonymous differences will be observed at 20 such loci is approximately 37% . on the other hand , the probability that no synonymous differences will be seen at 100 these calculations emphasize the need to examine a very large number of nucleotide sites to obtain a reliable estimate of nucleotide diversity and thus of the age of the most recent common ancestor in cases where the frequency of substitution is less than one in a thousand . even when the frequency of substitution is between one in a thousand and one in a hundred , substantial stochastic error is possible if the number of loci examined is small . thus , any study that estimates population parameters from nucleotide sequence data needs to survey a substantial number of loci . these considerations are particularly important in the case of pathogenic microorganisms , where a number of factors ( including both natural selection and horizontal gene transfer ) may lead to substantial differences among loci with respect to the level of nucleotide diversity . comparison of two complete genomes of m. tuberculosis showed a greater extent of sequence polymorphism than would be expected on the basis of previous studies , in turn suggesting that analysis of additional genomes will likely show further polymorphism . polymorphism in any species of pathogen may complicate therapeutic strategies because it implies the existence of variation on which selection can act , including selection imposed by human vaccines and pharmacologic agents ( 20 ) . on the other hand , known polymorphisms may prove useful to investigators in reconstructing the evolutionary relationships among clinical isolates and in providing markers for understanding the genetic basis of complex phenotypic traits .

comparison of the pattern of synonymous nucleotide substitution between two complete genomes of mycobacterium tuberculosis at 3,298 putatively orthologous loci showed a mean percent difference per synonymous site of 0.000328 0.000022 . although 80.5% of loci showed no synonymous or nonsynonymous nucleotide differences , the level of polymorphism observed at other loci was greater than suggested by previous studies of a small number of loci . this level of nucleotide difference leads to the conservative estimate that the common ancestor of these two genotypes occurred approximately 35,000 ago , which is twice as high as some recent estimates of the time of origin of this species . our results suggest that a large number of loci should be examined for an accurate assessment of the level of nucleotide diversity in natural populations of pathogenic microorganisms .

diabetic peripheral neuropathy ( dpn ) is a late - stage microvascular complication that develops in nearly 50% patients during the course of type 2 diabetes mellitus ( t2 dm ) , affecting particularly low extremities . dpn is characterized by irreversible nerve structural and functional changes due to demyelination , axonal atrophy , and diminished regenerative potential , clinically presenting as a symmetric chronic pain , paraesthesia , and sensory loss . the development of dpn often worsens the quality of life , and increases the risk of cardiovascular morbidity , foot ulcerations , amputations , and overall mortality . although exact mechanisms are not fully understood , dpn is generally regarded as the consequence of chronic hyperglycemia - induced endothelial dysfunction , resulting in impaired endoneurial blood flow , ischemia , and nerve hypoxia . metabolic - vascular interactions in dpn are however highly complex and include diverse molecular pathways , like activation of protein kinase c and polyol metabolism , glycation and glycoxidation , low - grade inflammation , and excessive formation of reactive oxygen species ( ros ) , such as superoxide and hydrogen peroxide . in vascular compartment these ros can react with intrinsic vasodilator nitric oxide , and while the fall of nitric oxide levels would cause vasoconstriction , the resulting peroxynitrite could trigger the downstream events leading to nerve damage . because normal oxygen supply is fundamental for all tissues , impaired vasodilation is often associated with cellular energy crisis and increased breakdown of purine nucleotide to uric acid ( ua ) via xanthine oxidoreductase . under physiological conditions this enzyme functions as dehydrogenase , but during hypoxia or after limited proteolysis it is converted to oxidase form ( xod ) . unlike dehydrogenase , xod more readily generates ros able to impair vascular relaxation . in diabetic patients , hyperglycemia - induced endothelial dysfunction is chronic and not limited solely to the blood vessels irrigating nerve tissue but to variable extent occurs throughout the body . moreover , xod has been previously linked to oxidative damage in diabetes [ 6 , 7 ] and diabetic cataract , as well as metabolic syndrome and its cardiovascular complications , and the role of xod was also proposed in an experimental model of diabetic neuropathy . as a molecular fingerprint of imminent hypoxia , serum ua was reported higher in t2 dm than in nondiabetic subjects , especially in those with dpn . during hypoxia , circulating serum albumin can undergo conformational changes at n - terminus aspartyl - alanyl - hystidyl - lysine sequence , which decrease its natural ability to bind cobalt and several other transition metal ions . the resulting ischemia - modified albumin ( i m a ) was originally associated with hypoxic conditions during myocardial ischemia but was thereafter reported also in t2 dm and some diabetic complications . however , there are missing data regarding serum xod activity or i m a in dpn . currently , there is no effective therapy to cure dpn , and even with good glycemic control the chance to develop dpn is relatively high among t2 dm patients . given that xod could be a significant source of ros in vascular compartment causing endothelial dysfunction , and a possible therapeutic target , this study was aimed at investigating the relationship between xod and dpn in t2 dm patients . this study enrolled 80 patients previously diagnosed with t2 dm , both sexes , who attended our local clinical hospital center on regular basis for diabetes control . not included were patients with significant motor deficits , recent cardiovascular or cerebrovascular events , overt renal or hepatic diseases , and retinopathy and those with foot ulcerations , amputations , and recent inflammatory disease or with known autoimmune or malignant disease . the control group was consisted of 30 age- and sex - matched subjects without dm , recruited from medical stuff and their relatives , who met the same exclusion criteria . this study was conducted in accordance with the declaration of helsinki , after informed consent from patients was provided . the institutional review board of the medical faculty pristina ( kosovska mitrovica ) has approved this study . dpn was defined as symmetrical sensorimotor polyneuropathy , diagnosed using a simplified scoring system for bedside examination , the diabetic neuropathy symptom ( dns ) score , followed by michigan neuropathy screening instrument ( mnsi ) scoring system . the mnsi consists of two parts : the first part is a 15-item yes or no questionnaire about history of sensory and motor dysfunction ( pain , temperature sensitivity , tingling , numbness , cramps , muscle weakness , feet ulcers or cracks , and amputation ) . the second part of mnsi encompasses examination of foot skin appearance , inspection for foot ulcers , examination of ankle reflexes , vibration perception testing using a 128-hz tuning fork test at the great toe , and fine touch sensation testing using a 10 g semmes - weinstein monofilament applied on the plantar sites of each foot . hypertension was defined as having systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg or being on antihypertensive medication . microalbuminuria , as a marker of incipient kidney damage , was defined as urinary albumin - to - creatinine ratio of 30300 mg / g . body mass index ( bmi ) was calculated as the ratio of body weight ( kg ) and square of body height ( m ) . venous blood was taken after an overnight fasting into vacutainer tubes without or with anticoagulant ( edta ) to obtain serum , plasma , or whole blood samples . the first morning urine sample taken into sterile urine containers during two months in three nonconsecutive days was provided for determination of urinary albumin and creatinine . serum xod activity was measured according to the method of roussos , as described earlier . xod activity was calculated after correction for preexisting uric acid , using molar absorbance of uric acid at = 293 nm , of = 1.26 10 l m cm . one unit of xod activity was defined as 1 mol / min uric acid formed at 37c . serum i m a was measured using a colorimetric method described by bar - or et al . . concentration of total serum thiols , as an indicator of protein oxidative damage , was measured using ellman 's reagent . concentration of serum age was determined spectrofluorometrically by the method of kalousov et al . and expressed as relative fluorescence units ( rfu ) . concentration of advanced oxidation protein products ( aopp ) , as marker of chronic oxidative albumin damage , was determined from lipid - depleted plasma samples to avoid interference with lipid status , by the method of anderstam et al . . serum xod , age , and aopp were assessed from aliquoted samples ( 0.5 ml ) kept at 80c . concentrations of serum and urinary creatinine , serum total proteins , albumin , triglycerides , total cholesterol , hdl - cholesterol , ua , and blood glycated hemoglobin ( hba1c ) were measured on cobas integra 400 biochemical analyzer , using standard protocols . urinary albumin was determined using tinaquant albumin gen.2 assay kit ( roche diagnostics gmbh , mannheim , germany ) . the average of urinary albumin excretion was calculated for each patient from results of three nonconsecutive urine samples . atherogenic index of plasma ( aip ) , as a surrogate marker of atherogenic dyslipidemia , was calculated according to dobiasova , as the logarithm of triglycerides to hdl - cholesterol concentration ratio . data were presented as either arithmetic mean sd , frequencies ( f ) , or median and 95% confidence interval of the median . differences between groups were tested by anova and student 's independent samples t - test , or chi - square test , where appropriate . the relationship between dpn , xod , and other risk factors was investigated by multivariable logistic regression analysis . basic demographical , clinical , and biochemical findings in controls and t2 dm patients are presented in table 1 . of 80 t2 dm patients enrolled in the study , 29 were with dpn ; the remaining ( n = 51 ) comprised the non - dpn group . there were no significant differences between control and groups with dm , in terms of age and sex distribution , smoking status , presence of hypertension , serum creatinine , and total protein levels ( table 1 ) . twenty - five t2 dm patients were with microalbuminuria , having urinary albumin to creatinine ratio within the range of 30300 mg / g . patients with t2 dm had significantly higher bmi than control subjects and were rather overweight ( n = 31 ) or obese ( n = 26 ) . concentrations of fasting blood glucose , total cholesterol , hdl - cholesterol , ldl - cholesterol , triglycerides , and aip significantly differed from controls in both groups with t2 dm ( table 1 ) . in comparison to non - dpn group , concentration of hdl - cholesterol was significantly lower in dpn group . in the overall sample the median xod activity was 16.4 u / l ( 95% ci 13.820.4 ) . serum xod activity and i m a concentration were higher in t2 dm patients than in control subjects and significantly differed between dpn and non - dpn groups ( table 2 ) . serum albumin and total thiols were lower in dm patients than in controls and differed between dpn and non - dpn groups . compared to controls , hba1c and serum age were increased in dm patients , being higher in dpn than in non - dpn group ; the occurrence of dpn also affected serum ua concentrations . serum aopp was higher in t2 dm patients than in controls , but the difference between diabetic groups was insignificant ( table 2 ) . in patients with t2 dm , serum xod activity was directly correlated to bmi , the presence of hypertension , and levels of hba1c , age , i m a , and ua and inversely correlated to serum albumin and total thiol groups concentrations ( table 3 ) . correlations between xod activity and age , duration of diabetes , aip , aopp , and microalbuminuria were not significant . we further analyzed the association between several clinical and biochemical variables and the occurrence of dpn . included were duration of diabetes , hba1c , aip , hypertension , serum xod , and ua levels . the multivariable logistic regression analysis ( b = 5.014 ; chi - square = 20.023 ; p = 0.0027 ) clearly revealed hba1c and serum xod activity as independent predictors of dpn , whereas the influence of diabetes duration was of the borderline significance ( table 4 ) . the major finding of the present study was significant elevation of serum xod activity in patients with t2 dm and an independent association between xod activity and the occurrence of dpn . serum xod activity was well correlated to the levels of i m a and some other biomarkers of increased ros formation . furthermore , xod activity was directly correlated to several risk factors relevant for the development of dpn , including hypertension , higher bmi , and concentrations of hba1c and ua , indicating the role of xod in the development of dpn . xod is a rate - limiting enzyme of purine catabolism to ua , during which high quantities of ros are produced . we observed that the formation of ros was specifically increased in dpn group , documented as decreased serum total free thiols and increased levels of oxidatively damaged molecules , like i m a and age , accompanied by higher concentration of serum ua ( table 2 ) . these findings are consistent with the concept that increased ros formation can contribute to the development of dpn [ 4 , 23 ] . serum total free thiol groups essentially originate from a single 34 cystein residue of albumin , which is abundantly present blood protein . acting as nonenzymatic antioxidant and reducing agent these free thiols are rapidly oxidized after exposure to ros , forming mixed disulfides and related sulfur - containing acids . in one previous study the loss of total serum thiols was found to correspond to the severity of diabetic microvascular complications and the development of dpn , and our current results support that finding . there was also a significant increase in serum i m a concentrations in t2 dm , especially in dpn group , in our study . several other studies reported elevated serum i m a levels in diabetes , correspondingly to the presence or severity of complications , including peripheral arterial disease . we observed that i m a concentrations were positively correlated to xod activity , indicating that oxidative damage of circulating biomolecules in dpn can be , at least partly , inflicted by xod - derived ros . although exact chemical mechanisms of i m a formation are still a matter of debate , it is generally accepted that i m a is formed due to oxidative damage of serum albumin n - terminus , specifically in hypoxic conditions , such as those created by dysfunctional blood vessels . in accordance to that , i m a is now regarded as a biomarker of widespread endothelial damage in a variety of pathological states , including t2 dm . however , in the absence of other biomarkers of endothelial damage these results should be cautiously interpreted . besides i m a , the occurrence of dpn was associated with higher serum ua and lower albumin levels , which are known risk factors of dpn . also , 31% of patients included in the study were microalbuminuric ; thus the coexistence of decreased ua elimination rate and increased urinary albumin excretion rate as confounding factors can not be excluded . still , increased serum levels of i m a and ua have been regarded as biomarkers widespread endothelial dysfunction in a variety of pathological states , including t2 dm [ 13 , 25 ] . tissues irrigated by dysfunctional vessels inevitably suffer hypoxic and even anoxic conditions , which promote the transition of xanthine dehydrogenase to xod form . within vascular compartment the enzyme is found mostly at xod form , attached to endothelial cell surface via sulfated glycosaminoglycan - rich receptors , and as a free enzyme in the blood . both bound and free xod can generate ros able to modify circulating blood and vascular wall constituents , such as albumin and endothelium - derived nitric oxide . the reaction of nitric oxide and superoxide anion radical diminishes nitric oxide levels and yields peroxynitrite , which can act as toxic mediator inducing nerve injury . at the same time , diminished availability of nitric oxide would exacerbate endothelial dysfunction , since it is implicated in regulation of vascular tone , as well as in prevention of platelet adhesion and aggregation and leukocytes adhesion to the vascular wall . moreover , augmented serum xod activity has been already linked to increased ros formation in metabolic syndrome and its cardiovascular complications , clinical and experimental diabetes , and diabetic ocular complications [ 69 ] . to the best of our knowledge the majority of vascular xod is believed to be of hepatic origin , nonspecifically released into the blood , particularly under hyperglycemic conditions . it was therefore not surprising that hba1c levels were directly correlated to xod activity in our and some previous studies [ 6 , 7 ] , thereby confirming that glycemic control plays critical role in modulating xod presence / activity within vascular compartment . besides hba1c , serum xod activity was also correlated to ages , which are chemically heterogeneous products of protein nonenzymatic glycation and subsequent oxidation reactions . ages not only are biomarkers of glycoxidative protein damage but also act as toxic mediators after binding to specific receptors , like multiligand rage or macrophage scavenger receptors . in peripheral nerves rage it is of note that age - rage interaction facilitated endoneurial vascular dysfunction in peripheral nerves , leading to microangiopathy . etiology of dpn is however highly complex , since diverse though interrelated metabolic lifestyles and genetic factors can be involved . for example , a prolonged ischemia and obesity can be both present in patients with t2 dm and associated with higher levels of serum ua . in fact , one recent study demonstrated that , beyond purine metabolism , xod also plays a role in differentiation of adipocytes acting as a regulator of the nuclear receptor peroxisome proliferator - activated receptor activity , which is the key factor controlling the induction and maintenance of adipogenesis . in the present study , serum xod activity was correlated to bmi as well as to ua levels ( table 3 ) . one probable explanation could be a high prevalence of overweight ( 38.7% ) and obese ( 23.5% ) diabetics , and since xod is abundantly expressed in adipocytes it might cause increased secretion of ua from adipose tissue , especially during ischemia . these results are consistent with those of feoli et al . , who reported a similar relationship in metabolic syndrome . it has been previously shown that ros formed in chronic hyperglycemia may induce unmyelinated and myelinated nerve fibers loss and impairment of endoneurial blood flow , resulting in nerve conduction velocity abnormalities . , macrophages can produce high quantities of ros via xod , thereby causing local endothelial dysfunction , further impairment of endoneurial blood flow , and myelin degeneration . overexpression of xod is actually necessary step for macrophages activation and regulation of proinflammatory mediators and chemokines secretion [ 31 , 32 ] . furthermore , ros generated by local macrophages via xod may induce nerve axonal loss and demyelination , as demonstrated in a murine model of neuroinflammation . because of high content of polyunsaturated fatty acids nervous tissue is exceptionally susceptible to ros ; thus tight control of prooxidant enzyme activity is of great importance . previously , inkster et al . reported that inhibition of xod with allopurinol , a structural analogue of hypoxanthine with both hypouricemic and anti - inflammatory effects , was able to prevent the loss of motor and sensory conduction velocity in an animal model of diabetes . their study demonstrated that inhibition of xod improved blood flow in sciatic and cervical nerve ganglion , thereby suggesting that upregulated xod present in nerve microvasculature , including endothelium and perivascular space , can cause neurovascular dysfunction leading to diabetic neuropathy . allopurinol also prevented cardiac ischemia and impaired relaxation in an experimental model of insulin resistance , normalized endothelial function in t2 dm patients with mild hypertension , alleviated oxidative injury and improved cardiovascular functions in diabetics in several intervention studies , and acted antinociceptive against various noxious stimuli in mice . on the other side , peripheral neuropathy can be a side effect of allopurinol treatment , and this drug failed to prevent the progression of dpn in type 1 diabetes mellitus patients with mild - to - moderate cardiovascular autonomic neuropathy . nonetheless , the current study has demonstrated that augmented xod activity was present within vascular compartment in t2 dm patients diagnosed with dpn . moreover , serum xod activity was closely correlated to risk factors relevant for the development of dpn , including poor glycemic control , obesity , and serum i m a and ua levels , which are considered as markers of generalized endothelial dysfunction in diabetics . our results therefore indicate that upregulated xod can be an additional source of ros in vascular compartment , implicated in chronic oxidative injury and endothelial dysfunction in t2 dm patients with poor glycemic control , contributing overtime to the development of dpn .

this study investigated the relationship between serum xanthine oxidase ( xod ) activity and the occurrence of diabetic peripheral neuropathy ( dpn ) in type 2 diabetes mellitus ( t2 dm ) patients . serum xod activity , ischemia - modified albumin ( i m a ) , uric acid ( ua ) , albumin , glycated hemoglobin ( hba1c ) , advanced glycation end products ( age ) , total free thiols , atherogenic index of plasma ( aip ) , and body mass index ( bmi ) were measured in 80 t2 dm patients ( 29 with and 51 without dpn ) , and 30 nondiabetic control subjects . duration of diabetes , hypertension , medication , and microalbuminuria was recorded . serum xod activities in controls , non - dpn , and dpn were 5.7 2.4 u / l , 20.3 8.6 u / l , and 27.5 10.6 u / l ( p < 0.01 ) , respectively . xod activity was directly correlated to i m a , ua , bmi , hba1c , and age , while inversely correlated to serum total free thiols . a multivariable logistic regression model , which included duration of diabetes , hypertension , aip , hba1c , ua , and xod activity , revealed hba1c [ or = 1.03 ( 1.001.05 ) ; p = 0.034 ] and xod activity [ or = 1.07 ( 1.001.14 ) ; p = 0.036 ] as independent predictors of dpn . serum xod activity was well correlated to several other risk factors . these results indicate the role of xod in the development of dpn among t2 dm patients .

during the course of sepsis pattern recognition receptors ( prrs ) of the innate immune system such as toll - like receptors ( tlrs ) have been shown to play a decisive role in the disease process . toll - like receptors bind pathogen associated molecular patterns ( pamps ) originating from non - self - sources as well as damage associated molecular patterns ( damps ) deriving from the host organism itself . in the human organism eleven tlrs are expressed , among those tlr4 the receptor for endotoxins has been described most extensively . however , in the last few years another tlr has entered the focus of scientific interest : the receptor for viral and bacterial dna tlr9 . bacterial dna is characterized by unmethylated cytosine - phosphate - guanine oligodesoxynucleotide ( cpg motifs ) , which are much less prevalent in mammalian dna . this receptor has been found to be of pivotal importance during polymicrobial sepsis [ 3 , 4 ] . the reason for its relevance may lie in the common appearance of cpg motifs in all bacterial and viral dna . therefore , tlr9 can be regarded as a central receptor for inflammatory response in any kind of sepsis . importantly , the presence of impaired cardiovascular function in sepsis is associated with significantly increased mortality [ 5 , 6 ] . hence , any successful intervention should aim to improve sepsis - dependent suppression of cardiovascular function . in vitro tlr9 signaling induced by the application of the synthetic stimulatory oligonucleotide 1668-thioate suppressed cardiac contractility via increased inos expression , which could be antagonized by s - methylisothiourea ( smt ) . in addition , myocytes with tlr9-deficiency ( tlr9-d ) proved to be insensitive to stimulation with a synthetic substitute for bacterial dna . thus , pharmacological antagonism of tlr9 might protect the cardiovascular system from the deleterious effects of bacterial dna . dilatation of blood vessels exposed to polymicrobial stimuli can be prevented by the synthetic odn ( h154-thioate ) inhibiting tlr9 . however , the in vivo effect of tlr9 antagonism on the heart has not yet been investigated . therefore , we wondered whether application of a synthetic oligodesoxynucleotide ( 1668-thioate ) would be sufficient to depress cardiac function in vivo . with respect to the importance of effective inhibition of tlr signaling in sepsis , the aim of the present study was to test different tlr9 inhibitors ( h154-thioate , irs954-thioate , and chloroquine ) for their effectiveness in protecting the cardiovascular system during systemic inflammation . in order to distinguish tlr9 signaling from other inflammatory pathways the specific tlr9 ligand 1668-thioate was chosen as stimulus . ten - to - twelve - week - old male c57bl/6 mice were purchased from charles river , sulzfeld / germany . the body weight varied from 20 to 25 g. tlr9-deficient mice ( tlr9-d ; backcrossed on c57bl/6 background ) were kindly provided by professor shizuo akira ( department of host defense , research institute for microbial diseases , osaka university , japan ) . all animal studies were approved by the state office for nature , environment and consumer affairs ( landesamt fr natur , umwelt und verbraucherschutz ) of north - rhine westphalia ( recklinghausen , germany ) and conformed to the guidelines for animal experimentation of the national institute of health ( nih publication no . buprenorphine was administered ( 0.1 mg / kg subcutaneously ) . in a pilot study cultured macrophages of the line raw 264.7 were stimulated with heat - inactivated feces of c57bl/6 mice for 24 h ( concentration 10 bacteria / ml ) . thereafter , tnf- protein was measured by elisa in the supernatant of the cell culture . stimulation led to a 30-fold increase of tnf- protein compared with control ( figures 1(a)1(c ) ) . this increase was used to test the suppressive effect of the tlr9 inhibitors h154-thioate , irs954-thioate , and chloroquine [ 810 ] . h154-thioate was applied simultaneously with the polymicrobial stimulus in three different concentrations ( 50 mg / l , 25 mg / l , and 0.5 mg / l ) . all applied concentrations of h154 were able to reduce the tnf- protein significantly in a concentration - dependent manner ( figure 1(a ) ) . comparable experiments were performed with irs954-thioate and chloroquine ( figures 1(b ) and 1(c ) ) . chloroquine was applied in four different concentrations ( 2.5 , 10 , 50 , and 100 mg / l ) ; the lowest effective concentration was 10 mg / l . in order to ensure efficaciousness of the antagonists a single dose of 8 mg / kg bw of h154- and irs954-thioate and 10 mg / kg of chloroquine was applied i.v . to the animals . all animals were initially treated with d - galactosaminen ( d - galn ; 1 g / kg bw , roth , karlsruhe , germany ) . d - galactosaminen was applied to slow down the hepatic degradation of cpg - odns [ 11 , 12 ] . in control experiments d - galn alone did not induce an inflammatory response . in order to induce a systemic inflammation thirty minutes later all mice were injected i.p . with 1668-thioate ( 5-tccatgacgttcctgatgct ; tibmolbiol , berlin , germany ; 2 nmol / g bw = 12.1 mg / kg bw ) in a total volume of 400 l pbs or with the same amount of pbs . another 30 minutes later the mice received intravenous ( i.v ) treatment with one of four agents : ( 1 ) a control odn ( 1612-thioate ; 5-gctagatgttagcgt-3 ; tibmolbiol , berlin , germany ; 2 nmol / g bw = 9.3 mg / kg bw ) , ( 2 ) with h154-thioate ( 5-cctcaagcttgagggg-3 ; tibmolbiol , berlin , germany ; 8 mg / kg bw ) , ( 3 ) with irs954-thioate ( 5-tgctcctggaggggttgt-3 ; tibmolbiol , berlin , germany ; 8 mg / kg bw ) , ( 4 ) with chloroquine ( 10 mg / kg bw ; bayer vital gmbh , leverkusen , germany ) . the mrna expression levels of tnf- , il-6 , and il-1 were determined using taqman real - time quantitative pcr ( rt - qpcr , applied biosystems , darmstadt , germany ) . upon excision of the hearts total rna was isolated ( trizol , applied biosystems ) and first - strand cdna was synthesized using the high - capacity cdna transcription kit ( applied biosystems ) with random hexameric primers according to the manufacturer 's protocol . rt - qpcr was performed and analyzed with cdna ( diluted 1 : 10 ) on an abi prism 7900 sequence detection system and sds2.2 software ( applied biosystems ) . target gene expression was normalized to an internal control ( glyceraldehyde-3-phosphate dehydrogenase , gapdh ) . relative rt - pcr was performed using taqman gene expression master mix ( part 4369016 ; applied biosystems ) with the following primers : gapdh ( mm99999915_g1 ) , tnf- ( mm00443258_m1 ) , il-1 ( mm99999061_g1 ) , and il-6 ( mm01210732_g1 ) . all murine primers were measured using fam tamra chemistry and the relative standard curve method . at the end of rt - qpcr cycle six hours after stimulation with 1668-thioate hemodynamic parameters which included left ventricular systolic pressure ( lvsp ) , stroke volume ( sv ) , left ventricular end - diastolic pressure ( lvedp ) , cardiac output ( co ) , and contractility indices ( dp / dtmax and dp / dtmin ) were recorded using a pressure - volume catheter according to the manufacturer 's manual ( millar instruments , houston tx ) . additionally , body temperature was monitored in representative mice using a rectal probe ( figure 2(a ) ) . for detailed descriptions significance testing included one - way anova followed by newman - keuls post hoc analysis . clinical appearance as well as body temperature was investigated in wt and tlr9-d mice up to 18 h after 1668-thioate stimulation . in addition , survival was monitored in all groups of stimulated wt mice . after only 2 h wt mice started to display sepsis - like symptoms such as ruffled fur , food refusal , and limited ability to respond to external stimuli . in the following neither the specific tlr9 antagonist irs954 nor the unspecific tlr9 inhibitor chloroquine improved the clinical appearance of wt mice stimulated with 1668-thioate in vivo . however , the application of h154-thioate improved clinical behavioral patterns obviously : none of the mice in this group showed external signs of systemic inflammation . in accordance with this tlr9-d mice also did not exhibit any clinical symptoms . six hours after 1668-thioate stimulation body temperature in both wt and tlr9-d mice significantly decreased ; however , body temperature in tlr9-d mice remained above that of wt mice ( figure 2(a ) ) . interestingly , wt mice cotreated with h154-thioate presented nearly the same temperature as tlr9-d mice ( figure 2(a ) ) . these findings concur with the observation that all wt animals treated with 1668-thioate were dead shortly after 6 hours . h154-thioate proved to be the most effective substance improving survival by 40% ( figure 2(b ) ) . furthermore , all tlr9-d animals survived longer than 18 hours after 1668-thioate application ( data not included in figure 2(b ) ) . two hours and six hours after 1668-thioate stimulation mediators of inflammation ( tnf- , il-1 , il-6 ) were monitored in cardiac tissue ( figures 3(a)3(f ) ) . in wt animals , 1668-thioate challenge resulted in a significant upregulation of all three proinflammatory cytokines at both time points with maximal expression at 2 h ( figures 3(a ) , 3(c ) , and 3(e ) ) . the additional application of the control oligonucleotide 1612-thioate led to a further increase in il-1 and il-6 mrna expression at 2 h ( figures 3(c ) and 3(e ) ) . four hours later , all three cytokines had decreased by at least 40% ( figures 3(b ) , 3(d ) , and 3(f ) ) . the application of h154-thioate diminished the 1668-thioate - dependent cytokine increase of all three mediators 2 h after stimulation . after 6 h this suppression was no longer detectable in neither tnf- nor il-1 ( figures 3(b ) and 3(d ) ) . neither irs954-thioate nor chloroquine suppressed cardiac tnf- or il-1 mrna expression at any time point ( figures 3(a)3(d ) ) . however , there was a significant downregulation of cardiac il-6 mrna expression in the 1668-thioate + irs954-thioate group 2 h after stimulation ( figure 3(e ) ) . chloroquine application induced a biphasic response of il-6 , enhancing its expression at 2 h and diminishing it at 6 h ( figures 3(e ) and 3(f ) ) . in cardiac tissue of tlr9-d animals , application of 1668-thioate did not influence the mrna expression of the investigated mediators . in clinical symptoms and survival as well as in cytokine parameters of cardiovascular performance monitored with a pressure - volume catheter in wt as well as in tlr9-d mice 6 h after stimulation with 1668-thioate are given in figures 4(a)4(f ) . there was a significant impairment of all measured functional parameters in stimulated wt mice compared to pbs controls . in agreement with cytokine mrna expression , additional application of h154-thioate significantly improved lv function heart rate ( hr ; data not shown ) , left ventricular systolic pressure ( lvsp ) , stroke volume ( sv ) , cardiac output ( co ) , end - diastolic volume ( edv ) , velocity of pressure increase ( dp / dtmax ) , and velocity of pressure decrease ( dp / dtmin ) were improved by h154-thioate in comparison to 1668-thioate + pbs ( figures 4(a)4(f ) ) . deficiency for tlr9 entirely prevented any deterioration of cardiac function ( figures 4(a)4(f ) ) . this study aimed to extend the in vitro finding that tlr9 stimulation decreases cardiomyocyte contractility to the in vivo setting . to our knowledge the present results are first to formally demonstrate that specific tlr9 stimulation with 1668-thioate lowers hemodynamic parameters in vivo in a murine model of systemic inflammation . the second attempt of the present investigation was to test possible tlr9 inhibitors ( h154-thioate , irs954-thioate , and chloroquine ) for their cardioprotective properties . the three tested inhibitors exhibited differential potencies ; that is , h154-thioate proved to be the most effective substance . 1668-thioate has been applied in various experimental settings for the induction of a tlr9-dependent systemic inflammation as well as organ dysfunction [ 7 , 11 , 14 , 16 , 17 ] . the effect of tlr9-dependent systemic inflammation as well as organ dysfunction seems to be dose dependent as a low concentration ( 0.25 nmol / g ) can serve as a mild stimulus for cardiac preconditioning , whereas higher concentrations in the range from 0.5 to 1 nmol / g induce sepsis - like inflammation [ 7 , 17 ] . in order to warrant clear changes in cardiac function the formerly applied dose of 1 nmol / g was doubled to 2 nmol / g bw in the current study . furthermore , the relatively high dose of 1668-thioate applied here seemed to be meaningful with respect to testing antagonizing strategies . treatment either with a control odn ( 1612-thioate ) or with one of the three inhibitors . application of the antagonists was to avoid a possible direct interaction between stimulating and inhibitory substances in the peritoneal cavity . specific tlr9 stimulation led to a time - dependent upregulation of cardiac tnf- , il-1 , and il-6 mrna expression with a peak 2 h after stimulation and a reduced level 4 h later . this time course as well as the tlr9-dependent cytokine induction confirms earlier findings of our group . an interesting new aspect is that costimulation with 1612-thioate increased the expression of all three pro - inflammatory mediators at 2 h , reaching significance in the case of il-1 and il-6 mrna . however , prestimulation with 1668-thioate may sensitize the organism to succeeding stimuli in a way that originally inert substances ( 1612-thioate ) are able to act as second hits , thereby further increasing the inflammatory response . a possible explanation for the enhanced mediator response may lie in the weak immune stimulatory properties of the charged phosphorothioate backbone of 1612-thioate . h154-thioate was the sole antagonist , which significantly depressed the upregulation of the three tested cytokines 2 h after stimulation with 1668-thioate . chloroquine failed to develop any protective influence at this time point , but even enhanced the expression of il-6 ( figures 3(a ) , 3(c ) , and 3(e ) ) . four hours later , the expression of the three cytokines had fallen to at least 60% , which was still significantly above the pbs and tlr9-d controls . also in the groups with additional application of antagonists the absolute levels of cytokine expression had fallen . significant antagonistic effects appeared only for the combinations of il-6 and h154-thioate as well as il-6 and chloroquine ( figures 3(b ) , 3(d ) , and 3(f ) ) . ligands applied from the extracellular space have to be internalized and transported to the endosomal compartment . chloroquine has been shown to disrupt this vesicle trafficking and acidification , which can explain the inhibition of the innate immune response . these data were mainly derived from in vitro experiments . however , there are reports that chloroquine is also effective in vivo [ 8 , 20 ] . in a model of polymicrobial sepsis , chloroquine improved survival and reduced renal injury as well as systemic inflammation . with respect to survival these findings appear to contradict ours . however , there are differences between the model in this study and that of yasuda et al . . we applied a specific tlr9 stimulus in a concentration high enough to induce a drastic systemic inflammation causing death of 100% of the animals shortly after 6 h , whereas yasuda et al . applied cecal ligation and puncture ( clp ) with a mortality of 50% after more than 48 h ; that is , the clp - dependent inflammation was less severe than the one in the present study . in addition to the inhibition of vesicle trafficking , it has been proposed that chloroquine decreases tlr9 protein . thus , tlr9 signaling itself may be a major target for the protective actions of chloroquine . this downregulation of tlr9 protein was detected 18 h after application of chloroquine ; this mechanism is possibly too slow to develop a beneficial influence in our model . in another attempt , hong et al . demonstrated that chloroquine at a dose of 30 mg / kg bw could protect mice from lethal challenge by 1668-thioate , whereas chloroquine at a dose of 25 mg / kg bw could decrease serum tnf- and il-6 in rats injected with sublethal doses of cpg - odn . in our hands , preliminary tests of a high chloroquine dose of 30 mg / kg bw caused a 100% mortality immediately after i.v . we therefore stayed with a dose of 10 mg / kg bw as explained in material and methods section . this divergence in chloroquine sensitivity may be attributed to differences in mouse strains ( balb / c versus c57bl/6n mice ) . taken together , in our model of cpg - induced sepsis the protective abilities of chloroquine could not be confirmed . synthetic oligonucleotides containing inhibitory properties such as h154-thioate and irs954-thioate are frequently used in experimental studies to suppress tlr signaling [ 21 , 22 ] . pisetsky 's group described synthetic oligonucleotides containing poly - g sequences , which block bacterial dna - induced activation [ 23 , 24 ] . furthermore , it has been suggested that suppressive odns interfere with the phosphorylation of signal transducer and activator of transcriptions 1 and 4 ( stat1 and stat4 , resp . ) , thereby blocking inflammation mediated by stat - associated signaling cascades . this interaction may be highly specific because suppressive odns do not bind to other molecules in the nfb and mapk regulatory cascade , and control odns do not bind to stat1 or stat4 [ 25 , 26 ] . furthermore , in vitro studies have demonstrated that inhibitory odns preferentially bind to the c - terminal of tlr9 , competing for cpg - odn binding . with respect to the structural details of inhibitory odns , ashman et al . demonstrated in vitro that a specific motif ( ccx(not - c)(not - c)xxggg ( x = any base ) ) provides the sequences required to block tlr9 in human b cells and hek cells transfected with human tlr9 . extending the sequence by four to five bases at the 5 end enhanced activity , even more so when a phosphorothioate backbone replaced the native phosphodiester backbone ( for further details , see [ 25 , 26 ] ) . in addition to the molecular composition of an inhibitory odn also the targeted cell type has been shown to influence the inhibitory potency of the respective odn ; that is , some inhibitory sequences that function well in macrophages have been shown to be inefficient in b cells . taken together , the workings of molecular blocking mechanisms of inhibiting odn have not yet been unraveled ; differences in the potency of h154-thioate and irs954-thioate up to now can not be attributed to their molecular structure . are generated in a highly controlled environment , which is not the case in living organisms . therefore , transfer from in vitro to in vivo can not be assumed to be self - evident . thus , the presented cardiodepressive effect of 1668-thioate stimulation in vivo enhances our knowledge of the understanding of tlr9 stimulation . this is further supported by the total insensitivity of tlr9-d mice to this stimulation . to our knowledge , this study is the first demonstration that specific tlr9 stimulation with 1668-thioate lowers hemodynamic parameters in vivo in a murine model of systemic inflammation . in our experimental setting h154-thioate there was a significant impairment of all measured functional parameters in stimulated wt mice compared to pbs controls . in agreement with cytokine mrna expression , additional application of h154-thioate significantly improved lv function when compared to mice treated with 1668-thioate + pbs . specifically , heart rate , left ventricular systolic pressure , stroke volume , cardiac output , end - diastolic volume , velocity of pressure increase , and velocity of pressure decrease were improved by h154-thioate in comparison to 1668-thioate + pbs ( figures 4(a)4(f ) ) . deficiency for tlr9 entirely prevented any deterioration of cardiac function ( figures 4(a)4(f ) ) . our finding that h154-thioate protected the heart against tlr9-stimulation complements the earlier observation that h154-thioate prevented tlr9-dependent vascular relaxation . the importance of tlr9 signaling in polymicrobial infection has already been shown by others and by our group [ 3 , 4 ] . a possible reason for this may lie in the ability of tlr9 to bind the dna of gram - positive as well as gram - negative bacteria and thus to react to a broad range of stimuli [ 3 , 30 , 31 ] . this may have contributed to the disappointing results of the application of eritoran ( e5564 ) , a tlr4 antagonist , evaluated in the access ( a controlled comparison of eritoran and placebo in patients with severe sepsis ) trial , a global , randomized , double - blind , placebo - controlled phase iii study . taken together , here we show that systemic tlr9 stimulation is able to depress cardiac function in vivo and that a pharmacological intervention is possible and may be a promising strategy for human clinical trials in future .

the involvement of toll - like receptor 9 ( tlr9 ) , a receptor for bacterial dna , in septic cardiac depression has not been clarified in vivo . thus , the aim of the study was to test possible tlr9 inhibitors ( h154-thioate , irs954-thioate , and chloroquine ) for their ability to protect the cardiovascular system in a murine model of cpg oligodeoxynucleotide- ( odn- ) dependent systemic inflammation . sepsis was induced by i.p . application of the tlr9 agonist 1668-thioate in c57bl/6 wild type ( wt ) and tlr9-deficient ( tlr9-d ) mice . thirty minutes after stimulation tlr9 antagonists were applied i.v . survival was monitored up to 18 h after stimulation . cardiac mrna expression of inflammatory mediators was analyzed 2 h and 6 h after stimulation with 1668-thioate and hemodynamic parameters were monitored at the later time point . stimulation with 1668-thioate induced a severe sepsis - like state with significant drop of body temperature and significantly increased mortality in wt animals . additionally , there was a time - dependent increase of inflammatory mediators in the heart accompanied by development of septic heart failure . these effects were not observed in tlr9-d mice . inhibition of tlr9 by the suppressive odn h154-thioate significantly ameliorated cardiac inflammation , preserved cardiac function , and improved survival . this suppressive odn was the most efficient inhibitor of the tested substances .

this study reports a rare patient of a rapid spontaneously resolving acute subdural hematoma . in addition , an analysis of potential clues for the phenomenon is presented with a review of the literature . a 1-year - and-2-month - old boy fell from a height of approximately 2 m. the patient was in a superficial coma with a glasgow coma scale of 8 when he was transferred to the authors hospital . computed tomography revealed the presence of an acute subdural hematoma with a midline shift beyond 1 cm . repeat imaging after 15 hours showed the evident resolution of the hematoma and midline reversion . progressive magnetic resonance imaging demonstrated the complete resolution of the hematoma , without redistribution to a remote site . even though this phenomenon has a low incidence , the probability of a rapid spontaneously resolving acute subdural hematoma should be considered when patients present with the following characteristics : children or elderly individuals suffering from mild to moderate head trauma ; stable or rapidly recovered consciousness ; and simple acute subdural hematoma with a moderate thickness and a particularly low - density band in computed tomography scans . a 1-year - and-2-month - old boy fell from a height of approximately 2 m and lost consciousness after he experienced head trauma . the patient was sent to a local hospital immediately . during the interval emergent computed tomography ( ct ) , the patient vomited several times . 1a ) revealed a right frontotemporal asdh that was approximately 9 mm in size with a subarachnoid hemorrhage in the longitudinal fissure and midline shift beyond 10 mm . he was in a superficial coma with the same gcs score that was documented in previous medical records . both of the patient 's pupils were equal in size 2 mm but exhibited a blunted response to light . 1b ) showed a decrease in hematoma density and diffuse cerebral edema with obviously squeezed ventricles ; the midline was still deviated compared with the first ct scan . given his consciousness and imaging findings , intracranial pressure monitoring was preferred . even though the laboratory features were not surgical contraindications , the boy 's guardians refused the placement of an intracranial pressure probe or a craniotomy . general monitoring aimed to maintain sao2 at > 95% , mean systolic blood pressure at > 90 mm hg , temperature at < 37c , and paco2 at 35 to 40 mm hg . owing to the lack of an accurate intracranial pressure assessment , mannitol was administered empirically at 0.5 g / kg every 6 hours . the third ct scan ( fig . 1c ) revealed evident resolution of the hematoma and midline shift improvement , with only a subarachnoid hemorrhage present in the posterior longitudinal fissure 24 hours after the trauma . after he had received 2 days of conservative management , the boy regained consciousness . c ) showed that the asdh was almost completely resolved on the fourth day after admission , without redistribution to the upper cervical spinal cord . ( a ) initial ct scan revealed right frontotemporal acute subdural hematoma ( arrow to show ) , with sah in anterior longitudinal fissure and midline shift exceeding 10 mm . ( b ) a second ct scan showed the decrease of hematoma ( arrow to show ) with obvious diffusion cerebral edema . ( c ) a third ct scan was taken after 24 hours of the injury revealed an evident resolution of hematoma ( arrow to show ) and midline revert with only sah left in posterior longitudinal fissure . ct , computed tomography ; sah , subarachnoid hemorrhage . magnetic resonance imaging on the fourth day of conservative management . transverse ( a ) , sagittal ( b ) , and coronal ( c ) planes were performed which revealed acute subdural hematoma complete resolution without redistribution to upper cervical spinal cord . the following 2 possible explanations were proposed : the asdh was diluted and washed out by cerebrospinal fluid ( csf ) after the tearing of the arachnoid membrane and the hematoma was redistributed to the subdural space . wong et al measured the hounsfield units of the subarachnoid space , which was close to normal , and confirmed the redistribution of a hematoma to the subdural space then to the subarachnoid space . the theory was based on the flow of liquefied blood , which was thought to be driven by the change in intraparenchymal pressure . in those patients without available definitive evidence of blood redistribution , csf flushing seems more likely . based on table 1 , rapid resolution occurred more frequently in children , less frequently in elders , and least often in middle - age individuals . in children and elders , asdh is often caused by mild or moderate injuries , such as falling or slipping . in elderly individuals , brain atrophy and a wide cavum of the subarachnoid space can cause an arachnoid membrane slit under external force . for middle - age individuals , well - developed brain tissue and narrow compensatory space results in an arachnoid membrane that is comparatively difficult to tear with a mild injury . wen et al proposed that gcs was higher than 8 in patients who exhibited this phenomenon . however , in table 1 , 7 of 18 patients exhibited a gcs score of less than 8 , and the minimum was 5 . in the process of the clinical evaluation , the dynamic change in consciousness is a more heavily weighted factor to consider than the initial gcs score . according to a review of all patients ( table 1 ) , most of those patients regained consciousness quickly . although some of those patients did not regain consciousness quickly , their conditions were stable . in our case , the patient 's consciousness improvement was even slower than the time of resolution according to the ct scan . thus , by comparing the initial gcs score after head trauma , we inferred that a rapid recovery or relative stabilization of consciousness is more persuasive in these patients . the evaluation of images from ct scans that were performed during different phases of our patient clearly demonstrated the rapid transition of hematoma density from a high density to a mixed density . some authors agreed that the potential sign in early ct of a rapid resolution was the presence of a low - density band . in our patient 's second ct scan ( fig . 1b ) , it was observed that remarkable cerebral edema could squeeze , in some way , the diluted hematoma . kuroiwa et al suggested that diffuse cerebral edema was necessary for the spontaneous resolution of a hematoma . we speculate that cerebral edema leads to resolution via both mechanisms , but it may not be the prerequisite for hematoma rapid resolution . indeed , in most patients , the diffuse edema was not even observed ( table 1 ) . we propose that the slice or middle thickness hematoma ( < 1.5 cm ) would be suitable in the consideration of rapid resolution . lee et al reported a patient with a hematoma thickness that was larger than 2.5 cm , yet it finally converted to a chronic subdural hematoma . the presence of simple asdh without contusion appears to be associated with rapid resolution . we suspect that this effect is due to the mass effect of the contusion , which impeded the flow of csf in the limited space . this patient has been reported because of clear evidence of csf dilution and a flush effect that led to resolution of the hematoma , and the finding that a distinctly swollen brain probably accelerated the process . there is no need for the deliberate evaluation of the phenomenon as a coincidence during treatment . we suggest that asdh rapid resolution should be considered when patients present with the following characteristics : children or elders suffering from mild to moderate head trauma ; stable or rapidly recovered consciousness ; and simple asdh with moderate thickness and a particularly low - density band in ct scan images .

introduction : this study reports a rare patient of a rapid spontaneously resolving acute subdural hematoma . in addition , an analysis of potential clues for the phenomenon is presented with a review of the literature.patient presentation : a 1-year - and-2-month - old boy fell from a height of approximately 2 m. the patient was in a superficial coma with a glasgow coma scale of 8 when he was transferred to the authors hospital . computed tomography revealed the presence of an acute subdural hematoma with a midline shift beyond 1 cm . his guardians refused invasive interventions and chose conservative treatment . repeat imaging after 15 hours showed the evident resolution of the hematoma and midline reversion . progressive magnetic resonance imaging demonstrated the complete resolution of the hematoma , without redistribution to a remote site.conclusions:even though this phenomenon has a low incidence , the probability of a rapid spontaneously resolving acute subdural hematoma should be considered when patients present with the following characteristics : children or elderly individuals suffering from mild to moderate head trauma ; stable or rapidly recovered consciousness ; and simple acute subdural hematoma with a moderate thickness and a particularly low - density band in computed tomography scans .

down syndrome ( trisomy 21 ) is the most common and best known of all malformation syndromes . it is an easily recognized congenital , autosomal anomaly characterized by generalized growth deficiency and mental deficiency affecting 1 in 600 - 1 in 1000 live births . a compromised immune system is characteristic of these individuals , and it is this immunodeficiency that contributes to an increased susceptibility to infections . various microorganisms are generally seen as normal commensals in the oral cavity , and these may become pathogenic under favorable conditions . candida is one such endogenous pathogen , the prevalence of which has been reported to be 45 - 65% in healthy children and 30 - 45% in healthy adults . opportunistic infections occur mainly when the host defenses are inadequate , candidiasis is the most common among them . streptococci form a large portion of the resident oral micro flora . in the general population , half the isolates from the tongue and saliva are streptococci , but in down syndrome individual 's oral streptococcal levels are relatively lower . this probably contributes to a disturbance in the normal microbial homeostasis and promotes the growth of other commensal organisms like candida . here , we evaluated the candidial / streptococci carriage and the various species of candida / streptococci isolated from the down syndrome patients and correlated it with normal oral micro flora from health subjects . fifty down syndrome individuals were selected for this study based on a karyotype of 47 xx , 21 + ( female ) and 47 xy , 21 + ( male ) . a total of 50 normal children / adolescents were selected as a control group . to isolate candida and streptococci , swab / saliva was used to culture and smear preparation for gram stain and periodic acid schiff ( pas ) stain . samples were taken from the oral cavity ( dorsum of tongue ) for each individual using a sterile cotton wool swab . sabouraud 's dextrose agar ( sda ) slope with antibiotic ( gentamicin / actidione ) was used for the isolation of candida . if colonies were white or cream to yellow , dull / shiny , pasty yeast like a smear was prepared from the colony , with a drop of saline . smear was then air - dried , heat fixed , and stained with crystal violet ( simple stain ) to confirm the presence of yeast . the isolates were then subjected to the following tests for identification of various species of candida : germ tube testmorphology on corn meal agar for chlamydospore formation.carbohydrate fermentation testcarbohydrate assimilation testchromagar candida culture characteristics morphology on corn meal agar for chlamydospore formation . carbohydrate fermentation test carbohydrate assimilation test chromagar candida culture characteristics smears taken from the oral cavity ( tongue ) were air - dried , heat fixed , and stained . samples were taken from the oral cavity ( dorsum of tongue ) for each individual using a sterile cotton wool swab . sabouraud 's dextrose agar ( sda ) slope with antibiotic ( gentamicin / actidione ) was used for the isolation of candida . if colonies were white or cream to yellow , dull / shiny , pasty yeast like a smear was prepared from the colony , with a drop of saline . smear was then air - dried , heat fixed , and stained with crystal violet ( simple stain ) to confirm the presence of yeast . the isolates were then subjected to the following tests for identification of various species of candida : germ tube testmorphology on corn meal agar for chlamydospore formation.carbohydrate fermentation testcarbohydrate assimilation testchromagar candida culture characteristics morphology on corn meal agar for chlamydospore formation . smears taken from the oral cavity ( tongue ) were air - dried , heat fixed , and stained . the study included a total of 100 ( 50 down syndrome , 50 controls ) subjects of both sexes . the study group of 50 down syndrome individuals comprised of 37 males and 13 females in the age range of 3 - 22 years and the mean age of 11.6 years . the control group of 50 normal individuals comprised of 34 males and 16 females in the age range of 3 - 18 years and a mean age of 9.5 years [ tables 1 and 2 ] . distribution of patients according to sex distribution of patients according to age of the 50 study group samples , which were cultured , 37 ( 74% ) showed growth of candida colonies , whereas in the 50 control samples only 18 ( 36% ) were positive for candida growth . in 4 sda culture slopes of the study group , more than one morphological type of colonies were observed [ figure 1 ] , and the colonies were considered as different isolates and subjected to further testing . a total of 42 isolates ( 1 isolate in 33 samples , 2 isolates in 3 samples , and 3 isolates in 1 sample ) were obtained from the 37 culture positive samples , in the study group and 18 isolates were obtained from the 18 culture positive samples in the control group ( 1 isolate each in 18 samples ) [ table 3 ] . sabouraud 's dextrose agar slope showing more than one type of colony ( white and cream ) candidal growth pattern in the study group and control group smears taken from the oral cavity ( tongue ) , for both the study group ( 50 ) and the control group ( 50 ) were stained with gram stain [ figure 2 ] and pas stain [ figure 3 ] . a tenfold increase in the positive staining for candida was observed in the study group when compared to the control group [ table 4 ] . gram - stained smear showing yeasts and pseudohyphae periodic acid schiff stained smear showing candidal hyphae and yeasts distribution of positive candidal staining in study group and control group of the 42 isolates from the 37 culture positive samples , 31 were germ tube positive , whereas all the 18 culture positive samples of the control group were positive for germ tube formation [ figure 4 ] . hence , 31 of the 42 isolates were candida albicans in the study group , and all 18 isolates of the control group were c. albicans . this was further confirmed by chlamydospore formation on corn meal agar [ figure 5 ] . all the isolates that were germ tube positive produced chlamydospores on corn meal agar further confirming c. albicans . of the 42 isolates and 37 culture positive samples , 31 isolates were c. albicans , 6 were candida tropicalis , and 5 were either candida parapsilosis / candida krusei / candida glabrata . of the 18 control samples , which were culture positive , all 18 were c. albicans . further confirmation and speciation was done by the carbohydrate assimilation test and supplemented with growth on chromagar candida . germ tube formation by candida albicans chlamydospore formation by candida albicans this test was done with sugars : glucose , maltose , sucrose , lactose , galactose , cellulose , and trehalose [ table 5 ] . carbohydrate assimilation reactions for different candida species of the 37 samples that were culture positive in the study group , 31 ( 84% ) samples had isolates of c. albicans , 6 ( 16% ) samples had isolates of c. tropicalis and 5 ( 14% ) had isolates of c. parapsilosis . 4 samples of the study group were colonized with more than one type of species . of the 18 control samples that were culture positive all 18 isolates were c. albicans . growth of all culture positive samples on chromagar candida was observed for the characteristic colony color [ table 6 ] . chromagar candida culture characteristics for different candida species in the chromagar candida , study sample showed 31 isolates of c. albicans , 6 of c. tropicalis , and 5 of c. parapsilosis [ figure 6 ] . in the control samples , chromagar candida showing green colonies of candida albicans and cream colonies of candida parapsilosis saliva samples collected from both the study group ( 50% ) and the control group ( 50 ) after inoculation on chocolate agar , and incubation at 37c for 24 h , the growth of alpha - hemolytic colonies were identified [ figure 7 ] . the colonies were checked for optochin resistance to classify it as streptococcus viridans group [ table 7 ] . primary culture showing predominantly alpha hemolytic colonies growth pattern of streptococcus the growth of alpha hemolytic s. viridans colonies in the primary culture was classified as in table 8 . distribution of viridans group of streptococci in control group and study group twenty - three out of 50 samples from study group had s. viridans colonies . in the 23 samples positive for streptococci 16 had many streptococcal colonies , and 7 had few streptococcal colonies in the primary culture . in the 32 samples , which were positive for streptococci , 29 had predominantly streptococcal colonies while 3 had few streptococcal colonies in the primary culture . the presence of candida with streptococci was compared in both the study group and the control group [ table 9 ] . comparison of candidal and streptococcal growth patterns in study group and control group candidal and streptococcal colonization was seen in 14 ( 28% ) individuals in the study group and 6 ( 12% ) individuals in the control group . candida colonization alone in the absence of streptococcal colonization was seen in 23 ( 46% ) individuals of the study group and 12 ( 24% ) of the control group . thus showing that candidal colonization occurred to a greater extent in the absence of streptococci ( p < 0.01 , z = 2.96 ) . our results showed that in the study group ( 74% ) candida colonization was comparatively higher when compared , with the control group ( 36% ) . of the 37 culture - positive samples , 42 isolates were obtained , four samples had more than one isolate ( one sample with three isolates , three samples with two isolates ) . the species isolated in the study group out of the 37 culture - positive samples were predominantly c. albicans ( 84% ) followed by c. tropicalis ( 16% ) and c. parapsilosis ( 14% ) . in the control group , only c. albicans were isolated . thus , out of the total 50 down syndrome subjects , 31 ( 62% ) showed colonization with c. albicans , 6 ( 12% ) with c. tropicalis , and 5 ( 10% ) with c. parapsilosis . in the comparative study of candida and streptococci , the oral findings of all the children and adolescents of the study group were recorded [ figures 810 ] . 12-year - old male showing fissured tongue high arched palatal vault angular cheilitis with prognathic mandible distribution of oral findings in down syndrome subjects in the 50 down syndrome individuals , 17 ( 34% ) of them showed an erythematous or a pseudomembranous lesion on the tongue ( 12 - erythematous , 5 - pseudomembranous ) , indicating possible oral candidiasis . this test was done with sugars : glucose , maltose , sucrose , lactose , galactose , cellulose , and trehalose [ table 5 ] . carbohydrate assimilation reactions for different candida species of the 37 samples that were culture positive in the study group , 31 ( 84% ) samples had isolates of c. albicans , 6 ( 16% ) samples had isolates of c. tropicalis and 5 ( 14% ) had isolates of c. parapsilosis . 4 samples of the study group were colonized with more than one type of species . of the 18 control samples that were culture positive all 18 isolates were c. albicans . growth of all culture positive samples on chromagar candida was observed for the characteristic colony color [ table 6 ] . chromagar candida culture characteristics for different candida species in the chromagar candida , study sample showed 31 isolates of c. albicans , 6 of c. tropicalis , and 5 of c. parapsilosis [ figure 6 ] . in the control samples , saliva samples collected from both the study group ( 50% ) and the control group ( 50 ) after inoculation on chocolate agar , and incubation at 37c for 24 h , the growth of alpha - hemolytic colonies were identified [ figure 7 ] . the colonies were checked for optochin resistance to classify it as streptococcus viridans group [ table 7 ] . primary culture showing predominantly alpha hemolytic colonies growth pattern of streptococcus the growth of alpha hemolytic s. viridans colonies in the primary culture was classified as in table 8 . distribution of viridans group of streptococci in control group and study group twenty - three out of 50 samples from study group had s. viridans colonies . in the 23 samples positive for streptococci 16 had many streptococcal colonies , and 7 had few streptococcal colonies in the primary culture . in the 32 samples , which were positive for streptococci , 29 had predominantly streptococcal colonies while 3 had few streptococcal colonies in the primary culture . the presence of candida with streptococci was compared in both the study group and the control group [ table 9 ] . comparison of candidal and streptococcal growth patterns in study group and control group candidal and streptococcal colonization was seen in 14 ( 28% ) individuals in the study group and 6 ( 12% ) individuals in the control group . candida colonization alone in the absence of streptococcal colonization was seen in 23 ( 46% ) individuals of the study group and 12 ( 24% ) of the control group . thus showing that candidal colonization occurred to a greater extent in the absence of streptococci ( p < 0.01 , z = 2.96 ) . our results showed that in the study group ( 74% ) candida colonization was comparatively higher when compared , with the control group ( 36% ) . of the 37 culture - positive samples , 42 isolates were obtained , four samples had more than one isolate ( one sample with three isolates , three samples with two isolates ) . the species isolated in the study group out of the 37 culture - positive samples were predominantly c. albicans ( 84% ) followed by c. tropicalis ( 16% ) and c. parapsilosis ( 14% ) . in the control group , thus , out of the total 50 down syndrome subjects , 31 ( 62% ) showed colonization with c. albicans , 6 ( 12% ) with c. tropicalis , and 5 ( 10% ) with c. parapsilosis . in the comparative study of candida and streptococci , the oral findings of all the children and adolescents of the study group were recorded [ figures 810 ] . table 10 shows in an ascending order the frequency of various findings . 12-year - old male showing fissured tongue high arched palatal vault angular cheilitis with prognathic mandible distribution of oral findings in down syndrome subjects in the 50 down syndrome individuals , 17 ( 34% ) of them showed an erythematous or a pseudomembranous lesion on the tongue ( 12 - erythematous , 5 - pseudomembranous ) , indicating possible oral candidiasis . down syndrome individuals are reported to exhibit an increased susceptibility to infections due to various factors such as increased activity of superoxide dismutase , increased activity of glutathione peroxidase , decreased levels of myeloperoxidase in leukocytes , defective neutrophil chemotaxis , abnormal serum immunoglobulin patterns and disturbed cell - mediated and humoral immunity . the present study determined the prevalence of oral candida carriage in down syndrome children and adolescents . of the 37 individuals in the study group who exhibited candida colonization , only 14 showed colonization with streptococci while 23 remained negative and showed no streptococcal colonization . thus , we concluded that the majority of the individuals showed candida colonization when there was no concomitant streptococcal colonization . the increased candida carriage in down syndrome individuals could probably be attributed to the relatively lower streptococcal levels . the increased susceptibility to candida infection may be a result of the disturbance in the microbial homeostasis . however , other factors such as a compromised immune system , poor oral hygiene , malnourishment and the like can not be completely ruled out and are also expected to play a significant role . it may be also possible that the candida and streptococci may produce mutually inhibitive factors in the form of toxins or enzymes , thus producing this inverse relationship . the most common predisposing factor resulting in candidiasis is the presence of a suppressed immune status , thus this infection is seen in a higher frequency in down syndrome subjects , where a compromised immune status is the hallmark . in addition to this , a variety of local factors may also play a role , the most common being an altered intra oral milieu . as observed in this study , it may be assumed that the presence of microbial imbalance in the oral cavity and the occurrence of fissured tongue could be some major local factors contributing to the increased candida carriage in down syndrome individuals . the oral cavity is an environment heavily colonized by microorganisms , which are generally in harmony with each other . the down syndrome subjects run a greater risk of having opportunistic infections with a possibility of systemic spread , and the associated morbidity and mortality is also higher . hence , to improve the quality - of - life of an individual with down syndrome , it becomes necessary to diagnose and treat these infections when they still remain localized .

aim : we aimed to evaluate the prevalence of candida and streptococci species in the oral cavity of down syndrome patients.materials and methods:50 children / adolescents with down syndrome with a karyotype of 47 xx , 21 + ( female ) and 47 xy , 21 + ( male ) , and 50 normal children / adolescents were included in our study . oral swab / saliva was used to culture and identify candida and streptococci species based on gram and periodic acid schiff staining.results:of the 50 study group samples , which were cultured , 37 ( 74% ) showed growth of candida colonies , whereas in the 50 control samples only 18 ( 36% ) were positive for candida growth . in 4 sabouraud 's dextrose agar culture slopes of the study group , more than one morphological type of colonies were observed . 23 out of 50 samples in our study group had streptococcus viridans colonies . in the 23 samples positive for streptococci 16 had many streptococcal colonies , and 7 had few streptococcal colonies in the primary culture . 32 out of 50 samples from the control group had s. viridans colonies . in these 32 samples positive for streptococci , 29 had predominantly streptococcal colonies while 3 had few streptococcal colonies in the primary culture.conclusion:the oral cavity is an environment heavily colonized by microorganisms , however , the down syndrome patients run a greater risk of having opportunistic infections especially from candida species . hence to improve the quality of life of an individual with down syndrome , it is necessary to diagnose and treat these infections by more frequent oral microbial assessment .

mucocele of the appendix ( collection of mucus within the appendiceal lumen ) is a rare lesion , found in only 0.2% to 0.3% of 43,000 appendectomies reviewed . currently , the assessment of pelvic masses relies heavily on usg as the primary diagnostic tool . in such cases , clinical findings and other investigative modalities are warranted to aid the diagnostic process . in spite of extensive preoperative investigations , the diagnosis may still remain elusive and may only be made at the time of surgery . some regard this lesion as benign , a result of obstruction of the proximal lumen by fibrosis ; others believe it to be a neoplasm of the appendix . is the method of choice in the management of simple mucocele and for cystadenoma with an intact base . several studies ( mostly case reports ) on laparoscopic resection of mucocele have been reported . a 60-year - old female presented with pain in lower part of abdomen and palpable tender lump in the right ileac fossa . ultrasound of the abdomen reports a cystic mass of size 12 15 cm with thin internal septations in the right adnexa . the pneumoperitoneum was created with veress needle using carbon dioxide and the pressure was kept at 11 mmhg . a 0 telescope was introduced through the umbilical port for the complete examination of the abdomen . diagnostic laparoscopy revealed approximately 14 15 cm large bluish mucocele of the appendix with omental adhesions . two 5-mm ports were placed in the supra pubic area below the pubic hair line as the working port . the mucocele of the appendix was isolated after separating the mesoappendix from it with the help of bipolar cautery . following this , mucocele of the appendix [ figure 1 ] was retrieved out in a plastic bag through the umbilical port . hemostasis was obtained and a suction drain left in situ which was removed when non - productive . cut section showed appendix was filled with mucin - like material [ figure 2 ] . she was started orally after 4 hours of operation and solid food on the next day . appendicular lump from the distal portion of appendix after removal the appendicular lump filled with mucinus material mucocele of the appendix is a descriptive term for an appendix distended by mucus , secondary to mucinous cystadenoma ( 63% ) , mucosal hyperplasia ( 25% ) , mucinous cystadenocarcinoma ( 11% ) , and retention cyst . clinical presentation may include right lower quadrant pain , change in bowel habits , per rectal bleeding , or a palpable mass . approximately 23 - 50% of patients are asymptomatic , with the lesions being discovered incidentally during surgery , radiological evaluations , or endoscopic procedures . the preoperative clinical diagnosis of appendiceal mucoceles can therefore be difficult because of this lack of clinical symptomotology . the initial detection of the lesion may be facilitated by radiological , sonographic , or endoscopic means . on barium enema , the lesion may be seen as a sharply outlined sub - mucosal or extrinsic mass indenting the cecum and laterally displacing it . purely cystic lesions with anechoic fluid , hypoechoic masses with fine internal echoes as well as complex hyperechoic masses can be seen depending on the contents . ct of the abdomen usually shows a cystic well - encapslated mass sometimes with mural calcification , in the expected location of the appendix . it may be causing extrinsic pressure on the cecal wall without any surrounding inflammatory reaction . colonoscopic findings include the volcano sign , the appendiceal orifice seen in the center of a firm mound covered by normal mucosa or a yellowish , lipoma - like submucosal mass . in our case , usg was unable to provide a preoperative diagnosis . in our case , the decision for excision of the appendiceal mucocele was made as a result of diagnostic laparoscopy and a need to rule out malignancy . therefore mucocele of the appendix can mimic an adnexal mass and prove to be a diagnostic challenge . in a woman presenting with right iliac fossa mass and with clinical features not indicative of gynecological pathology , an appendiceal origin should be considered in the differential diagnosis . surgery is the treatment of choice and should be done early as tumor can not be ruled out as the causative factor for the mucocele . pre - operative diagnosis is important to avoid unintended rupture and the development of pseudomyxoma peritonei during surgery . however , laparoscopic dissection , grasping of the appendix specimen , pneumoperitoneum , or transport of the specimen through the abdominal wall might contribute to peritoneal dissemination of a tumor , if present . these setbacks can be avoided by taking precautions like using bowel holding graspers ( non - traumatic ) to handle the mucocele and using a non - permeable bag to deliver the specimen out of the port . mucocele of the appendix can mimic an adnexal mass and prove to be a diagnostic challenge . laparoscopic resection of mucocele of the appendix is feasible in spite of the danger of malignancy , provided necessary precautions are taken .

mucocele of the appendix is an aseptic dilatation secondary to obstruction . the preoperative clinical diagnosis of appendiceal mucoceles can therefore be difficult because of this lack of clinical symptomotology . surgical excision is the treatment of choice in benign mucocele . we report a case presenting to the surgeons where initial clinical findings and investigations suggested a cyst in the right adnexa . diagnostic laparoscopy revealed mucocele of the appendix and laparoscopic appendicectomy was done .

the first pandemic reportedly occurred in 412 bc , , and the first attributed to influenza in 1580 . , since then , 31 influenza pandemics have been described ; the five most recent in 1889 , 1900 , 1918 , 1957 and 1968 , , , , , were separated respectively by 11 , 18 , 39 and 11 years . the present threat of a new influenza pandemic is at the origin of renewed interest in the 1918 spanish flu , as it was undoubtedly the most deadly influenza pandemic in modern history . recently , murray et al . extrapolated , in their paper published in 2006 , the potential global pandemic influenza mortality occurring in 2004 , based on data from the 19181920 influenza pandemic . a first american report in 1927 suggested that the main 19181919 wave was responsible for 21 millions of deaths worldwide . a revised estimation of influenza pandemic mortality given in 1991 was 247393 million , and another published in 2002 even set the death toll up to 100 million to take into account the lack of data in a large part of the world . the lack of reliable mortality data in a large part of the world leads to reliance on anecdotal evidence and extrapolation of mortality rates for entire countries from punctual information obtained in highly specific settings . , concerning europe , we are aware of only three published papers reporting estimates of the mortality burden . , , herein , we analyzed the mortality data obtained during the pandemic , in 14 european countries , which accounted for 75% of the european population in 1918 . monthly allcause deaths of civilians 19061936 were obtained from a book published , in 1954 , by the french national institute of demographic study , which compiled official demographic and vital statistics from several countries , including 14 european countries . for each country , allcause death counts were available on a monthly basis , except for england and wales , for which only quarterly data are available . the observed data were standardized to mortality rates per 10 000 inhabitants using the corresponding yearly populationcount estimates . we used a periodic equation to model the monthly baseline mortality rate ( b t ) of years 19061922 : with , respectively , n = 12 or n = 4 when monthly or quarterly data were analyzed . in this model , the baseline mortality rate depends on time t through a constant term 0 , a secular trend 1 , annual predictors 1 and 1 , semiannual predictors 2 and 2 and an error term t . the unknown parameters were estimated at maximum likelihood from the fit of a linear regression to data from a training period , extending from 1906 to 1917 . the estimation was based on the time series truncated from the values observed during the epidemic periods , defined as the 3 consecutive month period with the highest incidence . more precisely , from each annual august 1july 31 window , we removed the trimester with the highest mortality to take into account seasonal influenza , and the associated annual excess mortality . july period was used because seasonal influenza epidemics occur from fall to spring in europe . the baseline model described above we defined excessmortality periods as those starting when observed mortality exceeded the upper limit of the prediction interval [ threshold = e ( b t ) + 196 sd ( e t ) ] , for at least 2 consecutive months ( one trimester for england and wales ) and ending when the observed mortality fell below the upper limit of the prediction interval . excess mortality was calculated as the difference between observed and expected baseline deaths during the excessmortality period ( figure 1 ) . the crosshatched area represents excess mortality , calculated as the difference between observed ( black line ) and predicted ( continuous gray line ) mortality , from the beginning of the pandemic period ( light gray shading ; first month during which observed deaths exceeded the pandemic threshold , dashed gray line ) until the end ( first month during which observed deaths fell below the pandemic threshold ) . for finland , we subtracted from excess deaths , the 30 000 deaths attributed to the finnish civil war ( january may 1918 ) . data for the civilian mortality rates in germany presented a step pattern for the period 19141918 , probably reflecting mismatched definitions of population counts and deaths as a result of changing geographical borders . therefore , we first replaced the linear trend of mortality rates during 19141918 by those of the surrounding years ( 19061913 and 19191936 ) , and obtained excess mortality as described above . we also compared the excess mortality in each of the 14 countries during the 19181919 pandemic with the excess mortality during the rest of the years ( 19061917 ; 19201922 ) to determine how worse the 1918 period was compared with those seasons . cumulative excess deaths during the 19181919 period were 198 million in 14 countries in europe accounting for 75% of the population , an increase of 86% more deaths from baseline . extrapolating that figure to the rest of europe , the estimated total mortality in europe during the 1918 pandemic would be 264 ( 198/075 ) million deaths , i.e. 11% of the entire population estimated at 250 million in 1918 . the highest cumulative excess / predicted mortality ratio was observed in italy ( + 172% ) during the pandemic period ( peaked in october 1918 ) , following by bulgaria and portugal ( + 102% each ) , spain ( + 87% ) , the netherlands ( + 84% ) , sweden ( + 74% ) , germany ( + 73% ) , switzerland ( + 69% ) , france ( + 66% ) , norway ( + 65% ) , denmark ( + 58% ) , scotland ( + 57% ) and england and wales ( + 55% ) , with the lowest ratio being seen in finland ( + 33%)(table 1 ) . the highest excessmortality rate ( per 10 000 inhabitants ) cumulated throughout the entire excessmortality period was observed in portugal ( 233/10 000 inhabitants ) , followed by italy , spain , bulgaria , switzerland , finland , france , germany , sweden , netherlands , norway , england and wales , scotland and denmark ( figure 2 ) . excess mortality in 14 european countries represents the excess mortality calculated , based on expected baseline mortality . existence of a later peak in april 1920 ( not taken into account to calculate excess deaths throughout the pandemic period ) . south gradient in the progressively darker shades of gray , * ( /10 000 inhabitants ) . overall , the excess mortality found during the pandemic was 35 times higher than the excess mortality found in the rest of the 19061922 period . this ratio ranged between 16 ( finland ) and 61 ( portugal ) ( table 2 ) . excess mortality ( % ) comparison between the 19181919 pandemic periods and the rest of the years ( 19061917 ; 19201922 ) in each of the 14 countries we found a statistically significant negative correlation ( spearman s rank correlation = 066 ; p = 0013 ) between excess mortality and latitude ( figure 2 ) , meaning that northern countries experienced significantly less mortality , while southern europe suffered significantly more excess deaths . countries were clustered according to their mortality patterns ( figure 3 ) : countries with one sharp mortality peak ( e.g. portugal , spain , italy , bulgaria ) , countries with two major peaks ( e.g. switzerland , scotland , netherlands and france ) , country with several successive peaks ( only finland ) . monthly ( or trimesterly ) mortality rate * ( ordinate ) in 14 european countries from 1917 to 1921 ( abscise ) . lines and shading are as defined in the legend to figure 1 , * ( /10 000 inhabitants ) . excess mortality was time dependent : the start and stop months of the excessmortality period varied across the continent and occurred in four waves ( figure 4 , table 1 ) . moreover , an early first wave of deaths , occurring between march 1918 and july 1918 , was observed in six countries ( bulgaria , portugal , germany , finland , switzerland and spain ) ( figure 4 ) . importantly , the excessdeath curves could be temporally superposed , albeit not of the same intensity , for 19181919 , with mortality peaking in all countries within a 2month window ( october november 1918 , figure 3 ) , defined as the second wave ( figure 4 ) . in five countries ( spain , denmark , finland , germany and switzerland ) , a late peak between january 1920 and april 1920 was recorded . that fourth wave in 1920 was not taken into account in estimating the cumulative excessdeath rates for the entire pandemic period the limitation of any study focused on mortality burden at the epoch of the spanish pandemic depends on the exhaustiveness of the data . during this period , data were often scattered in various places or sources , making their collection for analyses difficult . this is the reason why earlier publications were based on various documents dating from 1918 to 1919 ( medical journals , daily newspapers and archives ) . in this study we took advantage of the existence of vital statistics compiled in european countries by demographers , using a homogeneous method . we used the information available on the monthly variations of death figures to estimate the pandemic mortality burden , based on the values of a 12 years , were for example computed by simply making the difference between the 1918 and 1920 yearly mortality data and the values observed in the preceding and succeeding 3year periods . the comparison of the datasets used in the different mortality assessments , and the results obtained , are shown in figure 5 . the changes in borders and the mass population movements related to world war 1 may be one reason for these discrepancies . for example , in germany , we checked that using the raw data ( not corrected for the mismatches of populations and deaths ) would have lowered our estimation of 426 574 to 340 000 , changing the excess mortality estimate from 73 to 67% . in total , however , the agreement between estimates are more remarkable than the discrepancies . for example , the total european burden was estimated at 2 300 000 in the two first reports , published in 1991 and 2002 , based respectively on 18 countries and 21 countries , at 2 005 569 deaths in the paper from murray et al . ( total of their 13 european values ) versus 1 980 950 deaths in this report . comparison of published mortality estimates in europe ( number of deaths ) during the 19181919 influenza pandemic . circle : johnson and mueller ( j ) ; dark circle : ansart et al . ( a ) ; square : patterson and pyle ( p ) ; triangle : murray et al . as the 14 european countries we have studied in this paper represented 75% of the european population at that time , one can deduce that 26 million excess deaths ( 11% of the total population ) occurred in europe during the period when spanish flu was circulating . american excess deaths during the same period were estimated at 550 000 ( corresponding to 065% of the american population ) by glezen wp . however , johnsonmueller put the us figure at 675 000 deaths and more recently murray et al . at only 400 000 deaths ( 047% of the total population ) . whatever is taken as estimation of the american pandemic death burden , it is well below the european estimations we provide and others have provided . a possible explanation is that , at the end of ww1 , europe was characterised by massive civilian and army movements , a health care system put at its minimum and frail populations . this has very likely heavily impacted the european death toll . finally , we measure the indirect impact of the influenza virus on the mortality . herein , we analyzed allcause mortality and not only influenza or pneumonia deaths . indeed , we were obliged to base our estimation on the available monthly data on concerned total mortality , not influenza and pneumonia ( p&i ) mortality however , there is a striking linear relationship between the overall annual influenza and pneumonia mortality records , for each of the 11 countries where such data were available , and the total number of excess deaths ( figure 6 ) . this is consistent with the hypothesis that p&i mortality is a relatively constant proportion of the total number of deaths , and supports our use of the total number of excess deaths to quantify the death toll of the pandemic . p&ideaths plotted versus allcause excess mortality in 11 european countries*. spearman s rank correlation = 094 ; p < 10 . * germany , spain , france , italy , norway , netherlands , portugal , sweden , switzerland , england and wales and scotland . there was a high level of variability between the excess of mortality experienced in the 14 countries we studied , with a minimal value of 33% in finland and a maximal value of 172% in italy . all factors that can be related to the mortality burden such as the existence of coinfectious diseases , especially bacterial pneumonia and tuberculosis , or socioeconomic status were unlikely to have a north south gradient . possible geographic determinants of the influenza mortality are cold temperature and vitamin d deficiency relative to low sunlight exposure , but they would have implied a higher mortality in northern countries , not the opposite as we observed . we observed a high degree of synchronism in europe , with the highest mortality peak occurring in all countries within a 2month window ( oct nov 1918 ) ( 3 , 4 ) . all but two countries ( denmark and italy ) experienced at least a twowave pattern . in addition to this common feature , six countries had an earlier first wave of excess mortality before october 1918 ( bulgaria , portugal , germany , finland , switzerland and spain ) and five countries ( spain , denmark , finland , germany and switzerland ) had one late excessmortality waves in february and march 1920 . the very peculiar profile of mortality in finland could be explained by their civil war that caused 30 000 deaths in early 1918 ( that could not be excluded directly from our analysis because of their unknown spatiotemporal distribution ) . a fourth wave having occurred in the early spring 1920 similar to the one we found was reported before in specific settings . including 6 additional months in the calculation of excess mortality ( until july 1920 ) , would have increased the burden estimates as follows : 405 800 ( + 153 744 ) deaths in spain , 60 500 ( + 5768 ) in finland , 13 600 ( + 3044 ) in denmark , 510 200 ( + 83 663 ) in germany and 39 000 ( + 9080 ) in switzerland . where the 1918 pandemic first emerged is still being debated . a recent analysis of the 1918 h1n1 genome failed to single out a particular location . , the origin has been successively proposed in asia , in a british army post in france in 1916 , , in usa , , , , or in spain . the european origin was not supported by taubenberger et al . in their 1997 report on the sequence of the 1918virus genome extracted from autopsy samples ( obtained from the armed forces institute of pathology in washington , dc ) of 28 us servicemen who died of the flu during their military service . our findings can provide clues as to the origin of the pandemic and do not make plausible an european origin : indeed , if the pandemic would have started somewhere in europe , we would have expected to see a spatiotemporal spread outwards from this hypothetical origin , while the data show that all the european countries reached simultaneously their epidemic peaks . all authors contributed to the study and agree with the contents of the manuscript . conceived and designed the study : sa , ajv , pyb , cp . collected the data : sa . contributed materials , analysis tools : fc , af . wrote the paper : sa , ajv .

background the origin and estimated death toll of the 19181919 epidemic are still debated . europe , one of the candidate sites for pandemic emergence , has detailed pandemic mortality information . objective to determine the mortality impact of the 1918 pandemic in 14 european countries , accounting for approximately threequarters of the european population ( 250 million in 1918 ) . methods we analyzed monthly allcause civilian mortality rates in the 14 countries , accounting for approximately threequarters of the european population ( 250 million in 1918 ) . a periodic regression model was applied to estimate excess mortality from 1906 to 1922 . using the 19061917 data as a training set , the method provided a nonepidemic baseline for 19181922 . excess mortality was the mortality observed above this baseline . it represents the upper bound of the mortality attributable to the flu pandemic . results our analysis suggests that 264 million excess deaths occurred in europe during the period when spanish flu was circulating . the method provided space variation of the excess mortality : the highest and lowest cumulative excess / predicted mortality ratios were observed in italy ( + 172% ) and finland ( + 33% ) . excessdeath curves showed high synchrony in 19181919 with peak mortality occurring in all countries during a 2month window ( oct nov 1918 ) . conclusions during the spanish flu , the excess mortality was 11% of the european population . our study highlights the synchrony of the mortality waves in the different countries , which pleads against a european origin of the pandemic , as was sometimes hypothesized .

serial analysis of gene expression ( sage ) is a powerful method for obtaining comprehensive and quantitative gene expression profiles from cell populations under selected physiological conditions . sage counts polyadenylated transcripts by sequencing a short 14 bp tag at the gene 's 3 end , adjacent to the last restriction site . the tag counts are than archived electronically for future analysis and comparisons . since the first publication introducing sage ( 1 ) , computational tools ( 27 ) and statistical methods ( 820 ) have been developed to correctly perform the analysis of a sage experiment . because sage determines absolute expression levels , comparisons between different sage libraries are relatively easy to perform . for this reason , sage has been selected as the major platform technology for the cancer genome anatomy project ( cgap ) . for 5 years , a large number of sage libraries , generated from diverse cancer and normal tissues in many laboratories , have been accessible via the national center for biotechnology information website ( ) . the sage genie website ( ) was constructed recently and is equipped with additional search and presentation tools ( 7 ) . these libraries were all constructed by using the same anchoring enzyme , thus all yielding tags likely to be 10 bp downstream from the 3 most nlaiii site in the transcript . the aim of most sage studies is to identify genes of interest by comparing the number of specific tags found in two different sage libraries . one of the most attractive applications of transcript profiling is to address the question of expression differences between normal and cancer samples or cancer and metastasis samples in order to define new diagnostic markers and therapeutic targets . over the past few years , several methods have been reported for determining the statistical significance of gene expression difference provided by the sage experiments . most of these methods have been incorporated into public database systems and analysis programs ( 24,68,1013,1820 ) . if within two types of cells y and z , a particular mrna species has unknown respective concentrations y and z and a total of a tags are sequenced from cell type y and b tags from cell type z , and among these , a and b tags , respectively , correspond to the mrna of interest , the question is : what inference may be made about the relative size of the actual concentrations , y and z ? they consider the null hypothesis h0 that y = z , and the alternative that y z , and derive formulas based upon the observed data for rejecting h0 with various degrees of confidence . if a / a and b / b differ significantly , one rejects h0 , concluding that the expression levels y and z are unequal . we have used their statistical approach and developed a useful and flexible tool ( websage ) that analyse and compare a large number of sage tags . the novelty of our tool compared with those available online ( 3,4 ) consists of the visualization of the results of the comparison of two sage libraries in a scatter plot . one of the libraries is presented on the x - axis and the second one on the y - axis . more than two libraries can not be visualized using our tool unless to pool libraries composed by the sum of all libraries from one group such as cancer group and all libraries from normal group . however , vncio et al . ( 20 ) showed clearly that this summing is an error and proposed a statistical analysis that accounts for the within class variability . in our tool , full sage data are very comprehensively represented in plots , which correspond to one or a set of tags , having the same p - value and the same apparition in two libraries . moreover , websage gives not only the identification but also the function of the gene . to gain insights into the molecular basis for metastasis , we compared the global gene expression profile of metastatic pancreatic cancer with that of primary cancer . the two cell lines were established from a 60-year - old woman with surgically removed pancreatic carcinoma that presented simultaneously metastatic liver lesion ( 21 ) . we isolated total cellular rna from pancreatic cancer cells and metastatic liver cells and constructed each sage library , using sage protocol version 1.0c ( 1 ) . afterwards , tags were extracted from the raw sequence data and repeated ditags were excluded using the sage software ( version 1 ) developed by kinzler and co - workers ( 1 ) . from the two sage libraries 12 090 tags were obtained , enabling us to compare the expression levels of 4807 unique transcripts . at this step , the question of interest is whether one sample has a significant change in expression relative to the other sample for each transcript . over the past few years , several methods have been developed for determining the statistical significance of gene expression difference derived from the sage experiments . zhang et al . ( 17 ) used a simulation approach to determine the probability of obtaining the observed difference and more extreme ones . other statistical approaches of the number of specific tags found in sage can be envisioned . ( 19 ) used a test for differences in tag numbers found in two experimental conditions , seemingly based on poisson distribution statistics . their approach suffers from the disadvantage that it can only be used reliably on tag libraries of similar size . the second approach is to look at the number of copies of a specific mrna per cell as a fraction or proportion of the total number of mrna molecules in that cell . the same proportion ( p ) of specific tags should be present in the sage library of all sequenced tags . in this approach , the number of tags found is binomially distributed because it is the result of the p of each tag to be identified as being the specific mrna or not . a bayesian method has been used by audic and claverie ( 8) and vncio et al . comparison of this test and the test based on poisson - distributed tag counts used by madden et al . ( 19 ) shows a difference in sensitivity , the test of madden et al . being more conservative ( 10 ) . to quantify the transcripts of the two cell line , we have used the significance test ( ) of audic and claverie ( 8) and developed a tool to visualizing the analysis of differentially expressed tags in a scatter - plot shape ( log / log coordinates ) . websage is the software that enables a rapid and comprehensive analysis of the sage data and integrates statistical data analysis methods using a database system . websage enables simple and rapid analysis of a file of tags and the determination of differentially expressed tags ( p - values ) . to provide tag - to - gene links , three files from the ncbi ftp site ( sage genie ) were stored in database ( mysql ) and used at each analysis , to get the tag - to - gene assignment , gene accession number ( unigene and genbank ) and gene function information ( kegg , biocarta and gene ontology databank ) , respectively . sage genie provides the best match between gene name and tag , although there are options for manually viewing an alternate tag 's expression data . furthermore , the information is stored in a table and visualized in a scatter plot . figure 1 shows an example of websage analysis of our file of tags . the total number of tags analysed included 5257 tags from pancreatic cancer cell line and 6833 from cell line derived from liver metastasis . websage computed a p - value and classified the tags into three groups : green , p 0.01 ( significant ) ; red , p > 0.05 ( not significant ) ; and yellow , p in ] 0.01 ; 0.05 ] ( require further analysis ) . the information computed for the query is stored in a temporary table during the session . next , the data are exported in the csv format ( compatibility with all spreadsheets ) . our application is a web tool , developed in the php language using jpgraph library ( ) that has been modified . , websage is a useful web service that performs statistical analysis on the sage data , identifies the tags differentially expressed and shows the results in a scatter plot . moreover , the user can query with a specific tag and get a tag - to - gene assignment and gene function from kegg , biocarta and gene ontology databank . a total of 4809 tags from pancreatic cancer lines and an isogenic liver metastasis cell line were analysed . each plot corresponds to one tag or a set of tags , having the same p - value and the same apparition on the two libraries . . once clicked , a web page is displayed with a list of tags and their identification . the identification consists of the name of the tag and the corresponding gene , the p - value and the unigene and genbank accession number .

the serial analysis of gene expression ( sage ) is a powerful method to compare gene expression of mrna populations . to provide quantitative expression levels on a genome - wide scale , the cancer genome anatomy project ( cgap ) uses sage . over 7 million sage tags , from 171 human cell types have been assembled . the growing number of laboratories involved in sage research necessitates the use of software that provides statistical analysis of raw data , allowing the rapid visualization and interpretation of results . we have created the first simple tool that performs statistical analysis on sage data , identifies the tags differentially expressed and shows the results in a scatter plot . it is freely available and accessible at .

kyphosis is a progressive curvature of the thoracic spine , which results in rounding or bowing of the back . a curved spine accompanied by restricted neck motion and poor lung reserve present challenges for an ophthalmic surgeon , who is used to patients neck and head resting flat on the operating table and the eye in the horizontal position . intraoperative difficulties include poor view , inability to maneuver surgical instruments properly and constant threat of elevated vitreous pressure . several innovative solutions have been published , including the maximal reverse trendelenburg position and pillows under the head and neck , use of a donut and wedge - shaped head positioner , allowing patients to remain in the upright sitting position while their head is positioned horizontally in a modified waiting room chair , performing surgery while standing [ 5 , 6 ] , using a 4-section orthopedic operating table , or laying the patients on their sides . however a 49-year - old obese male with controlled secondary glaucoma needed cataract surgery in the right eye . the past ocular history was positive for recurrent uveitis and glaucoma shunt surgery in the right eye . cataract density was + 4 , and the best - corrected visual acuity was 20/200 . the anterior chamber was shallow and the glaucoma shunt tube was visible at the 11 o'clock position . the patient was planned for pupillary dilation using iris retraction hooks , lens capsule staining with trypan blue and phacoemulsification with intraocular implantation under local anesthesia . the anesthesiologist was reluctant to use general anesthesia because of the patient 's disability and poor health . in the operating room , the patient could only recline to about 40 from the vertical axis despite maximal reverse trendelenburg position and a number of pillows under his back , neck , head and legs ( fig . 2 ) . while trying to focus the microscope , it became evident that by grasping the eye at the inferior limbus with a fine forceps and pulling it upwards , it could be moved to a more desirable horizontal position . a single 7/0 vicryl ( polyglactin ) corneal retraction suture was placed at the limbus at the 6 o'clock position to achieve the desired view ( fig . the assistant surgeon was able to rotate the eye to the desired positions during the whole procedure by relaxing or pulling the suture . patients with kyphosis may present several unique challenges to an eye surgeon , the foremost being the inability to lay the head and neck in the ideal horizontal position . the resulting poor view makes focusing and manipulation of tissues and instruments difficult . tilting the head end of the operating table down and raising the patient 's legs may compromise venous return from the orbit , causing venous engorgement and elevated posterior vitreous pressure during surgery . in addition , the patient 's poor pulmonary reserve itself may further impede orbital / jugular venous return , resulting in increased posterior vitreous pressure . other features of kyphosis that may adversely affect intraocular surgery are tenderness and stiffness of the back and difficulty in breathing . several practical solutions have been described by very experienced surgeons but complications may still occur . a wedge - shaped pillow with a built - in donut for the positioning of the head has been used by some surgeons . livingston and mackool recommended using more than one of these positioners in severe cases . another approach suggested performing cataract surgery while standing , using loupes and making an inferotemporal scleral tunnel [ 5 , 6 ] . the surgeons were unable to remove the subincisional cortex because of a shallow anterior chamber secondary to elevated posterior vitreous pressure . prasad et al . recommended using a 4-section orthopedic operating table to enable an extreme reverse trendelenburg position for cataract and glaucoma surgery . however , because of a blurred vision , these authors were unable to perform curvilinear capsulorhexis and had to resort to can - opener capsulotomy . in short , there is no single solution for the different groups of difficult patients . we have found that a single retraction suture at the inferior limbus can rotate the eye to any desired position . this may allow both the patient and surgeon to be in a comfortable position without compromising good view . it may be tried practically in all patients with kyphosis or other conditions that prevent them from reclining such as torticollis , chronic obstructive pulmonary disease , chronic congestive heart failure or morbid obesity . the surgeon can still sit on the preferred temporal side of the patient and perform intraocular surgery of choice . with this maneuver , it may be unnecessary to resort to extreme and uncomfortable tilting of the patient and avoid complications such as those described above . the authors have no financial or proprietary interest in the materials described in the article .

we describe a 49-year - old man with advanced kyphosis and dense cataract , who could only recline to about 40 from the vertical axis despite a maximal reverse trendelenburg position and pillows under the head , neck , shoulders and knees . with a single corneal retraction suture at 6 o'clock , the eye could be rotated horizontally , which enabled the surgeon to perform a complex cataract surgery despite prior glaucoma shunt , posterior synechiae , a small pupil and the need to stain the capsule . as the eye can be brought into any desired position with a retraction suture , patients with kyphosis or other conditions that prevent them from assuming a supine position can still have safe intraocular procedures . this maneuver reduces the need to tilt patients to an uncomfortable position that may cause pain , increased breathing difficulty and elevated posterior vitreous pressure .

amisulpride came into the indian market a few years back with hypes and hopes in the management of schizophrenia . its broad spectrum effectiveness with lower chances of extrapyramidal symptoms ( eps ) and metabolic syndrome did help psychiatrists to treat schizophrenia and related disorders more effectively . although this antipsychotic does not block serotonin receptors at all , it is a high - affinity and highly selective d3/d2 receptor antagonist with atypical properties . its selective affinity for dopamine receptors in the limbic structures , but not in the striatum , leads to a low risk of extrapyramidal side effects . all available reports suggest that chance of eps is very less with amisulpride at doses < 400 mg / day . however , there are sporadic reports of drug - induced eps including dystonia and akathisia even in patients receiving low doses of amisulpride . here a 30-year - old male with schizophrenia for the past 10 years now presented with predominantly negative symptoms . he was on olanzapine 15 mg / day for more than 6 months without much improvement . hence , amisulpride was instituted with a starting dose of 50 mg / day with a gradual increment up to 300 mg / day within 14 days . the patient came after 14 days to the casualty with features of parkinsonian syndrome such as slowed gait , mild rigidity , salivation , and bradykinesia . he was hospitalized , amisulpride was immediately stopped , and trihexyphenidyl 4 mg / day was given to manage the side effect . his eps gradually subsided and for negative symptoms , clozapine was introduced at a small dose of 25 mg / day and gradually increased to 200 mg / day over a period of 10 days . at the time of discharge , on the 14 day , he was free from parkinsonian symptoms . subsequent follow - up showed no parkinsonian symptoms and he had modest improvement in negative symptoms . a 48-year - old male with schizophrenia for the last 20 years was treated with various antipsychotics without much improvement . since the last 6 months , he was on olanzapine 15 mg / day . as there was no significant improvement , his olanzapine dose was gradually tapered and stopped over a period of 14 days and was started on amisulpride 100 mg / day and was increased to 200 mg / day over a period of 3 weeks . the patient returned on the 24 day with severe parkinsonian symptoms . in this patient also , there was no prior history of parkinsonism . we managed him with injection promethazine 25 mg intramuscular bid first 3 days along with trihexyphenidyl 2 mg bid after stopping amisulpride . after 7 days , parkinsonian symptoms improved considerably and clozapine was introduced at a dose of 25 mg / day which was subsequently increased to 100 mg / day on the 10 day and the patient was discharged . since the discovery that clozapine induces fewer eps and is more effective for negative symptoms than conventional antipsychotics for the treatment of schizophrenia , psychopharmacological research has focused on the development of drugs that block central 5-ht2 receptors more than d2 receptors . combined 5-ht2/d2 receptor antagonism is the most current explanation for the so - called atypical profile of some antipsychotics . amisulpride at low doses binds selectively to dopamine d2 , d3 autoreceptors , thereby enhancing dopaminergic transmission and thus might be effective for negative symptoms . it has no affinity for d1 , d4 , and d5 receptor subtypes . at higher doses , it blocks postsynaptic receptors , thus inhibiting dopaminergic hyperactivity . at the same time , amisulpride has greater specificity for the limbic system and thus has low incidence of eps . amisulpride binds more loosely than dopamine to the dopamine d2 receptor and is rapidly dissociated from the dopamine d2 receptor . low - dose therapy with amisulpride is associated with a significantly lower blockade of striatal dopamine d2 receptors than is seen during high - dose treatment . however , a significant striatal d2 blockade was demonstrated at therapeutically effective dose ranges , and a good relationship between the degree of striatal dopamine d2 receptor occupancy and the amisulpride plasma concentration or the administered dose was shown . in general , asians are slow metabolizers . low body weight and slow metabolism may increase the plasma concentration of drugs causing side effects . reported a low postsynaptic d2 occupancy in the striatum at low doses of amisulpride ( 50100 mg / day ) . it has also been suggested that extrastriatal binding could mediate the effect on negative symptoms . the probable causes of eps with low doses of amisulpride could be that it blocks postsynaptic d2 receptors significantly in striatum without much effect in the mesolimbic pathway . , the lower incidence of eps which is claimed by western researchers as well as pharmaceutical companies should be studied well in the indian context . we should at least keep this side effect in our minds while starting or increasing the doses .

amisulpride , recently introduced atypical antipsychotic , is well - known for its broad spectrum effectiveness and lower profile for extrapyramidal side effects ( eps ) . its selective affinity for dopamine receptors in the limbic structures , but not in the striatum , leads to a low risk of extrapyramidal side effects . here , we report two cases of eps associated with lower dose of amisulpride . the proposed mechanism for its causation is also discussed . authors invite more studies , specifically from the indian context to find out the incidence of eps and other associated side effects .

the prevalence of aggression is 8 - 20% in three- to six - year - old children . biological and genetic factors , environmental learning , cognitive processing and personal stimuli or motives are some important causes of aggression . treatment of aggressive behavior at an early age is very important , as aggression in early years of life sets the ground for many problems in personal and interpersonal areas of the lives of aggressive children . for instance , it can lead to a weak self - concept , being rejected by peers , poor academic performance and can serve as a predictor of delinquency , depression , academic failure , substance abuse , more inappropriate and aggressive reaction to social issues , and choosing more aggressive solutions for solving problems later in life . on the other hand , childhood exposure to aggression may also influence life - long health through biological mechanisms . also , the economic costs imposed on the community due to youth aggression , bullying and violence are extremely high . yet , social - learning theorists contend that environmental factors are responsible for learning aggressive behaviors and continuing them . therefore , the children who learn aggressive behaviors are able to avoid such behaviors by means of reinforcement behavior therapy . reinforcement behavior therapy is defined as the employment of reinforcement and extinction in order to increase the occurrence of desirable behaviors and decrease the incidence of behaviors that interfere with intended behavior . ( 2011 ) compared the effectiveness of two methods , namely reinforcement and reward behavioral therapy and ellis s cognitive therapy , in the degree of aggression among 89 parentless children . the reinforcement and reward behavioral therapy method led to a significant reduction in aggression mean score before and after applying the procedure , whereas ellis s cognitive therapy technique did not show any significant impact on lowering aggression . the study of khazaie and colleagues did suggest the effectiveness of behavioral therapy in aggression ; however , it was carried out by a psychotherapist . in many countries , factors , such as insufficient number of professional psychotherapists or expenses of the procedure , therefore , training kindergarten and day - care centers teachers can be an appropriate alternative for offering proper interventions under the supervision of a therapist , so that a greater number of preschool children can benefit from such procedures . school environment interventions that impact on a range of health risk behaviors , including aggression , are likely to be one of the most efficient ways of modifying population - level risk . schools approaches to discipline , behavior management and aggression prevention vary widely and are rarely evidence - based , and that further resources and research are urgently needed to combat aggressive behaviors . although a large number of studies have so far been conducted on the effectiveness of complimentary therapies in reducing children s aggression , most of them have fundamental limitations . some such limitations include a greater focus on aggressive boys than girls , more emphasis on overt forms of aggression in comparison to more subtle forms like relational aggression . another treatment alternative is family therapy which , although very effective in children s behavioral problems , is quite costly and time - consuming . there is , therefore , a pressing need to determine which interventions are effective in addressing aggression in preschoolers , and if the intervention can apply effectively by kindergarten teachers to scale up such interventions across local and national school networks especially in developing countries . since children spend a great part of their active hours in kindergarten in close contact with their teachers , training the teachers can make a significant contribution to the elimination of aggressive behaviors . moreover , given the fact that each teacher is in charge of several children , providing teachers with trainings will result in saving the time and cost . in addition , the existing studies have addressed almost all forms of aggression except for impulsive anger . therefore , the present study has focused on all forms of aggression and aims to identify the effect of reinforcement behavior therapy by kindergarten teachers on all types of aggressive behavior among kindergarten and preschool children . it was a cluster randomized control trial research with a pretest / post - test design and control group . the study compared two groups of aggressive children , the intervention group receiving reinforcement behavior therapy and routine care by kindergarten teachers and the control group only receiving routine care . mohr is located in iran , in the southernmost part of fars province , 360 km away from shiraz . currently , there are 8 urban and 27 rural kindergartens and daycare centers in this city and about 1000 children attend these centers . the research population consisted of all three- to six - year - old kindergarten and preschool children in mohr . the inclusion criteria of the study were parental consent , children s age range of 3 - 6 years , and obtaining a minimum aggression score of 117.48 for girls and 125.77 for boys ( according to preschoolers aggression questionnaire constructed by shahram vahedi et al . on the other hand , the children or families with emotional crisis during the 2 months of intervention 14 out of the 35 existing kindergartens were selected through cluster random sampling method , as recommended by a demographer . then , all the 14 kindergartens were randomly allocated into either the intervention ( n=7 ) or the control group ( n=7 ) through block randomization by the researcher s assistant . the preschool aggression scale was distributed among the teachers to complete it for all 3 - 6 year old children in 14 kindergartens . using a study entitled prevalence of behavioral problems in 3 to 6 year old children in hamadan city and based on effect size=0.95 , power of 0.8 , and =0.05 , a 54 subject sample size ( 27 subjects in each group ) was determined for the present study . considering the probable loss in the sample , more than 117.48 for girls were randomly selected by simple randomization procedure by a table of random numbers from the list of aggressive children and their parents were asked to complete the written informed consent forms . the teachers of the kindergartens ( no=14 ) in the intervention group received 4 educational sessions based on positive reinforcement behavior therapy , but the teachers of kindergartens in the control group did not receive any interventions during the research period . each teacher in the intervention and control groups had about 15 children in his / her class . during the study , consort flow diagram of study participants in this study , the data were collected using preschoolers aggression questionnaire . this questionnaire aimed to evaluate various types of aggression , including verbal aggression , physical aggression , relational aggression , and impulsive anger . the reliability and validity of preschool aggression scale was studied by shahram vahedi et al . ( 2007 ) in a research on evaluation of aggression in preschool children of urmia , iran . the cronbach s alpha was estimated to be 0.98 for the whole scale , 0.93 for verbal aggression , 0.92 for physical aggression , 0.94 for relational aggression , and 0.88 for impulsive anger . a factor analysis of this scale using principal component analysis with varimax rotation resulted in four elements of verbal aggression , physical aggression , relational aggression , and impulsive anger , which was indicative of the construct validity of the scale . therefore , this aggression measurement scale can be used as a valid and reliable instrument in educational and clinical settings . in this 43-item questionnaire , the first 14 questions are related to verbal aggression and the next 13 are about physical aggression ; also , there are 9 questions on relational aggression , and the last 7 items deal with impulsive anger . this questionnaire was filled out by the teacher using 5 options ( 0=never , 1=rarely , 2=once a month , 3=once a week , and 4=often ) . the scores of verbal aggression , physical aggression , relational aggression , and impulsive anger were between 0 and 56 , 0 and 52 , 0 and 36 , and 0 and 28 , respectively . the children whose aggression scores were two standard deviations above the mean ( 117.47 for girls and four training sessions , each lasting for two hours , were planned for the teachers in the intervention group who were ready to take part in the study by an interventionist who was unaware of the aim of the study and was professional in behavior therapy . in the first session , the teachers were given some information about mental , psychological , and physical characteristics of the preschool children , and gained an understanding of how to deal with them properly . in the second session , the teachers learned about aggression in children , its types , causes , importance of early treatment of aggressive children , and general treatment techniques and methods . the third session intended to make teachers familiar with behavioral therapy and its various techniques , with a focus on reinforcement behavior therapy . the fourth and final session was entitled practical work on reinforcement and reward behavior therapy , and aimed to demonstrate how this technique works in practice , how to write down the details , and how to prepare an anecdotal record . the teachers were also given a written and practical test on proper practice of the technique . then , the teachers were asked to practice what they had already learned during their four sessions of training for aggressive children in the intervention group for 8 weeks . they were also asked to complete anecdotal record for every aggressive event in children , so that the researcher could follow up any changes in the aggressive children as well as the way the teachers implemented the procedure . in these forms , the teachers were asked to describe their reactions towards the children s aggressive behaviors in details and free from any judgment . then , in his weekly visits to the kindergartens , the researcher would review these forms and discuss them together with teachers . afterwards , if necessary , the teacher s reactions were modified and the required instructions were given . the control group , however , received no interventions throughout the course of research . yet , the demographic information questionnaire and the preschoolers aggression questionnaire were filled out by the teachers . after this project was carried out for two months , the teachers were asked to complete the same preschoolers aggression questionnaire for the children in both intervention and control groups for the second time and the results were evaluated by the researcher . the outcome measures in this study included the children s scores in the preschoolers aggression . the study data were collected by kindergarten teachers before and after the eighth week of the intervention . the teachers completed a demographic information questionnaire and the preschoolers aggression questionnaire . in this study , the data were analyzed using statistical package for the social sciences ( version 15 , spss inc , chicago , il ) . to evaluate the homogeneity of the participants characteristics in the intervention and control groups , chi - square smirnov test , paired t test and student s t - test were used for statistical analysis , as well . besides , p<0.05 was considered as statistically significant . the study was conducted in accordance with the human subjects protection principles ( declaration of helsinki ) . ethics committee approval was obtained from the research ethics committee of shiraz university of medical sciences . a written informed consent was obtained from the teachers and family of children for participation in the study . it provided the subjects with some information about the study , such as the purpose , e procedure , possibility of sharing the study results after completion , and promised anonymity in the event of publication of the study results . in addition , the subjects were assured that participation / non - participation would not affect their received care . the participants were allowed to withdraw from the research at any stage of the process if they or their parents were unwilling to continue their cooperation . moreover , after completion of the study , a handbook was prepared and given to the teachers and parents of the control group children . mohr is located in iran , in the southernmost part of fars province , 360 km away from shiraz . currently , there are 8 urban and 27 rural kindergartens and daycare centers in this city and about 1000 children attend these centers . the research population consisted of all three- to six - year - old kindergarten and preschool children in mohr . the inclusion criteria of the study were parental consent , children s age range of 3 - 6 years , and obtaining a minimum aggression score of 117.48 for girls and 125.77 for boys ( according to preschoolers aggression questionnaire constructed by shahram vahedi et al . on the other hand , the children or families with emotional crisis during the 2 months of intervention 14 out of the 35 existing kindergartens were selected through cluster random sampling method , as recommended by a demographer . then , all the 14 kindergartens were randomly allocated into either the intervention ( n=7 ) or the control group ( n=7 ) through block randomization by the researcher s assistant . the preschool aggression scale was distributed among the teachers to complete it for all 3 - 6 year old children in 14 kindergartens . using a study entitled prevalence of behavioral problems in 3 to 6 year old children in hamadan city and based on effect size=0.95 , power of 0.8 , and =0.05 , a 54 subject sample size ( 27 subjects in each group ) was determined for the present study . considering the probable loss in the sample , more than 117.48 for girls were randomly selected by simple randomization procedure by a table of random numbers from the list of aggressive children and their parents were asked to complete the written informed consent forms . the teachers of the kindergartens ( no=14 ) in the intervention group received 4 educational sessions based on positive reinforcement behavior therapy , but the teachers of kindergartens in the control group did not receive any interventions during the research period . each teacher in the intervention and control groups had about 15 children in his / her class . during the study , this questionnaire aimed to evaluate various types of aggression , including verbal aggression , physical aggression , relational aggression , and impulsive anger . the reliability and validity of preschool aggression scale was studied by shahram vahedi et al . ( 2007 ) in a research on evaluation of aggression in preschool children of urmia , iran . the cronbach s alpha was estimated to be 0.98 for the whole scale , 0.93 for verbal aggression , 0.92 for physical aggression , 0.94 for relational aggression , and 0.88 for impulsive anger . a factor analysis of this scale using principal component analysis with varimax rotation resulted in four elements of verbal aggression , physical aggression , relational aggression , and impulsive anger , which was indicative of the construct validity of the scale . therefore , this aggression measurement scale can be used as a valid and reliable instrument in educational and clinical settings . in this 43-item questionnaire , the first 14 questions are related to verbal aggression and the next 13 are about physical aggression ; also , there are 9 questions on relational aggression , and the last 7 items deal with impulsive anger . this questionnaire was filled out by the teacher using 5 options ( 0=never , 1=rarely , 2=once a month , 3=once a week , and 4=often ) . the scores of verbal aggression , physical aggression , relational aggression , and impulsive anger were between 0 and 56 , 0 and 52 , 0 and 36 , and 0 and 28 , respectively . the children whose aggression scores were two standard deviations above the mean ( 117.47 for girls and 125.77 for boys ) were diagnosed as aggressive . four training sessions , each lasting for two hours , were planned for the teachers in the intervention group who were ready to take part in the study by an interventionist who was unaware of the aim of the study and was professional in behavior therapy . in the first session , the teachers were given some information about mental , psychological , and physical characteristics of the preschool children , and gained an understanding of how to deal with them properly . in the second session , the teachers learned about aggression in children , its types , causes , importance of early treatment of aggressive children , and general treatment techniques and methods . the third session intended to make teachers familiar with behavioral therapy and its various techniques , with a focus on reinforcement behavior therapy . the fourth and final session was entitled practical work on reinforcement and reward behavior therapy , and aimed to demonstrate how this technique works in practice , how to write down the details , and how to prepare an anecdotal record . the teachers were also given a written and practical test on proper practice of the technique . then , the teachers were asked to practice what they had already learned during their four sessions of training for aggressive children in the intervention group for 8 weeks . they were also asked to complete anecdotal record for every aggressive event in children , so that the researcher could follow up any changes in the aggressive children as well as the way the teachers implemented the procedure . in these forms , the teachers were asked to describe their reactions towards the children s aggressive behaviors in details and free from any judgment . then , in his weekly visits to the kindergartens , the researcher would review these forms and discuss them together with teachers . afterwards , if necessary , the teacher s reactions were modified and the required instructions were given . the control group , however , received no interventions throughout the course of research . yet , the demographic information questionnaire and the preschoolers aggression questionnaire were filled out by the teachers . after this project was carried out for two months , the teachers were asked to complete the same preschoolers aggression questionnaire for the children in both intervention and control groups for the second time and the results were evaluated by the researcher . the outcome measures in this study included the children s scores in the preschoolers aggression . the study data were collected by kindergarten teachers before and after the eighth week of the intervention . the teachers completed a demographic information questionnaire and the preschoolers aggression questionnaire . in this study , the data were analyzed using statistical package for the social sciences ( version 15 , spss inc , chicago , il ) . to evaluate the homogeneity of the participants characteristics in the intervention and control groups , chi - square , fisher exact test and student s t test were applied . smirnov test , paired t test and student s t - test were used for statistical analysis , as well . besides , p<0.05 was considered as statistically significant . the study was conducted in accordance with the human subjects protection principles ( declaration of helsinki ) . ethics committee approval was obtained from the research ethics committee of shiraz university of medical sciences . a written informed consent was obtained from the teachers and family of children for participation in the study . it provided the subjects with some information about the study , such as the purpose , e procedure , possibility of sharing the study results after completion , and promised anonymity in the event of publication of the study results . in addition , the subjects were assured that participation / non - participation would not affect their received care . the participants were allowed to withdraw from the research at any stage of the process if they or their parents were unwilling to continue their cooperation . moreover , after completion of the study , a handbook was prepared and given to the teachers and parents of the control group children . the two groups were compared in terms of the children s age , both parents age , both parents level of education , both parents occupation , and family income ; they were similar regarding all the features , except for father s age . the means ( sd ) of aggression score in boys and girls were compared and shown in table 2 . all of the teachers were female between 20 - 35 years old and most of them had a bs degree ( n=20 , 72% ) ; 28% of them had high school diploma level of education ( n=8 ) . comparison of the demographic characteristics in the control and intervention groups meansd scores of aggression among boys and girls in both groups moreover , no significant difference was found between the two groups regarding the total aggression score ( p=0.55 ) as well as the scores of the four subscales prior to the intervention ( table 4 ) the mean score of aggression after the intervention showed a statistically significant difference between the two groups in this regard ( p=0.01 ) ( table 3 ) . moreover , a statistically significant change was observed in the intervention group s mean score of aggression after the intervention ( p<0.02 ) ; also , such a difference was found in the control group ( p<0.023 ) , but the results showed that the mean score of aggression in the control group was increased in the post - test compared to the pre - test . furthermore , the results revealed a decrease in the mean scores of verbal aggression , physical aggression , relational aggression , and impulsive anger in the intervention group after the intervention . yet , the difference between the intervention and control groups was statistically significant only for physical and verbal aggression subscales ( p=0.02 , p<0.01 ) ( table 4 ) . comparison of the frequency distribution of the participants in the intervention and control groups based on demographic characteristics x = chi - square ; f = fisher exact test comparison of the changes in the mean scores of various types of aggression in the preschoolers in the intervention and control groups before and after the intervention the present study aimed to investigate the effect of training kindergarten teachers regarding reinforcement behavior therapy on reducing different forms of aggression in preschool children . the study findings indicated that using reinforcement behavior therapy by kindergarten teachers resulted in a significant decrease in the total aggression , verbal and physical aggression , scores in the intervention group compared to the control group . it did alleviate the children s covert aggression , including relational aggression and impulsive anger , as well , but this change was not statistically significant . this finding supported the results of a study that indicated the effectiveness of reinforcement behavior therapy on abatement of childhood aggression . since children spend a long time at kindergartens in close contact with their teachers , it is possible to reform , or even eliminate , many behaviors that are possibly shaped at home . in this regard , a meta - analysis was performed by smeet et al , ( 2014 ) , on twenty - five studies to identify predictors of treatment response regarding cbt . furthermore , the treatment setting and duration did not seem to influence the treatment effect , which shows the need for development of more cost - effective and less - invasive interventions . there are limited studies that show the effect of behavior therapy by kindergarten teachers but to support the effectiveness of behavior therapy on the children s aggression , both verbal and physical , several studies have been conducted ; their results are all consistent with the present research . verbally and physically aggressive children can be detected more easily . since preventing this form of aggression parents are more sensitive and try to rectify the child s behavior and even seek help and advice from teachers . the children who display relational aggression are less responsive to psychological treatments because of the nature of relational aggression compared to other types and the deeper mechanisms involved in it . relationally , aggressive children were popular among their peers , causing short - term treatments not to have noticeable effects . the place these children gain among their peers because of their behavior makes them more resistant to medium - term psychological treatments . the results of the present study demonstrated a decrease in impulsive anger after the intervention ; however , this reduction was not statistically significant . this can be due to the fact that impulsive anger is a covert form of aggression and , consequently , it is sometimes not considered as aggression at all . ( 2012 ) showed that group therapy using parent - child relationship also did not have any significant effects on covert aggression . utilizing a primary , secondary and tertiary intervention model from the public health perspective can help the organization to address violence in each of these three domains . in the absence of school nurses in kindergartens , the teachers are the best choice for working with aggressive children . in this regard , the psychiatric mental health nurses in their community based approach can arrange educational programs for teachers to facilitate and improve their ability in providing psychiatric interventions such as reinforcement behavior therapy . short duration of treatment , small sample size , all female teachers , potential contamination across schools and the fact that evaluation of children s aggression at home is not possible were some major limitations of the present study . filling out the questionnaire by teachers can affect the result . in conclusion , the results of this research emphasized the effectiveness of reinforcement behavior therapy in reducing physical and verbal aggression in preschool children . nevertheless , it was shown that this technique was not sufficient to alleviate relational aggression and impulsive anger . this reminds us of the necessity for continuation of the treatment or designing another type of intervention which is more appropriate for these types of behavioral problems in children . in this study , therefore , using other psychological interventions in aggressive children is suggested to be used in future studies . studying the effect of positive reinforcement therapy on aggression and its sub - scales for more than 8 weeks is also recommended . moreover , qualitative studies are recommended to be performed on aggression in preschoolers in future . to improve evidence - based nursing , further studies on the impact of this intervention

background : aggression is a kind of behavior that causes damage or harm to others . the prevalence of aggression is 820% in 36 years old children . the present study aimed to assess the effect of training kindergarten teachers regarding reinforcement behavior therapy on preschoolers aggression.methods:in this cluster randomized control trial , 14 out of 35 kindergarten and preschool centers of mohr city , iran , were chosen using random cluster sampling and then randomly assigned to an intervention and a control group . all 370 kindergarten and preschool children in 14 kindergarten were assessed by preschoolers aggression questionnaire and 60 children who obtained a minimum aggression score of 117.48 for girls and 125.77 for boys were randomly selected . the teachers in the intervention group participated in 4 educational sessions on behavior therapy and then practiced this technique under the supervision of the researcher for two months . preschoolers aggression questionnaire was computed in both intervention and control groups before and after a two - month period.results:the results demonstrated a significant statistical difference in the total aggression score ( p=0.01 ) , verbal ( p=0.02 ) and physical ( p=0.01 ) aggression subscales scores in the intervention group in comparison to the control group after the intervention . but the scores of relational aggression ( p=0.09 ) and impulsive anger ( p=0.08 ) subscales were not statistically different in the intervention group compared to the controls.conclusion:this study highlighted the importance of teaching reinforcement behavior therapy by kindergarten teachers in decreasing verbal and physical aggression in preschoolers.trial registration number : irct2014042617436n1

post - transplant lymphoproliferative disease ( ptld ) is a serious , often fatal complication after solid organ transplantation . the incidence of ptld is greatest among heart ( 213% ) , lung ( 12% ) , and heart / lung ( 59% ) transplant recipients , but occurs less frequently in liver ( 2% ) , renal ( 13% ) , and bone marrow ( 12% ) transplant recipients . the patient s age , transplanted organ , epstein - barr virus ( ebv ) status of the donor and recipient before transplantation and the dosage of immunosuppressive drugs are considered risk factors . these differences in the incidence of ptld may be partly attributed to the higher doses of immunosuppressive drugs necessary following heart and lung transplantation . other possible contributing factors may be bronchus - associated lymphoid tissue ( balt ) and nodal tissue harboring ebv in the donor transplant . ebv infection , either preexisting in the recipient or acquired from the donor , is strongly implicated in the pathogenesis of ptld . immunosuppression may permit uncontrolled proliferation of ebv - stimulated b cells by inhibition of suppressor t cells , from polyclonal to monomorphic and monoclonal proliferations . monoclonal proliferations can subsequently accrue mutations of oncogenes or tumor suppressor genes , and lead to gain a fully malignant behavior and loss of responsiveness to immune regulation . moreover , mutation of c - myc , n - ras and p53 genes has recently been implicated in the terminal progression of ptld . the peak incidence of ptld occurs 3 to 4 months after transplantation , but may develop as early as 6 days later . patients with early onset ptld , within the first year , have a better prognosis than those with one developed late onset . in lung transplant recipients , ptld may develop in the allograft lung , lymph nodes or other malt sites , which frequently involves extranodal locations , including the lung . a lung involved by ptld may present as nodular or diffuse reticulonodular infiltrates , as solitary or multiple lung masses , or as hilar and mediastinal lymphadenopathy , and pulmonary nodules often have foci of necrosis . histologically , ptld can range from polymorphic b cell hyperplasia to monomorphic b - cell proliferation that is indistinguishable from diffuse large cell or immunoblastic lymphomas . these lymphomas are generally high grade with diffuse large cell , immunoblastic , or small noncleaved cell burkitt or burkitt - like morphologies . herein , our experience of two recently encountered cases of ptld following a lung transplantation , which demonstrated an aggressive clinical behavior , is reported , with a review of the literature . a 42-year - old man was admitted with a fever and chills for 3 days . his past medical history included dyspnea on exertion , which had developed from childhood , and was diagnosed as having a large patent ductus arteriosus with eisenmenger syndrome at the age of 20 . six months prior to this presentation , the patient had undergone heart - lung transplantation due to progressive dyspnea , chest pain and peripheral cyanosis . his preoperative examination revealed cytomegalovirus ( cmv ) igm antibody negative , cmv igg antibody positive , herpes simplex virus ( hsv ) igm antibody negative , hsv igg antibody positive , varicella zoster virus ( vzv ) igm antibody negative and vzv igg antibody positive . the tests for ebv early antigen ( ea)-igm was positive , ea - igg negative and epstein - barr nuclear antigen ( eana ) igg positive . the donor was a 25-year - old male whose preoperative examination revealed cmv igm antibody negative and cmv igg antibody positive . the test for ebv ea - igm was negative and ea - igg positive . during the immediate postoperative period , two months after the transplantation , he developed spiking fever with right lower lung field haziness and an accompanying pleural effusion , which was treated under the impression of bacterial pneumonia . one month later , he suffered a sudden fever and chill , and was admitted for further evaluation . on physical examination his chest examination revealed coarse breathing sounds in both lung fields and a grade i / iv systolic murmur in the aortic and tricuspid areas . laboratory tests showed a white cell count of 11,090/l , hemoglobin 9.2 g / dl , platelet 315,000/l , wintrobe erythrocyte sedimentation rate ( esr ) 5 mm / hr ( reference range ; 010 mm / hr ) , serum protein 5.1 g / dl , albumin 2.4 g / dl , aspartate aminotransferase ( ast ) / alanine aminotransferase ( alt ) 22/21 iu / l ( reference range ; < 37/<43 iu / l , respectively ) , creatinine 0.8 mg / dl , cmv igm antibody positive , cmv igg antibody positive , urine cmv - pcr negative , hsv antibody igm negative , hsv antibody igg positive , vzv antibody igm negative and vzv antibody igg positive . the tests for ebv revealed ea - igm antibody positive , ea - igg antibody negative , ebna igg antibody positive and ebv - pcr negative . a plain chest radiograph and computerized tomography revealed multiple , variably sized nodular lesions at the apical segments of both upper lungs , anterior segment of the left lung , posterobasal segment of the left lower lobe , hilar portion of the right middle lobe and superior segment of the right lower lobe . irregular thickening of the alveolar septa and the perivascular connective tissue , due to diffuse infiltration of atypical lymphoid cells , were observed . the ebv - dna reaction was positive by in situ hybridization , but light chain restriction could not be confirmed ( figure 1d ) . thirty days after admission , the patient began to complain of visual disturbance , and was treated with intravitreal ganciclovir and foscarnet under the diagnosis of cmv retinitis . the chest lesion continued to progress , with multiple infectious foci , and multiorgan failure developed . a 33-year - old woman was admitted for evaluation of multiple air space nodules in the left lung field . seven years before this presentation , she began to suffer from progressive dyspnea and dry cough . four years before presentation , she was transferred to a general hospital and diagnosed as having severe emphysema ( fvc 1.34 l ( 35.0% ) , fev1 0.61l / sec ( 20.0% ) , dlco 53% of expected value ) . she was a healthy hepatitis b virus ( hbv ) carrier and her 1-antitrypsine levels were 189 , 381 and 225.3 mg / dl , by three separate tests . two years before presentation , she underwent left lung transplantation and had been administered cyclosporine a , azathioprine and corticosteroid for immunosuppression , and lamivudine for hbv management . preoperative serologic tests revealed cmv igm antibody negative , cmv igg antibody positive , hsv igm antibody negative , hsv igg antibody positive , vzv igm antibody negative and vzv igg antibody positive . there was no information regarding the donor . during 18 months of follow - up after the transplantation however , a routine follow up plain chest radiograph showed multiple air space nodules in the transplanted lung . on physical examination , she had a chronic ill - looking appearance and a moon face , although the lung sounds were clear in both lung fields without rales . laboratory tests revealed a white cell count of 5,440/l , hemoglobin 11.7 g / dl , platelet 232,000/l , c - reactive protein ( crp ) quantitation 25.9 ( reference range ; 08 mg / l ) , wintrobe esr 58 mm / hr , serum protein 5.6 g / dl , albumin 3.2 g / dl , ast / alt 19/12 iu / l , bun 33 mg / dl and creatinine 0.9 mg / dl . the ea - igm and igg antibodies were positive , and the ebna - igg antibody was positive , which is a typical reactivity pattern for ebv reactivation , e.g. ebv - associated lymphoproliferative diseases or epithelial carcinomas , such as nasopharyngeal carcinoma . chest high resolution computed tomogram revealed multiple air spaced nodules at the left upper lobe and collapse of the lingular segment . these nodules had speculated margins with accompanying peripheral ground glass opacity , suggesting inflammatory changes ( figure 2a2c ) . a comparison of inspiratory and expiratory phase images showed neither mosaic perfusion nor small airway disease . a percutaneous transthoracic needle biopsy was performed on a nodule in the left upper lobe . these nodules consisted of lymphocytes , which by immunohistochemical study revealed positive reactions to l-26 and negative reactions to uchl-1 , kappa , lambda , cytokeratin and ki-1 in the tumor cells . a malignant lymphoma , of high - grade large b - cell type , was confirmed ( figure 2d ) . she was given a chemotherapeutic regimen comprising of cytoxan 1200 mg , vincristine 2 mg and prednisone 100 mg for 5 days , and one - week later epirubicin 90 mg was administered before being discharged . two weeks later the patient developed sudden dyspnea and died of pulmonary edema and a pneumothorax . a 42-year - old man was admitted with a fever and chills for 3 days . his past medical history included dyspnea on exertion , which had developed from childhood , and was diagnosed as having a large patent ductus arteriosus with eisenmenger syndrome at the age of 20 . six months prior to this presentation , the patient had undergone heart - lung transplantation due to progressive dyspnea , chest pain and peripheral cyanosis . his preoperative examination revealed cytomegalovirus ( cmv ) igm antibody negative , cmv igg antibody positive , herpes simplex virus ( hsv ) igm antibody negative , hsv igg antibody positive , varicella zoster virus ( vzv ) igm antibody negative and vzv igg antibody positive . the tests for ebv early antigen ( ea)-igm was positive , ea - igg negative and epstein - barr nuclear antigen ( eana ) igg positive . the donor was a 25-year - old male whose preoperative examination revealed cmv igm antibody negative and cmv igg antibody positive . the test for ebv ea - igm was negative and ea - igg positive . during the immediate postoperative period , two months after the transplantation , he developed spiking fever with right lower lung field haziness and an accompanying pleural effusion , which was treated under the impression of bacterial pneumonia . one month later , he suffered a sudden fever and chill , and was admitted for further evaluation . on physical examination his chest examination revealed coarse breathing sounds in both lung fields and a grade i / iv systolic murmur in the aortic and tricuspid areas . laboratory tests showed a white cell count of 11,090/l , hemoglobin 9.2 g / dl , platelet 315,000/l , wintrobe erythrocyte sedimentation rate ( esr ) 5 mm / hr ( reference range ; 010 mm / hr ) , serum protein 5.1 g / dl , albumin 2.4 g / dl , aspartate aminotransferase ( ast ) / alanine aminotransferase ( alt ) 22/21 iu / l ( reference range ; < 37/<43 iu / l , respectively ) , creatinine 0.8 mg / dl , cmv igm antibody positive , cmv igg antibody positive , urine cmv - pcr negative , hsv antibody igm negative , hsv antibody igg positive , vzv antibody igm negative and vzv antibody igg positive . the tests for ebv revealed ea - igm antibody positive , ea - igg antibody negative , ebna igg antibody positive and ebv - pcr negative . a plain chest radiograph and computerized tomography revealed multiple , variably sized nodular lesions at the apical segments of both upper lungs , anterior segment of the left lung , posterobasal segment of the left lower lobe , hilar portion of the right middle lobe and superior segment of the right lower lobe . irregular thickening of the alveolar septa and the perivascular connective tissue , due to diffuse infiltration of atypical lymphoid cells , were observed . the ebv - dna reaction was positive by in situ hybridization , but light chain restriction could not be confirmed ( figure 1d ) . thirty days after admission , the patient began to complain of visual disturbance , and was treated with intravitreal ganciclovir and foscarnet under the diagnosis of cmv retinitis . the chest lesion continued to progress , with multiple infectious foci , and multiorgan failure developed . a 33-year - old woman was admitted for evaluation of multiple air space nodules in the left lung field . seven years before this presentation , she began to suffer from progressive dyspnea and dry cough . four years before presentation , she was transferred to a general hospital and diagnosed as having severe emphysema ( fvc 1.34 l ( 35.0% ) , fev1 0.61l / sec ( 20.0% ) , dlco 53% of expected value ) . she was a healthy hepatitis b virus ( hbv ) carrier and her 1-antitrypsine levels were 189 , 381 and 225.3 mg / dl , by three separate tests . two years before presentation , she underwent left lung transplantation and had been administered cyclosporine a , azathioprine and corticosteroid for immunosuppression , and lamivudine for hbv management . preoperative serologic tests revealed cmv igm antibody negative , cmv igg antibody positive , hsv igm antibody negative , hsv igg antibody positive , vzv igm antibody negative and vzv igg antibody positive . there was no information regarding the donor . during 18 months of follow - up after the transplantation however , a routine follow up plain chest radiograph showed multiple air space nodules in the transplanted lung . on physical examination , her body temperature , pulse and respiration rates were 36.2c , 74/min and 20/min . she had a chronic ill - looking appearance and a moon face , although the lung sounds were clear in both lung fields without rales . laboratory tests revealed a white cell count of 5,440/l , hemoglobin 11.7 g / dl , platelet 232,000/l , c - reactive protein ( crp ) quantitation 25.9 ( reference range ; 08 mg / l ) , wintrobe esr 58 mm / hr , serum protein 5.6 g / dl , albumin 3.2 g / dl , ast / alt 19/12 iu / l , bun 33 mg / dl and creatinine 0.9 mg / dl . the ea - igm and igg antibodies were positive , and the ebna - igg antibody was positive , which is a typical reactivity pattern for ebv reactivation , e.g. ebv - associated lymphoproliferative diseases or epithelial carcinomas , such as nasopharyngeal carcinoma . chest high resolution computed tomogram revealed multiple air spaced nodules at the left upper lobe and collapse of the lingular segment . these nodules had speculated margins with accompanying peripheral ground glass opacity , suggesting inflammatory changes ( figure 2a2c ) . a comparison of inspiratory and expiratory phase images showed neither mosaic perfusion nor small airway disease . a percutaneous transthoracic needle biopsy was performed on a nodule in the left upper lobe . these nodules consisted of lymphocytes , which by immunohistochemical study revealed positive reactions to l-26 and negative reactions to uchl-1 , kappa , lambda , cytokeratin and ki-1 in the tumor cells . a malignant lymphoma , of high - grade large b - cell type , was confirmed ( figure 2d ) . she was given a chemotherapeutic regimen comprising of cytoxan 1200 mg , vincristine 2 mg and prednisone 100 mg for 5 days , and one - week later epirubicin 90 mg was administered before being discharged . two weeks later the patient developed sudden dyspnea and died of pulmonary edema and a pneumothorax . the incidence of ptld after lung transplantation is 2-fold higher than that seen after any other type of organ transplantation . primary ebv infection has been implicated as the major risk factor for ptld , particularly for ebv - naive patients who seroconverted after the lung transplantation . a 20-fold increase in the risk of developing ptld was reported to occur in ebv - seronegative vs ebv - seropositive lung recipients after transplantation . in our cases , serologic ebv testing of the recipients showed that ea - igm was positive and ea - igg negative , suggesting a recent ebv infection prior to the transplantation . in the local community , ebv spreads by contact with oral secretion , but has been transmitted by blood transfusion and bone marrow transplantation in a hospital setting through the ebv receptor ( cd21 ) , which present on the surface of b cells and epithelial cells . the most likely source of ebv infection through solid organ transplantation is transmission through an organ from an ebv - positive to an ebv - seronegative recipient . the exact mechanism by which the donor virus is transmitted from the transplanted organ to the recipient is unknown , although in solid organ transplant recipients , virus - infected cells in ptld of recipient origin have been identified . thus , the donor virus may either be transmitted in a cell - free state or released by donor cells in the recipient . a sufficient number of ebv - carrying b cells may remain in the blood within the allograft and pass into the circulation of the recipient ; the virus would then enter the lytic cycle and infect the recipient b - cell population . alternatively , heart and lung tissue may contain foci of ebv - infected cells , which may act as sites of virus replication . ebv infection in a naive recipient , occurring in the setting of altered host immunity , leads to uncontrolled proliferation and transformation of recipient b cells infected with ebv . these possible patterns of events require identification of high - risk causes and the development of effective therapeutic modalities . a recent study found that continuous , specific anti - viral prophylaxis in high - risk ebv - seronegative recipients significantly reduced the incidence of ptld after lung transplantation in the absence of induction therapy . lymphoid proliferations that are morphologically indistinguishable from a malignant lymphoma occur with greater frequency in organ transplant recipients than in the general population ; but are unlikely to progress to malignant lymphomas in immunocompetent patients , but often regress if the immunosuppression is sufficiently reduced . polymorphic b - cell hyperplasia is associated with a mixture of small lymphocytes , plasma cells and immmunoblasts , without significant cytologic atypia . monomorphic ptld has sheets of large transformed monoclonal cells or immunoblasts . despite their overt malignant appearance , neither the histological appearance nor clonality can accurately predict the response to modulation of immunosuppression . over the past decades , numerous attempts have been made to classify these lesions using morphology , immunohistochemistry and monoclonality , but none of these tools alone or in combination was found to accurately predict the biological behavior . the first case was confirmed as polymorphic , and was treated by conservative means by adjusting the immunosuppressant and anti - viral agent used to treat the accompanying cmv infection , whereas the second case was suspected as monomorphic ptld , and treated aggressively using a chemo - therapeutic regimen , but did not respond to treatment . because ptld treatments are most effective in the early stage , prior to the occurrence of progression of monomorphic or monoclonal proliferations , not only the early detection of ptld , but also the identification of the clonality of ptld , are of importance in planning the means of treatment .

post - transplant lymphoproliferative disease ( ptld ) is a serious , often fatal complication after solid organ transplantation . the incidence of ptld is greater among heart ( 213% ) , lung ( 12% ) and heart / lung ( 59% ) transplant recipients than among liver ( 2% ) , renal ( 13% ) and bone marrow ( 12% ) transplants recipients . the difference in the incidence of ptld may be partly attributed to the higher dose of immunosuppressant therapy used for heart and lung transplantation . the epstein - barr virus ( ebv ) infection status of the donor and recipient before a transplant , and high dose of immunosuppressive drugs are considered major risk factors . recently , 2 cases of ptld in a single lung and a heart - lung transplantation recipient were encountered . both patients presented with multiple pulmonary nodules in the transplanted lung , which developed 6 months and 2 years after the transplantation , respectively . following a transthoracic lung biopsy for diagnostic confirmation , one patient underwent chemotherapy for ptld and the other conservative care for an accompanying viral infection . both patients showed rapid clinical deterioration , without response to treatment , and then rapidly succumbed . herein , our experiences are reported , with a review of the literature .

the increasing prevalence of methicillin - resistant staphylococcus aureus ( mrsa ) to newer antibacterial agents , in both community and healthcare settings , is a serious worldwide concern as therapeutic options are limited . hence , there is a need for new antimicrobials possessing potent activity against this pathogen . bacterial fatty acid biosynthesis ( fasii ) is a relatively new and unexploited target for antimicrobial treatment of s. aureus.1 afn-1252 , a potent inhibitor of staphylococcal enoyl - acyl carrier protein ( enoyl - acp ) reductase ( fabi ) , a critical enzyme required for bacterial fasii , is being developed by affinium pharmaceuticals , inc . ( toronto , on , canada ) , in both oral and intravenous formulations , for the treatment of s. aureus infections . in vitro , , with typical minimum inhibitory concentration ( mic90 ) values of 00080015 mg / l , up to 3 log reductions in bacterial viable count over 24 hours and low potential for resistance development.24 murine studies indicate a long elimination half - life 57 hours and excellent efficacy in models of infection.5,6 pharmacokinetic studies in man indicate the potential for once or twice daily dosing.7 the objective of this study was to utilize an in vitro pharmacodynamic model simulating human pharmacokinetics to evaluate potential therapeutic regimens of afn-1252 against s. aureus , including both methicillin - susceptible s. aureus ( mssa ) and mrsa . bacterial s. aureus isolates utilized were atcc 29213 , a standard reference mssa and s186 , a clinical mrsa isolate obtained from the bloodstream of an infected patient at the buffalo veterans affair health system of western new york . , were prepared in 100% dimethyl sulphoxide and diluted at least 100-fold in mueller hinton broth ( difco laboratories , detroit , mi , usa ) supplemented with calcium ( 25 g / ml ) and magnesium ( 125 mg / l ) for mics determinations8 and use in the in vitro pharmacodynamic model . bacterial quantification of all samples was determined using tryptic soy agar with 5% sheep blood ( becton - dickinson , mississauga , on , canada ) . the in vitro pharmacodynamic model was as previously described.8 human pharmacokinetic profiles of afn-1252 were based on phase 0 studies.9 simulated therapeutic regimens of afn-1252 were based on free drug area under the concentration time curve to mic ratio over mic ( fauc / mic ) as detailed in table 1 . the initial bacterial inoculum was adjusted spectrophotometrically to achieve a final concentration of 10 colony - forming units / ml . bacterial samples were taken at 0 , 2 , 4 , 6 , 8 , 24 , 28 , 32 , and 48 hours and the viable counts were determined . to investigate the impact of escalating exposures on selection of resistant isolates , samples taken at 0 , 24 , and 48 hours were also plated on tryptic soy agar containing 4 and 6 mics of afn-1252 to detect and amplify resistant subpopulations . bacterial s. aureus isolates utilized were atcc 29213 , a standard reference mssa and s186 , a clinical mrsa isolate obtained from the bloodstream of an infected patient at the buffalo veterans affair health system of western new york . , were prepared in 100% dimethyl sulphoxide and diluted at least 100-fold in mueller hinton broth ( difco laboratories , detroit , mi , usa ) supplemented with calcium ( 25 g / ml ) and magnesium ( 125 mg / l ) for mics determinations8 and use in the in vitro pharmacodynamic model . bacterial quantification of all samples was determined using tryptic soy agar with 5% sheep blood ( becton - dickinson , mississauga , on , canada ) . the in vitro pharmacodynamic model was as previously described.8 human pharmacokinetic profiles of afn-1252 were based on phase 0 studies.9 simulated therapeutic regimens of afn-1252 were based on free drug area under the concentration time curve to mic ratio over mic ( fauc / mic ) as detailed in table 1 . the initial bacterial inoculum was adjusted spectrophotometrically to achieve a final concentration of 10 colony - forming units / ml . bacterial samples were taken at 0 , 2 , 4 , 6 , 8 , 24 , 28 , 32 , and 48 hours and the viable counts were determined . to investigate the impact of escalating exposures on selection of resistant isolates , samples taken at 0 , 24 , and 48 hours were also plated on tryptic soy agar containing 4 and 6 mics of afn-1252 to detect and amplify resistant subpopulations . the afn-1252 mics for mssa 29213 and mrsa s186 were both 0008 g / ml . the activity of afn-1252 in the in vitro pharmacodynamic model is shown in fig . d . for all dosage regimens , reductions in viable count over the first 6 hours were generally 12 logs and maximal reductions in viable count ( e.g. 2 to 3 logs ) generally achieved at fauc / mic of 100200 . greater reductions in viable count were observed with q12h regimens than corresponding q24h regimens , against both mssa 29213 and mrsa s186 at 24 and 48 hours . the greatest reductions in viable count against mssa 29213 and mrsa s186 were approximately 4 logs , achieved by 450 mg q12h ( fauc / mic = 1875 ) dosing at 28 hours . further analysis of the pharmacodynamic responses revealed an excellent correlation in the hill model10,11 between log reduction in viable count and fauc / mic fits ( r values 09980999 ) . afn-1252 did not develop resistance at any time point throughout the 48-hour experiment as no growth was present on afn-1252-containing agar . in vitro pharmacodynamic model experiments of ( a ) afn-1252 q24h versus atcc 29213 , ( b ) afn-1252 q12h versus atcc 29213 , ( c ) afn-1252 q24h versus mrsa s186 and ( d ) afn-1252 q12h versus mrsa s186 . fabi is the sole form of enoyl - acp reductase present in s. aureus , s. epidermidis , and other staphylococci . no alternative enzyme or rescue pathway , e.g. exogenous fatty acids , for fabi in staphylococci has been identified suggesting that fabi is essential to cell viability in staphylococcus spp and therefore has the potential to become a significant new target for the treatment of staphylococcal infections.4 afn-1252 is a highly potent and specific inhibitor of fabi with exquisite activity against staphylococci in extensive mic studies,24 and superior activity to linezolid in the mrsa murine thigh lesion model.5 pharmacokinetic studies in human volunteers indicate a good safety profile and the potential for once or twice a day dosing.7 rate of kill studies demonstrate that afn-1252 typically achieves a 12 log reduction in viable count within 24 hours and therefore is not bacteriostatic but does not meet the clsi criteria of bactericidal.12 other studies have also indicated that afn-1252 can achieve a > 2 log reduction in bacterial count over more than 24 hours4 and hence its action may be best described as slowly bactericidal. although other triclosan - based fabi inhibitors cg400462 , cg400549,13 and mut05639914 are under investigation , they appear to be less potent than afn-1252 and are less advanced . in this current investigation , we studied the bactericidal activity and pharmacodynamics of afn-1252 , using an in vitro pharmacodynamic model , against two strains of s. aureus to determine optimal therapeutic regimens including comparisons of once and twice daily dosing . as a result of these studies , the pharmacodynamic profile of afn-1252 was adequately characterized with the fauc / mic well related to antibacterial killing , which is also in agreement with what others have shown.15 twice daily dosing achieved marginally greater reductions in bacterial viable counts than once daily dosing , approaching the clsi definition of bactericidal . . it will be interesting to perform further pharmacokinetic / pharmacodynamic studies using hollow fibre and animal infection models to see if this affects the degree of bactericidal activity .

afn-1252 , a potent enoyl - acp reductase ( fabi ) inhibitor , is under development for the treatment of staphylococcus aureus infections . the activity of afn-1252 against two isolates of s. aureus , mssa 26213 and mrsa s186 , was studied in an in vitro pharmacodynamic model simulating afn-1252 pharmacokinetics in man . reductions in bacterial viable count over the first 6 hours were generally 12 logs and maximal reductions in viable count were generally achieved at fauc / mic ratios of 100200 . maximum reductions in viable count against mssa 29213 and mrsa s186 were approximately 4 logs , achieved by 450 mg q12h ( fauc / mic = 1875 ) dosing at 28 hours . staphylococcal resistance to afn-1252 did not develop throughout the 48-hour experiments . as multidrug resistance continues to increase , these studies support the continued investigation of afn-1252 as a targeted therapeutic for staphylococcal infections .

halorhodospira halochloris is an anoxygenic photosynthetic halophile that was isolated from the hypersaline wadi natrun lakes in egypt , residing in the mats near the sediments 8 . the genus halorhodospira was formed by separating species h. halophila , h. halochloris and h. abdelmalekii from genus ectothiorhodospira based on their 16s rrna sequences 4,6 . its cells are vibroid , motile by bipolar flagella and have internal photosynthetic membranes as lamellar stacks 13 . h. halochloris exhibits growth over an unusually wide range of medium nacl concentrations and is capable of growth down to 5% nacl , which is unusual for extremely halophilic bacteria . halophilic bacteria employ two differing strategies to protect their cytoplasmic volume against osmotic movement of water to the hypersaline environment 12 . , organic compounds are accumulated in the cytoplasm - these osmoprotectants are known as compatible solutes . the second biochemically , more radical adaptation involves the selective influx of k ions into the cytoplasm . h. halochloris accumulates glycine betaine ( n , n , n - trimethylglycine ) , a compatible solute as its osmoprotectant 5 . in addition to its osmoprotectant activity , glycine betaine also provides protection against mutagenic compounds and radiation - induced damage 9 . glycine betaine can either be taken up directly from the environment , or be synthesized de novo 11 . we recently used isoelectric focusing of total cell proteins to demonstrate that h. halochloris does not exhibit an acidic proteome , matching its inability to accumulate k 2 . in striking contrast we found that a closely related organism h. halophila accumulates molar concentrations of kcl when grown in high salt medium and has an acidic proteome . comparative genomics of h. halochloris and h. halophila promises to provide insights into this issue , which has implications both for genome - wide evolutionary processes and the mechanisms of halophilic adaptations . these considerations led us to determine the genome of h.halochloris , which we report on here . recently we reported the complete genome of h. halophila 1 . in this study , we report the draft genome sequence of h. halochloris , which was obtained through standard roche 454 pyrosequencing using the roche 454-junior instrument . the raw data obtained were trimmed at either end based on the quality score analysis performed using fastqc tool . poor or bad quality bases , probably originating from sequencing mis - calls , were trimmed off before subjecting it to the assembly software . we performed genome assembly using three different assemblers , namely newbler 16 , mira 17 and phrap 18 , with the default set of parameters . after comparisons of these assembly attempts based on contig sizes , genome representation and its functional elements , the output of mira 3.4.1 was selected to proceed with further analysis . the final output had some low quality contigs in terms of length and average coverage . all contigs of length less than 1kbp and average coverage < 10 were removed as being uninformative , from annotation point of view , and subjected to an individual annotation check using blast 14 . most of these individual contigs yielded relatively high e - value or no scores with halophiles . processed contigs were mapped against a distant reference genome ( thioalkalivibrio sulphidophilus ) , as no true known reference genome is currently available , based on 16s analysis , using contiguator 15 . the assembled scaffold comprises 137 contigs , 3,460,134 bases at 20 fold coverage and has a gc content of 63% . for comparison the genome of h. halophila is 2.7 mb in size and has gc content of 67% 1 . the jgi img / er annotation pipeline ( http://img.jgi.doe.gov/er ) was employed for gene annotation with img submission i d and img project i d , 15725 and 50543 respectively . the numbers of trna and rrna genes were predicted as 46 and 8 , respectively . a total of 3,301 putative protein coding genes ( cdss ) or open reading frames ( orfs ) were predicted with a total gene count of 3,376 . the genome has been submitted in public databases , ncbi , genbank ( gi number : 589289709 , genbank accession number : cp007268 ) . the draft genome information reported here provides opportunity for further research into the mechanism involved in halophilic adaptations and allow organisms to thrive in hyper saline environments , how these evolve and how they differ for bacteria and archaea 10 .

halorhodospira halochloris is an extremely halophilic bacterium isolated from hypersaline wadi nantrun lakes in egypt . here we report the draft genome sequence of this gammaproteobacteria ( gi number : 589289709 , genbank accession number : cp007268 ) . the 3.5-mb genome encodes for photosynthesis and biosynthesis of organic osmoprotectants . comparison with the genome of h.halophila promises to yield insights into the evolution of halophilic adaptations .

health care workers ( hcws ) are at the front line for acquiring blood - borne viruses ( hepatitis b virus , hbv ; hepatitis c virus , hcv and human immunodeficiency virus , hiv ) infections . the worldwide hbv infection rate is higher in dentists than other blood borne viruses like hcv and hiv . hbsag prevalence among dentists reported to be 0.6% in the usa ( 1 ) , 2.4% in malaysia ( 2 ) and 13% in korea ( 3 ) . likewise , among health care workers , dentists experience the highest chance of hbv infection and hbv incidences increase with duration of clinical experience of dentistry ( 4 , 5 ) . on the other hand , past ( anti - hbc positivity ) or present ( hbsag positivity ) hbv infection rate in dentists are usually higher than the general population , regardless of hbv endemicity in those area ( 6 ) . among blood - borne viruses , despite being effective in decreasing the hbv prevalence , the extended program on immunization ( epi ) only targeted newborns and adults in general populations , as well as high - risk groups , including dentists . however , dental health care workers are not fully covered by hbv immunization programs . estimated of a 100-fold reduction in the incidence of hbv infection in vaccinated individuals compared to non - vaccinated individuals , regardless of the vaccine response ( 7 ) , indicates that dental care workers should be advised to receive hepatitis b vaccine and it should be confirmed if they have acquired immunity to hbv by testing the level of anti - hbs ( 1 , 4 , 5 , 8 - 10 ) . reports from different countries indicated that hbv vaccine coverage rate in dental health care workers ranged between 26% and 96.6% ( 8 , 9 ) . in iran , this coverage rate ranged between 74.8% and 94.9% ; on average 70% of dentists care workers received at least one dose of hbv vaccine ( 11 ) . as it shown , the reported response to hbv vaccine has not been reached 100% among dentists . response to hbv vaccine ( i.e. anti - hbs levels > 10 iu / ml ) between iranian dental workers has been studied extensively and 89.2% to 94.4% of dentists showed reasonable levels of anti - hbs following one to three doses of hbv vaccine ( 11 - 14 ) . in the largest iranian survey , of 598 participants , 35 ( 5.9% ) were nonimmune ( anti - hbs < 10 iu / l ) , 101 ( 16.9% ) were relatively immune ( anti- hbs > 1099 iu / l ) and 462 ( 77.3% ) were completely immune ( anti - hbs > 100 the objectives of the present study were to assess the hbv vaccine coverage and investigate the responsiveness to hbv vaccine as well as socio - demographic data , health - related and occupational factors and other correlates of vaccine responsiveness in iranian dentists and dental staff . this was a cross - sectional survey of dentists attending the 51st annual international congress of iranian dental association held on 10 to 13 may 2011 . individuals who met our inclusion criteria and willingness to give blood samples and knowing their vaccination history were recruited . an informed consent was obtained from all enrolled participants and a questionnaire was distributed to collect data . at a special booth in the conference building , potential participants gave oral consent and completed the questionnaire . the items on the questionnaire included : demographic information , such as age , gender and marital status ; occupational information , comprising years of dental practice and place of practice and vaccination data , which included the number of doses , time interval between doses , last dose date and checking the titer of antibody after vaccination . among 1665 participants , history of immunization was extracted for 1612 individuals ; hence 53 cases were excluded from the study . the samples were tested for antibodies against hepatitis b ( anti - hbs ) and anti - hbc using commercially available enzyme linked immunosorbent assays ( elisa ) ( m.b.s s.r.l . anti - hbs were measured in iu / ml , and the results were classified into two groups as follows : 1 ) titers below 10 iu / l as no immunity and 2 ) titers above 10 iu/ ml as complete immunity . data was analyzed using the statistical package for social sciences ( ibm corp . released 2011 . ibm spss statistics for windows , version 20.0 , armonk , ny : ibm corp ) . all statistical comparisons were performed by simple and multiple linear regression analyses on logarithm ( base 10 ) of the anti - hbs antibody values . 1612 participants including 1300 ( 80.7% ) general practitioner , 155 ( 9.6% ) students , 120 ( 7.4% ) specialists and 37 ( 2.3% ) clinical dental assistants were studied ( table 1 ) . the subjects comprised 1058 ( 65.6% ) males and 554 ( 34.4% ) females with a mean age of 40.4 years ( range 19 - 75 years , results not shown ) . the number of years in practice ranged from 0 to 55 years ( excluding students ) with a median of 15 years ( results not shown ) . the demographic characteristics of the study sample according to job classifications are shown in table 1 . ( % ) . of total 1538 vaccinated individuals , 176 ( 11.5% ) were nonimmune ( anti - hbs < 10 iu / ml ) and 1362 ( 88.5% ) were immune ( anti - hbs > 10 iu/ ml ) regardless of the number of doses and time after the last dose and time intervals between doses . no significant associations were found between the levels of anti - hbs and the kind of dental job ( table 2 ) . according to subjects reports , 55 ( 3.7% ) , 126 ( 8.4% ) and 1309 ( 87.9% ) had received one , two and full three doses of vaccine , respectively ( see table 1 for more details ) . fifty - eight ( 3.59% ) of participants did not receive any hbv vaccine at all ; however , they had positive results for anti - hbs , indicating a past hbv infection . however , anti - hbs mean value was 12.96 ( 95% ci : 5.72 - 29.39 ) for those who did not receive hbv vaccine . nevertheless , the mean anti - hbs titers were 24.89 ( 95% ci : 11.16 - 55.52 ) , 100.32 ( 95% ci : 70.52 - 142.72 ) and 107.04 ( 95% ci : 96.82 - 118.33 ) for subjects who received one , two and three doses of vaccine , respectively ( p values between 1 and 2 ; 2 and 3 doses : 0.006 , and 0.98 , respectively , results not shown ) . therefore , no significant difference was found between antibody levels and receiving second or third doses of vaccine ; whereas , this association was significant between individuals who received only one versus those who received the second dose . among total participants , 1033 who knew their exact time of vaccination history , 542 ( 52.5% ) mentioned that they received vaccination within the past five years , while others ( 491 ; 47.5% ) reported having received the last dose of vaccine more than five years prior this study . 356/542 ( 65.7% ) of dentists who had received their third dose of vaccination less than five years before the study were completely immune ( anti - hbs > 100 iu / ml ) ; this rate was significantly higher than individuals who had completed all three recommended doses in a period more than 5 years prior to the study 279/491 ( 56.8% ) ( p = 0.003 ) ( table 1 ) . a significant relation was found between gender and anti - hbs antibody titer ; females showed a higher level of anti - hbs ( p = 0.022 ) , ( table 1 ) . accordingly , the median of antibody titer was significantly higher in the age group < 45 years compared to the age group > 45 years ( p < 0.001 ) . furthermore , statistically significant associations were found between the median titer of anti - hbs following vaccination and duration of dental practice engagement ( p < 0.001 ) ( table 1 ) . eighty - one ( 5% ) of participants had positive results for anti - hbc , of whom 66 ( 81.4% ) had a history of vaccination ( results not shown ) ; 55 ( 83.3% ) had protective levels of anti - hbs ( 10 iu / ml ) and 11 ( 16.7% ) had inadequate anti - hbs levels ( < 10 iu / ml ) . although 13 ( 16.5% ) of anti - hbc positive dentists did not receive vaccine , seven had protective levels of anti - hbs and six had inadequate levels , indicating a possible past hbv infection . data are presented as mean se . to determine possible risk factors and anti - hbs levels , no significant associations were found between levels of antibody titers and number of needle stick as well as other risk factors such as trauma , suspicious sexual contact and a history of liver disease ( table 2 ) . however , a significant negative association was found between the history of transfusion and low levels of anti - hbs ( p = 0.016 ) ( table 2 ) . of 1385 cases with known cigarette smoking history , there were 211 ( 15.2% ) dentists who were smokers . the median of years for smoking was 14 and the median of pack - years of smoking was 3.75 ( results not shown ) . however , there was no significant association between antibody titer and history , duration and pack - years of smoking ( p = 0.1 , 0.38 and 0.37 , respectively ) . among participants , anti - hbs levels were not different according to consistent use of gloves ( p = 0.322 ; table 2 ) . on the other hand , anti - hbs levels for those who regularly used mask , glasses and shield were higher than those who used them irregularly or not at all , although they were not significant statistically ( p = 0.093 , 0.158 and 0.127 , respectively ; table 2 ) . 1612 participants including 1300 ( 80.7% ) general practitioner , 155 ( 9.6% ) students , 120 ( 7.4% ) specialists and 37 ( 2.3% ) clinical dental assistants were studied ( table 1 ) . the subjects comprised 1058 ( 65.6% ) males and 554 ( 34.4% ) females with a mean age of 40.4 years ( range 19 - 75 years , results not shown ) . the number of years in practice ranged from 0 to 55 years ( excluding students ) with a median of 15 years ( results not shown ) . the demographic characteristics of the study sample according to job classifications are shown in table 1 . of total 1538 vaccinated individuals , 176 ( 11.5% ) were nonimmune ( anti - hbs < 10 iu / ml ) and 1362 ( 88.5% ) were immune ( anti - hbs > 10 iu/ ml ) regardless of the number of doses and time after the last dose and time intervals between doses . no significant associations were found between the levels of anti - hbs and the kind of dental job ( table 2 ) . according to subjects reports , 55 ( 3.7% ) , 126 ( 8.4% ) and 1309 ( 87.9% ) had received one , two and full three doses of vaccine , respectively ( see table 1 for more details ) . fifty - eight ( 3.59% ) of participants did not receive any hbv vaccine at all ; however , they had positive results for anti - hbs , indicating a past hbv infection . however , anti - hbs mean value was 12.96 ( 95% ci : 5.72 - 29.39 ) for those who did not receive hbv vaccine . nevertheless , the mean anti - hbs titers were 24.89 ( 95% ci : 11.16 - 55.52 ) , 100.32 ( 95% ci : 70.52 - 142.72 ) and 107.04 ( 95% ci : 96.82 - 118.33 ) for subjects who received one , two and three doses of vaccine , respectively ( p values between 1 and 2 ; 2 and 3 doses : 0.006 , and 0.98 , respectively , results not shown ) . therefore , no significant difference was found between antibody levels and receiving second or third doses of vaccine ; whereas , this association was significant between individuals who received only one versus those who received the second dose . among total participants , 1033 who knew their exact time of vaccination history , 542 ( 52.5% ) mentioned that they received vaccination within the past five years , while others ( 491 ; 47.5% ) reported having received the last dose of vaccine more than five years prior this study . 356/542 ( 65.7% ) of dentists who had received their third dose of vaccination less than five years before the study were completely immune ( anti - hbs > 100 iu / ml ) ; this rate was significantly higher than individuals who had completed all three recommended doses in a period more than 5 years prior to the study 279/491 ( 56.8% ) ( p = 0.003 ) ( table 1 ) . a significant relation was found between gender and anti - hbs antibody titer ; females showed a higher level of anti - hbs ( p = 0.022 ) , ( table 1 ) . accordingly , the median of antibody titer was significantly higher in the age group < 45 years compared to the age group > 45 years ( p < 0.001 ) . furthermore , statistically significant associations were found between the median titer of anti - hbs following vaccination and duration of dental practice engagement ( p < 0.001 ) ( table 1 ) . eighty - one ( 5% ) of participants had positive results for anti - hbc , of whom 66 ( 81.4% ) had a history of vaccination ( results not shown ) ; 55 ( 83.3% ) had protective levels of anti - hbs ( 10 iu / ml ) and 11 ( 16.7% ) had inadequate anti - hbs levels ( < 10 iu / ml ) . although 13 ( 16.5% ) of anti - hbc positive dentists did not receive vaccine , seven had protective levels of anti - hbs and six had inadequate levels , indicating a possible past hbv infection to determine possible risk factors and anti - hbs levels , no significant associations were found between levels of antibody titers and number of needle stick as well as other risk factors such as trauma , suspicious sexual contact and a history of liver disease ( table 2 ) . however , a significant negative association was found between the history of transfusion and low levels of anti - hbs ( p = 0.016 ) ( table 2 ) . of 1385 cases with known cigarette smoking history , there were 211 ( 15.2% ) dentists who were smokers . the median of years for smoking was 14 and the median of pack - years of smoking was 3.75 ( results not shown ) . however , there was no significant association between antibody titer and history , duration and pack - years of smoking ( p = 0.1 , 0.38 and 0.37 , respectively ) . among participants , anti - hbs levels were not different according to consistent use of gloves ( p = 0.322 ; table 2 ) . on the other hand , anti - hbs levels for those who regularly used mask , glasses and shield were higher than those who used them irregularly or not at all , although they were not significant statistically ( p = 0.093 , 0.158 and 0.127 , respectively ; table 2 ) . this study was performed to determine the anti - hbs antibody titers of iranian dental care workers and to investigate the possible correlation between demographic features as well as details of vaccination schedule with anti - hbs antibody titer in this population . moreover , risk factors related to immune status of subjects together with protective measurement were considered in this investigation . although the study sample was not selected randomly , our sample size was the largest among iranian researches . in the present investigation , 1538 hbv vaccine recipients were anti - hbs - positive , of whom 1362 ( 88.5% ) subjects developed adequate levels of antibody to hbv infection and 176 ( 11.5% ) were non - immune . a similar study on dentists in iran showed that 69%-77% of participants were completely immune , while 17% were relatively immune and 6 - 13% were non - immune ( 12 , 14 ) . the number of those who had received their three recommended doses of vaccine was 1312 ( 87.9% ) . furthermore , statistically significant correlations were found between the median titer of anti - hbs following vaccination and time after the last vaccine injection ( p < 0.001 ) . regarding very low to moderate levels of anti - hbs ( < 10 - 100 iu / ml ) in 481 ( 31.3% ) participants , they are at increased risk for hbv infection ( 15 - 17 ) . there are two possibilities : ( i ) decline in the antibody titer with the passage of time despite initial adequate levels of anti - hbs . in the present study , the difference between the times lasting from the third dose of vaccine was statistically significant ( > 5 years vs. < 5 years ) . similar studies showed that in individuals who respond adequately to vaccination , anti - hbs antibody levels decrease over time and may fall below protective levels . basically , administering a booster dose of hbs ag vaccine results in a vigorous anamnestic response , demonstrating that immune memory against hbv infection lasts longer than anti - hbs antibodies ( 18 , 19 ) . on the other hand , ( ii ) the other possibility is nonresponsiveness to the vaccine . several factors were reported to influence the response to hbv vaccine in nonresponders such as genetic background , older age , obesity ( 20 , 21 ) and smoking ( 9 , 22 ) . for those who do not respond to the primary vaccination series , an additional regimen of ordinary vaccines ( either administration of a higher dose or a second course of three doses of hbv recombinant vaccine ) usually gives rise to about 15% to 25% and 30% to 50% of responsiveness to one and three additional doses , respectively ( 23 , 24 ) . likewise , still more than 50% of non - responders are not able to acquire protective levels of anti - hbs despite administration of at least two additional booster recombinant vaccines ( 25 , 26 ) . alternate schedule includes intradermal vaccine administration ( 27 ) or third generation vaccines ( that contained pre - s1/pre - s2 proteins through recombinant technology in mammalian cell lines ) with higher immunogenicity and more seroconversion rate compared to the second generation vaccines ( 28 , 29 ) . otherwise , for those persistent nonresponders , it is recommended to avoid epp ( exposure - prone procedures ) and they should be noticed that they may be susceptible to hbv and that they should receive hepatitis b immune globulin ( hbig ) following hbv exposure ( 30 ) . we did not check anti - hbc status of participants , hence anti - hbs level in the study does not necessarily differentiate rising of antibody following vaccination or past infection with hbv . in the present study , the rate of incomplete vaccinations was 13% , hence more efforts should be made to persuade all dentists to receive the three doses of vaccine . unfortunately , as worldwide , no mandatory hbv vaccination program exists for dentists in iran , which may cause a low rate of compliance in voluntary vaccination program(s ) now available for hcws at health offices . it is of some concern that dentists are willing to accept significant degree of personal risk , despite recorded danger from hepatitis b , either by failing to ensure immunization against hepatitis b or by failing to check the presence of hepatitis b antibodies following immunization . in conclusion , hbv vaccine coverage and infection control measures were satisfactory among iranian dental personnel in this study . since dental care workers have a high risk of exposure to hepatitis virus , a compulsory vaccination for hepatitis b virus is desirable for all dental care workers .

background : studies showed that hbv vaccination and consequent level of antibody are not completely adequate among dentists despite performance of highly exposure prone procedures.objectives:the objectives of the study were to evaluate the levels of responsiveness to hbv vaccine and to determine the occupational factors associated among dental staff.materials and methods : in total , 1612 dental health care workers were recruited . the level of anti - hbs was tested using a commercially enzyme - linked immunosorbent assay ( elisa ) . data on demographic , risk factors associated with dental practice and level of protective procedures and occupational exposure aspects were collected through self - reported questionnaires.results:of 1538 vaccinated individuals , 55 ( 3.7% ) , 126 ( 8.4% ) and 1309 ( 87.9% ) had received one , two and full three doses of vaccine , respectively . one - hundred - seventy - six ( 11.5% ) were nonimmune ( anti - hbs < 10 iu / ml ) and 1362 ( 88.5% ) were immune ( anti - hbs > 10 iu/ ml ) . 392/542 ( 72.3% ) of dentists who received their third dose of vaccination less than five years before the commencement of study were completely immune compared to those who had completed all three recommended doses in a longer period ( 308/491 , 64.3% ) ( p = 0.001 ) . fifty - eight ( 3.59% ) of participants did not receive any hbv vaccine at all ; however , they had positive results for anti - hbs , indicating a past hbv infection . statistically , the levels of anti - hbs were significantly associated with gender , age , duration of dental practice engagement and regularly use of mask , glasses and shield.conclusions:since dental care workers have a high risk of exposure to hepatitis virus , they should be advised to receive hepatitis b vaccine and it should be confirmed if they have acquired immunity to hbv by testing the level of anti - hbs .

this may cause inconveniences with regard to activities of daily living ; thus physiotherapy is often used to treat problems related to joint movement limitation . in previous studies , we have reported the histopathological changes that occur in the rat knee joint components during immobilization ; however , the changes have not been fully clarified . joint immobilization induces muscle atrophy , articular cartilage loss , and proliferation of connective tissue within the joint space1 , 7 . immobilization of joints surrounded by edematous soft tissue often produces joint stiffness more quickly and more severely than immobilization of nonedematous extremities8 . after two weeks of joint immobilization , the number of synoviocytes in the posterior synovial intima was increased compared with a control group9 . watanabe reported atrophy of fat cells , proliferation of fibroblasts , and narrowing of the spaces between collagen fibers in the rat knee joint after immobilization10 . some studies reported changes in joint components caused by remobilization after immobilization8 , 11,12,13,14,15 . the range of motion of rat knees immobilized for 8 weeks remained substantially reduced after a 4-week period of unassisted remobilization16 . but histopathological findings of the joint components were not described in that study . in the precedent studies , histopathological changes in joint components after remobilization and/or reloading during joint contracture were observed . a few studies have reported the effect of exercise during an immobilization period17 , 18 . tatsuta related that exercise during immobilization periods did not improve range of motion but also that granulation tissue did not infiltrate into the articular cavity19 . in this study , we used external fixation in order to immobilize the joint and subjected the rats to exercise during the immobilization period . the purpose of this study was to clarify the effects of the rom exercise on joint components according to histopathological analysis . the protocol for these experiments were approved by the animal care committee and institutional ethics committee of kanazawa university , and all procedures for animal care and treatment were performed in accordance with the guidelines for the care and use of laboratory animals at kanazawa university . in total , twenty - six 9-week - old adult male wistar rats ( weighing 250275 g ) were used in this study . the rats were randomly divided into three groups , the immobilization group ( n=10 ) , exercise group ( n=10 ) , and control group ( n=6 ) . the animals were kept under normal conditions for one week before the start of experiments in order to acclimatize them to the environment . they were housed , 1 per cage , in a room maintained under a 12-hour light - dark cycle . the immobilization group and exercise group were anaesthetized ( pentobarbital sodium , 40 mg / kg bw , ip ) and operated on under sterile conditions . the right knee joints of the rats in the immobilization group and exercise group were immobilized with external fixation at 120 degrees of flexion . a 2-mm longitudinal incision was made in the right hind thigh skin ; the right femur and tibia were pierced by kirschner wire with a diameter of 0.8 mm , and the wire was then bent ( fig . the right femur and tibia were pierced by kirschner wire with a diameter of 0.8 mm , and the wire was then bent ( x - ray ) . ) . kirschner wires were fixed outside the wounds in a knee flexion posture with screw ( 4 mm diameter ) and nuts . the instruments for external fixation weighed 6 to 7 g. the rat s knee flexion angle ( 120 degrees ) was determined by the method described in our previous studys13 , 21 , 22 . this immobilization method is low cost , and immobilization can be reversed to perform exercise and then reestablished easily . although the right hindlimb knee joint was immobilized , the rats were able to move freely without restriction in their respective cages and had free access to food and water . the right femur and tibia were pierced by kirschner wire with a diameter of 0.8 mm , and the wire was then bent ( x - ray ) . range of motion exercise ( rom ex ) was started from the day after immobilization . rom ex was performed in the exercise group once a day for 3 minutes , 6 days a week , for 2 weeks . the mean torque necessary for the rat to passively extend the knee joint was 1 n , and this value was adopted as the strength during the rom ex . the exercise consisted in 18 cycles of 10 seconds of exercise ( a total of 3 minutes ) . the exercise time was determinate according to the results of a preliminary experiment . in the first 5 seconds of the exercise cycle , the rat s right hind limb was maintained in 120 degrees of hip flexion , and in the next 5 seconds , the limb was pulled in the caudal direction with 1 n force applied by a manual force gauge . the rom exercise intensity was controlled using a force gauge continuously during the 3 minutes . after each exercise , the rat knee was re - immobilized by external fixation at 120 degrees of knee flexion . the control group did not undergo surgery and was not subjected to exercises . after 2 weeks of intervention , the roms of all rat knees ( extension limitation ) were measured under anaesthesia . in the rats in the immobilization and exercise groups , the external fixation was removed , and shortly after the tight hind limbs of the rats were later pulled with 1 n force in the caudal direction . after angle measurement , the knees of the rats were re - immobilized at 120 degrees of flexion by external fixation . later , the rats were sacrificed by intraperitoneal injection of an overdose of pentobarbital sodium . immediately after euthanasia , their right hind limbs the right knees of the rats were fixed in 10% buffered formalin and decalcified . after decalcification , they knees were cut in the sagittal plane at the level of the anterior and posterior cruciate ligament . following neutralization with 5% sodium sulfate solution , the specimens were fixed , decalcified , and neutralized at 4 c for 72 hours . the specimens were embedded in paraffin , sectioned at 3 m , and mounted on microscope slides . all sections were stained with hematoxylin and eosin and toluidine blue stains and used for observation in a histopathological examination . histopathological analysis was performed by examining the synovial membrane , the joint capsule , and the joint cartilage . knee limitation data were statistically analyzed using ibm spss statistics for windows ( version 19.0.1 , ibm corp . rom was analyzed using one - factor analysis of variance ( anova ) with bonferroni post - hoc multiple comparisons . the histopathological analysis showed no swelling or signs of infection in the knees as a result of the intervention . the mean values of knee rom ( extension limitation angle ) were 19.33.0 degrees , 77.37.4 degrees , and 51.04.9 degrees in the control group , immobilization group , and exercise group , respectively , and each groups showed a statistically significant difference ( p<0.05 ) . the mean body weights were 326.08.8 g , 310.616.6 g , and 304.57.7 g in the control group , immobilization group , and exercise group , respectively . there were statistically significant differences ( p<0.05 ) between the control group and other groups . in the control group , the joint capsule was typically composed of coarse and relatively loose fibrous connective tissues . the joint capsule in the immobilization group and exercise group showed narrowing of the collagen bundles in interstitial spaces but was less dense in the control group ( fig . the joint capsule in the control group was typically composed of coarse and relatively loose fibrous connective tissues , but those in the immobilization group and exercise group showed narrowing of the collagen bundles of interstitial spaces ; the joint capsule was less dense in the control group . a , control group ; b , immobilization group ; c , exercise group . the surface of the articular cartilage in animals of the control group was smooth , and the hyaline cartilage was directly exposed to the articular cavity ( fig . the surface of the articular cartilage was smooth , and the hyaline cartilage was directly exposed to the articular cavity ( a ) ; the cartilage matrix of the hyaline cartilage was stained with toluidine blue ( b ) . f , femur ; t , tibia ; m , meniscus . he stain 200 ) . the cartilage matrix of the hyaline cartilage was stained with toluidine blue . in the immobilized group , the articular cartilages were covered with the proliferating tissue composed of fibroblast - like spindle - shaped cells ( fig . 4 . hyperplastic tissues infiltrated into the articular cavity and adhered to the surface of the articular cartilage ( white arrow head ) , and infiltration of vessels ( black arrows ) was observed . conversely , in the exercise group , hyperplasia of membrane - like tissue was localized to the synovial membrane , and the infiltration of fibroblast - like spindle - shaped cells was localized to the articular cavity ( fig . 5 . hyperplasia of tissue was localized to the meniscus ( black arrows ) , and the surface of the articular cartilage was smooth ( a ) . the cartilage matrix of the hyaline cartilage was stained with toluidine blue ( b ) . notably , the presence of erythrocytes that had leaked into the articular cavity was observed in some exercise group animals ( n=6 ) , suggesting the presence of hemorrhage after rom ex . the joint capsule in the control group was typically composed of coarse and relatively loose fibrous connective tissues , but those in the immobilization group and exercise group showed narrowing of the collagen bundles of interstitial spaces ; the joint capsule was less dense in the control group . a , control group ; b , immobilization group ; c , exercise group . the surface of the articular cartilage was smooth , and the hyaline cartilage was directly exposed to the articular cavity ( a ) ; the cartilage matrix of the hyaline cartilage was stained with toluidine blue ( b ) . he stain 200 hyperplastic tissues infiltrated into the articular cavity and adhered to the surface of the articular cartilage ( white arrow head ) , and infiltration of vessels ( black arrows ) was observed . he stain ( a ) , toluidine blue stain ( b ) 200 hyperplasia of tissue was localized to the meniscus ( black arrows ) , and the surface of the articular cartilage was smooth ( a ) . the cartilage matrix of the hyaline cartilage was stained with toluidine blue ( b ) . proliferation of intracapsular connective tissue and the formation of adhesions are primary responses to limitation of motion5 . in this study , we observed the presence of granulation tissue - like organization and infiltration in the joint cavity after two weeks of knee immobilization in rats . the cartilage appeared to be more or less confluent with the overlying connective tissue20 . these findings are consistent with those reported in our previous study21 , 22 . during the first two weeks of immobilization , rom limitation caused damage in the myogenic component , and after two weeks , the arthrogenic component constituted more than 80% of the total restriction in rom20 . this suggests that in our contracture model , the myogenic factor was stronger than the arthrogenic factor for rom limitation . rom limitation was significantly decreased by exercise , presumably due to its property that contributes to the maintenance of muscle extensibility . however , changes were found not only in the muscular component but also in the joint component . proliferation of intracapsular connective tissue and the formation of adhesions are primary responses to limitation of motion20 . in the present study , extensive hyperplasia of fibroblasts was observed in all animals in the immobilization group , but it was less severe and was focally distributed in the animals in the exercise group . this finding may suggest that rom exercise induces some change within the joint components and tissue metabolism . connective - tissue proliferation was present in all three of the knees immobilized for fifteen days , and it was well established at thirty days20 . the changes in the joint capsule were observed from the early period , and there were no important differences between the immobilized group and exercised group . in our model , we did not observe a significant influence of exercise on the joint capsule ; however we can not rule out that some changes may be observe in response to other exercise methods . we observed slight intra - articular hemorrhage , which indicates that the exercises used in this study might cause laceration of granulation tissue and adhesion . in this study , it was clarified that rom ex maintains range of motion and reduces the changes in the joint components . therefore , it is necessary to examine several kinds of frequency and the exercise strength in a future study . a number of studies have performed remobilization following a joint immobilization period , and some have subjected animals to exercise during an immobilization period .

[ purpose ] the aim of this study was to clarify the effects of the rom exercise on joint components according to histopathological analysis . [ subjects and methods ] in total , twenty - six 9-week - old adult male wistar rats were used in this study . the rats were randomly divided into three groups , the immobilization group ( n=10 ) , exercise group ( n=10 ) , and control group ( n=6 ) . the immobilization group and exercise group were anaesthetized and operated on under sterile conditions . the right knee joints in the immobilization group and exercise group were immobilized with external fixation at 120 degrees of flexion . range of motion exercise was started from the day after immobilization . rom exercise was performed in the exercise group once a day for 3 minutes , 6 days a week , for 2 weeks . [ result ] the joint capsule in the immobilization group and exercise group showed narrowing of the collagen bundles in interstitial spaces but was less dense in the control group . in the immobilized group , a hyperplastic reaction was associated with infiltration into the articular cavity and adhesion to the surface of the articular cartilage . conversely , in the exercise group , hyperplasia of tissue was localized to the synovial membrane . [ conclusion ] this finding may suggest that rom exercise induces some changes within the joint components and tissue metabolism .

ectopic thyroid tissue is a common abnormality and results from abnormal embryologic development and migration of the thyroid gland . such tissue is usually found along the path of descent of the thyroid gland in the anterior midline of the neck . a lingual thyroid is the most common presentation of thyroid ectopy along with thyroglossal duct remnants . ectopic thyroid tissue has been described in other parts of the head and neck such as the submandibular [ 3 , 4 ] and parotid salivary glands . there are case descriptions of thyroid tissue identified in diverse locations such as the axilla , trachea [ 710 ] , adrenal , small intestine , and porta hepatis . the most frequent noncervical location for ectopic thyroid tissue is the thoracic cavity [ 1418 ] . , the authors describe a case of ectopic anterosuperior mediastinal thyroid mass excised through a cervical incision and the subsequent investigation to exclude malignancy . an 80-year - old nonsmoker female patient presented with an incidental finding of a mediastinal mass on mri scan for investigation of vertigo ( figures 1 and 2 ) . thyroid stimulating hormone ( tsh ) and free thyroxine ( ft4 ) were within the normal range , and routine preoperative blood tests were normal . an anteroposterior chest radiograph demonstrated deviation of the intrathoracic trachea around the mass ( figure 3 ) . a subsequent ultrasound scan demonstrated a superior mediastinal mass separate from the inferior pole of the right thyroid lobe measuring 4.8 cm in maximal diameter ( figure 4 ) . the first sample was inadequate ( thy1 ) , the second sample demonstrated only colloid and a few follicular cells . since malignancy could not be definitively ruled out , the mass was excised through a cervical incision . under general anesthesia , the patient was placed in the supine position with the neck extended , prepared , and draped . endotracheal laryngeal nerve monitoring was used with nim contact emg endotracheal tube and nim - response 2.0 monitor ( medtronic usa , inc.6743 southpoint drive north , jacksonville , florida , usa , 32216 - 0980 ) . through a standard 5 cm midline cervical incision , the right thyroid lobe was dissected , found to be grossly normal , and excised . the right recurrent laryngeal nerve was identified in the normal position , confirmed functioning with nerve stimulation at 2 ma , and preserved . separate and inferior to the right thyroid lobe a discrete encapsulated mass was identified in the superior , mediastinum . this extended across the anterior surface of the trachea to the left side , adjacent to the thymus . the mass was separated from the thyroid gland by fat , in the same tissue plane but with no identifiable connection to the cervical region . the mass was excised through the cervical incision . during excision of the mass , a double vascular pedicle , arising from inferiorly in the mediastinum , histopathologic evaluation of the excised mediastinal specimen demonstrated a 5.3 cm by 2 cm thyroid tissue mass with large oedematous loose areas and foci of calcification and fibrosis ( figure 5 ) . the excised right thyroid lobe had a nodular architecture composed of follicles of varying sizes with focal lymphoid aggregates.thyroid function tests at 3 months were normal . further investigation with whole body radioiodine i123 scintigraphy demonstrated expected residual uptake in the remaining left thyroid lobe and surgical bed but no uptake suggestive of other ectopic thyroid tissue or metastatic disease . ultrasound surveillance of a 9 mm nodule in the remaining left lobe demonstrated no change in size over 6 months . in humans , the thyroid gland begins to develop at the 24th day of gestation . the thyroid is the first endocrine gland to develop and originates from between the first and second branchial arches . an invagination of endodermal epithelial cells begins at the midline of the developing pharyngeal floor forming a diverticulum . this site , known as the foramen caecum , lies between the tuberculum impar ( median tongue bud ) and the hypobranchial eminence ( copula ) . the foramen caecum can be observed in adults as a small pit at the base of the tongue where the tongue is divided into an oral anterior two - thirds and pharyngeal posterior third by the sulcus terminalis . the initial path of descent of the bilobed thyroid diverticulum is anterior to the pharyngeal gut , the hyoid bone , and the laryngeal cartilages . at the same time embryological studies have shown that the strap muscles pull downward on the hyoid bone during development causing a forward tilt . this tilt pulls the tract posteriorly and causes it to be hooked up behind the hyoid bone . this explains the importance of excision of the median portion of the hyoid bone in the sistrunk procedure to excise a thyroglossal duct cyst . the gland reaches its final location anterior to the trachea by the 7th week gestation . by this stage , it has acquired a median isthmus and two lateral lobes . as it descends , it is joined by the ultimobranchial bodies which form the parafollicular c cells . thyroid function begins at around the end of the 3rd month when the follicular cells commence production of colloid and follicles appear . during migration , from the 5th week gestation , the thyroid is still connected to the tongue by the thyroglossal duct . in normal development , this tubular structure subsequently obliterates entirely at approximately the 8 to 10th week . in some individuals , however , abnormalities in the embryologic development and migration of the thyroid gland can result in ectopic thyroid tissue . these remnants can appear at any point along the migratory path and are always located at or near the midline . the majority of remnants are found at , or just inferior to , the body of the hyoid bone as a thyroglossal cyst . remnants can also be found in the tongue base as a lingual thyroid or close to the thyroid cartilage . the inferior end of the thyroglossal duct may fail to obliterate , and in at least half of individuals , a pyramidal lobe of the thyroid can be seen to persist . this pyramidal lobe itself may be attached to the hyoid bone or may be incorporated into a thyroglossal duct cyst . ectopic thyroid tissue in other locations is rare and generally the subject of single case reports . the most important diagnosis to exclude is metastatic lymph node deposits of well - differentiated thyroid carcinoma . indeed , it is generally accepted that early reports of lateral aberrant thyroid tissue may represent papillary thyroid carcinoma metastases . another hypothesis is that a thyroid nodule may become detached from the gland . in this case , the vascular supply should come from branches of the superior or inferior thyroid artery . there are reports of thyroid deposits in the soft tissues of the neck representing surgical implantation of thyroid neoplasms . in one case , infiltrating thyroid tissue in muscle and fibrous tissue presented 3 years after major blunt trauma to the neck . the tissue resembled that in a disrupted thyroid nodule present in the gland itself and was regarded as traumatically implanted . there are , however , a number of ectopic thyroid masses reported which do not have features consistent with these theories and are considered truly developmental . animal studies suggest that ectopic location of thyroid tissue may be related to vascular development . the normal arterial supply of the thyroid gland consists of paired superior and inferior thyroid arteries . the superior thyroid artery is generally considered to be present in 100% of cases , and its absence has only been reported once . an unusually large superior thyroid artery may replace the contralateral vessels or the inferior thyroid artery . the inferior thyroid artery when unusually small or absent may be replaced or supplemented by a thyroidea i m a artery . published reports place the incidence of a thyroidea i m a artery between 2% and 12% . in the case the ectopic mass was separate from the thyroid gland but in the same anatomical plane . careful histological examination of the mass and the excised ipsilateral thyroid lobe demonstrated no evidence of neoplasia , and exhaustive investigations showed no evidence of thyroid malignancy elsewhere . the authors therefore conclude that this represents developmentally ectopic sequestered thyroid tissue in the anterior mediastinum . despite this , the authors suggest that abnormally situated thyroid tissue , other than that in the central neck along the path of embryological descent of the thyroid gland , should be excised for careful histological analysis in order to exclude metastatic disease .

an 80-year - old female presented with an incidental finding of a retrosternal mass on magnetic resonance imaging . ultrasound demonstrated a mediastinal lesion adjacent to but separate from the inferior pole of the right thyroid lobe . fine needle aspiration cytology demonstrated colloid and follicular cells . at surgery , the right thyroid lobe was found to be normal . a discrete 5 cm nodule was found in the anterior mediastinum separate from the thyroid and just anterior and to the right of the trachea and thymus . the nodule had a vascular pedicle arising from the mediastinum . the differential diagnosis included metastatic thyroid carcinoma . histology was consistent with a benign ectopic sequestered thyroid nodule . extensive investigations demonstrated no sign of a thyroid malignancy .

previous studies of traditional approaches to cognitive remediation in schizophrenia have been confounded by various methodological issues ( see refs 11,12 ) : small subject samples , open - label conditions , treatment- as - usual control groups , unblinded assessments , and unspecified plans for statistical analysis . studies that avoided these issues have tended to find only small to moderate effect sizes . nonetheless , the following findings point to some interesting next steps for the field : with the exception of verbal learning and memory , the meta - analysis by mcgurk et al found no significant heterogeneity in effect sizes on various matricsdefined cognitive domains based on either the number of hours of training or the method employed . this indicates that for the majority of cognitive domains , neither the training method , nor the amount of training ( several hours to over 100 hours ) has been a key moderating variable . thus , although previous cognitive remediation approaches have provided modest nonspecific cognitive benefits , further refinement of the intervention and the use of rigorous study designs are critical next steps for the field . meta - analyses have also shown that in verbal memory , larger effect sizes are obtained when computerized training is given in a drill - and - practice approach for a large number of hours . significant synergy occurs when cognitive remediation is combined with a psychosocial intervention , such as vocational rehabilitation or social skills training . this indicates that appropriate cognitive training can prepare the individual with schizophrenia to benefit from ecologically meaningful learning events , and underscores the fact that optimal treatment in schizophrenia will necessitate multimodal approaches . over the past decade , clinical neuroscience research has unequivocally demonstrated that declarative memory ( those processes involved in recollection of facts and events ) is impaired in schizophrenia ; thus , in keeping with the meta - analysis findings discussed above , it seems clear that strategy coaching and other direct instruction methods are likely to be of only limited benefit for patients . further , the sensory processing deficits of schizophrenia , which demonstrate a strong association with higher - order cognitive dysfunction , may confer a bottleneck in the response to behavioral interventions . a neuroscience - guided approach to cognitive training in schizophrenia should therefore take into account the following factors : the use of both implicit learning , through which skills and abilities are acquired indirectly and without direct awareness , and repetitive practice , may be crucial for maximizing patients ' response to cognitive training . in addition , attention to sensory processing deficits may be necessary in order to drive an optimal response to cognitive treatments . basic experimental work with motor skill learning and motor cortex remapping indicates that significant cortical synaptogenesis and reorganization of task - specific representations occurs after an animal reaches the flat congruent with the meta - analysis findings described earlier , this suggests that dosing and intensity of training is important : in order to drive maximally enduring and neurologically reliable cognitive gains , subjects must perform large numbers of learning trials and must train at threshold ( ie , training must be individually adapted to the capacities of each learner ) . during learning , the brain selectively promotes both bottom - up and top - down neural activity patterns that represent meaningful stimuli and behaviors ; successful learning is most efficiently driven by exercises which target all of the specific component skills of a given cognitive process . for example , intensive computerized frequency - sweep discrimination exercises markedly improve the ability of language - impaired children to recognize and respond to speech stimuli . for patients with schizophrenia , intensive training in a wide range of basic cognitive operations is likely to be necessary to improve higher - order functions ( eg , it may be necessary to train the representational fidelity of early sensory data ; vigilance ; working memory ; etc . before achieving significant gains in executive functions ) . learning - based plasticity is profoundly influenced by neuromodulatory neurotransmitters ; therefore , learning trials must be designed that are closely attended by the subject and that involve a heavy reward schedule . moreover , some of the medications currently used in schizophrenia may adversely affect the response to cognitive training strategies . we have been investigating the efficacy of a set of neuroscience - based cognitive training exercises designed with the considerations described above ( software developed by posit science , inc ) . subjects were randomly assigned to either 50 hours ( 1 hour per day , 5 days per week ) of a computer games control condition , or to 50 hours of computerized training that places implicit , increasing demands on auditory perception and accurate aural speech reception . frequency discrimination and phoneme recognition exercises targeted aspects of early auditory processing deficits of schizophrenia , which have been shown to affect higher - order cognitions such as verbal memory , reading ability , and social - emotional recognition . this psychophysical training was embedded within increasingly complex auditory and verbal working memory / verbal learning exercises that progress from simple frequency discrimination to phoneme identification and then recall of verbal instructions and narrative details . compared with age- and education - matched subjects in the computer games control condition , we found that subjects who underwent targeted cognitive training showed significant increases in working memory , verbal learning and memory , and global cognition ( figure 1 , table i ) . these data suggest that an intensive dose of computerized cognitive training focused on auditory and verbal processing results in improved verbal learning and memory in adults with schizophrenia . cognitive training subjects showed significantly greater improvement in verbal learning and memory measures from baseline to a 6-month follow - up assessment , indicating the durability of the cognitive training effects beyond the immediate post - training period . furthermore , improved cognition was significantly associated with improvements in quality of life at 6 months . serum anticholinergic activity ( saa ) , reflecting medication - induced anticholinergic burden , showed a significant negative correlation with cognitive improvement after training . consistent with basic science research , this suggests that the anticholinergic burden reduces the efficacy of cognitive training in driving cognitive improvement . subjects in the cognitive training condition showed a significant increase in serum brain - derived neurotrophic factor ( bdnf ) levels compared with the computer games control group . this suggests that bdnf may be a peripheral biomarker for the effects of intensive cognitive training , and provides an indication of neurobiological response induced by the training . normalization of physiological response patterns in auditory cortex within the cognitive training group but not the computer games control group - indicating that adaptive plastic changes in auditory processing systems can be induced in schizophrenia patients in response to a behavioral training intervention . compared with age- and education - matched subjects in the computer games control condition , we found that subjects who underwent targeted cognitive training showed significant increases in working memory , verbal learning and memory , and global cognition ( figure 1 , table i ) . these data suggest that an intensive dose of computerized cognitive training focused on auditory and verbal processing results in improved verbal learning and memory in adults with schizophrenia . cognitive training subjects showed significantly greater improvement in verbal learning and memory measures from baseline to a 6-month follow - up assessment , indicating the durability of the cognitive training effects beyond the immediate post - training period . furthermore , improved cognition was significantly associated with improvements in quality of life at 6 months . serum anticholinergic activity ( saa ) , reflecting medication - induced anticholinergic burden , showed a significant negative correlation with cognitive improvement after training . consistent with basic science research , this suggests that the anticholinergic burden reduces the efficacy of cognitive training in driving cognitive improvement . subjects in the cognitive training condition showed a significant increase in serum brain - derived neurotrophic factor ( bdnf ) levels compared with the computer games control group . this suggests that bdnf may be a peripheral biomarker for the effects of intensive cognitive training , and provides an indication of neurobiological response induced by the training . normalization of physiological response patterns in auditory cortex within the cognitive training group but not the computer games control group - indicating that adaptive plastic changes in auditory processing systems can be induced in schizophrenia patients in response to a behavioral training intervention . although these early data using a neuroscienceinformed approach to cognitive training in schizophrenia are promising , they require replication with larger , more representative samples across multiple treatment sites . in addition , they raise many crucial questions for future studies : what are the necessary and sufficient ingredients essential for successful cognitive training in schizophrenia ? what are the optimal methods , cognitive domains , and sequence of training ? what is the minimum amount of cognitive training that results in meaningful cognitive improvement in patients ? at what minimal frequency can training be delivered ? what is the relationship between individual patient profiles at baseline ( eg , genotype , biomarkers , neurocognitive profile ) and their ability to realize and retain benefits from cognitive training ( see , for example , refs 41 - 43 ) ? what is the influence of commonly prescribed anticholinergic and antidopaminergic medications on cognitive training outcomes ? how can we maximize the synergistic benefits of combining cognitive training with psychosocial rehabilitation treatments ? can targeted cognitive training be used to remit preexisting cognitive deficits and to promote recovery of function in young individuals who are in the very earliest phases of schizophrenia ? if the promising initial findings we describe here are replicated , we will enter an exciting time for the field of schiz - ophrenia treatment , one which will require active collaborations between basic and clinical neuroscientists with expertise in neuroplasticity ; researchers who perform clinical trials as well as experts in psychosocial remediation ; clinical and research psychopharmacologists , and designers of computer games . we will enter a time of paradigm shift , and we will have the privilege of developing novel beneficial treatments for our patients .

meta - analytic data from over a decade of research in cognitive remediation , when combined with recent findings from basic and clinical neuroscience , have resulted in a new understanding of the critical elements that can contribute to successful cognitive training approaches for schizophrenia . some of these elements include : the use of computerized repetitive practice methods , high dosing schedules , a focus on sensory processing , and carefully constrained and individually adapted learning trials . in a preliminary randomized controlled trial of cognitive training exercises based on these principles , we demonstrated significant improvements in working memory , verbal learning and memory , and global cognition in patients with schizophrenia . these cognitive improvements were accompanied by neurobiological findings suggestive of learning - induced cortical plasticity . future directions for research and essential remaining questions are discussed .

von willebrand disease ( vwd ) is the most common inherited bleeding disorder , with a prevalence of approximately 1% according to population studies,1 but clinically relevant cases have a tenfold lower prevalence.2 the 2011registro nazionale delle coagulopatie congenite [ national registry of congenital coagulopathy],3 which reports data relative to 51 of 54 hemophilia centers in italy , indicated that a total of 8,411 subjects are affected by coagulation disorders 25% by vwd ; 43% by hemophilia a ; 9% by hemophilia b ; 14% by disorders of other coagulation factors ; and 9% by platelet disorders , carrier hemophilia a / b , or other disorders . vwd is caused by a deficiency or abnormality of the von willebrand factor ( vwf ) , a multimeric adhesive glycoprotein with a key role in platelet adhesion ; it is also the carrier and stabilizer of the factor viii coagulant moiety ( fviii : c ) , thus indirectly contributes to the coagulation process.4 type 1 and 2 vwd usually display a mild hemorrhagic phenotype ( partial deficiency and qualitative defect , respectively ) , whereas patients with type 3 vwd ( complete deficiency ) are affected by a severe bleeding tendency . the goal of the therapy for vwd is to correct the dual defects of hemostasis , abnormal platelet adhesion ( due to low vwf adhesive activity ) and abnormal intrinsic coagulation pathway ( due to low fviii : c).4 two main options are available to manage vwd patients : desmopressin acetate ( ddavp ) , which induces the release of endogenous vwf from endothelial compartments type 1 patients and a fraction of type 2 patients usually respondreplacement therapy , which involves the transfusion of exogenous vwf contained in plasma - derived fviii concentrates enriched with vwf ( vwf / fviii concentrates ) . vwf / fviii concentrates are the first choice for the treatment of patients with type 3 vwd , for patients with type 2 b ( because ddavp can induce transient thrombocytopenia ) , and for those patients with type 1 and 2 who are unresponsive to ddavp or have contraindications to its use . desmopressin acetate ( ddavp ) , which induces the release of endogenous vwf from endothelial compartments type 1 patients and a fraction of type 2 patients usually respond replacement therapy , which involves the transfusion of exogenous vwf contained in plasma - derived fviii concentrates enriched with vwf ( vwf / fviii concentrates ) . vwf / fviii concentrates are the first choice for the treatment of patients with type 3 vwd , for patients with type 2 b ( because ddavp can induce transient thrombocytopenia ) , and for those patients with type 1 and 2 who are unresponsive to ddavp or have contraindications to its use . therapy with vwf / fviii concentrates can be administered either on demand , through infusion of the amount of factors determined by the severity of the disease and by the patient s body weight , in order to stop occasional bleedings , or as prophylaxis in the more severe forms of the disease , through multiple weekly infusions , in order to control recurrent bleeding and to prevent life - threatening hemorrhages.5 patients with severe forms of vwd may have frequent bleeding episodes , especially in those cases with fviii levels below 20 iu / dl , occurring in type 3 vwd and in some cases with severe forms of type 1 and 2 . in these cases large doses of vwf / fviii concentrates are required to control the bleeding . prophylaxis with vwf / fviii concentrates is considered a potential approach for those patients with severe bleeding tendency.58 the wide heterogeneity of the bleeding tendency that impacts heavily on quality of life ( qol ) contributes to the uncertainties about the potential candidates , the optimal dose of concentrates , and regimes for prophylaxis . furthermore , patients with vwd have an intact endogenous production of factor viii ( fviii : c ) and long - term exposure to vwf - enriched fviii concentrates may confer thromboembolic potential to prophylaxis.912 this observation suggests a particular care in the use of vwf / fviii concentrates in patients with severe vwd and the need for regular fviii : c level monitoring in order to maintain an appropriate hemostatic balance . in this regard , there is a growing interest in replacement therapy with vwf concentrates almost devoid of fviii , especially in severe vwd patients , in whom therapies with ddavp and dual concentrates are not effective , not tolerated , or contraindicated ( when a rise in fviii concentration could predispose the patient to thrombotic events ) in order to allow the control of bleeding episodes . the objective of the analysis presented here is to assess the cost benefit ratio of vwf with a low fviii content ( wilfactin , laboratoire francais du fractionnement et des biotechnologies , les ulis , france ) when compared with the vwf / fviii concentrates treatments ( ie , haemate p , zlb behring , marburg , germany , and fanhdi , instituto grifols , barcelona , spain ) currently applied in italy for long - term prophylaxis in patients with severe vwd , based on data from four case reports . the decision to use single case reports for the pharmacoeconomic analysis depended on two aspects : there are very few patients requiring long - term prophylaxis due to the difficulty in controlling bleeding episodes , and there are even fewer cases in which treatment with vwf with a low fviii content has been utilized in italy . a cost consequence analysis was adopted in order to assess the economic impact of the treatment of vwd from the perspective of the national health service ( nhs ) and society . the use of a cost consequence approach depends on the fact that this analysis measures the health consequences without combining them into one summary effectiveness measure . this kind of analysis describes the value of a health care intervention and has the advantage of being more readily understandable and more likely to be applied by health care decision - makers , leaving the decision regarding the relative importance of different outcomes to the decision - maker.13,14 the analysis described in this paper was conducted on case reports in disaggregated form , as a pooled analysis would not give a robust statistical approach . the analysis has been based on four case reports of type 3 ( n=1 ) , type 2 m ( n=1 ) and type 1 ( n=2 ) vwd from hemophilia centers at the molinette hospital of turin and university hospital of catania on the long - term treatment of vwd with vwf / fviii concentrates and vwf with a low fviii content . these centers have clinical experience with patients with severe bleeding tendency receiving long - term prophylaxis ( started at least 6 months before ) who were switched from vwf / fviii concentrates to vwf with a low fviii content because of frequent recurrence of bleeding at the same sites , in order to obtain a better control of bleedings and a better efficacy of the therapy . patients were included in the analysis if they had severe forms of vwd with periodic bleeding episodes , generally presented a low basal level of fviii : c and fviii : ristocetin - cofactor ( ri.cof ) before starting prophylaxis with vwf with a low fviii content , and had been treated with vwf / fviii concentrates . the analysis includes direct costs associated with drug acquisition , hospital admissions , monitoring visits , and the cost of transfused units of red blood cell concentrates ( packed red blood cells ) , and indirect costs evaluated as the number of working days lost per bleeding episode ( loss of productivity ) . in order to collect real - world evidence , a questionnaire addressed to clinicians experienced in the treatment of bleeding disorders was developed . in the analysis , outcomes were evaluated in terms of treatment of minor or major bleeding episodes and related costs . the comparators used in the pharmacoeconomic evaluation were vwf / fviii concentrates ( haemate p and fanhdi ) , as these are the most used vwf / fviii concentrates in the italian clinical practice setting for long - term prophylaxis in the treatment of vwd . both medicinal products , vwf / fviii concentrates and vwf with a low fviii content , are authorized in italy for vwd . the concentrates were administered for prophylaxis at the following dosage : vwf / fviii concentrates ( fandhi ) 40 iu / kg three times per week ; vwf / fviii concentrates ( haemate p ) from 35 to 50 iu / kg three times per week ; vwf with a low fviii content from 30 to 50 iu / kg twice per week , and , in one case , 30 iu / kg three times per week . a single bleeding episode was treated with replacement therapy that is , 3050 iu / kg / day per 3 days of concentrates , according to national guidelines and summary of product characteristics.1 the unitary cost of concentrates was obtained from telematic pharmaceutical compendium 2013 http://www.farmadati.it/ and is equivalent to the ex - factory price that represents the maximum cost for the nhs structures . the cost of outpatients visits , transfusion of blood components , and laboratory and diagnostic tests are set in the national outpatient tariffs ministerial decree [ dm ] 1997 . the absorption of health care resources was calculated considering the diagnosis related group ( drg ) version 24 in order to assess drg refund value for day hospital and for ordinary hospitalization . the cost of hospitalization and day hospital was derived from national hospital tariffs dm 18th october , 2012 . the human capital method was adopted for the determination of the cost due to loss of productivity . based on this approach , each day of absence from work , results in a simultaneous reduction in production . on the basis of this methodology , a value equivalent to the value generated from work for the same time period should be given to the working days lost due to illness.15 here , all working days lost ( in 1 year ) due to disease - related medical and diagnostic investigations , are considered . the number of working days lost was estimated on the basis of the average annual income.16 pharmaceutical cost includes both the yearly cost of treatment for long - term prophylaxis and the cost of treatment of bleeding episodes with concentrates ( replacement therapy ) . health care cost includes monitoring visits , diagnostic and laboratory tests , transfusion requirements , and hospitalizations ( table 1 ) . although the cost of long - term prophylaxis was found to be higher with vwf with a low fviii content , the cost per bleeding episodes per year was considerably reduced ( in case 1 ) or eliminated ( in cases 24 ) with vwf with a low fviii content with respect to vwf / fviii concentrates ( table 1 , column 6 : cost of bleeding episodes per year ) . indeed , in these four cases , vwf with a low fviii content was able to reduce the number of bleeding episodes and consequently to minimize the cost of replacement therapy . in all cases , health care costs were found to be substantially lower with vwf with a low fviii content with respect to vwf / fviii concentrates ( table 1 , column 17 : health care cost per year without cost of treatment ) . indirect costs were evaluated in terms of number of working days lost ( table 2 ) . these data show that replacement therapy with vwf with a low fviii content was able to reduce the number of working days lost per bleeding episode together with the discomfort related to bleedings and , consequently , to improve the qol in all these patients . in one case report ( case 4 ) , the cost of loss of productivity was not calculated , as the patient was retired . despite this , the days of well - being lost due to illness may be considered and have an impact on the qol of this patient . the results show data of pharmaceutical costs , health care costs , and indirect costs ( loss of productivity ) relative to each case ( tables 36 and figures 14 ) . these results show that the replacement therapy , both with vwf / fviii concentrates and vwf with a low fviii content , accounts for most of the total cost . although the dose to achieve full control of bleeding episodes depends on many factors , including in particular patient - related factors , it is possible to observe a minimization of doses of concentrates after the switch to vwf with a low fviii content ( table 1 , column 3 : concentrates iu / year ) . in the four cases , long - term prophylaxis with vwf with a low fviii content allowed the achievement of good control of bleedings , minimizing the cost of hospital admittances and transfusions of blood components . the reduction of these events with prophylaxis with vwf with a low fviii content was found to afford a reduction in the health care costs per patient per year in all the case reports analyzed ( table 7 , column 2 : total health care costs without pharmaceutical costs avoided with vwf with a low fviii content per patient / year ) . long - term prophylaxis with vwf with a low fviii content , allowed in all cases the use of a lower quantity of this concentrate with respect to the vwf / fviii concentrates . nevertheless only in one case report ( case 2 ) was it possible to observe a reduction in pharmaceutical costs per patient per year ( table 7 , column 3 : pharmaceutical cost avoided with vwf with a low fviii content per patient / year ) . furthermore these results show a reduction in indirect costs , evaluated as a reduction of the number of working days lost per bleeding episode , with a related positive impact on qol ( table 7 , column 4 : indirect cost avoided with vwf with a low fviii content per patient / year ) . the analysis of these four case reports shows that vwf with a low fviii content seems be a cost effective treatment option for patients with severe vwd phenotype , in whom therapies with ddavp and dual concentrates are partially or not effective , non - tolerated or contra - indicated ( when a rise of fviii concentration could predispose the patient to thrombotic events ) , aimed to obtain a better control of bleeding episodes and consequently to improve the qol . the treatment of a bleeding episode in vwd patients requires correction of the vwf deficiency ( the defect in primary hemostasis ) and the impaired secondary hemostasis ( to increase the potentially low fviii : c level ) . apart from the administration of ddavp in mild / moderate cases , replacement therapy with concentrates is the therapy of choice for bleeding situations or for short - term prophylaxis during surgery or clinical interventions,1719 while for long - term prophylaxis few retrospective or prospective data are available to date.2026 the greatest experience with secondary long - term prophylaxis in italy has been with prophylaxis implemented in a cohort of italian patients with vwd . in the study of federici et al,24 among 89 patients who needed treatment with vwf / fviii concentrates during the previous 2 years because of one or more bleeding episodes , eleven were included in a prophylaxis program because of frequent recurrence of bleeding at the same sites . when prophylaxis was compared with previous on - demand regimens , in all the eleven cases , the annual total consumption of concentrates , the number of transfused blood units , and the number of days spent in hospital were found to be significantly reduced . similar results were obtained in a swedish study by berntorp and petrini.22 in that study , 35 patients with mostly type 3 vwd , prophylaxis was associated with a substantial decrease in the annual number of bleeding events . as regards vwf with a low fviii content , as far as we are aware , only one prospective study is available that includes , as additional experience , data on long - term prophylaxis in four patients with type 3 vwd . the study of borel - derlon et al26 investigated the efficacy and safety of vwf with a low fviii content to treat patients with clinically severe vwd by merging the results of two comparable protocols conducted prospectively in five european ( italy is included ) and twelve french centers . in that study , four patients received secondary long - term prophylaxis ( of 17 months duration ) with vwf with a low fviii content in order to prevent skin and musculoskeletal bleedings . after the long - term prophylaxis , the number of bleedings was substantially reduced . in our analysis , we report on four adult patients ( two with type 1 vwd , one with type 2 m vwd , and one with type 3 vwd ) who switched the long - term prophylaxis from vwf / fviii concentrates to vwf with a low fviii content in order to obtain better control of bleeding episodes and better efficacy of the treatment . in all these cases , vwf with a low fviii content treatment was found to be an effective tolerated modality that was highly beneficial for the patients both in terms of a reduction in bleeding episodes and in terms of cost benefit ratio . to the best of our knowledge , this is the first analysis to have investigated the impact of secondary long - term therapies with vwf with a low fviii content and the most used vwf / fviii concentrates ( ie , haemate p and fanhdi ) , in the italian clinical practice setting , on clinical outcome ( ie , bleeding frequency , hospitalizations ) and indirect / social outcomes ( reduction in number of working days lost per bleeding ) . as no previous studies have addressed this aspect , it is difficult to make any comparison . however long - term prophylaxis with vwf with a low fviii content was found to be associated with a significant reduction of the number of days spent on patient care ( hospitalizations , outpatient visits , laboratory and other diagnostic examinations ) together with a decrease in total concentrate consumption for bleedings , in all case reports considered . the main limitation of the analysis is that it was based on single case reports , because few patients are in long - term prophylaxis with vwf / fviii concentrates or vwf with a low fviii content in the italian clinical practice setting . further case reports of long - term prophylaxis treatment with concentrates in vwd are necessary to support the results obtained in this primary analysis . despite the limitations of the use of single case reports for the analysis , it seems that long - term prophylaxis with vwf with a low fviii content is likely to be a cost - effective approach with a favorable impact on the reduction of health care resource consumption and that also allows the patient to resume their normal life and work activities ( the number of working days lost was found to be considerably reduced after switching to vwf with a low fviii content [ table 2 , column 2 ] ) . these data are consistent with the improvement in the qol of the patients , which is a primary objective of any health care intervention . this analysis shows that vwf with a low fviii content was more able to control bleeding episodes and , consequently , to reduce the health care interventions necessary , with respect to vwf / fviii concentrates , in four patients with severe vwd . although these are preliminary data , they are likely to contribute to establishing the role of specific concentrates for the long - term prophylaxis of severe cases of vwd . nevertheless , a large collection of clinical data is needed so as to assess regimens , comparative effectiveness , and the cost - effectiveness of long - term prophylaxis treatment in patients with vwd , in order to evaluate which of the available concentrates has a better cost benefit ratio in patients with severe vwd .

purposeprophylaxis with von willebrand factor ( vwf)/factor viii ( fviii ) concentrates is a potential approach for patients with severe von willebrand disease ( vwd ) . as far as we are aware , to date there have been no pharmacoeconomic analyses in order to assess the economic impact of treatments for severe vwd . the analysis presented here estimates the cost benefit ratio of vwf with a low fviii content when compared with vwf / fviii concentrates currently used in italy for long - term prophylaxis in patients with severe vwd.methodsa cost consequence analysis was undertaken to assess the economic impact of the treatment of severe vwd from the perspective both of the italian national health service and society . the analysis was based on four case reports of long - term prophylaxis with vwd with vwf / fviii concentrates and vwf with a low fviii content . the costs per patient included direct and indirect costs for each treatment.resultsconsidering the four case reports , health care costs ( without cost of treatment ) and indirect costs per patient per year were lower with vwf with a low fviii content than vwf / fviii concentrates . the total health care costs ( without cost of treatment ) and indirect costs avoided with vwf with a low fviii content per patient per year ranged from 2,295 to 17,530 and from 1,867 to 4,978 , respectively.conclusionvwf with a low fviii content seems to be a cost - effective treatment option for patients with severe vwd . although the drug cost per se is higher , the use of vwf with a low fviii content is associated with decreased consumption of hospital resources and fewer lost working days due to bleedings and consequently with an improvement of the quality of life of the patients .

percutaneous coronary intervention ( pci ) revolutionized treatment of coronary artery disease ( cad ) ; to date , it is the most commonly used myocardial revascularization method in cardiology [ 1 - 3 ] . drug - eluting stents ( dess ) can markedly reduce restenosis and have become the most commonly used devices in interventional cardiology for treatment of coronary stenosis . although this technique does not completely abolish restenosis , a drastic reduction due to significant decrease in neointimal hyperplasia has been reported . on the other hand , the rate of restenosis bare - metal stent ( bms ) varies according to clinical setting , patient and angiographic characteristics , with rates as high as 60% [ 5 , 6 ] . oral drugs for reduction of restenosis are not a practice in interventional cardiology because studies performed did not support it [ 6 , 7 ] . several studies that evaluated oral administration of sirolimus or immunosuppressants after bms deployment have reported reduced neointimal hyperplasia compared with bms alone ; however , this reduction was not sufficient to allow their clinical use [ 7 , 8 ] . increased knowledge of restenosis pathophysiology after stent implantation in the past decade has helped to explain why certain drugs were not effective when administered orally . methotrexate ( mtx ) is a folate antagonist that blocks the s - phase of cell division , consequently blocking mitosis . this drug was initially developed for cancer treatment ; however , it has been used to treat rheumatic diseases including rheumatoid arthritis ( ra ) and psoriasis . in this study , the primary objective was to evaluate the safety of oral administration of mtx to patients with severe cad and the secondary goal was to evaluate the possibility that mtx has an impact on restenosis after bms deployment . this was a transversal , prospective and descriptive study that recruited 16 patients in whom pci was planned from january to december 2016 . this study followed the ethical principles in clinical research and was approved by ethics committee and all patients signed the informed consent . the inclusion criteria were : age > 18 years , one vessel disease with de novo lesion > 70% and moderate or severe ischemia according to myocardium scintigraphy for which optimal medical therapy was taken , and one of the following : diabetes mellitus ( dm ) ; reference vessel diameter < 2.5 mm ; or chronic kidney disease stages iii associated with reference diameter of the target vessel < 2.75 mm . the exclusion criteria were : contraindications to mtx administration ; severe lung disease ; liver or kidney disease ; and inability to undergo cine coronary angiography at follow - up . patients with stenosis 70% , requiring pci , were potentially candidates to be enrolled and the study was explained to them and those that accepted assigned the informed consent . mtx was administered to patients at a dose of 5 mg / week for 2 weeks before pci and 8 weeks after pci . patients were monitored clinically every 15 days during the first 2 months after the procedure and monthly until 9 months after pci . clinical restenosis was defined as the occurrence of acute coronary syndrome or stable coronary disease with positive ischemia detection test ( moderate or severe ischemia ) . angiographic restenosis was defined as 50% in - stent stenosis or within 10 mm proximal or distal to stent [ 6 , 10 ] . the complications associated with the procedures evaluated included hematoma , allergic reaction , pseudoaneurysm , infections , acute myocardial infarction ( ami ) , stroke , emergency surgery , retroperitoneal bleeding , arteriovenous fistula , and death . the following complications associated with the use of mtx were evaluated : erythematous rash , pruritus , urticaria , photosensitivity , depigmentation , alopecia , ecchymosis , acne , furunculosis , bone marrow depression , leukopenia , thrombocytopenia , anemia , hypogammaglobulinemia , hemorrhage , septicemia , gingivitis , pharyngitis , stomatitis , anorexia , vomiting , diarrhea , hematemesis , melena , gastrointestinal ulceration , enteritis , liver disease , fatty change , renal failure , azotemia , cystitis , hematuria , menstrual dysfunction , abortion , birth defects , severe nephropathy , interstitial pneumonitis , headache , drowsiness and blurred vision , hemiparesis , aphasia , paresis , and convulsions . a descriptive analysis was performed and the numerical variables were presented as mean and standard deviation after the normal distribution of the data has been tested by shapiro - wilks . there was a predominance of male participants , the average age was 62.4 8.3 years , and majority of the patients had an elementary school level of education . analysis of coronary angiograms revealed 26 severe , 21 moderate , and 18 mild stenosis cases . of note , eight patients had single - vessel disease , five had two - vessel disease , and three had multi - vessel disease . coronary artery stenosis was more prevalent in the left anterior descending artery ( lda ) ( table 3 ) . the average diameter and length of the stents were 3.0 0.4 and 18.1 5.9 mm , respectively , as shown in table 4 . adverse events and side effects due to use of mtx occurred in three patients ( prevalence 18.7% ) . of note , two patients had skin desquamation on the extremities of the upper limbs and one patient had dryness of the oral mucosa . only one patient had angiographic restenosis ( prevalence was 6.2% ; however , her myocardial perfusion imagining did not show ischemia ) . based on the few side effects observed , this study demonstrated the safety of mtx administration in this study population ( patients with severe cad ) . in addition , these complications did not result in treatment withdrawal . mtx has been used for treatment of patients with rheumatic disease and the rate of side effects is low . there were no reported cases of clinical restenosis . these results are quite satisfactory considering the clinical and angiographic characteristics of the patients enrolled . vascular injury occurs , followed by a reparative process , in which migration of vascular smooth muscle cells ( vsmc ) occurs from the medial layer into the intima , cellular multiplication , and cellular secretion of molecules . dess have been used as a platform to carry and release drugs at the site of injuries resulting from pcis . the drugs most commonly used in clinical practice are sirolimus ( and its analogues ) and paclitaxel . these types of des reduce the chance of restenosis up to 70% compared to bms [ 12 - 18 ] . dess with some cell mitosis inhibitors , such as dexamethasone and tacrolimus , have not shown sufficiently positive effects in pre - clinical and clinical studies to allow their use in clinical practice . the prevalence of angiographic restenosis after bms deployment is up to 60% . despite many attempts to reduce restenosis rates , only reduction of the stent structure thickness reduction of restenosis was attempted by oral drug administration . however , the results of the drugs tested were not satisfactory enough to warrant their incorporation into clinical practice [ 7 , 20 ] . some studies , including the oral sirolimus to inhibit recurrent in - stent stenosis ( osiris ) , oral rapamune to inhibit restenosis ( orbit ) , and prospective , randomized oral rapamycin in argentina ( orar ) studies , as well as other investigations , evaluated oral use of sirolimes and reported an intermediate efficacy however , these drugs caused many major side effects , which may have contributed to the lack of interest for phase - iii clinical trials [ 20 - 23 ] . the high prevalence of side effects reported in these studies was surprising considering that sirolimus ( rapamycin ) has been used in transplanted patients and this high frequency of side effects had not been previously reported . authors evaluated the neointimal hyperplasia of transplanted kidney patients ( taking immunosuppressive therapy ) that underwent bms implantation and reported that neointimal hyperplasia was minimal and determined 9% of vessel obstructions . some studies evaluated the use of mtx and restenosis after pci . in this context , in 1992 , an animal study evaluated , among other aspects , whether mtx would have any effect on neointimal hyperplasia after stent deployment . the authors concluded that the amount of neointima did not differ from the amount obtained after using stents alone [ 25 , 26 ] . two decades after this study , some considerations need to be made about these findings . the stent used in this study was a coil . nowadays , this type of stent is out of market because it did not have good outcomes in clinical studies . the kinetic of drug release was not appropriate because the majority of the drug had been released from the stent during the first hour after deployment . according to stent design , these release kinetics are considered unacceptable considering recent standards for any experiment involving dess . in this study , it is known that this kind of model is related to higher possibility of great amount of hyperplasia . therefore , the large amount of drug delivered in the first hour , the small amount of drug delivered to the vessel wall , and the balloon - artery ratio are factors that may raise doubts about the authors conclusions . in 2004 , a study was published that compared mtx - eluting stents to sae - coated stents which was done in pigs . in this experiment , the stent - artery ratio was 1:1 and 50% of the drug had been released by 24 h. the results showed that mtx - eluting stents reduced neointimal hyperplasia ( 1.22 0.34 vs. 2.25 1.28 mm , p < 0.01 ) and in - stent obstruction ( 21 8% vs. 36 21% , another aspect that deserves attention was the finding that mtx had no effect on cell proliferation in vitro . however , this conclusion was based on an experiment that used cells derived from rabbit aortas and not from stent - treated pigs , which were the basis of the study . we should imagine how neointimal hyperplasia could have been reduced in this study if mtx had no effect on vsmc proliferation . therefore , we hypothesize that other mechanisms leading to hyperplasia reduction , e.g. inactivation of the secretory function of vsmc or the effect of mtx on the proliferation of vsmc from rabbit aortas was different from that on pig cells may justify this matter . one case - control study evaluated 228 lesions in patients with ra taking mtx and 677 lesion controls treated by pci . both groups were followed for a mean of 3.8 years . in the ra group , davis et al evaluated the relationship between polymorphisms in the enzyme methylenetetrahydrofolate reductase ( mthfr ) and cardiovascular events in 1,047 subjects . they did not find any type of association between mthfr and cardiovascular events in ra , while mtx use was protective against these events . in addition , our results allow us to hypothesize that mtx may have beneficial effects on restenosis . it is of note that administration of mtx for 15 days before pci and its maintenance for 60 days aimed to block inflammation , considered one of the triggers for formation of the neointimal hyperplasia , and mitosis . at the time of the previous studies on mtx , the efficacy of des is high and its safety has improved in second- and third - generation devices . however , high costs have limited the introduction of this type of material on a large scale in underdeveloped or developing countries [ 29 - 31 ] . therefore , the search for low - cost therapies that have a positive effect on restenosis is extremely important for patients living in countries with limited financial resources . furthermore , the feasibility of effective and safe therapies that are not used in the population due to their high cost should be addressed . mtx was safe in the study population and raised the possibility that a low - cost drug may have positive effects on restenosis after bms implantation . however , studies with larger sample sizes and other imagine modalities ( intravascular ultrasound and/or optical coherence tomography ) are required to confirm this hypothesis . mtx was safe in the study population and raised the possibility that a low - cost drug may have positive effects on restenosis after bms implantation . however , studies with larger sample sizes and other imagine modalities ( intravascular ultrasound and/or optical coherence tomography ) are required to confirm this hypothesis .

backgroundpercutaneous coronary intervention ( pci ) revolutionized treatment of coronary artery disease . drug - eluting stents are effective and safe but their cost is high , especially for some countries . the primary objective was to evaluate the safety of methotrexate ( mtx ) in patients who underwent pci and the secondary goal was to evaluate the possibility that mtx has an impact on restenosis.methodsthis was a transversal , prospective and descriptive study that recruited 16 patients in whom pci was planned . mtx was administered to patients at a dose of 5 mg / week for 2 weeks before pci and 8 weeks after pci . bare - metal stent ( bms ) deployment was performed according to standard practice . patients were monitored clinically every 15 days during the first 2 months after the procedure and monthly until 9 months after pci.resultsthere were no immediate or late complications associated with pci . adverse events and side effects due to mtx occurred in three patients ( prevalence 18.7% ) . these side effects are classified as minor complications . mtx was not discontinued due to these side effects . there were no reported cases of clinical restenosis.conclusionsmtx was safe in the study population and raised the possibility that a low - cost drug may have positive effects on restenosis after bms implantation . however , studies with larger sample sizes and other imagine modalities ( intravascular ultrasound and/or optical coherence tomography ) are required to confirm this hypothesis .

macrophages ( m ) are one of the resident cell types in synovial tissue , along with fibroblasts . while quiescent in health , m become activated in the inflamed joint , where they make up around 3040% of the cellular content , and regulate secretion of pro - inflammatory cytokines and enzymes involved in driving the inflammatory response and joint destruction ( firestein and zvaifler , 1990 ) . their position throughout the sub - lining layer and lining layer at the cartilage it is estimated that rheumatoid arthritis ( ra ) and psoriatic arthritis ( psa ) each affects approximately 1% of the population ( firestein , 2003 ; gladman , 2009 ) , leading to patient pain and disability as well as contributing to a great economic burden in terms of lost working days and patient health services ( cooper , 2000 ) and therefore is an area of intense investigation . as our understanding of inflammation progresses , including the recent concept that resolution of inflammation is an active process rather than a passive return to homeostasis , the role of m is increasingly appreciated . the inability to resolve acute inflammation may lead to a chronic inflammatory state . depending on their phenotype , m can secrete either pro- or anti - inflammatory cytokines and mediate matrix destruction or deposition . synovial m participate in many of the events driving inflammation including the stimulation of angiogenesis , leukocyte and lymphocyte recruitment , fibroblast proliferation , and protease secretion leading to eventual joint destruction ( burmester et al . , 1997 ; vallejo et al . , 2003 ; abeles and pillinger , 2006 ) . while ra and psa are considered more inflammatory than osteoarthritis ( oa ) , it can still contain an inflammatory component , of which m play a large part . in all of these conditions depletion of m from both ra and oa synovial cell cultures leads to reduced synovial fibroblast responses such as cytokine and mmp production ( janusz and hare , 1993 ; bondeson et al . , both macrophages and fibroblasts display an activated cell phenotype with increased cell surface expression of hla - dr and leukocyte adhesion molecules ( athanasou et al . , 1988 ; interaction of m with t - cells potentiates the expression of several pro - inflammatory mediators such as il-1 and and mmps ( mcinnes et al . important pro - inflammatory cytokines like tnf and il - l are abundant in the inflamed synovium and are characteristically released by classically activated ( m1 ) m . the importance of m in driving the inflammatory response has been highlighted by several quantitative microscopic studies , where they have shown that m number ; correlates with disease activity ( tak et al . , 1997 ) , has potential use as a biomarker for disease ( kruithof et al . , 2006 ; bresnihan et al . , 2009 ) and declines in response to therapy ( goedkoop et al . , 2004 ; canete et al . , , a prominent feature of the inflamed joint , promotes the survival of monocytes / macrophages and induces their anaerobic adaptations including glycolysis ( roiniotis et al . , it is long appreciated that m play an important role in the pathogenesis of arthritis and this observation was supported by studies showing that the number of m was increased in clinically affected joints compared to non - affected joints ( kraan et al . , 1998 ) . several studies also linked the number of synovial m to inflammatory cytokine production joint destruction ( mulherin et al . , the culmination of this work has led to sub - lining cd68 positive synovial m currently being the only validated biomarker for disease severity ( tak et al . , 1997 ) and response to therapy in arthritis ( haringman et al . , 2005 ) , further confirming their importance in the pathogenesis of this disease , a finding which is independent of treatment type ( haringman et al . , 2005 ; thurlings et al . , several studies have concluded that m number is decreased in psa synovial tissue following therapy ( goedkoop et al . besides the abundant pro - inflammatory cytokines and chemokines present in inflamed synovial tissue , activation , and survival of m can be achieved through acetylation or de - acetylation of histones . downstream effects of tnf and other molecules results in the induction of histone acetyltransferase ( hat ) activity in m which causes acetylation of histones and subsequent modulation of transcriptional activity . two recent studies have found evidence of depressed hdac activity in ra , particularly in synovial macrophages and fibroblasts . the ratio of hdac : hat activity was significantly lower in ra synovial tissue compare to healthy controls . in combination with this , hdac inhibition decreases il-10 production from whole tissue synovial explants cultures , indicating a negative effect on anti - inflammatory pathways , which would lead us to believe that a lack of hdac may contribute to perpetuation of inflammation ( huber et al . , 2007 ; grabiec et al . hdac inhibitors reduced il-6 production from tnf stimulated m and induced apoptosis of ra synovial fluid ( sf ) m , even in the presence of a pro - inflammatory stimulus ( grabiec et al . , 2010 ) . this is of interest considering the ability of synovial cells and infiltrating cells to evade apoptosis during joint inflammation contributing to synovial hypercellularity ( salmon et al . , 1997 ; the potential use of hdac inhibitors has been further promoted by their success in suppressing synovial inflammation and cartilage destruction in a cia mouse model ( nasu et al . , 2008 ) . toll like receptors ( tlr ) are pattern recognition receptors that mediate response to infection . however , it is becoming apparent that some of these receptors may become activated by non - infectious agents from within the body and may therefore play a role in autoimmune conditions such as ra . engagement of tlrs induces signaling through a well defined pathway involving myd88 that leads to transcriptional activation ( joosten et al . , tlr knockout and arthritis mouse models , or a combination of both , have highlighted the position of tlrs in the pathogenesis of arthritis . in a model of spontaneous arthritis due to il-1 receptor antagonist knockout , simultaneous knockout of tlr4 attenuated inflammation while tlr2 knockout produced a more severe arthritis . knockout of tlr9 had no effect ( abdollahi - roodsaz et al . , 2008 ) . however the role of tlr2 seems less defined as other studies have shown that knockdown of tlr2 produces beneficial effects in arthritis ( joosten et al . , 2003 ) . further to this , many tlr ligands have been identified in synovial inflammation ( okamura et al . , 2001 ; park et al . , 2004 ) . acute serum amyloid a ( saa ) , which is significantly upregulated in arthritis and propagates pro - inflammatory effects similar to tnf ( ohara et al . , 2000 ; mullan et al . , 2006 ; connolly et al . , 2011 ) , is a functional ligand for tlr2 and may contribute to the deleterious effects of saa in arthritis ( cheng et al . , 2008 ) . ra m are more responsive to stimulation than m from other forms of inflammatory arthritis , despite no difference in m number ( huang et al . , 2007 ) . therefore , engagement of tlr2 and 4 may contribute to m activation and a sustained m response in ra . rheumatoid factor ( rf ) is one of the diagnostic criteria for ra and can help to distinguish ra from similar arthropathies like psa . classification of ra as an autoimmune disease came initially from the discovery of igg auto - antibodies in the blood of patients ( waaler , 1940 ; franklin et al . , 1957 ) . rf is mostly igm - rf , but igg - rf and iga - rf can also be detected in some patients . the cellular receptors for igg are the fc receptors , fcri ( cd64 ) , fcrii ( cd32 ) , and fcriii ( cd16 ) . fcriii has been demonstrated to play a role in the development of arthritis through animal models . mice deficient in fcriii are protected from the development of collagen induced arthritis without alteration of their humoral response , and therefore the protection is not due to alterations in t - cell responses ( sthl et al . , 2002 ; andrn et al . , polymorphisms in fc receptors are associated with incidence of ra as well as response to therapy ( morgan et al . , 2006 ; canete et al . , 2009 ; thabet et al . in the immune system m are effective antigen presenting cells with phagocytic activity which respond to lymphocyte derived cytokines . however , the responses elicited by m are variable and depend entirely on the tissue environment . dedicated reviews on this topic discuss in more detail the cytokines and chemokines involved in promoting one phenotype over another ( mantovani et al . , 2004 ; murray and wynn , 2011 ) but an overview of the main components are outlined in figure 1 . classically activated m1 m have a pro - inflammatory phenotype , producing high levels of tnf , il-1 , il-6 , il-12 , il-23 , reactive oxygen species , and low levels of il-10 . alternatively activated m , of which there are three subsets ( mantovani et al . , 2004 ; martinez et al . , 2008 ) , display and anti - inflammatory phenotype , producing high levels of il-10 , il-1 receptor antagonist , decoy il-1rii , tgf , and low levels of il-12 . an interesting , and potentially useful , property of these m is that they remain plastic and polarization into one phenotype does preclude re - polarization ( stout et al . , 2005 ) . therefore , if we could elucidate the exact pathways and transcription factors involved in promoting one phenotype over the other in vivo , this system could be exploited for therapeutic gain . ifn along with lps or tnf drive polarization of m1 ( classically activated ) macrophages which participate in pro - inflammatory activities . on the other hand , il-4 + il-13 , il-10 , or immune complexes drive m2 ( alternatively activated ) macrophages , which participate in anti - inflammatory responses . there appears to be a lack of evidence for m polarization in either direction in the inflamed joint . it has been suggested that spondyloarthropathies such as psa display a more m2 profile compared to ra patients and that m1 mediators correlate with joint inflammation in ra ( vandooren et al . , 2009 ) . however , in general , most studies of m in arthritis focus on important m functions and not polarization . the mediators that can control m polarization are indeed present in the synovium and some show potential as therapeutic targets . synovial lining layer thickness is greater in ra , compared to psa or healthy control subjects , which is associated with an increase in synovial m and fibroblasts . ( 2000 ) also found similar levels of il-10 in ra and psa synovium , despite the difference in synovial lining layer m numbers , however levels were described as being quite low . it is difficult to determine if this lack of il-10 is a contributor to or consequence of the overwhelming inflammation in the joint . a study by mottonen et al . ( 1998 ) found that 68% of m isolated from ra sf were cd86 positive and that sf m can take on a dendritic cell phenotype when exposed to a combination of il-4 and gm - csf and that these cells were more effective at activating t - cells than control or tnf stimulated m . the effects of il-4 + gm - csf were mediated through cd86 , a marker of classically activated m . il-10 was able to inhibit the observed effects with il-4 + gm - csf as it downregulated the expression of cd86 , as well as cd-40 and hla - dr which also participate in m mediated t - cell activation . this is consistent with the classification that m2c m , which are driven by il-10 are involved in suppression of the immune response ( mantovani et al . , 2004 ) . these results may appear confusing as il-4 along with il-13 drive the m2a or alternative m phenotype which should be involved in anti - inflammatory responses . however gm - csf drives the m1 phenotype in monocyte derived macrophages so this may be the driving force for inflammatory responses in these experiments . the m1 m phenotype is induced by interferon- ( ifn ) in combination with either lipopolysaccharide ( lps ) or tnf. ifn is highly expressed in ra synovial tissue and its levels significantly correlate with disease severity ( milman et al . , 2010 ) . exposure to inf increases the response of m exposed to other stimuli by either upregulating pro - inflammatory cytokines , like tnf , or downregulating anti - inflammatory cytokines , like il-10 ( erwig et al . tnf is a master cytokine in inflammation and as such is a potent inducer of other pro - inflammatory cytokines ( nawroth et al . , 1986 ; butler et al . , 1995 ) , is chemotactic for leukocytes , is a potent inducer of angiogenesis ( leibovich et al . , 1987 ) , stimulates adhesion molecule expression in sfc in vitro ( marlor et al . , 1992 ) , and lymphoid migration into inflamed synovial tissue in vivo ( wahid et al . , 2000 ) . within the inflamed joint macrophages , fibroblasts , lymphocytes , and endothelial cells produce tnf. an important role for tnf in arthritis was confirmed by studies which showed its potential to degrade both cartilage ( dayer et al . , 1985 ) and bone ( bertolini et al . , 1986 ) . further rationale for the involvement of tnf in the progression of inflammatory arthritis was provided when transgenic mice expressing a modified human tnf gene spontaneously developed arthritis which exhibited increased human tnf protein , joint inflammation , bone erosion , and cartilage destruction . in this study , antibodies specific for human , but not mouse tnf reduced disease severity ( keffer et al . , 1991 ) . in subsequent studies administration of a monoclonal antibody to tnf ameliorated inflammation and joint damage after disease onset in a cia model of arthritis ( williams et al . , 1992 ) . tnf cytokine targeted therapies have now been developed for inflammatory arthritis . the first clinical trial was undertaken in the uk in 1992 and demonstrated that targeted biologic therapy decreased serum il-6 levels , swollen joint numbers and levels of the acute phase proteins crp and a - saa which are markers of inflammation ( elliott et al . , 1993 ) . alternatively , anti - inflammatory and m2 polarizing cytokines like il-10 are lowly expressed in arthritis as its signaling is blocked during fc receptor ligation ( ji et al . , 2003 ) , and treatment with the pro - resolving mediator annexin a1 stimulates release of il-10 ( ferlazzo et al . , 2003 ) . treatment of pbmc with il-10 caused a change in the ratio of th17:treg cells in favor of treg cells and decreased production of the pro - inflammatory cytokine il-17 ( heo et al . , 2010 ) . animal models of arthritis have also demonstrated how treatment with il-10 can suppress the development and progression of joint inflammation , even in established disease ( walmsley et al . , 1996 the cytokines involved in promoting polarization are well defined , however less is known about which transcription factors are utilized to induce polarization . irf5 ( interferon regulatory factor 5 ) has been implicated in driving the m1 phenotype as well as actively suppressing m2 polarization and driving th1 and th17 responses ( krausgruber et al . , ( 2011 ) was not performed in synovial m , animal studies suggest that inflammation in ra is driven by th1 cytokines such as ifn , which is upregulated early in the disease process ( miltenburg et al . , 1992 ; schulze - koops and kalden , 2001 ) and a rapid growth in interest in the th17 pathway and indeed il-17 itself in the last few years would suggest that this would warrant investigation in the inflamed joint . recent reports confirm that alterations in the irf5 gene confers susceptibility to ra ( dieguez - gonzalez et al . 2011 ) as well as many related illnesses such as inflammatory bowel disease , sjogrens syndrome , and systemic lupus erythematosus ( dideberg et al . , 2007 ; , 2006 ) and irf4 ( satoh et al . , 2010 ) have been implicated in promoting m polarization in other disease settings , and irf family members contribute to determination of dendritic cell fate ( tamura et al . , 2005 ) . these findings make the irf family attractive candidates to study in the context of m s in arthritis . nr4a is part of the orphan nuclear receptor superfamily which have roles in lipid metabolism and inflammation ( desreumaux et al . , 2001 ; receptors in the same superfamily as nr4a are downregulated in arthritic tissue and their activation appears to play a role in inhibiting disease progression ( bonnelye et al . however members of the nr4a subfamily appear to have less clearly defined effects to the anti - inflammatory family members liver x receptor and peroxisome - proliferator - activator receptor and drive inflammation in human synovial tissue ( murphy et al . , 2001 ) . the role of nr4a receptors specifically in m polarization has not yet been elucidated , however , any role for nr4a in m polarization would be an interesting finding due to the modulation of nr4a by both dexamethasone and methotrexate , which are effective treatments for joint inflammation in some patients . , 2006 ) where it promotes transcription of pro - inflammatory genes . in arthritis , and inflammation in general , nfb can be considered a master transcription factor as it is utilized by many ligand tnf and il-1 which are abundant in the inflamed joint employ this transcription factor and in turn can be regulated by it . nfb activation has been detected prior to the clinical onset of arthritis in animal models ( tsao et al . , 1997 ; han et al . , 1998 ) and the nfb pathway has been directly targeted as a treatment method for ra confirming its essential role in the pathogenesis of this disease ( wakamatsu et al . , 2005 ) . immunohistochemical staining has confirmed nuclear expression of nfb subunits in synovial m ( handel et al . , 1995 ) . the nfb family consists of five proteins ; p50 , p52 , rela ( p65 ) , relb , and c - rel . rela , relb , and c - rel contain a transcriptional activation domain and therefore upregulate gene expression , however p50 and p52 do not contain the transcriptional activation domain and homodimers of these proteins can sometimes have a repressing function ( bohuslav et al . , nfb p50 activation has been linked to promoting m2 polarizing genes in m ( porta et al . , 2009 ) . this study found that knockout of the nfb p50 subunit prevented the development of tolerance in lps challenged m by restoring m1 mediators and inhibiting m2 cytokines . ( 2006 ) found that accumulation of the p50 homodimer occurred in the nuclei of tumor associated m and that these m expressed an m2 phenotype . therefore due to the prominence of the nfb pathway in ra there is a growing understanding of the molecular drivers of inflammation and an appreciation that the resolution of inflammation is an active process rather than a passive return to homeostasis . endogenously produced mediators that actively promote the resolution of inflammation are now under investigation for their therapeutic use . lipoxin a4 ( lxa4 ) is an eicosanoid produced by the transcellular metabolism of arachidonic acid by 15/5- or 5/12-lipoxinagese ( serhan et al . , 1984 ) . its biosynthesis is co - incident with the resolution phase of inflammation and many of its bioactions are mediated through ligation of its receptor , alx / fpr2 ( fiore et al . , 1994 ) . lxa4 is produced in inflamed synovial tissue ( thomas et al . , 1995 ) where it can downregulate pro - inflammatory activities of activated fibroblasts and upregulate anti - inflammatory activities , even in the presence of a pro - inflammatory stimulus which acts through the same receptor ( sodin - semrl et al . , 2004 ; kronke et al . , 2009 ; chan and moore , 2010 ) . in other disease models , lxa4 has been shown to induce anti - inflammatory / pro - resolving actions such as inhibition of neutrophil recruitment and activation ( filep et al . , 1999 ) , regulation of nfb activation ( decker et al . , 2009 ) , and the clearance of apoptotic cells by m ( godson et al . , 2000 ) once these cells have carried out their functions in regard to host defense they are programmed to die by apoptosis . resolution of inflammation and return to homeostasis involves phagocytosis of apoptotic neutrophils to prevent the persistence to necrosis and leakage of cellular contents , which may itself begin an inflammatory reaction . despite the lack of apoptosis occurring in all cell types in the inflamed synovium , resident synovial m retain the capacity to phagocytose apoptotic cells , even at an early timepoint after arthritis induction ( van lent et al . , 2001 ) . if normal apoptosis and phagocytosis could be induced in the inflamed synovium , possibly by native lxa4 or its stable analogs , this process may trigger a normal resolution of inflammation . in order to encourage phagocytosis , apoptotic cells release mediators that attract phagocytes toward them , essentially flagging themselves for engulfment . one such mediator is the anti - inflammatory compound annexin a1 ( arur et al . , 2003 ; annexin a1 is a 37-kda protein of the annexin superfamily where all family members contain a similar core region and a distinct n - terminal region which confers specificity of function . generally annexin a1 is localized to the cytoplasm where , upon stimulation , it is mobilized to the cell membrane and secreted ( for an extensive review on annexin a1 see perretti and dalli , 2009 ) . interestingly annexin a1 also signals through alx / fpr2 , the same receptor utilized by lxa4 and saa . immunohistochemical analysis has demonstrated an increased expression of annexin a1 in the ra synovial lining layer macrophages and fibroblasts compared to oa and normal joints . this may , however , be due to the increased lining layer thickness in this condition ( goulding et al . , 1995 ) as other studies have shown decreased binding of annexin a1 to several cell types in ra ( goulding et al . glucocorticoid stimulation causes annexin a1 mobilization to the cell surface and secretion where it mediates glucocorticoid induced anti - inflammatory effects . this is of particular interest in arthritis as glucocorticoid therapy is one of the current treatments for this condition ( flower , 1988 ; podgorski et al . , 1992 ; yang et al . , 1998 , 1999 ; maderna et al . , 2005 ) . however , as is increasingly the case for many mediators , the role of annexin a1 may not be as unambiguous as initially described and it may also potentiate pro - inflammatory actions in arthritis . ( 2008 ) has revealed synergistic actions with tnf and annexin a1 in terms of mmp production from synovial fibroblast cells . they saw firstly that tnf can induce expression of endogenous annexin a1 and secondly that tnf along with the annexin a1 mimetic peptide ac2 - 26 enhanced secretion of mmp-1 which was dependent on fpr2/alx , erk , jnk , and nfb ( tagoe et al . , as mentioned , this study was not performed in synovial m , but as they have similar actions to synovial fibroblasts , the same results may be produced by these cells once investigated . further to this , it has also been shown that administration of human recombinant annexin a1 during the immunization phase of the collagen induced arthritis model perpetuated the development of the signs and symptoms of arthritis . this may have been due to the increased t - cell activation and skewing toward a th1 phenotype by annexin a1 acting through fpr2/alx ( dacquisto et al . , 2007 ) . t - cells from ra patients 48 h post steroid therapy demonstrated depressed expression of annexin a1 ( dacquisto et al . , 2008 ) further lending support to the possibility that annexin a1 may also mediate pro - inflammatory actions . however we must be careful to acknowledge the actions of specific cleavage products from full length annexin . one such cleavage product has been identified as causing neutrophil extravasation , an important event in inflammation , where other truncated forms of annexin can not ( williams et al . , 2010 ) . in the study of inflammation and our efforts to promote its normal resolution , m remain to the fore of our interest . in the inflamed joint , m will continue to be a focal point for therapeutic intervention which , currently , centers around cytokine blockade but now has the possibility of extending into m re - programming . this remains an interesting and a yet to be fully explored option in terms of treatment for synovial inflammation . 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 .

synovial macrophages are one of the resident cell types in synovial tissue and while they remain relatively quiescent in the healthy joint , they become activated in the inflamed joint and , along with infiltrating monocytes / macrophages , regulate secretion of pro - inflammatory cytokines and enzymes involved in driving the inflammatory response and joint destruction . synovial macrophages are positioned throughout the sub - lining layer and lining layer at the cartilage pannus junction and mediate articular destruction . sub - lining macrophages are now also considered as the most reliable biomarker for disease severity and response to therapy in rheumatoid arthritis ( ra ) . there is a growing understanding of the molecular drivers of inflammation and an appreciation that the resolution of inflammation is an active process rather than a passive return to homeostasis , and this has implications for our understanding of the role of macrophages in inflammation . macrophage phenotype determines the cytokine secretion profile and tissue destruction capabilities of these cells . whereas inflammatory synovial macrophages have not yet been classified into one phenotype or another it is widely known that tnf and il - l , characteristically released by m1 macrophages , are abundant in ra while il-10 activity , characteristic of m2 macrophages , is somewhat diminished . here we will briefly review our current understanding of macrophages and macrophage polarization in ra as well as the elements implicated in controlling polarization , such as cytokines and transcription factors like nfb , irfs and nr4a , and pro - resolving factors , such as lxa4 and other lipid mediators which may promote a non - inflammatory , pro - resolving phenotype , and may represent a novel therapeutic paradigm .

several clinically significant antibiotics as well as widely used drugs against common diseases have been derived from this unique genus affiliated with the order actinomycetales . diverse members of this order including streptomyces have been isolated from previously unexplored natural habitats to nourish the current microbial antibiotic searching programs [ 2 , 3 ] . jaj06 is a gram - positive , moderately halophilic streptomyces strain , which has previously been isolated from hypersaline coastal solar saltern at tuticorin , india . this strain produces an antimicrobial polyketide compound which has been reported to have potent antimicrobial activity against a set of bacteria and yeast with significant minimal inhibitory concentrations . production of antibiotic compound in jaj06 has been reported to have seawater dependence . like many other complex medium components , seawater has nondefined composition which might affect the reproducibility of the production profile of microorganisms . to maintain a reproducible production profile , media components and their optimum levels are critical to the secondary metabolites produced by microorganisms . in the field of antibiotics , much effort was directed toward optimizing production rates and directing the product spectrum . production of microbial secondary metabolites in a microbial system can be improved by optimization of physical parameters and nutritional constituents of production medium . the optimization experiments are usually performed using nonstatistical one - factor - at - a - time [ 9 , 10 ] and statistical experimental design approaches [ 11 , 12 ] . however , the former one is highly laborious and time consuming than the statistical methods . consequently , statistical experimental design techniques especially plackett - burman design ( pbd ) and response surface methodology ( rsm ) are widely used to select the significant variables and obtain the optimal levels , respectively [ 7 , 1118 ] . the application of these statistical experimental design techniques in media optimization can result in improved product yields , reduced process variability , reduced time , and overall costs , compared with conventional practice of single factor optimization [ 7 , 13 ] . plackett - burman design has been applied by several researchers to select influencing factors among the constituents of complex medium [ 1517 ] . optimization of selected , highly influencing factors can be done using response surface methodology with either central composite design ( ccd ) or box - behnken design experiments . in present work , efforts taken to expel the seawater from the production medium of jaj06 , with incorporation of chemically defined salt formulations . further , in order to enhance the antibiotic production by jaj06 , production medium was optimized using a successive optimization strategy , selection of media components that significantly influence the antibiotic production using plackett - burman design and optimization of these media components using rsm with box - behnken design . jaj06 was previously isolated from a coastal solar saltern soil and extensively studied for their antibacterial secondary metabolite . the strain was maintained over the surface of isp4 agar slants supplemented with 3% of sea salt ( w / v ) . a nutrient medium contained 10 g of starch , 4 g of yeast extract , 2 g of peptone , 1 g of mgso4 , 2 g of caco3 , 0.04 g of feso47h2o , and 0.1 g of kbr in 1 l of sterile seawater was taken as basal production medium for further modification and statistical optimization . in order to replace the chemically nondefined seawater , two defined salt formulations were screened and compared with seawater to support antibiotic compound in jaj06 . according to previous reports , two salt formulations were prepared with some modifications : salt formulation i contained 12 g of nacl , 0.35 g of kcl , 0.22 g of cacl22h2o , 10.7 mg of h3bo3 , 7.3 mg of srcl2 , 1.3 mg of naf , and 26 g of cocl26h2o in 1 liter of deionized water ; salt formulation ii contained 15 g of kcl , 0.22 g of cacl22h2o , 10.7 mg of h3bo3 , 7.5 mg of srcl2 , 1.3 mg of naf , and 26 g of cocl26h2o in 1 liter of deionized water . spore suspension of jaj06 was prepared in distilled water from cultures grown on modified isp-4 medium supplemented with 3% of sea salt ( w / v ) at 30c for 7 days . the suspension was then added to isp-2 broth in 250 ml erlenmeyer flask at a rate of 10 spores in 50 ml liquid medium . cultures were incubated on a shaker at 120 rpm at 30c for 3 days and used as seed stocks . for antibiotic production , strain jaj06 was inoculated into production medium and incubated on a shaker at 120 rpm for 8 days at 30c . cell - free supernatant of fermentation broth was recovered by centrifuging it at 10000 rpm for 10 min . ethyl acetate was added to the supernatant in 1 : 1 proportion and the mixture was agitated for 10 min . the solvent layer containing antibiotic substance was separated from broth and it was further centrifuged at 5000 rpm for 15 min to remove traces of fermentation broth . the antimicrobial crude extract was concentrated tenfold using a rotational vacuum concentrator and used for antibacterial assay . the extracted crude substance was assayed in triplicate for their antimicrobial activity against bacillus subtilis mtcc 441 by agar diffusion plate assay [ 7 , 20 ] . the crude extract was loaded on 6 mm sterile discs , dried , and placed on nutrient agar plate inoculated with b. subtilis suspension adjusted to a mcfarland standard of 0.5 , which is equivalent to 1.5 10 cfu / ml . the plates were incubated at 37c for 24 h and the inhibition zone formed around the disc was measured with transparent ruler in millimeter . . confirmed that the size of the zones of inhibition can be considered as measure of antibiotic titre . therefore , the antibiotic activity was expressed as units of activity per millilitre the crude substance of culture , where 1 u was defined as a 1.0 mm annular clearing around the antibiotic disk . plackett - burman design ( pbd ) was employed for screening the most significant medium components for growth and antimicrobial compound production by streptomyces sp . , pa , usa ) was used for the experimental designs and subsequent analysis of the experimental data . in the experimental design , 7 medium components ( independent variables ) were screened by representing them at two levels , low ( ) and high ( + ) in 12 trials . table 1 shows media components , symbol code , and actual low and high level of the variables . table 2 shows the detail of the design , each row represents a trial , and each column represents an independent variable . the experiment was carried out in triplicate and the average antimicrobial activity against b. subtitles was taken as the response . the variables with confidence levels above 90% were considered to have significant effect on antimicrobial compound production and thus used for further optimization . response surface methodology ( rsm ) was used with box - behnken design to optimize the selected media constituents : starch , kbr , and caco3 for enhanced antibiotic production in jaj06 . the three medium components ( independent variables ) were studied at three different levels : ( ) , ( 0 ) , and ( + ) for low , intermediate , and high concentrations , respectively ( table 3 ) . the experiment was carried out in 17 trials ( table 4 ) with five replicates at the centre point and the values of responses were the mean of two replications . for statistical calculations , coding of the factors was done according to the following equation : ( 1)xi = xix0xi , i=0,1,2,3, ,n , where xi was the coded value of an independent factor , xi was the actual value of an independent factor , x0 was the actual value of an independent factor at the center point , and xi was the step change value . for predicting the optimal point , a second - order model was fitted to correlate the relationship between independent variables and response . the behaviour of the system was explained by the following quadratic equation : ( 2)y=0+ixi+ijxixj+iixi2 , where y is the predicted response , 0 is the intercept term , i is the linear coefficient , ij is the quadratic coefficient , ii is the interaction coefficient , and xixj represent the independent variables . the design expert trial package ( version 7.0 ) the statistical significance of the model was verified by applying the analysis of variance ( anova ) . overall model significance was determined using fisher 's f - test and its associated probability p(f ) . lack of fit values lower than 0.05 indicates that there might be a contribution to the variables - response relationship that the model does not take into account . the quality of the polynomial model equation was judged statistically by coefficient of determination ( r ) and adjusted r. the fitted polynomial equation was then expressed in the form of three - dimensional response surface plots , to illustrate the relationship between the responses and the experimental levels of each independent variable . the design expert 's numerical optimization method was employed to optimize the level of each variable for maximum response . the combination of different optimized variables , which yielded the maximum response , was experimentally validated by culturing jaj06 in optimized and unoptimized production medium . the cell - free culture broths were collected and extracted with equal volume of ethyl acetate and the top organic layer was dried for further analysis . the dried ethyl acetate extracts were resuspended in methanol and assayed as above for antibiotic activity . the maximal antibiotic activity of jaj06 in three different media prepared separately with nondefined sterile seawater and two chemically defined salt formulations was examined in shake flask cultures . growth and antibiotic activity exerted by streptomyces sp . however growth rate in both seawater and sodium chloride based salt formulation seemed to be the same , while the growth rate and antibiotic activity are slightly lower in salt formulation ii than those of the other two . the main effect of each variable on antibiotic activity was calculated to determine the medium components which influence the antimicrobial compound production by streptomyces sp . table 6 represents the effect , standard error , t - value , p value , and confidence level of each component from the result of antibiotic assay given in table 2 . the medium components were screened at the confidence level of 92.5% on the basis of their effects . it was indicated that starch ( x1 ) , kbr ( x5 ) , and caco3 ( x7 ) had apparent influences in antibiotic production by jaj06 . confidence levels of these three variables were higher than 90% which indicates their significant contributions than those of other media components . the same was confirmed from the pareto graph ( figure 1 ) in which higher effects were presented in the upper portion and then progress down to the lower effects . it directly shows that the most important factors influencing antimicrobial compound production were starch , kbr , and caco3 . based on the results of the plackett - burman design , starch , kbr , and caco3 were chosen as most influencing media components and further optimised using response surface methodology . in this approach , the batch runs were conducted in box - behnken design experiments and results to determine the effect of independent factors on the response along with the predicted values are shown in table 4 . the regression equation coefficients were calculated and the data was fitted to a second - order polynomial equation . the response of antibiotic activity ( y ) can be expressed in terms of the following regression equation : ( 3)y ( antibiotic activity ) = 165.34 + 13.54a+7.29b 5.83c0.43ab+2.50ac+0.85bc 14.13a221.63b218.71c2 , where y represented the response of antibiotic activity , and a , b , and c were the coded values of starch , kbr , and caco3 , respectively . the anova was performed to inspect the second - order response surface model and results are given in table 7 . the anova showed that the model was highly significant , as it was evident from the low p value ( < 0.0001 ) of the fisher 's f - test . significance of model was also supported by statistically insignificant lack of fit , as was evident from the lower calculated f value ( 4.01 ) . the model r of 0.9831 implied that model equation could explain 98.31% of the total variation which suggested a good agreement between predicated values and experimental data . diagnostic plots were drawn to judge the model adequacy and clarify the signs of any problems in the experimental data . plot of observed response ( antibiotic activity ) versus predicted reponse is shown in figure 2(a ) . in this case , predicted values were in agreement with observed ones in the range of the operating variables . the normal probability plot of the studentized residuals was used to check for normality of residuals ( figure 2(b ) ) . a linear pattern observed in this plot suggests that there was no sign of any problem in the experimental data . figure 2(c ) represents a plot of studentized residuals versus predicted values to check for constant error . residuals displayed randomness in scattering and suggested that the variance of the original observation was constant . the three - dimensional ( 3d ) response surface plots were drawn to illustrate the individual and interactive effects of starch , kbr , and caco3 on antibiotic production by jaj06 ( figure 3 ) . each 3d plot presented the effects of two variables while the rest one was held at middle level . there was insignificant mutual interaction between starch and kbr as well as starch and caco3 ( figures 3(a ) and 3(b ) ) , respectively . with the increase of the concentration of starch from 2 to 17.5 g / l ( coded value , 1.0 to 0.75 ) , the antibiotic activity significantly increased at moderate concentration of kbr ( coded value , 0.5 ) ; however , decreased antibiotic activity was observed with any further increase of kbr even at high concentration of starch ( figure 3(a ) ) . when the concentration of caco3 was just beyond the middle level ( coded value , 0.25 ) , the antibiotic activity increased with increasing concentration of starch from 2 to 15 g / l ( coded value , 0.1 to 0.5 ) and further increase of starch to its high level resulted mild decrease in antibiotic production ( figure 3(b ) ) . with the increase of the concentration of kbr from 0.02 to 1.1 g / l ( coded value , 1 to 0.15 ) , the antibiotic activity significantly increased to certain level at a low concentration of caco3 ( coded value , 1 ) and further slightly increased at a moderate level of caco3 ( coded value , 0.25 ) . lower concentration of caco3 was found to be supportive for antibiotic activity ; however the activity was suppressed when the concentration of caco3 was higher in production medium . on the basis of numerical optimization , the quadratic model predicted that the maximum antibiotic activity was 169.07 u / ml , when the optimal values of test factors in the coded units were starch = 0.37 , kbr = 0.24 , and caco3 = 0.20 ( figure 4 ) , which were 7.4 g / l starch , 0.048 g / l kbr , and 0.8 g / l caco3 , respectively . the validation of the statistical results using the optimized medium was accomplished by carrying out shake - flask experiments in triplicate . the maximum antibiotic activity unit obtained experimentally was found to be 173.3 u / ml , ( table 8) which is in close agreement with the prediction value ( 169.07 u / ml ) . therefore , the developed model was considered to be accurate and reliable for predicting the production of antibiotic by streptomyces sp . the final optimized medium contained 7.4 g of starch , 4 g of yeast extract , 2 g of peptone , 1 g of mgso4 , 0.8 g of caco3 , 0.04 g of feso47h2o , and 0.048 g of kbr in 1 l of sodium chloride based salt formulation i. their antibiotic producing capability is not a static property and it can be significantly affected by constituents of production medium [ 24 , 25 ] . . undefined nature of seawater can affect the reproducibility of the production profile of actinomycetes . in this study , the seawater has been expelled from the production medium with incorporation of chemically defined salt formulations . among the two defined salt formulation , this type of chemically defined salt formulation has already been reported for consistent production of bioactive compounds from seawater dependent salinispora tropica strain nps21184 . small manipulations in the culture medium composition can exert significant effect on secondary metabolites biosynthesis in microorganisms [ 7 , 26 ] . several researchers working on antibiotics discovery programs have applied pbd and rsm as statistical tools to recognize , manipulate and optimize influencing medium constituents and recorded the increased antibiotic production . for instance , wang et al . , applied rsm approach for medium optimization for antibiotic production by xenorhabdus bovienii and recorded 37.8% increase in antibiotic activity . recently , chen et al . reported 2.7-fold increase in antibiotic production by bacillus sp . zjuibe-076 using rsm approach . in the present study , pbd and rsm were applied for medium optimization for antibiotic production by streptomyces sp . jaj06 and recorded 26.8% increase in antibiotic activity . the results of pbd revealed that the crucial media components related to the antibiotic production by jaj06 were starch , kbr , and caco3 . raytapadar and paul reported starch as a significant media component for production of antibiotic from streptomyces aburaviensis 1da-28 . similarly , caco3 has been identified as a crucial ingredients related to the production of cyclic hexapeptide antibiotic by streptomyces alboflavus . rsm was found to be very effective in optimizing the selected medium components evident from positive diagnostic plots ( figure 2 ) and r value 0.9831 which was comparable with the earlier reports [ 7 , 27 ] . jaj06 as a function of various salt compositions and levels of ingredients in production medium . pbd and rsm were found to be very effective in selecting and optimizing the medium components in manageable number of experimental trials with overall 26.8% increase in antibiotic activity . moreover , the optimum culture medium obtained in this experiment will be useful for further study with large scale fermentation in a fermenter for the efficient production of antibiotic from this streptomyces sp .

streptomyces sp . jaj06 is a seawater - dependent antibiotic producer , previously isolated and characterised from an indian coastal solar saltern . this paper reports replacement of seawater with a defined salt formulation in production medium and subsequent statistical media optimization to ensure consistent as well as improved antibiotic production by streptomyces sp . jaj06 . this strain was observed to be proficient to produce antibiotic compound with incorporation of chemically defined sodium - chloride - based salt formulation instead of seawater into the production medium . plackett - burman design experiment was applied , and three media constituents , starch , kbr , and caco3 , were recognised to have significant effect on the antibiotic production of streptomyces jaj06 at their individual levels . subsequently , response surface methodology with box - behnken design was employed to optimize these influencing medium constituents for the improved antibiotic production of streptomyces sp . jaj06 . a total of 17 experiments were conducted towards the construction of a quadratic model and a second - order polynomial equation . optimum levels of medium constituents were obtained by analysis of the model and numerical optimization method . when the strain jaj06 was cultivated in the optimized medium , the antibiotic activity was increased to 173.3 u / ml , 26.8% increase as compared to the original ( 136.7 u / ml ) . this study found a useful way to cultivate streptomyces sp . jaj06 for enhanced production of antibiotic compound .

stat3 is a transcription factor that is activated by extracellular ligands , e.g. , the cytokines il-6 , il-10 , il-21 , il-27 , g - csf and leptin , but also by the growth factors egf and hgf , through specific binding to transmembrane receptors and the induction of receptor associated and cytoplasmic tyrosine kinases . stat3 activation can be observed in multiple organs and cell types , e.g. , in immune cells , mammary epithelial cells , adipocytes , neural cells , cardiomyocytes , hepatocytes , stem cells and tumor cells , and is correlated with such diverse cellular phenotypes as differentiation , proliferation , apoptosis regulation , angiogenesis , malignant transformation , metastasis formation and drug responsiveness . how can a single transcription factor influence so many different functions ? the cellular contexts , defined by the activity of interacting signaling pathways and the epigenetic state most likely play determining roles , but the discrete levels and the duration of stat3 signaling also cooperate and contribute to the manifestation of distinct cellular outcomes . in skeletal muscle cells for example , it reaches a maximum after about 1 h of cytokine stimulation and dampening mechanisms cause a return of activated stat3 to basal levels within about 2 h. in tumor cells , a different activation pattern can be observed and strong activation of stat3 is being maintained over long periods of time . persisting and high levels of stat3 signaling seem to be associated with cellular proliferation and transformation . since the strength of stat3 signaling and the duration of its activation appear as central determinants of the phenotypic cellular functions of this transcription factor , these parameters are tightly controlled . several mechanisms have been described that regulate these parameters and either affect the activation step of stat3 or the subsequent deactivation events . the diversity of the molecular mechanisms contributing to the finetuning of stat3 signaling probably reflects the importance of the quantitative aspects of this signaling pathway for the cellular physiology , but also provides vulnerability and possibilities for disturbance . enhanced stat3 signaling output can be traced to two basic steps in the regulation of extent and duration of stat3 activation : ( 1 ) mechanisms that enhance the activation step . they can be based on genetic alterations in molecular components that result in intracellular gains of function or effects on intercellular communication events that result in the exposure of cells to high levels of kinase activating signals . ( 2 ) mechanisms and mutations that impede the negative regulation of stat3 , i.e. , alterations that result in a loss of function of molecular components that are involved in the downregulation , e.g. , the dephosphorylation or intracellular utilization , of activated stat3 . the enhancement of stat3 upregulation can be attributed to mutational events that directly alter known pathway components : ( 1 ) c - src is a non - receptor tyrosine kinase closely related to v - src , an oncogene encoded by the rous sarcoma virus . v - src lacks the regulatory c - terminal domain of c - src that , together with several point mutations , results in a constitutively active kinase molecule with high transforming activity . phosphorylation of stat3 has also been observed in cells upon the expression of a several other oncoproteins . ( 2 ) a somatic mutation in the jak2 tyrosine kinase ( v617f ) is present in most patients with myeloproliferative neoplasms , like polycythemia vera ( pv ) , essential thrombocythemia ( et ) and chronic idiopathic myelofibrosis . the mutation causes a gain of kinase function , and jak2 v617f induces , for example , the expansion of erythroid progenitors accompanied by activation of stat3 , stat5 , akt and erk signaling in an erythropoietin - independent manner . ( 3 ) small deletions in the il-6 binding site of the gp130 subunit of the il-6 receptor have been found in cells of inflammatory hepatocellular adenomas . this results in a gain of function , il-6 independent receptor activation and stat3 signaling . about 12% of inflammatory hepatocellular adenomas express a stat variant that is constitutively activated , independently of il-6 stimulation of the cells . the mutation at position y640 occurs in the dimerization domain of stat3 and promotes its activation status . a similar observation has been made in t - cell large granular lymphocytic leukemia cells . this lymphoproliferative disorder is characterized by a high percentage of clonal cd3cd8 cytotoxic t lymphocytes ( ctls ) and is associated with autoimmune disorders and immune - mediated cytopenias . forty percent of the patients with large granular lymphocytic leukemia express constitutively activated stat3 variants with mutations in the dimerization of stat3 and aberrant activation . the inappropriate activation of stat3 , however , can also be explained by the deregulation of linked signaling events that are not directly attributable to distinct mutations in components of the jak stat pathway , but by deregulated expression levels of accessory protein . ( 1 ) g - csf receptor activation controls survival , proliferation and differentiation of myeloid progenitor cells via jaks . jaks in turn control the levels of cytokine receptor expression and increased jak expression can confer growth factor independent stat3 activation and hematopoietic cell transformation . ( 2 ) elevated expression of sphingosine-1-phosphate receptor-1 ( s1pr1 ) , a g protein - coupled receptor for the lysophospholipid sphingosine-1-phosphate ( s1p ) , has been found in tumors with activated stat3 . stat3 induces the transcription of the s1pr1 gene and enhanced s1pr1 expression activates stat3 and il-6 expression . these reciprocal regulatory events are thought to maintain the persistent activation stat3 in cancer cells . ( 3 ) autocrine and paracrine mechanisms , especially the secretion of il-6 by tumor cells and cells of the tumor microenvironment , contribute to stat3 activation . ras activation , e.g. , induces the secretion of il-6 and can act in a paracrine fashion to promote angiogenesis and tumor growth . similarly , egfr activation in lung carcinoma cells causes il-6 expression and secretion , and thus the paracrine and autocrine stimulation of stat3 in cells of the tumor microenvironment . finally , stat3 activation induces il-6 gene transcription and thus establishes a positive feedback loop in tumor cells in vitro and tumor tissues in vivo . mechanisms resulting in the enhancement of stat3 signaling are not restricted to the activation steps , but can also be founded in the molecular events governing the downregulation of activated stat3 and the cessation of signaling . these events are based on diverse components and their loss of function can result in the maintenance of the activated state and the persistence of stat3 signaling . ( 1 ) the direct reversion of stat3 activation can be accomplished by protein tyrosine phosphatases , ptp . at least three of them have been identified that can catalyze stat3 dephosphorylation , tc - ptp , shp1 and shp2 . the loss of ptp function has been observed in tumor cells with inappropriately activated stat3 . the receptor protein tyrosine phosphatase delta ( ptprd ) , for example , is frequently inactivated in glioblastoma multiforme ( gbm ) , head and neck squamous cell carcinomas and lung cancer . the inactivation of the ptp can be the result of intragenic deletions or of epigenetic silencing by promoter methylation . the suppressor of cytokine signaling ( socs ) 1 and 3 genes are stat3 targets and their products bind to jak or cytokine receptors , thereby suppressing further signaling . socs-1 and socs-3 are strong inhibitors of jaks , with kinase inhibitory regions at their n - terminus . hypermethylation of the socs-3 promoter and transcriptional silencing was frequently detected in lung and breast cancer and mesotheliomas . restoration of socs-3 expression in lung cancer cells resulted in the downregulation of activated stat3 , induction of apoptosis and growth suppression . pasteurella multocida toxin ( pmt ) is a highly mitogenic protein that affects cellular signaling through its modulation of heterotrimeric g proteins . enhanced stat3 activity seems to be promoted by the induction of the serine / threonine kinase pim-1 , which in turn phosphorylates socs-1 . this modification disrupts the interaction with the elongin bc complex , which normally allows the socs proteins to shuttle activating components of stat signaling to the proteasome . the negative regulatory function of socs-1 is subverted by pim-1 dependent phosphorylation and results in the sustained activity of stat3 . ( 4 ) a small family of proteins , most descriptively named pias ( protein inhibitors of activated stat ) , directly interacts with their targets , and pias3 has shown specificity for stat3 recognition . this interaction results in the inhibition of stat3 mediated gene activation most likely by blocking the dna binding activity . the expression of pias3 correlates with stat3 activation , and pias3 controls the extent and the duration of stat3 activity in normal cells . in cancer cells , the expression of the pias3 protein is post - transcriptionally suppressed and promotes the oncogenic effects of activated stat3 . ( 5 ) the lymphocyte adaptor protein ( lnk ) is a negative regulator of thrombopoietin and erythropoietin mediated jak2 activation . deletion mutations in this gene were observed in patients with myeloproliferative neoplasms ( mpns ) . these lnk mutants caused augmented and sustained thrombopoietin dependent signaling and stat3 activation due to the loss of lnk negative feedback regulation . advanced dna sequencing technology allows the comparison of the genomes of normal cells and tumor cells and bioinformatic analysis the determination and interpretation of mutations consistently associated with cellular transformation . the investigation of dna derived from 77 patients with t - cell large granular lymphocytic leukemia , a lymphoproliferative disorder characterized by the presence of a large fraction of clonal cd3cd8 cytotoxic t lymphocytes ( ctls ) , revealed that in 40% of the cases mutations in the stat3 gene could be detected . the mutations were all clustered in exon 21 encoding the sh2 domain of stat3 and resulted in a more hydrophobic dimerization domain . the most frequent mutation found was y640f in 17% of the cases , followed by d661v in 9% , d661y in 9% and n647i 4% . the mutant stat3 molecules were preferentially phosphorylated on tyrosine 705 and present in the nucleus of the leukemic cells . the y640f and d661v variants were further analyzed and exhibited enhanced transactivation potential for known stat3 target genes . the mutation at position 661 is very reminiscent of the stat3 variant , which was originally obtained in mutagenesis experiments and defined stat3 as an oncogene . why would the expansion of cd8 t cells be affected by inappropriately strong stat3 signaling ? non - redundant functions in the immune system and development of lymphocytes have been assigned to stat signaling . they influence cell fate decisions of differentiating naive t cells and regulate the intensity and duration of inflammatory responses . t helper cell differentiation , th1 , th2 , th17 and treg , requires the functions of stat1 , stat3 , stat4 , stat5 and stat6 and stat3 determines the differentiation of naive t cells into the regulatory ( treg ) or inflammatory ( th17 ) t cell lineages . th17 cells produce il-17 , act in the host defense against bacteria and fungi and contribute to autoimmune diseases . stat3 also regulates cell growth , apoptosis and the transcription of inflammatory genes and contributes to the development of chronic inflammatory diseases and malignant and neurodegenerative diseases . targeted deletion of the stat3 gene in the cd4 t cell compartment of mice impaired the experimental induction of autoimmune conditions , for example , uveoretinitis or encephalomyelitis , most likely because of a reduction in the expression of activated 4/1 integrins on cd4 t cells . insights into the role of stat3 signaling in cd8 t cells have recently be gained from studies in mice and humans in which stat3 signaling was negatively impaired through genetic manipulation of mice or a stat3 gene mutation in human patients . cd8 t cells can be distinguishes into short lived effector cd8 t cells important for immediate pathogen control and memory cd8 t cells , which can self - renew , persist and provide for long - term immunity . stat3 activation , through the cytokines il-10 and il-21 , seems to be directly involved in the cell fate decision of activated t cells , their differentiation into functional cd8 memory t cells and the maintenance of this cell pool . these conclusions were derived from experiments in a mouse model in which the stat3 gene had been conditionally deleted in activated cd8 t cells . they were corroborated by observations in a cohort of patients with autosomal dominant hyper ige syndrome ( ad - hies ) in which a dominant negative mutation in the stat3 gene has been detected . these patients show a reduced number of central memory cd4 and cd8 t cells when compared with healthy controls and a decreased ability to control bacterial and viral infections . although the systems employed to arrive at the conclusion that stat3 is a central signaling factor in the establishment and maintenance of memory cd8 t cells both rely on diminished stat3 signaling , they complement the observations reported by koskela et al . enhanced and prolonged stat3 signaling , emanating from the mutated stat3 variant , seems to mimic the functions of il-10 and il-21 and result in the expansion of a stable cd8 memory t cell pool . this may eventually lead to t cell large granular lymphocytic leukemia , a clonal disorder of large granular lymphocytes . the observation that patients with activating stat3 mutations frequently also suffered from neutropenia and rheumatoid disorders indicate that the expansion of the cd8 t cell population can trigger adverse autoimmune reactions . the summary of the molecular mechanisms responsible for the enhanced activation and the diminished deactivation of stat3 , shown in tables 1 and 2 , indicates that the fine tuning of the extent and duration of stat3 activation can easily be derailed and that this deregulation is associated with diverse pathological states . the variants that have been detected in the stat3 gene in t - cell large granular lymphocytic leukemia and in inflammatory hepatocellular adenomas gene have been designated as constitutively active and the ones found in the autosomal dominant hyper ige syndrome ( ad - hies ) as dominant negative . the constitutively active variant is probably still partially regulated by cytokine signaling and affected by the deactivation mechanisms ; similarly , the dominant negative variant is probably not entirely inactive and still has some residual activity . such observations have also been made with hyperactive and muted variants of stat5 and emphasize the quantitative aspects of stat regulation . the presence of an inappropriately strong stat3 signal is sufficient to trigger the clonal expansion of cd8 t cells , the emergence of large granular lymphocytic leukemia and the occurrence of autoimmune disorders .

multiple molecular mechanisms have been identified that are responsible for the deregulation of the quantitative aspects of jak - stat signaling . these mechanisms enhance the extent and the duration of , e.g. , stat3 activation and have profound consequences on the phenotypes of the affected cells . the fine tuning of stat3 signaling is required to maintain its physiological functions and its deregulation is associated with diverse pathological states . deregulation can be exerted by the gain of function of components mediating the activation of stat3 or the loss of function of molecules involved in the deactivation steps of stat3 . gain of function mutations can involve tyrosine kinases that phosphorylate stat3 , mutations in cytokine and growth factor receptors causing their ligand independent activation , mutations in stat3 that enhance and prolong its tyrosine phosphorylation and the autocrine or paracrine production and secretion of cytokines , most notably il-6 . diminished deactivation of phosphorylated stat3 can be due to the reduced expression of tyrosine phosphatases , inactivating mutations in these enzymes , silencing or functional inactivation of socs molecules , post - transcriptional inhibition of pias3 expression or deletion mutations in the lymphocyte adaptor protein , lnk . stat3 variants that exhibit autonomous transactivation potential have been detected in 40% of patients with t - cell large granular lymphocytic leukemia in clonally expanded cd8 + t cells . these patients also were preferentially affected by neutropenia and rheumatoid disorders and the results suggest that activating stat3 mutations in t lymphocytes could be a cause of autoimmune diseases .

the design and baseline characteristics of the whi have been described in detail elsewhere ( 8) . in brief , the whi enrolled postmenopausal women 5079 years of age who provided written informed consent and had an expected survival and local residency of 3 years . exclusion criteria included current alcoholism , drug dependency , dementia , or other conditions that would limit full participation in the study . a total of 161,808 women ( 68,132 in clinical trials and 93,676 in an observational study ) were enrolled in the whi between 1993 and 1998 . data used in this report were obtained from women at baseline and then at periodic follow - up points over an average of 10.4 years . the protocol and consent forms were approved by the institutional review boards of all participating institutions . prevalent diabetes was defined as a self - report at baseline of ever having received a physician diagnosis of diabetes when not pregnant . incident diabetes was based on data collected at annual follow - up visits at which participants were asked , since the date given on the front of this form , has a doctor prescribed any of the following pills or treatments ? choices included cases of incident treated diabetes reported as of 31 august 2009 were included in the analysis . a study of the accuracy of self - reported diabetes conducted in a subset of whi participants indicated that self - reported disease status was a reasonably valid indicator of diagnosed diabetes when compared with medication and laboratory criteria ( 9 ) . however , self - report fails to identify undiagnosed diabetes and tends to underestimate diabetes prevalence and incidence in our study group ( 9 ) . at baseline , whi participants self - reported their race / ethnicity , choosing from non - hispanic white ( referred to hereafter as white ) , non - hispanic black ( referred to hereafter as black ) , hispanic , american indian / alaska native , asian ( ancestry was chinese , indo - chinese , korean , japanese , pacific islander , or vietnamese ) , and other . we excluded 1,849 women who reported race / ethnicity as other and 413 women without race / ethnicity information . the sample size was limited among american indians ( n = 714 ) ; therefore , analyses were restricted to whites , blacks , hispanics , and asians . body weight , height , and waist circumference were measured at baseline and year 3 . demographic and health history data were self - reported at baseline and included age , years of education , cigarette smoking status , family history of diabetes , and hormone therapy use . the baseline physical activity questionnaire asked about usual frequency and duration of several types of recreational and household activities using a standardized classification of physical activity intensity ( 10 ) . total energy expenditure was summarized in metabolic equivalent ( met ) hours per week ( met - h / week ) , computed as the summed product of frequency , duration , and intensity for reported activities . participants also completed a standardized food frequency questionnaire ( ffq ) developed for the whi to estimate average daily nutrient intake over the 3-month period prior to baseline visit ( 11 ) . dietary quality , assessed by the alternate healthy eating index ( ahei ) ( 12,13 ) , was computed based on food items and nutrients derived from the ffq , including 1 ) fruit , 2 ) vegetables , 3 ) nuts and legumes , 4 ) ratio of white to red meat , 5 ) total dietary fiber , 6 ) trans - fat , 7 ) ratio of polyunsaturated fat to saturated fat , 8) alcohol , and 9 ) multivitamin use . the reliability and validity of physical activity measurements were assessed among a random sample of 536 participants by a second measure of physical activity 10 weeks after the first measure . the test - retest reliability ( weighted ) for the physical activity variables ranged from 0.53 to 0.72 , and the intraclass correlation for the total physical activity variable was 0.77 ( 8) . for the ffq , we assessed bias and precision of the ffq by comparing the intake of 30 nutrients estimated from the ffq with means from four 24-h dietary recalls and a 4-day food record from 113 women who participated in the whi ( 11 ) . for most nutrients , means estimated by the ffq were within 10% of the records or recalls . energy - adjusted correlation coefficients ranged from 0.2 ( vitamin b12 ) to 0.7 ( magnesium ) , with a mean of 0.5 . we concluded that the ffq produced nutrient estimates , which were similar to those obtained in other studies comparing short - term dietary recall and recording methods . we acknowledge that the reliability and validity are not perfect ; however , they provided reasonable measures in a large clinical trial , as published by several of our authors in high - impact articles ( 1416 ) . racial / ethnic differences in the prevalence of diabetes were assessed using logistic regression and are expressed as odds ratios ( ors ) and associated 95% cis , with whites used as the reference group . we present four logistic regression models : 1 ) unadjusted , including only race / ethnicity as a covariate ; 2 ) age - adjusted ; 3 ) adjusted for multiple potential confounding factors , including study arm , baseline age , bmi , waist circumference , physical activity , dietary quality , and smoking status ; and 4 ) adjusted for the same covariates as in model 3 plus educational attainment . among women who were free of diabetes at baseline , incidence rate was calculated as the number of newly reported postbaseline diabetes cases divided by total follow - up time in person - years . the time to diabetes ( i.e. , time to event ) was calculated as the interval between enrollment date and the earliest of the following : 1 ) date of annual medical history update when new diagnosis of diabetes and initiation of treatment for diabetes were ascertained ( observed event ) ; 2 ) date of last annual medical update during which participants were identified to be without diabetes ( censored event ) ; or 3 ) date of death from any cause ( censored event ) . cox proportional hazards models were used to estimate the hazard ratios ( hrs ) and associated 95% ci of incident diabetes , with whites used as the reference group . four cox regression models that parallel those described for the analysis of diabetes prevalence are presented . to identify determinants of diabetes incidence that might vary by racial / ethnic group , the impact of several covariates was evaluated by assessing the extent to which the hr estimate for a specific race / ethnicity group changed when each covariate was added individually to the unadjusted model . the percentage change in hr between the model with and without the covariate was used to describe the contribution of each covariate , considered singly , to diabetes risk estimated by race / ethnic group . to identify the most parsimonious prediction model , a stepwise cox proportional hazards regression analysis was conducted ( p values for entry = 0.25 and for retaining in the model = 0.05 ) . subgroup analyses also were conducted by race / ethnicity . weight change from baseline to 3 years was evaluated in relation to risk of incident disease by race / ethnicity among women who reported being free of diabetes at the 3-year measurement point . a similar analysis was conducted for waist circumference . to assess how the combined role of healthy lifestyle habits in diabetes prevention may vary by race / ethnicity ( 6,7 ) , categories of four modifiable lifestyle factors were defined as follows : physical activity ( upper vs. lower two tertiles ) , dietary quality score ( upper vs. lower two tertiles ) , smoking ( nonsmokers vs. past and former smokers combined ) , and bmi ( < 25 vs. 25 kg / m ) . in subgroup analyses by race / ethnicity , hrs and 95% cis were calculated for diabetes incidence for each lifestyle risk factor individually and several lifestyle factors in aggregate , adjusting for age , family history of diabetes , hormone therapy use , study arm , and lifestyle risk factors not already included in the model . the design and baseline characteristics of the whi have been described in detail elsewhere ( 8) . in brief , the whi enrolled postmenopausal women 5079 years of age who provided written informed consent and had an expected survival and local residency of 3 years . exclusion criteria included current alcoholism , drug dependency , dementia , or other conditions that would limit full participation in the study . a total of 161,808 women ( 68,132 in clinical trials and 93,676 in an observational study ) were enrolled in the whi between 1993 and 1998 . data used in this report were obtained from women at baseline and then at periodic follow - up points over an average of 10.4 years . the protocol and consent forms were approved by the institutional review boards of all participating institutions . prevalent diabetes was defined as a self - report at baseline of ever having received a physician diagnosis of diabetes when not pregnant . incident diabetes was based on data collected at annual follow - up visits at which participants were asked , since the date given on the front of this form , has a doctor prescribed any of the following pills or treatments ? choices included cases of incident treated diabetes reported as of 31 august 2009 were included in the analysis . a study of the accuracy of self - reported diabetes conducted in a subset of whi participants indicated that self - reported disease status was a reasonably valid indicator of diagnosed diabetes when compared with medication and laboratory criteria ( 9 ) . however , self - report fails to identify undiagnosed diabetes and tends to underestimate diabetes prevalence and incidence in our study group ( 9 ) . at baseline , whi participants self - reported their race / ethnicity , choosing from non - hispanic white ( referred to hereafter as white ) , non - hispanic black ( referred to hereafter as black ) , hispanic , american indian / alaska native , asian ( ancestry was chinese , indo - chinese , korean , japanese , pacific islander , or vietnamese ) , and other . we excluded 1,849 women who reported race / ethnicity as other and 413 women without race / ethnicity information . the sample size was limited among american indians ( n = 714 ) ; therefore , analyses were restricted to whites , blacks , hispanics , and asians . body weight , height , and waist circumference were measured at baseline and year 3 . demographic and health history data were self - reported at baseline and included age , years of education , cigarette smoking status , family history of diabetes , and hormone therapy use . the baseline physical activity questionnaire asked about usual frequency and duration of several types of recreational and household activities using a standardized classification of physical activity intensity ( 10 ) . total energy expenditure was summarized in metabolic equivalent ( met ) hours per week ( met - h / week ) , computed as the summed product of frequency , duration , and intensity for reported activities . participants also completed a standardized food frequency questionnaire ( ffq ) developed for the whi to estimate average daily nutrient intake over the 3-month period prior to baseline visit ( 11 ) . dietary quality , assessed by the alternate healthy eating index ( ahei ) ( 12,13 ) , was computed based on food items and nutrients derived from the ffq , including 1 ) fruit , 2 ) vegetables , 3 ) nuts and legumes , 4 ) ratio of white to red meat , 5 ) total dietary fiber , 6 ) trans - fat , 7 ) ratio of polyunsaturated fat to saturated fat , 8) alcohol , and 9 ) multivitamin use . the reliability and validity of physical activity measurements were assessed among a random sample of 536 participants by a second measure of physical activity 10 weeks after the first measure . the test - retest reliability ( weighted ) for the physical activity variables ranged from 0.53 to 0.72 , and the intraclass correlation for the total physical activity variable was 0.77 ( 8) . for the ffq , we assessed bias and precision of the ffq by comparing the intake of 30 nutrients estimated from the ffq with means from four 24-h dietary recalls and a 4-day food record from 113 women who participated in the whi ( 11 ) . for most nutrients , means estimated by the ffq were within 10% of the records or recalls . energy - adjusted correlation coefficients ranged from 0.2 ( vitamin b12 ) to 0.7 ( magnesium ) , with a mean of 0.5 . we concluded that the ffq produced nutrient estimates , which were similar to those obtained in other studies comparing short - term dietary recall and recording methods . we acknowledge that the reliability and validity are not perfect ; however , they provided reasonable measures in a large clinical trial , as published by several of our authors in high - impact articles ( 1416 ) . racial / ethnic differences in the prevalence of diabetes were assessed using logistic regression and are expressed as odds ratios ( ors ) and associated 95% cis , with whites used as the reference group . we present four logistic regression models : 1 ) unadjusted , including only race / ethnicity as a covariate ; 2 ) age - adjusted ; 3 ) adjusted for multiple potential confounding factors , including study arm , baseline age , bmi , waist circumference , physical activity , dietary quality , and smoking status ; and 4 ) adjusted for the same covariates as in model 3 plus educational attainment . among women who were free of diabetes at baseline , incidence rate was calculated as the number of newly reported postbaseline diabetes cases divided by total follow - up time in person - years . the time to diabetes ( i.e. , time to event ) was calculated as the interval between enrollment date and the earliest of the following : 1 ) date of annual medical history update when new diagnosis of diabetes and initiation of treatment for diabetes were ascertained ( observed event ) ; 2 ) date of last annual medical update during which participants were identified to be without diabetes ( censored event ) ; or 3 ) date of death from any cause ( censored event ) . cox proportional hazards models were used to estimate the hazard ratios ( hrs ) and associated 95% ci of incident diabetes , with whites used as the reference group . four cox regression models that parallel those described for the analysis of diabetes prevalence are presented . to identify determinants of diabetes incidence that might vary by racial / ethnic group , the impact of several covariates was evaluated by assessing the extent to which the hr estimate for a specific race / ethnicity group changed when each covariate was added individually to the unadjusted model . the percentage change in hr between the model with and without the covariate was used to describe the contribution of each covariate , considered singly , to diabetes risk estimated by race / ethnic group . to identify the most parsimonious prediction model , a stepwise cox proportional hazards regression analysis was conducted ( p values for entry = 0.25 and for retaining in the model = 0.05 ) . subgroup analyses also were conducted by race / ethnicity . weight change from baseline to 3 years was evaluated in relation to risk of incident disease by race / ethnicity among women who reported being free of diabetes at the 3-year measurement point . a similar analysis was conducted for waist circumference . to assess how the combined role of healthy lifestyle habits in diabetes prevention may vary by race / ethnicity ( 6,7 ) , categories of four modifiable lifestyle factors were defined as follows : physical activity ( upper vs. lower two tertiles ) , dietary quality score ( upper vs. lower two tertiles ) , smoking ( nonsmokers vs. past and former smokers combined ) , and bmi ( < 25 vs. 25 kg / m ) . in subgroup analyses by race / ethnicity , hrs and 95% cis were calculated for diabetes incidence for each lifestyle risk factor individually and several lifestyle factors in aggregate , adjusting for age , family history of diabetes , hormone therapy use , study arm , and lifestyle risk factors not already included in the model . at baseline , the average age of the 158,833 women with evaluable data was 63 years . the racial / ethnic distribution was 84.1% white , 9.2% black , 4.1% hispanic , and 2.6% asian . blacks and hispanics tended to have more risk factors , whereas asians tended to have fewer ( table 1 ) . baseline characteristics of participants in the whi 19932009 ( n = 158,833 ) at enrollment , diabetes prevalence was highest among blacks ( 12.2% ) , followed by hispanics ( 7.2% ) , asians ( 5.9% ) , and whites ( 3.3% ) ( table 2 ) . compared with the unadjusted ors , which were significantly higher in all three race / ethnic groups when compared with whites , the age - adjusted and multivariable - adjusted ors were attenuated for blacks and hispanics but strengthened for asians . additional adjustment for educational attainment further attenuated the ors for blacks and hispanics ; however , ors for all three racial / ethnic minority groups remained significantly higher than that of whites . prevalence and incidence of diabetes in the whi 19932009 during an average of 10.4 years ( sd = 3.2 ) of follow - up , 14,604 new cases of diabetes were reported ( 11,127 whites , 2,181 blacks , 879 hispanics , and 417 asians ) , with incidence being highest in blacks and lowest in whites . compared with whites , unadjusted analyses showed a significantly higher diabetes incidence of 136% in blacks , 114% in hispanics , and 43% in asians . after adjusting for age , study arm , bmi , physical activity , smoking status , and educational attainment , 1 ) , the highest cumulative incidence of diabetes was seen among women who had both high bmi and low levels of physical activity . the cumulative incidence of diabetes was 23.4% among black women who were both obese and in the lowest tertile of physical activity , but decreased to 8.8% in those who had a healthy weight and exercised . white women who were overweight ( bmi = 2529.9 kg / m ) and in the lowest tertile of physical activity had a cumulative incidence of 8.7% , whereas white women who were obese ( bmi 30 kg / m ) and in the lowest tertile of physical activity had a cumulative incidence of 18.6% . across all racial / ethnic groups , women who were normal weight ( bmi < 25 kg / m ) and in the highest tertile of physical activity had less than one - third to one - sixth the incidence of diabetes compared with women with bmi 30 kg / m and in the lowest tertile of physical activity . cumulative incidence of diabetes by race / ethnicity , physical activity level , and bmi category . analyses conducted to estimate the influence of individual factors that may account for the observed racial / ethnic variation in diabetes incidence by comparing hrs obtained from unadjusted versus fully adjusted models ( results not shown ) revealed that if asian women had the same waist circumference , bmi , and dietary quality intake as whites , their hr for diabetes would be increased by 44 , 29 , and 3.5% , respectively . among blacks , if they had the same bmi , waist circumference , family history of diabetes , dietary quality intake , and physical activity levels as whites , their hr for diabetes would be decreased by 25 , 19 , 11 , 7 , and 6% , respectively . among hispanics , if they had the same educational attainment , bmi , family history of diabetes , dietary quality intake , and physical activity levels as whites , their hr for diabetes would be decreased by 14 , 10 , 9 , 7 , and 6% , respectively . results from the stepwise cox proportional hazards regression analysis conducted to identify the most parsimonious predictive model for diabetes incidence , fitting candidate predictors plus race / ethnicity , showed that race / ethnicity , age , bmi , waist circumference , education , smoking status , physical activity , family history of diabetes , and dietary quality score entered the model . hrs of incident diabetes corresponding to single and specific combinations of lifestyle risk factors in race / ethnicity subgroups in the whi 19932009 subgroup analyses by race / ethnicity ( results not shown ) conducted to assess the effect of interval ( i.e. , baseline to 3 years ) changes in factors found to be significant in the cox proportional hazards models revealed a significant effect of 5% increased risk of diabetes for each 5-cm increase in waist circumference ( hr 1.05 [ 95% ci 1.041.07 ] ) . the observed effect was consistent across all race / ethnicity groups . an alternative model fit to assess if weight gain could explain some of the observed racial / ethnic variation in diabetes incidence revealed an 3% increase in subsequent risk for each 1-kg / m increment in bmi ( 1.03 [ 1.021.04 ] ) . higher levels of physical activity , better diet , and having a healthy weight tended to be associated with a significantly lower risk of diabetes in each racial / ethnic group , with some exceptions . whites , blacks , and hispanics with all factors in the low - risk category ( 4.0 , 1.1 , and 2.2% ) had 60 , 69 , and 63% lower risk for incident diabetes ( table 3 ) . healthy weight ( bmi < 25 kg / m ) demonstrated the greatest role in reducing risk of diabetes in each racial / ethnic group : 66% in whites , 55% in blacks , 64% in hispanics , and 66% in asians . at baseline , the average age of the 158,833 women with evaluable data was 63 years . the racial / ethnic distribution was 84.1% white , 9.2% black , 4.1% hispanic , and 2.6% asian . blacks and hispanics tended to have more risk factors , whereas asians tended to have fewer ( table 1 ) . at enrollment , diabetes prevalence was highest among blacks ( 12.2% ) , followed by hispanics ( 7.2% ) , asians ( 5.9% ) , and whites ( 3.3% ) ( table 2 ) . compared with the unadjusted ors , which were significantly higher in all three race / ethnic groups when compared with whites , the age - adjusted and multivariable - adjusted ors were attenuated for blacks and hispanics but strengthened for asians . additional adjustment for educational attainment further attenuated the ors for blacks and hispanics ; however , ors for all three racial / ethnic minority groups remained significantly higher than that of whites . prevalence and incidence of diabetes in the whi 19932009 during an average of 10.4 years ( sd = 3.2 ) of follow - up , 14,604 new cases of diabetes were reported ( 11,127 whites , 2,181 blacks , 879 hispanics , and 417 asians ) , with incidence being highest in blacks and lowest in whites . compared with whites , unadjusted analyses showed a significantly higher diabetes incidence of 136% in blacks , 114% in hispanics , and 43% in asians . after adjusting for age , study arm , bmi , physical activity , smoking status , and educational attainment , 1 ) , the highest cumulative incidence of diabetes was seen among women who had both high bmi and low levels of physical activity . the cumulative incidence of diabetes was 23.4% among black women who were both obese and in the lowest tertile of physical activity , but decreased to 8.8% in those who had a healthy weight and exercised . white women who were overweight ( bmi = 2529.9 kg / m ) and in the lowest tertile of physical activity had a cumulative incidence of 8.7% , whereas white women who were obese ( bmi 30 kg / m ) and in the lowest tertile of physical activity had a cumulative incidence of 18.6% . across all racial / ethnic groups , women who were normal weight ( bmi < 25 kg / m ) and in the highest tertile of physical activity had less than one - third to one - sixth the incidence of diabetes compared with women with bmi 30 kg / m and in the lowest tertile of physical activity . cumulative incidence of diabetes by race / ethnicity , physical activity level , and bmi category . analyses conducted to estimate the influence of individual factors that may account for the observed racial / ethnic variation in diabetes incidence by comparing hrs obtained from unadjusted versus fully adjusted models ( results not shown ) revealed that if asian women had the same waist circumference , bmi , and dietary quality intake as whites , their hr for diabetes would be increased by 44 , 29 , and 3.5% , respectively . among blacks , if they had the same bmi , waist circumference , family history of diabetes , dietary quality intake , and physical activity levels as whites , their hr for diabetes would be decreased by 25 , 19 , 11 , 7 , and 6% , respectively . among hispanics , if they had the same educational attainment , bmi , family history of diabetes , dietary quality intake , and physical activity levels as whites , their hr for diabetes would be decreased by 14 , 10 , 9 , 7 , and 6% , respectively . results from the stepwise cox proportional hazards regression analysis conducted to identify the most parsimonious predictive model for diabetes incidence , fitting candidate predictors plus race / ethnicity , showed that race / ethnicity , age , bmi , waist circumference , education , smoking status , physical activity , family history of diabetes , and dietary quality score entered the model . hrs of incident diabetes corresponding to single and specific combinations of lifestyle risk factors in race / ethnicity subgroups in the whi 19932009 subgroup analyses by race / ethnicity ( results not shown ) conducted to assess the effect of interval ( i.e. , baseline to 3 years ) changes in factors found to be significant in the cox proportional hazards models revealed a significant effect of 5% increased risk of diabetes for each 5-cm increase in waist circumference ( hr 1.05 [ 95% ci 1.041.07 ] ) . the observed effect was consistent across all race / ethnicity groups . an alternative model fit to assess if weight gain could explain some of the observed racial / ethnic variation in diabetes incidence revealed an 3% increase in subsequent risk for each 1-kg / m increment in bmi ( 1.03 [ 1.021.04 ] ) . higher levels of physical activity , better diet , and having a healthy weight tended to be associated with a significantly lower risk of diabetes in each racial / ethnic group , with some exceptions . whites , blacks , and hispanics with all factors in the low - risk category ( 4.0 , 1.1 , and 2.2% ) had 60 , 69 , and 63% lower risk for incident diabetes ( table 3 ) . kg / m ) demonstrated the greatest role in reducing risk of diabetes in each racial / ethnic group : 66% in whites , 55% in blacks , 64% in hispanics , and 66% in asians . previous reports , mainly focused on younger men and women , have indicated significant racial / ethnic disparities in diabetes in the u.s . ( 1,4,5 ) . the 20072009 national health interview survey found that 7.1% of whites , 8.4% of asians , 11.8% of hispanics , and 12.6% of blacks reported having a diagnosis of diabetes ( 1 ) . the whi allows a unique opportunity to deepen understanding of the patterns and determinants of diabetes in older women , including racial / ethnic minorities that represent growing segments of the u.s . we found that the prevalence and incidence of diabetes ranged from approximately two to three times higher in blacks and approximately two times higher in hispanics and in asians , compared with whites . observed racial / ethnic differences in diabetes incidence were explained , in large part , by modifiable lifestyle factors that included diet quality , physical activity , and smoking status or factors resulting from lifestyle behaviors including bmi and waist circumference . adjustment for differences in these variables indicates that asians have the highest inherent risk , though women of all four racial / ethnic groups would experience a large reduction in diabetes risk by maintaining a healthy body weight , healthy diet , and a physically active lifestyle . maintaining a bmi < 25 kg / m appears to be particularly important , and interval changes in both bmi and waist circumference predicted newly incident disease . in this study , both bmi and waist circumference were found to be related to prevalent diabetes , and interval changes were associated with risk of incident disease . because waist circumference reflects centralized obesity , and the propensity toward large waist circumference varies according to race / ethnicity , its effect may differ from that of bmi ( 17,18 ) . although bmi and waist circumference among asian , postmenopausal women in the whi were relatively lower than whites , asians were at higher risk of diabetes at lower levels of bmi and with smaller waist circumferences ( i.e. , by 44 and 29% , respectively ) compared with whites , a result consistent with that seen in the multi - ethnic study of atherosclerosis ( 19 ) . our results on asians suggest that additional risk factors , which might be biological , social , or a combination , drive the higher prevalence and incidence of diabetes in this population . diabetes risk in blacks with the same bmi and waist circumference as whites was lower by 25 and 17% , respectively . similarly , diabetes risk in hispanics with the same bmi and waist circumference as whites was lower by 10 and 2% , respectively . we noted that within each group , bmi was the most important determinant for diabetes incidence . physical inactivity also was found to be associated with increased risk of diabetes in all groups . in a previous publication using whi data , we reported the association between physical inactivity and development of diabetes ( 20 ) . our present study showed that , compared with whites , given the same level of physical activity , the risk for diabetes for blacks and hispanics is 6% lower . several studies have found differences in dietary intake by race / ethnicity ( 26,27 ) . although their diet quality was better than that of whites , we found , for example , if their overall dietary quality were to decrease to that of whites , their risk of diabetes would increase by 4% relative to whites . in fact , if blacks and hispanics improved their diet to a quality similar to that of whites , their risk of diabetes would be 7% lower . although previous studies demonstrated that diabetes is preventable by relatively simple lifestyle modifications ( 6,7 ) , our data suggest that some minority groups might obtain greater benefit from improving lifestyle factors ( i.e. , blacks and hispanics ) than others ( i.e. , asians ) due to differences in the amount of change possible or the vulnerability to these factors . the divergent findings in asian women are of interest , suggesting that additional approaches to prevention deserve attention . for example , asians may need to achieve even greater weight loss to have the same low risk of diabetes as nonoverweight whites , and the world health organization already uses a lower bmi cut point for asians . the fact that asians in the whi had much healthier body weights at baseline set the stage for much lower overall diabetes risk . however , further research with this population is warranted because our statistical models , with additional covariates , may be unstable and less reliable due to small numbers . we found that blacks and hispanics are more sensitive to lifestyle modifications and weight loss than whites , and this is corroborated by our previous lifestyle intervention study results from a hispanic population ( 28 ) . hispanic sensitivity to the development of an insulin - resistant state or diabetes with lower weight gain is well described ( 29 ) . if confirmed in weight - loss studies , the sensitivity to modest weight loss that we observed in high - risk groups in the whi will have important clinical and public health implications . it will also be important to explore possible social and genetic underpinnings for such population sensitivity . two recent studies indicate that social economic status ( ses ) may account for some of the observed race / ethnicity disparities in diabetes prevalence ( 30,31 ) . similarly , in our study , adjustment for educational attainment resulted in the largest decrease in diabetes incidence in hispanics ; if hispanics had achieved the same education levels as whites , their risk of diabetes would be 14% lower . however , education is only one component of ses , and thus consideration of an adjustment for other ses parameters ( e.g. , income and occupation ) would likely account for a greater proportion of the disparities observed in black and hispanic women . first , observed prevalence of diabetes in the whi was lower than expected , as relatively healthy postmenopausal women were enrolled in the study . second , reported education level among black women in the whi was considerably higher than education level among black women in the u.s . the education differential for blacks and hispanics is much more extreme than that observed in whites , and this may underlie some of the other observations in the data ( 32,33 ) . however , observed patterns are consistent with the population - based literature in terms of race / ethnic patterns of diabetes prevalence and incidence of diabetes . third , asian women appear to have the greatest inherent risk in that their risk factor profile was in fact better than whites , and large increases in diabetes risk would occur if the risk factors deteriorate . ( 19 ) , which showed that asians had a higher diabetes risk per unit increase in bmi and waist circumference . recognizing that each of the racial / ethnic groups is heterogeneous due to differences by national origin and other relevant parameters fourth , it is encouraging to note the effect of interval changes in weight amounting to a 3% reduction in risk of diabetes for each unit decrease in bmi . individual efforts to reduce weight and increase physical activity are difficult to sustain ; however , current efforts are underway to tackle weight regulation through a variety of approaches that go beyond the individual . lessons from the tobacco literature indicate that behavior change is influenced by our social and political structure ; thus , multilevel approaches are likely needed . in the u.s . , the race / ethnicity categories used most often in medical and public health research are from self - report , the same as the u.s . census categories . genetic race / ethnicity information known as admixture data ( i.e. , ancestry - informative markers ) from another study indicates great diversity within all four of the groups that we examined ( 34 ) . yet , few studies obtain such data due to issues concerning cost , feasibility , practicality , and comparability . thus , the differences observed and reported here reflect any inherent biological differences across the groups studied as well as differences in life experiences ( e.g. , exposure to specific environments and stressors ) , which may also contribute to acquired physiological differences in reactivity , and in turn diabetes risk ( 35 ) . the challenges for interpreting the results associated with studying self - reported racial / ethnic groups will likely increase in future studies , as more people identify themselves as belonging to multiple racial / ethnic groups . although geographically diverse , racial / ethnic groups vary in their representation of the general population . data for whites show that many characteristics of the whi participants are similar to white women participating in the national health and nutrition examination survey ( 11 ) ; however , ethnic groups are underrepresented in the whi . women from parts of the country where we see large disparities in certain minorities ( e.g. , rural southern blacks ) were not represented . thus , both prevalence and racial differentials are smaller in the whi than what we might expect to see in the u.s . as a whole . second , only self - reported prevalence of diabetes and treated incident diabetes were ascertained ; thus , prevalence and incidence of diabetes may be underestimated . we acknowledge that this is a limitation , and we did not account for nontreated diabetes . however , self - reported diabetes in the whi was found to be reliable and sufficiently accurate to allow its use in epidemiologic studies ( 9 ) . third , there could be other factors for which we did not control that further contribute to racial / ethnic disparities , such as health care access ( 36 ) . however , > 90% of the whi participants had insurance coverage , and diabetes prevalence and incidence were assessed at regular study visits . fourth , although incident diabetes in older women is likely to be type 2 diabetes , the whi question did not specify type of diabetes . other limitations include missing data ; however , the rates of retention in the whi were > 95% during an average of 7 years of follow - up ( 37 ) . balancing the limitations , there are several major strengths . in addition , the whi collected detailed information on a comprehensive range of diabetes risk factors relevant to this investigation , with a 10-year follow - up for diabetes outcome . in conclusion , significant disparities exist between the major ethnic groups in diabetes prevalence and incidence in postmenopausal women ; these differences withstand adjustment for a very comprehensive group of physiological and behavioral risk factors . although these results highlight the potential benefits of tailored diabetes prevention strategies directed at those specific factors that are most likely to increase the risk of diabetes among each racial / ethnic group , it is prudent to recommend avoidance of weight gain , weight loss , a healthy diet , and adequate levels of physical activity to all postmenopausal women for the purpose of diabetes risk reduction .

objectiveto examine determinants of racial / ethnic differences in diabetes incidence among postmenopausal women participating in the women s health initiative.research design and methodsdata on race / ethnicity , baseline diabetes prevalence , and incident diabetes were obtained from 158,833 women recruited from 19931998 and followed through august 2009 . the relationship between race / ethnicity , other potential risk factors , and the risk of incident diabetes was estimated using cox proportional hazards models from which hazard ratios ( hrs ) and 95% cis were computed.resultsparticipants were aged 63 years on average at baseline . the racial / ethnic distribution was 84.1% non - hispanic white , 9.2% non - hispanic black , 4.1% hispanic , and 2.6% asian . after an average of 10.4 years of follow - up , compared with whites and adjusting for potential confounders , the hrs for incident diabetes were 1.55 for blacks ( 95% ci 1.471.63 ) , 1.67 for hispanics ( 1.541.81 ) , and 1.86 for asians ( 1.682.06 ) . whites , blacks , and hispanics with all factors ( i.e. , weight , physical activity , dietary quality , and smoking ) in the low - risk category had 60 , 69 , and 63% lower risk for incident diabetes . although contributions of different risk factors varied slightly by race / ethnicity , most findings were similar across groups , and women who had both a healthy weight and were in the highest tertile of physical activity had less than one - third the risk of diabetes compared with obese and inactive women.conclusionsdespite large racial / ethnic differences in diabetes incidence , most variability could be attributed to lifestyle factors . our findings show that the majority of diabetes cases are preventable , and risk reduction strategies can be effectively applied to all racial / ethnic groups .

vascular lesions of the small intestine and colon are common causes of acute or chronic gastrointestinal hemorrhage , and arteriovenous malformations ( avms ) are an important vascular cause of obscure and intermittent gastrointestinal bleeding ( 1 ) . histologically , avms are persistent communications between thick - walled arteries and veins without intervening capillaries and are divided into true or congenital and acquired forms according to their locations and age at their presentations . although the endoscopic appearances of avms occurring in gastrointestinal tracts are not distinctive , some reported cases were observed as flat or mildly elevated hemorrhagic spots or erosions , while others have been appeared as polypoid masses . avms are usually small , single , and are located mainly in the rectum and descending colon in case of congenital or true avms , unlike acquired avms which are located mainly within the right colon . avms are relatively uncommon in the small bowel , and it is extremely rare that they present as a circumferential , transmural , and dilated vascular structure encasing only the terminal ileum and mimicking intestinal varicosis . we report a rare case in which a transmural avm mimicking intestinal varicosis and encasing the terminal ileum circumferentially . a 30-yr - old man was transferred to our hospital for the investigation and management of intermittent hematochezia for a 5-yr duration . on admission , he was not being prescribed a nonsteroidal anti - inflammatory drug , nor was suffering from portal hypertension or liver disease . there was no history of a peptic ulcer , and examinations of skin , oral , and anal mucosa revealed no abnormal findings . subsequent colonoscopy revealed a circumferential erythematous , nodular , and elevated lesion with hematocystic spots in the terminal ileum resembling varicosis ( fig . 1 ) . computed tomography ( ct ) scans with 3-dimensional angiographic reconstruction showed a vascular tuft located around the terminal ileum ( fig . 2 ) . to exclude other synchronous intraluminal sources of bleeding in the small bowel we performed enteroclysis , which revealed a segmental , nodular , and uneven elevated lesion with a length of 10 cm in the terminal ileum ( fig . the patient was treated by ileocecectomy , which encompassed the nodular and dilated vascular structure in the terminal ileum that resembled a varicosis lesion on gross inspection ( fig . 4 ) . surgical biopsy specimens revealed dilated vascular channels with a thickened wall involving the entire bowel wall and secondary arterialization of veins consistent with an avm ( fig . 5 ) . his postoperative course was uneventful , and the patient has been followed - up without a further episode of hemorrhage . despite dramatic improvements in diagnostic and therapeutic modalities , it is not always possible to determine the appropriate diagnosis and treatment for vascular lesions , such as , vascular ectasia , hemangiomas , dieulafoy lesions , and avms . in particular in the case of avms , no distinctive endoscopic or radiologic appearance has been elucidated because of their rarity , terminologic inconsistencies , and the diverse natures of their endoscopic morphologies . the presented case describes an unusual avm that involved the full thickness of the terminal ileum , mimicking intestinal varicosis on endoscopic and gross inspection . endoscopic finding of our case which demonstrates dilated and tortuously distended vascular tufts with red markings is a typical feature of intestinal varicosis ( 2 , 3 ) . the majority of avms reported are focal , flat , or mildly elevated with erythematous spots or erosions , and a minority of avms have polypoid or pedunculated masses ( 4 - 6 ) . a medline search revealed that only a small proportion of avms mimic intestinal varicosis with transmural involvement of the small and/or large bowel , and no endoscopic pictures were provided ( 7 - 9 ) . we found only one case of an avm with complete endoscopic pictures and gross pictures of dilated , tortuous , vascular tufts similar to our case . it involved the entire colon without displaying a vascular texture by angiography ( 10 ) . although avms can occur in jejunum ( 11 ) , the most common sites of involvement of true congenital avms are the rectum or sigmoid colon . many reports have suggested that they are most common in the cecum and ascending colon , but the terminologic confusion concerning the differentiation of angiodysplasia and acquired avms , which mainly develop in right colon , from congenital avms may have influenced the results . true avms are developmental and mainly occur in the rectum or sigmoid colon of patients younger than 50 yr , and correspond to type 2 lesions according to moore 's classification ( 12 ) . acquired avms or angiodysplasia correspond to type 1 according to this classification and occur in patients older than 50 yr . avms in younger patients tend to occur at atypical sites such as in the small bowel , but no segmental form of avm has been previously reported to involve only the terminal ileum in a cylindrical manner . the usefulness of ct for the detection of sources of gastrointestinal bleeding has been recently described in the context of imaging modality developments ( 13 ) . in the present case , we performed ct with a 3-dimensional angiographic reconstruction instead of conventional mesenteric arterial angiography because we did not consider invasive radiological therapeutic intervention as a potential elective hemostatic modality , and because we required an evaluation of the relation between the vascular architecture and the bowel wall . ultimately , surgical resection offers the best chance of curative treatment in such situations . in conclusion , we experienced a rare case of a segmental transmural avm involving the terminal ileum and mimicking intestinal varicosis , which presented as a case of recurrent lower gastrointestinal hemorrhage , and which was successfully treated by surgical resection .

we report on an exceptional vascular cause of gastrointestinal hemorrhage . a 30-yr - old man was admitted because of recurrent hematochezia . colonoscopy showed circumferential , erythematous , and nodular vascular distensions with hematocystic spots in the terminal ileum resembling varicosis and subsequent computed tomography with 3-dimensional angiographic reconstruction revealed a vascular architecture around the terminal ileum . no other potential source of bleeding was identified . the patient was treated by ileocecectomy and the final diagnosis was of an arteriovenous malformation confined to the terminal ileum . he has been followed - up without a further hemorrhagic episode .

this variation can be quantified by determining the gene frequencies of alleles at segregating loci which characterize one population and distinguish with another . tasting ability to phenylthiocarbamide ( ptc ) by an individual is considered as a useful and important tool to study the genetic diversity in human populations . taste and smell affects food preferences and dietary habits , thereby directly influencing the eating behavior of an individual . as taste threshold increases with age , abnormality in taste function may contribute to poor dietary intake in the elderly ( 1 ) . bitter taste perception is a conserved chemical sense against the ingestion of naturally toxic substances in mammals ( 2 ) . the experience of bitterness occurs after certain chemicals contact taste receptors located in cells on the surface of the tongue . some investigators hypothesize that this sense provides information so that people do not ingest bitter - tasting toxic chemicals ( 3 ) . studies of sensitivity to the bitter tasting anti thyroid compound , ptc have shown this to be an inherited trait and non - taster status has been linked to a variety of medical and health disorders ( 4 ) . a high incidence of non - tasters has been reported among patients with nodular goiter ( 56 ) , congenital athyreotic cretinism ( 7 , 8) , and dental caries ( 9 ) . bitter perception generally occurs through bitter taste receptors located on the surface of taste cells of the tongue ( 10 ) . these receptors are encoded by t2r genes that show 2589% amino acid sequence identity between the 25 different members of this gene family . these differences presumably allow a wide variety of different chemical shapes , sizes , and functionalities to be bound by these receptors and perceived as bitter . in humans , responses to some bitter compounds the best - studied example of these is the ability to taste ptc and other structurally related compounds ( 11 ) . the importance of the ability to taste bitter chemical compound ptc was realized by fox ( 12 ) . thereafter , synder ( 13 ) showed that the inheritance of the ability to taste ptc was dependent on a single autosomal dominant gene . ptc is a bitter tasting , harmless chemical compound ( 14 ) , which is a member of a class of compounds known as these compounds are having the chemical group n - c = s , which is responsible for their characteristic bitter taste ( 15 , 16 ) . 17 ) have identified a small region on chromosome 7q , and harbours a gene that encodes a member of the tas2r bitter taste receptor family . a major locus on chromosome 7q35-q36 and a secondary locus on chromosome 16p tasters are those who taste the substance ( ptc ) while non - tasters can not taste at all . the ability to taste ptc is a dominant genetic trait , and the test to determine ptc sensitivity is one of the most commonly used genetic tests on humans . the strong genetic basis for sensitivity to ptc has been used as a tool to trace family lineages and population migration patterns ( 11 , 19 ) . it was previously used in paternity testing before the advent of dna markers ( 20 ) . a review of the literature reveals that human populations show a tremendous variation in the frequency of tasters which ranges from 10% to 98% ( 2123 ) . among population groups of india , the frequency of taster allele ( t ) is higher among population groups of islands , followed by people in north and south india and is low in the people of west and central india , as well as among scheduled tribes ( 24 ) . the aim of the present study was to analyze the gene frequency , relative fitness value and threshold distribution for phenylthiocarbamide among some muslim populations of uttar pradesh , india to show their genetic heterogeneity . males and females of different populations have also been compared for the taste sensitivity of this trait . we also tried to find out the status of ptc tasters and non - tasters in different muslim populations of north india . muslims of india comprise more than 12% of the population , yet their genetic structure has not been well investigated . muslims belong to two major sects : sunnis and shias , while each sect has different biradaries , which are grouped under ashraf and ajlaf ( 25 ) . the former comprise higher rank muslims like syeds , sheikhs , pathans and mughuls while the latter comprise qureshis , ansaris , and saifis . a large number of the ajlaf may also be converted from local indigenous population of other faiths ( 26 ) . the aligarh city in uttar pradesh is situated between latitude 27.28 to 28.10 north , and 77.29 to 78.36 east longitude and its total area is 34.05 km . the annual average rainfall in the district is 594.1mms and maximum temperature recorded is 44c . hindus comprise brahmins , jats , banias , thakurs and balmikis while muslims comprise syed , sheikh , pathan , shia , sherwani , ansari , saifi and qureshi populations . a cross - sectional , analytical and randomized study was done to find out phenylthiocarbamide taste sensitivity and threshold distribution among different muslim populations of aligarh district , from march 2011 to october 2011 . out of a total number of about 1124 individuals , a survey was conducted among healthy , normal and unrelated individuals with the age range of 16 - 45 years which were randomly selected from six populations viz . ; syed , sheikh , pa - than , shia , sherwani and ansari . his or her general particulars ( address , age , sex , ethnic group ) were recorded . the prior informed written consent was taken from the individuals and their parents before their inclusion in the study . the questionnaire was designed to collect information about food preferences , socio economic factors , health status and medical history of family . the samples were collected from the upper court , civil lines , hamdard nagar , friends colony , amu campus , sir syed nagar and jamalpur areas of aligarh . the method to distinguish tasters from non - tasters was adopted as per the sorting technique with serial dilutions of harris and kalmus ( 27 ) , because of its superiority in discerning the threshold of the individual with near perfection . a solution of 0.13% of ptc was prepared by dissolving 130 mg of the ptc in 100ml of water ( solution 14 ) . the serial dilution from 1 through 14 was prepared taking 50ml of solution and adding 50ml of distilled water to it to make solution 13 which is diluted as half 14 . if an individual did not taste even the solution 14 ( strongest ) , then he was designated as non - taster . taster and non taster status was also entered into the proforma and presented in the form of tables . after the test , the participant was asked to spit out the chemical and to rinse the mouth with water . the distribution of the frequency of tasters and non - tasters is usually bi - modal with antimode recording the lowest frequency separates the two distributions . the anit - modal point was taken to classify the subjects as tasters or non - tasters . the phenotypes were recorded for ptc taste sensitivity for each individual , and the allele frequencies were calculated according to hardy - weinberg law ( 28 ) using a gene counting method . the level of heterozygosity was calculated using the formula , hetrozygosity= 1 - ho where ho is the homozygosity of the allele , ho=pi chi - square test : it is used for the measurement of the size of the discrepancy between the observed and expected values at particular degrees of freedom . muslims of india comprise more than 12% of the population , yet their genetic structure has not been well investigated . muslims belong to two major sects : sunnis and shias , while each sect has different biradaries , which are grouped under ashraf and ajlaf ( 25 ) . the former comprise higher rank muslims like syeds , sheikhs , pathans and mughuls while the latter comprise qureshis , ansaris , and saifis . a large number of the ajlaf may also be converted from local indigenous population of other faiths ( 26 ) . the aligarh city in uttar pradesh is situated between latitude 27.28 to 28.10 north , and 77.29 to 78.36 east longitude and its total area is 34.05 km . the annual average rainfall in the district is 594.1mms and maximum temperature recorded is 44c . hindus comprise brahmins , jats , banias , thakurs and balmikis while muslims comprise syed , sheikh , pathan , shia , sherwani , ansari , saifi and qureshi populations . a cross - sectional , analytical and randomized study was done to find out phenylthiocarbamide taste sensitivity and threshold distribution among different muslim populations of aligarh district , from march 2011 to october 2011 . out of a total number of about 1124 individuals , only 821 individuals consented to participate in this study . a survey was conducted among healthy , normal and unrelated individuals with the age range of 16 - 45 years which were randomly selected from six populations viz . ; syed , sheikh , pa - than , shia , sherwani and ansari . his or her general particulars ( address , age , sex , ethnic group ) were recorded . the prior informed written consent was taken from the individuals and their parents before their inclusion in the study . the questionnaire was designed to collect information about food preferences , socio economic factors , health status and medical history of family . the samples were collected from the upper court , civil lines , hamdard nagar , friends colony , amu campus , sir syed nagar and jamalpur areas of aligarh . the method to distinguish tasters from non - tasters was adopted as per the sorting technique with serial dilutions of harris and kalmus ( 27 ) , because of its superiority in discerning the threshold of the individual with near perfection . a solution of 0.13% of ptc was prepared by dissolving 130 mg of the ptc in 100ml of water ( solution 14 ) . the serial dilution from 1 through 14 was prepared taking 50ml of solution and adding 50ml of distilled water to it to make solution 13 which is diluted as half 14 . if an individual did not taste even the solution 14 ( strongest ) , then he was designated as non - taster . taster and non taster status was also entered into the proforma and presented in the form of tables . after the test , the participant was asked to spit out the chemical and to rinse the mouth with water . the distribution of the frequency of tasters and non - tasters is usually bi - modal with antimode recording the lowest frequency separates the two distributions . the anit - modal point was taken to classify the subjects as tasters or non - tasters . the phenotypes were recorded for ptc taste sensitivity for each individual , and the allele frequencies were calculated according to hardy - weinberg law ( 28 ) using a gene counting method . the level of heterozygosity was calculated using the formula , hetrozygosity= 1 - ho where ho is the homozygosity of the allele , ho=pi chi - square test : it is used for the measurement of the size of the discrepancy between the observed and expected values at particular degrees of freedom . a well - defined bimodal distribution of the taste sensitivity was observed in all the communities investigated as shown in ( fig . 1 ) . fig . 2 presents the threshold values among six populations which ranged from 8.29 to 9.59 in males , 7.76 to 9.03 in females and 8.09 to 8.95 as combined . the means and standard deviations of the thresholds for males , females and combined population groups were calculated as 9.08 0.195 , 7.99 0.163 , 8.51 0.13 respectively . graph showing threshold distribution of ptc trait among combined population groups of north india graph showing of mean threshold values of males , females and combined populations for ptc trait of north india the pathan shows the highest threshold value ( 9.59 in males and 8.35 in females ) while the syed the lowest ( 8.47 in males and 7.76 in females ) and the sheikh , sherwani , shia and ansari showed the intermediate threshold values . in overall population , it is interesting to note that the males show a higher mean threshold value than females . out of 821 subjects studied 545 ( 66.38% ) were tasters and 276 ( 33.62% ) were non - tasters to ptc . among 400 males , 140 ( 35.00% ) were non - tasters and among 421 females , 136 ( 32.30% ) were non - tasters , and this shows more males were observed to be non - tasters of ptc as compared to females . again females were having more tasters ( 285 , 67.70% ) than males ( 260 , 65.00% ) . 3 presents the phenotypic frequencies of tasters and non - tasters for the male , female and total combined populations . the phenotype frequency of taster showed that the percentage of taster was higher as compared to non- tasters , and is statistically significant ( = = 5 , p = 0.036 ) . it is observed that the highest phenotypic frequencies for ptc tasters were found among syed ( 73.75% in males and 72.83% in females ) while least among ansari ( 52.33% in males and 61.04% in females ) . the highest non - tasters frequencies were found among syed ( 26.25% in males and 27.17% in females ) while least among ansari ( 47.67% in males and 38.96% in females ) . we also observed that more females were ptc tasters than males which is statistically significant ( = 31.20 , df= 5 , p < 0.00001 ) . phenotype frequency for ptc tasting ability in north indian muslim populations the values for ptc tasting ability in north indian muslim populations table 3 shows the allelic frequencies for ptc tasters and non - tasters among male , female and combined populations . allele frequency for the non - taster ( t ) varies in different populations . the allelic frequency for the non - taster ( t ) for males and females the highest allelic frequency for , non - taster ( t ) was found among ansaris for being males and females 0.6904 and 0.6242 respectively . in the syed population the t - allele frequencies for males and females were found to be least i.e. 0.5123 and 0.5212 respectively . the difference in allelic frequencies for different population was non - significant because there is no rigid caste system in muslims , only biradaries are there . allele frequency for ptc tasting ability in north indian muslim populations table 4 and fig . 4 present the homozygosity and heterozygosity among male , female and combined population groups . the pooled heterozygosity for males and females is found to be 0.4832 and 0.4907 respectively . the highest heterozygosity was found among syed i.e. 0.4994 and the least among ansari i.e. 0.4489 . heterozygosity and homozygosity for ptc tasting ability in north indian muslim populations fitness values of different traits from the total population are presented in ( table 5 ) . the highest fit genotype is taken as having fitness value of one , and the fraction of fitness of each genotype with fitness of highest trait is taken as relative fitness of the other genotypes ayala ( 29 ) . the highest fitness value of t trait was found among syed ( 1.0 ) followed by sherwani ( 0.96 ) , shia ( 0.93 ) , sheikh ( 0.92 ) , pathan ( 0.88 ) and ansari ( 0.77 ) . graph showing phenotype frequencies for ptc tasting ability among different muslim populations of north india graph showing heterozygosity and homozygosity for ptc tasting ability among different muslim populations of north india relative fitness value ( w ) for ptc tasting ability in north indian muslim populations a well - defined bimodal distribution of the taste sensitivity was observed in all the communities investigated as shown in ( fig . 1 ) . fig . 2 presents the threshold values among six populations which ranged from 8.29 to 9.59 in males , 7.76 to 9.03 in females and 8.09 to 8.95 as combined . the means and standard deviations of the thresholds for males , females and combined population groups were calculated as 9.08 0.195 , 7.99 0.163 , 8.51 0.13 respectively . graph showing threshold distribution of ptc trait among combined population groups of north india graph showing of mean threshold values of males , females and combined populations for ptc trait of north india the pathan shows the highest threshold value ( 9.59 in males and 8.35 in females ) while the syed the lowest ( 8.47 in males and 7.76 in females ) and the sheikh , sherwani , shia and ansari showed the intermediate threshold values . in overall population , it is interesting to note that the males show a higher mean threshold value than females . out of 821 subjects studied 545 ( 66.38% ) were tasters and 276 ( 33.62% ) were non - tasters to ptc . among 400 males , 140 ( 35.00% ) were non - tasters and among 421 females , 136 ( 32.30% ) were non - tasters , and this shows more males were observed to be non - tasters of ptc as compared to females . again females were having more tasters ( 285 , 67.70% ) than males ( 260 , 65.00% ) . 3 presents the phenotypic frequencies of tasters and non - tasters for the male , female and total combined populations . the phenotype frequency of taster showed that the percentage of taster was higher as compared to non- tasters , and is statistically significant ( = = 5 , p = 0.036 ) . it is observed that the highest phenotypic frequencies for ptc tasters were found among syed ( 73.75% in males and 72.83% in females ) while least among ansari ( 52.33% in males and 61.04% in females ) . the highest non - tasters frequencies were found among syed ( 26.25% in males and 27.17% in females ) while least among ansari ( 47.67% in males and 38.96% in females ) . we also observed that more females were ptc tasters than males which is statistically significant ( = 31.20 , df= 5 , p < 0.00001 ) . phenotype frequency for ptc tasting ability in north indian muslim populations the values for ptc tasting ability in north indian muslim populations table 3 shows the allelic frequencies for ptc tasters and non - tasters among male , female and combined populations . allele frequency for the non - taster ( t ) varies in different populations . the allelic frequency for the non - taster ( t ) for males and females the highest allelic frequency for , non - taster ( t ) was found among ansaris for being males and females 0.6904 and 0.6242 respectively . in the syed population the t - allele frequencies for males and females were found to be least i.e. 0.5123 and 0.5212 respectively . the difference in allelic frequencies for different population was non - significant because there is no rigid caste system in muslims , only biradaries are there . table 4 and fig . 4 present the homozygosity and heterozygosity among male , female and combined population groups . the pooled heterozygosity for males and females is found to be 0.4832 and 0.4907 respectively . the highest heterozygosity was found among syed i.e. 0.4994 and the least among ansari i.e. 0.4489 . fitness values of different traits from the total population are presented in ( table 5 ) . the highest fit genotype is taken as having fitness value of one , and the fraction of fitness of each genotype with fitness of highest trait is taken as relative fitness of the other genotypes ayala ( 29 ) . the highest fitness value of t trait was found among syed ( 1.0 ) followed by sherwani ( 0.96 ) , shia ( 0.93 ) , sheikh ( 0.92 ) , pathan ( 0.88 ) and ansari ( 0.77 ) . graph showing phenotype frequencies for ptc tasting ability among different muslim populations of north india graph showing heterozygosity and homozygosity for ptc tasting ability among different muslim populations of north india relative fitness value ( w ) for ptc tasting ability in north indian muslim populations human infants show an innate pleasure response to sweet taste , but dislike bitterness and reject bitter - tasting foods ( 30 ) . whereas sweetness serves as a sensory cue for energy rich foods , bitterness often predicts toxicity ( 30 ) . the human sense of taste can be categorized into five basic tastes sweet , bitter , sour , salty and umami , that are critical for nutrition and survival . bitter perception has a particularly significant role , as it protects us from ingesting naturally toxic substances which are typically bitter in taste ( 3 ) . the best known example of variation in sensitivity to a bitter compound is that of phenylthiocar - bamide ( ptc ) . in 1931 fox ( 12 ) observed that to some individuals the simple chemical compound phenylthiocarbamide ( ptc ) , has an intensely bitter taste , while to others it is tasteless . harris and kalmus ( 27 ) found that the distribution of ptc tasting thresholds was bimodally distributed , but there were some intermediate individuals . the major gene tas2r38 on chromosome 7 responsible for this trait was identified as a member of the tas2r bitter taste receptor gene family consisting of a single coding exon 1002 bp long , encoding a 333 amino acid , 7-transmembrane domain g protein- coupled receptor ( 17 ) , that responds to bitter stimuli ( 3135 ) and the milestones of this discovery have been summarized ( 36 ) . bufe et al . ( 31 ) demonstrated that alleles of htas2r38 codes for functionally different receptor types that directly affect perception of bitterness containing compounds . two major forms of this bitter receptor gene were identified in most of the world s populations , designated as the these two forms differ in 3 amino acid positions , numbers 49 , 262 , and 296 ( 37 ) . the ability or inability to taste the ptc is a classic inherited mendelian trait that has long been known to vary in human population . this trait is of genetic , epidemiologic and evolutionary interest and has been shown to correlate with a number of dietary preferences and thus have important implications for human health ( 31 , 38 , 39 ) . the main objective of this study was to investigate the threshold distribution and determine the gene frequencies of dominant and recessive alleles for ptc taste sensitivity among different muslim populations of uttar pradesh , north india . the present study provides brief information on the distribution of ptc tasters and non - tasters in different populations of north indian muslims . some studies on muslim populations have been attempted earlier in uttar pradesh ( 40 ) . the present work shows some differences from earlier studies on ptc taste blindness among muslim populations of north india . data on ptc taste ability is vast and a great deal of work has been done over the world . many studies have reported that in world population , approximately 30% of them are ptc non tasters and 70% are tasters ( 11 , 41 ) . the prevalence of taste blindness or an inability to taste bitter chemicals ranges from 3% in west africa , to 6 - 23% in china , 40% in india and 30% in us population ( 11 , 42 ) . the average frequency of t allele among indian populations is 53.4 percent ( varies from 8.8% among scheduled caste of andhra pradesh to 89.2% in munda of ranchi , bihar ) while in european populations it varies from 25 to 57% which is little higher but similar to that of south west asian ( 43 ) . for ptc taste ability the overall frequency of allele t for muslims is 51.3% ( 44 ) , which is 57.98% in our case . for the ansari from bihar , the frequency of the t allele is 71% ( 26 ) , which is 65.99% in the present study . aarzoo and afzal ( 45 ) , reported that the overall frequency of allele t is 58% for muslims which is similar to the present data . in our study we found the percentage frequency of 33.62% for non - tasters and the allele frequency t , as 0.5798 . singh and singh ( 46 ) , observed percentage frequency of 31.3% and allele frequency t as 0.56 among the muslims of ambedkar nagar district of eastern uttar pradesh , north india . the value of non - taster allele t varies from 0.44 - 0.56 among muslims of eastern uttar pradesh ( 46 ) . in india the frequency of t - allele shows variation in different ethnic groups and castes ( 45 , 24 ) . in the present study the overall frequency of tasters was 66.38% and it varies in different populations of india ( 47 ) . the frequency of allele t among indian population is 45.7% ( 48 ) . in general frequency of allele t is higher among population groups with mongoloid affinities from the himalayan region but lower from the mongoloid populations from the east and southeast asia and lowest among scheduled tribes ( 24 ) . in the present study the frequency of taster ( t ) is about 0.50 among european populations ( 5 ) . in the present investigation the frequency of t allele ranged from 0.34 - 0.48 and the non - taster allele t ranged from 0.52 - 0.65 among muslims of uttar pradesh , north india . our observations on ptc taste perception revealed that there is significant higher percentage frequency of tasters as compared to non - tasters among different populations in the present study which supports other studies ( 11 , 41 , 34 ) . in the present study , we found the ratio of non - tasters among males was more than females as reported by others ( 24 , 49 ) . previous studies suggest that the frequency values for non - taster of ptc may be unique to the specific populations studied ( 50 ) . we found that females are more ptc tasters as compared to males which is in total conformity with other studies ( 50 , 51 ) . ptc taste thresholds vary in different populations and females are found to taste ptc at lower concentrations of thresholds than males , a small number of specific differences in taste ability have long been known and well - studied ( 11 ) . it also provides a clear cut distribution of ptc taster and non - taster phenotypes among different muslim populations of north india . acceptance and rejection of bitter fruits and vegetables , as well as sweet foods , added fats , spicy foods and alcoholic beverages had an association with ptc / prop taste sensitivity ( 5254 ) . people who can taste ptc ( taster ) are more sensitive to salt , sweet foods , sharp tasting foods and spicy foods ( 55 ) . anatomical studies reported that tasters actually have more taste buds than non - tasters ( 56 ) . keller et al . ( 57 ) suggested that non tasters prefer high fat diet more , than low fat diet , while tasters show this lack of preference for food . the prop / ptc tasters also have other oral sensations for example , the prop tasters perceive more fattiness in foods whereas non - tasters can not discriminate , between high and low fat containing foods and consequently be more obese ( more weight gain / higher body mass ) . hence , the variation in genetic taste sensitivity may also play important role in dietary patterns and food habits . aside its importance in genetic and anthropological studies , ptc taste sensitivity has been shown to be important in food selection , which may affect individual metabolism and physiology ( 58 ) . the effect of genetic factors on food selection has acquired a new importance in cancer research . if ptc and prop taster status helps to identify food preferences and food consumption , it might turn out to be a genetic marker for some of the major diet related chronic diseases ( 16 ) . on a larger scale , the ptc gene may be illustrative of ancient genetic variation that has been proposed to underlie common disease in modern populations ( 59 ) . in addition , the mapping of the ptc genes will provide a powerful tool to examine the genetic basis for food preferences and the relationship between taste status and health outcomes ( 11 ) . finally , ptc presents a unique opportunity in the field of bitter taste transduction . having a known gene with a strong effect on pheno - type in vivo provides many opportunities for studies of taste physiology , biochemical function , and the molecular structure elucidation in the human sense of taste . the present paper reports the threshold distribution and gene frequencies of ptc taste sensitivity among different muslim populations of north india . we also tried to find out the status of ptc tasters and non - tasters among these populations . our observations reveal that the percentage of ptc tasters is greater than that of non - tasters and the , females have lower non - taster phenotypes than males . although ptc itself has not been found in nature , the ability to taste ptc is strongly correlated with the ability to taste other naturally occurring bitter substances , many of which are toxic ( 60 , 55 ) . such studies have a great significance in understanding the adaptability of the populations to the same region which results in their varying response to threshold of sensitivity of the same genetic trait ( 6163 ) . further , the adaptive fitness of tasters is higher than that of non - tasters for all phenotypes , which speaks of adaptive value of taster pheno - type . therefore , understanding the nature of the variation in bitter taste perception and its relationship to diet and other behavior aspects may have important implications for human health . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .

abstractbackgroundthe ability to taste phenylthiocarbamide ( ptc ) , a bitter organic compound , described as a bimodal autosomal trait is widely used to know the heritable trait in both genetic and anthropological studies . the present study was carried out to analyze the prevalence of ptc taste sensitivity and to determine the gene frequencies among some muslim populations of uttar pradesh , india . this study has some physiological relevance to highlight the adaptability of endogamous groups to behavioral traits in the same place.methodsunrelated , healthy individuals of both sexes ( male-403 , female-418 ) belonging to different populations of uttar pradesh , india were randomly selected with the age range of 16 - 45 years observed for phenylthiocarbamide to taste sensitivity . ptc tasting ability was measured by using a serial dilution method of harris and kalmus.resultsthe phenotypic frequency of tasters was higher as compared to non - tasters , and the same is statistically significant ( 2= 11.92 , df = 5 , p = 0.036 ) . there were more females among tasters ( 67.94% ) than males ( 64.76% ) . this observation was statistically significant ( 2 = 14.79 , df = 5 , p = 0.011).conclusionthe frequency of ptc tasters is greater than non - tasters and the females have lower non - taster pheno - types as compared to males . this type of study will provide background information about genetic structure of population and serves as useful interaction of genetics , food preferences and dietary patterns .

heterotopic pregnancy is the coexistence of a living or dead intrauterine pregnancy , single or multiple , and extrauterine pregnancy located in the oviduct , ovary , uterine corner , cervix or peritoneal cavity . the first case was described in 1708 at that time the diagnosis was established during autopsy . in the latter years together with assisted reproductive techniques and the ultrasound techniques improvement , the incidence of heterotopic pregnancies increased from 1:500 to 1:100 of pregnancies . in this study the case of the coexistence of inter- and extrauterine pregnancies has been presented in a patient , who had been stated no increased risk of heterotopic pregnancy and who conceived in the course of natural menstrual cycle . a 25-year - old primipara in 6 week of the second pregnancy ( the first one ended with miscarriage in 6 week 6 months before ) came do the clinic at night ( 1 november 2007 ) because of lasting for 24 hours vaginal bleeding and abdominal pain . in medical interview regular menstrual cycles every 30 days , quite abundant , lasting 5 days , with accompanying abdominal pain . the patient had not undergone surgeries , suffered from no chronic diseases , no extrauterine pregnancy risk factors were stated in the medical interview . on admission do the clinic satisfactory general condition , arterial blood pressure 130/70 mm hg , pulse 78/min , body temperature 36.5c . the patient before the admission to the ward had been consulted by a surgeon who did not state indications for urgent surgical procedure . in physical examination the abdomen was soft , tender to palpation , mainly in the right hypogastric area , with no pathological resistance and surgical abdomen symptoms , peristalsis slow . in gynecological examination : perineum and vulva as in nullipara , smooth - walled vagina , brownish discharge in vagina visible in speculum examination , with no symptoms of active bleeding from cervix canal . bimanual examination : uterus in retroflexion , slightly tender , slightly enlarged , adnexa bilaterally with no pathological resistance , painless . laboratory tests on admission : wbc 14.210/l , rbc 3.5010/l , hb 11.3 g / dl , hct 32.8% , plt 28610/l , normal general urine test . in the transvaginal ultrasound examination the following was stated : gestational sac in the uterine cavity of the diameter of 9 mm , with a yolk sac of 3.1 mm , no embryo visualized ; left ovary 4730 mm ; right ovary 3021 mm , translocated behind the uterus , over the superior pole a well - defined thick - walled vesicle visible , of the diameter of 20 mm , with visible peripheral vascularization of medium resistance flow character which can refer to the gestational sac implanted in the oviduct . because of the suspicion of intra- and extrauterine pregnancy the patient was admitted to the gynecology ward . the following treatment was introduced : duphaston , kaprogest , no - spa . on the next day the patient in satisfactory general condition , cardiovascularly and respiratorily stable , arterial blood pressure 100/60 mm hg , pulse 80/min , slight vaginal bleeding sustained . laboratory tests : wbc 13.910/l , rbc 3.5910/l , hb 11.7 g / dl , crp 46 mg / l , -hcg 23 173 miu / ml . tvs examination was performed one more time : in the uterine cavity the gestation sac of the diameter of 11 mm visible , with yolk sac of the diameter of 3.5 mm , the embryonic structures were still not visualized ; ovaries of normal size bilaterally , over the superior pole a well - defined thick - walled vesicle visible , of the diameter of 20 mm , with an increased vascularization ; no pathological changes over left adnexa ; trace amount of fluid in the pouch of douglas ultrasound image suggests the coexistence of inter- and extrauterine pregnancy on the right side , of the gestational age about 5/6 weeks ( fig . on the 4 november 2007 control -hcg 27 393 miu / ml , in the us examination a gestational sac containing a single living embryo was stated in the uterine cavity , crl ( crown - rump length ) 4.2 mm , which referred to the 6 week of gestation , fhr(+ ) 110/min , with no trophoblast shearing off symptoms , in the area of the right adnexa of the uterus visible thick - walled vesicle of the diameter of 30 mm , with quite abundant peripheral vascularization ; with the absence of free fluid collection in the pouch of douglas ( figs . 2 , 3 ) . because of the diagnosis of living intrauterine pregnancy and the suspicion of the coexistence of the right extrauterine pregnancy , the patient was qualified for surgical treatment . intraoperatively the following was stated : body of the uterus enlarged , smooth , mobile , with the size referring to the 6 week of pregnancy ; right oviduct livid , dilated to 23 cm , right ovary normal , left adnexa of normal size , with no pathological lesions ; abdominal cavity organs macroscopically normal . because of macroscopic symptoms of tubal pregnancy presence , the right oviduct was removed . postoperative course not complicated : the patient in satisfactory general condition , cardiovascularly and respiratorily stable , normal body temperature , no vaginal bleeding . on the 5 day after surgery in tvs examination the following was stated : body of the uterus enlarged on the whole surface , of even outline , gestation sac visible in uterine cavity gs equals 25 mm , with visible embryo of crl 10 mm , which refers to 7 week of gestation , with regular heart rate of 140 bpm , trophoblast with no symptoms of shearing off . ultrasound image of intrauterine and extrauterine pregnancy located in the right oviduct registered embryonic heart rate in the intrauterine pregnancy peripheral trophoblast vascularization in the extrauterine pregnancy basing on the studies of natural cycle stated that it can refer to 1/7963 of cases . among the risk factors of intra- and extrauterine pregnancies one can list : chronic inflammation in the region of lesser pelvis , surgical procedures on adnexa of uterus , undergone extrauterine pregnancies , sexually transmitted diseases , the use of intrauterine device and assisted reproductive techniques ( art ) . walker at al . proved that in patients treated because of infertility and who underwent in vitro fertilization the risk of heterotopic pregnancy increases 400-fold in comparison to natural conception . it is also thought that the incidence of this pathology after the use of art oscillates from 0.75% to 1.3% . the most frequent location of extrauterine pregnancy coexisting with intrauterine pregnancy are oviducts ( 93.9% ) , definitely more rarely the pregnancy is located in the ovary ( 6% ) . more frequent incidence of pregnancies in the left oviduct in comparison to the right oviduct has been observed ( 31.8% vs. 36.3% ) . in the described case the authors also report about incidences of single or multiple intrauterine pregnancy and extrauterine pregnancy localized in cervix , uterine corner or peritoneal cavity and also the coexistence of pathological intrauterine pregnancy , e.g. blighted ovum and living extrauterine pregnancy . heterotopic pregnancy diagnosis is one of the most difficult clinical problems . to the most frequent symptoms body of the uterus enlargement 42% , vaginal bleeding 32% . according to tal et al . the major symptoms are abdominal pain 83% , surgical abdomen symptoms and shock 13% and vaginal bleeding 50% of cases . in the presented case the majority of symptoms occurred the patient reported abdominal pain and vaginal bleeding . because of the early period of pregnancy development no pathological resistance in gynecological examination was stated . nevertheless , precise imaging of the adnexa of the uterus area by means of transvaginal ultrasound enabled to visualize a pathological structure which might have referred to a pregnancy implanted in the oviduct ( thick - walled , peripherally vascularized fluid reservoir , characteristic for implanting trophoblast ) . this confirms the opinion that the basic diagnostic method in case of heterotopic pregnancy is transvaginal ultrasound examination , particularly recommended in patients after art between 4 and 6 week of pregnancy , however , some authors say that the sensitivity of this method is insufficient and equals about 56% . report that heterotopic pregnancies in 70% of cases are diagnosed between 5th and 8th week of gestation , similarly as in the pregnancy described by us , in 20% between 9 and 10 week and in 10% in 11 week of gestation . it seems that the assessment of beta unit of chorionic gonadotropin in the diagnostics of such cases is little useful because the dynamics of concentration changes of this marker is not characteristic for this abnormality and additionally the presence of developing intrauterine pregnancy makes the result interpretation difficult . it should be hence thought that the most efficient diagnostic method , confirming final diagnosis and being at the same time a form of treatment is laparoscopy , more rarely laparotomy , performed in cases of intraperitoneal bleeding with shock symptoms . it should be also mentioned about the possibilities of conservative treatment in selected cases . among these methods one can number inter alia fine needle aspiration of the embryo from the oviduct with simultaneous application of 50% glucose under the us control and in case of intramural pregnancy localized in the uterine corner local methotrexate application ; in cervical pregnancy there are trials of placing hemostatic sutures or local hypertonic nacl solution . there are also reports about the use of prostaglandins . in these cases , however , because of their teratogenicity , this method is not widely accepted . it is estimated that intrauterine pregnancy after the abortion of extrauterine pregnancy develops normally in 6592% of cases . reece et al . submitted for analysis 37 patients with diagnosed heterotopic pregnancy after surgical treatment of extrauterine pregnancy 75.6% gave birth around their expected delivery date , 16.2% prematurely and 3% of pregnancies ended with a miscarriage . on the other hand , it should be remembered that the coexistence of extrauterine pregnancy with intrauterine pregnancy may be threatening not only for the developing fetus in the uterus but also for the mother because the mortality of women in such cases equals 1% . concluding , we think that the visualization of normal pregnancy in the uterus in ultrasound examination does not release a doctor from a necessity of precise imaging of adnexa of the uterus . thanks to this we can establish the diagnosis earlier and introduce adequate procedure , limiting the possibility of complication occurrence and assuring optimal conditions for the developing intrauterine pregnancy .

heterotopic pregnancy is the coexistence of living or dead intrauterine pregnancy , single or multiple , and extrauterine pregnancy located in the oviduct , ovary , uterine corner , cervix or peritoneal cavity . this condition is very rare ( 1:30 000 pregnancies ) . nevertheless , in the latter years because of the development and accessibility of assisted reproductive techniques , the incidence of heterotopic pregnancies increased to 1:100 of pregnancies . the aim of this report is to present a case of early recognized intrauterine and extrauterine pregnancy . the case refers to 25-year - old patient , pregnant for the second time , in more or less 6th week of gestation , who had not been stated any heterotopic pregnancy incidence risk factors . after the observation lasting for several days in the clinic , the presence of a living intrauterine and coexisting extrauterine pregnancy located in the right oviduct was stated . the patient had her right oviduct removed by means of laparoscopy . after the surgery the patient with the living intrauterine pregnancy was released from the clinic . normal further course of intrauterine pregnancy . the patient gave birth around her expected delivery date . the presented case indicates the significance of correctly and carefully performed ultrasound examination . moreover , it is a warning for the doctors performing ultrasound examinations in the early weeks of pregnancy the visualization of a normal pregnancy in the ultrasound examination does not release the examiner from a necessity of precise imaging of adnexa of the uterus . early diagnosis of this pathology , thanks to a precise ultrasound examination , decreases the risk of complication incidence as well as women mortality .

glaucoma and ocular hypertension are progressive , vision - threatening conditions associated with increased intraocular pressure ( iop ) . quigley and broman have reported that , by 2020 , more than 28 million people in asia and nearly 60 million people worldwide will be diagnosed with open - angle glaucoma.1 in japan , glaucoma is the leading cause of visual impairment.2 reducing iop is currently the only established treatment for slowing or preventing progression of open - angle glaucoma and ocular hypertension,3 which , if untreated can lead to visual field defects and blindness.4 multiple classes of iop - lowering agents have been developed for therapeutic use , including carbonic anhydrase inhibitors and beta - adrenergic receptor antagonists ( blockers).3 systemic administration of carbonic anhydrase inhibitors and blockers , among other agents , can produce adverse effects , including malaise , fatigue , and sleep disturbances;5,6 as a result , ophthalmic instillation has been pursued as a route of administration for treatment of glaucoma and ocular hypertension . many patients require multiple ocular hypotensive agents to achieve sufficient iop reduction.7 topical treatment with the nonselective blocker timolol 0.5% ( tim ) and an adjunctive agent to lower iop has become increasingly common.3,8 concomitant therapy with topical brinzolamide 1% ( brinz ) , a carbonic anhydrase inhibitor , and tim has been demonstrated to significantly decrease iop in patients with glaucoma or ocular hypertension.911 both brinz and tim , individually and in combination , are generally well tolerated.1014 concomitant administration of multiple topical agents has been associated with drug washout , increased complexity of administration , and decreased treatment compliance ; these factors can decrease delivery of effective doses of ocular hypotensives.1517 fixed - combination pharmacotherapies of iop - lowering agents eliminate risk of washout and simplify drug administration.16,17 the safety and efficacy of fixed - combination brinzolamide 1%/timolol 0.5% ( brinz / tim - fc ) have been established in adult patients with glaucoma or ocular hypertension ; because brinz / tim - fc has been marketed in the european union , earlier studies included mostly white or black patients and relatively few asian patients.1822 in a double - masked , parallel - group study , brinz / tim - fc achieved significantly superior iop reduction compared with brinz or tim monotherapy.22 furthermore , patients transitioned to brinz / tim - fc ( usually because of insufficient iop reduction with or intolerance to their previous treatment ) achieved significant iop reductions from baseline after 46 weeks of therapy , and nearly 90% of patients judged the tolerability of brinz / tim - fc positively.19 the objective of this randomized , double - masked , multicenter , parallel - group , controlled study was to evaluate the safety and efficacy of brinz / tim - fc compared with concomitant therapy of unfixed brinz and tim ( brinz + tim ) in japanese patients with open - angle glaucoma or ocular hypertension . the main hypothesis tested was that the iop - lowering efficacy of brinz / tim - fc is similar to that of brinz + tim . this was a phase iii , randomized , double - masked , multicenter , parallel - group , positive - controlled study ( figure 1 ) conducted at 34 sites in japan . at the screening visit ( week 4 ) , patients gave their written informed consent to participate in the study and were evaluated for inclusion and exclusion criteria and use of prohibited or restricted concomitant drugs . use of other iop - lowering agents was suspended for the duration of the study . patients were instructed to administer tim twice daily at 9 am ( 30 minutes ) and 9 pm ( 30 minutes ) until the baseline visit ( observation phase ) . at the baseline visit ( week 0 ) , conducted 287 days after screening , eligibility was confirmed based on inclusion and exclusion criteria and compliance with tim therapy during the observation phase . patients were randomized to brinz / tim - fc or brinz + tim treatment groups and instructed to instill the appropriate active agent(s ) or placebo ( ie , brinz / tim - fc and matched placebo or brinz and tim ) twice daily , at 9 am ( 30 minutes ) and 9 pm ( 30 minutes ) , for 8 weeks ( treatment phase ) . efficacy and safety endpoints were assessed at 9 am ( before instillation of ophthalmic solutions ) and 11 am ( 2 hours post instillation ) at baseline , week 4 , and week 8 . a total of 24 sites also performed efficacy and safety assessments at 4 pm ( 7 hours post instillation ) . the institutional review board of each participating institution reviewed and approved the study protocol , and the study was conducted according to the declaration of helsinki . study participants were japanese patients aged $ 20 years with a diagnosis of open - angle glaucoma ( primary open - angle glaucoma , exfoliation glaucoma , pigmentary glaucoma ) or ocular hypertension for whom tim monotherapy provided insufficient iop reduction . patients were eligible for participation if iop in at least one eye was in the range of 1836 mmhg at both 9 am and 11 am at the baseline visit ( week 0 ) , with neither eye > 36 mmhg . key exclusion criteria included a history of chronic or recurrent severe ocular inflammatory disease , ocular trauma or intraocular surgery within 6 months or laser eye surgery within 3 months of screening , ocular infection or endophthalmitis , retinal disease , hypersensitivity to study drugs , or use of confounding ophthalmic or systemic drugs ( unless on a stable dosage regimen with no new administration from screening to the end of the treatment phase ) . additionally , patients with maximum corrected visual acuity 0.2 ( decimal acuity ) or an anterior chamber angle grade < 2 in either eye were excluded . during the observation phase ( week 4 to week 0 ) , all patients instilled one drop of tim aqueous ophthalmic solution in each eye twice daily . throughout the treatment phase ( week 0 to week 8) , patients instilled either a placebo ( an aqueous ophthalmic solution containing no active ingredient ) followed by brinz / tim - fc ophthalmic suspension ( azarga ; alcon laboratories , inc , fort worth , tx , usa ) or tim followed by brinz ophthalmic suspension ( alcon laboratories , inc ) , with concomitant instillations 5 minutes apart twice daily . at the screening visit , patients were provided with a journal and requested to record the conditions of investigational drug instillation and any changes in concomitant drugs . the primary efficacy endpoint was mean iop change from baseline at 11 am ( 2 hours post instillation ) at the week 8 visit . similarity of the iop - lowering efficacy of brinz / tim - fc versus brinz + tim was evaluated ( margin of noninferiority , + 1.1 mmhg ) . iop was measured by goldmann applanation tonometry at screening and at 9 am , 11 am , and 4 pm at baseline , week 4 , and week 8 . adverse events were assessed at the baseline , week 4 , and week 8 visits . solicited and unsolicited adverse events were recorded at each visit and coded using the medical dictionary for regulatory activities japanese translation , version 14.1 . the following safety parameters were assessed at screening , baseline , week 4 , and week 8 : best corrected visual acuity ( decimal acuity scale ) , slit lamp examination ( cornea , eyelid / conjunctiva , iris / anterior chamber , lens ) , blood pressure , and pulse rate . funduscopy ( vitreous , retina / macula / choroid , optic nerve , optic nerve cup - to - disc ratio ) , gonioscopy ( anterior chamber angle grade ) , static perimetry ( visual field stage , defined using greve s modification of the aulhorn classification23 ) , and laboratory tests ( hematology , serum chemistry , and qualitative urinalysis ) were performed at screening and week 8 . mean changes in iop from baseline for each group and the difference between groups were estimated with 95% confidence intervals ( cis ) by a repeated - measures analysis of covariance model with baseline iop values used as the covariate . significance of the primary endpoint , ie , iop reduction from baseline at the 11 am week 8 assessment , was determined by paired t - tests . descriptive statistics were obtained for mean standard deviation iop and iop change from baseline throughout the study . noninferiority of brinz / tim - fc versus brinz + tim was considered to be proven if the upper limit of the one - sided 97.5% ci of the difference between treatment groups in mean iop change from baseline at 11 am at week 8 was < 1.1 mmhg . post hoc analyses of treatment group differences in incidence of adverse events were performed using fisher s exact tests . group sizes and statistical power were determined before initiation of the study . assuming a standard deviation of 3.0 mmhg for the iop change from baseline , sample sizes of 135 patients per group were determined to be sufficient to detect a difference in iop $ 1.1 mmhg within the coverage probability of the 97.5% upper ci with 85% power . efficacy was analyzed in the per - protocol population ( defined as all patients who received study medication in the treatment phase , had post - administration tests and observation data , and satisfied the protocol criteria ) and the intent - to - treat population ( defined as all patients who received study medication in the treatment phase and who had post - administration tests and observation data ) . safety variables were analyzed in the safety population ( defined as all patients who received study medication ) . this was a phase iii , randomized , double - masked , multicenter , parallel - group , positive - controlled study ( figure 1 ) conducted at 34 sites in japan . at the screening visit ( week 4 ) , patients gave their written informed consent to participate in the study and were evaluated for inclusion and exclusion criteria and use of prohibited or restricted concomitant drugs . use of other iop - lowering agents was suspended for the duration of the study . patients were instructed to administer tim twice daily at 9 am ( 30 minutes ) and 9 pm ( 30 minutes ) until the baseline visit ( observation phase ) . at the baseline visit ( week 0 ) , conducted 287 days after screening , eligibility was confirmed based on inclusion and exclusion criteria and compliance with tim therapy during the observation phase . patients were randomized to brinz / tim - fc or brinz + tim treatment groups and instructed to instill the appropriate active agent(s ) or placebo ( ie , brinz / tim - fc and matched placebo or brinz and tim ) twice daily , at 9 am ( 30 minutes ) and 9 pm ( 30 minutes ) , for 8 weeks ( treatment phase ) . efficacy and safety endpoints were assessed at 9 am ( before instillation of ophthalmic solutions ) and 11 am ( 2 hours post instillation ) at baseline , week 4 , and week 8 . a total of 24 sites also performed efficacy and safety assessments at 4 pm ( 7 hours post instillation ) . the institutional review board of each participating institution reviewed and approved the study protocol , and the study was conducted according to the declaration of helsinki . study participants were japanese patients aged $ 20 years with a diagnosis of open - angle glaucoma ( primary open - angle glaucoma , exfoliation glaucoma , pigmentary glaucoma ) or ocular hypertension for whom tim monotherapy provided insufficient iop reduction . patients were eligible for participation if iop in at least one eye was in the range of 1836 mmhg at both 9 am and 11 am at the baseline visit ( week 0 ) , with neither eye > 36 mmhg . key exclusion criteria included a history of chronic or recurrent severe ocular inflammatory disease , ocular trauma or intraocular surgery within 6 months or laser eye surgery within 3 months of screening , ocular infection or endophthalmitis , retinal disease , hypersensitivity to study drugs , or use of confounding ophthalmic or systemic drugs ( unless on a stable dosage regimen with no new administration from screening to the end of the treatment phase ) . additionally , patients with maximum corrected visual acuity 0.2 ( decimal acuity ) or an anterior chamber angle grade < 2 in either eye were excluded . during the observation phase ( week 4 to week 0 ) , all patients instilled one drop of tim aqueous ophthalmic solution in each eye twice daily . throughout the treatment phase ( week 0 to week 8) , patients instilled either a placebo ( an aqueous ophthalmic solution containing no active ingredient ) followed by brinz / tim - fc ophthalmic suspension ( azarga ; alcon laboratories , inc , fort worth , tx , usa ) or tim followed by brinz ophthalmic suspension ( alcon laboratories , inc ) , with concomitant instillations 5 minutes apart twice daily . at the screening visit , patients were provided with a journal and requested to record the conditions of investigational drug instillation and any changes in concomitant drugs . the primary efficacy endpoint was mean iop change from baseline at 11 am ( 2 hours post instillation ) at the week 8 visit . similarity of the iop - lowering efficacy of brinz / tim - fc versus brinz + tim was evaluated ( margin of noninferiority , + 1.1 mmhg ) . iop was measured by goldmann applanation tonometry at screening and at 9 am , 11 am , and 4 pm at baseline , week 4 , and week 8 . adverse events were assessed at the baseline , week 4 , and week 8 visits . solicited and unsolicited adverse events were recorded at each visit and coded using the medical dictionary for regulatory activities japanese translation , version 14.1 . the following safety parameters were assessed at screening , baseline , week 4 , and week 8 : best corrected visual acuity ( decimal acuity scale ) , slit lamp examination ( cornea , eyelid / conjunctiva , iris / anterior chamber , lens ) , blood pressure , and pulse rate . funduscopy ( vitreous , retina / macula / choroid , optic nerve , optic nerve cup - to - disc ratio ) , gonioscopy ( anterior chamber angle grade ) , static perimetry ( visual field stage , defined using greve s modification of the aulhorn classification23 ) , and laboratory tests ( hematology , serum chemistry , and qualitative urinalysis ) were performed at screening and week 8 . mean changes in iop from baseline for each group and the difference between groups were estimated with 95% confidence intervals ( cis ) by a repeated - measures analysis of covariance model with baseline iop values used as the covariate . significance of the primary endpoint , ie , iop reduction from baseline at the 11 am week 8 assessment , was determined by paired t - tests . descriptive statistics were obtained for mean standard deviation iop and iop change from baseline throughout the study . noninferiority of brinz / tim - fc versus brinz + tim was considered to be proven if the upper limit of the one - sided 97.5% ci of the difference between treatment groups in mean iop change from baseline at 11 am at week 8 was < 1.1 mmhg . post hoc analyses of treatment group differences in incidence of adverse events were performed using fisher s exact tests . group sizes and statistical power were determined before initiation of the study . assuming a standard deviation of 3.0 mmhg for the iop change from baseline , sample sizes of 135 patients per group were determined to be sufficient to detect a difference in iop $ 1.1 mmhg within the coverage probability of the 97.5% upper ci with 85% power . efficacy was analyzed in the per - protocol population ( defined as all patients who received study medication in the treatment phase , had post - administration tests and observation data , and satisfied the protocol criteria ) and the intent - to - treat population ( defined as all patients who received study medication in the treatment phase and who had post - administration tests and observation data ) . safety variables were analyzed in the safety population ( defined as all patients who received study medication ) . a total of 366 patients were enrolled in the observation phase . of these , 319 continued into the treatment phase ( brinz / tim - fc , n=158 ; brinz + tim , n=161 ; mean age 6412 years ) and were included in the intent - to - treat data set ( ie , all patients administered an investigational drug for whom post - administration tests and observation data existed ) . a total of 309 completed the study and were included in the per - protocol population . forty - seven patients discontinued the study during the observation phase ; the most frequent reasons for discontinuation from the observation phase were selection criteria ( n=25 ) , meeting exclusion criteria ( n=1 0 ) , and adverse events ( n=8 ) . baseline demographic and diagnosis information for the per - protocol population are presented in table 1 . the majority of patients ( 56% ) were diagnosed with primary open - angle glaucoma , followed by ocular hypertension ( 41% ) . efficacy for the per - protocol and intent - to - treat populations was similar throughout the study ; therefore , the efficacy data presented here are only from the per - protocol data set . mean baseline iop values were similar between the brinz / tim - fc and brinz + tim treatment groups at 9 am ( 21.2 mmhg and 21.0 mmhg , respectively ) , 11 am ( 20.8 mmhg and 20.8 mmhg ) , and 4 pm ( 19.8 mmhg and 20.3 mmhg ) . mean iop reductions from baseline with brinz / tim - fc and brinz + tim ranged from 2.5 mmhg to 3.4 mmhg and from 2.7 mmhg to 3.3 mmhg , respectively , and were similar in the brinz / tim - fc and brinz + tim groups at all time points ( figure 3 ) . treatment comparisons of iop values for patients who had assessments at two time points ( ie , 9 am and 11 am ) and those who had assessments at three time points ( ie , 9 am , 11 am , and 4 pm ) are presented in tables 2 and 3 , respectively . reductions in iop from baseline at the 11 am week 8 assessment ( the primary efficacy endpoint ) were significant in both the brinz / tim - fc and brinz + tim treatment groups ( least squares mean iop reduction : brinz / tim - fc , 3.4 mmhg , n=150 ; brinz + tim , 3.3 mmhg , n=149 ; both p<0.0001 ) . the difference in least squares mean iop reduction from baseline between treatments ( brinz / tim - fc minus brinz + tim ) ranged from 0.0 mmhg to 0.3 mmhg , and the upper limit of the 97.5% one - sided ci of the difference between groups at 11 am in week 8 was 0.4 mmhg , which was within the margin of noninferiority of < 1.1 mmhg . during the treatment phase , descriptive statistics for mean iop values were similar for brinz / tim - fc and brinz + tim ( range 17.118.3 mmhg and 17.518.2 mmhg , respectively ; figure 4 ) . no differing trends in iop - lowering efficacy of brinz / tim - fc or brinz + tim were found in patients diagnosed with primary open - angle glaucoma versus ocular hypertension . mean iop at baseline was similar between patient subgroups ( primary open - angle glaucoma , 20.521.0 mmhg ; ocular hypertension , 20.421.3 mmhg ) . mean iop reductions from baseline with brinz / tim - fc were 2.9 mmhg to 3.6 mmhg and 2.0 mmhg to 3.0 mmhg in patients with primary open - angle glaucoma and ocular hypertension , respectively ; iop reductions with brinz + tim were 2.7 mmhg to 3.7 mmhg and 2.3 mmhg to 2.8 mmhg . in total , 318 patients received an investigational drug during the treatment phase ( safety population ; brinz / tim - fc , n=157 ; brinz + tim , n=161 ) . one or more adverse events were observed in 21% ( n=68/318 ) of patients ( brinz / tim - fc , n=29/157 [ 18% ] ; brinz + tim , n=39/161 [ 24% ] ; p=0.22 ; table 4 ) . most adverse events were mild or moderate ; one serious adverse event , ie , coronary spastic angina , for which a causal relationship with treatment could not be ruled out , occurred in a patient in the brinz + tim group . a post hoc analysis showed that significantly fewer treatment - related adverse events occurred in brinz / tim - fc - treated patients ( 3% ) compared with brinz + tim - treated patients ( 12% ; p=0.0029 ; table 4 ) . the most frequently reported treatment - related adverse events were blurred vision and eye irritation ( table 4 ) . the most common treatment - emergent adverse event ( observed in 2% of patients ) with brinz / tim - fc was nasopharyngitis ( n=6/157 [ 4% ] ) ; with brinz + tim , the most common treatment - emergent adverse events were blurred vision ( n=5/161 [ 3% ] ) , eye irritation ( n=4/161 [ 2% ] ) , and allergic conjunctivitis ( n=4/161 [ 2% ] ) . additional visual and ophthalmic safety parameters , including best corrected visual acuity , visual field , optic nerve cup - to - disc ratio , angle grade , and intraocular and external features assessed by slit - lamp and funduscopy were largely unchanged from baseline to week 8 . deterioration in visual field score from baseline was observed in 3% ( n=5/156 ) of patients who received brinz / tim - fc and 7% ( n=11/161 ) of patients who received brinz + tim . there were no substantial variations in hematology , serum chemistry , or urinalysis parameters in either treatment group over time . a total of 366 patients were enrolled in the observation phase . of these , 319 continued into the treatment phase ( brinz / tim - fc , n=158 ; brinz + tim , n=161 ; mean age 6412 years ) and were included in the intent - to - treat data set ( ie , all patients administered an investigational drug for whom post - administration tests and observation data existed ) . a total of 309 completed the study and were included in the per - protocol population . forty - seven patients discontinued the study during the observation phase ; the most frequent reasons for discontinuation from the observation phase were selection criteria ( n=25 ) , meeting exclusion criteria ( n=1 0 ) , and adverse events ( n=8 ) . baseline demographic and diagnosis information for the per - protocol population are presented in table 1 . the majority of patients ( 56% ) were diagnosed with primary open - angle glaucoma , followed by ocular hypertension ( 41% ) . efficacy for the per - protocol and intent - to - treat populations was similar throughout the study ; therefore , the efficacy data presented here are only from the per - protocol data set . mean baseline iop values were similar between the brinz / tim - fc and brinz + tim treatment groups at 9 am ( 21.2 mmhg and 21.0 mmhg , respectively ) , 11 am ( 20.8 mmhg and 20.8 mmhg ) , and 4 pm ( 19.8 mmhg and 20.3 mmhg ) . mean iop reductions from baseline with brinz / tim - fc and brinz + tim ranged from 2.5 mmhg to 3.4 mmhg and from 2.7 mmhg to 3.3 mmhg , respectively , and were similar in the brinz / tim - fc and brinz + tim groups at all time points ( figure 3 ) . treatment comparisons of iop values for patients who had assessments at two time points ( ie , 9 am and 11 am ) and those who had assessments at three time points ( ie , 9 am , 11 am , and 4 pm ) are presented in tables 2 and 3 , respectively . reductions in iop from baseline at the 11 am week 8 assessment ( the primary efficacy endpoint ) were significant in both the brinz / tim - fc and brinz + tim treatment groups ( least squares mean iop reduction : brinz / tim - fc , 3.4 mmhg , n=150 ; brinz + tim , 3.3 mmhg , n=149 ; both p<0.0001 ) . the difference in least squares mean iop reduction from baseline between treatments ( brinz / tim - fc minus brinz + tim ) ranged from 0.0 mmhg to 0.3 mmhg , and the upper limit of the 97.5% one - sided ci of the difference between groups at 11 am in week 8 was 0.4 mmhg , which was within the margin of noninferiority of < 1.1 mmhg . during the treatment phase , descriptive statistics for mean iop values were similar for brinz / tim - fc and brinz + tim ( range 17.118.3 mmhg and 17.518.2 mmhg , respectively ; figure 4 ) . no differing trends in iop - lowering efficacy of brinz / tim - fc or brinz + tim were found in patients diagnosed with primary open - angle glaucoma versus ocular hypertension . mean iop at baseline was similar between patient subgroups ( primary open - angle glaucoma , 20.521.0 mmhg ; ocular hypertension , 20.421.3 mmhg ) . mean iop reductions from baseline with brinz / tim - fc were 2.9 mmhg to 3.6 mmhg and 2.0 mmhg to 3.0 mmhg in patients with primary open - angle glaucoma and ocular hypertension , respectively ; iop reductions with brinz + tim were 2.7 mmhg to 3.7 mmhg and 2.3 mmhg to 2.8 mmhg . in total , 318 patients received an investigational drug during the treatment phase ( safety population ; brinz / tim - fc , n=157 ; brinz + tim , n=161 ) . one or more adverse events were observed in 21% ( n=68/318 ) of patients ( brinz / tim - fc , n=29/157 [ 18% ] ; brinz + tim , n=39/161 [ 24% ] ; p=0.22 ; table 4 ) . most adverse events were mild or moderate ; one serious adverse event , ie , coronary spastic angina , for which a causal relationship with treatment could not be ruled out , occurred in a patient in the brinz + tim group . a post hoc analysis showed that significantly fewer treatment - related adverse events occurred in brinz / tim - fc - treated patients ( 3% ) compared with brinz + tim - treated patients ( 12% ; p=0.0029 ; table 4 ) . the most frequently reported treatment - related adverse events were blurred vision and eye irritation ( table 4 ) . the most common treatment - emergent adverse event ( observed in 2% of patients ) with brinz / tim - fc was nasopharyngitis ( n=6/157 [ 4% ] ) ; with brinz + tim , the most common treatment - emergent adverse events were blurred vision ( n=5/161 [ 3% ] ) , eye irritation ( n=4/161 [ 2% ] ) , and allergic conjunctivitis ( n=4/161 [ 2% ] ) . additional visual and ophthalmic safety parameters , including best corrected visual acuity , visual field , optic nerve cup - to - disc ratio , angle grade , and intraocular and external features assessed by slit - lamp and funduscopy were largely unchanged from baseline to week 8 . deterioration in visual field score from baseline was observed in 3% ( n=5/156 ) of patients who received brinz / tim - fc and 7% ( n=11/161 ) of patients who received brinz + tim . there were no substantial variations in hematology , serum chemistry , or urinalysis parameters in either treatment group over time . the primary treatment approach for glaucoma and ocular hypertension is to reduce iop and prevent disease progression.3,7 clinical studies have demonstrated that multiple hypotensive agents are often required to achieve sufficient iop reduction.7,24 increasing use of multiple anti - glaucoma medications over time was previously observed in japanese patients.25 fixed - combination pharmacotherapies of multiple ocular hypotensive agents , including brinz / tim - fc , were shown to reduce iop in patients with glaucoma and ocular hypertension , with greater efficacy than their component monotherapies.22,2628 furthermore , compared with unfixed combination therapies ( ie , multiple medication bottles ) , fixed - combination therapies can decrease cumulative patient exposure to preservatives , reduce risk of drug washout , and simplify drug administration.1517,29 the goal of the current study was to evaluate the safety and efficacy of brinz / tim - fc compared with unfixed concomitant brinz + tim in japanese patients with open - angle glaucoma ( primary open - angle , exfoliation , pigmentary ) or ocular hypertension . both brinz / tim - fc and brinz + tim produced significant iop reductions from baseline over 8 weeks of treatment ; iop reductions with brinz / tim - fc were numerically equal to or greater than those with brinz + tim at all time points . the iop - lowering efficacy of brinz / tim - fc and brinz + tim was similar in patients diagnosed with primary open - angle glaucoma versus ocular hypertension . adverse events occurring with brinz / tim - fc and brinz + tim were mostly of mild or moderate severity , and post hoc analysis suggested that fewer treatment - related adverse events occurred with brinz / tim - fc compared with brinz + tim . no substantial or clinically relevant aggravations of visual and ophthalmic safety parameters of disease progression were observed with either treatment . the safety and efficacy of brinz / tim - fc and brinz + tim in this study were consistent with those reported in previous studies of these agents as combination therapy and monotherapy in other patient populations.10,11,18,19,21,22,30,31 similar to the current study , which demonstrated comparable efficacy of brinz / tim - fc and unfixed concomitant brinz + tim , other clinical assessments of fixed - combination therapies have demonstrated that fixed - combination pharmacotherapies containing tim and dorzolamide , a carbonic anhydrase inhibitor , or latanoprost , a prostaglandin analog , have efficacy similar to that of concomitant treatment with their active components.3235 fixed - combination dorzolamide 1%/tim is approved for use in japan ; there are currently no comparative data for brinz / tim - fc versus the fixed combination of dorzolamide 1%/tim . however , in a previous multinational noninferiority study conducted in europe , brinz / tim - fc produced iop reductions similar to those achieved with fixed - combination dorzolamide 2%/tim.20 further , studies conducted in the us and south america indicate that among patients with a treatment preference , brinz / tim - fc was favored by patients over fixed - combination dorzolamide 2%/tim.21,31 this result may be attributable to the increased severity and duration of ocular discomfort with dorzolamide 2%/tim compared with brinz / tim - fc.18,21,31,36 patients in the current study reported a low incidence of dysgeusia , a result that may be related to adequate informed consent ( including explanation of adverse events ) before study participation . based on the similar iop - lowering efficacy of brinz / tim - fc and brinz + tim , brinz / tim - fc is expected to provide a benefit to patients with glaucoma as a substitute for unfixed concomitant brinz + tim in terms of both treatment convenience and compliance . a potential limitation of the current study is that the study population did not include any patients with normal - tension or closed - angle glaucoma ; both of these conditions are common in people of japanese descent.1,37 additionally , future studies of patients with pigmentary or exfoliation open - angle glaucoma are needed ; because of the small numbers of patients with these glaucoma etiologies in the current study , conclusions regarding the safety and efficacy of brinz / tim - fc in these patients should be made with caution . treatment with twice - daily brinz / tim - fc significantly reduced iop throughout this 8-week study in japanese patients with open - angle glaucoma or ocular hypertension . iop - lowering efficacy was similar with brinz / tim - fc and brinz + tim . further , the safety profiles of brinz / tim - fc and brinz + tim were similar , and both therapies were well tolerated .

backgroundthe purpose of this study was to assess the safety and efficacy of fixed - combination brinzolamide 1%/timolol 0.5% ( brinz / tim - fc ) compared with concomitant brinzolamide 1% and timolol 0.5% ( brinz + tim ) in japanese patients with open - angle glaucoma ( primary open - angle , exfoliation , pigmentary ) or ocular hypertension.methodsthis randomized , double - masked , multicenter , parallel - group , positive - control , phase iii study was conducted in japan and included patients aged 20 years . baseline intraocular pressure was assessed after 4 weeks of treatment with timolol 0.5% . patients were randomized to twice - daily brinz / tim - fc or brinz + tim for 8 weeks ( treatment phase ) . the primary endpoint was mean intraocular pressure reduction from baseline to week 8 at 11 am , at which time noninferiority of brinz / tim - fc versus brinz + tim was evaluated . data were analyzed using repeated - measures analysis of covariance and t - tests . adverse events and ophthalmic / physiologic variables were assessed.resultsin total , 319 patients of mean age 6412 years were enrolled in the treatment phase . brinz / tim - fc and brinz + tim were associated with reductions in mean intraocular pressure from baseline throughout the study ( ranges 2.5 to 3.4 mmhg and 2.7 to 3.3 mmhg , respectively ) . mean between - group differences in intraocular pressure reduction ranged from 0 to 0.3 mmhg ; the upper limit of the 97.5% confidence interval for week 8 at 11 am was < 1.1 mmhg , indicating noninferiority of brinz / tim - fc . treatment - related adverse events were observed in 3% and 12% of patients receiving brinz / tim - fc and brinz + tim , respectively . no substantial changes in other safety parameters were reported.conclusiontwice-daily brinz / tim - fc reduced intraocular pressure by levels similar to concomitant brinz + tim in japanese patients with open - angle glaucoma or ocular hypertension and was noninferior to brinz + tim . both treatments were well tolerated .

understanding the physical interactions governing the structure and dynamics of ribonucleosides should improve the accuracy of simulations of ribonucleic acid ( rna ) molecules . methods for simulating biological systems include residue - centered force fields ( coarse - grained),(1 ) atom - centered force fields ( amber,(2 ) charmm , gromos ) , approximate quantum mechanics , and mixed quantum mechanics / molecular mechanics methods ( qm / mm ) . with advances in computer power , it is possible to run simulations at least as long as milliseconds and microseconds with coarse - grained and atom - centered potentials , respectively . they have provided satisfactory descriptions of structural and thermodynamic properties for some rna and deoxyribonucleic acid ( dna ) systems , while some challenging systems still provide difficulty . predictions for the individual ribonucleosides have not been extensively used as benchmarks for amber force fields . a fundamental understanding of nucleosides is crucial to simulate the behavior of residues in single strands , noncanonical base pairs , and hairpins . due to limitations of computer power , small model systems were used to parametrize the glycosidic dihedral angle in the amber94 force field.(2 ) in this article , the glycosidic dihedral angle , , of ribonucleic acids is reparameterized by extending the quantum mechanical ( qm ) fitting protocol , and new parameters are used in a revised force field , amber99. structural and thermodynamic results are extracted from molecular dynamics ( md ) simulations using amber99(33 ) and amber99 force fields . previous experimental work on nucleosides and nucleotides has classified the behavior of individual torsion angles . structures of modified and unmodified nucleosides / nucleotides have been interrogated by one - dimensional ( 1d ) h nuclear magnetic resonance ( nmr ) and steady - state 1d h nuclear overhauser effect ( noe ) difference spectroscopy ( ssnoe ) . in this work , transient 1d h noe spectroscopy(51 ) and sugar proton coupling constants extracted from 1d h nmr spectra for cytidine ( c ) and uridine ( u ) are used to quantitatively deduce the preferred conformations of the glycosidic dihedral angle and the sugar pucker , respectively . the amber99 force field overestimates the fraction of syn conformations for the base orientation and of c2-endo sugar puckering of the pyrimidines , while the results of amber99 are more consistent with that of the experimental nmr data . simulations on adenosine ( a ) and guanosine ( g ) show that amber99 prefers high anti conformations around 310 , while amber99 prefers anti conformations around 185. the latter is more consistent with qm energy profiles and is the typical anti region seen in crystal structures of nucleic acids . solutions of 0.2 , 1 , and 5 mm nucleosides were made in h2o with an nmr buffer consisting of 80 mm nacl , 10 mm sodium phosphate , and 0.5 mm disodium edta at a ph of 7.0 . two lyophilizations were performed on each sample , reconstituting each time with 99.9% d2o ( cambridge isotopes laboratories ) . one final lyophilization was performed , and each sample was reconstituted with 99.990% d2o ( sigma aldrich ) . chemical shift data were extracted from 1d h nmr ( see supporting information ) . for a , the chemical shifts of h8 , h2 , h1 , and h2 protons vary with concentration , implying that there is base stacking and/or base pairing interactions ( see supporting information).(52 ) the 5-guanosine monophosphate is known to form quadruplex structures and other kinds of aggregates in solution , and presumably , guanosine does the same . thus , the nmr spectra for nucleosides of a and g were not interpreted , except that j spinspin couplings of 0.2 mm samples were measured as a function of temperature ( see supporting information ) . for c and u , transient 1d noe measurements were performed with a selective inversionrecovery experiment in which the frequency of the selective inversion pulse was alternated between on resonance with the h6 proton and 2000 hz downfield , where no resonances are present . the on / off resonance spectra were subtracted , and the integral of the resulting noe peaks was divided by peak integrals in a 1d spectrum to obtain percent enhancement . steady - state 1d noe spectra were acquired in a similar manner with the inversionrecovery replaced by low - power irradiation for 10 s that was on / off the h6 resonance . initial geometries were chosen to represent experimental conformations . the dihedral angle ( o5c5c4c3 ) was set to 54 , which is the observed value for a - form rna . the dihedral angle ( c5c4c3o3 ) was set to either 140 or 81 , which is c2- or c3-endo sugar pucker , respectively . the o4c1c2c3 dihedral was set to either 32 or 24 to force the sugar pucker to stay in c2- or c3-endo conformations , respectively . in ribonucleosides , there are three oh groups ( 5 , 3 , and 2 ) that are free to rotate in solution . the 3 oh group will not interact with the base as much as the 5 and 2 oh groups . thus , different conformations of 5 and 2 oh groups were included in the fitting . for each nucleoside ( figure 1 ) , four different sugar conformations ( table 1 ) were chosen for qm calculations with gaussian03.(56 ) for each sugar conformation , a potential energy surface ( pes ) scan was done around the glycosidic dihedral angle with increments of 5 , yielding 4 72 = 288 conformations for each nucleoside . for each conformation in the pes scan , the structures were first optimized with hf/6 - 31 g * level of theory . during the optimization , most dihedrals were frozen in order to have a smooth energy profile with respect to the torsion angle ( see supporting information ) . then , qm energies , eqm , were calculated with mp2/6 - 31 g * level of theory . atom notations of nucleosides : ( a ) cytidine , ( b ) uridine , ( c ) adenosine , and ( d ) guanosine . for c and u , is the dihedral angle defined by o4c1n1c2 , and for a and g , is defined by o4c1n9c4 . the molecular mechanics ( mm ) energies , emm(nochi ) , of each conformation were calculated by restraining the dihedral angles to the values of the optimized qm geometries with a force constant of 1500 kcal / mola using the amber99(33 ) force field parameters , except torsion parameters were set to zero ( see supporting information ) . amber9(57 ) was used to calculate the mm energies , which use the default 14 vdw and electrostatic screening factors of 2.0 and 1.2 , respectively . the energy difference , eqm emm(nochi ) , represents the potential energy due to torsion : for each nucleoside , the 4 72 = 288 data points from eq 1 were fitted by linear least - squares to the fourier series shown in eq 2 . here , 1 and 2 are the dihedral angles of o4c1n1c6 ( o4c1n9c8 ) and c2c1n1c6 ( c2c1n9c8 ) , respectively . vn1 and vn2 are the potential energy barriers of o4c1n1c6 ( o4c1n9c8 ) and c2c1n1c6 ( c2c1n9c8 ) torsions . for each nucleoside , a separate fitting each structure was created with the xleap module of amber9.(57 ) two conformations were used as initial structures : c3-endo sugar puckering with base orientations of anti or syn . c , u , a , and g were solvated with tip3p water molecules(58 ) in a truncated octahedral box , having 458 , 451 , 427 , and 430 water molecules , respectively . the structures were minimized in two steps : ( i ) with the nucleoside held fixed with a restraint force of 500 kcal / mol , steepest descent minimization of 500 steps was followed by a conjugate gradient minimization of 500 steps . ( ii ) with all restraints removed , steepest descent minimization of 1000 steps was followed by a conjugate gradient minimization of 1500 steps . after minimization , two steps of pressure equilibration were done with the sander module in amber9 : ( i ) nucleosides were held fixed with a restraint force of 10 kcal / mol . a total of 20 ps of md were run with a 2 fs time step . ( ii ) the above conditions were chosen , except the constant pressure dynamics with isotropic position scaling was turned on . a total of 100 ps of md were run with a 2 fs time step . the production run was similar to the second step of the pressure equilibration described above . , a total of 30 ns of md were run with a 1 fs time step . for cytidine with amber99 force field , the simulation time was 120 ns for convergence purposes . in production runs , simulations were carried out with the pmemd module in amber9.(57 ) trajectory files were written at each 250 fs time step . simulations were performed for systems prepared with the amber99 and amber99 force fields . for c , u , a , and g , and each force field , 10 separate simulations of 30 ns each were run at 300 k yielding a total of 300 ns of explicit solvent md simulation ( see supporting information ) . five of the 10 md simulations had a starting structure of anti type , while the other five had a starting structure of syn type ( see supporting information ) . for c with amber99 force field , the simulations were extended to 11 separate simulations with 120 ns each ( see supporting information ) . the fractions of anti and syn conformations observed were essentially independent of the starting structure as were values obtained for c when the time for each of the 11 simulations was extended from 30 to 60 ns and 120 ns ( see supporting information ) . ultrasonic relaxation studies in aqueous solution revealed a relaxation time of 3 ns for a and no relaxation signal for pyrimidines . evidently , 300 ns of md simulations of the nucleosides is sufficient to sample adequately the synanti transformation . in solution , nucleosides have two important regions that describe their structures : ( i ) the glycosidic dihedral angle , and ( ii ) the sugar pucker . nmr noe experiments were done to analyze the structures of c and u. the magnitudes of noes are proportional to 1/(rij ) , where rij is the distance between the protons of i and j. when the base of a pyrimidine is oriented in an anti conformation , the h6 proton is about 3.5 from the h1 proton , essentially independent of sugar pucker.(62 ) thus , irradiation of h6 yields a moderate noe to h1. when the base of a pyrimidine is oriented in a syn conformation , however , the h6 proton is about 2.1 from h1 , yielding a strong noe to h1 when h6 is irradiated.(62 ) in pyrimidines , the distance between the h5 and h6 protons is constant at 2.48 , which can be used as a reference for calculating interproton distances from noesy or transient noe experiments according to eq 3:(63 ) here , noeij is the noe between protons i and j , noeh5h6 is the noe between h5 and h6 protons , and rh5h6 is the distance between the h5 and h6 protons , i.e. 2.48 . transient noe spectroscopy(51 ) with different mixing times was used to quantitatively analyze the preferences for anti / syn populations , and the results are presented in table 3 ( also see supporting information ) . transient noe is similar to noesy nmr except that it is 1d . to minimize spin diffusion effects and maximize signal - to - noise ratio , mixing times in the linear region of intensity vs mixing time plots were used to estimate distances between protons ( see supporting information ) . a two - state model described by the following equation , which assumes that the structure is in either syn or anti conformations , was used to determine the proportions of each conformation : for a transformation of ab , gab = rtln(k ) , where r = 1.987 cal k mol , t is the temperature in kelvins , and k is the ratio of the concentrations of each species , [ b]/[a ] ( see supporting information ) . measurements of the syn / anti proportions of pyrimidines were extracted from transient noe experiments at 10 c , while the simulations were done at 300 k ( 27 c ) . nmr spectra for c at 2 and 10 c indicate essentially no temperature dependence for the synanti equilibrium ( see supporting information ) , so all g s were calculated at 300 k. these values are for 30 c ( see supporting information ) . these values are for 0.2 mm samples of a and g at 30 c where there may be some association ( see supporting information ) . these values represent populations of high anti conformations with 310 ( see supporting information and figure 7 ) . these values represent populations of anti conformations with 185 ( see supporting information and figure 7 ) . here , noeh1h6 is the noe between the protons of h1 and h6 , fanti and fsyn are the fractions of anti and syn conformations satisfying fanti + fsyn = 1 , rh1h6,anti and rh1h6,syn are the distances between the protons of h1 and h6 when the structures are in anti and syn conformations , respectively , which are 3.48 and 2.12 , corresponding to the distances extracted from the minimum energy structures of the pes scans for c and u ( see methods section ) . as can be seen from table 3 , the anti orientation is favored over syn . comparison of nmr results for c at 2 and 10 c show that the fraction of anti base orientation is essentially independent of temperature ( supporting information ) . higher temperature could not be used because of the overlap of the h1 and h5 peaks ( see supporting information ) . ssnoe spectroscopy confirms that anti is favored over syn base orientation ( see supporting information ) . sugar proton coupling constants extracted from 1d h nmr spectra were used to determine the sugar puckering on the basis of the following equation:(64 ) where j12 and j34 are j spinspin couplings between h1 and h2 and between h3 and h4 protons , respectively . the proportion of c2-endo sugar puckering is equal to ( 1 fraction of c3-endo ) . sugar pucker ( 2% ) is independent of temperature from 5 to 40 c ( supporting information ) , and results at 30 c are presented in table 3 . figures 25 show the qm , mmamber99 , mmamber99 , and mmode(65 ) energy profiles with respect to the glycosidic dihedral angle of all the structures used in the fitting protocol for the nucleosides , where amber99 , amber99 , and ode(65 ) force fields were used to calculate mmamber99 , mmamber99 , and mmode energies , respectively . in all the plots , energy profiles of the amber99 force field describe the qm energy profiles best , although the ode force field s energy profile is also similar to the qm energy profiles . the differences between the predictions of the amber99 and ode force fields is likely due to the ode force field using ch3 , h2cch3 and h2coch3 as model systems to represent the sugar , while the amber99 force field used the entire ribose with four different sugar conformations to calculate the torsional parameters . , both ode and amber99 force fields should provide similar predictions for structural and/or dynamical properties of rna . comparisons of the force fields to qm calculations on eight sugar conformations not included in the fitting showed that amber99 also describes those qm energy profiles better than amber99 and ode force fields ( see supporting information ) . total energy ( in kcal / mol ) vs o4c1n1c6 of cytidine with amber99 ( black ) , amber99 ( red ) , qm ( green ) , and ode force field ( blue ) for : ( a ) sc 1 , ( b ) sc 2 , ( c ) sc 3 , and ( d ) sc 4 ( see table 1 ) . for visualization purposes , minimum energies of each curve anti , high anti , and syn base orientations correspond to x - axis ranges of 070 , 100180 , and 200300 , respectively , because the x - axis is + 180 to be consistent with the amber94 force field.(2 ) total energy ( in kcal / mol ) vs o4c1n1c6 of uridine with amber99 ( black ) , amber99 ( red ) , qm ( green ) , and ode force field ( blue ) for : ( a ) sc 1 , ( b ) sc 2 , ( c ) sc 3 , and ( d ) sc 4 ( see table 1 ) . for visualization purposes , minimum energies of each curve anti , high anti , and syn base orientations correspond to x - axis ranges of 070 , 100180 , and 200300 , respectively , because the x - axis is + 180 to be consistent with the amber94 force field.(2 ) total energy ( in kcal / mol ) vs o4c1n9c8 of adenosine with amber99 ( black ) , amber99 ( red ) , qm ( green ) , and ode force field ( blue ) for : ( a ) sc 1 , ( b ) sc 2 , ( c ) sc 3 , and ( d ) sc 4 ( see table 1 ) . for visualization purposes , minimum energies of each curve anti , high anti , and syn base orientations correspond to x - axis ranges of 070 , 100180 , and 200300 , respectively , because the x - axis is + 180 to be consistent with the amber94 force field.(2 ) total energy ( in kcal / mol ) vs o4c1n9c8 of guanosine with amber99 ( black ) , amber99 ( red ) , qm ( green ) , and ode force field ( blue ) for : ( a ) sc 1 , ( b ) sc 2 , ( c ) sc 3 , and ( d ) sc 4 ( see table 1 ) . for visualization purposes , minimum energies of each curve are set to zero . anti , high anti , and syn base orientations correspond to x - axis ranges of 070 , 100180 , and 200300 , respectively , because the x - axis is + 180 to be consistent with the amber94 force field.(2 ) for comparison with nmr results , predictions of population distributions of dihedral angle and sugar pucker were analyzed for c , u , a , and g using the combined trajectories of the 10 individual md simulations with amber99 and amber99 force fields ( see methods ) . population distribution plots in 2d of dihedral and pseudorotation angles for each nucleoside are shown in figures 6 and 7 . table 3 shows the force field predictions of base orientation and sugar pucker for each nucleoside ( also see table 4 and supporting information ) . analyses of the individual md simulations show at least seven synanti transformations for each ( see supporting information ) . population distribution of cytidine and uridine using amber99 ( a and b , respectively ) and amber99 ( c and d , respectively ) force fields . pse ( y - axis ) and chi ( x - axis ) stand for the pseudorotation and dihedral angles . pse angles of 18 and 162 correspond to c3-endo and c2-endo sugar pucker , respectively . angles of 200 , 300 , and 60 correspond to anti , high - anti , and syn conformations , respectively.(70 ) population distribution of adenosine and guanosine using amber99 ( a and b , respectively ) , and amber99 ( c and d , respectively ) force fields . pse ( y - axis ) and chi ( x - axis ) stand for the pseudorotation and dihedral angles . table 4 shows the predicted populations of ( i ) , ( ii ) , ( iii ) , ( iv ) , ( v ) , and ( vi ) . pse angles of 18 and 162 correspond to c3-endo and c2-endo sugar pucker , respectively . angles of 200 , 300 , and 60 correspond to anti , high - anti , and syn conformations , respectively.(70 ) regions of ( i ) syn / c2-endo , ( ii ) syn / c3-endo , ( iii ) anti / c2-endo , ( iv ) anti / c3-endo , ( v ) high anti / c2-endo , and ( vi ) high - anti / c3-endo ( figures 6 and 7 ) . table 3 shows the experimental results for c and u as well as the predictions of amber99 and amber99 force fields of the base orientation and the sugar pucker for c , u , a , and g. for the synanti equilibrium of c and u , nmr indicates 87% and 93% anti conformation , respectively , corresponding to gsynanti of 1.07 and 1.45 kcal / mol . the amber99 force field predicts 30% and 28% anti conformation , respectively , corresponding to gsynanti of 0.49 and 0.55 kcal / mol . in comparison , the amber99 force field predicts 66% and 83% anti conformation , respectively , corresponding to gsynanti of 0.45 and 0.95 kcal / mol , closer to the nmr results . evidently , amber99 overestimates the syn conformations of c and u ( see figure 6 ) . for the c2-endoc3-endo equilibrium of c and u , nmr indicates 60% and 56% c3-endo sugar puckering at 30 c , respectively , corresponding to free energy differences , gc2c3 , of 0.24 and 0.15 kcal / mol ( table 3 ) . the percentages are essentially independent of temperature from 5 to 40 c ( see supporting information ) . the amber99 force field predicts 27% and 35% c3-endo sugar pucker at 27 c , respectively , corresponding to gc2c3 of 0.58 and 0.36 kcal / mol . in comparison , the amber99 force field predicts 54% and 55% c3-endo sugar pucker at 27 c , respectively , corresponding to gc2c3 of 0.11 and 0.13 kcal / mol , which is close to the experimental values . evidently , amber99 underestimates c3-endo sugar puckering of c and u ( see figure 6 ) . the amber99 force field predicts a and g to have 13% and 24% anti conformation ( table 3 ) , respectively , with a dihedral angle around 185 , which is consistent with qm calculations and typical of the anti region seen in crystal structures of rna.(66 ) the amber99 force field predicts 15% and 11% anti conformation ( table 3 ) , respectively , but with a dihedral angle around 310 ( figure 7 ) , which is the high anti region . qm pes scans did not find any minimum around 310 but rather between 180250 for three different sugar puckers for a and g ( figures 45 and supporting information , where the x - axis , however , is + 180 ) . the concentration dependence of chemical shifts for a and g indicated aggregation at concentrations required to determine noes with enough signal - to - noise to determine the base orientation quantitatively . pioneering studies of 2- and 3- amp and gmp at high concentrations , however , indicated syn populations well over 50% . the amber99 force field predicts a and g to have 24% and 35% c3-endo sugar puckering , respectively , while amber99 predicts 32% and 54% . chemical shift data of 0.2 , 1.0 , and 5.0 mm a implies base stacking that differs with concentration . the differences of the chemical shifts between 0.2 and 1.0 mm samples are small , however . therefore , the 0.2 mm samples of a and g were used to calculate j spinspin couplings to estimate the sugar puckering ( see supporting information ) . at room temperature , the c3-endo sugar puckering of a and g is about 40% ( table 3 and supporting information ) . for a , both force fields predictions are similar to the experimental results . for g , it is known , however , that guanosine monophosphate forms quadruplex structures and other aggregates in solution . thus , it is not conclusive whether 0.2 mm g can be used to reveal the sugar puckering of monomer g. there are several reasons why the amber99 force field improves predictions for nucleosides . when the torsions were parametrized for amber99 , model systems for adenosine and thymidine were used , and the results were generalized for all dna / rna residues.(2 ) moreover , the model systems mimicked deoxyribose c2-endo sugar puckering . at that time , qm calculations were limited by computer power and only 89 data points were used in the qm fitting . also , in the amber99 force field , the original cornell force field parameters for torsions were changed without doing any fitting . the v2 term of torsion parameters was zeroed to improve the c2-endo sugar puckering phase angle for dna residues.(69 ) this effect , however , changes the whole predicted potential energy surface of the nucleosides , which , therefore , does not represent the qm energy surface well . for the amber99 force field , the torsions of c , u , a , and g were reparameterized individually . a multiconformational fitting that included the entire nucleoside with different sugar puckering was done to provide the torsion parameters . in the pes scan , a total of 4 72 = 288 data points were used in the fitting protocol for each nucleoside . the new parameter set was tested on 12 different sugar conformations ( four separate conformations for each of c2-endo , c3-endo , and o4-endo sugar puckering ) for each nucleoside and shown to predict well the qm energy surface for these conformations ( see figures 25 and supporting information ) . the shape of the qm energy surfaces of these conformations is also predicted well by the ode force field,(65 ) although not quite as well as by amber99 ( see figures 25 and supporting information ) . as a result , there should not be any big difference between amber99 and ode force field(65 ) predictions for structural and thermodynamic properties of nucleosides . many reasonable combinations of parameters were tested for approximating the qm pes representing the four major conformations of each nucleoside . for instance , we tried fitting to two dihedrals with three cosine terms , four dihedrals with two cosine terms , and four dihedrals with three cosine terms . two dihedrals with four cosine terms provided excellent fits , and more terms gave minimal improvement . as a comparison , the ode force field(65 ) uses 3 dihedrals ( a total of 13 vi parameters ) to represent the torsions , while we use 2 dihedrals ( a total of 8 vi parameters ) , but comparisons of the force fields to the qm potential energy surfaces shown in figures 25 and supporting information reveal that amber99 provides a better fit . it is crucial to use a force field that appropriately models the true behavior of rna systems . otherwise , during md simulations , sampling space will include unphysical regions , which will cause errors in predictions . with the amber99 modification , significant improvements are seen in the structural and thermodynamic predictions for cytidine and uridine in solution ( table 3 ) . this modification should be particularly important for non - watsoncrick regions and terminal base pairs because sampling will not include unrealistic populations of syn conformations or of c2-endo sugar puckering . in watsoncrick regions , the torsion is restricted by hydrogen bonding in base pairs , so little effect should be seen there . thus , the amber99 force field should improve structural and thermodynamic predictions for rna .

a reparameterization of the torsional parameters for the glycosidic dihedral angle , , for the amber99 force field in rna nucleosides is used to provide a modified force field , amber99. molecular dynamics simulations of cytidine , uridine , adenosine , and guanosine in aqueous solution using the amber99 and amber99 force fields are compared with nmr results . for each nucleoside and force field , 10 individual molecular dynamics simulations of 30 ns each were run . for cytidine with amber99 force field , each molecular dynamics simulation time was extended to 120 ns for convergence purposes . nuclear magnetic resonance ( nmr ) spectroscopy , including one - dimensional ( 1d ) 1h , steady - state 1d 1h nuclear overhauser effect ( noe ) , and transient 1d 1h noe , was used to determine the sugar puckering and preferred base orientation with respect to the ribose of cytidine and uridine . the amber99 force field overestimates the population of syn conformations of the base orientation and of c2-endo sugar puckering of the pyrimidines , while the amber99 force field s predictions are more consistent with nmr results . moreover , the amber99 force field prefers high anti conformations with glycosidic dihedral angles around 310 for the base orientation of purines . the amber99 force field prefers anti conformations around 185 , which is more consistent with the quantum mechanical calculations and known 3d structures of folded ribonucleic acids ( rnas ) . evidently , the amber99 force field predicts the structural characteristics of ribonucleosides better than the amber99 force field and should improve structural and thermodynamic predictions of rna structures .

the most important goal of rehabilitation for a lower limb amputee is the independent performance of daily activities through restoration of functional walking ability . miller et al . reported that 52% of prosthesis users experienced falls and 49% of amputees have a fear of falling . it also said that the fear of falling limits the daily movement of amputees and causes further deterioration of the balance ability required for walking1 . therefore , physical therapy for lower limb amputees should be focused on the prevention of falls during movement / walking for the improvement of everyday activities . for this purpose , it is necessary to properly evaluate the balance ability of amputees and to perform training to improve the balance ability based on the results of that evaluation . the objectives of the present study were to elucidate what type of compensatory movement and postural strategy are used by lower limb amputees for posture control during the period from prosthesis fitting to hospital discharge by analyzing the muscle activity patterns of the lower limb muscles and to elucidate the changes over time in the muscle activity pattern during the course of physical therapy . elderly patients also have a higher risk of falling than younger patients because of biomechanical , physiological , and psychological differences . however , no study has yet focused on comparing time - dependant changes in muscle activity patterns between younger and elderly patients . therefore , the other purpose of this study was to reveal the differences in muscle activity patterns during standing between younger and elderly patients . a young male , 34 years old , and an elderly female , 84 years old , who had a lower limb amputated were the subjects . neither of the patients had a history of orthopedic or neurological disease in the unaffected lower limb or the trunk . a patient who undergoes below - knee amputation due to diabetes has a normal deep sensation in the unaffected ankle joint . in both of our cases , the fitting between the socket and the stump on wearing was well adjusted with no pain in the stump . the prosthesis used by the lower limb amputees was a total surface bearing liner type and a sach foot ( table 1table 1 . characteristics of the subjectssubjectssexage ( year)height ( cm)weight ( kg)amputation sitesocketankle jointcauseweek to start pt after amputationweek to wear prosthesis after amputationweek to discharge after wearing prosthesis for the first timeyoungm3317572right bkatsbsachtumor174elderf8515045left bkatsbsachdiabetes1514 m : male , f : female , bka : below knee amputee , tsb : total surface bearing , sach : solid ankle cushion heel ; j - foot m1170 , pt : physical therapy ) . surface electromyography ( emg ) measurements were performed of the gastrocnemius medial head ( ga ) of the unaffected lower limb . the electrodes were placed on the most prominent bulge of the ga muscle following seniam guidelines . after sufficient treatment with skinpure to reduce the inter - electrode resistance to no more than 10 k on the skin , disposable electrodes ( blue sensor , nf-50-k / w , ambu ) were attached at an inter - electrode distance of 2 cm . the emg signals were amplified ( to 1.0 mv ) and band - pass filtered between 201,000 hz using a synact mt-11 ( nec corp . , tokyo , japan ) . the emg signals were digitized using a waveform analysis system , power lab 16s ( ad instruments ltd , hasting , uk ) , at a sampling frequency of 1,000 hz , and stored on a personal computer for off - line analysis . after data input , waveform processing was performed using waveform analysis software , chart36.8 , ad instruments . two different tasks of maintaining the static standing position were performed on the floor . the test subjects were asked to stand on the floor with their feet shoulder - width apart and look at a point set at 1 m distance to the front at the height of the eye for 10 seconds . the first task , reference task , was to maintain upright standing position on the prosthesis while holding parallel bars . the second task , experimental task , was forward weight shift while maintaining the standing position without grasping the parallel bars . the recorded data were smoothed using the root mean square ( rms ) value of every 100 msec using waveform analysis software . the reference contraction is judged to be reliable by being reproducible , giving approximately equal results within subjects over trial . therefore , we used the emg of another task as a reference contraction for emg normalization . the normalized level of % rmsemg was determined using the following formula . m : male , f : female , bka : below knee amputee , tsb : total surface bearing , sach : solid ankle cushion heel ; j - foot m1170 , pt : physical therapy % rmsemg = experimental task rmsemg ( second task ) / reference task rmsemg ( first task ) 100% maximum walking speed was measured in 10 m walking . depending on the condition at the time of the measurements , subjects were allowed to use a crutch , or a single cane on the unaffected side while walking . the surface muscle activity and the walking speed were measured once a week , a total of four occasions , starting on the day of prosthesis fitting ( first week ) . this study was conducted after approval was obtained from the ethics committee of tohoku university graduate school of medicine . the study patients were sufficiently informed about the purpose and methods of the study , and their consent to participation was obtained . when the time - dependent changes in muscular activity of ga , which is the most active muscle in the standing position , were investigated in the forward weight shift task , two unique patterns were observed . for the younger subject , no muscle activity of ga was observed in the standing position tasks in the first week , but the amount of muscular activity of ga gradually increased over time . on the other hand , for the elderly subject , increased activity of ga was observed in the forward weight shift task in the first week , when the prosthesis was worn for the first time , followed by a gradual decrease in activity over time ( table 2table 2 . time shift of activity in ga ( % rmsemg ) and walking speed ( m / min)week%rmsemg ( % ) mws ( m / min)youngerelderlyyoungerelderlyfirst0.6618.4348.5815.12second4.7116.4873.8924.57third5.9812.6580.3224.33fourth8.855.4110043.17%rmsemg : % root mean square electromyography , mws : maximum walking speed ) . the maximum walking speed gradually increased over the period from when the prosthesis was worn for the first time to when the subjects were discharged from the hospital ( table 2 ) . the basic elements for the smoothest and most energy efficient walk are proper balance ability and posture control function2 . in lower limb amputation , the contralateral lower limb function influences subsequent ability to perform daily living and transfer activities , and standing balance on the unaffected side is an important determinant of whether walking with a prosthesis is possible or not3 . reported that the muscle of the unaffected lower limb of prosthesis wearers tended to adopt a balancing strategy to compensate for the loss of the ankle joint and muscles due to amputation4 . therefore , lower limb amputees need to adapt to asymmetrical body weight and muscular strength when they exert effort to maintain static standing while wearing a prosthesis . in the present study , we evaluated the changes in surface emg activity of the gastrocnemius during standing , and the walking speed with a prosthesis . the muscular activity of ga is most conspicuous when the amputee is standing with the prosthesis for the first time . when the static standing position is maintained , the ga is activated in preparation for the forward movement of the center of pressure5 , 6 . we found two unique muscle activity patterns were exhibited by the two below - knee amputees . when the prosthesis was first worn , the younger subject was so careful about maintaining the standing posture that adequate forward movement of the center of gravity could not be achieved . for the elderly subject , however , marked activity of the ga was found , and ga activity at the final assessment was lower than that of the initial measurement , especially during the forward weight shift task . these time - dependent changes in the muscular activity of ga indicate how younger and elderly amputees adapt muscular force to maintain standing posture , and it might also be as a cause of falls by elderly patients in the early stage of standing or gait training . the subjects learned the method for shifting the center of gravity forward in a stable manner , resulting in the gradual reduction or increase of ga muscle activity . there are many factors known to contribute to the improvement of the walking speed of amputees . as far as the results of this study are concerned , it can be concluded that one important factor is regaining the ability to be able to shift the center of gravity in the static standing position through the efficient muscle activity of the unaffected lower limb . in conclusion , the trend of muscle activities over time should contribute to the development of effective rehabilitation programs . due to the limited number of subjects included in this study , a statistical analysis of sufficient power could not be performed . furthermore , there were obvious differences in biomechanical , physiological , and psychological attributes because of age differences . biomechanical or physiological perspective approaches are very important . in this study , however , there was no history of disorders of the trunk , such as lordosis or kyphosis , so we only focused on the differences in emg . further studies with larger numbers of subjects stratified by the causes and types of amputation are needed to clarify the specific factors associated with the acquisition of the ability to walk by amputees , and the development of efficient methods for physical therapy and programs .

[ purpose ] dysfunction of lower extremity muscles is one risk factor of falls for amputee patients . however , the change in muscle activity pattern and balance ability of amputees who have no experience in standing with prosthesis during the period from prosthesis fitting to regaining the ability to walk has never been studied . therefore , the objectives of the present study were to elucidate changes over time in the muscle activity pattern and walking speed from first prosthesis fitting to hospital discharge . we also investigated the differences of muscle activity during standing between younger and elderly amputee patients . [ methods ] electromyography measurements were performed on the gastrocnemius of the intact leg during standing . the test subjects were asked to shift their center of gravity forward . [ results ] two unique patterns of gastrocnemius activities were observed over time . in a younger patient , the amount of muscular activity of the gastrocnemius gradually increased over time . in an elderly patient , however , the amount of muscular activity of gastrocnemius gradually decreased over time . [ conclusion ] the time - dependent changes in gastrocnemius muscle activities are indicative postural control ability . therefore , understanding the time - dependent changes in muscle activities during rehabilitation and the differences of postural control between younger and elderly patients would contribute to the development of effective rehabilitation programs for each patient .

natural killer t ( nkt ) cells are recently discovered innate - like subset of lymphocytes expressing both nk and t cell markers . nkt cells are a phenotypically and functionally diverse subset of t cells that recognize self- and microbial lipids [ 1 , 2 ] . most nkt cells are restricted by mhc - i like molecule cd1 , which can further distributed into two major subsets : type i and type ii nkt cells ( table 1 ) . type i nkt cells are also called invariant nkt ( inkt ) , expressing exclusively limited t cell receptor ( tcr ) and tcr receptors , that is , v14-j18 predominately paired with v8.2 , v7 , or v2 in mice and v24-j18 paired with v11 in human [ 2 , 3 ] . inkt cells secrete a wide array of cytokines and chemokines immediately upon activation through tcr engagement by glycolipids . inkt cells exert their regulatory and cytotoxic activities also through direct contact , granzyme b , or fasl - induced mechanisms [ 46 ] . hence , inkt cells exert both regulator and effector cell function and bridge the innate and adaptive immune responses . recently , inkt cells have been further classified into nkt1 , nkt2 , and nkt17 lineages based on their cytokine profiles and distinct transcription factors , t - bet , gata-3 , and rort , as conventional t helper 1 ( th1 ) , th2 , and th17 cells [ 8 , 9 ] . on the other hand , type ii nkt cells , which are also called non - inkt or variant nkt ( vnkt ) cells , express more diverse tcr and tcr receptors . there still exists a minor group of cd1 nonrestricted nkt cells , referred to as nkt - like cells [ 11 , 12 ] . a hallmark of inkt cells is their capacity to rapidly produce copious amounts of cytokines and chemokines upon tcr stimulation , which endows these cells with potent immunomodulatory activities for a wide variety of immune responses and diseases ( figure 1 ) . inkt cells exhibit potent effector functions and play critical roles in antimicrobial defense , cancer immunosurveillance , and modulation of immune - mediated disorders [ 1316 ] . as inkt cells recognize glycolipids that are enriched in microbes and parasites , they are believed to play important roles in the infections caused by these pathogens . hygiene hypothesis , which tries to explain the declined infections with rising autoimmune and atopic diseases in the recent two to three decades [ 17 , 18 ] . in this review , we summarize the roles of inkt cells in parasitic infections , with particular emphasis on the involvement of inkt cells in the hygiene hypothesis and underlying mechanisms . albeit being widely studied during viral and bacterial infections , the role of inkt cells during parasite infections remains largely unexplored . as helminth infections can usually induce th2-dominated immune responses and inkt cells can rapidly produce copious amounts of various cytokines including interleukin-4 ( il-4 ) , these cells might be important players in the initial steps leading to th2 responses during helminthiasis . recent reports have indicated that nkt cells are involved in the pathogenesis of several parasite infections in animal models and patients , playing , in most cases , protective or regulatory roles towards hosts . it is caused by digenetic blood trematodes , of which there are three main species : schistosoma mansoni , s. japonicum , and s. haematobium . previous studies by others and us indicated that egg deposit in the liver was a determining factor to drive th2 response in s. mansoni and s. japonicum infections in mice [ 20 , 21 ] . nkt cells may contribute to regulating the cytokine secretion profiles during infections . in mice infected with s. mansoni , inkt cells exhibited an activated phenotype . hepatic inkt cells produced both interferon- ( ifn- ) and il-4 following schistosome egg deposited in the liver . further studies revealed that s. mansoni activated both inkt and non - inkt cells in vivo . inkt cells contributed to th1 cell differentiation , whereas non - inkt cells might be mostly implicated in th2 cell differentiation in response to this parasite . luo and colleagues reported that nk and nkt cells were activated and expanded from draining mesenteric lymph node ( mln ) in mice 57 wk after infection with s. japonicum . . however , the kinetics of nkt cells and their precise roles in immunomodulation during schistosome infections remain unclear . this is also true to nematode infections , where nkt cells remains ill - defined in their pathogenesis . a pioneer work from balmer and colleagues found an expansion of nkt ( cd3nk1.1 ) as early as 24 hours following the infection with brugia pahangi . however , depletion of nk1.1-expressing cell had no effect on the th2 development during the gastrointestinal nematode trichuris muris infection . inkt cells have been reported playing crucial roles in the pathogenesis of protozoan infections . in malaria , early interactions between blood - stage plasmodium parasites and cells of the innate immune system , including innate - like nkt cells , are important in the timely control of parasite replication and in the subsequent elimination and resolution of the infection . the lipid extracts from murine malaria parasites could actually be loaded onto cd1 molecules to stimulate inkt cell by the use of artificial antigen - presenting beads . the level of protective antimalaria immunity was greatly enhanced by coadministration of -galactosylceramide ( -galcer ) with suboptimal doses of irradiated sporozoites or recombinant viruses expressing a malaria antigen in mice . inkt cells were increased in numbers and played critical roles through the secretion of ifn- in reducing liver - stage burden to a secondary infection by murine malaria plasmodium yoelii . -c - galcer displayed a superior inhibitory activity against the liver stages of the rodent malaria parasite p. yoelii compared to its parental glycolipid , -galcer . this adjuvant effect depended on nkt cell activation , which was able to boost ifn- production by nk cells and memory cd8 t cells . visceral leishmaniasis ( kala - azar ) is a deadly disease caused by the parasitic protozoa leishmania donovani . inkt cells are involved in the pathogenesis of leishmaniasis . in patients with visceral leishmaniasis , bone - marrow - derived non - inkt cells dominantly produced ifn- in response to l. donovani antigen in vitro . post - kala - azar dermal leishmaniasis is a chronic dermal complication that occurs usually after recovery from visceral leishmaniasis . there was a raised proportion of circulating nkt cells in these patients compared to health controls . karmakar and colleagues isolated a natural ligand of nkt cells , -(14)-galactose terminal glycosphingophospholipid ( gspl ) from this parasite to treat infected balb / c mice . this immunotherapy with gspl induced ifn- through the cooperative action of tlr4 and nkt cells , which contributed to the effective control of acute parasite burden in the infected animals . by use of inkt cell - deficient ( j18 ) c57bl/6 mice , another study demonstrated that inkt cells played a role in early and sustained proinflammatory cytokine response warranting efficient organization of hepatic granulomas and parasite clearance of l. donovani . nkt cell activation by -galcer during intradermal dnap36 priming was highly protective against murine cutaneous leishmaniasis , resulting in the heightened activation and development of cd4 and cd8 effector and memory t cells . conversely , activation of inkt cells exacerbated , rather than ameliorated , experimental visceral leishmaniasis by l. donovani , which was correlated with a bias towards increased il-4 production by inkt . this may illustrate the double - edged sword of nkt cell - based therapy in leishmaniasis . inkt cells protect mice against toxoplasma gondii infection . by oral infection of mildly virulent strain me49 t. gondii cysts , most cd1d - deficient c57bl/6 mice died within 2 wk of infection compared to no death in wt mice . after activation with t. gondii , nkt cells were important mediators of the immune response via a robust ifn--mediated effect that limited parasite replication and allowed for parasite clearance . nevertheless , this strong th1 response , when uncontrolled , can mediate the lethal ileitis . treatment of mice with a single injection of -galcer one day before infection activated intestinal nkt and led to a shift in cytokine secretion towards a th2 profile and a dramatic increase in treg cells in mlns , which alleviated intestinal lesions and increased survival of mice . on the other hand , inkt cells may negatively regulate the immune response against t. gondii infection possibly by producing il-4 and suppressing the induction of heat shock protein 65 . the latter is induced in host macrophages by t cells and plays an essential role in protective immunity in this infection . nkt cells are involved in the pathogenesis of some other protozoan infections , providing protection against infections in most cases . cd8 nkt cells were able to activate macrophages to kill trypanosoma congolense through the production of nitrogen oxides , whereas treg cells prevented the activation of the cd8 nkt cells . however , another report indicated that loss of inkt cells did not affect the susceptibility or resistance in cd1d c57bl/6 mice to the infections with virulent african trypanosomes , t. congolense or t. bruce . lotter and colleagues identified a lipopeptidophosphoglycan from entamoeba histolytica membranes ( ehlppg ) as a possible inkt natural ligand . ehlppg treatment , similar to -galcer application , induced protective ifn- but not il-4 production from inkt cells and significantly reduced the severity of amebic liver abscess in mice infected with e. histolytica . by the use of cd1d ko mice , it was found that inkt cells contributed to resistance against this protozoan and to the control of inflammation in the colitis induced by the infection . inkt cells play important roles in the pathogenesis of some other parasitic diseases , as well as of a wide range of microbe infections , as seen in recent nice reviews [ 10 , 46 , 47 ] . nkt cells play protective role against a wide range of parasite infections as discussed above , whereas the underlying molecular mechanisms are not fully elucidated . shifting of host 's cytokine secretion profiles may account for the protective or , in some cases , pathogenic effects of nkt cells on parasitic infections . activated inkt cells can also transactivate many other immune cells or attract these cells to the sites of infection to exert their regulatory roles . like nk cells , activated nkt cells can also mediate cytotoxic activity , possibly involving both perforin / granzyme and fas / fasl pathways [ 5 , 6 , 48 ] . parasites are enriched in lipid , which may contain natural ligands for nkt cells as discussed in the next section . therefore , it is not surprising that inkt cells participate in the pathogenesis of a range of different parasitic infections . the role of inkt cells in some parasitic infections and possible effect mechanisms are summarized in table 2 . the hygiene hypothesis was proposed in 1989 by strachan to explain the dramatic increase in the prevalence of autoimmune and allergic diseases over the past two to three decades . according to this hypothesis , reduced exposure to microorganisms and parasites in childhood is the main cause for the increased incidence of both th1-mediated autoimmune diseases and th2-mediated allergic diseases . currently , the hypothesis is becoming more accepted with accumulating epidemiological and clinical evidences to support [ 5052 ] . although the exact scientific underpinnings for the hygiene hypothesis and the underlying mechanisms by which infections affect the immune system to prevent diseases have remained a puzzle over the years for both scientists and clinicians , there is increasing recognition that exposure to infectious agents evokes fundamental effects on the development and behavior of the immune system [ 18 , 53 ] . the core of this hypothesis consists in the notion that the microbial environment interfaces with the innate immune system and modulates its ability to impart instructions to adaptive immune responses , particularly when such interactions occur in utero and/or in early life . many chronic infections , both microbial and parasitic , induce forms of immune suppression or downmodulation . recent studies have indicated that infectious agents stimulate a large variety of regulatory t cells , such as th2 , treg , tr1 , and nkt cells , which secrete immunosuppressive cytokines / chemokines , such as il-10 and tgf- to alter the th1/th2 balance . infections may induce the generation of regulatory macrophages , dendritic cells , innate lymphoid cells ( ilc ) , nk , and b cells [ 5658 ] . tlr - myd88 pathway is believed to be involved in the induction of different subsets of regulatory t cells , such as treg and nkt cells . the ability of infectious agents to regulate the immune system of their host is an increasingly fascinating topic . helminths , as long - lived parasites , are remarkable for their ability to manipulate host immunity , protecting themselves from elimination and minimizing severe pathology in the host [ 53 , 56 , 59 , 60 ] . immunomodulation by parasitic helminths is a general phenomenon that is conserved across species , classes , and even phyla . therefore , parasitic infections are a major theme in the hygiene hypothesis . allergies and autoimmune diseases are less prevalent in countries with higher burdens of helminths and other parasitic organisms . there are strong epidemiological evidences to support the premise that the dramatic increase in atopic disease in the developed world is a direct consequence of the eradication of helminth infections . at least some helminthes seem to have antiallergic or anti - inflammatory effects in humans . experimental evidences have also shown the significant suppression for the development of airway hyperresponsiveness ( ahr ) in mice infected with numerous helminths , including blood fluke schistosoma japonicum , filaria litomosoides sigmodontis , nematode heligmosomoides polygyrus , and nippostrongylus brasiliensis . these mice show attenuated airway inflammation with reduced infiltration of eosinophilia in the bal and lung and allergen - specific ige in sera . experimental studies have also shown protective effects of helminth infections in animal models of autoimmunity . surprisingly , helminths have been shown to suppress various types of autoimmune disease , such as collagen - induced arthritis , experimental autoimmune encephalomyelitis , and type 1 diabetes in murine models as reviewed recently . helminth infections might be beneficial to the induction of multiple regulatory mechanisms , including various regulatory cell populations , inhibitory receptors , blocking antibodies , and two prominent cytokines : il-10 and tgf- [ 61 , 66 ] . thus , it is not surprising that helminths can modulate immunopathology , whether in the context of allergic inflammation or autoimmune disease , either directly or indirectly . a study shows that serum antibodies to toxoplasma gondii tend to be negatively associated with allergic sensitization to food and aeroallergens in children from different geographical areas in greece , netherlands , china , india , and russia . a negative association also exists between t. gondii infection and the presence of multiple sclerosis . the literature is limited regarding the involvement of inkt cells in this hypothesis . on the other hand , many microorganisms and parasites contain various lipids in their structures . the bacterial and parasitic cell wall is a cellular structure highly enriched in a variety of glycolipids and lipoproteins . along with -galcer , growing evidences suggest that some microorganisms , including mycobacteria , sphingomonas , borrelia , helicobacter pylori , streptococcus pneumoniae , and group b streptococcus , can produce cd1d - restricted ligands capable of activating a proportion of inkt cells . hence , inkt cells can respond directly by recognizing the glycolipid antigens expressed by these bacteria . inkt cells can also respond indirectly to many other bacteria such as salmonella enterica and staphylococcus aureus . of note , some protozoan and helminthic parasites also contain natural ligands of nkt cells and several candidates have been successfully isolated . the excretory and secretory ( es ) products , which are often glycosylated , are found in the bloodstream of infected hosts and dictate particular functional immune responses that allow persistence of the parasite , typically by inducing th2-associated cytokines and expansion of various regulatory cell subsets , including nkt cells [ 22 , 56 , 69 ] . the adult worms of schistosoma mansoni express a range of glycoconjugates , such as galactosylceramide and glucosylceramide , which may contain natural ligands of nkt cells . infection with s. mansoni or exposure to eggs from this helminth inhibited the development of type 1 diabetes in nod mice . in addition , soluble extracts from worms or eggs of this schistosome possess the similar ability as infection to prevent the onset of diabetes if injection is given at early age ( 4 wk old ) . soluble adult worm antigen or soluble egg antigen may expand the inkt cell population in nod mice , although the lipids binding to inkt cells have not been targeted in this study . lipid extracts from murine malaria parasites can actually be loaded onto cd1 molecules to expand inkt cells . lotter and colleagues have identified a lipopeptidophosphoglycan from e. histolytica membranes as a possible inkt natural ligand , which can stimulate inkt cells to produce ifn- to exert protective role against this infection . karmakar and colleagues isolated a natural ligand of nkt cells , -(14)-galactose terminal glycosphingophospholipid ( gspl ) , from l. donovani to treat infected balb / c mice . these investigations pave the way to identify natural ligands of inkt cells for the development of novel therapeutic agents . inkt cells may exert their immunomodulatory effects in hygiene hypothesis through the interaction with treg , th17 , and other immune cells . upon activation with microbial and parasitic lipid antigens , inkt cells rapidly produce a wide range of cytokines and chemokines , transactivating many immune cell types , such as th1 , th2 , and treg . in eae model , inkt cells th17 cells in the mlns are greatly reduced in cd1 mice or j281 mice , which lack inkt cells . inkt cells induce the conversion of nave diabetogenic bdc2.5 t cells into foxp3(+ ) treg cells in the pancreatic lymph nodes accumulating in the pancreatic islets . in addition , inkt cells can suppress both antigen - induced acute arthritis and collagen - induced chronic arthritis , likely via inhibition of arthritogenic th1 cells . in toxoplasma gondii infection , activation of inkt cells by -galcer can lead to a shift to th2 cytokine profile and a significant increase in treg cells in mlns , which exerts protective role and increases survival of mice . the recently defined innate lymphoid cells ( ilc ) share some features with inkt cells . upon activation without the need of prior sensitization , both cells can release copious amounts of th1 , th2 , and/or th17 cytokines that shape subsequent innate and adaptive immune responses . although sparse up to now , there exists experimental evidence for direct interactions of ilcs and nkt cells possibly via their effector cytokines . for example , nkt cells , as well as alveolar macrophages , secrete endogenous il-33 that enhance il-5 production from ilc2 in lungs during influenza virus infection . the interaction of inkt cells with other t cell subsets and underlying mechanisms remain to be elucidated . taken together , microbe and parasite infections , especially at early lifetime , parasitic worms are able to survive in their mammalian host for many years due to their ability to manipulate the immune response . the underlying mechanisms regarding how infections affect the immunity of hosts remain to be clarified . upregulation of regulatory t cell subsets , such as treg , and induction of inhibitory cytokines and/or chemokines are the common findings . given the fact that many microbes and parasites are enriched in lipid antigens and nkt cells are unique t cell subset that can recognize lipid antigens , it is reasonable to speculate that nkt cells play key roles in this hypothesis ( figure 2 ) . inkt cells are unique innate - like t cell subset that bridge between innate and acquired immunity systems . inkt cells exert both effector and regulatory functions through direct contact or quick secretion of copious amounts of cytokines , chemokines , and other mediators upon their tcr engagement by glycolipid antigens . these cytokines and chemokines critically regulate the downstream differentiation of th1 , th2 , th17 , and other cells . therefore , inkt cells have been postulated to have an important proximal immunoregulatory role and influence both innate and acquired immune systems . they participate in the host 's immunity and immunopathogenesis of a wide range of parasite infections as discussed above . more thorough investigation is clearly necessary to better define their mode of activation and their regulatory functions in parasitic infections . although the involvement of inkt cells in the hygiene hypothesis and the contribution to autoimmunity and allergic inflammation remains to be fully elucidated , exploiting inkt cells in helminth immunomodulatory mechanisms may lead to opening a new avenue to develop novel safer therapeutic agents for these diseases based on the manipulation of inkt cell function .

invariant natural killer t ( inkt ) cells are unique subset of innate - like t cells recognizing glycolipids . inkt cells can rapidly produce copious amounts of cytokines upon antigen stimulation and exert potent immunomodulatory activities for a wide variety of immune responses and diseases . we have revealed the regulatory effect of inkt cells on autoimmunity with a serial of publications . on the other hand , the role of inkt cells in parasitic infections , especially in recently attractive topic hygiene hypothesis , has not been clearly defined yet . bacterial and parasitic cell wall is a cellular structure highly enriched in a variety of glycolipids and lipoproteins , some of which may serve as natural ligands of inkt cells . in this review , we mainly summarized the recent findings on the roles and underlying mechanisms of inkt cells in parasite infections and their cross - talk with th1 , th2 , th17 , treg , and innate lymphoid cells . in most cases , inkt cells exert regulatory or direct cytotoxic roles to protect hosts against parasite infections . we put particular emphasis as well on the identification of the natural ligands from parasites and the involvement of inkt cells in the hygiene hypothesis .

cancer genomics has gone through a dramatic period of progress due to the availability of genome - wide technologies . ( tcga , https://cancergenome.nih.gov/ ) and the international cancer genome consortium ( icgc , http://icgc.org/ ) , involve thousands of patients and have generated petabytes of data . one of the major assets of such projects is the public availability of the data allowing their integration with data from other initiatives . in that way , data from these initiatives can push a more focused and deeper analysis either in a specific gene set or in a specific cohort of patients / samples . the human surfaceome , the collection of cell surface proteins in human cells , has been defined and studied by us previously . by using bioinformatics pipeline and an experimental approach based either on real - time pcr or on other gene expression technologies , we were able to identify potential new biomarkers for few tumor types and have characterized new cell surface putative cancer - testis ( ct ) antigens [ 1 , 2 ] . relevant roles of surface proteins include nutrient and ion transport , adhesion to substrates , signaling , and intercellular interaction . due to these roles and their subcellular localization , easily accessible to therapeutic agents , surface proteins are important targets for cancer intervention . since our original publication few reports have further explored the human surfaceome [ 26 ] , mostly in the context of a mass - spectrometry - based characterization of the cell surface of tumor cells . data from tcga / icgc allow the development of new metrics that evaluate the frequency of gene alterations in different cancer types . recently , we developed a new scoring system for the identification and prioritization of cancer genes . the s - score method integrates information derived from different omics technologies to generate a gene - specific score that indicates whether that specific gene is a tumor suppressor ( negative s - score ) or an oncogene ( positive s - score ) . the numerical value indicates the frequency in which that gene is altered in the cohort of samples used in the calculation . we have used the s - score metric to identify cancer genes in a set of human homologs of yeast genes characterized as suppressors of genome instability in yeast . the availability of the s - score system provides a quantitative way to identify and prioritize cancer genes in a particular set of samples . here , we capitalize on the availability of data from the tcga project to further and deeper investigate the status of the human surfaceome in 15 tumor types , including gbm and colorectal and breast tumors , all analyzed in our previous publications [ 1 , 2 ] . this generated a pan - cancer landscape of the human surfaceome with the identification of shared and tumor - specific markers . furthermore , the use of the s - score system allowed us to identify gene signatures associated with overall survival in breast cancer patients . these signatures can be ultimately used in the development of new and more efficient diagnostic and therapeutic protocols . the protein - coding sequences of all human genes were obtained from the ncbi refseq ( release 64 ) . the illumina human bodymap 2.0 dataset was obtained from embl - ebi expression atlas ( https://www.ebi.ac.uk/gxa/experiments/e-mtab-513 ) . tcga data was retrieved from both , the tcga web site ( https://cancergenome.nih.gov/ ) and the cbio cancer genomics portal ( http://www.cbioportal.org/ ) . to predict plasma membrane subcellular localization , the ncbi reference sequence dataset was submitted to tmhmm version 2.0 ( http://www.cbs.dtu.dk/services/tmhmm/ ) . all sequences containing at least one tm domain were selected . to avoid false positives , sequences containing only one tm domain in the first 50 residues , which could be a signal peptide , were excluded and classified as secreted protein . furthermore , sequences were also filtered based on the identification of signal peptide cleavage sites by signalp , release 4.1 ( http://www.cbs.dtu.dk/services/signalp/ ) . since tm domains are not exclusive to cell surface proteins , the sequences were grouped according to subcellular localization as defined by gene ontology . we excluded sequences that were exclusively located at the following cellular compartments : lysosome , endoplasmic reticulum , mitochondria , cytoskeleton , endosome , liposome , nucleolus , nucleus , and ribosome . this step was conducted using in - house perl scripts . to validate the obtained list of surfaceome genes , we classified these genes as belonging to the following classes : g - protein - coupled receptors ( gpcrs ) , solute carrier ( slc ) proteins , and cluster of differentiation ( cd ) antigens . the gpcr genes were obtained from gpcrdb ( http://gpcrdb.org ) , while cd and slc genes were collected from hgnc ( http://www.genenames.org/genefamilies/a-z#r ) . s - scores were calculated for the human surfaceome and for the 15 tumor types as previously defined . the distribution of s - scores was used to calculate z - scores for all genes using r statistical package . the go enrichment analyses of the surfaceome gene cluster were conducted using clusterprofiler , implemented in r , with p values < 0.01 as a cutoff . genes with extreme s - score ( s - score < 2 and > 2 for breast tumors ) were selected to test any putative association with overall survival in breast cancer samples derived from tcga ( without subtype distinction ) . the altered set comprised samples within which the respective genes were differentially expressed ( z - score > 2 or z - score < 2 , as reported by tcga ) , amplified or deleted , or presenting deleterious mutations ( nonsense , frameshift , and splice - site ) . after that , for each gene , the survival analysis was performed using the kaplan - meier method and the difference in survival curves was tested for statistical significance using the log rank test p value . we then selected a nonredundant set of 20 genes with the lowest p value ( cutoff of 0.05 ) and tested all possible groups of three genes . first of all , we decided to reannotate the set of human genes coding for putative cell surface proteins . this was required due to ( i ) an improvement in the annotation of gene and protein databases regarding a protein 's subcellular localization and ( ii ) the inclusion of new human genes in the reference sequence collection . the same approach used by da cunha et al . ( 2009 ) generated now a set of 3,758 human genes , composing the human surfaceome ( figure 1 ) . the complete list of human genes coding for cell surface proteins is provided as supplementary table s1 , in supplementary material available online at http://dx.doi.org/10.1155/2016/8346198 . as expected , the great majority ( 85% ) of surfaceome genes present in our dataset in 2009 remained classified as such in 2016 . new genes were added ( 585 ) , mostly due to their inclusion in the reference sequence collection and some other genes ( 529 ) were excluded due mainly to new functional annotation that classified their protein products as belonging to other subcellular compartments . to assess the robustness of our approach , we performed the same analysis reported by us in our original 2009 paper checking the representation of three known families of cell surface proteins ( g - protein - coupled receptors ( gpcrs ) , solute carrier ( slc ) proteins and cluster of differentiation ( cd ) antigens ) . since these are large and well - studied families of cell surface proteins , we envisaged that they would be appropriate for a benchmark analysis . for gpcrs , 98% of their known members were represented in our dataset . for slc proteins and cd antigens we found 77% and 88% represented in the surfaceome set , respectively . overall , 90% of members of these three families were represented in our present surfaceome set , compared to 83% in our previous analysis . this improvement is expected due to a better annotation of the sequences in public databases . capitalizing on the availability of surfaceome sets derived from mass - spectrometry analysis , we decided to compare our dataset to the dataset from bausch - fluck et al . . although this type of comparison is problematic for different reasons , including ( i ) the nonexhaustive nature of the wet - based approach ( due to the method itself and the samples screened ) and ( ii ) the different premises of both methods ( the requirement of at least one tm domain per protein in our pipeline and the lack of such requirement in which allowed the authors to characterize gpi - anchored proteins , e.g. ) , the analysis may be illuminating in the sense that it can highlight important differences in both methodologies . we found that 66.6% ( 664 out of 996 ) of the proteins classified by bausch - fluck et al . were present in our dataset while only 17.6% ( 664 out of 3758 ) of our proteins were present in their dataset . this was expected due to the issues raised above . to illustrate the complex nature of this comparison , 23.8% of all cell surface proteins found in have no tm domain , as identified by tmhmm . next , the s - score method was used to identify cancer genes within the surfaceome set . s - score threshold was defined for each tumor type as the s - score representing the average s - score plus / minus three standard deviations ( z - score 3 or 3 ) . the list of all cancer genes coding for cell surface proteins in all 15 tumors types is shown in supplementary table s1 . using the above z - score threshold , we found 248 surfaceome genes classified as a cancer gene in at least one tumor type . in the heatmap representation of the surfaceome cancer genes ( figure 2(a ) ) we can clearly identify three distinct clusters based on the s - score values for all 15 tumor types . although all three groups have a variety of oncogenes and suppressors , some features deserve further comments . for example , the first group is mainly composed of suppressors , especially in melanoma and colorectal and lung adenocarcinoma and uterine corpus endometrial carcinoma . genes in this group include several members of the cadherin superfamily ( pcdhgb3 , pcdha2 , pcdha7 , pcdh15 , pcdhgb5 , pcdh11x , pcdhac1 , fat1 , fat2 , and fat4 ) . there is a set of oncogenes in group 2 shared by almost all tumors and involving 30 genes , including ephb1 and ephb3 . there is no clear pattern in group 3 and oncogenes and suppressors seem to be distributed evenly across all tumors . to better understand the pattern presented in figure 2(a ) , we performed a gene ontology ( go ) enrichment analysis ( using the biological process ontology ) for the three different clusters . as expected , all three groups shared go categories associated with the cell surface such as transmembrane transport and cell surface receptor signaling pathway . more interestingly , however , is the fact that specific go categories were enriched in individual groups ( figure 2(b ) ) . go categories exclusively found in group 1 were clearly associated with nervous system including neuromuscular process ; memory ; and neuronal action potential . the same pattern was observed for group 2 although the go categories represented different aspects of nervous system including the following : sensory perception of pain and other categories related to axonogenesis . regarding group 3 , go analysis lent further support for the current concept of ion transport associated with cancer , including manganese ion transport . additionally , this group presented genes related to antigen processing and presentation , highlighting that the interplay between immune and tumor cells is complex . several of the identified surfaceome cancer genes are known for their involvement in different aspects of cancer biology , especially the ones classified in group 2 . abcc5 , a cell surface transporter , was involved in resistance to anticancer drugs and overexpression of atp11b has been linked to drug resistance in ovarian cancer . both genes were regarded as oncogenic by our work especially in lung squamous cell carcinoma and ovarian cancer . on the other hand , ephb3 has already been suggested as a candidate target gene for both lung small cell carcinoma and colorectal cancer . finally , a transferrin receptor ( tfrc ) has shown an increased expression in many malignant tumors and was also found to be highly oncogenic in this work . as previously discussed by us , the s - score method allows the prioritization of cancer genes based on clinical parameters . for example , we have identified genes associated with both short- and long - term survival in ovarian cancer . to test whether we could identify genes in the surfaceome set associated with clinical parameters , we decided to look at overall survival in breast tumors , since this type of tumor is the one with the largest cohort in tcga . for this specific analysis a more relaxed threshold ( z - score < 2 or > 2 ) was used to classify a gene as a cancer gene in breast tumor to increase the number of genes under test without compromising the quality of the classification ( a heatmap , similar to figure 2(a ) and generated using the dataset with a more relaxed threshold , is presented in supplementary figure 1 ) . for each surfaceome gene classified as oncogene or suppressor in breast tumor , we split the breast cancer samples into two groups : altered ( genes with differential expression , genes amplified / deleted , or genes mutated ) and unaltered . for each gene , the two groups were then compared by a kaplan - meier analysis to evaluate whether they had significantly different overall survival . twenty - three genes , seven oncogenes and 16 suppressors , were significantly ( p - value < 0.05 ) associated with differences in overall survival in breast cancer patients ( supplementary table 2 ) . these genes are involved in cell adhesion and ion transport , two of the main categories enriched in our gene ontology analysis . next , all possible combinations of these genes were similarly tested for differences in overall survival . although we found several combinations with statistically significant differences in overall survival , we have focused on wnt5a , cnga2 , and igsf9b due to statistical significance ( it is the most significant three - gene set in supplementary table 2 ) and novelty . patients in which one of the three genes was altered had a significantly shorter survival ( p value = 1.82e)compared to patients where these three genes were unaltered ( figure 3 ) . the wnt5a , cnga2 , and igsf9b genes have negative s - scores in breast cancer ( 2.59 , 3.39 , and 2.56 , resp . ) , demonstrating a tumor suppressor profile . , the loss of wnt5a has been associated with poor prognosis , in agreement with the suppressor status defined by the respective s - score . on the other hand , wnt5a was recently reported to promote cancer cell lines invasion and proliferation , a feature typical of oncogenes . wnt5a is present in pathways where wnt signaling is involved through interaction with frizzleds ( fzd10 , e.g. ) and dishevelled . cnga2 , a homotetrameric channel in olfactory sensory neurons , has not been reported in association with cancer . however , cnga2 represents the alpha subunit of a cyclic nucleotide - gated olfactory channel possessing a role in calcium signaling pathway acting through calmodulin - like 6 and calcium / calmodulin - dependent protein kinase iv directly involved with protein kinase a ( pka ) , a biological target in cancer therapy . igsf9b was only recently identified as an inhibitory synaptic adhesion molecule and no link with cancer was found in the literature . we have updated the set of human genes coding for cell surface proteins , the human surfaceome . using tcga data for 15 tumor types and a new method of cancer genes classification that integrates information from different omics technologies and allows a ranking based on clinical parameters ( s - score ) , we identified several potential therapeutic targets within the surfaceome set . furthermore , we present evidence that a three - gene set wnt5a , cnga2 and igsf9b was associated with shorter survival in breast cancer patients . our results clearly show the importance of large - scale genomics datasets from cancer patient cohorts , like the one provided by tcga . we envisage that the data we provide here will be extremely useful to researchers who aim to characterize cell surface targets for a variety of tumor types .

it is estimated that 10 to 20% of all genes in the human genome encode cell surface proteins and due to their subcellular localization these proteins represent excellent targets for cancer diagnosis and therapeutics . therefore , a precise characterization of the surfaceome set in different types of tumor is needed . using tcga data from 15 different tumor types and a new method to identify cancer genes , the s - score , we identified several potential therapeutic targets within the surfaceome set . this allowed us to expand a previous analysis from us and provided a clear characterization of the human surfaceome in the tumor landscape . moreover , we present evidence that a three - gene set wnt5a , cnga2 , and igsf9b can be used as a signature associated with shorter survival in breast cancer patients . the data made available here will help the community to develop more efficient diagnostic and therapeutic tools for a variety of tumor types .

participants were drawn from the minnesota twin family study , a community - based sample of adolescents and their families . this sample was utilized because the participants were representative of minnesota families with children living at home ( based on the 2000 us census ) and because it included rigorous , developmentally - timed assessments of key study variables ( see above ) . of eligible families , youth were born between 1977 and 1982 ( males ) or 1981 and 1985 ( females ) . participants ( 752 males , 760 females ) were first recruited and assessed when the youth were 11 ( mean=11.7 , sd=.4 ) and were invited back to return to the study at ages 14 ( mean=14.8 , sd=.5 ) , 17 ( mean=18.2 , sd=.7 ) , 20 ( mean=21.5 , sd=.8 ) , and 24 ( mean=25.3 , sd=.7 ) . although twin zygosity was not considered in this report , both monozygotic ( n=487 ) and dizygotic ( n=269 ) pairs comprised the sample . this study was approved by the university of minnesota irb , and participants gave informed consent ( if 18 or over or parent on behalf of a minor child ) or assent ( for minors ) . consistent with the population of the state of minnesota at the time these youth were born , approximately 95% of the sample was white . mdd was assessed in youth younger than age 17 using the diagnostic interview schedule for children and adolescents and in youth ages 17 and older using the structured clinical interview for dsm - iii - r . diagnostic interviews were conducted in person by trained interviewers with bachelor s or master s degree in psychology , reviewed by teams of advanced clinical psychology doctoral students who achieved consensus agreement for each assessed symptom of mdd , and diagnosed using computer algorithms following dsm rules ( kappas = .78 or better ) . for youth 17 and younger , diagnoses reported by either the mother or the child were counted as present ( the best - estimate method ) . definite and probable ( exhibiting all symptoms necessary for the diagnosis except one ) diagnoses were used . at the initial assessment , lifetime diagnoses were assessed ; after that , mdd was assessed since the last visit to the study . diagnoses reported at either age 11 or age 14 were combined into an assessment of mdd during early adolescence . if mdd was first reported at 17 or 20 , this was considered to represent late adolescent - onset mdd . if mdd was first reported at 24 , this was considered to represent an onset of mdd during early adulthood . body mass index ( bmi ) was calculated using the standard formula ( weight in kilograms divided by height in meters squared ) . for youth at age 20 and 24 , the standard bmi cutoff of 30 was used to define obesity . for youth younger than age 20 , growth curves from the center for disease control were used to determine obesity cutoffs ( 95 percentile ) for each age and sex based on the average participant ages at each assessment ( age 11 : 23.90 for males , 24.89 for females ; age 14 : 26.71 for males , 27.99 for females ; age 17 : 28.90 for males , 30 for females ) . analogous to mdd , obesity first occurring at age 11 or age 14 was combined into an assessment of obesity during early adolescence . if obesity was first present at 17 or 20 , this if obesity was first present at 24 , this was considered to represent an onset of obesity during early adulthood . sas version 9.2 was employed to compute generalized estimating equations ( gees ; proc genmod ) to account for the correlated observations in this sample ( twins nested within families ) . there was little missing data because if a participant missed an early assessment , the relevant data for the missed assessment were obtained in a subsequent assessment . in cases where a data point was missing first , prevalences of the onset of each disorder during each developmental period , as well as anytime prior to the final assessment , were computed and compared across sex using gees . next , in order to examine cross - sectional associations between these disorders , tetrachoric correlations were conducted ( reported with asymptotic standard errors ) to describe the associations between mdd and obesity with onsets occurring within each developmental stage ( early adolescence , late adolescence , and early adulthood ) , as well as onsets anytime prior to the final assessment . for the prospective ( i.e. , across time ) analyses that were the focus of our study , because of our interest in disorder onsets ( rather than the recurrence or persistence of disorders ) , some participants were eliminated from certain analyses due to their having earlier onset of the disorder . for example , if a participant experienced mdd prior to age 14 , he / she was not included in analyses predicting obesity onsets in early adulthood from mdd onsets between 14 and 20 ( because the mdd onset had been experienced prior to that ) . for the prospective analyses , the first examined the overall effect of one disorder on the other , adjusting for the effect of gender . the second included a disorder - by - gender interaction term in order to assess the significance of gender differences in the effect of the independent variable on the dependent variable . first , we examined whether the occurrence of obesity by age 14 predicted the development of mdd ( 1 ) during late adolescence , and ( 2 ) during early adulthood . second , we examined the association between obesity first developing during late adolescence and the onset of mdd in early adulthood . next , we examined the opposite directions of effect : mdd by age 14 predicting the development of obesity ( 1 ) during late adolescence and ( 2 ) during early adulthood ; and mdd first developing during late adolescence and the onset of obesity in early adulthood . for inclusion in the analysis , participants were required to have data at the oldest assessment point within each developmental period ( age 14 for early adolescence , age 20 for late adolescence , and age 24 for early adulthood ) . data were occasionally missing at the younger assessment point ( age 11 for early adolescence and age 17 for late adolescence ) . if a participant missed one of these assessments , when he or she returned , mdd was assessed since the previous visit to the study ; therefore , these entire developmental periods were covered . for obesity , the only way a case would have been missed would be if the participant first became obese and then became non - obese all within a 6-year period . participants were drawn from the minnesota twin family study , a community - based sample of adolescents and their families . this sample was utilized because the participants were representative of minnesota families with children living at home ( based on the 2000 us census ) and because it included rigorous , developmentally - timed assessments of key study variables ( see above ) . of eligible families , youth were born between 1977 and 1982 ( males ) or 1981 and 1985 ( females ) . participants ( 752 males , 760 females ) were first recruited and assessed when the youth were 11 ( mean=11.7 , sd=.4 ) and were invited back to return to the study at ages 14 ( mean=14.8 , sd=.5 ) , 17 ( mean=18.2 , sd=.7 ) , 20 ( mean=21.5 , sd=.8 ) , and 24 ( mean=25.3 , sd=.7 ) . although twin zygosity was not considered in this report , both monozygotic ( n=487 ) and dizygotic ( n=269 ) pairs comprised the sample . this study was approved by the university of minnesota irb , and participants gave informed consent ( if 18 or over or parent on behalf of a minor child ) or assent ( for minors ) . consistent with the population of the state of minnesota at the time these youth were born , approximately 95% of the sample was white . mdd was assessed in youth younger than age 17 using the diagnostic interview schedule for children and adolescents and in youth ages 17 and older using the structured clinical interview for dsm - iii - r . diagnostic interviews were conducted in person by trained interviewers with bachelor s or master s degree in psychology , reviewed by teams of advanced clinical psychology doctoral students who achieved consensus agreement for each assessed symptom of mdd , and diagnosed using computer algorithms following dsm rules ( kappas = .78 or better ) . for youth 17 and younger , diagnoses reported by either the mother or the child were counted as present ( the best - estimate method ) . definite and probable ( exhibiting all symptoms necessary for the diagnosis except one ) diagnoses were used . at the initial assessment , lifetime diagnoses were assessed ; after that , mdd was assessed since the last visit to the study . diagnoses reported at either age 11 or age 14 were combined into an assessment of mdd during early adolescence . if mdd was first reported at 17 or 20 , this was considered to represent late adolescent - onset mdd . if mdd was first reported at 24 , this was considered to represent an onset of mdd during early adulthood . body mass index ( bmi ) was calculated using the standard formula ( weight in kilograms divided by height in meters squared ) . for youth at age 20 and 24 , younger than age 20 , growth curves from the center for disease control were used to determine obesity cutoffs ( 95 percentile ) for each age and sex based on the average participant ages at each assessment ( age 11 : 23.90 for males , 24.89 for females ; age 14 : 26.71 for males , 27.99 for females ; age 17 : 28.90 for males , 30 for females ) . analogous to mdd , obesity first occurring at age 11 or age 14 was combined into an assessment of obesity during early adolescence . if obesity was first present at 17 or 20 , this was considered to represent late adolescent - onset obesity . if obesity was first present at 24 , this was considered to represent an onset of obesity during early adulthood . mdd was assessed in youth younger than age 17 using the diagnostic interview schedule for children and adolescents and in youth ages 17 and older using the structured clinical interview for dsm - iii - r . diagnostic interviews were conducted in person by trained interviewers with bachelor s or master s degree in psychology , reviewed by teams of advanced clinical psychology doctoral students who achieved consensus agreement for each assessed symptom of mdd , and diagnosed using computer algorithms following dsm rules ( kappas = .78 or better ) . for youth 17 and younger , diagnoses reported by either the mother or the child were counted as present ( the best - estimate method ) . definite and probable ( exhibiting all symptoms necessary for the diagnosis except one ) diagnoses were used . at the initial assessment , lifetime diagnoses were assessed ; after that , mdd was assessed since the last visit to the study . diagnoses reported at either age 11 or age 14 were combined into an assessment of mdd during early adolescence . if mdd was first reported at 17 or 20 , this was considered to represent late adolescent - onset mdd . if mdd was first reported at 24 , this was considered to represent an onset of mdd during early adulthood . body mass index ( bmi ) was calculated using the standard formula ( weight in kilograms divided by height in meters squared ) . for youth at age 20 and 24 , younger than age 20 , growth curves from the center for disease control were used to determine obesity cutoffs ( 95 percentile ) for each age and sex based on the average participant ages at each assessment ( age 11 : 23.90 for males , 24.89 for females ; age 14 : 26.71 for males , 27.99 for females ; age 17 : 28.90 for males , 30 for females ) . analogous to mdd , obesity first occurring at age 11 or age 14 was combined into an assessment of obesity during early adolescence . if obesity was first present at 17 or 20 , this was considered to represent late adolescent - onset obesity . if obesity was first present at 24 , this was considered to represent an onset of obesity during early adulthood . sas version 9.2 was employed to compute generalized estimating equations ( gees ; proc genmod ) to account for the correlated observations in this sample ( twins nested within families ) . there was little missing data because if a participant missed an early assessment , the relevant data for the missed assessment were obtained in a subsequent assessment . in cases where a data point was missing first , prevalences of the onset of each disorder during each developmental period , as well as anytime prior to the final assessment , were computed and compared across sex using gees . next , in order to examine cross - sectional associations between these disorders , tetrachoric correlations were conducted ( reported with asymptotic standard errors ) to describe the associations between mdd and obesity with onsets occurring within each developmental stage ( early adolescence , late adolescence , and early adulthood ) , as well as onsets anytime prior to the final assessment . for the prospective ( i.e. , across time ) analyses that were the focus of our study , because of our interest in disorder onsets ( rather than the recurrence or persistence of disorders ) , some participants were eliminated from certain analyses due to their having earlier onset of the disorder . for example , if a participant experienced mdd prior to age 14 , he / she was not included in analyses predicting obesity onsets in early adulthood from mdd onsets between 14 and 20 ( because the mdd onset had been experienced prior to that ) . for the prospective analyses , the first examined the overall effect of one disorder on the other , adjusting for the effect of gender . the second included a disorder - by - gender interaction term in order to assess the significance of gender differences in the effect of the independent variable on the dependent variable . first , we examined whether the occurrence of obesity by age 14 predicted the development of mdd ( 1 ) during late adolescence , and ( 2 ) during early adulthood . second , we examined the association between obesity first developing during late adolescence and the onset of mdd in early adulthood . next , we examined the opposite directions of effect : mdd by age 14 predicting the development of obesity ( 1 ) during late adolescence and ( 2 ) during early adulthood ; and mdd first developing during late adolescence and the onset of obesity in early adulthood . for inclusion in the analysis , participants were required to have data at the oldest assessment point within each developmental period ( age 14 for early adolescence , age 20 for late adolescence , and age 24 for early adulthood ) . data were occasionally missing at the younger assessment point ( age 11 for early adolescence and age 17 for late adolescence ) . if a participant missed one of these assessments , when he or she returned , mdd was assessed since the previous visit to the study ; therefore , these entire developmental periods were covered . for obesity , the only way a case would have been missed would be if the participant first became obese and then became non - obese all within a 6-year period . prevalences of the onset of each disorder during early adolescence , late adolescence , and early adulthood , as well as at any time prior to age 24 , are presented in table 1 separately by gender . turning to the bottom 3 rows , by age 24 , lifetime rates of mdd ( 26.5% males , 36.4% females ) and co - occurring mdd and obesity ( 7.8% males , 11.9% females ) , but not obesity considered alone ( 25.7% males , 27.5% females ) , differed by gender , with women having higher rates than men . turning to specific developmental stages , gender differences were evident for mdd in late adolescence ( 12.0% males , 18.9% females ) and early adulthood ( 7.6% males , 12.3% females ) , with females showing higher rates than males . as can be seen from table 1 , the co - occurrence of the onsets of the two disorders at each developmental stage was uncommon , never attaining a rate higher than 1.8% for either gender , and thus did not occur with sufficient frequency to justify computing odds ratios . by age 24 , the lifetime occurrence of obesity and mdd was significantly correlated ( r=.14 , se=.05 , p<.05 ) . however , of particular interest was how the correlation between these conditions varied with development ; we expected that there would be stronger correlations at younger , compared to older , ages . mdd and obesity were significantly correlated if both occurred by early adolescence ( r=.20 , se=.08 , p<.05 ) , but the correlation was not significant when mdd and obesity first occurred in late adolescence ( r=.03 , se=.09 ) or in early adulthood ( r=.09 , se=.11 ) . as can be seen from table 2 , obesity that developed by early adolescence did not significantly predict the onset of mdd anytime during the follow - up period ( during late adolescence or early adulthood ) , though there was a trend for early adolescent obesity to predict the onset of mdd during the late adolescent period . obesity that developed in late adolescence did predict the onset of mdd in early adulthood among females ( a gender - by - obesity interaction effect ; figure 1 ) . as can be seen in table 2 , mdd that developed by early adolescence predicted the onset of obesity in late adolescence among females ( a gender - by - mdd interaction effect ; figure 2 ) . mdd that developed in late adolescence did not predict the onset of obesity in early adulthood . prevalences of the onset of each disorder during early adolescence , late adolescence , and early adulthood , as well as at any time prior to age 24 , are presented in table 1 separately by gender . turning to the bottom 3 rows , by age 24 , lifetime rates of mdd ( 26.5% males , 36.4% females ) and co - occurring mdd and obesity ( 7.8% males , 11.9% females ) , but not obesity considered alone ( 25.7% males , 27.5% females ) , differed by gender , with women having higher rates than men . turning to specific developmental stages , gender differences were evident for mdd in late adolescence ( 12.0% males , 18.9% females ) and early adulthood ( 7.6% males , 12.3% females ) , with females showing higher rates than males . as can be seen from table 1 , the co - occurrence of the onsets of the two disorders at each developmental stage was uncommon , never attaining a rate higher than 1.8% for either gender , and thus did not occur with sufficient frequency to justify computing odds ratios . by age 24 , the lifetime occurrence of obesity and mdd was significantly correlated ( r=.14 , se=.05 , p<.05 ) . however , of particular interest was how the correlation between these conditions varied with development ; we expected that there would be stronger correlations at younger , compared to older , ages . mdd and obesity were significantly correlated if both occurred by early adolescence ( r=.20 , se=.08 , p<.05 ) , but the correlation was not significant when mdd and obesity first occurred in late adolescence ( r=.03 , se=.09 ) or in early adulthood ( r=.09 , se=.11 ) . as can be seen from table 2 , obesity that developed by early adolescence did not significantly predict the onset of mdd anytime during the follow - up period ( during late adolescence or early adulthood ) , though there was a trend for early adolescent obesity to predict the onset of mdd during the late adolescent period . obesity that developed in late adolescence did predict the onset of mdd in early adulthood among females ( a gender - by - obesity interaction effect ; figure 1 ) . as can be seen in table 2 , mdd that developed by early adolescence predicted the onset of obesity in late adolescence among females ( a gender - by - mdd interaction effect ; figure 2 ) . mdd that developed in late adolescence did not predict the onset of obesity in early adulthood . the results of this study indicate that there appear to be developmental as well as gender differences in the association between mdd and obesity . mdd occurring by early adolescence predicted the development of obesity in late adolescence among females . conversely , obesity with an onset during late adolescence predicted the onset of mdd in early adulthood among females . in addition , mdd and obesity developing by early adolescence were cross - sectionally associated with each other ; this effect was not present at later ages . the results of this study were consistent with our expectations and highlight the importance of the adolescent period in the development of comorbidity between these disorders . regarding cross - sectional associations , we anticipated that these disorders would be more strongly related at younger , compared to older , ages , and mdd and obesity with onsets during childhood and early adolescence were cross - sectionally related while disorders with later onsets were not . it is important to note that within this key developmental period ( i.e. , by age 14 ) , we do not know which disorder developed first , or whether they developed simultaneously . the prospective results were also consistent with expectations , with each disorder during adolescence predicting the later onset of the other disorder among females . the earliest - onset cases of mdd appear to be most predictive of later obesity for girls ; this could be for any of the reasons discussed earlier , including the still - developing eating and activity habits of young people or the symptom differences between childhood mdd and mdd in adults . interestingly , the earliest - onset cases of obesity were not the most predictive of later - onset mdd , though they were concurrently associated with mdd . although obesity by early adolescence ( age 14 ) did not significantly predict the later onset of mdd ( though this effect was significant at a trend level , with obese early adolescents being at 1.5 times the risk of non - obese adolescents for developing mdd in late adolescence , and no significant gender difference in this trend ) , obesity developing during late adolescence ( 1420 ) was particularly predictive of the later onset of mdd among females . perhaps body - related insecurities and/or peer pressure relating to body shape and weight are at a height during this late adolescent period ( when the pressure to conform to peer norms is high and dating relationships are forming ) and therefore young women who experience the onset of obesity during this time are particularly vulnerable to the subsequent onset of mdd . considering lifetime diagnoses by age 24 , both mdd and co - occurring mdd and obesity were more prevalent among women . significant gender interaction effects were found for prospective associations in both directions , with the increased risk for the other disorder being found among young women specifically . for example , obesity may be a more stigmatized condition among females and/or women may be more likely to eat to cope with negative feelings than men . interestingly , inspection of prevalences ( figures 1 and 2 ) indicates that males with either earlier disorder were at slightly decreased risk for the development of the other disorder ; this may indicate the presence of significantly different pathways for the development of these disorders among males and females . the results of this study are broadly consistent with prior prospective research that used study - assessed mdd and obesity diagnoses . specifically , richardson and colleagues found that mdd between 11 and 15 was not associated with obesity at 26 , though later mdd ( between 18 and 21 ) was for females . we also found that effects were strongest on the adjacent developmental period ( early to late adolescence for the mdd to obesity pathway ) . however , we did not replicate their effect of mdd between 18 and 21 ( similar to the latter half of our late - adolescent period ) on later obesity in females ; the reasons for this difference are unclear but may relate to richardson et al.s examination of the occurrence of mdd and obesity , not specifically the first onsets of these disorders . the results of this study imply that prevention efforts aimed at both of these disorders in childhood and adolescence may be fruitful in decreasing the prevalence of this form of comorbidity . in particular , preventing mdd by early adolescence has the potential to decrease the later onset of obesity , and preventing the development of obesity during later adolescence ( and perhaps early adolescence as well , given the trend - level effect found for that period ) has the potential to decrease the prevalence of early adult - onset mdd , particularly among women . for example , if decreased activity among depressed youth moderates this association , then encouraging young girls with depression to exercise and/or participate in sports as they are being treated for depression may prevent the later onset of obesity . although we found differences in the associations between these disorders depending on whether we examined the period from early adolescence to late adolescence or late adolescence to early adulthood , our methodology did not allow us to directly test for the presence of developmental differences ( e.g. , by examining age interaction effects , as we did to test for gender differences ) . therefore , it is possible that the differences in significance that we found by developmental stage are not indicative of true developmental differences . for instance , although obesity first occurring in late adolescence was strongly associated with increased odds of mdd ( or=2.83 ) , obesity occurring by early adolescence showed a weak association with mdd ( or=1.53 ) that just missed significance ( see table 2 ) . as we have noted above , from this we would not conclude that risk is only elevated in late adolescence . however , a somewhat different picture emerged for mdd as a risk factor for obesity , especially in girls . the interaction effect for mdd by early adolescence leading to obesity was significant , indicating that the risk was especially elevated for girls ( or=3.76 ) . for mdd first occurring in late adolescence , the interaction effect was not only not significant , but in the opposite direction , indicating that for girls , mdd was associated with non - significant but slightly reduced risk of obesity . although this pattern of results can not rule out that mdd first occurring in late adolescence is associated with elevated risk for obesity , it nonetheless offers plausible support backing the hypothesis that the key risk period in the mdd to obesity association is in early adolescence for girls in sum , until other research directly examines interaction effects by age , it is important to consider the developmental differences we found to be tentative . it is not clear how these results would generalize to other samples ; however , other research has not found different associations between these disorders among african - american and white adolescents and our sample was representative of the state of minnesota at the time these participants were born . in sum , the results of this study indicate that among women , mdd occurring by early adolescence predicts the later onset of obesity . conversely , adolescent - onset obesity among women ( perhaps particularly obesity with an onset in late adolescence ) predicts the later onset of mdd . in addition , the onsets of these disorders are cross - sectionally associated in childhood and early adolescence , but not later . research investigating possible mechanisms accounting for these differing associations in different developmental periods would be useful . in addition , studies examining prevention and treatment efforts focusing on early - onset cases of mdd and adolescent - onset cases of obesity are warranted , as these may be most likely to reduce risk for the later development of the other disorder .

objectiveobesity and major depressive disorder ( mdd ) are associated , but evidence about how they relate over time is conflicting . the goal of this study was to examine prospective associations between depression and obesity from early adolescence through early adulthood.methodsparticipants were drawn from a statewide , community - based , minnesota sample . mdd and obesity with onsets by early adolescence ( by age 14 ) , late adolescence ( between 14 and 20 ) , and early adulthood ( ages 20 to 24 ) were assessed via structured interview ( depression ) and study - measured height and weight.resultscross-sectional results indicated that depression and obesity with onsets by early adolescence were concurrently associated , but the same was not true later in development . prospective results indicated that depression by early adolescence predicted the onset of obesity ( odds ratio=3.76 , confidence interval= 1.3310.59 ) during late adolescence among females . obesity that developed during late adolescence predicted the onset of depression ( odds ratio=5.89 , confidence interval= 2.3115.01 ) during early adulthood among females.conclusionsfor girls , adolescence is a high risk period for the development of this comorbidity , with the nature of the risk varying over the course of adolescence . early adolescent - onset depression is associated with elevated risk of later onset obesity , and obesity , particularly in late adolescence , is associated with increased odds of later depression . further investigation into the mechanisms of these effects and the reasons for the observed gender and developmental differences is needed . prevention programs focused on early - onset cases of depression and adolescent - onset cases of obesity , particularly among females , may help in reducing risk for this form of comorbidity .

cardiac resynchronisation therapy ( crt ) is most effective in heart failure patients with left bundle branch block as it specifically aims to resynchronise the delayed electrical activation of the left ventricle [ 12 ] . however , crt is indicated in a much broader spectrum of patients with heart failure . a large european survey among 141 hospitals demonstrated that approximately 25 % of crt implantations are upgrade procedures . patients with an existing cardiac implantable electronic device ( cied ) have a class i indication for upgrading to crt when they experience a high percentage of ventricular pacing and remain in new york heart association ( nyha ) class iii to ambulatory iv with a left ventricular ejection fraction ( lvef ) 35 % . other reasons for upgrading to crt include ( i ) worsening of heart failure ( class ) in patients with pre - existing wide qrs complexes , ( ii ) electrical remodelling due to heart failure or antiarrhythmic drug therapy with widening of the qrs complex , and ( iii ) evolving crt guidelines . previous literature has shown that cardiac device replacement or upgrade procedures are associated with more complications and are more complex than de novo implantations due to the possibility of venous thrombosis and the risk of damaging or extracting old leads [ 5 , 6 ] . the fear of a complicated procedure might prevent upgrading to crt in patients with a clear indication . moreover , reluctance about the need for an upgrade procedure in the future might make clinicians more willing to implant a de novo crt device in heart failure patients with only a class iib indication for crt but a clear indication for a conventional ( single or dual chamber ) device . we aimed to evaluate procedural data and periprocedural and long - term complication rates of upgrade procedures compared with de novo crt implantations in our university medical centre . this retrospective study analysed data from all patients who had undergone an upgrade procedure from a single or dual chamber pacemaker or implantable cardioverter defibrillator ( icd ) to a crt device at the university medical centre of utrecht ( umcu ) in the period 20062012 . an equally sized reference group of de novo implantations was randomly constructed out of patients implanted in the same period ( n = 358 ) . procedures were performed by experienced operators , defined as performing at least 75 icd / pacemaker implantations annually with at least one - third of these being crt device implantations . periprocedural complications ( 30 days ) and long - term device - related complications ( 1 year ) were divided into complications with and without consequences . complications with consequences were defined as those in need of invasive procedures ( e.g. lead revision / replacement , extraction of the system , pericardiocentesis ) or any other action deviating from standard clinical care ( prolonged hospitalisation , postponement of implant after start , transfer to the coronary or intensive care unit or the need to start anticoagulants ) . categorical variables are presented as percentages and the p - value was calculated using pearson s chi - square test . if distribution was skewed , variables are presented as median with 25th to 75th percentile interquartile range ( iqr ) and the p - value was calculated by the mann - whitney u test . normally distributed variables are presented as means with a standard deviation and a p - value calculated by the student s t test . the occurrence of specific periprocedural complications was compared between de novo and upgrade procedures using a chi - square test . the hazard ratio for the risk of any periprocedural complication with consequences for the type of procedure ( upgrade versus de novo implantation ) was calculated by a crude logistic regression analysis . to identify any confounding effect of baseline characteristics on the risk of periprocedural complications after an upgrade procedure and de novo crt implantation , multiple bivariate logistic regression analyses were performed , including procedure type and one baseline characteristic . the crude hazard ratio for procedure type and risk of periprocedural complications was adjusted for all baseline characteristic differences in a multivariable logistic regression analysis . the comparison of the occurrence of long - term complications by procedure type was performed by the chi - square test . patients with censored data due to loss to follow - up or death within 1 year of follow - up were excluded from this comparison . this retrospective study analysed data from all patients who had undergone an upgrade procedure from a single or dual chamber pacemaker or implantable cardioverter defibrillator ( icd ) to a crt device at the university medical centre of utrecht ( umcu ) in the period 20062012 . an equally sized reference group of de novo implantations was randomly constructed out of patients implanted in the same period ( n = 358 ) . procedures were performed by experienced operators , defined as performing at least 75 icd / pacemaker implantations annually with at least one - third of these being crt device implantations . periprocedural complications ( 30 days ) and long - term device - related complications ( 1 year ) were divided into complications with and without consequences . complications with consequences were defined as those in need of invasive procedures ( e.g. lead revision / replacement , extraction of the system , pericardiocentesis ) or any other action deviating from standard clinical care ( prolonged hospitalisation , postponement of implant after start , transfer to the coronary or intensive care unit or the need to start anticoagulants ) . categorical variables are presented as percentages and the p - value was calculated using pearson s chi - square test . if distribution was skewed , variables are presented as median with 25th to 75th percentile interquartile range ( iqr ) and the p - value was calculated by the mann - whitney u test . normally distributed variables are presented as means with a standard deviation and a p - value calculated by the student s t test . the occurrence of specific periprocedural complications was compared between de novo and upgrade procedures using a chi - square test . the hazard ratio for the risk of any periprocedural complication with consequences for the type of procedure ( upgrade versus de novo implantation ) was calculated by a crude logistic regression analysis . to identify any confounding effect of baseline characteristics on the risk of periprocedural complications after an upgrade procedure and de novo crt implantation , multiple bivariate logistic regression analyses were performed , including procedure type and one baseline characteristic . the crude hazard ratio for procedure type and risk of periprocedural complications was adjusted for all baseline characteristic differences in a multivariable logistic regression analysis . the comparison of the occurrence of long - term complications by procedure type was performed by the chi - square test . patients with censored data due to loss to follow - up or death within 1 year of follow - up were excluded from this comparison . this analysis included 134 patients who underwent an upgrade procedure to a crt device and 134 randomly selected patients receiving a de novo crt device . of these 268 patients , 264 ( 98.5 % ) received a crt defibrillator ( crt - d ) , three ( 2.2 % ) patients were upgraded to a crt pacemaker ( crt - p ) and one ( 0.7 % ) patient received a de novo crt - p . fifty - four ( 40 % ) patients where upgraded from a double chamber device and 81 ( 60 % ) from a single chamber device . seventy - six ( 57 % ) upgrade patients were paced from the right ventricle at baseline . upgrade patients were more often males ( p = 0.011 ) and of older age ( p = 0.001 ) than the random sample of patients receiving a de novo crt implantation . they more often had ischaemic cardiomyopathy ( p = 0.049 ) and a history of atrial fibrillation ( p = 0.008 ) and were more often on amiodarone ( p < 0.001 ) and a vitamin k antagonist ( p = 0.022 ) . no baseline characteristic had a significant confounding effect on the hazard ratio for procedure type and the risk of periprocedural complications.table 1baseline comparison between patients receiving an upgrade to a crt device and those receiving a de novo crt devicebaseline characteristicstotal population ( n = 268)upgrade(n = 134)de novo implants ( n = 134 ) p - value age ( years ) 69(6174)71(6375)67(6072 ) 0.001 male ( n(%))202( 75)110( 82)92( 69 ) 0.011 bmi26(2429)26(2429)26(2430)0.450 nyha class ( n , % ) 0.080 i2(1)1(1)1(1 ) ii36(13)13(10)23(17 ) iii213(80)110(82)103(77 ) iv17(6)10(8)7(5)ischaemic cmp ( n , % ) 148(55)82(61)66(49 ) 0.049 lvef ( % ) 23 ( 7)24 ( 7)23 ( 7)0.239bnp141(68262)146(73273)130(50226)0.112atrium fibrillation ( n , % ) 60(22)39(29)21(16 ) 0.008 cardiac medication ( n , % ) diuretic231(88)117(88)114(87)0.816beta - blocking201(76)100(76)101(76)0.940ace inhibiting227(86)110(83)117(89)0.158 other medication amiodarone54(21)39(29)15(11 ) < 0.001 use of anticoagulation ( n , % ) vitamin - k antagonist191(72)104(78)87(65 ) 0.022 other57(21)23(17)34(25)0.101inr ( n = 191 ) 1.7(1.32.1)1.7(1.42.1)1.5(1.31.9)0.053 comorbidity diabetes mellitus ( n , % ) 62(23)29(22)33(25)0.540copd ( n , % ) 34(13)17(13)17(13)0.981renal function ( egfr)57(19)55(20)59(19)0.090 subclavian / anonyma vein thrombosis ( n , % ) 11(4)11(8)0(0 ) 0.001 time since initial cied implantation ( months ) 57(31115) standard deviation deviation , range ; 25th to 75th percentile inter quartile range . ace angiotensin i converting enzyme , bmi body mass index , bnp brain natriuretic peptide , cied cardiac implantable electronic device , cmp cardiomyopathy , copd chronic obstructive pulmonary disease , inr international normalised ratio , lvef left ventricular ejection fraction , nyha new york heart association , sd standard deviation . nyha iii and iv versus i , ii baseline comparison between patients receiving an upgrade to a crt device and those receiving a de novo crt device standard deviation deviation , range ; 25th to 75th percentile inter quartile range . ace angiotensin i converting enzyme , bmi body mass index , bnp brain natriuretic peptide , cied cardiac implantable electronic device , cmp cardiomyopathy , copd chronic obstructive pulmonary disease , inr international normalised ratio , lvef left ventricular ejection fraction , nyha new york heart association , sd standard deviation . nyha iii and iv versus i , ii procedural data are presented in table 2 . seventy - three ( 27 % ) procedures were extended by study - related lv pressure ( lv dp / dt max ) measurements to optimise lead positions and device settings . median radiation time was 25 ( 20 ) minutes and median hospitalisation after implantation , including rehospitalisation for another attempt to complete crt implantation after initial lv lead placement failure , was 2 ( 2 ) days . there were no significant differences in these procedural data between upgrade and de novo crt implantation procedures . at baseline , ten ( 8 % ) patients undergoing an upgrade procedure had a subclavian ( or anonyma ) vein thrombosis . in six of these patients the vein was recanalised , in two a contralateral implant was chosen and in two patients the occlusion could be passed . in 124 ( 92 % ) ten ( 8 % ) patients did not receive a right atrial lead because of permanent atrial fibrillation . of the upgrade patients 91 ( 68 % ) received two new leads , three leads were placed in 16 ( 12 % ) patients and 27 ( 20 % ) patients only received an additional lv lead.table 2procedural dataprocedural data ( median , iqr)totalupgradede novo p - valuelength procedure ( min)156(78)145(71)163(81)0.062standard procedure ( n = 194)139(69)135(75)142(61)0.515procedure with lv dp / dt max measurements 187(66)171(72)195(42)0.176radiation time ( min)25(20)25(19)25(21)0.595total hospitalisation time ( days)2(2)1(2)2(2)0.418 leads added or implanted ( intention to ) ( n , % ) < 0.001127(20 % ) 0(0)291(68 % ) 10(8 % ) 316(12 % ) 124(92 % ) n of operating specialists 1.000 n = 1 ( experienced cardiologist)224(85)112(85)112(85 ) n = 2 ( fellow with experienced cardiologist or two experienced cardiologists)40(15)20(15)20(15 ) of which 39.7 % upgrade procedures . starting after implantation including additional hospitalisation for later lv lead placement after initial failure or re - hospitalisation due to implantation or device related complications . of which 39.7 % upgrade procedures . starting after implantation including additional hospitalisation for later lv lead placement after initial failure or re - hospitalisation due to implantation or device related complications . 4 patients had missing data on implanting physician . the periprocedural complication rate is presented in table 3 . in total 21 ( 7.8 % ) patients experienced a complication with consequences . the crude hazard ratio for periprocedural complications ( with consequences ) for upgrade procedures compared with de novo crt implantations was 1.692 ( p = 0.260 ) and when adjusted for baseline differences between the two groups it was 1.779 ( p = 0.249 ) ( table 4 ) . one patient developed acute decreased saturation during an upgrade procedure probably based on cardiac asthma . saturation did not improve with pharmacological treatment or intubation , and resuscitation was required , which the patient did not survive . in seven ( 2.6 % ) patients transvenous lv lead placement was unsuccessful due to difficulty in visualising or cannulating the coronary sinus . phrenic nerve stimulation was seen in 14 ( 5.3 % ) patients and usually managed by adjustments in the pacing configuration , but in one case lead revision was needed shortly after implantation . pocket haematoma was seen in 12 ( 4.5 % ) of the total 268 patients ; of these four upgrade patients needed to be re - hospitalised or needed blood transfusion . table 3periprocedural complicationscomplication ( n , % ) total ( n = 268)upgrade ( n = 134)de novo ( n = 134 ) perforation / dissection entrance vein 2(0.7)1(0.8)1(0.8 ) cs dissection conservative8(3.0)4(3.0)4(3.0)with consequences cardiac tamponade conservative1(0.4)1(0.8)pericardiocentesis1(0.4)1(0.8 ) cardiac arrest during implant 1(0.4)1(0.8 ) haemorrhage during implant 2(0.7)2(1.5)conservative1(0.4)1(0.8)transfer to ccu lv lead placement failure ( transvenous ) 7(2.6)2(1.5)5(3.7 ) pneumothorax conservative2(0.7)2(1.5)drainage3(1.1)1(0.8)2(1.5 ) pocket haematoma conservative8(3.0)4(3.0)4(3.0)re - hospitalisation / transfusion 4(1.5)4(3.0)0(0 ) pns conservative13(4.9)4(3.0)9(6.7 ) lead revision1(0.4)1(0.8 ) thrombosis v. subclavian post implant 1(0.4)1(0.4) death ( implant related ) 1(0.4)1(0.8) total 56(20.9)24(17.9)32(23.9 ) with consequences 21(7.8)9(6.7)12(9.0 ) ccu coronary care unit , cs coronary sinus , lv left ventricular , pns phrenic nerve stimulation . leading to postponement of implant or conversion to another entrance vein . due to temporary av block without an escape rhythm for which a short period of resuscitation followed by temporary pacing by the icd lead after which recovery of conduction periprocedural complications ccu coronary care unit , cs coronary sinus , lv left ventricular , pns phrenic nerve stimulation . due to temporary av block without an escape rhythm for which a short period of resuscitation followed by temporary pacing by the icd lead after which recovery of conduction p < 0.05 for comparison between de novo and upgrade procedures . table 4hazard ratio for upgrade versus de novo crt implantation procedures and the risk of periprocedural complications , adjusted for differences in baseline characteristics between upgrade and de novo patients . hazard ratio for upgrade versus de novo crt implantation procedures and the risk of periprocedural complications , adjusted for differences in baseline characteristics between upgrade and de novo patients . ten ( 4.2 % ) patients had a long - term device - related complication with consequences ; there was no difference between upgrade ( 3.5 % ) and de novo ( 4.9 % ) patients . lead dysfunction in need of lead replacement occurred once in a de novo and an upgrade patient . lead dislodgment in need of lead revision was seen in three de novo patients and in one upgrade patient . device - related endocarditis requiring extraction of the system was seen once in both a de novo and in an upgrade patient.table 5long - term device - related complications during 1 year of follow - upcomplication ( n , % ) total n = 236 upgrade n = 113de novo n = 123 pns conservative11(4.7)6(5.3)5(4.1)lead revision1(0.4)1(0.9) lead dysfunction conservative6(2.5)3(2.7)3(2.4)lead replacement2(0.8)1(0.9)1(0.8 ) lead dislodgement conservative4(1.7)1(0.9)3(2.4)lead revision4(1.7)1(0.9)3(2.4 ) infection conservative2(0.8)-2(1.6)ab / hospitalisation1(0.4)-1(0.8)extraction2(0.8)1(0.9)1(0.8 ) total 33(14.0)14(12.4)19(15.4 ) with consequences 10(4.2)4(3.5)6(4.9)loss to fu514htx33death 27198n before death or htx long - term complication 3 2 1 ab antibiotics , htx heart transplantation , fu follow - up , pns phrenic nerve stimulation . excluded were those patients who were loss to follow up ( n=5 ) or died / received an htx during follow up before any long term complication could occur ( n = 32 ) . one upgrade patient died during procedure. p < 0.05 for comparison between de novo and upgrade procedures . long - term device - related complications during 1 year of follow - up ab antibiotics , htx heart transplantation , fu follow - up , pns phrenic nerve stimulation . excluded were those patients who were loss to follow up ( n=5 ) or died / received an htx during follow up before any long term complication could occur ( n = 32 ) . this analysis included 134 patients who underwent an upgrade procedure to a crt device and 134 randomly selected patients receiving a de novo crt device . of these 268 patients , 264 ( 98.5 % ) received a crt defibrillator ( crt - d ) , three ( 2.2 % ) patients were upgraded to a crt pacemaker ( crt - p ) and one ( 0.7 % ) patient received a de novo crt - p . fifty - four ( 40 % ) patients where upgraded from a double chamber device and 81 ( 60 % ) from a single chamber device . seventy - six ( 57 % ) upgrade patients were paced from the right ventricle at baseline . upgrade patients were more often males ( p = 0.011 ) and of older age ( p = 0.001 ) than the random sample of patients receiving a de novo crt implantation . they more often had ischaemic cardiomyopathy ( p = 0.049 ) and a history of atrial fibrillation ( p = 0.008 ) and were more often on amiodarone ( p < 0.001 ) and a vitamin k antagonist ( p = 0.022 ) . no baseline characteristic had a significant confounding effect on the hazard ratio for procedure type and the risk of periprocedural complications.table 1baseline comparison between patients receiving an upgrade to a crt device and those receiving a de novo crt devicebaseline characteristicstotal population ( n = 268)upgrade(n = 134)de novo implants ( n = 134 ) p - value age ( years ) 69(6174)71(6375)67(6072 ) 0.001 male ( n(%))202( 75)110( 82)92( 69 ) 0.011 bmi26(2429)26(2429)26(2430)0.450 nyha class ( n , % ) 0.080 i2(1)1(1)1(1 ) ii36(13)13(10)23(17 ) iii213(80)110(82)103(77 ) iv17(6)10(8)7(5)ischaemic cmp ( n , % ) 148(55)82(61)66(49 ) 0.049 lvef ( % ) 23 ( 7)24 ( 7)23 ( 7)0.239bnp141(68262)146(73273)130(50226)0.112atrium fibrillation ( n , % ) 60(22)39(29)21(16 ) 0.008 cardiac medication ( n , % ) diuretic231(88)117(88)114(87)0.816beta - blocking201(76)100(76)101(76)0.940ace inhibiting227(86)110(83)117(89)0.158 other medication amiodarone54(21)39(29)15(11 ) < 0.001 use of anticoagulation ( n , % ) vitamin - k antagonist191(72)104(78)87(65 ) 0.022 other57(21)23(17)34(25)0.101inr ( n = 191 ) 1.7(1.32.1)1.7(1.42.1)1.5(1.31.9)0.053 comorbidity diabetes mellitus ( n , % ) 62(23)29(22)33(25)0.540copd ( n , % ) 34(13)17(13)17(13)0.981renal function ( egfr)57(19)55(20)59(19)0.090 subclavian / anonyma vein thrombosis ( n , % ) 11(4)11(8)0(0 ) 0.001 time since initial cied implantation ( months ) 57(31115) standard deviation deviation , range ; 25th to 75th percentile inter quartile range . ace angiotensin i converting enzyme , bmi body mass index , bnp brain natriuretic peptide , cied cardiac implantable electronic device , cmp cardiomyopathy , copd chronic obstructive pulmonary disease , inr international normalised ratio , lvef left ventricular ejection fraction , nyha new york heart association , sd standard deviation . nyha iii and iv versus i , ii baseline comparison between patients receiving an upgrade to a crt device and those receiving a de novo crt device standard deviation deviation , range ; 25th to 75th percentile inter quartile range . ace angiotensin i converting enzyme , bmi body mass index , bnp brain natriuretic peptide , cied cardiac implantable electronic device , cmp cardiomyopathy , copd chronic obstructive pulmonary disease , inr international normalised ratio , lvef left ventricular ejection fraction , nyha new york heart association , sd standard deviation . seventy - three ( 27 % ) procedures were extended by study - related lv pressure ( lv dp / dt max ) measurements to optimise lead positions and device settings . median radiation time was 25 ( 20 ) minutes and median hospitalisation after implantation , including rehospitalisation for another attempt to complete crt implantation after initial lv lead placement failure , was 2 ( 2 ) days . there were no significant differences in these procedural data between upgrade and de novo crt implantation procedures . at baseline , ten ( 8 % ) patients undergoing an upgrade procedure had a subclavian ( or anonyma ) vein thrombosis . in six of these patients the vein was recanalised , in two a contralateral implant was chosen and in two patients the occlusion could be passed . in 124 ( 92 % ) ten ( 8 % ) patients did not receive a right atrial lead because of permanent atrial fibrillation . of the upgrade patients 91 ( 68 % ) received two new leads , three leads were placed in 16 ( 12 % ) patients and 27 ( 20 % ) patients only received an additional lv lead.table 2procedural dataprocedural data ( median , iqr)totalupgradede novo p - valuelength procedure ( min)156(78)145(71)163(81)0.062standard procedure ( n = 194)139(69)135(75)142(61)0.515procedure with lv dp / dt max measurements 187(66)171(72)195(42)0.176radiation time ( min)25(20)25(19)25(21)0.595total hospitalisation time ( days)2(2)1(2)2(2)0.418 leads added or implanted ( intention to ) ( n , % ) < 0.001127(20 % ) 0(0)291(68 % ) 10(8 % ) 316(12 % ) 124(92 % ) n of operating specialists 1.000 n = 1 ( experienced cardiologist)224(85)112(85)112(85 ) n = 2 ( fellow with experienced cardiologist or two experienced cardiologists)40(15)20(15)20(15 ) of which 39.7 % upgrade procedures . starting after implantation including additional hospitalisation for later lv lead placement after initial failure or re - hospitalisation due to implantation or device related complications . of which 39.7 % upgrade procedures . starting after implantation including additional hospitalisation for later lv lead placement after initial failure or re - hospitalisation due to implantation or device related complications . the periprocedural complication rate is presented in table 3 . in total 21 ( 7.8 % ) patients experienced a complication with consequences . the crude hazard ratio for periprocedural complications ( with consequences ) for upgrade procedures compared with de novo crt implantations was 1.692 ( p = 0.260 ) and when adjusted for baseline differences between the two groups it was 1.779 ( p = 0.249 ) ( table 4 ) . one patient developed acute decreased saturation during an upgrade procedure probably based on cardiac asthma . saturation did not improve with pharmacological treatment or intubation , and resuscitation was required , which the patient did not survive . in seven ( 2.6 % ) patients transvenous lv lead placement was unsuccessful due to difficulty in visualising or cannulating the coronary sinus . phrenic nerve stimulation was seen in 14 ( 5.3 % ) patients and usually managed by adjustments in the pacing configuration , but in one case lead revision was needed shortly after implantation . coronary sinus dissection ( 3 % ) pocket haematoma was seen in 12 ( 4.5 % ) of the total 268 patients ; of these four upgrade patients needed to be re - hospitalised or needed blood transfusion . table 3periprocedural complicationscomplication ( n , % ) total ( n = 268)upgrade ( n = 134)de novo ( n = 134 ) perforation / dissection entrance vein 2(0.7)1(0.8)1(0.8 ) cs dissection conservative8(3.0)4(3.0)4(3.0)with consequences cardiac tamponade conservative1(0.4)1(0.8)pericardiocentesis1(0.4)1(0.8 ) cardiac arrest during implant 1(0.4)1(0.8 ) haemorrhage during implant 2(0.7)2(1.5)conservative1(0.4)1(0.8)transfer to ccu lv lead placement failure ( transvenous ) 7(2.6)2(1.5)5(3.7 ) pneumothorax conservative2(0.7)2(1.5)drainage3(1.1)1(0.8)2(1.5 ) pocket haematoma conservative8(3.0)4(3.0)4(3.0)re - hospitalisation / transfusion 4(1.5)4(3.0)0(0 ) pns conservative13(4.9)4(3.0)9(6.7 ) lead revision1(0.4)1(0.8 ) thrombosis v. subclavian post implant 1(0.4)1(0.4) death ( implant related ) 1(0.4)1(0.8) total 56(20.9)24(17.9)32(23.9 ) with consequences 21(7.8)9(6.7)12(9.0 ) ccu coronary care unit , cs coronary sinus , lv left ventricular , pns phrenic nerve stimulation . leading to postponement of implant or conversion to another entrance vein . due to temporary av block without an escape rhythm for which a short period of resuscitation followed by temporary pacing by the icd lead after which recovery of conduction . periprocedural complications ccu coronary care unit , cs coronary sinus , lv left ventricular , pns phrenic nerve stimulation . leading to postponement of implant or conversion to another entrance vein . due to temporary av block without an escape rhythm for which a short period of resuscitation followed by temporary pacing by the icd lead after which recovery of conduction . . table 4hazard ratio for upgrade versus de novo crt implantation procedures and the risk of periprocedural complications , adjusted for differences in baseline characteristics between upgrade and de novo patients . hazard ratio for upgrade versus de novo crt implantation procedures and the risk of periprocedural complications , adjusted for differences in baseline characteristics between upgrade and de novo patients . ten ( 4.2 % ) patients had a long - term device - related complication with consequences ; there was no difference between upgrade ( 3.5 % ) and de novo ( 4.9 % ) patients . lead dysfunction in need of lead replacement occurred once in a de novo and an upgrade patient . lead dislodgment in need of lead revision was seen in three de novo patients and in one upgrade patient . device - related endocarditis requiring extraction of the system was seen once in both a de novo and in an upgrade patient.table 5long - term device - related complications during 1 year of follow - upcomplication ( n , % ) total n = 236 upgrade n = 113de novo n = 123 pns conservative11(4.7)6(5.3)5(4.1)lead revision1(0.4)1(0.9) lead dysfunction conservative6(2.5)3(2.7)3(2.4)lead replacement2(0.8)1(0.9)1(0.8 ) lead dislodgement conservative4(1.7)1(0.9)3(2.4)lead revision4(1.7)1(0.9)3(2.4 ) infection conservative2(0.8)-2(1.6)ab / hospitalisation1(0.4)-1(0.8)extraction2(0.8)1(0.9)1(0.8 ) total 33(14.0)14(12.4)19(15.4 ) with consequences 10(4.2)4(3.5)6(4.9)loss to fu514htx33death 27198n before death or htx long - term complication 3 2 1 ab antibiotics , htx heart transplantation , fu follow - up , pns phrenic nerve stimulation . excluded were those patients who were loss to follow up ( n=5 ) or died / received an htx during follow up before any long term complication could occur ( n = 32 ) . one upgrade patient died during procedure. p < 0.05 for comparison between de novo and upgrade procedures . long - term device - related complications during 1 year of follow - up ab antibiotics , htx heart transplantation , fu follow - up , pns phrenic nerve stimulation . excluded were those patients who were loss to follow up ( n=5 ) or died / received an htx during follow up before any long term complication could occur ( n = 32 ) . our single - centre experience did not find any significant differences in either procedure length , radiation duration , total hospitalisation , or in periprocedural and long - term device - related complication rates when comparing upgrade procedures with de novo crt implantations . previously , the raft study showed that crt - d reduced rates of death and hospitalisation for heart failure when compared with icd therapy only . however , device - related hospitalisation was significantly higher for the crt - d group compared with the icd - only group . the increased risk of complications of a triple chamber device compared with conventional single and dual chamber pacemakers or icds was also highlighted by a large nationwide cohort of 5918 danish patients who underwent a cied implantation , in which patients receiving a crt device showed the highest complication rate . therefore , the decision to implant a triple chamber device needs to be carefully considered . for patients with a class iib indication for crt but a clear indication for a conventional icd the alternative to a de novo crt implantation is to await the potential need for an upgrade to crt . however , as was shown by poole et al . , crt upgrade is a high - risk procedure as well , with an 18.7 % risk of any complication during a follow - up of six months . the fear of complications associated with an upgrade procedure might prevent the upgrade to crt in patients with a clear indication . this was suggested by the results of scott et al . who performed a single - centre retrospective observational study evaluating the implantation of icds for a period of 5 years . in this period 549 new icds were implanted of which 73 % single or dual chamber icds and 27 % crt - d . when they applied alternative indication criteria ( lvef 30 % , qrs 130 ms , nyha i iv ) , 42.6 % of the icd recipients met the criteria for crt at initial implant . however , the upgrade rate to crt at 5 years was only 5.1 % . this is in line with the results of the european survey which showed that there were no significant differences in complication rates when comparing 692 upgrades with 1675 de novo crt procedures at 141 centres in europe . however , this was a randomised trial and such trials commonly report less complications than in a real - life setting due to more strict patient selection and more experienced operators . on the contrary , the previously mentioned danish population - based cohort of cied implantations , including 445 crt - d implantations , showed that crt upgrade procedures or lead revisions were associated with more complications than crt de novo implantations . as both our study and the danish cohort study reflect the results of a real - life setting the differences are not likely to be based on patient selection . in our retrospective study , patient selection was equal for both de novo and upgrade procedures in that it followed standard clinical care in which the benefit of crt is balanced against the risk of complications . it might be that the operators in our tertiary centre were more experienced in implanting crt devices . although most procedures in the danish cohort were also performed by operators with an annual volume of at least 75 icd or pacemaker implantations , it is not clear whether the experience in implanting a crt device was as high as in our centre . another point of concern for many clinicians is the fear of a more complex procedure when a single or dual chamber pacemaker or icd needs to be upgraded , compared with a de novo implantation of a crt device , due to the possibility of damaging or extracting old leads . four upgrade patients experienced lead dysfunction after the procedure , which is comparable with numbers in previous literature [ 5 , 10 ] . these were three old leads ( two right atrial leads and one right ventricular lead ) and one newly placed right atrial lead . moreover , this was done due to damage to the old lead during the procedure in only one of 16 upgrade patients who received a completely new system . therefore , our data seem to indicate that when an upgrade procedure is performed by an experienced operator the risk of damaging old leads is small . furthermore , we found no significant difference in procedure duration comparing upgrade procedures with de novo implantations , but there was a slight trend towards an even shorter procedure length for upgrade procedures . this suggests comparable complexity as one would expect the upgrade procedure to require a smaller amount of time , as in most upgrades less leads are added than implanted during a de novo implantation . complicating factors such as subclavian vein thrombosis only slightly increased the median procedure duration compared with standard procedures ( 159 ( 95 ) minutes versus 139 ( 69 ) minutes ) . moreover , in our upgrade group the procedure success was 98.5 % and did not significantly differ for the randomly selected de novo crt implantation procedures . a meta - analysis of 9082 patients in 25 crt trials showed an implantation success rate of 94.4 % indicating that the success rate of upgrade procedures was most definitely not lower compared with de novo procedures . decisions on device type ( conventional or triple chamber pacemaker or icd ) can be difficult in patients with an indication for a conventional pacemaker or icd without a clear class i or iia indication for crt . based on fulfilment of indication criteria for crt the chance of benefit from crt in these patients is low and the complication rate increases with the number of leads implanted . however , when a conventional pacemaker or icd is opted for the need for upgrading can eventually arise . this means the patient is exposed to two procedures , where it is generally believed that an upgrade procedure is more complex and associated with a higher complication rate . this single - centre experience of a dutch university hospital shows neither a higher complexity nor a higher complication rate of upgrade procedures to crt compared with de novo crt implantations . therefore , crt upgrade procedures should be performed when indicated without fear for a higher complication rate . however , as upgrading means an additional procedure is needed , future research should focus on identifying patient characteristics that predict the necessity for upgrading to crt , especially for those patients in need of upgrading within the life expectancy of the initial device . due to the retrospective design of the study only complications properly documented in the electronic patient file were identified and not all baseline data were available for all patients . however , the advantage of the retrospective design is that it reflects a real - life setting of these device implantations . furthermore , although none of the baseline characteristics showed a confounding effect on the risk of periprocedural complications , it is possible that some data that were not collected confounded the results , for instance steroid use . finally , as this study shows the results of a single - centre experience , the results can only be applicable for a comparable centre . in a device implantation centre with experienced operators , crt upgrade procedures are not associated with more complications nor do they seem more complex compared with de novo crt implantations .

objectivethe objective of the study was to examine whether cardiac resynchronisation therapy upgrade procedures are more complex and associated with more complications than de novo implantations.methodwe retrospectively compared 134 upgrade procedures performed between 20062012 with a random , equally sized , sample of de novo crt device implantations in the same period . procedural data and the occurrence of periprocedural ( 30 days ) and long - term device - related ( 1 year ) complications were analysed . complications with consequences were defined as those in need of adjustment of standard care.resultsmedian time to upgrade was 57 ( 31115 ) months . there were no significant differences in procedure duration , radiation time or total hospitalisation between upgrades and de novo implantations . perioperative complications occurred in 6.7 % of upgrade patients and in 9.0 % of de novo patients . the most frequently seen complications were phrenic nerve stimulation , coronary sinus dissection and pocket haematoma . procedure success was comparable ( upgrade : 98.5 % versus de novo : 96.3 % ) . a total of 236 patients completed 1 year of follow - up . ten ( 4.2 % ) patients had a long - term device - related complication with consequences including phrenic nerve stimulation , lead dislodgement / dysfunction , and infection ( upgrade : 3.5 % versus de novo : 4.9 % ) .conclusionupgrade procedures are not more complex nor associated with more complications than de novo crt implantations .

pursuing the goal of improved health literacy requires more alliances between health and education sectors to improve literacy levels in the population . it is important for health educators to know about knowledge transfer to meet their needs in transferring their knowledge to public . knowledge transfer , which means the synthesis , exchange and ethically application of knowledge within a complex system of relationship among researchers and users , has become one of the recent priorities in research centers . the word the method of knowledge transfer , apart from its characteristics , requires active interactions between researchers and users . caplan proposes the two- communities theory indicating a gap between researchers and policymakers , whereas these days another gap has been formed between researchers and other users . in addition to illiteracy in developing countries that hampers effective health education , there are multitudes of non - medical specialists who do not have enough information about daily health affairs , and this lack is more remarkable in countries that welcome immigrants . immigrants often have significant language and health literacy difficulties , which are further exacerbated by cultural barriers . alongside with sophisticated methods like computer and internet , using simple methods help us bring to achieve our health goals[68 ] . also , the lack of health literacy needs more attention when it is manifested in caregivers . it is believed that some of the undesirable health outcomes in children are because of inadequate health knowledge among caregivers . in the usa , therefore researchers have considered different methods to come up with this deficiency by having children to cooperate in their health programs . a research group in india endeavored to transfer knowledge on leprosy in cooperation with children and informed their parents through them . jacob et al ( 1994 ) started conducting a similar research that yields promising results . rimal and flora ( 1998 ) express that parental dietary behavior is partially affected by children . the findings from studies in this field have encouraged other experts to apply similar methods to conduct their research projects , allahverdipour and bashirian , onyango - ouma and mwangagain from the influence of children in order to teach different parts of their society[1316 ] . despite the researcher 's trends in using novel methods in this field , none of them have used childish poem as a medium for knowledge transferring . in this study , we try to raise families health knowledge about infectious diseases by a method that uses children as health agents . infectious diseases are still the leading cause of mortality in children less than 60 months in developing countries . juvenile age is accompanied by learning childish poems , which can be sung contnuously at home and potentially become as part of both children 's and parents memories . we decided to transfer health knowledge to families through altering this information to childish poems and teaching children at kindergartens . in this method the outcome of transferring health knowledge through childish poems has been assessed in this study . in this study , we use the method of interventional pre and post series to conduct our research . the proposal of this non - invasive project was approved by the research committee of hamadan medical university in january 2009 . it was also accepted by hamadan welfare organization , which is the responsible organization of kindergartens in hamadan . our methodology is briefly explained as follows . at first , a sub specialist in pediatric infectious diseases provides seven short texts about health and common pediatric infectious diseases prevention . then , a poet transfers these conceptions to childish poems for the first time ( appendix 1&2 ) . in the process of preparing an accurate questionnaire , at first 30 questions with three choices of , false and do not know were made , but 24 items of those are confirmed as valid questions by two experts of health education and infectious diseases . to assess the reliability of the questionnaire a pilot study on a 40 participant sample was performed and cronbach 's alpha of the questionnaire was determined as 83 percent . in addition , we defined knowledge mark ( km ) as a parent mark in both the pre - test and/or post - tests . among 35 kindergartens in hamadan , we selected 7 kindergartens by simple random method . the parents ( either father or mother , the one who is responsible to take her / his child ) of all five to six year old children of the selected kindergartens are asked to participate in the survey and a verbal consent is obtained from those who accepted . we excluded the following children from our sample : those whose parents are physicians , nurses , health - care workers , and those children whose parents work at the kindergarten ( exclusion criteria ) . we asked the parents of the sample survey to participate in a pretest by answering to a questionnaire when they are in the kindergartens . also , they were asked to come to the kindergartens personally one more time when it is required ( to answer to the post test ) . then , we asked our kindergarten tutors to teach children seven musical poems about hydatid cyst , antibiotic misuse , botulinum toxin in home - canned foods , dysentery , the importance of sixth tooth , brucellosis and tetanus . in this step , children were not allowed to take home these texts , but they were asked to sing the poems at home and want their parents to re - write the poems on a paper and give them the papers to take to the kindergarten . the teaching stage took between three to four months . during the next stage , parents took a post - test survey with the same 24 questions . parents of 115 kids participated in the pre - test and 103 of them completed the post - test . statistical analysis is done on 103 who completed both tests by paired t - test . the sample data consists of 103 parents who provided solutions to both the pre- and post - test question survey . of the participants 77 were female and 26 male . from the level of education point of view , 19 were below high school diploma , 45 had high school diploma , 8 had associate degree , 27 had ba , 3 had ma , and 1 had phd . the results of the survey show a significant difference between the correct answers in both pre - test and post - test stages ( fig . 1 ) . the frequency of correct answers to the tests table 1 shows the mean of the correct answers in the pre - test stage is 59.22 while the same figure for the post - test step is 81 ( p < 0.001 ) . in addition , we define knowledge mark ( km ) as a parent mark in both the pre - test and/or post - test stages . the mean km shows a significant increase ( 5.01 ) from 13.62 in the pre - test to 18.63 in the post - test ( table 2 ) . mean percent of correct answers to pre - test and post - test categories analyzed by paired t - test sd : standard deviation mean of knowledge mark in pre - test and post test analysed by paired t - test the difference of km promotion among females and males was not significant ( 21.6120.17 in males and 20.331.61 in females , p=0.8 ) . the km was increased to 19.9325.06 among parents who at most had high school diploma and 23.4720.84 among parents with upper educational levels ( p=0.5 ) . health has been the concern of poets such as fiona sampson for years , however , these poems never had educational purposes . the results of this study confirm our hypothesis about the role of children in raising family 's knowledge on health . the results show that neither gender nor the grade of knowledge degree ( below and upper high school diploma or below and upper ba ) affect answers . therefore , people with different educational status have received the information similarly implying that this method can be extended to general cases in a straightforward manner . 1 shows that the percentage of correct answers has increased in 23 out of 24 questions . the percent - age of correct answer to post - test has decreased only in one item ( item 11 ) which compares the risk of brucellosis transmission via unpasteurized milk and yogurt . although the term yogurt has not been mentioned directly in the related poem , but there is a hint about boiling mechanism and we expect that the audience discover the answer by logical thinking about the necessitation of boiling milk for preparing yogurt . the decrease in answering is 2 percent which is not significant ; however , it shows that it would be better to mention the conceptions directly in these types of poems in order to avoid any misconceptions . moreover , the percentage of correct answers to the questions of selected topics in pre - test and post - test has been compared ( table 1 ) and shows that parents have done better in post - test about all topics except one . the difference of correct answers to the questions about brucellosis does not show a significant increase in post - test . this finding is probably due to higher basic knowledge about brucellosis among parents . in other words , parents have had acceptable information about brucellosis even without our educational program , so we observe lower contrast between pre - test and post - test about brucellosis . it should be taken into consideration that brucellosis is an endemic disease in hamadan and people have been educated in different ways about this disease in recent years . although the lowest contrast between pre - test and post - test correct answers belongs to a question about botulism , the overall assessment shows that parent 's knowledge about this topic has been promoted significantly ( p<0.001 , table 1 ) . the significant difference between the pre - test and post - test km , based on the results of table 2 , is the sign of successful knowledge transfer through childish poems . the researcher 's method is impressively successful ; however , the fact that he mentions that some mothers can not distinguish the semantic difference between information and advice , needs serious attention . in addition , since daughters may misunderstand the information , it would be probable to transfer the misconceived information to their mothers and this fact threats the successfulness of study in applying . while , in our study ; firstly , childish poems do not imply sententious , secondly , if the poem is sung wrong , the disturbance in rhythm will be appeared , so the information which is transferred through poems is not at risk of alteration . in evans research , parents get familiar with educated topics with the help of their children in homework assignments designed for asthma management , and are taught about asthma indirectly . the advantage of our study in comparison with evans is that in evans method knowledge transfer brings to bear via homework assignment , while in our study there is no obligation for doing home works , and parents are educated while enjoying their child 's singing . on account of the fact that making communication with younger children is not as difficult as with adolescents for parents , we are sure that knowledge transfers within a family through a continuous and friendly communication will work . christensen expressed the importance of children 's role in promoting the family health status . in his article , he emphasized on the activities that children can perform to enhance their health and promote their family health situation via the health efforts for themselves . we showed that children , additionally to what christensen mentioned , are able to affect their family 's health status directly . they can improve their family 's health as little teachers in health . it should be taken into consideration that the limitations of this study was the impact of other media on participant 's knowledge , which was not preventable . one of the acceptable results of this survey was publishing the poems as a book for children which was republished and welcomed by public . this study 's results suggest that health knowledge transfer to families through childish poems is an applicable method that has many advantages . by applying this joyful , cost - effective and easy - to - use method however , it should be taken into consideration that this method is applicable for families with trainable children , for teaching in larger scales more general ways should be added to this method . moreover , the simplicity of poems and expressing the topics in a less elaborative form are the key factors that affect the successfulness of this way .

objectivethe purpose of this study is to propose an innovative method of knowledge transfer that aims to improve health literacy about pediatric infectious diseases prevention in families . children have an appreciable role in this scheme.methodsthis study is a before and after trial that has been conducted in hamedan in 2009 . after changing seven infectious disease topics into childish poems , we selected five kindergartens randomly and taught these poetries to the children . teaching process held after a pretest containing 24 questions that examined 103 of parents about mentioned topics . the same post - test was given after 4 months of teaching process.findingsthe mean of correct answers to the pretest was 59.22% comparable with 81.00% for post - test ( p<0.00 ) . gender and knowledge degree could not change the results significantly . assuming one 's correct answers to the questions as his / her knowledge mark , the mean of this variable increased to 5.32 by this method.conclusionthis cost - effective and joyful method had successful results in promoting health knowledge . children are able to play an active role in family 's health situation . learning within family atmosphere without any obligations makes our scheme a solution for paving the knowledge transferring way .

the principal risk factors are tobacco smoking and alcohol consumption , and the association of both factors is highly synergistic . genetic factors , dietary habits , human papillomavirus ( hpv ) infection , consumption of hot mate ( traditional south american infused drink ) , and poor oral health are also risk factors [ 35 ] . as lifestyles and human behaviour are directly affected by these factors , social inequalities are related to increases in risk . the most recent oropharyngeal cancer incidence data of spain reveals that the country is situated amongst those that present elevated incidence in men , with rates of 3.6 cases per 100,000 inhabitants / year ( ci 95% = 3.43.8 ) , along with france , slovenia , switzerland , and germany . for women , spain is amongst the countries with lowest incidences , with a rate of 0.2 cases per 100,000 inhabitants / year ( ci 95% = 0.2 - 0.3 ) . oropharyngeal cancer is rarely described separately from others head and neck tumours . the anatomical proximity between the oral cavity and the oropharynx , along with the fact that the risk factors are the same , besides , some publications analyse these locations in conjunction with other types of cancers that present different behavior and risk factors , such as nasopharynx and hypopharynx cancers , and denominate the entire group as oral cavity and pharynx cancer . other publications separate between oral cavity and oropharynx but include base of tongue cancers in the oral cavity classification , when the base of tongue should be analysed as a sublocation of the oropharynx . all these problems in the classification of oral cavity and oropharynx cancers hinder a more detailed analysis of the changes in epidemiological profiles , of the effect of treatment , and consequently , of the survival rates of both types of cancers . recently , studies have proved that some risk factors are more related to specific locations , such as hpv infection and oropharynx locations , and tobacco and alcohol with oral cavity . the objective of this study is to determine oropharyngeal cancer survival from the population - based cancer registry ( pbcr ) of zaragoza , spain . cancer survival analysis from population - based data constitutes a valuable tool for the evaluation of welfare services offered and allows for the orientation towards diagnose and treatment strategies . the population studied was diagnosed with oropharyngeal cancer in the province of zaragoza , spain , between january 1 , 1978 and december 31 , 2002 . data was actively collected , analysed , and monitored by the pbcr of zaragoza until december 31 , 2007 . the following locations were included : base of tongue , lingual tonsils , soft palate , uvula , tonsils , and oropharynx ( icd codes : c01.9 , c02.4 , c05.1 , c05.2 , c09.0 , c09.1 , c09.8 , c09.9 , c10.0c10.4 , c10.8 y c10.9 ) . the difficulty in establishing survival rates at a population level lies firstly in obtaining reliable data on cancer incidence for the population , and secondly , in carrying out the monitoring with accuracy and integrity . monitoring of cases was carried out by the cancer registry itself through the mortality registry of the aragon government , which is administered by the statistical institute of aragon . all new cases registered as major salivary gland cancer were included in the analysis , except for the cases diagnosed through death certificates . the pbcr data of zaragoza was the first spanish data to be published in cancer incidence in five continents , appearing in this publication since volume iii . a set of quality indicators evaluates the data prior to publication in ci5 ; the registry data of zaragoza fulfils the established quality standards and presents excellent quality [ 13 , 14 ] . calculation of the survival rate was carried out by the kaplan - meier method , and relative survival was calculated through the webpage of the catalan institute of oncology ( cio ) . the relative survival rate is defined as the relationship between the observed survival and the expected survival in a group of healthy people of similar age and gender . in practice , the survival of people without cancer is difficult to predict , and for this reason the general mortality rate of the population is used . the cio webpage uses the hakulinen method to calculate the relative survival of the database sent by the user and is based on the mortality tables of the mortality registries of the autonomous communities and provinces of spain [ 15 , 16 ] . when the number of risk patients was lower than 15 , these results were not considered in the analysis , as the final estimations were unstable . survival was studied by gender , age groups ( 4064 and over 65 years of age ) and location ( base of tongue , tonsils , soft palate , and oropharynx ) . in order to study the dynamics of survival , the data was stratified in three study periods ( 19781986 , 19871994 , and 19952002 ) , and the survival indicators were compared . the effect of each prognostic factor ( sex , age group , location , and time period ) on the survival rates was evaluated by the log - rank test . the log - rank test is a statistical hypothesis contrast test , used to compare two or more survival curves , and the null hypothesis is that the survival of the groups under comparison is the same . the distribution of cases , number of deaths , and percentage of censored cases according to sex , age , and period are presented in table 1 . the number of incidence cases included in the study was 380 , after exclusion of the cases registered through death certificates . it was observed that 87.6% of cases occurred in men ; the censored percentage was 26.6% ( 24.3% men and 42.6% women ) . the observed survival after one year of diagnosis of oropharyngeal cancer was 61.3% ( ci 95% : 56.466.2 ) . one - year relative survival was 61.9% ( ci 95% : 56.867.5 ) in men and 64.3% ( ci 95% : 51.970.8 ) in women ; five - year relative survival was 34.2% ( ci 95% : 29.240.1 ) in men and 53.3% ( ci 95% : 40.370.5 ) in women ( table 2 ) . comparison of survival rates by sex revealed statistically significant differences ( p value = 0.017 ) with better survival in women . there were no differences when comparing the three age groups ( p value = 0.61 ) , the locations ( p value = 0.25 ) , and the three studied time periods ( p value = 0.17 ) . the continuous increase in the number of cancer survivors along with population ageing in spain , with a consequent increase in the number of cases , translate into new challenges that have to be overcome by the health care system . nowadays in spain , more than 50% of cancer patients are alive after five years of diagnosis and the trend is to increase this percentage . as a consequence of improvements in diagnose tests and treatments , an increase in cancer survivors is expected , and this situation creates new demands in welfare services , which must consider such complexity for patient monitoring . it is estimated that in 2015 in spain , 136,002 new cancer cases are diagnosed in men and 85,818 in women . actually , knowledge on the health situation of long - term survivors is still limited . it is known that the adoption of healthy lifestyles , selfcare attitude , and sociocultural and psychological aspects play an important role in the duration and life quality of patients [ 20 , 21 ] . the eurocare studies are the most important survival data used for comparison purposes against this study . eurocare is a multicentric project that gathers population - based cancer registries for european countries . survival data from this study shows that the highest survival rates are in western europe , independently from the period studied . for the 19901994 period , average survival for europe was 28.7% ( ci 95% = 26.031.5 ) in men and 43.5% in women the highest survival rates occurred in sweden ( 46.6% , ci 95% = 40.753.4 in men and 56.2% , ci 95% = 47.566.4 in women ) , and the lowest rates were obtained in the czech republic , estonia , and slovakia . spanish data is within the european average , with a 29% five - year survival rate for men ( ci 95% = 24.035.0 ) and 44.1% ( ci spanish data originates from grenada , majorca , murcia , navarre , and tarragona . in zaragoza , the data encountered for the 19871994 period corroborates these findings , with a survival percentage of 30.4% ( ci , the average for all european countries reveals that 37.6% of men and 49.6% of women survived after five years of diagnosis . sweden and denmark presented the highest survival rates , with five - year survival rates of 45.3% ( ci 95% = 26.042.0 ) , and the lowest survival rates were found in portugal and northern ireland . the economic situation of countries and the amount of resources destined to welfare assistance are the main reasons for such a variation in survival rates and explain the lowest survival rates found in eastern europe . nevertheless , low survival rates in countries such as northern ireland or other regions of the united kingdom could also be associated with other aspects , such as the structure of the health care system , comorbidity , and risk factor patterns [ 24 , 25 ] . regarding gender , the results for zaragoza indicate that women present higher survival rates and most part of the studies found results in the same direction . the reasons for these higher survival rates in women could be associated with a biological superiority of women in response to illness and treatment , or a higher awareness in women concerning their bodies , and consequently , a higher percentage of early - state diagnosis [ 24 , 26 ] . higher survival rates in women were also found by another study , which investigated 89 population - based cancer registries in several european countries for the head and neck regions . oropharyngeal cancer results demonstrate highest relative survival rates in women ( 47.67% ) than in men ( 37.67% ) , in younger ages and in northern europe . the fact that along with an increase in age there is a significant decrease in survival has been published in all eurocare studies . the reasons are comorbidity and the therapeutics used in elder patients , where many times surgical treatments are not indicated [ 22 , 23 ] . the results for the population of zaragoza did not show significant differences when comparing survival rates in the age groups of 4064 and over 65 years of age . the low number of diagnosed cases in patients with 39 years of age or less prevented the survival rate analysis for this group . regarding location , it was observed that in zaragoza the highest survival rate was found for cancers located in the tonsils and the lowest , for those located in the tongue . a recent study carried out with data from the surveillance , epidemiology end results ( seer ) , a program of the united states national cancer institute , compared cancers located in the oropharynx with those in the oral cavity . the results revealed a higher survival rate for cancers located in the oral portion of the tongue than for cancers located in the tonsils and base of tongue . this division was adopted to differentiate those locations that supposedly present a higher risk for the development of cancers associated with hpv , such as oropharyngeal cancer , from those situated in the oral cavity . when comparing the three studied period , no statistically significant differences were found in the survival rates for oropharyngeal cancer in zaragoza . spanish results published in international studies do not demonstrate significant improvement in survival rates [ 22 , 23 ] . an investigation using canadian cancer registry data has studies changes in survival rates for oropharyngeal and head and neck cancers for patients diagnosed between 1992 and 2001 , totaling 10,860 cases in men and 4002 cases in women . the results revealed significant improvements only in men , with an increase of 13.5% in the survival rates after five years . the data presented herein suggest that zaragoza presents survival rates similar to other spanish registries already published . nevertheless , no statistically significant changes have been identified when dividing and comparing the total study period of 25 years in three study periods . these results must be interpreted with caution because it is difficult to followup cases in such a long span study . although there are limitations , the authors consider that that survival studies using data from population - based cancer registries must be carried out and published , as they allow for the evaluation of the results obtained when treating the illness in the studies population . investigations such as the one presented herein can be the first step for the development of more effective treatment , prevention and control programs for cancer , improvement in the follow - up process of patients and future research . advances in the treatment of cancer and the increase in the number of survivors call for a progressively wider monitoring of this part of population studies on cancer survival need to increase follow - up time and broaden perspectives , with the objective of knowing the physical , psychological , and social aspects associated with this illness [ 18 , 30 ] .

objective . this paper aims at studying oropharyngeal cancer survival from the population - based cancer registry of zaragoza , spain , for the 19782002 period . methods . the survival rates were calculated by the kaplan - meier method , and the automated calculation method of the catalan institute of oncology was utilized to obtain the relative survival . results . the oropharyngeal cancer survival rate was 61.3% in the first year and 33.9% in the fifth year . one - year relative survival was 62.2% ( ci 95% : 57.467.4 ) , and five - year relative survival was 36.6% ( ci 95% : 31.842.1 ) . comparison of survival rates by sex revealed statistically significant differences ( p value = 0.017 ) with better survival in women . there were no differences when comparing the three age groups and the three studied time periods 19781986 , 19871994 , and 19952002 . conclusions . the data suggests that there were no significant changes in oropharyngeal cancer survival in the province of zaragoza throughout the years .

wiskott aldrich syndrome ( was ) is a monogenic x - linked primary immunodeficiency characterized by the classic triad of microthrombocytopenia , eczema and recurrent infections . multiple autoimmune manifestations and tumors are serious complications and the life expectancy of was patients is severely reduced , unless they are successfully cured by hematopoietic stem cell transplantation . was is caused by mutations in the was gene impairing the expression and/or function of the was protein ( wasp ) , a hematopoietic - specific regulator of cytoskeletal reorganization also involved in signal transduction of cells . the absence or reduction of wasp compromises multiple processes of different immune cell types involved in innate and adaptive responses , resulting in a variable and progressive immunodeficiency . susceptibility of was patients to develop autoimmune diseases has been mainly attributed to the breakdown of self tolerance sustained by dysfunction of both natural t regulatory and effector t cells ( reviewed in ref . ) . however , b - cell intrinsic defects have been demonstrated to critically contribute to was - associated autoimmunity in was mouse model . in humans , the contribution of b - cell defects in the pathogenesis of was has been partially investigated . b cells from patients exhibit lower motility , migratory and adhesive capacities , likely due to defective f - actin nucleation . in contrast , despite the role of wasp in b - cell receptor ( bcr ) signaling , abnormalities in b - cell activation still remain controversial . a skewed distribution of serum immunoglobulin ( ig ) classes and the inability to mount a proper antibody response , particularly to t - cell independent ( ti ) antigens , suggest defects in b - cell effector function . previous findings in was patients show phenotypical b - cell perturbations in the periphery . in order to evaluate whether an abnormal b - cell development might generate a b - cell repertoire unable to unsure full protection against pathogens and tolerance against self - antigens , to this end , we have combined a detailed phenotypical analysis of b - cell maturation stages , from the bone marrow ( bm ) to the periphery , with a molecular study of ig repertoire and in vivo b - cell maturation processes in a large cohort of was pediatric patients . our data show that wasp - deficiency affects critical stages of central and peripheral b - cell differentiation contributing to abnormalities in humoral immunity and b - cell tolerance in humans . the diagnoses were clinically defined and confirmed by genetic analysis . a description of all patients is reported in supplementary table 1 . human samples were obtained according to the code of ethics of the world medical association ( declaration of helsinki ) with the approval of the local medical ethical committees of the erasmus mc and the san raffaele scientific institute internal review board ( tiget02 ) . all results obtained from samples of was patients were compared to age and sex matched healthy donors ( hds ) . the composition of the precursor b - cell compartment was analyzed by flow cytometric immunophenotyping as described in the supplementary material . for the analysis of replication history and somatic hypermutation , four b - cell subsets were isolated from thawed peripheral blood mononuclear cells ( pbmcs ) using a facs diva cell sorter ( bd biosciences ) . gating on cd19 cells , transitional ( cd27cd24cd38 ) , mature nave ( cd27igdcd24cd38 ) , natural effector ( cd27igd ) and memory ( cd27igd ) b - cell subsets were sorted with a purity of > 95% for all fractions . for intracytoplasmic detection of human wasp , cells were fixed and permeabilized using a cytofix / cytoperm kit ( bd pharmingen , oregon , usa ) . the anti - wasp antibody 503 ( a kind gift from prof h. d. ochs , seattle , wa , and l. d. notarangelo , boston , ma ) was used , followed by detection with pacific blue - labeled anti - rabbit igg secondary antibody ( invitrogen , san diego , usa ) . cd20 positive cells were purified from pbmcs of pediatric was patients and age - matched hds by immunomagnetic beads ( miltenyi biotec , germany ) or facs sorting . the purity of the isolated cells were analyzed by facs and ranged from 84% to 98% . after isolation , cells were left overnight at 37 c in culture medium composed of rpmi-1640 , 10% fbs , 2 mm glutamine , 100 iu / ml penicillin and 100 g / ml streptomycin ( lonza , basel , switzerland ) . in vitro chemotaxis assay was performed using 5 m pore - size transwell inserts ( costar corporation , corning , ny , us ) in 24-well plates . filters were prewet 30 min at 37 c in presence of 600 l of medium supplemented with 250 ng / ml of recombinant human stromal cell - derived factor ( sdf)-1 ( cxcl12 ; peprotech , rocky hill , us ) . fifty thousand cd20 positive or negative cells were resuspended in 100 l of culture medium , seeded in the upper chamber and incubated at 37 c for 3 h. transmigrated cells , collected in the lower chamber , were counted for viable cells and stained with anti - cd19 , anti - cd24 , anti - cd38 , anti - cd27 and anti - cd3 ( bd biosciences ) for the phenotypical analysis by facs . migration frequency was estimated as the [ ( cell n at the lower chamber / the initial b cell input in the upper chamber ) 100 ] . levels of b - cell activating factor ( baff ) were measured in duplicate in plasma samples of was patients and hds using a quantikine human baff / blys / tnfsf13b immunoassay kit ( r&d systems , minneapolis , usa ) . the assay was performed according to manufacturer 's instructions and the od was determined using a microplate reader set to 450 nm . ig and igh gene rearrangements were amplified and analyzed as described in the supplementary material . dna was extracted from sorted b - cell subsets with the genelute mammalian total dna miniprep kit ( sigma aldrich ) and used to perform the ig-deleting recombination excision circles ( krec ) assay . the amounts of coding and signal joints of the ig-deleting rearrangement were measured by real - time quantitative pcr on an abi prism 7000 sequence detection system ( applied biosystems ) . ig restriction enzyme - based hot - spot mutation assay ( igrehma ) was performed on genomic dna isolated from sorted b - cell subsets . briefly , v3 - 20-j rearrangements were pcr - amplified and the pcr products ( 500 bp ) were first digested using the restriction enzyme fnu4hi and then kpni . the unmutated gene products can be visualized as 244 or 247 bp hex - coupled fragments and the mutated as 262 bp hex - coupled fragment . comparisons between proportions were calculated by using the chi - square test ( test ) ( with continuity correction ) . until now , the composition of the b - cell compartment in the bm of was patients has not been reported . to evaluate the effect of wasp - deficiency on b - cell development in the bm , we characterized five stages of b - cell differentiation in three was patients . we observed a decreased frequency of immature b lymphocytes in was patients ( fig . this observation was further assessed in four additional patients , focusing on late stages of b - cell differentiation based on the expression of cd10 and cd20 ( gating strategy is shown in suppl . we confirmed a significant decrease in the frequency of immature b cells compared with age - matched hds ( fig . in addition , at the earliest differentiation stages , we observed a significant increase of small pre - b - ii cells in was patients ; while no statistical difference was detected in large pre - b - ii cell frequency ( fig . these findings might reflect a partial block in the bm b - cell differentiation between the small pre - b - ii and immature b - cell stages in was , or an early egress of immature b cells into the periphery . next we examined the distribution of b - cell subsets in the peripheral blood of was patients and age - matched hds . when available , we evaluated the absolute count of total b cells , which was found significantly reduced in patients less than three years old ( < 3y ) , while no differences were observed in the older was group ( fig . four b - cell subsets were identified according to the expression of cd24 , cd38 , igd and cd27 : transitional , mature nave , memory and plasmablasts ( fig . transitional b cells ( cd24cd38 ) are the most immature peripheral b - cell population that were significantly increased in both was age groups as frequency ( fig . 2c ) and absolute number ( suppl . the next maturation stage , represented by mature nave b cells ( cd27igdcd24cd38 ) , was significantly decreased only in was patients less than 3 years old both in frequency ( fig . finally , the frequency of plasmablasts ( cd24cd38 ) was significantly increased in the older group of patients ( fig . 2c ) , while the absolute number of both memory b cells ( cd27igdcd24cd38 ) and plasmablasts were significantly decreased in the younger group of was patients ( suppl . altogether , these findings demonstrate that was patients have a perturbed peripheral b - cell distribution . the overrepresentation of transitional b cells in the blood of was patients led us to hypothesize that an early egress of immature b cells from bm could occur . cxcr4 , the chemokine receptor for sdf-1 , has an important role in retaining b - cell precursors within the bm in order to prevent premature migration to the periphery [ 2224 ] . we tested the chemotactic response to sdf-1 of b lymphocytes of was patients and hds by transwell migration assay . as shown in fig . 3 , both transitional and mature peripheral b cells were less responsive to sdf-1 in the absence of wasp ( fig . this defect was specific for b lymphocytes since cd3 positive cells of was patients showed a normal migratory capacity to sdf-1 ( suppl . thus , we hypothesize that a diminished retention signal mediated by cxcr4/sdf-1 could explain the premature egress of immature b cells from the bm leading to an increase of transitional b cells in periphery . to further characterize the peripheral b - cell distribution , we focused our analysis on b - cell subsets found expanded in autoimmune diseases and potentially autoreactive [ 2528 ] . we found a significant increase of cd19cd21cd35 subset in both was age groups ( fig . 4a ) , confirming data previously reported by park and colleagues . we also analyzed the frequency of cd19cd21cd38 cells ( referred to as cd21 ) described to be expanded in autoimmune diseases and immunodeficiencies . interestingly , we observed that the frequency of this unusual population was markedly increased in was patients , both in the younger and older groups ( fig . in addition , higher levels of soluble baff were found in the plasma of was patients as compared to pediatric hds ( fig . this finding was also associated with a decreased expression of its receptor , baffr , in transitional b cells of was patients ( fig . because of the role of baff in b - cell homeostasis and peripheral b - cell tolerance , high baff levels may affect the stringency of peripheral b - cell selection thus favoring the survival of b - cell subsets containing potentially autoreactive clones . in order to evaluate the effect of wasp - deficiency in an antigen dependent context , we analyzed in detail the memory b - cell compartment of was patients . we identified six distinct memory b - cell subsets ( gating strategy is shown in suppl . igm - only ) and cd27igg deriving from primary germinal center ( gc ) responses ; igg cd27 and iga cd27 switched memory b cells originating from secondary gc responses ; cd27igd ( named natural effector ) and cd27iga generated in a ti manner in the gastrointestinal tract or in the splenic marginal zone area , respectively . we did not observe differences in memory b - cell populations deriving from both primary and secondary gc responses between was patients and hds ( fig . a marked reduction in natural effector b cells was found in both age groups of was patients both as frequency ( fig . the frequency of cd27iga b cells was decreased in 312 years old patients compared with hds ( fig . , the absence of wasp mainly affects ti responses of b cells , whereas only mildly impairs the generation of early memory b cells in gc without influencing the class - switched memory b - cell compartment . we determined the in vivo b - cell proliferation history by krec assay and in parallel shm by igrehma in sorted transitional , mature nave , natural effector and memory b cells . as expected , transitional b lymphocytes did not undergo cell divisions and mature nave b cells had a limited number of cell divisions in hds ( fig . both b - cell subsets were characterized by the absence of shm ( fig . 6b ) and we did not find differences in transitional and mature nave b cells of was subjects ( fig . in contrast , the memory compartment showed less proliferation in was memory b cells in association with a reduced shm level ( fig . interestingly , the defect in maturation was even more pronounced in natural effector was b lymphocytes . to study shm also in the ig heavy chain ( igh ) , we analyzed the frequency of mutated nucleotides in rearranged variable region of igh ( ighv ) genes by cloning and sequencing the most frequent ighv subgroups , the ighv3 and ighv4 gene families , of both - and -chain of constant regions ( c and c ) . the mutational frequency of was b cells was significantly lower for both c and c transcripts in all domains of v region ( fig . thus , the decreased in vivo proliferation and shm levels of was memory b cells could be responsible of a lower protection against infections and persistence of pathogens that finally lead to autoimmunity . was b cells showed an increased frequency of ighv3 - 30 in both ig classes ( fig . 7a ) and the absence of ighv3 - 48 genes in c sequences ( suppl . 7a ) . in ighv4 transcripts , we noticed a dominant usage of the ighv4 - 34 genes in was c ( fig . moreover , the antibody repertoire of was b cells was devoid of ighv4 - 59 , a gene commonly used in b cells from hds . the distribution of d families was only slightly altered in was patients ( suppl . finally , we evaluated the csr process in total b cells by analyzing the frequency of ig subclasses in sequenced igh transcripts . a preferential usage of -chain c region 3 ( c3 ) and c1 , igh - proximal genes ( the igh locus is schematized in fig . 7d ) , accompanied by a reduction in the expression of distal c2 and c4 , were detected in was patients ( fig . the analysis of iga transcripts showed no significant difference in the subclass usage ( fig . 7e ) . in conclusion was patients show a skewed ig gene usage suggesting an altered selection of the antibody repertoire during b - cell development . we reported herein that wasp - deficiency affects many aspects of b - cell development , first in the bm and then in the periphery . indeed , precursor b - cell as well as memory b - cell development and selection appear affected by the absence of functional wasp . in the bm we have observed a decreased frequency of immature b cells , while in the periphery an overrepresentation of transitional b cells likely due to a reduced retention in the bm and/or a decreased migration to peripheral lymphoid tissues . the interaction of the chemokine receptor cxcr4 with its ligand sdf-1 is required for the retention of developing b cells in the bm . wasp is actively involved in cxcr4 signaling [ 3739 ] and was murine b cells show defective migratory response to sdf-1 . consistently , here we demonstrate in was patients that b cells migrate less to sdf-1. thus , was immature b cells could be unable to properly sense retention signals derived from cxcr4 in the bm leading to a premature release in the periphery . the increase of transitional b cells could also reflect an altered migration to the peripheral lymphoid organs resulting in a prolonged persistence in the circulation . consistently , our data allow us to exclude that the expansion of transitional b cells is due to homeostatic proliferation since we demonstrated the absence of replication history in this subset . transitional b cells are found expanded in immunodeficient conditions or autoimmune diseases and represent a reservoir of autoreactive b cells . the cohort of patients analyzed here did not show any overt sign of autoimmunity , probably due to their young age . however , the analysis of serum autoantibodies performed in four patients showed positivity for anti - nuclear antibodies in three patients . one of them also showed the presence of anti - platelet antibodies ( data not shown ) . these cells are enriched in anergic autoreactive clones and expanded in systemic lupus erythematosus ( sle ) , rheumatoid arthritis and in common variable immunodeficiency groupia patients developing autoimmune syndromes . baff levels and signals through baffr coordinate the maintenance of the primary b - cell pool and the fate of self - reactive b cells . herein , we report for the first time increased baff levels and decreased baffr expression in was patients . elevated baff serum levels are often present in immunodeficiencies , autoimmune diseases and viral infections and may lower the thresholds for the survival of autoreactive b cell clones . the enrichment of cd21 b cells and the alterations in baff levels and expression of its receptor suggest that the mechanisms of b - cell selection could be altered in was patients . our data also suggest the presence of a defective selection of was b cells producing high - affinity antibodies . indeed , we noticed a restricted or null presence of ighv3 - 48 and ighv4 - 59 gene families and a preferential usage of ighv3 - 30 and ighv4 - 34 . in particular , ighv3 - 48 gene is selectively used against polysaccharide antigens and its decrease in was could account for an inefficient antibody response . in contrast , ighv3 - 30 is highly represented among anti - platelet autoantibodies from patients with idiopathic thrombocytopenic purpura and in sle patients . additionally , ighv4 - 34 encoded antibodies are intrinsically autoreactive when unmutated , as observed in our patients . this peculiar antibody repertoire reflects an altered selection of both protective and autoreactive ig gene families . the perturbation of b - cell homeostasis present in was patients supports the administration of anti - cd20 mab in the conditioning regimen of gene therapy to deplete b cells . in the memory b - cell compartment , the frequency of isotype - switched igg b cells we observed a normal frequency of memory b cells and of switched cd27igg and cd27iga b cells in our cohort of was patients . this suggests that t - cell dependent antigen response is induced normally in was patients . in contrast , the frequency and the shm level of ti memory b - cell subsets were reduced , likely due to their reduced in vivo proliferation . the marked reduction in the natural effector b - cell subset , resembling marginal zone b cells in the spleen , mirrors the decreased number of splenic marginal zone b cells and the histological defects in the marginal zone area of the spleen already described in was mice and patients . in addition , both the reduction in natural effector and igacd27 b cells , which are generated during ti responses , provide evidence of a defect in b - cell function independent from the cross - talk with t lymphocytes . the mutational status of both heavy and light ig chains is reduced in was in presence of a diminished rate of proliferation in the total memory b - cell pool . in addition , the analysis of ig gene selection showed a preferential use of igh - proximal genes ( ighg1 and ighg3 ) accompanied by reduced shm suggesting that was b cells have undergone less gc reactions . reduced igg2 switching in was patients could account for the poor response to ti antigens which is typical of the syndrome . the cytokine profile of was patients shows an impairment in the production of th1 cytokines that could explain their reduced switching to igg2 subclass . moreover , the isotype - switching outcome is also influenced by two additional factors : cell division and antibody affinity to antigens . the defective proliferation of memory and natural effector b cells and the reduced affinity maturation found in was might contribute to these alterations in class switching . our results in was patients show an early egress of immature b cells from bm leading to an overrepresentation of transitional b cells in the periphery . the memory compartment is characterized by a reduced maturation status that could affect the b - cell effector functions contributing to a lower clearance of pathogens and leading to chronic inflammation that can break tolerance . in conclusion , our results add novel immunological features of was b - cell phenotype that can complement the evaluation of the efficacy of various treatment approaches .

wiskott aldrich syndrome protein ( wasp ) regulates the cytoskeleton in hematopoietic cells and mutations in its gene cause the wiskott aldrich syndrome ( was ) , a primary immunodeficiency with microthrombocytopenia , eczema and a higher susceptibility to develop tumors . autoimmune manifestations , frequently observed in was patients , are associated with an increased risk of mortality and still represent an unsolved aspect of the disease . b cells play a crucial role both in immune competence and self - tolerance and defects in their development and function result in immunodeficiency and/or autoimmunity . we performed a phenotypical and molecular analysis of central and peripheral b - cell compartments in was pediatric patients . we found a decreased proportion of immature b cells in the bone marrow correlating with an increased presence of transitional b cells in the periphery . these results could be explained by the defective migratory response of was b cells to sdf-1 , essential for the retention of immature b cells in the bm . in the periphery , we observed an unusual expansion of cd21low b - cell population and increased plasma baff levels that may contribute to the high susceptibility to develop autoimmune manifestations in was patients . was memory b cells were characterized by a reduced in vivo proliferation , decreased somatic hypermutation and preferential usage of ighv4 - 34 , an immunoglobulin gene commonly found in autoreactive b cells.in conclusion , our findings demonstrate that wasp - deficiency perturbs b - cell homeostasis thus adding a new layer of immune dysregulation concurring to the increased susceptibility to develop autoimmunity in was patients .

it has been proven to be very effective and became a standard of care in the treatment of most cases with severe prolapsus . sacropexy is a surgical repair technique that restores pelvic anatomy by attaching synthetic graft material into the vagina and sacrum . erosion of vaginal wall or bowel can be seen as a long term complication . in this paper , we present a patient suffering from mesh migration into the rectum after abdominal sacral colpopexy . a 69-year - old woman was admitted to the hospital with a complaint of sensation of fullness and a feeling of a foreign material protruding during defecation . she had been diagnosed with uterine prolapsus and stress incontinence in 2008 and underwent total abdominal hysterectomy , bilateral salpingo - oophorectomy and sacral colpopexy with prolene mesh . the patient was admitted to our clinic five years after the procedure with complaints mentioned above . there was a foreign material palpated in rectum with digital examination . prolene mesh was detected in sacral region but resection of the mesh could not be conducted because of high levels of adhesions in that region . genital prolapse or genital hernia is described as the protrusion of pelvic organs along the vagina . it is one of the common gynecological conditions that affect the quality of life in women . it may be seen in up to 50% of multipara women , and its incidence increases with age . high rates of recurrence with traditional techniques led to the development of new surgical techniques . the use of synthetic mesh has become more popular surgical approach in cystocele and rectocele repair . mesh migration is a well - known clinical pathology and have been reported in literature . yolen and grossman suggested that intra - abdominal foreign bodies ( like mesh ) transmigrate into the small or large bowel by triggering an inflammatory reaction . persistent inflammatory reaction causes an opening into a hollow organ assisted by the peristaltic movement of the bowel . insufficient fixation of a mesh is another factor for migration of synthetic materials in some patients . larger pores greater than 75 nm permit the migration of macrophage and leukocyte migration and reduce the infection rate . large pores also improves flexibility of the mesh and cause tissue ingrowths and healthy collagen deposition . complications reported after sacropexy include ileus , intraoperative vessel injury , ureter injuries , recurrent descensus and mesh tearing . the use of a mesh as a graft material results in higher success rates but also causes a higher number of complications , such as mesh erosions or chronic infections . taoka reported a case of rectal migration of mesh in a 64-year - old woman who presented with a recto - cutaneous fistula 11 months after a tension - free vaginal ( tvm ) repair ; the patient was treated by removal of the infected mesh and closure of the rectal wall defect under cover of a temporary colostomy . by contrast with the troublesome symptoms reported in such patients in the literature , the only presenting complaint of our patient was protrusion of foreign material from the rectum . in conclusion , mesh migration is a serious complication after sacral colpopexy . sometimes surgical resection of migrated mesh with laparotomy can be difficult due to dense adhesions . scu was responsible for writing , conception and design of the study ; ob contributed toward analysis and interpretation of data ; nas , oa , aa- performed acquisition of data ; bk drafted the manuscript .

introductionpelvic organ prolapse ( pop ) is a common gynecological problem . repair with synthetic materials such as prolene mesh has become a popular approach in prolapsus surgery . migration of synthetic materials can cause serious complications.presentation of casea 69-year - old woman was admitted to the hospital with a complaint of sensation of fullness and a feeling of a foreign material protruding during defecation . the patient underwent exploratory laparotomy . prolene mesh was detected in sacral region but resection of the mesh could not be conducted because of dense adhesions causing frozen pelvis . the migrated prolene mesh was resected transanally.discussiongenital prolapse or genital hernia is described as the protrusion of pelvic organs along the vagina . it is one of the common gynecological conditions that affect the quality of life in women . mesh migration is a well - known clinical pathology.conclusionmesh migration is a serious complication after sacral colpopexy . surgical resection of migrated mesh can be difficult due to dense adhesions .

eccentric exercise was shown to provoke skeletal muscle damage leading to delayed onset of muscle soreness ( doms)1,2,3 . acute muscle damage can cause discomfort at the site of injury , inflammation , oxidative stress , edema , and loss of muscular function and strength4 , 5 . many researchers have attempted to ameliorate symptoms by various treatments , such as nutritional interventions3 , 6 , pharmacological interventions7 , massage8,9,10 , low - intensity exercise10 , ultrasound application11 , or cryotherapy12 . even though some positive effects have been reported , several studies did not find any reductions in the symptoms of muscle injuries . beside these treatments , static or pulsed electromagnetic field therapy have been used for the treatment of doms13,14,15 . again , some authors reported positive effects on doms symptoms , whereas others were not able to find benefits13 , 14 . application of a pulsating electrostatic field ( pesf ) , which is a comparable but not identical technique , might be a further method capable of reducing the symptoms of muscle soreness . a pesf was shown to increase vasomotion and as a consequence blood flow16 , to reduce the formation of rouleaux , and to increase metabolic activity16 , 17 . furthermore , a pesf may function as an ion pump able to move calcium ions , the concentration of which was shown to be increased after eccentric exercise with detrimental effects2 , 18 . due to these effects , it might be speculated that a pesf could prevent the development of doms and muscle strength loss after muscle damaging exercise . therefore , the aim of the present study was to investigate the effect of pesf application on sensation of muscle soreness and muscle strength after eccentric exercise . nine sport students ( 5 males , 4 females ) without any medical problems and not adapted to eccentric exercise were recruited for the study . all of them were informed about the study aims and gave written informed consent for participation . two participants ( one male and one female ) had to be excluded from the final analysis , one because of a pre - start muscle soreness score of already 4 and one because of a pre - start ck value of 333 u / l . therefore , 7 participants ( 4 males and 3 females ; weight , height , and age : 74.35.7 kg and 71.05.3 kg , 183.09.9 cm and 172.36.4 cm , and 26.52.4 years and 24.31.2 years , respectively ) were included in the final analysis . the study was carried out in conformity with the ethical standards of the declaration of helsinki and was approved by institutional review board of the department of sport science , university of innsbruck . the study was designed as a single - blinded , placebo - controlled cross - over intervention study . all participants performed 2 downhill running sessions on a treadmill separated by at least 4 weeks . before and at different time points after the sessions , muscle soreness score , creatine kinase ( ck ) , and jump ability were assessed as described in detail below . the first running session was carried out in a slightly modified form according to kingsley et al5 . running sessions started with a 3 min stage of horizontal running at 5 km / h ( warm - up ) . thereafter , the inclination was set at 17.5% , and the speed was increased every minute by 1 km / h until reaching approximately 70% of the age - predicted maximal heart rate ( hr ) ( calculated as 220 age ) . overall , the running duration was 30 minutes , not including the warm - up time . during the second test , the speed was regulated in the same way as during test one but independent of hr in order to have identical test speeds and a comparable muscular stimulus . immediately after each run , half of the subjects were treated in a sitting position with a pesf for 45 min , and half of the subjects were exposed to a sham treatment . after at least 4 weeks , in which participants were advised not to change their physical activity habits , the subjects that were treated with a pesf during the first session received the sham treatment and those that were treated with the sham treatment during the first session received the pesf treatment . the ( negative ) pesf was generated with a new health 9000 device ( akern srl , pontassieve , italy ) and was applied through a mat with a field intensity of 9000 v , a current of < 9 ma , and a pulsatile frequency of 50 hz . the electrostatic field was confined to the ion mat surface , and it has been proven to be safe17 . the general rating of muscle soreness was assessed as described by trombold et al.3 before and immediately after the runs , immediately after the 45 min treatment or sham treatment , and 48 hours after treatment . participants subjectively rated the degree of soreness using a visual analog scale of 0 to 10 , with 0 describing no soreness and 10 describing unbearable soreness . jump ability ( jump and reach test ) was assessed before running , after the 45 min session , and 48 hours after the running . ck activity was assessed before running and 48 hours after running from capillary blood samples with a reflotron sprint device ( reflotron sprint , mannheim , germany ; reference range for men : 24195 u / l at a measurement temperature of 37 c ) . delta values ( ) were calculated as post values ( at different time points ) minus pre values for both the pesf application and sham setting . differences at baseline , changes in the course of pesf or sham treatment , and differences between conditions ( values for the pesf and sham treatment ) were assessed by paired student s t - tests or the wilcoxon test as appropriate . spearman correlation analyses were used to calculate associations between ck and cmj changes and muscle soreness score . when compared with the pre values , the muscle soreness score was increased at all time points and under both conditions ( p<0.05 ) . jump performance was reduced immediately after the sham application ( p=0.038 ) and tended to be reduced immediately after the pesf application ( p=0.054 ) . table 1table 1.values for jump height , creatine kinase , and muscle soreness score in the courses of the pesf and sham treatmentspreafterrunafter45 min 24 hafter48 hafterjump and reach ( cm)pesf276.28.7273.07.03.33.9273.010.53.33.6sham276.521.6272.023.54.55.3272.023.24.56.2creatine kinase ( u / l)pesf133.632.1171.971.338.358.7sham100.141.0271.7274.5171.6285.8muscle soreness scorepesf1.41.43.61.72.12.04.02.02.61.65.10.93.71.64.61.53.12.0sham1.01.04.01.43.01.74.11.73.12.26.71.75.72.26.42.45.43.2pesf : pulsating electrostatic field . the table represents values of the same participants receiving the pesf and sham treatments ( n=7 , crossover design ) . there was a trend toward significant differences in delta values ( = after values ( i.e. 45 min , 24 h , and 48 h after ) minus pre value ) between treatments ( pesf vs. sham ) . shows values for ck , muscle soreness score , and jump height in the courses of the pesf and sham treatments . twenty - four and 48 hours after downhill running , the muscle soreness score ( mss ) tended to be less after pesf administration when compared with the sham treatment ( mss : 3.71.6 vs. 5.72.2 after 24 h and 3.12.0 vs. 5.43.2 after 48 h , respectively ; 0.05<p<0.01 ) . furthermore , the highest muscle soreness scores were found after the sham session , with one subject having a score of 9 and two subjects having a score of 8 after 24 hours . after 48 h , one subject had a score of 10 , one subject had a score of 9 , and one subject had a score of 7 . after 48 hours , one subject had a score of 7 , whereas all others had lower values . the table represents values of the same participants receiving the pesf and sham treatments ( n=7 , crossover design ) . there was a trend toward significant differences in delta values ( = after values ( i.e. 45 min , 24 h , and 48 h after ) minus pre value ) between treatments ( pesf vs. sham ) . the main finding of the present investigation was that pesf application tended to reduce muscle soreness scores without significantly affecting ck increase and jump performance decrease . to the best of our knowledge , this is the first study to use a pesf after downhill running for the prevention of muscle soreness . in the literature , different methods , including cold or infrared therapy , massage , pharmacological intervention , etc . , have been applied to reduce muscle pain after eccentric exercise7 , 8 , 10 , 12 . the mechanisms causing beneficial effects are as diverse as the methods applied . for cold application , the main beneficial effect is thought to be the cold - related vasoconstriction , which may limit vessel permeability and thus inflammatory processes12 . the mechanism by which infrared therapies might induce potential positive effects is mainly based on the increased peripheral flow due to warmth - related vasodilatation , which could enhance the evacuation of edema , limiting inflammation and perceived pain12 . the mechanisms mediating the biological benefits of nutritional interventions are not clear but may be linked to the attenuation of oxidative stress and inflammation3 . with respect to electromagnetic field therapy , it was suggested that the treatment influences muscle immune and inflammatory responses by an unknown mechanism , may disrupt afferent input from free nerve endings of nociceptors , and may influence excitation - contraction coupling processes13 , 15 , 19 . in the present study additionally , the improvement of vasomotor performance might have provoked some beneficial effects16 , 17 . comparable to the effects of infrared application , the increased blood flow might have enhanced the removal of edema , limited the inflammatory response , and reduced perception of pain12 . a further mechanism might be the function of a pesf as an ion pump moving positively charged ions , e.g. , calcium and hydrogen16 . the calcium concentration was shown to be elevated in skeletal muscle cells following eccentric contractions18 . increased calcium concentrations within the sarcoplasm have the potential to activate many different molecular pathways in skeletal muscle , including the phospholipase - prostaglandin pathway and the calpain proteolytic pathway , which might lead to breakdown of the cell membrane and myofibrils2 . however , whether or not such mechanisms might be involved in processes of recovery from exercise - induced muscle damage still needs to be determined . it has to be mentioned that although there was a trend toward a decrease in muscle soreness score , ck and jump performance did not seem to be affected by pesf application . in this regard , the small sample size certainly is a main limitation of the present study and outlines its nature as a pilot study . nonetheless , when looking at the mss and ck values in table 1 , some beneficial effects clearly seem to be apparent . this is supported by the finding that the highest mss values were found after the sham treatment . additionally , pesf application for only 45 min may be considered a short exposure time , and it is not known if a longer pesf application time would have been more effective . in conclusion , application of a pesf might be a promising treatment to reduce muscle soreness after exercise - induced muscle damage . however , further studies are needed to confirm the present outcomes and to establish the mechanism by which a pesf may reduce muscle pain .

[ purpose ] the aim of the study was to establish whether pulsating electrostatic field application , shown to increase blood flow and metabolic activity and to function as an ion pump , is able to reduce muscle pain after exercise - induced muscle damage . [ subjects and methods ] seven participants ( 4 males , 3 females ) performed two sessions of downhill running separated by at least 4 weeks . after the running sessions , participants were either treated for 45 min with a pulsating electrostatic field ( field intensity , 9000 v ; current , < 9 ma ; frequency , 50 hz ) or a sham treatment . the order of the intervention was random , and the condition was blinded for the participants . muscle soreness score , creatine kinase , and jump ability were assessed before and up to 48 hours after running . [ results ] twenty - four and 48 hours after the downhill running , the muscle soreness score tended to be less increased after pulsating electrostatic field administration when compared with the sham setting ( changes in muscle soreness score : 3.71.6 vs. 5.72.2 after 24 h and 3.12.0 vs. 5.43.2 after 48 h , respectively ) . no further differences were detected . [ conclusion ] the outcomes show that a pulsating electrostatic field might be a promising treatment to reduce muscle soreness after exercise - induced muscle damage . however , further studies are needed to confirm the present outcomes and to establish the mechanism by which a pulsating electrostatic field may reduce muscle pain .

on the somewhat nominal date of 31 october 2011 , the world human population was deemed to have reached 7 billion ( 7 10 ) , prompting speculation as to whether everyone on the planet can be adequately fed . that currently a poor job of it is being made is not in dispute : the number of people estimated to be undernourished in 2010 was close to 1 billion ( fao , 2011a ) . the most obvious signs of undernutrition are mortality , morbidity , stunting , underweight and wasting . less immediately apparent are the effects on immune function , cognitive ability and work productivity , with profound effects at individual , family and societal levels ( underwood , 2000 ; caulfield et al . , 2006 ; it is estimated that maternal and child undernutrition accounts for 11% of total global disability - adjusted life years ( daly ; a measure of overall disease burden , expressed as the numbers of years lost due to ill - health , disability or early death ) , with dire consequences for development ( black et al . , 2008 ) . , it is anticipated that the global population will rise to 9 billion ( un department of economic and social affairs , population division , 2011 ) . satisfying the food and nutrition needs of the growing population , let alone its demands , is likely to require a profound change in what and how much is eaten , and where and how food is produced . failure to do so could profoundly affect the biosphere in ways that further erode the life - support system through biodiversity loss , changes in ecosystem services supply and exacerbated global warming ( battisti & naylor , 2009 ; foley et al . , 2011 ; phalan et al . , 2011 ; royal society , 2012 ) . in 2009 , c. 95 10 t of fishes were consumed by humans ( fao , 2012b ; fao fishstat , 2012 ) . more than 1 billion poor people obtain most of their average per capita intake of animal protein from fish ( tacon & metian , 2009 ) . fao data for 2009 ( updated with data from tacon & metian , 2009 ) show that africa has the lowest average per capita supply of total energy ( 10 711 kj day ) , protein ( 666 g day ) , non - fish animal protein ( 151 g day ) and fish protein ( 92 kg year ) of any region ( fao faostat , 2013 ) . nevertheless , fish contributed 9% of total animal energy intake here , the highest of any region , and 18% of total animal protein consumption ( second only to asia at 19% ) . global wild fish catches have for some time been at or near the limits of what aquatic ecosystems can be expected to naturally provide ( fao , 2012a ; unhrc , 2012 ) . meeting the world 's demand for fish has thus depended on the spectacular growth of aquaculture . in 2011 , c. 41% of fish consumed came from farming ( fao , 2012b ; fao fishstat , 2012 ) . a growing body of research studies shows the importance of fish in the supply of not only protein but also more importantly of essential fatty acids and micronutrients ( kawarazuka & bn , 2011 ) . the importance of fish as a rich source of essential fats , crucial for brain development and cognition , is highlighted in the implementation of the scaling up nutrition framework and roadmap [ a private and public sector and civil society partnership that seeks to better understand the crisis of undernutrition in early life ( first 1000 days ) and elevate nutrition on the global agenda ; http://www.scalingupnutrition.org ] . discussion of the rise of aquaculture has largely focused on its contribution to global aquatic animal food supplies , largely ignoring the resultant changes in species composition of the fishes consumed or how it is farmed and the consequences for food and nutrition security ( kawarazuka & bn , 2010 ; unhrc , 2012 ) . this paper sets out to redress the situation and considers the technological and policy innovations needed to ensure that fish cultivation fulfils its potential to meet the global population 's food and nutrition needs . this paper focuses on fishes that account for two - thirds of gross global aquatic animal source food supplies , and make particular reference to africa and asia . fish is an excellent source of high quality animal protein and essential fatty acids , especially long - chain polyunsaturated fatty acids ( lcpufa ) and micronutrients , which are much greater in fishes than in terrestrial animal - source foods ( table 1 ) . drawing on such evidence , a recent fao who expert consultation group concluded that among the general population , fish consumption is beneficial for individual growth and development , while consumption of certain amount of fish ( fatty fishes in particular ) is associated with reduced risk of coronary heart disease and stroke ( fao who , 2011 ) . people are generally encouraged to increase consumption of fatty fishes two to three - fold in order to obtain sufficient quantities of lcpufas ( surette , 2008 ; jenkins et al . there is no convincing evidence , however , for increased risk of heart disease linked with methylmercury while the potential cancer risks from dioxins are concluded to be well below coronary heart disease benefits associated with fish consumption ( fao who , 2011 ) . nutrient content of selected fishes and other foods ( modified from kawarazuka & bn , 2011 ) . where no figure is given , no analysis was carried out pufa , polyunsaturated fatty acid ; epa , eicosapentaenoic acid ; dha , docosahexaenoic acid . the benefits of fish consumption for vulnerable groups are also increasingly recognized and there is a growing interest in its potential to better contribute to food and nutrition security objectives through supplemental feeding and other food - based strategies ( roos et al . , 2002 , 2007c ; gibson et al . , 2003 ) . a controlled study in malawi , for example , showed that individuals fed intervention diets containing significantly more soft - boned fishes had lower incidences of anaemia and common infections than those in the control group ( gibson et al . , 2003 ) . recognizing that eating fish is beneficial and encouraging people to do so are clearly insufficient to overcome undernutrition ; issues of food availability , food access and food utilization these imperatives are codified in the definition of food security adopted by the world food summit in 1996 , which states that food security exists when all people , at all times , have physical and economic access to sufficient , safe and nutritious food to meet their dietary needs and food preferences for an active and healthy life. the term food and nutrition security incorporates the other pillars of good nutrition : care , health , hygiene water and sanitation ( klennert , 2009 ) . a conceptual model showing how nutritional status is determined by these various causal factors is presented in fig . individual nutritional status can be seen as an immediate outcome of nutrient intake , care and health . nutrient intake is , in turn , governed by use and utilization , the former being dependent upon the food that is purchased by and shared within households , while the latter refers to the body 's ability to utilize the nutrients consumed . the underlying causes of health status are in part not only environmental , determined by factors such as air quality , but also related to health care , housing , sanitation and access to clean water , sometimes worsened by inadequate nutrition , which predisposes individuals to disease . availability and access to food and nutrients , however , change over time and adequacy of dietary intake is affected by critical life stages such as pregnancy , lactation and early childhood . determinants of nutritional status ( modified from unicef , 1991 ; kawarazuka & bn , 2010 ) . for a comprehensive analysis of food and nutrition security , all of the contributory factors shown in fig . , however , it is its potential as a driver of change in availability ( abundance ) , access and the nutritional quality of fish being produced that warrants particular attention . the availability of food - fish is a function of production and trade , less that used for non - food purposes . the term food - fish is applied here exclusively to fish and does not include shellfish , as is usually the case , used directly for human consumption . unless otherwise stated , fao data for 2009 ( fao faostat , 2013 ) is used as the most recent set of figures that allows calculation of regional per capita food - fish supplies ( excluding molluscs and crustaceans ) in which fish imports and exports can also be taken into account . global fish supplies continue to expand , primarily as a result of the sustained , rapid growth in aquaculture production . supplies of food - fish from capture and farming increased by c. 62% , from 86 to 139 kg year , between 1961 and 2009 ( fig . increases during the 1960s1980s were largely due to increased capture fisheries landings while those since are largely attributable to aquaculture . per capita aquatic food ( , total fish and aquatic resources ; , total fish ) and fishes by species group ( , freshwater and diadromous fishes ; , demersal fishes ; , pelagic fishes ; , marine fishes ) by decade ( 19612001 ) and by year ( 20022009 ) . the statistics , however , mask enormous inter - regional differences in production and trade ( table 2 ) . asia , including china , accounts for c. 90% of global aquaculture production while production in least developed countries is < 1% ( fao , 2012a ) . seafood is the most highly traded food commodity internationally and exports of seafood from developing countries exceed the total value of coffee , cocoa , tea , tobacco , meat and rice combined ( smith et al . , 2010 ; trade increased in value by an average of nearly 11% per annum between 1976 and 2009 ( fao , 2012b ) . the effect of trade on food - fish availability at regional level can be assessed from the fao food - fish balance statistics that account for production plus imports , minus fish used for non - food uses ( e.g. fishmeal ) and exports ( fao fishstat , 2012 ) . the data for 2009 show that regional per capita food - fish supplies are greatest in oceania ( 189 kg year ) , followed by europe ( 174 kg year ) and asia ( 150 kg year ) , with africa ( 92 kg year ) trailing last ( table 2 ) . high food - fish availability in north america and europe is in part sustained by net imports from asian countries such as china and vietnam , accounting for > 20 and 26% of supplies in north america and europe , respectively ( fao fishstat , 2012 ) . low production costs and relatively high international prices , combined with liberal trade policies , have increased exports of farmed white - fleshed fishes such as tilapia oreochromis spp . and catfishes pangasius spp . , substituting for dwindling supplies from domestic white - fish fisheries ( norman - lpez & bjorndal , 2009 ; beveridge et al . , 2010 ; little et al . even africa is a net food - fish importer , imports of mainly cheap , oily pelagic fishes increasing net food - fish availability by 14% . total and per capita food - fish supplies by region and economic grouping , 2009 ( from fao , 2011b ; fao fishstat , 2012 ) data for central and south america and caribbean combined . while trade data do not indicate whether the fish is of farmed origin , with key commodities such as oreochromis spp . and pangasius spp . allison ( 2011 ) found that effects of fish trade on poverty and food and nutrition security ranged from large and positive to negative and small . with the exception of tuna ( thunnini ) , increases in both per capita food - fish supplies and the value of trade only occurred when there was strong growth in aquaculture ( e.g. in bangladesh , indonesia and vietnam ) . increases in aquaculture enhance food - fish availability in countries where sector growth is strong . although annual per capita food - fish availability in low - income food - deficit countries ( lifdcs ) grew from 45 kg in 1961 to 105 kg in 2009 , it remains comparatively low ( fao , 2011b ; fao fishstat 2012 ) ( table 2 ) . moreover , in africa , where fish constitutes a particularly large proportion of animal - source foods , per capita food availability of fish has increased little over the past 20 years , in large measure due to the limited growth of aquaculture . it is not just the availability of food - fish per se that is of concern , however , rather the nutrients that fish provide . qualitatively , fish supplies have changed markedly over the past 50 years . in 1961 , 32% of food fishes were of demersal origin , 26% were pelagic , 16% were fresh water and diadromous and 12% comprised other ( i.e. non - demersal and pelagic ) marine fishes ( fig . the availability of demersal and other marine fishes had fallen in both absolute and relative terms , that of pelagic fishes had increased in terms of biomass but had fallen as a proportion of total food - fish supplies , while that of freshwater and diadromous fishes had increased by c. 250% . annual per capita freshwater and diadromous food - fish supplies in 2009 were 61 kg , equivalent to one - third of supplies ( fig . the changes largely result from stagnation in capture fisheries production and the large rise in carps ( cypriniformes ) , oreochromis spp . and salmonid aquaculture . the species being farmed while changes in the fishes eaten are likely to have some effect on protein and energy supplies , they may have an even greater effect on the availability of dietary lipids ( see table 1 ) . demersal white fishes ( gadoids ) typically have low levels ( < 2% ) of fat . pelagic herring , anchovies and sardines ( clupeids ) , as well as salmonids , mackerels ( scombrids ) and eel ( anguilla spp . ) , are oily fishes ( > 2% oil content ) , with levels in mackerel scomber scombrus l. 1758 seasonally exceeding 20% ( wallace , 1991 ) . while species is a major determinant of farmed fish nutrient content , nutrient composition is also influenced by how the animal is farmed . farm environmental conditions , especially water temperature , quantitatively and qualitatively influence lipid deposition ( haard , 1992 ) . while the majority of farmed fishes are freshwater omnivores and herbivores , there has nonetheless been a trend towards intensification of production methods , involving increasing reliance on nutritionally complete feeds ( tacon & metian , 2008 ) . traditional pond - based fish farming methods use inorganic and organic fertilizers to stimulate autotrophic and heterotrophic production ( hepher , 1988 ) . production is further enhanced by the administration of small quantities of supplemental feeds , generally derived from long - chain carbohydrate - based agricultural by - products ( e.g. rice and wheat bran ) ( hepher , 1988 ; avinmelech , 1999 ) . the intensification of rearing methods responsible for increasing production per unit land and water is primarily driven by economics : the growing scarcity and thus value of land and fresh water , together with the price of feed and the strong market for fish ( van der zijpp et al . , 2007 ) . total lipid content of farmed fishes is typically higher due to differences between wild and farmed environments in terms of feed availability and composition and animal activity ( vliet & katan , 1990 ; haard , 1992 ; grigorakis et al . , 2002 ; palstra & planas , 2011 ) . also have lower -3:-6 fatty acids , the intensity of production methods ( i.e. the amount and composition of external feed used ) having a particularly important influence on fish lipid levels and composition ( grigorakis et al . , 2002 ; karapanagiotidis et al . , 2006 ; weaver et al . , 2008 ) in short , while aquaculture has increased global food - fish supplies , it has also radically changed the availability of certain food - fish types , species and nutrients consumed via fishes . freshwater species , in particular cyprinids and tilapines , increasingly dominate food - fish supplies . while this may have marginal effects on protein supplies per unit food - fish , it changes the quantities and types of lipid . farming methods have a further effect on fish lipid content and composition : intensification of production methods tends to increase fat levels and , where plant - based diets in particular are used , alter fat composition . the significance of these changes for food and nutrition security can only be determined in the context of individual food consumption and health . availability alone is insufficient to ensure household food security : access to food , determined by power relations , poverty , lack of assets and prices that are increasingly governed by globalized markets , is also critical ( fig . although global food supplies undoubtedly have been improving , growing competition for use of farm land for biofuels , lack of key inputs in some places , natural disasters and increasing dependence of food production systems on energy have , since 2006 , resulted in high and volatile food prices , a situation that is likely to continue for some time ( fao , 2011a ) . poor , food import - dependent countries , with inadequate resources and capacity to put into practice policies targeted at incomes and expenditure , markets and prices or implement food safety nets , will continue to be disproportionally affected ( fao , 2011a ) . fisheries and aquaculture can improve household food security ( prein & ahmed , 2000 ; dey et al . , 2006 ; thilsted , 2012 ) . the pathways by which this occurs have been elaborated by kawarazuka & bn ( 2010 ) : directly through consumption of fish and indirectly through increased purchasing power from the sale of fish ( i.e. increased access ) ( fig . the latter was found to be especially true when women engage in aquaculture and fisheries - related activities such as processing and trading , which results in a greater proportion of income being spent on food ( i.e. improved use ) ( kawarazuka & bn , 2010 ) . worldwide , an estimated 120 10 people are engaged in fishing ( world bank , 2010 ) and 23 10 in aquaculture ( valderrama et al . , 2010 ) , increasing their physical access to and , it is hypothesized , consumption of fish . the great majority of people in developing countries do not of course produce their own fish and physical access is largely determined by distance from source . drying , salting and smoking extend the life of this highly perishable commodity , enabling distribution over far greater areas , albeit at some deterioration in nutritional quality ( roos et al . , 2002 ) . there have been few specific studies and no comprehensive review of how increases in farmed - fish availability affect access and use by poor consumers ( kawarazuka & bn , 2010 ; allison , 2011 ) . the growth of aquaculture has undoubtedly increased access in production hotspots such as the nile delta , egypt , but despite the volumes produced , physical access in distant upper egypt remains poor ( macfadyen et al . moreover , in subsaharan africa , the fish aquaculture sector remains dominated by smallholder , subsistence - type operations and physical access to the relatively small amounts of fish produced is largely limited to producer households and neighbours ( dey et al . , 2006 ; brummett et al . the greatest influence on access to food is prices , which are largely determined by markets and incomes ( fig . the consumption of meat and fish , which are expensive compared to starchy staples such as rice or maize , is highly linked to wealth ( speedy , 2003 ) . low - income consumers therefore preferentially purchase less expensive fishes , such as small pelagic species , high in lcpufas ( tacon & metian , 2009 ; kabahenda et al . , 2012 ) . in southern africa , for example , the cheapest fishes are the universally available small , dried kapenta limnothrissa spp . quantities as little as a few tens of grams are purchased and used with vegetables to prepare relishes that accompany the staple maize porridge . a review of studies of fish price elasticities of demand in western markets concluded that demand responds closely to price ( gallet , 2009 ) . in developing countries , price is likely to be an even greater determinant of whether poor consumers buy fish or not . where aquaculture production dominates the market , farmed fish tends to be cheaper , increasing economic access . in egypt , for example , farmed fish now account for two - thirds of all fish consumed and is considerably less expensive than most wild - caught fish ( gafrd , 2010 ) . in bangladesh , a decade ago , farmed fish was more expensive than wild - caught fish ( thompson et al . , 2002 ) . today , due to a doubling of farmed - fish production , however , farmed fish is generally less expensive than wild - caught fish ( b. belton , pers . although unsupported by rigorous study , it is hypothesized that aquaculture producers in developing countries tend to target the production of larger - sized fish , aimed at middle - class urban regional and international markets ( unhrc , 2012 ) , presumably in the expectation that the higher absolute and relative prices such fish command increase profits . it is proposed here that there is a largely unarticulated and increasingly unmet demand for economically accessible fish by poor consumers , who are in the majority in developing countries . given any particular species , and irrespective of whether fresh or frozen , smaller fish are generally less expensive . in a study conducted in bangladesh throughout 2011 , for example , market prices of farmed 250 g oreochromis spp . were 2030% less per kg than that of 500 g fish ( b. belton , unpubl . while production of smaller , less expensive fish undoubtedly increases access of poor consumers , from a food and nutrition security perspective the point is not how much fish is eaten but that fish consumption fulfils its potential to help meet nutritional needs , especially of poor and vulnerable consumers . the most important role that fish plays in diets dominated by starchy staples is the provision of essential fatty acids and highly bioavailable micronutrients ( gibson et al . , 2003 ; roos et al . , 2003 , 2007a , b , c , d ; hambraeus , 2009 ; kawarazuka & bn , 2010 , 2011 ) from this perspective , oily fishes and small fishes that are eaten whole are superior . small fish species , however , are generally not farmed , an exception being in bangladesh , where small species , e.g. mola amblypharyngodon mola ( hamilton 1822 ) and chanda parambassis baculis ( hamilton 1822 ) , are allowed to enter freshwater fish ponds from adjacent water bodies ( roos et al . , 2002 ) . while incorporation of small fish species into cyprinid - based pond polycultures increases overall production and revenues , seed remains unavailable from commercial hatcheries ( alim et al . , 2004 ; kadir et al . , the absence of obvious candidate small fish species for aquaculture and the likely high associated seed costs may make their culture unprofitable , except where self - recruitment occurs . there also seems to be little market for very small - sized ( i.e. that can be eaten whole ) commonly farmed fishes , such as oreochromis spp . or cyprinids . a healthy diet depends on the consumption and absorption of the appropriate amounts of energy in the form of carbohydrate , fats and protein , as well as essential fatty acids , amino acids , minerals and vitamins ( fig . inadequate food intake , compounded by the body 's inability to utilize needed nutrients , results in undernutrition . nutritional requirements are determined by genetic makeup , sex , age and health status , as well as physiological demands associated with factors such as pregnancy and physical labour . what individuals choose to eat has always been determined not only by availability and access but also by taste , nutritional value , culture and religion . personal perspectives and social contexts ( life experiences , reliance on foods away from home , food advertising , marketing and promotion ) are increasingly influential . the result is that a complex mix of product attributes such as convenience , quality , freshness , health and nutrition beliefs , environmental and animal welfare concerns and social relationships now determines western consumer food choices ( furst et al . , 1996 ; lennerns et al . , 1997 ) . it is no surprise then that the importance of fish in household food baskets varies enormously geographically and temporally . demographic characteristics , education , place of purchase and generic advertising have been found to determine beliefs and attitudes about aquaculture production ( fernndez - polanco & lunes , 2012 ) . a survey of women consumers in belgium , for example , found that consumption of wild - caught fish was influenced by sustainability and ethical considerations whilst that of farmed fish by perceptions of quality ( verbeke et al . , 2007 ) . in bangladesh , much of the increase in aquaculture production has been of exotic species , such as oreochromis spp . , which are less preferred than the increasingly scarce indigenous wild fishes ( b. belton , pers . comm . ) . it seems , therefore , that there can be consumer resistance to farmed fish , especially unfamiliar exotic species . use may change over time , especially where availability and economic access increase . at the household level , how food is handled , prepared and cooked influences the nutritional value of what is consumed ( kabahenda et al . , 2012 ) . the quantities of food distributed among family members depend on cultural and power determined norms with respect to sex and age in particular ( wheeler , 1991 ) . traditional products such as dried , smoked , salted and fermented small fishes are usually cooked as a mixed curry or stew dish , with a little oil , vegetables and spices . such dishes , as well as fish paste and fish sauces , are more readily shared among family members than larger farmed fishes . individual health status determines how effectively the body utilizes what is consumed ( caulfield et al . , 2006 ) . the institutions and policies that regulate air quality , adequacy of housing ( especially in relation to provision of clean water and sanitation ) and access to health care influence the prevalence of infectious , diarrhoeal and acute respiratory diseases among the poor in developing countries ( cairncross & valdmanis , 2006 ) . while only the flesh and sometimes the skin or heads of larger fishes tend to be consumed , small fishes are often eaten whole . the micronutrient content of small fishes is often high ( see table 1 ) but the micronutrients are concentrated in the bones , heads and gut , and these fishes must be eaten whole to derive full nutritional benefit ( steiner - asiedu et al . , 1993 ; the contribution of fish as rich sources of vitamins and minerals is poorly documented and often overlooked . studies from rural bangladesh have shown that a number of widely occurring small tropical freshwater fish species , such as a. mola , p. baculis , dhela osteobrama cotio cotio ( hamilton 1822 ) and darkina esomus danricus ( hamilton 1822 ) , have high contents of pre - formed vitamin a ( thilsted et al . , 1997 ; roos et al . , 2007a ) . because most of the small fish species are eaten whole , they are also a very rich source of highly bioavailable calcium ( hansen et al . , 1998 ; esomus danricus and trey changwa plieng esomus longimanus ( lunel 1882 ) from cambodia have a high iron and zinc content ( roos et al . , 2007c ) . a traditional daily meal of rice and sour soup made with e. longimanus can meet 45% of the daily iron requirement of a cambodian woman ( roos et al . table 1 shows the vitamin a , calcium , iron and zinc content of some selected common small fishes , as well as two commonly farmed large freshwater fishes . cooking affects the macro and micronutrient content of fish ( kabahenda et al . , 2012 ) . minerals are generally conserved during cooking but vitamins such as thiamine , cobalamine and niacin are often lost or denatured ( holland et al . , 1991 ) . consuming fish as part of a meal also confers additional benefits in terms of increasing the bioavailability of iron and zinc from other foods in a meal ( aung - than - batu et al . , 1976 ) . vulnerability , a product of exposure to risk factors such as seasonality in supply , conflict , rising prices and disease , and the ability to cope with such shocks , as established by assets ( physical and educational ) and degree of social marginalization , determines the stability of food and nutrition security ( fig . inadequate food access and utilization on a periodic basis , especially during key life stages such as the first 1000 days of life , can have long - term consequences for an active and healthy life ( caulfield et al . , 2006 ; ruel et al . , 2008 ) farming improves the continuity of global fish supplies , but locally , export - oriented aquaculture increasingly falls into a gilded trap in focusing on economic rather than socio - ecological aspects ( steneck et al . , 2011 ) , which can undermine rather than improve local access to fish food . the availability of food - fish is a function of production and trade , less that used for non - food purposes . the term food - fish is applied here exclusively to fish and does not include shellfish , as is usually the case , used directly for human consumption . unless otherwise stated , fao data for 2009 ( fao faostat , 2013 ) is used as the most recent set of figures that allows calculation of regional per capita food - fish supplies ( excluding molluscs and crustaceans ) in which fish imports and exports can also be taken into account . global fish supplies continue to expand , primarily as a result of the sustained , rapid growth in aquaculture production . supplies of food - fish from capture and farming increased by c. 62% , from 86 to 139 kg year , between 1961 and 2009 ( fig . increases during the 1960s1980s were largely due to increased capture fisheries landings while those since are largely attributable to aquaculture . per capita aquatic food ( , total fish and aquatic resources ; , total fish ) and fishes by species group ( , freshwater and diadromous fishes ; , demersal fishes ; , pelagic fishes ; , marine fishes ) by decade ( 19612001 ) and by year ( 20022009 ) . the statistics , however , mask enormous inter - regional differences in production and trade ( table 2 ) . asia , including china , accounts for c. 90% of global aquaculture production while production in least developed countries is < 1% ( fao , 2012a ) . seafood is the most highly traded food commodity internationally and exports of seafood from developing countries exceed the total value of coffee , cocoa , tea , tobacco , meat and rice combined ( smith et al . , 2010 ; deutsch et al . , 2011 ) . trade increased in value by an average of nearly 11% per annum between 1976 and 2009 ( fao , 2012b ) . the effect of trade on food - fish availability at regional level can be assessed from the fao food - fish balance statistics that account for production plus imports , minus fish used for non - food uses ( e.g. fishmeal ) and exports ( fao fishstat , 2012 ) . the data for 2009 show that regional per capita food - fish supplies are greatest in oceania ( 189 kg year ) , followed by europe ( 174 kg year ) and asia ( 150 kg year ) , with africa ( 92 kg year ) trailing last ( table 2 ) . high food - fish availability in north america and europe is in part sustained by net imports from asian countries such as china and vietnam , accounting for > 20 and 26% of supplies in north america and europe , respectively ( fao fishstat , 2012 ) . low production costs and relatively high international prices , combined with liberal trade policies , have increased exports of farmed white - fleshed fishes such as tilapia oreochromis spp . and catfishes pangasius spp . , substituting for dwindling supplies from domestic white - fish fisheries ( norman - lpez & bjorndal , 2009 ; beveridge et al . even africa is a net food - fish importer , imports of mainly cheap , oily pelagic fishes increasing net food - fish availability by 14% . total and per capita food - fish supplies by region and economic grouping , 2009 ( from fao , 2011b ; fao fishstat , 2012 ) data for central and south america and caribbean combined . while trade data do not indicate whether the fish is of farmed origin , with key commodities such as oreochromis spp . and pangasius spp . allison ( 2011 ) found that effects of fish trade on poverty and food and nutrition security ranged from large and positive to negative and small . with the exception of tuna ( thunnini ) , increases in both per capita food - fish supplies and the value of trade only occurred when there was strong growth in aquaculture ( e.g. in bangladesh , indonesia and vietnam ) . increases in aquaculture enhance food - fish availability in countries where sector growth is strong . although annual per capita food - fish availability in low - income food - deficit countries ( lifdcs ) grew from 45 kg in 1961 to 105 kg in 2009 , it remains comparatively low ( fao , 2011b ; fao moreover , in africa , where fish constitutes a particularly large proportion of animal - source foods , per capita food availability of fish has increased little over the past 20 years , in large measure due to the limited growth of aquaculture . it is not just the availability of food - fish per se that is of concern , however , rather the nutrients that fish provide . qualitatively , fish supplies have changed markedly over the past 50 years . in 1961 , 32% of food fishes were of demersal origin , 26% were pelagic , 16% were fresh water and diadromous and 12% comprised other ( i.e. non - demersal and pelagic ) marine fishes ( fig . the availability of demersal and other marine fishes had fallen in both absolute and relative terms , that of pelagic fishes had increased in terms of biomass but had fallen as a proportion of total food - fish supplies , while that of freshwater and diadromous fishes had increased by c. 250% . annual per capita freshwater and diadromous food - fish supplies in 2009 were 61 kg , equivalent to one - third of supplies ( fig . the changes largely result from stagnation in capture fisheries production and the large rise in carps ( cypriniformes ) , oreochromis spp . and salmonid aquaculture . the species being farmed are largely determined by ease of culture and profitability . while changes in the fishes eaten are likely to have some effect on protein and energy supplies , they may have an even greater effect on the availability of dietary lipids ( see table 1 ) . demersal white fishes ( gadoids ) typically have low levels ( < 2% ) of fat . pelagic herring , anchovies and sardines ( clupeids ) , as well as salmonids , mackerels ( scombrids ) and eel ( anguilla spp . ) , are oily fishes ( > 2% oil content ) , with levels in mackerel scomber scombrus l. 1758 seasonally exceeding 20% ( wallace , 1991 ) . while species is a major determinant of farmed fish nutrient content , nutrient composition is also influenced by how the animal is farmed . farm environmental conditions , especially water temperature , quantitatively and qualitatively influence lipid deposition ( haard , 1992 ) . more important , however , is the role of food . while the majority of farmed fishes are freshwater omnivores and herbivores , there has nonetheless been a trend towards intensification of production methods , involving increasing reliance on nutritionally complete feeds ( tacon & metian , 2008 ) . traditional pond - based fish farming methods use inorganic and organic fertilizers to stimulate autotrophic and heterotrophic production ( hepher , 1988 ) . production is further enhanced by the administration of small quantities of supplemental feeds , generally derived from long - chain carbohydrate - based agricultural by - products ( e.g. rice and wheat bran ) ( hepher , 1988 ; avinmelech , 1999 ) . the intensification of rearing methods responsible for increasing production per unit land and water is primarily driven by economics : the growing scarcity and thus value of land and fresh water , together with the price of feed and the strong market for fish ( van der zijpp et al . , 2007 ) . total lipid content of farmed fishes is typically higher due to differences between wild and farmed environments in terms of feed availability and composition and animal activity ( vliet & katan , 1990 ; haard , 1992 ; grigorakis et al . also have lower -3:-6 fatty acids , the intensity of production methods ( i.e. the amount and composition of external feed used ) having a particularly important influence on fish lipid levels and composition ( grigorakis et al . , 2002 ; karapanagiotidis et al . while aquaculture has increased global food - fish supplies , it has also radically changed the availability of certain food - fish types , species and nutrients consumed via fishes . freshwater species , in particular cyprinids and tilapines , increasingly dominate food - fish supplies . while this may have marginal effects on protein supplies per unit food - fish , it changes the quantities and types of lipid . farming methods have a further effect on fish lipid content and composition : intensification of production methods tends to increase fat levels and , where plant - based diets in particular are used , alter fat composition . the significance of these changes for food and nutrition security can only be determined in the context of individual food consumption and health . availability alone is insufficient to ensure household food security : access to food , determined by power relations , poverty , lack of assets and prices that are increasingly governed by globalized markets , is also critical ( fig . although global food supplies undoubtedly have been improving , growing competition for use of farm land for biofuels , lack of key inputs in some places , natural disasters and increasing dependence of food production systems on energy have , since 2006 , resulted in high and volatile food prices , a situation that is likely to continue for some time ( fao , 2011a ) . poor , food import - dependent countries , with inadequate resources and capacity to put into practice policies targeted at incomes and expenditure , markets and prices or implement food safety nets , will continue to be disproportionally affected ( fao , 2011a ) . fisheries and aquaculture can improve household food security ( prein & ahmed , 2000 ; dey et al . , 2006 ; thilsted , 2012 ) . the pathways by which this occurs have been elaborated by kawarazuka & bn ( 2010 ) : directly through consumption of fish and indirectly through increased purchasing power from the sale of fish ( i.e. increased access ) ( fig . the latter was found to be especially true when women engage in aquaculture and fisheries - related activities such as processing and trading , which results in a greater proportion of income being spent on food ( i.e. improved use ) ( kawarazuka & bn , 2010 ) . worldwide , an estimated 120 10 people are engaged in fishing ( world bank , 2010 ) and 23 10 in aquaculture ( valderrama et al . , 2010 ) , increasing their physical access to and , it is hypothesized , consumption of fish . the great majority of people in developing countries do not of course produce their own fish and physical access is largely determined by distance from source . drying , salting and smoking extend the life of this highly perishable commodity , enabling distribution over far greater areas , albeit at some deterioration in nutritional quality ( roos et al . , 2002 ) . there have been few specific studies and no comprehensive review of how increases in farmed - fish availability affect access and use by poor consumers ( kawarazuka & bn , 2010 ; allison , 2011 ) . the growth of aquaculture has undoubtedly increased access in production hotspots such as the nile delta , egypt , but despite the volumes produced , physical access in distant upper egypt remains poor ( macfadyen et al . , 2012 ) . moreover , in subsaharan africa , the fish aquaculture sector remains dominated by smallholder , subsistence - type operations and physical access to the relatively small amounts of fish produced is largely limited to producer households and neighbours ( dey et al . the greatest influence on access to food is prices , which are largely determined by markets and incomes ( fig . the consumption of meat and fish , which are expensive compared to starchy staples such as rice or maize , is highly linked to wealth ( speedy , 2003 ) . low - income consumers therefore preferentially purchase less expensive fishes , such as small pelagic species , high in lcpufas ( tacon & metian , 2009 ; kabahenda et al . , 2012 ) . in southern africa , for example , the cheapest fishes are the universally available small , dried kapenta limnothrissa spp . quantities as little as a few tens of grams are purchased and used with vegetables to prepare relishes that accompany the staple maize porridge . a review of studies of fish price elasticities of demand in western markets concluded that demand responds closely to price ( gallet , 2009 ) . in developing countries , price is likely to be an even greater determinant of whether poor consumers buy fish or not . where aquaculture production dominates the market , farmed fish tends to be cheaper , increasing economic access . in egypt , for example , farmed fish now account for two - thirds of all fish consumed and is considerably less expensive than most wild - caught fish ( gafrd , 2010 ) . in bangladesh , a decade ago , farmed fish was more expensive than wild - caught fish ( thompson et al . , 2002 ) . today , due to a doubling of farmed - fish production , however , farmed fish is generally less expensive than wild - caught fish ( b. belton , pers . although unsupported by rigorous study , it is hypothesized that aquaculture producers in developing countries tend to target the production of larger - sized fish , aimed at middle - class urban regional and international markets ( unhrc , 2012 ) , presumably in the expectation that the higher absolute and relative prices such fish command increase profits . it is proposed here that there is a largely unarticulated and increasingly unmet demand for economically accessible fish by poor consumers , who are in the majority in developing countries . given any particular species , and irrespective of whether fresh or frozen , smaller fish are generally less expensive . in a study conducted in bangladesh throughout 2011 , for example , market prices of farmed 250 g oreochromis spp . were 2030% less per kg than that of 500 g fish ( b. belton , unpubl . while production of smaller , less expensive fish undoubtedly increases access of poor consumers , from a food and nutrition security perspective the point is not how much fish is eaten but that fish consumption fulfils its potential to help meet nutritional needs , especially of poor and vulnerable consumers . the most important role that fish plays in diets dominated by starchy staples is the provision of essential fatty acids and highly bioavailable micronutrients ( gibson et al . , 2003 ; roos et al . , 2003 , 2007a , b , c , d ; hambraeus , 2009 ; kawarazuka & bn , 2010 , 2011 ) from this perspective , oily fishes and small fishes that are eaten whole are superior . small fish species , however , are generally not farmed , an exception being in bangladesh , where small species , e.g. mola amblypharyngodon mola ( hamilton 1822 ) and chanda parambassis baculis ( hamilton 1822 ) , are allowed to enter freshwater fish ponds from adjacent water bodies ( roos et al . , 2002 ) . while incorporation of small fish species into cyprinid - based pond polycultures increases overall production and revenues , seed remains unavailable from commercial hatcheries ( alim et al . the absence of obvious candidate small fish species for aquaculture and the likely high associated seed costs may make their culture unprofitable , except where self - recruitment occurs . there also seems to be little market for very small - sized ( i.e. that can be eaten whole ) commonly farmed fishes , such as oreochromis spp . or cyprinids . a healthy diet depends on the consumption and absorption of the appropriate amounts of energy in the form of carbohydrate , fats and protein , as well as essential fatty acids , amino acids , minerals and vitamins ( fig . inadequate food intake , compounded by the body 's inability to utilize needed nutrients , results in undernutrition . nutritional requirements are determined by genetic makeup , sex , age and health status , as well as physiological demands associated with factors such as pregnancy and physical labour . what individuals choose to eat has always been determined not only by availability and access but also by taste , nutritional value , culture and religion . personal perspectives and social contexts ( life experiences , reliance on foods away from home , food advertising , marketing and promotion ) are increasingly influential . the result is that a complex mix of product attributes such as convenience , quality , freshness , health and nutrition beliefs , environmental and animal welfare concerns and social relationships now determines western consumer food choices ( furst et al . , 1996 ; lennerns et al . , 1997 it is no surprise then that the importance of fish in household food baskets varies enormously geographically and temporally . demographic characteristics , education , place of purchase and generic advertising have been found to determine beliefs and attitudes about aquaculture production ( fernndez - polanco & lunes , 2012 ) . a survey of women consumers in belgium , for example , found that consumption of wild - caught fish was influenced by sustainability and ethical considerations whilst that of farmed fish by perceptions of quality ( verbeke et al . , 2007 ) . in bangladesh , much of the increase in aquaculture production has been of exotic species , such as oreochromis spp . , which are less preferred than the increasingly scarce indigenous wild fishes ( b. belton , pers . comm . ) . it seems , therefore , that there can be consumer resistance to farmed fish , especially unfamiliar exotic species . use may change over time , especially where availability and economic access increase . at the household level , how food is handled , prepared and cooked influences the nutritional value of what is consumed ( kabahenda et al . , 2012 ) . the quantities of food distributed among family members depend on cultural and power determined norms with respect to sex and age in particular ( wheeler , 1991 ) . traditional products such as dried , smoked , salted and fermented small fishes are usually cooked as a mixed curry or stew dish , with a little oil , vegetables and spices . such dishes , as well as fish paste and fish sauces , are more readily shared among family members than larger farmed fishes . individual health status determines how effectively the body utilizes what is consumed ( caulfield et al . , 2006 ) . the institutions and policies that regulate air quality , adequacy of housing ( especially in relation to provision of clean water and sanitation ) and access to health care influence the prevalence of infectious , diarrhoeal and acute respiratory diseases among the poor in developing countries ( cairncross & valdmanis , 2006 ) . while only the flesh and sometimes the skin or heads of larger fishes tend to be consumed , small fishes are often eaten whole . the micronutrient content of small fishes is often high ( see table 1 ) but the micronutrients are concentrated in the bones , heads and gut , and these fishes must be eaten whole to derive full nutritional benefit ( steiner - asiedu et al . , 1993 ; larsen et al . , 2000 ; roos et al . , 2002 , 2003 , 2007d , ) the contribution of fish as rich sources of vitamins and minerals is poorly documented and often overlooked . studies from rural bangladesh have shown that a number of widely occurring small tropical freshwater fish species , such as a. mola , p. baculis , dhela osteobrama cotio cotio ( hamilton 1822 ) and darkina esomus danricus ( hamilton 1822 ) , have high contents of pre - formed vitamin a ( thilsted et al . , 1997 ; roos et al . , 2007a ) . because most of the small fish species are eaten whole , they are also a very rich source of highly bioavailable calcium ( hansen et al . , 1998 ; esomus danricus and trey changwa plieng esomus longimanus ( lunel 1882 ) from cambodia have a high iron and zinc content ( roos et al . , 2007c ) . a traditional daily meal of rice and sour soup made with e. longimanus can meet 45% of the daily iron requirement of a cambodian woman ( roos et al . table 1 shows the vitamin a , calcium , iron and zinc content of some selected common small fishes , as well as two commonly farmed large freshwater fishes . cooking affects the macro and micronutrient content of fish ( kabahenda et al . , 2012 ) . minerals are generally conserved during cooking but vitamins such as thiamine , cobalamine and niacin are often lost or denatured ( holland et al . , 1991 ) . consuming fish as part of a meal also confers additional benefits in terms of increasing the bioavailability of iron and zinc from other foods in a meal ( aung - than - batu et al . , 1976 ) . vulnerability , a product of exposure to risk factors such as seasonality in supply , conflict , rising prices and disease , and the ability to cope with such shocks , as established by assets ( physical and educational ) and degree of social marginalization , determines the stability of food and nutrition security ( fig . inadequate food access and utilization on a periodic basis , especially during key life stages such as the first 1000 days of life , can have long - term consequences for an active and healthy life ( caulfield et al . , 2006 ; ruel et al . , 2008 farming improves the continuity of global fish supplies , but locally , export - oriented aquaculture increasingly falls into a gilded trap in focusing on economic rather than socio - ecological aspects ( steneck et al . , 2011 ) , which can undermine rather than improve local access to fish food . the availability of food - fish is a function of production and trade , less that used for non - food purposes . the term food - fish is applied here exclusively to fish and does not include shellfish , as is usually the case , used directly for human consumption . unless otherwise stated , fao data for 2009 ( fao faostat , 2013 ) is used as the most recent set of figures that allows calculation of regional per capita food - fish supplies ( excluding molluscs and crustaceans ) in which fish imports and exports can also be taken into account . global fish supplies continue to expand , primarily as a result of the sustained , rapid growth in aquaculture production . supplies of food - fish from capture and farming increased by c. 62% , from 86 to 139 kg year , between 1961 and 2009 ( fig . increases during the 1960s1980s were largely due to increased capture fisheries landings while those since are largely attributable to aquaculture . per capita aquatic food ( , total fish and aquatic resources ; , total fish ) and fishes by species group ( , freshwater and diadromous fishes ; , demersal fishes ; , pelagic fishes ; , marine fishes ) by decade ( 19612001 ) and by year ( 20022009 ) . the statistics , however , mask enormous inter - regional differences in production and trade ( table 2 ) . asia , including china , accounts for c. 90% of global aquaculture production while production in least developed countries is < 1% ( fao , 2012a ) . seafood is the most highly traded food commodity internationally and exports of seafood from developing countries exceed the total value of coffee , cocoa , tea , tobacco , meat and rice combined ( smith et al . , 2010 ; deutsch et al . , 2011 ) . trade increased in value by an average of nearly 11% per annum between 1976 and 2009 ( fao , 2012b ) . the effect of trade on food - fish availability at regional level can be assessed from the fao food - fish balance statistics that account for production plus imports , minus fish used for non - food uses ( e.g. fishmeal ) and exports ( fao fishstat , 2012 ) . the data for 2009 show that regional per capita food - fish supplies are greatest in oceania ( 189 kg year ) , followed by europe ( 174 kg year ) and asia ( 150 kg year ) , with africa ( 92 kg year ) trailing last ( table 2 ) . high food - fish availability in north america and europe is in part sustained by net imports from asian countries such as china and vietnam , accounting for > 20 and 26% of supplies in north america and europe , respectively ( fao fishstat , 2012 ) . low production costs and relatively high international prices , combined with liberal trade policies , have increased exports of farmed white - fleshed fishes such as tilapia oreochromis spp . and catfishes pangasius spp . , substituting for dwindling supplies from domestic white - fish fisheries ( norman - lpez & bjorndal , 2009 ; beveridge et al . even africa is a net food - fish importer , imports of mainly cheap , oily pelagic fishes increasing net food - fish availability by 14% . total and per capita food - fish supplies by region and economic grouping , 2009 ( from fao , 2011b ; fao fishstat , 2012 ) data for central and south america and caribbean combined . while trade data do not indicate whether the fish is of farmed origin , with key commodities such as oreochromis spp . and pangasius spp . allison ( 2011 ) found that effects of fish trade on poverty and food and nutrition security ranged from large and positive to negative and small . with the exception of tuna ( thunnini ) , increases in both per capita food - fish supplies and the value of trade only occurred when there was strong growth in aquaculture ( e.g. in bangladesh , indonesia and vietnam ) . increases in aquaculture enhance food - fish availability in countries where sector growth is strong . although annual per capita food - fish availability in low - income food - deficit countries ( lifdcs ) grew from 45 kg in 1961 to 105 kg in 2009 , it remains comparatively low ( fao , 2011b ; fao moreover , in africa , where fish constitutes a particularly large proportion of animal - source foods , per capita food availability of fish has increased little over the past 20 years , in large measure due to the limited growth of aquaculture . it is not just the availability of food - fish per se that is of concern , however , rather the nutrients that fish provide . qualitatively , fish supplies have changed markedly over the past 50 years . in 1961 , 32% of food fishes were of demersal origin , 26% were pelagic , 16% were fresh water and diadromous and 12% comprised other ( i.e. non - demersal and pelagic ) marine fishes ( fig . the availability of demersal and other marine fishes had fallen in both absolute and relative terms , that of pelagic fishes had increased in terms of biomass but had fallen as a proportion of total food - fish supplies , while that of freshwater and diadromous fishes had increased by c. 250% . annual per capita freshwater and diadromous food - fish supplies in 2009 were 61 kg , equivalent to one - third of supplies ( fig . the changes largely result from stagnation in capture fisheries production and the large rise in carps ( cypriniformes ) , oreochromis spp . and salmonid aquaculture . the species being farmed are largely determined by ease of culture and profitability . while changes in the fishes eaten are likely to have some effect on protein and energy supplies , they may have an even greater effect on the availability of dietary lipids ( see table 1 ) . demersal white fishes ( gadoids ) typically have low levels ( < 2% ) of fat . pelagic herring , anchovies and sardines ( clupeids ) , as well as salmonids , mackerels ( scombrids ) and eel ( anguilla spp . ) , are oily fishes ( > 2% oil content ) , with levels in mackerel scomber scombrus l. 1758 seasonally exceeding 20% ( wallace , 1991 ) . while species is a major determinant of farmed fish nutrient content , nutrient composition is also influenced by how the animal is farmed . farm environmental conditions , especially water temperature , quantitatively and qualitatively influence lipid deposition ( haard , 1992 ) . more important , however , is the role of food . while the majority of farmed fishes are freshwater omnivores and herbivores , there has nonetheless been a trend towards intensification of production methods , involving increasing reliance on nutritionally complete feeds ( tacon & metian , 2008 ) . traditional pond - based fish farming methods use inorganic and organic fertilizers to stimulate autotrophic and heterotrophic production ( hepher , 1988 ) . production is further enhanced by the administration of small quantities of supplemental feeds , generally derived from long - chain carbohydrate - based agricultural by - products ( e.g. rice and wheat bran ) ( hepher , 1988 ; avinmelech , 1999 ) . the intensification of rearing methods responsible for increasing production per unit land and water is primarily driven by economics : the growing scarcity and thus value of land and fresh water , together with the price of feed and the strong market for fish ( van der zijpp et al . , 2007 ) . total lipid content of farmed fishes is typically higher due to differences between wild and farmed environments in terms of feed availability and composition and animal activity ( vliet & katan , 1990 ; haard , 1992 ; grigorakis et al . also have lower -3:-6 fatty acids , the intensity of production methods ( i.e. the amount and composition of external feed used ) having a particularly important influence on fish lipid levels and composition ( grigorakis et al . , 2002 ; karapanagiotidis et al . while aquaculture has increased global food - fish supplies , it has also radically changed the availability of certain food - fish types , species and nutrients consumed via fishes . freshwater species , in particular cyprinids and tilapines , increasingly dominate food - fish supplies . while this may have marginal effects on protein supplies per unit food - fish , it changes the quantities and types of lipid . farming methods have a further effect on fish lipid content and composition : intensification of production methods tends to increase fat levels and , where plant - based diets in particular are used , alter fat composition . the significance of these changes for food and nutrition security can only be determined in the context of individual food consumption and health . availability alone is insufficient to ensure household food security : access to food , determined by power relations , poverty , lack of assets and prices that are increasingly governed by globalized markets , is also critical ( fig . 1 ) . although global food supplies undoubtedly have been improving , growing competition for use of farm land for biofuels , lack of key inputs in some places , natural disasters and increasing dependence of food production systems on energy have , since 2006 , resulted in high and volatile food prices , a situation that is likely to continue for some time ( fao , 2011a ) . poor , food import - dependent countries , with inadequate resources and capacity to put into practice policies targeted at incomes and expenditure , markets and prices or implement food safety nets , will continue to be disproportionally affected ( fao , 2011a ) . fisheries and aquaculture can improve household food security ( prein & ahmed , 2000 ; dey et al . , 2006 ; thilsted , 2012 ) . the pathways by which this occurs have been elaborated by kawarazuka & bn ( 2010 ) : directly through consumption of fish and indirectly through increased purchasing power from the sale of fish ( i.e. increased access ) ( fig . 1 ) . the latter was found to be especially true when women engage in aquaculture and fisheries - related activities such as processing and trading , which results in a greater proportion of income being spent on food ( i.e. improved use ) ( kawarazuka & bn , 2010 ) . worldwide , an estimated 120 10 people are engaged in fishing ( world bank , 2010 ) and 23 10 in aquaculture ( valderrama et al . , 2010 ) , increasing their physical access to and , it is hypothesized , consumption of fish . the great majority of people in developing countries do not of course produce their own fish and physical access is largely determined by distance from source . drying , salting and smoking extend the life of this highly perishable commodity , enabling distribution over far greater areas , albeit at some deterioration in nutritional quality ( roos et al . there have been few specific studies and no comprehensive review of how increases in farmed - fish availability affect access and use by poor consumers ( kawarazuka & bn , 2010 ; allison , 2011 ) . the growth of aquaculture has undoubtedly increased access in production hotspots such as the nile delta , egypt , but despite the volumes produced , physical access in distant upper egypt remains poor ( macfadyen et al . , 2012 ) . moreover , in subsaharan africa , the fish aquaculture sector remains dominated by smallholder , subsistence - type operations and physical access to the relatively small amounts of fish produced is largely limited to producer households and neighbours ( dey et al . , 2006 ; brummett et al . , 2008 ; kawarazuka & bn , 2010 ) . the greatest influence on access to food is prices , which are largely determined by markets and incomes ( fig . the consumption of meat and fish , which are expensive compared to starchy staples such as rice or maize , is highly linked to wealth ( speedy , 2003 ) . low - income consumers therefore preferentially purchase less expensive fishes , such as small pelagic species , high in lcpufas ( tacon & metian , 2009 ; kabahenda et al . , 2012 ) . in southern africa , for example , the cheapest fishes are the universally available small , dried kapenta limnothrissa spp . and other associated freshwater pelagic species . quantities as little as a few tens of grams are purchased and used with vegetables to prepare relishes that accompany the staple maize porridge . a review of studies of fish price elasticities of demand in western markets concluded that demand responds closely to price ( gallet , 2009 ) . in developing countries , price is likely to be an even greater determinant of whether poor consumers buy fish or not . where aquaculture production dominates the market , farmed fish tends to be cheaper , increasing economic access . in egypt , for example , farmed fish now account for two - thirds of all fish consumed and is considerably less expensive than most wild - caught fish ( gafrd , 2010 ) . in bangladesh , a decade ago , farmed fish was more expensive than wild - caught fish ( thompson et al . , 2002 ) . today , due to a doubling of farmed - fish production , however , farmed fish is generally less expensive than wild - caught fish ( b. belton , pers . although unsupported by rigorous study , it is hypothesized that aquaculture producers in developing countries tend to target the production of larger - sized fish , aimed at middle - class urban regional and international markets ( unhrc , 2012 ) , presumably in the expectation that the higher absolute and relative prices such fish command increase profits . it is proposed here that there is a largely unarticulated and increasingly unmet demand for economically accessible fish by poor consumers , who are in the majority in developing countries . given any particular species , and irrespective of whether fresh or frozen , smaller fish are generally less expensive . in a study conducted in bangladesh throughout 2011 , for example , market prices of farmed 250 g oreochromis spp . were 2030% less per kg than that of 500 g fish ( b. belton , unpubl . while production of smaller , less expensive fish undoubtedly increases access of poor consumers , from a food and nutrition security perspective the point is not how much fish is eaten but that fish consumption fulfils its potential to help meet nutritional needs , especially of poor and vulnerable consumers . the most important role that fish plays in diets dominated by starchy staples is the provision of essential fatty acids and highly bioavailable micronutrients ( gibson et al . , 2003 ; roos et al . , 2003 , 2007a , b , c , d ; hambraeus , 2009 ; kawarazuka & bn , 2010 , 2011 ) . from this perspective , oily fishes and small fishes that are eaten whole are superior . small fish species , however , are generally not farmed , an exception being in bangladesh , where small species , e.g. mola amblypharyngodon mola ( hamilton 1822 ) and chanda parambassis baculis ( hamilton 1822 ) , are allowed to enter freshwater fish ponds from adjacent water bodies ( roos et al . , 2002 ) . while incorporation of small fish species into cyprinid - based pond polycultures increases overall production and revenues , seed remains unavailable from commercial hatcheries ( alim et al . , 2004 ; kadir et al . , the absence of obvious candidate small fish species for aquaculture and the likely high associated seed costs may make their culture unprofitable , except where self - recruitment occurs . there also seems to be little market for very small - sized ( i.e. that can be eaten whole ) commonly farmed fishes , such as oreochromis spp . or cyprinids . a healthy diet depends on the consumption and absorption of the appropriate amounts of energy in the form of carbohydrate , fats and protein , as well as essential fatty acids , amino acids , minerals and vitamins ( fig . inadequate food intake , compounded by the body 's inability to utilize needed nutrients , results in undernutrition . nutritional requirements are determined by genetic makeup , sex , age and health status , as well as physiological demands associated with factors such as pregnancy and physical labour . what individuals choose to eat has always been determined not only by availability and access but also by taste , nutritional value , culture and religion . personal perspectives and social contexts ( life experiences , reliance on foods away from home , food advertising , marketing and promotion ) are increasingly influential . the result is that a complex mix of product attributes such as convenience , quality , freshness , health and nutrition beliefs , environmental and animal welfare concerns and social relationships now determines western consumer food choices ( furst et al . , 1996 ; it is no surprise then that the importance of fish in household food baskets varies enormously geographically and temporally . demographic characteristics , education , place of purchase and generic advertising have been found to determine beliefs and attitudes about aquaculture production ( fernndez - polanco & lunes , 2012 ) . a survey of women consumers in belgium , for example , found that consumption of wild - caught fish was influenced by sustainability and ethical considerations whilst that of farmed fish by perceptions of quality ( verbeke et al . , 2007 ) . in bangladesh , much of the increase in aquaculture production has been of exotic species , such as oreochromis spp . , which are less preferred than the increasingly scarce indigenous wild fishes ( b. belton , pers . comm . ) . it seems , therefore , that there can be consumer resistance to farmed fish , especially unfamiliar exotic species . use may change over time , especially where availability and economic access increase . at the household level , how food is handled , prepared and cooked influences the nutritional value of what is consumed ( kabahenda et al . , 2012 ) . the quantities of food distributed among family members depend on cultural and power determined norms with respect to sex and age in particular ( wheeler , 1991 ) . traditional products such as dried , smoked , salted and fermented small fishes are usually cooked as a mixed curry or stew dish , with a little oil , vegetables and spices . such dishes , as well as fish paste and fish sauces , are more readily shared among family members than larger farmed fishes . individual health status determines how effectively the body utilizes what is consumed ( caulfield et al . , 2006 ) . the institutions and policies that regulate air quality , adequacy of housing ( especially in relation to provision of clean water and sanitation ) and access to health care influence the prevalence of infectious , diarrhoeal and acute respiratory diseases among the poor in developing countries ( cairncross & valdmanis , 2006 ) . while only the flesh and sometimes the skin or heads of larger fishes tend to be consumed , small fishes are often eaten whole . the micronutrient content of small fishes is often high ( see table 1 ) but the micronutrients are concentrated in the bones , heads and gut , and these fishes must be eaten whole to derive full nutritional benefit ( steiner - asiedu et al . , 1993 ; , 2000 ; roos et al . , 2002 , 2003 , 2007d , ) . the contribution of fish as rich sources of vitamins and minerals is poorly documented and often overlooked . studies from rural bangladesh have shown that a number of widely occurring small tropical freshwater fish species , such as a. mola , p. baculis , dhela osteobrama cotio cotio ( hamilton 1822 ) and darkina esomus danricus ( hamilton 1822 ) , have high contents of pre - formed vitamin a ( thilsted et al . because most of the small fish species are eaten whole , they are also a very rich source of highly bioavailable calcium ( hansen et al . , 1998 ; larsen et al . , 2000 ) . esomus danricus and trey changwa plieng esomus longimanus ( lunel 1882 ) from cambodia have a high iron and zinc content ( roos et al . , 2007c ) . a traditional daily meal of rice and sour soup made with e. longimanus can meet 45% of the daily iron requirement of a cambodian woman ( roos et al . , 2007b table 1 shows the vitamin a , calcium , iron and zinc content of some selected common small fishes , as well as two commonly farmed large freshwater fishes . cooking affects the macro and micronutrient content of fish ( kabahenda et al . , 2012 ) . minerals are generally conserved during cooking but vitamins such as thiamine , cobalamine and niacin are often lost or denatured ( holland et al . , 1991 ) . consuming fish as part of a meal also confers additional benefits in terms of increasing the bioavailability of iron and zinc from other foods in a meal ( aung - than - batu et al . , 1976 ) . vulnerability , a product of exposure to risk factors such as seasonality in supply , conflict , rising prices and disease , and the ability to cope with such shocks , as established by assets ( physical and educational ) and degree of social marginalization , determines the stability of food and nutrition security ( fig . inadequate food access and utilization on a periodic basis , especially during key life stages such as the first 1000 days of life , can have long - term consequences for an active and healthy life ( caulfield et al . farming improves the continuity of global fish supplies , but locally , export - oriented aquaculture increasingly falls into a gilded trap in focusing on economic rather than socio - ecological aspects ( steneck et al . , 2011 ) , which can undermine rather than improve local access to fish food . fish is a preferred food item in the diets of many , especially poor , consumers . fish provides energy and is a superior source of protein to other animal source foods in terms of total protein and essential amino acid content and digestibility ( kawarazuka & bn , 2011 ) . it is as a source of essential fatty acids and micronutrients , superior in both qualitative and quantitative terms to other animal - source foods ( table 1 ) , that may be of greatest importance in food and nutrition security terms . aquaculture accounts for an increasing proportion of global food - fish supplies and , indeed , has increased global per capita food - fish supplies . market demand , resulting more from urbanization and increasing wealth than from population growth , is likely to continue to drive expansion of global aquaculture production , albeit that the rate of increase is reducing as constraints on and costs of resources and essential ecosystem services ( feedstuffs , space and capacity to disseminate and assimilate wastes ) increasingly affect the sector ( hall et al . , 2011 ; merino et al . , fish farming is largely absent from those parts of the world where it is most needed , especially sub - saharan africa , however , despite favourable soils and climate . here , arguably , there has been an over - emphasis on promotion of subsistence aquaculture , which , while improving resilience of poor smallholders to external shocks ( dey et al . , 2006 ) , has proved costly to implement and has had limited effect on incomes or food and nutrition security ( brummett et al . , 2008 ) . there is , however , an increasing focus on market - based aquaculture that is resulting in marked increases in aquaculture production ( beveridge et al . , 2010 ) , although whether this will suffice to meet growing demand is a moot point . in much of sub - saharan africa , growth in demand between 2007 and 2015 is expected to be 2550% ( cai , 2011 ) . assuming little change in capture fisheries or import supplies , demand must be met by aquaculture , but the required rates of sector growth , in the order of 40140% ( cai , 2011 ) , are much higher than are taking place . in addition to increasing availability , especially in those parts of the world where fish is most needed , aquaculture must also increase access . a number of economists have identified low - income markets as potential growth areas ( prahalad , 2004 ; diamandis & kotler , 2012 ) . there are many good economic and environmental reasons to produce smaller - sized herbivorous and omnivorous fishes or fish products for poorer consumers . in addition to increased economic access , production per unit land use ( productivity ) is higher and it is more energy efficient and cheaper per unit biomass to produce smaller - sized fishes , a result of better food conversion ratios ( fcr ) and lower production costs ( robinson & li , 2010 ; tlusty et al . , 2011 ) . because it takes less time to produce smaller fishes , farmers may be able to produce several harvests per season . moreover , risks ( mortality , price changes and natural disasters ) are lower ( pascoe et al . , 2002 ; bauer & schlott , 2009 ) and the cost of any borrowing is also reduced . surprisingly , little research has been done on production economics and on these so - called bottom - of - the - pyramid markets . access to food can also be increased by greater policy focus on incomes and expenditure , markets and prices ( fao , 2011a ) . while such instruments are often used to increase access to starchy staples , more evidence is needed to show why increasing access to fish is an effective and cost - efficient way to improve food and nutrition security ( allison , 2011 ; kawarazuka & bn , 2011 ) . while production of smaller , less expensive fishes may increase access by poor consumers , from a food and nutrition security perspective ( unhrc , 2012 ) the point is not how much fish is eaten but that the farmed fish meets the nutrition needs of poor and vulnerable consumers . farmed fish is an excellent source of animal protein but because of species , size and method of rearing it is often inferior to small wild fish species as a source of essential fatty acids and micronutrients . inclusion of fish - derived products , especially fish oil , in aquaculture diets can help improve the nutritional value of the finished product . given the importance of wild pelagic fishes in the diets of poor consumers , however , it is important that the growth of aquaculture does not increase the amount of fish of low economic value converted into fish meal and fish oil . there are strong economic drivers to reduce dietary fish oil content and over the past decade , much has been replaced by plant ( soy , flax and corn ) oils ( olsen , 2011 ) . judicious use of diets with higher fish oil inclusion rates in the weeks prior to harvesting can change fish fat content and fatty acid profile ( young , 2009 ) . the feed sector must continue to seek alternative feedstuffs that do not compromise the nutritional quality of farmed fishes . recent studies have also increased the understanding of fish lipid metabolism , indicating the possibility of selecting for strains of some species of farmed fishes with improved lcpufa biosynthesis capabilities ( leaver et al . , 2008 ) .

people who are food and nutrition insecure largely reside in asia and sub - saharan africa and for many , fish represents a rich source of protein , micronutrients and essential fatty acids . the contribution of fish to household food and nutrition security depends upon availability , access and cultural and personal preferences . access is largely determined by location , seasonality and price but at the individual level it also depends upon a person 's physiological and health status and how fish is prepared , cooked and shared among household members . the sustained and rapid expansion of aquaculture over the past 30 years has resulted in > 40% of all fish now consumed being derived from farming . while aquaculture produce increasingly features in the diets of many asians , it is much less apparent among those living in sub - saharan africa . here , per capita fish consumption has grown little and despite the apparently strong markets and adequate biophysical conditions , aquaculture has yet to develop . the contribution of aquaculture to food and nutrition security is not only just an issue of where aquaculture occurs but also of what is being produced and how and whether the produce is as accessible as that from capture fisheries . the range of fish species produced by an increasingly globalized aquaculture industry differs from that derived from capture fisheries . farmed fishes are also different in terms of their nutrient content , a result of the species being grown and of rearing methods . farmed fish price affects access by poor consumers while the size at which fish is harvested influences both access and use . this paper explores these issues with particular reference to asia and africa and the technical and policy innovations needed to ensure that fish farming is able to fulfil its potential to meet the global population 's food and nutrition needs .

a sound , agreeable or disagreeable , is a stimulus discerned by the sense of hearing . disagreeable or undesired sounds were described as noises , which may cause undesirable masking of sounds , may interfere with speech and communication , may produce pain , injury and brief or perpetual loss of hearing . the acoustic environment of learning - teaching activities at a dental college is characterized by high noise levels in relation to other teaching areas , due to the exaggerated noise produced by the use of dental equipment 's by many users at the same time . the sources of dental sounds that can be treated as potentially damaging to hearing are high - speed turbine handpieces , low - speed handpieces , high - velocity suction , ultrasonic instruments and cleaners , vibrators and other mixing devices , model trimmers and also worth mentioning are air conditioners . in dental learning areas , teachers and students exposure to noise constitutes a health risk and has both auditory and non - auditory effects . the non - auditory effects include hypertension , sleep disturbance , decreased learning performance , stress reactions , interference with communication and concentration , annoyance , mental fatigue and a reduction in efficiency . while auditory effects may include permanent hearing loss , and short term exposure to loud noise can cause a temporary change in hearing or a tinnitus . hearing loss caused by noise is referred to as noise induced hearing loss . according to the national institute for occupational safety and health ( niosh ) , exposure to noise levels above 80 db is associated with these consequences , which depends on the intensity of the noise , distance to the source , total duration of noise and the individual 's age , physical condition and sensitivity . furthermore , noise has an adverse impact on patients as a factor that may cause fear . exposure to noise is measured in units of sound pressure levels called decibels , named after alexander graham bell , using a - weighted sound levels [ db(a ) ] . the a - weighted sound levels closely match the perception of loudness by the human ear . niosh has recommended that all worker exposures to noise should be controlled below a level equivalent to 85 db(a ) for 8 h / day to minimize occupational noise induced hearing loss . noise pollution is one of the most important situations requiring a solution by the contemporary world . niosh has recognized noise as 1 of the 10 leading causes of work related diseases and injuries . in dental learning areas , teachers and students are vulnerable to different noise levels while working and teaching in dental clinics and laboratories . the aims of this study were to measure noise levels produced during the different dental learning clinics , by equipment 's used in dental learning areas under different working conditions and by used and brand new handpieces under different working conditions . in pre - clinical and clinical areas , the microphone was placed at ear level at a distance of 15 cm from a main noise source to simulate the auditory position of the operator . the noise levels of the equipment 's were measured at the corner of the learning area , which may consider the less noisy part of the learning area to eliminate as much as possible the interference with the external noise . the noise levels were measured over about 20 s interval and the maximum and minimum intensities in decibel was recorded . this was repeated three times sequentially in the same day , so we recorded 6 measurements for each equipment , one maximum and one minimum for each time interval . the noise levels of equipment 's used in laboratories , pre - clinical and clinical areas at the dental college of damascus university were measured under different working conditions . the laboratories , pre - clinical and clinical areas will be henceforth referred to as dental learning areas in this study . the equipment 's of which the noise levels were measured in clinical areas were : ultrasonic scaler , turbine , contra angle handpiece , micro motor handpiece , low volume suction pump , high volume suction pump and amalgamator ( capsule ) . the measurements were taken with the equipment only turned on ( without cutting ) and during cutting operations . ultrasonic scalers with or without suction pump and suction pumps running free and when they touch mucosa were measured for noise levels . the noise level of micro motor handpiece was measured by setting it at 35.000 rpm . the noise levels of different dental learning clinics was measured by placing the noise level meter at the center of the clinic during the middle - third of working time ( between 11 a.m. and 1 p.m. ) , which nearly represents the highest noise hours . the number of equipments , which were used at the same time , was 20 equipments . noise levels were measured in seven clinics : operative , fixed prosthodontics , removable prosthodontics , endodontics , pedodontics , oral surgery and periodontics . the sound levels were measured with a precision sound level meter ( beha unitest 93517 , germany ) with a microphone in dental learning areas . the sound level is measured on the a scale , which was designed to mimic the response of the human ear . at the dental laboratories , the microphone was placed near the technician 's ear at a distance of 15 cm from a main noise source to simulate the noise intensity reaching the eardrum and another reading was taken 2 m away . this was to simulate the person within a 2 m radius of the operator who is also exposed to the same noise . the equipments of which the noise levels were measured in the dental laboratories were : stone trimmer , automatic molding machine , manual molding machine and sandblaster . the data were collected , tabulated and statistically analyzed using t - tests with significance level set at 5% using the statistical package for the social sciences program version 13(spss inc . , chicago , il , usa ) . the results of the noise levels of equipments measured in dental laboratories at two distances of 15 cm and 2 m are shown in table 1 . noise levels [ db(a ) ] of dental laboratory engines the noisiest laboratory equipment recorded in this study was by the sandblaster with an la(eq ) of 93.32 1.99 at 15 cm distance . the results of the noise levels of the equipments measured in pre - clinical and clinical areas are shown in table 2 . noise levels [ db(a ) ] of the equipments measured in pre - clinical and clinical areas the results indicated that the maximum sound levels of equipments in dental clinics and laboratories were 92.2 db and 96 db , respectively . in dental clinics , the highest noise was produced by the micro motor handpiece while cutting on acrylic ( 92.2 db ) and the lowest noise ( 51.7 db ) was created by the ultrasonic scaler without suction pump . the highest noise in laboratories was caused by the sandblaster ( 96 db at a distance of 15 cm ) and the lowest noise by the stone trimmer while only turned on ( 61.8 db at a distance of 2 m ) . there was significant differences in noise levels of the equipments used in dental laboratories and dental learning clinics ( p = 0.007 ) . the mean noise levels of the equipments used in dental laboratories were much higher than those used in dental clinics . the mean noise level for dental laboratories engines at a distance of 15 cm from a main noise source was 81.62 db , compared with the mean value of 73.75 db for dental clinic equipments ( p = 0.009 ) . the laboratory engines had the highest noise levels ( at a distance of 15 cm from a main noise source-81.62 db ) , whereas the noise levels in high - speed turbine handpieces ( 75.44 db ) ( p = 0.003 ) and the low - speed contra angle handpieces ( 68.31 db ) ( p < 0.01 ) were decreased . the noise level of a contra angle handpiece at the clinical areas was significantly lower than at the pre - clinical areas ( p = 0.019 ) . noise levels of turbine in cutting activities compared to non - cutting showed that the noise levels measured during the cutting activities were significantly higher to those found when only turned on ( p < 0.01 ) . the average value of the difference was equal to 5.38 db(a ) and 19.45 db(a ) for brand new and used respectively . there was no significant difference in noise levels of contra angle handpiece in cutting activities compared to non - cutting activities in both pre - clinical and clinical areas for brand new and used ones . the average value of difference for turbine and contra angle handpieces ( only turned on ) was equal to 6.09 db(a ) . furthermore , there was no significant difference in noise levels of micro motor handpiece in cutting activities compared to non - cutting activities . there was no significant difference in noise levels between low and high volume suction pump . furthermore , there was no significant difference in noise levels of ultrasonic scaler without suction pump and with suction pump . noise levels of used equipments compared with brand new equipments in the clinics showed that the noise levels produced by used turbine were significantly higher than those produced by the brand new turbine ( p = 0.045 ) [ figure 1 ] . there was no significant difference in noise levels between brand new and used contra angle handpieces in both pre - clinical and clinical areas . average noise levels of used and brand new dental turbine compared in this study , the high - speed turbine was significantly noisier than low - speed contra angle ( p = 0.001 ) . the average value of difference for the high - speed turbine against low - speed contra angle handpiece was equal to 7.14 db(a ) [ figure 2 ] . mean noise levels of high - speed turbine and contra angle handpieces the results of the noise levels at the center of the dental learning clinics are shown in table 3 . the highest noise level for all dental clinics was at the pedodontic clinic ( 67.37 5.56 db(a ) ) ( p = 0.019 ) . in this study , noise levels of the equipments used in dental learning areas under different working conditions were measured . the noise levels measured in this study were similar to that measured in other international studies of noise in dentistry . noise levels for suction pump were 69.41 - 81.51 db(a ) in this study , whereas in the united kingdom they were 68 - 70 db(a ) , in portugal they were 70 - 74 db(a ) and in india they were 79 - 81 db(a ) . noise levels for the turbine were 66.72 - 84.16 db(a ) in this study , whereas in the united kingdom , portugal , india and saudi arabia were 70 - 75 db(a ) , 68 - 76 db(a ) , 75 - 81 db(a ) and 69 - 76d b(a ) , respectively ; for contra angle handpiece they were 66.12 - 70.5 db(a ) in this study , whereas in united kingdom , portugal , india and saudi arabia were 72 - 75 db(a ) , 69 - 75 db(a ) , 70 - 76 db(a ) and 65 - 71 db(a ) , respectively . the noise levels of turbine measured during the cutting activities were significantly higher to those found when only turned on . presented average values of + 10 db(a ) , in similar conditions of measurement in portugal and saudi arabia , respectively . the significant level of difference in noise levels of used turbines compared to brand new turbines could be an indication of bearing failure . the bearing resistance is affected by wear , not only of the metal surfaces , but also of the ball - cages , when roughness contributes to friction . in general , the used turbine was noisier at an average of about 9.11 db(a ) difference more than the brand new one [ figure 1 ] ; therefore , the hearing damage risk may be lesser among dentists who use brand new turbine . in this study , the high - speed turbine was the noisiest equipment compared to low - speed contra angle . this is concordant with antecedent studies mentioning that the high - speed turbine handpiece generates a higher noise level than the low - speed handpiece . maximum sound pressure levels of the noise created by the dental drill was 91.9 db by the brand used dental turbine while cutting on a tooth , which has a risk of damage to the dentists hearing . the noise level of a contra angle handpiece at the clinical areas was lower than at the pre - clinical areas , which may have been because students rarely used the maximum speed of the air contra angle handpiece during dental treatment , while in the pre - clinical area it was always used at the higher speeds . . dental laboratories in dental teaching institutions were the areas of highest noise levels when compared to other dental learning areas . the effect of noise on learner comfort affecting the work performance and mental efficiency has been researched . noise can induce learned helplessness , increase arousal , alter the choice of task strategy and decrease attention to the task . suggest the classification given by cavanaugh to set a limit value in places of learning in dental teaching institutions . accordingly , 56 db(a ) could be adequate as the upper limit value for a relaxed communication at a normal tone at 3 m. all the evaluated areas presented a value higher than this maximum . the highest noise level recorded for all dental clinics was at the pedodontic clinic . this may be due to the children who are normally crying during the oral health treatment . comparison of noise levels in this study with some european limits indicated that they did not comply with these laws . some international legal limits for equipment noise levels in la(eq ) ( db[a ] ) are : italy 40 , france 38 , sweden 35 , portugal 46 and india 50 . in this study , there were some high recorded measurements of noise levels , which have a risk of damage to the dentists hearing ( exceeding the limit of risk of hearing loss of 85 db[a ] ) such as stone trimmer , manual molding machine , sandblaster ( at distance 15 cm and 2 m ) , low volume suction pump , turbine ( brand used ) and micro motor handpiece . therefore , a necessary reduction of exposure in sound levels is required for acoustic comfort . based on the above study it can be concluded that the noise levels detected in this study were considered to be close to the limit of risk of hearing loss ( 85 db(a ) ) ; a necessary reduction of exposure in sound levels is required for acoustic comfort .

background : in dental practical classes , the acoustic environment is characterized by high noise levels in relation to other teaching areas . the aims of this study were to measure noise levels produced during the different dental learning clinics , by equipments used in dental learning areas under different working conditions and by used and brand new handpieces under different working conditions.materials and methods : the noise levels were measured by using a noise level meter with a microphone , which was placed at a distance of 15 cm from a main noise source in pre - clinical and clinical areas . in laboratories , the microphone was placed at a distance of 15 cm and another reading was taken 2 m away . noise levels of dental learning clinics were measured by placing noise level meter at clinic center . the data were collected , tabulated and statistically analyzed using t - tests . significance level was set at 5%.results : in dental clinics , the highest noise was produced by micro motor handpiece while cutting on acrylic ( 92.2 db ) and lowest noise ( 51.7 db ) was created by ultrasonic scaler without suction pump . the highest noise in laboratories was caused by sandblaster ( 96 db at a distance of 15 cm ) and lowest noise by stone trimmer when only turned on ( 61.8 db at a distance of 2 m ) . there was significant differences in noise levels of the equipment 's used in dental laboratories and dental learning clinics ( p = 0.007 ) . the highest noise level recorded in clinics was at pedodontic clinic ( 67.37 db).conclusions : noise levels detected in this study were considered to be close to the limit of risk of hearing loss 85 db .

defects in many of the molecules that regulate the cell cycle have been implicated in cancer initiation and progression . these include p53 , rb , p107 and prb2/p130 , and cdk inhibitors ( p15 , p16 , p18 , p19 , p21 , p27 ) , all of which act to prevent cell cycle progression until all repairs to damaged dna have been completed . in this section , we will review tumor suppressor genes that regulate cell cycle and apoptosis that have been found to be methylated in gastric cancers . tumor suppressor genes which are most recently found to be methylated in gastric cancers regulate cell cycle progression and apoptosis . kim et al . reported that promoter methylation of early b - cell factor-3 ( ebf3 ) was detected in 40.4% ( 42/104 ) of gastric cancer tissues but not in normal gastric tissues . the ebfs are a group of four highly conserved , dna - binding transcription factors with an atypical zinc - finger domain and a helix - loop - helix domain . functional analysis demonstrates that ebf3 represses gastric cancer cell growth and migration but activates cell cycle arrest and apoptosis . ebf3 up - regulates p21 and p27 in gastric cancer cells but represses cdk2 , cyclin d1 , cyclin d2 , and rb as previously reported in different types of cancers , . interestingly , in addition to their central roles in apoptosis regulation , the bcl-2 family of proteins influences the cell cycle , specifically the transition between quiescence and proliferation . bcl2l10 methylation was detected in 44.5% of gastric cancers and 21.34% of normal gastric mucosae ( p < 0.05 ) by bisulphite sequencing . the pro - apoptotic effect of bcl2l10 and growth promotion by sirna targeting bcl2l10 in gastric cancer cells suggests that bcl2l10 may be a tumor suppressor by inducing apoptosis through mitochondrial pathways . the iroquois homeobox protein 1 ( irx1 ) gene is located on chromosome 5p15.33 , which is a cancer susceptibility locus . irx1 transcription was suppressed by hypermethylation in gastric cancer , and restoring irx1 expression in sgc-7901 and nci - n87 gastric cancer cells inhibited tumor growth , invasion , and tumorigenesis in vitro and in vivo . irx1 directly targets bradykinin receptor b2 ( bdkrb2 ) , an angiogenesis - related gene , as well as histone h2b type 2-e ( hist2h2be ) and fibroblast growth factor 70 ( fgf7 ) , cell proliferation and invasion related genes . hypermethylation of irx1 was detected not only in primary gastric cancer tissues but also in the peripheral blood cells of gastric cancer patients , suggesting that irx1 could potentially serve as a biomarker for gastric cancer . the cklf - like marvel transmembrane domain - containing family ( cmtm ) is a novel family of proteins linking chemokines and the transmembrane-4 superfamily . wang et al . reported that cmtm3 is frequently methylated in many carcinoma cell lines and some primary tumors including gastric cancer , resulting in loss of its expression at both the mrna and protein levels . ectopic restoration of cmtm3 expression in tumor cells leads to the suppression of cell growth and induction of apoptosis with caspase-3 activation , suggesting that cmtm3 may function as a tumor suppressor . hypermethylation signals were also observed in some cultured and primary gastric cancers with little or no expression of transcription factor sox2 , a sox transcription factor . among the 52 patients with advanced gastric cancers who were tested , those having sox2 gene methylation in their cancer tissues had a significantly shorter survival than did those without the methylation ( p = 0.006 ) . exogenous expression of sox2 inhibited cell growth through apoptosis and cell cycle arrest . sox2-overexpressing cells exhibited characteristics of apoptosis , such as dna laddering and caspase-3 activation . cell cycle arrest was associated with decreased levels of cyclin d1 and phosphorylated rb as well as an increased p27 level . reported that zinc - finger transcription factor znf382 functions as a tumor suppressor in many types of carcinomas including gastric cancer . ectopic expression of znf382 in tumor cells in which the gene was silenced significantly reduced clonogenicity and proliferation and induced apoptosis . further found that znf382 inhibited nf-b and ap-1 signaling and down - regulated the expression of multiple oncogenes , including myc , mitf , hmga2 , cdk6 , stat3 , stat5b , id1 , and ikbke , most likely through heterochromatin silencing . a , pyrosequencing ; b , q - msp ; c , high - resolution melting ( hrm ) analysis ; d , methylight ; e , bisulfit sequencing . methylation of the ubiquitin carboxyl - terminal hydrolase l1 ( uchl1 ) was detected in primary digestive tumors , including 77% ( 53/69 ) of gastric carcinomas , but not in or occasionally in paired adjacent non - tumor tissues . restoring uchl1 expression in cell lines where the gene had been silenced significantly inhibited their growth and colony formation ability by inhibiting cell proliferation , causing cell cycle arrest in g2/m phase , and inducing apoptosis through the intrinsic caspase - dependent pathway similarly , sdr - related gene product that binds to c - kinase ( hsrbc ) , another novel methylated tumor suppressor gene in gastric cancer , increased the stability of p53 and expression of p53 target genes , such as p21 , puma , and noxa . hsrbc - mediated cell cycle arrest and apoptosis were abolished by blockade of p53 function . these results suggested that epigenetic inactivation of hsrbc contributes to the malignant progression of gastric tumors , in part , through attenuated p53 response to stress . moreover , opioid binding protein / cell adhesion molecule - like gene ( opcml ) is also a stress- and p53-responsive gene , but this response is epigenetically impaired when the opcml promoter becomes methylated . ecotopic expression of opcml led to significant inhibition of both anchorage - dependent and -independent growth of carcinoma cells in which the gene had been silenced . as reviewed above , many methylated tumor suppressor genes in gastric cancer contribute to the dysregulation of the important aspects of tumorigenesis : cell cycle and apoptosis . the understanding of these molecular mechanisms will provide accesses to novel molecular therapeutic strategies for inhibiting oncogenic activity of signaling pathways . morbidity in most cancer patients is not due to primary cancer but to metastatic disease . the high death rate of gastric cancer strikingly correlates with the high metastatic capacity of most gastric cancers . thus , understanding tumor progression to the metastatic state and changes in highly aggressive cells is important for the development of novel approaches to diagnose and treat aggressive malignancies . detachment of cells with increased motility from a primary tumor is the first step of cancer invasion and metastasis . gastric cancer cells with fibroblastoid morphological changes show increased motility and invasiveness due to decreased cell - cell adhesion , which is reminiscent of epithelial - mesenchymal transition ( emt ) during embryonic development . the dynamic regulation of cell - cell adhesion is crucial for developmental processes including tumorigenesis . with reduced cell - cell adhesiveness , cancer cells can disobey the social order and result in destruction of the histological structure . a common and early requirement for cell motility is actin polymerization , which drives the formation of cell protrusions that are used to adhere to the extracellular matrix , define the direction of migration and initiate cell crawling . animal cells respond to signaling at the plasma membrane by remodeling their actin cytoskeleton . on the other hand , the movement of cells into a tightly woven extracellular matrix also require an active proteolytic system , which can cleave a path for cell migration . so the genes regulating the host cell cytoskeleton rearrangement and proteolytic activity also have important functions in cell morphogenesis and motility . our group reported that epigenetic inactivation of phospholipase c delta 1 ( plcd1 ) is common and tumor - specific in gastric cancer and that plcd1 acts as a functional tumor suppressor in gastric carcinogenesis . located at the important tumor suppressor locus 3p22 , plcd1 encodes an enzyme that regulates energy metabolism , calcium homeostasis , and intracellular movements . ectopic expression of plcd1 in gastric tumor cells with silenced plcd1 dramatically inhibited clonogenicity and migration , possibly through down - regulation of mmp7 expression and hampered cyto - skeletal reorganization via phosphorylation and inactivation of cofilin . deleted in liver cancer 1 ( dlc1 ) is another tumor suppressor gene involved in the regulation of cytoskeletal organization and other functions and is frequently methylated in many cancers , including gastric cancer , . recently , its new isoform , dlc1 isoform 4 ( dlc1-i4 ) , was also found to be silenced epigenetically , to have tumor inhibitory properties in multiple carcinomas , and to be regulated by p53 . . the down - regulation of e - cadherin may favor dissociation of cancer cells from one another , facilitating their invasion through the basal membrane . although mutation and allelic loss have been confirmed as major mechanisms for e - cadherin ( cdh1 ) gene inactivation in many malignancies , cdh1 promoter methylation could be frequently detected in gastric carcinoma . as a co - partner of e - cadherin , -catenin not only is critical for cell adhesion in the membrane and cytoplasm of cells , but also plays a role as a transcription activating protein in nuclei . aberrant activation of the wnt/-catenin signaling pathway is frequently found in many cancers , including gastric cancer , . in addition to genetic deletions and point mutations , a number of negative regulators of wnt signaling , including secreted frizzled - related protein ( sfrp ) , dickkopf homolog 2 ( dkk2 ) , dickkopf homolog 2 ( dkk3 ) and wnt inhibitory factor 1 ( wif1 ) , were frequently methylated in gastric cancer. recently , other genes involving wnt pathway were also reported as methylated tumor suppressor genes in gastric cancer . for example , sry - box containing gene 17 ( sox17 ) was reported to be indispensable for embryonic development and a candidate tumor suppressor gene that antagonizes the canonical wnt/-catenin signaling pathway in colorectal cancer . treatment with a demethylating agent induced sox17 expression in gastric cancer cells , thus indicating the down - regulation of sox17 by methylation . transgenic expression of sox17 suppressed dysplastic tumor development in the stomach of k19-wnt1/c2me mice by suppressing wnt activity . these results suggested that sox17 protects benign tumors from malignant progression at an early stage of tumorigenesis , and down - regulation of sox17 contributes to malignant progression through promotion of wnt activity . the popeye domain - containing ( popdc ) genes bves , popdc2 , and popdc3 encode proteins that regulate cell - cell adhesion and cell migration during development . bves and popdc3 were reported to be hypermethylated in 69% and 64% of the gastric cancer tissues , respectively . promoter hypermethylation is a causal event for long - term repression of bves and popdc3 , whereas egf stimulation is an immediate repression mechanism for both genes in gastric cancer . bves , popdc3 , and e - cadherin mrnas were down - regulated and snail mrna expression was up - regulated in egf - induced emt in snu-216 cells . interestingly , accumulating evidence suggests that a major subfamily within the cadherin superfamily , protocadherins , frequently act as tumor suppressor genes . recent studies have shown that the main structural and functional properties of protocadherins are distinct from those of classical cadherins . they may not have the strong cell - cell adhesion activity but do have other functions , such as mediating specificity of cell - cell interactions and signal transduction . yu et al . reported that protocadherin 10 ( pcdh10 ) was silenced or down - regulated in 94% ( 16 of 17 ) of gastric cancer cell lines . furthermore , pcdh10 methylation was detected in 82% ( 85 of 104 ) of gastric tumors compared with 37% ( 38 of 104 ) of paired non - tumor tissues ( p < 0.001 ) . re - expression of pcdh10 reduced colony formation in vitro and tumor growth in vivo . it also inhibited cell proliferation , induced cell apoptosis , and repressed cell invasion , possibly through up - regulation of the pro - apoptosis genes fas , caspase 8 , jun , and cdkn1a ; the anti - proliferation gene fgfr ; and the anti - invasion gene htatip2 . in cancer cells , glucose is often converted into lactic acid , which is known as the warburg effect . the reason that cancer cells have a higher rate of aerobic glycolysis , but not oxidative phosphorylation , remains largely unclear . recently , fructose-1,6-bisphosphatase-1 ( fbp1 ) , which functions to antagonize glycolysis , was reported to be down - regulated through the nf-b pathway in ras - transformed nih3t3 cells . fbp1 was hypermethylated in 57% ( 4/7 ) of gastric cancer cell lines and 33% ( 33/101 ) of gastric carcinomas . restoration of fbp1 expression suppressed anchorage - independent growth , indicating the relevance of fbp1 down - regulation in carcinogenesis . leucine - rich repeat - containing 3b ( lrrc3b ) is an evolutionary highly conserved leucine - rich repeat - containing protein , but its biological significance is unknown . recently , lrrc3b was identified as a putative tumor suppressor gene and is reportedly silenced in gastric cancers by epigenetic mechanisms . stable transfection of lrrc3b in gastric cancer cell line snu-601 inhibited anchorage - dependent and anchorage - independent colony formation . moreover , lrrc3b expression suppressed tumorigenesis in nude mice . microarray analysis of lrrc3b - expressing xenograft tumors showed induction of immune response - related genes and ifn signaling genes . hematoxylin and eosin ( h&e)-stained sections of lrrc3b - expressing xenograft tumors showed lymphocyte infiltration in the region . these results suggested that lrrc3b silencing in cancer may play an important role in tumor escape from immune surveillance . as reviewed above , aberrant dna methylation of promoter cpg islands is one of the major inactivating mechanisms of tumor suppressor genes and is deeply involved in gastric carcinogenesis . moreover , in gastric cancer , dna methylation from the pre - malignant till the most aggressive stage of cancer can be defined . for instance , many genes such as thbd , lox , hrasls , flnc , and hand1 were found to be infrequently methylated in non - cancerous gastric mucosae , in addition to their frequent methylation in cancers . similar findings were reported for cdh1 , dapk , p14 , thbs1 , and timp-1 . the presence of trace amounts of methylation in non - cancerous gastric mucosae suggested that some gastric carcinogens could have induced the methylation . the most important gastric carcinogenic factor is helicobacter pylori ( h. pylori ) infection , which increases the risk of developing gastric cancers by 2.2- to 21.0-fold , . recent studies to quantify dna methylation changes induced by the h. pylori infection in light of imflammatory reactions have been performed in both animal models and in the clinical setting . the findings suggest that h. pylori infection induces aberrant methylation in gastric mucosae and that levels of accumulated methylation are associated with gastric cancer risk . notably , although h. pylori infection is important to trigger inflammation capable of inducing aberrant dna methylation , some inflammation processes appear to be critical in induction of aberrant dna methylation. indeed , the inflammation induced by h. pylori infection , not h. pylori itself , was critically involved in methylation induction . h. pylori may induce methylation of promoters containing cpg islands by release of reactive oxygen species ( ros ) and nitric oxide ( no ) and by activation of dna methyltransferase , . more recently , sepulveda et al . suggested that permanent dna methylation after h. pylori eradication may occur in stem cells , thus promoting tumorigenesis . considering the significant involvement of aberrant dna methylation of cpg islands in human cancers , further identification of the inducing factors and mechanisms can be expected to provide novel targets for cancer prevention . in addition to regional hypermethylation , global hypomethylation is also purported to be a hallmark of cancer cells and is known to cause chromosomal instability as an early event during gastric carcinogenesis . however , yoshida et al . reported that h. pylori infection potently induces alu and sata hypomethylation in gastric mucosae as an early event during gastric carcinogenesis , whereas global hypomethylation is present only in some individuals . thus , the use of hypomethylation as a risk marker has not been considered realistic . aberrant hypermethylation of promoter regions of specific genes is a key event in the formation and progression of cancer . an accumulating number of studies have reported that gene silencing by dna methylation may be established at an early stage in the multistep process of carcinogenesis . methylations of specific genes or methylation patterns of groups of genes were found to be associated with chemotherapy response and prognosis . thus , aberrant dna methylation can be applied to cancer diagnostics in three ways : as a marker to detect cancer cells or cancer - derived dna , as a marker to predict prognosis , and as a biomarker for the assessment of therapeutic response . methylation analysis has an advantage in that it can be performed using chemically stable dna ( compared to rna ) . moreover , detecting gene methylation is easier than detecting gene mutation because the exact location of a mutation is usually unknown , making it difficult to specifically amplify dna molecules with an embedded mutation in excess of wild - type molecules . detection of aberrant methylation can provide confirmation of the presence of intact cancer cells or cancer - derived dna in bodily fluids , such as blood , urine , sputum , saliva , and stools . recently , the methylations of bnip3 , chfr , cyp1b1 , mint25 , sfrp2 , and rassf2 were analyzed in 107 specimens of peritoneal fluid by quantitative methylation - specific polymerase chain reaction ( msp ) . the results showed that dna methylation in periotoneal fluid is a possible marker for detecting occult neoplastic cells on the peritoneum . methylation analysis along with a cytological examination might , therefore , improve the positive detection of cancer cells in the peritoneal fluid of gastric cancer patients . the development of the bisulfate conversion technique that reproducibly changes unmethylated cytosines to uracil but leaves methylated cytosines unchanged rapidly increased progress in dna methylation detection . bisulfite sequencing , msp , and combined bisulfite restriction analysis ( cobra ) were all developed on the basis of bisulfite conversion among these technologies , msp is a subjective , gel - based assay and can not provide quantitative information . to date , several real - time msp methods , such as bisulfite treatment in combination with methylight , quantitative multiplex - msp(qm - msp ) , , or pyrosequencing , have been developed and used in dna methylation studies . for example , a recent study showed that one pyrosequencing analysis of global and site - specific dna methylation in peripheral blood samples from 105 gastric cancer patients provides quantitative dna methylation values that may serve as important prognostic indicators . however , prior to clinical application , these findings require validation in prospective clinical studies . , an increasing number of drugs targeting dna methylation have been improved to increase efficacy and to decrease toxicity . 5-azacyti - dine , the most successful epigenetic drug to date , is currently recommended as the first - line treatment of high - risk myelodysplastic syndromes ( mds ) . new methods for gene - specific epigenetic modification are being developed and tested in solid tumors . gastric cancer is a biologically heterogeneous disease with various molecular tumor subsets that likely respond distinctly to therapy . methylation of genes involved in dna repair and maintaining genome integrity ( e.g. mgmt , hmlh1 , wrn , and fancf ) , as well as genes involved in cell cycle checkpoints ( e.g. chfr , 14 - 3 - 3 , cdk10 , and p73 ) all reportedly influence sensitivity to chemotherapeutic drugs , suggesting that dna methylation could serve as a molecular marker for predicting tumor responsiveness to chemotherapy . an investigation of dna methylation using specialized high - throughput platforms can potentially be applied to further stratify patients to individualized therapies . methylated tumor suppressor genes are being intensively investigated in gastric cancer , but the underlying functions and mechanisms need to be carefully examined . future investigations of the connections between h. pylori infection , gastric cancer , and epigenetic changes will greatly expand our understanding . we believe that information obtained from studying dna methylation will have an impact on cancer prevention , diagnostics , and treatment , and will contribute to cancer elimination . however , proper selection of methylation markers is crucial for sensitive and specific detection , and further technological advances are necessary in the future .

gastric cancer is one of the most common malignancies and a leading cause of cancer mortality worldwide . the pathogenesis mechanisms of gastric cancer are still not fully clear . inactivation of tumor suppressor genes and activation of oncogenes caused by genetic and epigenetic alterations are known to play significant roles in carcinogenesis . accumulating evidence has shown that epigenetic silencing of the tumor suppressor genes , particularly caused by hypermethylation of cpg islands in promoters , is critical to carcinogenesis and metastasis . here , we review the recent progress in the study of methylations of tumor suppressor genes involved in the pathogenesis of gastric cancer . we also briefly describe the mechanisms that induce tumor suppressor gene methylation and the status of translating these molecular mechanisms into clinical applications .

the technical details of the arfem system used in this study have been described in detail previously . briefly , the system essentially is comprised of three core components , a femtosecond laser , a dual element ultrasound transducer , and a sample chamber . santa clara , ca , usa ) that produces 130 fs pulses at a wavelength of 800 nm . the ultrasound transducer has two elements , an outer low frequency , high intensity force - generating element , and in an inner low intensity , high frequency tracking , or a - scan element . the outer force - generating , or pushing , element has a central frequency of 1.5 mhz and a spatial peak , pulse average intensity ( isppa ) of 125 watts / cm . the a - scan element is driven by a commercial pulser - receiver ( panametrics model 5072pr , waltham , ma , usa ) at a pulse repetition frequency of 5 khz . the pushing element is driven by an arbitrary function generator ( model 3314a ; agilent technologies , palo alto , ca , usa ) feeding into a 50 db amplifier ( eni model 240l ; mks instruments , wilmingtom , ma , usa ) . the focal point of the laser is fixed in space , while the ultrasound transducer can be moved in three dimensions . to ensure that the optical and ultrasonic elements are confocal , the a - scan signal from a laser - created bubble was maximized by adjusting the alignment of the ultrasonic transducer . the 3-d sample stage then is moved independently of the optical and ultrasonic components , which are focused to the same location . the corneal sample is embedded in gelatin within the sample chamber , while the rest of the chamber is filled with degassed , distilled water for acoustic coupling . the chamber itself is mounted to a 3-d microstage , allowing for the placement of the microbubble anywhere within the cornea . the laser is focused through a 0.3 na objective creating a microbubble ( d = 22 2 m ) within the cornea with a single pulse . once the bubble has been created and confirmed via a - scan , the measurement sequence is initiated manually and the outer element of the ultrasound transducer generates an acoustic radiation force chirp 2 ms in duration . the acoustic force displaces the microbubble within the cornea , while the displacement of the bubble is tracked by the inner ultrasound element with an a - scan at a pulse repetition frequency of 5 khz . thus , the position / movement of the bubble in response the acoustic radiation force is measured every 200 s . the elastic modulus is inversely proportional to the bubble displacement and is calculated using : where i is the acoustic intensity , a is the bubble radius , the acoustic intensity and bubble radius are calibrated before the experiment , while maximum bubble displacement is measured experimentally with an a - scan . the raw a - scan rf data are filtered using a 10 mhz high pass filter , and then processed using 1-d cross - correlation between a reference bubble a - scan and the remaining pulse - echoes . the speed of sound m / s ) was used in all calculations in place of the speed of sound in cornea for convenience . six human cadaver eyes from three donors were obtained from the san diego eye bank . samples were refrigerated and measurements were performed within 3 days of death on average , but no later than 5 days . basic donor data are provided in the table . before the experiment , corneas were thinned to a physiologic thickness of 500 to 570 m by inflating the intact globe with 20% dextran solution at 20 mm hg iop . the samples were placed into a suction holder whereby the posterior portion of the globe created a seal with the holder allowing a mild suction force to hold the sample in place . the sample was oriented such that the central corneal surface was orthogonal to the ultrasonic tip of the pachymeter ( reichert , inc . , the cornea then was excised , leaving a 2-mm scleral rim , and embedded in a 7.5% gelatin gel within the sample chamber . experiments were performed only when the corneal thickness was in the physiologic 500 to 570 m range . we obtained arfem measurements in the central cornea ( 0 mm ) , at 2.5 mm ( mid ) , and at 5 mm ( peripheral ) toward the limbus . at each of these locations , the cornea was placed on the gelatin surface in such a way that half of the cornea was flat against the gelatin surface , with the other half curving upward . this was necessary due to the fact that the corneal surface must be perpendicular to the laser beam for optimal bubble creation . laying the half cornea flat against the gel allowed measurements to be made in the central and peripheral regions without having to reorient the cornea . during the measurements , the cornea was oriented so that the acoustic force and resultant microbubble displacements were perpendicular to the corneal surface . corneal orientation and arfem measurement locations . measurements are made at 0 ( center ) , 2.5 ( mid ) , and 5 ( peripheral ) mm . at each radial position , the sample is flattened against the surface of the gel so that measurements can be made at multiple locations without having to reposition the cornea . the technical details of the arfem system used in this study have been described in detail previously . briefly , the system essentially is comprised of three core components , a femtosecond laser , a dual element ultrasound transducer , and a sample chamber . santa clara , ca , usa ) that produces 130 fs pulses at a wavelength of 800 nm . the ultrasound transducer has two elements , an outer low frequency , high intensity force - generating element , and in an inner low intensity , high frequency tracking , or a - scan element . the outer force - generating , or pushing , element has a central frequency of 1.5 mhz and a spatial peak , pulse average intensity ( isppa ) of 125 watts / cm . the a - scan element is driven by a commercial pulser - receiver ( panametrics model 5072pr , waltham , ma , usa ) at a pulse repetition frequency of 5 khz . the pushing element is driven by an arbitrary function generator ( model 3314a ; agilent technologies , palo alto , ca , usa ) feeding into a 50 db amplifier ( eni model 240l ; mks instruments , wilmingtom , ma , usa ) . the focal point of the laser is fixed in space , while the ultrasound transducer can be moved in three dimensions . to ensure that the optical and ultrasonic elements are confocal , the a - scan signal from a laser - created bubble was maximized by adjusting the alignment of the ultrasonic transducer . the 3-d sample stage then is moved independently of the optical and ultrasonic components , which are focused to the same location . the corneal sample is embedded in gelatin within the sample chamber , while the rest of the chamber is filled with degassed , distilled water for acoustic coupling . the chamber itself is mounted to a 3-d microstage , allowing for the placement of the microbubble anywhere within the cornea . the laser is focused through a 0.3 na objective creating a microbubble ( d = 22 2 m ) within the cornea with a single pulse . once the bubble has been created and confirmed via a - scan , the measurement sequence is initiated manually and the outer element of the ultrasound transducer generates an acoustic radiation force chirp 2 ms in duration . the acoustic force displaces the microbubble within the cornea , while the displacement of the bubble is tracked by the inner ultrasound element with an a - scan at a pulse repetition frequency of 5 khz . thus , the position / movement of the bubble in response the acoustic radiation force is measured every 200 s . the elastic modulus is inversely proportional to the bubble displacement and is calculated using : where i is the acoustic intensity , a is the bubble radius , the acoustic intensity and bubble radius are calibrated before the experiment , while maximum bubble displacement is measured experimentally with an a - scan . the raw a - scan rf data are filtered using a 10 mhz high pass filter , and then processed using 1-d cross - correlation between a reference bubble a - scan and the remaining pulse - echoes . the speed of sound m / s ) was used in all calculations in place of the speed of sound in cornea for convenience . arfem experimental setup . six human cadaver eyes from three donors were obtained from the san diego eye bank . samples were refrigerated and measurements were performed within 3 days of death on average , but no later than 5 days . basic donor data are provided in the table . before the experiment , corneas were thinned to a physiologic thickness of 500 to 570 m by inflating the intact globe with 20% dextran solution at 20 mm hg iop . the samples were placed into a suction holder whereby the posterior portion of the globe created a seal with the holder allowing a mild suction force to hold the sample in place . the sample was oriented such that the central corneal surface was orthogonal to the ultrasonic tip of the pachymeter ( reichert , inc . , the cornea then was excised , leaving a 2-mm scleral rim , and embedded in a 7.5% gelatin gel within the sample chamber . experiments were performed only when the corneal thickness was in the physiologic 500 to 570 m range . we obtained arfem measurements in the central cornea ( 0 mm ) , at 2.5 mm ( mid ) , and at 5 mm ( peripheral ) toward the limbus . at each of these locations , the cornea was placed on the gelatin surface in such a way that half of the cornea was flat against the gelatin surface , with the other half curving upward . this was necessary due to the fact that the corneal surface must be perpendicular to the laser beam for optimal bubble creation . laying the half cornea flat against the gel allowed measurements to be made in the central and peripheral regions without having to reorient the cornea . during the measurements , the cornea was oriented so that the acoustic force and resultant microbubble displacements were perpendicular to the corneal surface . measurements are made at 0 ( center ) , 2.5 ( mid ) , and 5 ( peripheral ) mm . at each radial position , the sample is flattened against the surface of the gel so that measurements can be made at multiple locations without having to reposition the cornea . the mean elastic moduli were 4.2 1.2 , 3.4 0.7 , and 1.9 0.7 kpa in the central , mid , and peripheral regions , respectively . the mean elastic moduli were 2.3 0.7 , 1.6 0.3 , and 2.9 1.2 kpa in central , mid , and peripheral regions , respectively . while the anterior cornea was approximately twice as stiff as the posterior cornea in the central and mid cornea , the anterior elastic modulus decreased with respect to the posterior elastic modulus in the peripheral cornea . multiple comparisons were performed using anova in matlab ( mathworks , natick , ma , usa ) to determine which location - specific elastic moduli means were different from the others . statistically significant differences in elasticity could be established between the following regions : the central anterior cornea is stiffer than any other region ( p = 0.05 ) ; the mid anterior cornea is stiffer than the mid posterior , central posterior , and peripheral anterior cornea ( p = 0.05 ) ; and the peripheral posterior cornea is stiffer than the central posterior , mid posterior , and peripheral anterior cornea ( p = 0.05 ) . no statistically significant differences could be established when comparing arfem elasticity measurements in between all other regions . elastic modulus in the anterior 150 m of cornea from the center to the periphery . elastic modulus in the posterior 150 m of cornea from the center to the periphery . the arfem technique used in this study has the significant limitation of being unable to measure the elasticity distribution in the mechanically - loaded cornea and intact globe . given the nature of the current arfem device , the cornea must be excised from the globe and placed between the laser objective and ultrasound . additionally , the cornea is laid flat against the gelatin surface in such a way that half of the cornea curves unnaturally upward . the mechanical effect of this curvature is assumed to be negligible based on previous measurements of the central cornea with the natural radius of curvature preserved showing the same 2:1 ratio in elasticity between the anterior and posterior regions . we found the elasticity in the central anterior cornea to be nearly twice the elasticity in the central posterior cornea . similar results were found using needle indentation , which revealed the same 2:1 ratio in elastic modulus between the anterior and posterior central cornea . likewise , the mid anterior cornea was twice as stiff as the mid posterior cornea . structurally , the anterior cornea possesses highly interwoven collagen structure compared to the relative homogeneity in the posterior . despite this apparent correlation between microstructure and elasticity in the central and mid cornea the peripheral anterior cornea was approximately half as stiff as the central anterior , while the peripheral posterior cornea was approximately 30% stiffer than the central posterior cornea . it is interesting to note that interlamellar cohesive strength in the stroma is twice as strong in the periphery as it is centrally . though arfem does not measure interlamellar cohesive strength , both results suggest a biomechanical change in the peripheral cornea . furthermore , the peripheral anterior elasticity is approximately 2/3 that of the peripheral posterior cornea . it appears as though in the periphery , the anterior and posterior corneas nearly reverse elastic moduli relative to each other . structural data in the literature anterior collagen interweaving , as measured by collagen lamellae angle relative to the corneal surface using nonlinear optical high resolution macroscopy ( nlo - hrmac ) , did not detect a significant difference between the central and peripheral cornea . the wrinkles on the posterior surface in figure 6a are artifacts from the mounting of the sample . figures 6b to 6d show that the anterior interweaving relative to posterior collagen orientation is preserved from the center to the periphery . structural differences in the periphery of the cornea may be masked by the shifting orientation of lamellae from central to peripheral . structurally , the cornea is changing as it nears the limbus , with collagen fibers adopting a more tangential ( circumferential ) orientation in the periphery , as imaged by wide angle x - ray scattering . collagen fibers in this orientation would not have been detected in the previous work by nlo hrmac , as the technique does not image en face collagen fibers . while the posterior lamellae continue uninterrupted into the sclera , the anterior interwoven region seems to end abruptly at the limbus . ( b d ) center , middle , and peripheral measurement locations , respectively . recently , micro focus x - ray scattering data were used to determine the inclination angles of lamellae relative to the corneal surface as a measure of lamellar interweaving . as expected , inclination angles in the central anterior cornea were the greatest . the same study found that inclination angles in the posterior peripheral cornea were high when compared to the posterior central cornea , suggesting more interweaving in the posterior peripheral cornea . this finding seems to support our observation of an increasing elastic modulus in the peripheral posterior cornea . the effect of increasing inclination angle in the peripheral stroma along with the effect of tangentially oriented collagen fibers on local elasticity is unknown . as the lamellae exit the biomechanical effects of the transition or insertion of collagen lamellae into the sclera also are unknown . furthermore , the periphery may be subject to different swelling conditions because the peripheral edge of the sample is exposed directly to the gelatin medium . the cornea is , indeed , thicker in the periphery and hydration differences between the anterior and posterior could be exacerbated to the proximity of the measurements to the edge of the sample . in addition to collagen microstructure , proteoglycan ( pg ) distribution throughout the cornea also may have an effect on local elasticity as measured by arfem . specifically , it is pertinent to explore any differences in pg distribution between the anterior and posterior cornea , centrally and peripherally . a pg consists of a core protein with one or more covalently attached glycosaminoglycans ( gag ) . the gag is highly polar , negatively charged , and , as such , binds water . the concentration of keratan sulfate , the most prominent pg in the cornea , increases from anterior to posterior in the central cornea . furthermore , stromal swelling is caused almost entirely by the gel pressure ( swelling pressure ) generated by negatively charged proteoglycans . when these findings are considered along with the highly interwoven structure of the anterior cornea , it seems reasonable that the anterior cornea is resistant to edema compared to the posterior cornea . our results also indicated that the peripheral anterior corneal elasticity is approximately half that of the central anterior cornea . with studies showing conflicting accounts of the amount of interweaving in the periphery , , it would be interesting to know the trend in pg distribution in the peripheral cornea . it is possible that local pg concentration and the resultant swelling pressure have an effect on local elasticity as measured by arfem . unfortunately , the literature does not reveal many telling details about the differences in pg concentration between the central and peripheral cornea . one study looked at total soluble protein concentration between the anterior and posterior cornea , traversing across the cornea from the limbus to the central cornea . they found that total soluble protein concentration in the peripheral anterior cornea was nearly twice that of the central anterior cornea . though it may appear that this result corroborates our findings between central and peripheral elasticity , the correlation could be entirely coincidental since they were not specifically measuring pg distribution . to truly resolve this issue , a specific measurement of the axial pg gradient in the peripheral cornea would need to be performed . we found that there is a unique distribution of elasticity axially and radially throughout the cornea . while arfem results in the central cornea are in reasonable agreement with other techniques , including needle indentation and torsional rheometry , lack of mechanical data in the peripheral cornea makes comparisons difficult . however , it is clear that the full picture of corneal biomechanics is quite broad , from complex and inhomogenous collagen microstructure to varying concentration of pg throughout the cornea . a better understanding of these variables along with elasticity measurements , especially in the peripheral cornea , would certainly help in completing the picture . future modifications of arfem to automate the measurement procedure , specifically the movement of the sample with respect to the confocal volume , will result in an elasticity map with greater spatial density and may help provide this information .

purposethe biomechanical properties of the cornea have an important role in determining the shape of the cornea and visual acuity . since the cornea is a nonhomogeneous tissue , it is thought that the elastic properties vary throughout the cornea . we aim to measure a map of corneal elasticity across the cornea.methodsan acoustic radiation force elasticity microscope ( arfem ) was used to create a map of corneal elasticity in the human cornea . this arfem uses a low frequency , high intensity acoustic force to displace a femtosecond laser - generated microbubble , while using a high frequency , low intensity ultrasound to monitor the position of the microbubble within the cornea . from the displacement of the bubble and the magnitude of the acoustic radiation force , the local value of corneal elasticity is calculated in the direction of the displacement . measurements were conducted at 6 locations , ranging from the central to peripheral cornea at anterior and posterior depths.resultsthe mean anterior elastic moduli were 4.2 1.2 , 3.4 0.7 , and 1.9 0.7 kpa in the central , mid , and peripheral regions , respectively , while the posterior elastic moduli were 2.3 0.7 , 1.6 0.3 , and 2.9 1.2 kpa in the same radial locations.conclusionswe found that there is a unique distribution of elasticity axially and radially throughout the cornea .

sacubitril / valsartan is a combination of a neprilysin inhibitor and an angiotensin ii receptor blocker , indicated to decrease the risk of cardiovascular death and hospitalization for heart failure in patients with chronic heart failure.a patient previously stable on atorvastatin developed severe rhabdomyolysis and an elevation of transaminases within 26 days of initiation of sacubitril / valsartan . rhabdomyolysis is a syndrome characterized by skeletal muscle cell damage , leading to elevated creatine kinase ( ck ) , lactate dehydrogenase ( ldh ) , and aspartate aminotranferase ( ast ) , which can result in severe sequelae , including renal failure , cardiac arrhythmia , hyperthermia and death . associated elevations in transaminases may be seen , even in the absence of significant liver injury . clinically , rhabdomyolysis classically presents with muscle pain , weakness and dark , red or tea - colored urine ; however , less than 10% of patients will demonstrate all three components of this triad . rhabdomyolysis may be triggered by hereditary and/or acquired mechanisms , with approximately 75% of initial episodes being a result of acquired causes . in particular , a large number of prescription drugs and drugs of abuse can cause rhabdomyolysis , with statins being one of the top three prescription drugs most commonly responsible for rhabdomyolysis for this syndrome . myopathy and myositis have been reported in conjunction with all of the currently available statins and are considered to be a class effect of these medications . in addition , it has been recognized that statin - associated muscle complaints are dose - related and drug drug interactions that cause an elevation of statin levels have been identified . subgroups of patients at greater risk of statin - associated muscle pathology have been identified and include age > 70 years , impaired renal function , and impaired hepatic function . entresto is a combination of sacubitril , a neprilysin inhibitor , and valsartan , an angiotensin ii receptor blocker ( arb ) , indicated to decrease the risk of cardiovascular death and hospitalization for heart failure in patients with chronic heart failure ( new york heart association [ nyha ] class ii sacubitril / valsartan is usually administered in conjunction with other heart failure drug regimens instead of an angiotensin - converting enzyme inhibitor ( acei ) or another arb . the commonly occurring ( 5% ) adverse reactions reported are hypotension , hyperkalemia , cough , dizziness , and renal failure [ 6 , 7 ] . sacubitril / valsartan has not been associated with rhabdomyolysis and is commonly used in patients who are also receiving statin therapy . a 63-year - old guyanese woman with hypertension , hyperlipidemia , congestive heart failure ( chf ) , atrial fibrillation , and peripheral vascular disease presented to her primary care provider s office with a complaint of an episode of weakness at home associated with general malaise . she was receiving atorvastatin 40 mg daily , carvedilol 25 mg twice daily , digoxin 0.125 mg daily , furosemide 40 mg daily , spironolactone 25 mg daily , rivaroxaban 15 mg daily , and sacubitril / valsartan 24/26 mg twice daily . she was initiated on sacubitril / valsartan 22 days before her clinical presentation and had been on all of her other concurrent medications for more than 1 year . on the review of systems , the patient reported generalized weakness , pain in her knees , and an ill - defined stinging pain in the skin of her arms and legs . her physical examination was non - focal , with no muscle weakness or tenderness of either the skin or muscles noted . laboratory tests performed on presentation were significant for elevated ast / alanine aminotransferase ( alt ) of 351 u / l/131 be having a reaction to her statin therapy prescribed for hyperlipidemia and therefore the atorvastatin was withheld . she was scheduled to return in 4 days for repeat laboratory testing and re - evaluation . when the patient returned in 4 days , she complained of dark urine , generalized weakness , and increased muscle and skin pain throughout her body . she was then found to have rhabdomyolysis , with ck at 58,349 u / l ( > 50 the upper limit of normal ) . sacubitril / valsartan was stopped at this time as it was the only new medication in her regimen . the ck level decreased steadily and her symptoms resolved over the course of a 5-day hospitalization ( fig . 1 ) . her renal function was preserved throughout and her bilirubin and transaminases briefly increased and also subsequently steadily decreased ( figs . 2 , 3 ) . barr virus , hepatitis a , herpes simplex virus , hiv , and cytomegalovirus ( cmv ) . barr virus , and herpes simplex virus , and had negative hepatitis a and b , hiv , hsv . and cmv tests.fig . ast aspartate aminotransferase , alt alanine aminotransferase creatine kinase values during and post hospitalization . ast aspartate aminotransferase , alt alanine aminotransferase by 23 days after her initial presentation , the patient no longer had any muscle or skin pain and her ck and liver function test ( lft ) levels were completely normalized . the patient has not had any reoccurrence of rhabdomyolysis and elevated transaminases for 46 days , and her bilirubin and transaminase levels also returned to normal ( figs . 2 , 3 ) . one week later , the patient was started on an alternate statin at a low dose ( rosuvastatin 5 mg ) and the ck levels and lfts have remained stable . this patient had been on a variety of statins for more than 7 years , including atorvastatin , simvastatin , and rosuvastatin . her baseline ck and lfts were normal prior to the initiation of sacubitril / valsartan . she had previously experienced two transient elevations in ck , to a maximum of 1608 u / l , while hospitalized for an implantable defibrillator infection and explanation complicated by an acute chf exacerbation and a recurrent chf exacerbation in the 3 years prior to the current admission . her ck levels returned to normal after her acute illnesses , without change in her lfts . in this case , the immediate cause of her rhabdomyolysis was unknown , but the new medication in her profile seemed to be the likely trigger . due to the severity of her rhabdomyolysis the naranjo adverse drug reaction probability scale score was 5 , making it probable that the adverse drug reaction was precipitated by sacubitril / valsartan . the drug interaction probability scale score was 3 , consistent with a possible interaction as a cause for the reaction , with sacubitril / valsartan as the precipitant drug and atorvastatin as the object drug . of note , one of the concurrent medications , carvedilol , is a p - glycoprotein inhibitor that may increase the serum concentration of atorvastatin , a p - glycoprotein substrate ; however , the patient was stable on this combination for 7 years before sacubitril / valsartan was initiated . none of the other medications taken by this patient are known to cause rhabdomyolysis or an elevation in transaminases . a literature search regarding a drug interaction between sacubitril and statins yielded no reports of rhabdomyolysis with statins . as per a recent study , coadministration of sacubitril / valsartan with atorvastatin led to a twofold increase in the maximum concentration ( c max ) of atorvastatin and its metabolites . in addition , the us and canadian package inserts of sacubitril / valsartan cite in vitro data showing sacubitril inhibiting organic anion transporting polypeptides ( oatp ) 1b1 and 1b3 , resulting in increased c max and area under the curve ( auc ) of atorvastatin with coadministration of sacubitril / valsartan [ 6 , 7 ] . the canadian version of the product insert further states that sacubitril / valsartan may increase the systemic exposure of oatp1b1 and oatp1b3 substrates , such as statins . sacubitril / valsartan increases the c max of atorvastatin and its metabolites by up to twofold , and auc by up to 1.3-fold . the canadian insert recommends that sacubitril / valsartan must be coadministered cautiously with statins , especially simvastatin , a sensitive oatp1b1/1b3 substrate , and a decrease in dose of simvastatin and atorvastatin may be considered when coadministered with sacubitril / valsartan . the interaction between atorvastatin and sacubitril is mentioned in the us product package insert as a theoretical possibility . there is currently no recommendation to increase monitoring or empirically decrease the dose of any statin on initiation of sacubitril / valsartan . a number of medications are known to inhibit oatps and have been shown to increase statin plasma concentrations . many single nucleotide polymorphisms ( snps ) have been found within the slco1b1 and/or slco1b3 genes encoding oatp1b1 and oatp1b3 proteins , respectively , and were shown to effect the pharmacokinetics and/or pharmacodynamics of statins [ 12 , 13 ] . the clinical consequences of the drug interactions may vary based on the genetic variation in oatp - encoding genes and inhibition of oatp function . the slco1b1 * 1b haplotype appears to be associated with enhanced hepatic uptake and reduced plasma concentrations of oatp1b1 substrates . discontinuation of the statin alone did not result in a decrease in the patient s lfts . it was not until sacubitril / valsartan was also discontinued , and treatment for rhabdomyolysis was initiated , that the patient s symptoms began to improve and her ck and lfts returned to baseline . this could be due to either a direct effect of sacubitril / valsartan or the increased serum concentration of atorvastatin , as described in the literature . because of the above - stated pharmacodynamics , we believe that the more likely mechanism is the increased serum concentration of atorvastatin . currently , there is no recommendation regarding the initiation or continuation of statins in patients with nyha class ii iv heart failure as there is insufficient information on which to base recommendations for or against statin treatment . in these patients , the choice of statin dose , and using a statin for cardiovascular risk reduction benefit , must be weighed against adverse effects , drug drug interactions , precautions and contraindications to statin therapy . we would have liked to have arranged slcob testing for the patient but were unable to . further research will be useful in identifying patients at risk for this adverse reaction , and will likely include genetic testing . this case of severe rhabdomyolysis should stimulate further investigation of the potential negative effects of the initiation of sacubitril / valsartan in patients receiving statin therapy . since sacubitril / valsartan is targeted for patients with heart failure , we predict that this would comprise a large number of the candidates for treatment . we recommend consideration of obtaining baseline ck levels and lfts and close observation of patients . in this case , rhabdomyolysis developed within 3 weeks of initiation of sacubitril / valsartan and evaluation within a shorter time frame may be appropriate . future reports of this reaction may help to define high - risk patients , but it should be noted that our patient had no known high - risk characteristics for the development of rhabdomyolysis , except for prior mild elevations of ck during hospitalizations for chf exacerbations . we encourage the reporting of any similar reactions to the us fda , the manufacturer , and the medical community . eve s. faber , madhavi gavini , ronald ramirez and richard sadovsky declare that they have no conflicts of interest that are directly relevant to the content of this report . a copy of the written consent may be requested for review from the corresponding author .

a 63-year - old woman previously stable on a regimen of atorvastatin 40 mg daily , carvedilol 25 mg twice daily , digoxin 0.125 mg daily , furosemide 40 mg daily , spironolactone 25 mg daily , rivaroxaban 15 mg daily , and enalapril 20 mg twice daily for heart failure developed rhabdomyolysis 26 days after enalapril was stopped and sacubitril / valsartan ( entresto ) started . the patient received sacubitril / valsartan at 24/26 mg twice daily for heart failure ; however , after 26 days she developed muscle and skin pain . investigations revealed elevated creatine kinase and liver function tests , and rhabdomyolysis with raised transaminases was diagnosed . sacubitril / valsartan and atorvastatin were discontinued and the patient was hydrated . she returned to baseline in 23 days and has not had any reoccurrence of rhabdomyolysis and elevated transaminases for 46 days . a naranjo assessment score of 5 was obtained , indicating a probable relationship between the patient s rhabdomyolysis and her use of sacubitril / valsartan . the drug interaction probability scale score was 3 , consistent with a possible interaction as a cause for the reaction , with sacubitril / valsartan as the precipitant drug and atorvastatin as the object drug .

the use of sodium hypochlorite as toilet detergent is related to a red - brown urine discolouration in patients taking mesalamine.it seems that the red - brown discolouration relates to a harmless reaction.polymerisation of mesalamine and/or metabolites could be a theoretical mechanism of this discolouration . the exact working mechanism of mesalamine is not fully understood , but it appears to have anti - inflammatory properties . the drug blocks interleukin-1 and tumour necrosis factor-. mesalamine also inhibits the cyclo - oxygenase pathway , leading to inhibition of prostaglandin e2 in inflamed intestine . the most common adverse drug reactions ( adrs ) , as described in the summary of product characteristics ( smpc ) , are headache , rash and gastrointestinal symptoms , including diarrhoea , nausea , vomiting and abdominal pain . mesalamine is also associated with the renal adrs of renal failure , interstitial nephritis and haematuria [ 1 , 3 ] . in the period from august 2009 to april 2015 , the netherlands pharmacovigilance centre lareb received two case reports of red - brown urine discolouration in association with mesalamine . both cases were reported by patients and the urine discoloured after contact with the toilet bowl . this case concerned a 38-year - old male with a history of crohn s disease . the patient reported separate several episodes of a red - brown deposit in the toilet bowl after the start of mesalamine in 1999 ( pentasa slow - release 500 mg tablet ) . the latest episode occurred in april 2015 , 2 days after the use of mesalamine . the patient had a mild form of crohn s disease that had been stable for 68 years and only used mesalamine during episodes of exacerbations . due to an exacerbation in september 2014 , the daily dose of mesalamine was increased from 2 to 4 g. during this dose escalation , the patient recognised the same red - brown deposit that had occurred during previous use . in january 2015 , the mesalamine was tapered over a period of 2 months to 2 g once daily and he remained stable . he observed that the urine initially had a normal colour and developed the red - brown colour after contact with the water in the toilet bowl . most of the time the persistent deposit did not appear directly after contact with the toilet water but developed after a few days , despite flushing the toilet . of note , the deposit in the toilet bowl was more intense during the use of an increased dose of 4 g a day . the patient wondered whether the use of detergents could have caused this kind of discolouration and therefore he cleaned his toilet in the morning with a chlorine - containing detergent . in the evening a red - brown deposit had developed in the toilet bowl and on the seat and the water was unaffected . as the patient wanted more information on the possibility of the discolouration being the result of a reaction with the sodium hypochlorite detergent , he reported it to lareb . after the report had been assessed , the pharmacovigilance centre lareb asked the patient to collect his urine and to add household bleach as a test , to which he agreed ( fig . 1 ) . the urine ( 40 ml ) was mixed with 20 ml of sodium hypochlorite bleach and within a few minutes the discolouration developed . after shaking the mixture , 1 red brown discolouration after adding sodium hypochlorite bleach to normal - appearing urine ( left , right ) . agitation discoloured the urine completely ( right ) red brown discolouration after adding sodium hypochlorite bleach to normal - appearing urine ( left , right ) . agitation discoloured the urine completely ( right ) this case concerned a 36-year - old woman with ulcerative colitis , for which she is treated with 2 g of mesalamine ( pentasa sachet 2 g prolonged - release granules ) twice daily . since she started taking mesalamine in june 2009 , her urine appeared red - brown in the toilet bowl , but only when she had recently cleaned the toilet with sodium hypochlorite detergent . when the patient added a drop of sodium hypochlorite detergent to her normal - coloured urine in the toilet , the same red - brown discolouration occurred immediately . the patient is still taking mesalamine and the phenomenon is still present when cleaning the toilet . this case concerned a 38-year - old male with a history of crohn s disease . the patient reported separate several episodes of a red - brown deposit in the toilet bowl after the start of mesalamine in 1999 ( pentasa slow - release 500 mg tablet ) . the latest episode occurred in april 2015 , 2 days after the use of mesalamine . the patient had a mild form of crohn s disease that had been stable for 68 years and only used mesalamine during episodes of exacerbations . due to an exacerbation in september 2014 , the daily dose of mesalamine was increased from 2 to 4 g. during this dose escalation , the patient recognised the same red - brown deposit that had occurred during previous use . in january 2015 , the mesalamine was tapered over a period of 2 months to 2 g once daily and he remained stable . he observed that the urine initially had a normal colour and developed the red - brown colour after contact with the water in the toilet bowl . most of the time the persistent deposit did not appear directly after contact with the toilet water but developed after a few days , despite flushing the toilet . of note , the deposit in the toilet bowl was more intense during the use of an increased dose of 4 g a day . the patient wondered whether the use of detergents could have caused this kind of discolouration and therefore he cleaned his toilet in the morning with a chlorine - containing detergent . in the evening a red - brown deposit had developed in the toilet bowl and on the seat and the water was unaffected . as the patient wanted more information on the possibility of the discolouration being the result of a reaction with the sodium hypochlorite detergent , he reported it to lareb . after the report had been assessed , the pharmacovigilance centre lareb asked the patient to collect his urine and to add household bleach as a test , to which he agreed ( fig . 1 ) . the urine ( 40 ml ) was mixed with 20 ml of sodium hypochlorite bleach and within a few minutes the discolouration developed . after shaking the mixture , 1 red brown discolouration after adding sodium hypochlorite bleach to normal - appearing urine ( left , right ) . agitation discoloured the urine completely ( right ) red brown discolouration after adding sodium hypochlorite bleach to normal - appearing urine ( left , right ) . this case concerned a 36-year - old woman with ulcerative colitis , for which she is treated with 2 g of mesalamine ( pentasa sachet 2 g prolonged - release granules ) twice daily . since she started taking mesalamine in june 2009 , her urine appeared red - brown in the toilet bowl , but only when she had recently cleaned the toilet with sodium hypochlorite detergent . when the patient added a drop of sodium hypochlorite detergent to her normal - coloured urine in the toilet , the same red - brown discolouration occurred immediately . the patient is still taking mesalamine and the phenomenon is still present when cleaning the toilet . likewise , the disordered haem production in patients with porphyria can result in red - brown discolouration . a red - brown urine discolouration could be perceived as a sign of possible renal impairment or other diseases ; however , neither of the described patients are known to have an impaired renal function or physical abnormalities . furthermore , various drugs are associated with red and/or brown discolouration , e.g. rifampicin , doxorubicin , metronidazole , methyldopa , levodopa and sulfasalazine . a notable difference from the described mesalamine cases is that the urine of patients taking the above - mentioned drugs , such as sulfasalazine , is already discoloured at micturition . a pubmed search was conducted and it was found that two cases of red - brown urine associated with mesalamine have been reported previously by sacks and davis . both of the male adolescents in these reports experienced the same red - brown urine discolouration after contact with sodium hypochlorite bleach . a similar experiment was performed to that performed by patient a , and after adding sodium hypochlorite bleach to the collected urine the discolouration developed spontaneously . to our knowledge , there are no other published reports of this association after a chemical reaction with drugs structurally related to mesalamine ( sulfasalazine , olsalazine and 4-aminosalicylic acid ) . notably , there has been discussion regarding this phenomenon in several patient forums and blogs . case reports from altmann and mansell do , however , describe urine discolouration in patients using methyldopa or levodopa after contact with lavatory bleach and exposure to light . normal biotransformation of methyldopa produces a variety of metabolites ; presumably , these metabolites have properties that form melanin by spontaneous polymerisation , which results in darkening of the urine . melanins are polymers of phenolic compounds ( e.g. dopaquinone , benzothiazole ) that are formed by an extensive bio pathway . melanin is an aggregate of smaller component molecules such as pheomelanin and eumelanin ; both of these are initially formed by oxidation of tyrosine and then undergo cysteinylation and cyclisation , respectively . the end product is ultimately formed by polymerisation of several different derivatives . altmann and mansell found that urine samples that were made alkaline in patients treated with methyldopa or levodopa turned black on exposure to sunlight . mesalamine is acetylated by the enzyme n - acetyltransferase 1 ( nat1 ) in the liver and gut mucosal wall into the metabolite n - acetyl-5-aminosalicylic acid ( n - ac-5asa ) . evidence from the literature indicates that nat1 is polymorphic , but that these polymorphisms are associated with relatively minor effects on acetylation function . it is unknown whether the patients in our reports have a nat1 polymorphism , but if so we would expect no significant effect on the propensity of discolouring . furthermore , this acetylation is minimal when mesalamine and n - ac-5asa are both secreted back into the lumen by the drug efflux pump p - glycoprotein , excreted in the faeces , absorbed via the colon into the blood and , lastly , eliminated in the urine . the theory is that n - ac-5asa and/or mesalamine react with the sodium hypochlorite - containing bleach . sodium hypochlorite usually has a ph of 1113 and could therefore initiate the reaction as described by altmann and mansell . according to the cases reported by altmann and mansell , it is also possible that a similar polymerisation of mesalamine and/or n - ac-5asa could cause the discolouration of the urine . perhaps due to the alkaline environment , partial deprotonation of n - ac-5asa and mesalamine could also contribute to this reaction . since the introduction of the national spontaneous reporting system in 2003 to the general public , consumers have now become the largest group of reporters in the netherlands . the quality of consumer reports are generally good and these reports highlight via first - hand information the real - life experience of an adr . being unconstrained with regards to the probability of causality , consumers may report relevant adrs that healthcare professionals may not recognise initially or consider to be unlikely . patient b s gastroenterologist recognised the phenomenon , but it is conceivable that not every gastroenterologist will establish a direct relationship between urine discolouration , mesalamine therapy and the use of a specific detergent . the two reports received provide important information about a harmless reaction as this phenomenon probably concerns a lot of other patients using mesalamine . based on the described reports and the case reports in the literature , we suggest a causal relationship between red brown discolouration of urine after contact with sodium hypochlorite detergent and the use of mesalamine . the naranjo assessment scores for cases a and b were 9 and 6 , indicating a certain and probable relationship , respectively . this seems to be a harmless reaction as the patients experienced no physical complaints and in both the lareb cases and those described by sacks and davis , the patients were under the care of a gastroenterologist . in the differential diagnosis it is important to be aware that the use of sodium hypochlorite as a toilet detergent could be the cause of coloured deposits in and on the toilet bowl . the presence of sodium hypochlorite does not exclude the possibility of renal impairment ; therefore , it is important to distinguish already discoloured urine from normal - coloured urine that only discoloured after contacting the toilet bowl . thus , to inform and reassure patients , it might be helpful to mention red - brown discolouration in the patient leaflet for mesalamine . no financial support was received for the conduct of this study or preparation of this manuscript .

a 38-year - old male and a 36-year - old female experienced red - brown urine discolouration after 2 and 3 days , respectively , during the use of mesalamine for inflammatory bowel disease . both patients mentioned that the urine discoloured after contact with sodium hypochlorite detergent in toilet water . mesalamine and the inactive metabolite n - acetyl-5-aminosalicylic acid are primarily excreted in the urine . we hypothesised a possible reaction with sodium hypochlorite and/or light . naranjo assessment scores of 9 and 6 were obtained for the reports , indicating a certain and probable relationship , respectively , between the red - brown urine discolouration and the use of the suspect drug mesalamine . knowledge of this harmless reaction is desirable to avoid unnecessary physical examination and worry .

the study was conducted at the drug deaddiction and treatment center ( ddtc ) , department of psychiatry , postgraduate institute of medical education and research ( pgimer ) , chandigarh - a multispecialty tertiary - care teaching hospital providing services to a major area of north india . patients with any substance dependence were recruited from outpatient setting at their first contact at ddtc from 1 january , 2010 - 31 december , 2010 . a cross - sectional design was employed and written informed consent was obtained from the patients taken up for the study . diagnoses were made by consultant psychiatrist as per international classification of diseases - classification of mental and behavioral disorders - clinical descriptions and diagnostic guidelines tenth revision ( icd-10 ) . all of our patients were males as because of lack of separate facilities in this center , we admitted female substance users in psychiatric inpatient unit under the same department . after enrolment into the study , a semistructured proforma was used to assess demographic and substance use details . body weight was measured in kilogram ( kg ) and height in centimeters ( cm ) by a calibrated scale . waist circumference , in centimeters ( cm ) , was measured midway between the inferior costal margin and the superior border of the iliac crest , at the end of normal expiration in standing position . at least two readings at 5 min intervals were recorded for blood pressure ( bp ) using standard mercury manometer in supine position . if blood pressure was found to be high ( 140/90 ) then a third reading was taken after 30 min ; the lowest of these readings was taken . fasting venous blood sample was collected under aseptic condition to measure the blood glucose ( fbs ) , triglycerides ( tg ) , and high density lipoprotein ( hdl ) levels . ms was diagnosed using international diabetes federation ( idf ) criteria . according to idf criteria a person is considered to have ms if he has high waist circumference ( 80 cm for females and 90 cm for males of asian origin ) along with two of the following criteria : systolic blood pressure 130 mmhg and/or diastolic blood pressure 85 mmhg ( or on treatment for hypertension ) , tg levels 150 mg / dl ( or on specific treatment for this abnormality ) , hdl cholesterol < 40 mg / dl for male and < 50 mg / dl for females ( or on specific treatment for this abnormality ) , fbs 100 mg / dl ( or on treatment for diabetes mellitus ) . all patients and healthy controls with metabolic abnormalities were informed and educated about the need for proper diet and regular exercise , and referred for specialist care whenever required . analysis was done using the statistical package for social sciences ( spss ) version 14.0 for windows ( chicago , illinois , usa ) . frequencies with percentages were calculated for categorical variables and mean and standard deviation ( sd ) were calculated for continuous variables . chi - square test and t - test and one - way analysis of variance ( anova ) were used for comparisons . body weight was measured in kilogram ( kg ) and height in centimeters ( cm ) by a calibrated scale . waist circumference , in centimeters ( cm ) , was measured midway between the inferior costal margin and the superior border of the iliac crest , at the end of normal expiration in standing position . at least two readings at 5 min intervals were recorded for blood pressure ( bp ) using standard mercury manometer in supine position . if blood pressure was found to be high ( 140/90 ) then a third reading was taken after 30 min ; the lowest of these readings was taken . fasting venous blood sample was collected under aseptic condition to measure the blood glucose ( fbs ) , triglycerides ( tg ) , and high density lipoprotein ( hdl ) levels . ms was diagnosed using international diabetes federation ( idf ) criteria . according to idf criteria a person is considered to have ms if he has high waist circumference ( 80 cm for females and 90 cm for males of asian origin ) along with two of the following criteria : systolic blood pressure 130 mmhg and/or diastolic blood pressure 85 mmhg ( or on treatment for hypertension ) , tg levels 150 mg / dl ( or on specific treatment for this abnormality ) , hdl cholesterol < 40 mg / dl for male and < 50 mg / dl for females ( or on specific treatment for this abnormality ) , fbs 100 mg / dl ( or on treatment for diabetes mellitus ) . all patients and healthy controls with metabolic abnormalities were informed and educated about the need for proper diet and regular exercise , and referred for specialist care whenever required . analysis was done using the statistical package for social sciences ( spss ) version 14.0 for windows ( chicago , illinois , usa ) . frequencies with percentages were calculated for categorical variables and mean and standard deviation ( sd ) were calculated for continuous variables . chi - square test and t - test and one - way analysis of variance ( anova ) were used for comparisons . a total of 250 subjects , all men , were included in the study . by the substance of dependence opioid ( n = 52 ) , alcohol + opioid ( n = 49 ) , and other substance , with or without alcohol or opioid ( others ) ( n = 52 ) . among the groups ; 68 , 73.1 , 87.8 , and 80.8 subjects respectively were currently using tobacco in dependent or nondependent pattern . a typical study subject was aged 34.5 1.04 years , married ( 68.8% ) , working ( 66.8% ) , from urban area ( 60% ) , hindu ( 54.4% ) , educated up to matriculation ( 49.6% ) , and from nuclear family ( 47.2% ) . compared to others , alcohol group was more often married , currently employed ( p < 0.001 ) and older ( p < 0.01 ) , alcohol and others groups were more often hindu ( p < 0.001 ) , and others group was more often from urban areas ( p < 0.05 ) . majority of the subjects were from urban background in all the groups except in the alcohol + opioid group , who were from rural background [ table 1 ] . ms criteria were met in 13.6% of the subjects and highest in alcohol group ( 21.6% ) and the lowest in others group subjects ( 7.7% ) . among the metabolic abnormalities , the commonest were increased tg ( 54% ) and waist circumference ( 36.8% ) , and the least common were increased fbs level ( 6.4% ) and low hdl ( 7.6% ) . groups differed for hypertension by systolic or diastolic criteria ( p < 0.001 ) , high systolic and diastolic blood pressures ( p < 0.01 ) , duration of dependence and number of subjects with increased waist circumference ( p < 0.01 ) , and the prevalence of ms ( p < 0.05 ) . the groups were similar for the number of subjects with low hdl levels , high tg levels or high fbs . nearly two - third of the subjects ( 62.8% ) in entire sample was fulfilling one or two ms components [ table 2 ] . clinical profile of total sample and substance dependent groups comorbid psychiatric diagnosis present in 9.6% cases ( n = 24 ) , included depression ( n = 6 ) , and psychotic illness and bipolar affective disorder ( bpad ; n = 5 each ) . comorbid physical diagnosis were present in 14% ( n = 35 ) , included seizure disorder ( n = 12 ) and diabetes mellitus and alcoholic liver disease ( n = 5 each ) , while 13 subjects have other physical diagnosis . compared to those without ms , the subjects with ms were more often currently working ( 82.4 vs 55.1% ; p < 0.01 ) , married ( 85.3 vs 66.2% ; p < 0.05 ) , and older ( 38.2 vs 33.9 years ; p < 0.05 ) ; and had higher body weight ( 79 vs 72 kg ; p < 0.005 ) , body mass index ( 27.7 vs 21.6 ; p < 0.01 ) , and obesity ( bmi 25 ) ( 76.5 vs 18.5% ; p < 0.01 ) . simple binary logistic regression analysis with enter method was used to study the relationship among independent variables which were more frequently present in subjects with ms in entire sample and individual groups . as shown in table 3 , significant predictors of ms were greater age , higher body weight , higher body mass index and obesity ( body mass index 25 ) in entire group . of all predictors , odds ratio was highest ( or = 14.3 ) for obesity . in individual groups , significant ms predictors were body weight , bmi and obesity in alcohol group ; bmi and obesity in opioid and others groups . a total of 250 subjects , all men , were included in the study . by the substance of dependence opioid ( n = 52 ) , alcohol + opioid ( n = 49 ) , and other substance , with or without alcohol or opioid ( others ) ( n = 52 ) . among the groups ; 68 , 73.1 , 87.8 , and 80.8 subjects respectively were currently using tobacco in dependent or nondependent pattern . a typical study subject was aged 34.5 1.04 years , married ( 68.8% ) , working ( 66.8% ) , from urban area ( 60% ) , hindu ( 54.4% ) , educated up to matriculation ( 49.6% ) , and from nuclear family ( 47.2% ) . compared to others , alcohol group was more often married , currently employed ( p < 0.001 ) and older ( p < 0.01 ) , alcohol and others groups were more often hindu ( p < 0.001 ) , and others group was more often from urban areas ( p < 0.05 ) . majority of the subjects were from urban background in all the groups except in the alcohol + opioid group , who were from rural background [ table 1 ] . ms criteria were met in 13.6% of the subjects and highest in alcohol group ( 21.6% ) and the lowest in others group subjects ( 7.7% ) . among the metabolic abnormalities , the commonest were increased tg ( 54% ) and waist circumference ( 36.8% ) , and the least common were increased fbs level ( 6.4% ) and low hdl ( 7.6% ) . groups differed for hypertension by systolic or diastolic criteria ( p < 0.001 ) , high systolic and diastolic blood pressures ( p < 0.01 ) , duration of dependence and number of subjects with increased waist circumference ( p < 0.01 ) , and the prevalence of ms ( p < 0.05 ) . the groups were similar for the number of subjects with low hdl levels , high tg levels or high fbs . nearly two - third of the subjects ( 62.8% ) in entire sample was fulfilling one or two ms components [ table 2 ] . clinical profile of total sample and substance dependent groups comorbid psychiatric diagnosis present in 9.6% cases ( n = 24 ) , included depression ( n = 6 ) , and psychotic illness and bipolar affective disorder ( bpad ; n = 5 each ) . comorbid physical diagnosis were present in 14% ( n = 35 ) , included seizure disorder ( n = 12 ) and diabetes mellitus and alcoholic liver disease ( n = 5 each ) , while 13 subjects have other physical diagnosis . compared to those without ms , the subjects with ms were more often currently working ( 82.4 vs 55.1% ; p < 0.01 ) , married ( 85.3 vs 66.2% ; p < 0.05 ) , and older ( 38.2 vs 33.9 years ; p < 0.05 ) ; and had higher body weight ( 79 vs 72 kg ; p < 0.005 ) , body mass index ( 27.7 vs 21.6 ; p < 0.01 ) , and obesity ( bmi 25 ) ( 76.5 vs 18.5% ; p < 0.01 ) . simple binary logistic regression analysis with enter method was used to study the relationship among independent variables which were more frequently present in subjects with ms in entire sample and individual groups . as shown in table 3 , significant predictors of ms were greater age , higher body weight , higher body mass index and obesity ( body mass index 25 ) in entire group . of all predictors , odds ratio was highest ( or = 14.3 ) for obesity . in individual groups , significant ms predictors were body weight , bmi and obesity in alcohol group ; bmi and obesity in opioid and others groups . our typical study subject was a married , working , male in forth decade from urban area with 7.16 years of substance dependence . study consisted of all male subjects as more than 99% of the subjects that present to us for treatment are males . the prevalence rate of ms at 13.6% in the present study was within the range of 5 - 31% reported for subjects taking alcohol by the western studies . in our study , the prevalence of ms was found to be highest in the alcohol dependent group and it was also in the range of ms reported earlier . compared to the previous study from our center which was conducted in inpatient setting , our study is from outpatient setting with bigger sample size ( 250 vs 100 ) ; ms rate is similar in alcohol group ( 21.6 vs 24.6% ) , but lower in opioid group ( 9.6 vs 29.3% ) . our finding is also consistent with the reported increase in tg and decrease in hdl with heroin use by a study from the west . the most common abnormality in the present study being increased tg ( 54% ) was similar to previous studies . the significant difference among various groups for the number of subjects with increased waist circumference , hypertension , and prevalence of ms ; signifies that the substance dependent population is a heterogeneous one and needs to be studied separately . the subjects with ms being older and more likely to be married and employed in the present study , was a finding similar to that from other studies from our center in subjects with substance dependence . our subjects with ms was older and had more weight , higher bmi , and obesity ; compared to subjects without ms and similar variables remained significant predictors of ms . these facts point to the need for physical examination and measurement of weight , waist circumference , and body mass index to be an essential part of the assessment of patients seeking deaddiction , especially for those aged > 30 years . being a hospital based sample , it was not a true representation of the community . besides presence of ms , a significant proportion of patients had one or two metabolic abnormality . this suggests that clinicians should not just focus on patients who have ms , but also look at this high risk population , which can convert to ms positive cases . hence , any patient who fulfills a single criterion of ms should be considered at risk for development of ms and the preventive strategies should be in place . older age , higher body weight , and higher body mass index are certain risk factors for development of ms . however , these findings need further validation with larger samples , prospective , and longitudinal designs .

background : substance abuse , alcohol in particular , is associated with increased risk of diabetes and metabolic syndrome ( ms ) . the relationship between the substance abuse and ms is complex and the literature is sparse.objectives:the present research was aimed to study the prevalence and predictors of ms among outpatients with substance dependence.materials and methods : patients with substance dependence were recruited from a deaddiction center in north india , who attended outpatient clinic from 1st january , 2010 - 31st december , 2010 . ms was assessed using international diabetes federation ( idf ) criteria.results:out of 250 subjects , 34 ( 13.6% ) of the subjects met the idf criteria for ms and highest being in alcohol group ( 21.6% ) . the commonest abnormality was increased triglycerides ( tg ; 54% ) and increased waist circumference ( 36.8% ) . age , body weight , body mass index , and obesity were significant predictor of ms.conclusion:ms was highest in subjects with alcohol dependence with the commonest abnormality of tg and blood pressure . hence , routine screening is advisable in this population to address emerging ms .

traditionally , the emotional needs of dementia family caregivers are conceptualized with the transactional model of stress and coping ( lazarus and folkman , 1984 ; pearlin et al . , 1990 ) . in this model , the focus is specifically on the emotion of stress , with emphasis on improving coping skills and lowering stress level . this has translated into caregiver services like disease education , skills training , stress management and respite care . however , stress is only one of the emotions experienced by caregivers , and there are a complex range of other emotions , such as guilt , denial , sadness and anger . from the recent evidence , this whole array of emotions can be better understood using the concept of predeath grief ( blandin and pepin , 2015 ) . predeath grief is the emotional response as dementia family caregivers mourn for the psychologically absent patient and anticipate impending losses ( blandin and pepin , 2015 ) . it is prevalent among dementia caregivers ( chan et al . , 2013 ) and can be more overwhelming than handson care issues ( frank , 2007 ) . caregivers who do not recognize the presence of predeath grief tend to deny the losses they experienced and attempt to control the uncontrollable progression of dementia . they become more paternalistic in their communication with the patients and more authoritarian in making decisions related to the patients . such behaviours create inequity in the dyadic patient caregiver relationship , with the caregivers feeling more helpless and the patients losing the sense of autonomy ( piiparinen and whitlatch , 2011 ) . with this understanding , it is not surprising that predeath grief has been linked to negative consequences like caregiver burden ( holley and mast , 2009 ) , caregiver depression ( sanders and adams , 2005 ; chan et al . , 2013 ) and caregivers ' desire to institutionalize the patient prematurely ( walker et al . , 1995 ) . however , the published literature on predeath grief is derived primarily from the caucasian population ( chan et al . , 2013 ) . because the expression of grief varies with culture ( eisenbruch , 1984 ) this study investigates the existence ( primary aim ) and the characteristics ( exploratory aim ) of predeath grief in a multiethnic asian population using a wellestablished predeath grief scale the marwit meuser caregiver grief inventory ( mmcgi ) . ethical approval was obtained from the domain specific review board of national healthcare group , singapore . the following inclusion criteria were used : ( i ) spouses or children of patient with dementia ; ( ii ) caring for a patient with dementia not residing in nursing home ; ( iii ) able to read in english ; and ( iv ) aged at least 21 years . the other scales of related construct , such as prolonged grief scale ( pg12 ) , zarit burden interview ( zbi ) and centre for epidemiologic studies depression scale ( cesd ) , were also included to contrast with mmcgi . meuser caregiver grief inventory is a predeath grief scale developed empirically from extensive qualitative interview of dementia caregivers ( marwit and meuser , 2002 ) . its 50 items are assessed through 5point scales and summed to generate a total score and three subscale scores corresponding to the different dimensions of loss personal sacrifice burden , heartfelt sadness and longing , and worry and felt isolation . in the original us study ( marwit and meuser , 2002 ) , mmcgi showed high internal consistency reliability , with cronbach 's alpha ranging from 0.90 to 0.96 . it also had good construct validity , with strong correlation with anticipatory grief scale ( pearson 's correlation coefficient , r = 0.798 ) . meanwhile , personal sacrifice burden subscale correlated with caregiver strain index ( r = 0.730 ) , heartfelt sadness and longing subscale with anticipatory grief scale ( r = 0.666 ) and worry and felt isolation subscale with wellbeing scale ( r = 0.718 ) . the authors of mmcgi proposed using one standard deviation above the normative mean of a population to indicate high predeath grief and a need for further interventions . in the us population , this translates to cutoff scores of > 175 for mmcgi , > 68 for personal sacrifice burden subscale , > 59 for heartfelt sadness and longing subscale and > 52 for worry and felt isolation subscale . such normative values were not available for the singapore population at the time of this study . prolonged grief scale is a 12item predeath grief scale for caregivers modified from a postdeath grief scale , the inventory of complicated grief . it is based on the diagnostic criteria of prolonged grief disorder proposed for icd11 ( prigerson et al . , 2009 ) . its first 11 items are rated on 5point scales and summed to produce a quantifiable total score , while item 12 is scored dichotomously for the absence or presence of sociooccupational dysfunction . it assesses the perceived burden experienced by caregivers of older persons ( zarit et al . , 1980 ) . zbi has five domains burden in the relationship , emotional wellbeing , social and family life , finances and loss of control over one 's life ( rankin et al . , 1994 ) . zbi has been validated in singapore ( seng et al . , 2010 ) , with good reliability and construct validity . centre for epidemiologic studies depression scale is a 20item depression scale , assessed through 4point scales . its four domains are depressed affect , positive affect , somatic symptoms and interpersonal problems ( radloff , 1977 ) . cesd was previously validated in singapore ( stahl et al . , 2008 ) , with cronbach 's alpha of 0.700.79 and diagnostic performance of 6482% . the three dementia severities in the revised third edition of diagnostic and statistical manual of mental disorders ( american psychiatric association , 1987 ) were used as a brief measure of dementia staging . from the three options , participants chose the description that best described the patient with dementia still capable of independent living ( mild stage ) , needs some assistance with daily living ( moderate stage ) or needs roundtheclock supervision ( severe stage ) . this brief measure was previously shown to have reasonable agreement with clinical dementia rating scale ( = 0.560.6 ) ( forsell et al . , 1992 ; juva et al . , if predeath grief exists in the asian context ( primary aim ) , it should be measurable on mmcgi with high internal consistency reliability and with convergent and discriminant validities consistent with the original caucasian constructs ( marwit and meuser , 2002 ) . we had four hypotheses regarding convergent validity : mmcgi measures the experience of predeath grief . therefore , it should show stronger correlation with another predeath grief scale , pg12 , than with zbi or cesd.personal sacrifice burden subscale captures the experience of individual losses due to caregiving . it resembles caregiver burden measured by zbi and should correlate stronger with zbi than with pg12 or cesd.worry and felt isolation subscale measures the feelings of losing connection with others . it is a feature of grief reaction and should correlate stronger with pg12 than with zbi or cesd.heartfelt sadness and longing subscale resembles the traditional concepts of grief , that is , one 's intrapersonal reactions to lost relationship . therefore , it should show stronger correlation with another predeath grief scale , pg12 , than with zbi or cesd . it resembles caregiver burden measured by zbi and should correlate stronger with zbi than with pg12 or cesd . it is a feature of grief reaction and should correlate stronger with pg12 than with zbi or cesd . heartfelt sadness and longing subscale resembles the traditional concepts of grief , that is , one 's intrapersonal reactions to lost relationship . hence , it should correlate stronger with pg12 than with zbi or cesd . we made two hypotheses regarding discriminant validity : heartfelt sadness and longing subscale should correlate poorer with depression scale cesd , consistent with ample evidence demonstrating the difference between grief and depression ( prigerson et al . , 2009).mmcgi should correlate poorly with finances subscale of zbi and positive affect subscale of cesd , because they are distinctly different constructs . heartfelt sadness and longing subscale should correlate poorer with depression scale cesd , consistent with ample evidence demonstrating the difference between grief and depression ( prigerson et al . mmcgi should correlate poorly with finances subscale of zbi and positive affect subscale of cesd , because they are distinctly different constructs . first , linear regression was performed , with mmcgi as dependent variable , to identify factors associated with predeath grief . all variables with p 0.075 in univariate regression were entered into multivariate regression , and variables with p > 0.05 in multivariate regression were removed through stepwise backward selections . second , the mean scores of mmcgi and its subscales were compared with those from the original us study ( marwit and meuser , 2002 ) using onesample ttest . we calculated the sample size using bonett and wright 's formulas ( bonett and wright , 2000 , 2015 ) , setting the 95% confidence interval width at 0.2 . referencing to the original study ( marwit and meuser , 2002 ) , we expected the cronbach 's alpha of mmcgi to approximate 0.90 , corresponding to a minimum sample size of 11 ; spearman 's correlation coefficient between mmcgi and pg12 should approximate 0.8 , corresponding to a minimum sample size of 72 . ethical approval was obtained from the domain specific review board of national healthcare group , singapore . the following inclusion criteria were used : ( i ) spouses or children of patient with dementia ; ( ii ) caring for a patient with dementia not residing in nursing home ; ( iii ) able to read in english ; and ( iv ) aged at least 21 years . the other scales of related construct , such as prolonged grief scale ( pg12 ) , zarit burden interview ( zbi ) and centre for epidemiologic studies depression scale ( cesd ) , were also included to contrast with mmcgi . marwit meuser caregiver grief inventory is a predeath grief scale developed empirically from extensive qualitative interview of dementia caregivers ( marwit and meuser , 2002 ) . its 50 items are assessed through 5point scales and summed to generate a total score and three subscale scores corresponding to the different dimensions of loss personal sacrifice burden , heartfelt sadness and longing , and worry and felt isolation . in the original us study ( marwit and meuser , 2002 ) , mmcgi showed high internal consistency reliability , with cronbach 's alpha ranging from 0.90 to 0.96 . it also had good construct validity , with strong correlation with anticipatory grief scale ( pearson 's correlation coefficient , r = 0.798 ) . meanwhile , personal sacrifice burden subscale correlated with caregiver strain index ( r = 0.730 ) , heartfelt sadness and longing subscale with anticipatory grief scale ( r = 0.666 ) and worry and felt isolation subscale with wellbeing scale ( r = 0.718 ) . the authors of mmcgi proposed using one standard deviation above the normative mean of a population to indicate high predeath grief and a need for further interventions . in the us population , this translates to cutoff scores of > 175 for mmcgi , > 68 for personal sacrifice burden subscale , > 59 for heartfelt sadness and longing subscale and > 52 for worry and felt isolation subscale . such normative values were not available for the singapore population at the time of this study . prolonged grief scale is a 12item predeath grief scale for caregivers modified from a postdeath grief scale , the inventory of complicated grief . it is based on the diagnostic criteria of prolonged grief disorder proposed for icd11 ( prigerson et al . , 2009 ) . its first 11 items are rated on 5point scales and summed to produce a quantifiable total score , while item 12 is scored dichotomously for the absence or presence of sociooccupational dysfunction . it assesses the perceived burden experienced by caregivers of older persons ( zarit et al . , 1980 ) . zbi has five domains burden in the relationship , emotional wellbeing , social and family life , finances and loss of control over one 's life ( rankin et al . , 1994 ) . zbi has been validated in singapore ( seng et al . , 2010 ) , with good reliability and centre for epidemiologic studies depression scale is a 20item depression scale , assessed through 4point scales . its four domains are depressed affect , positive affect , somatic symptoms and interpersonal problems ( radloff , 1977 ) . cesd was previously validated in singapore ( stahl et al . , 2008 ) , with cronbach 's alpha of 0.700.79 and diagnostic performance of 6482% . the three dementia severities in the revised third edition of diagnostic and statistical manual of mental disorders ( american psychiatric association , 1987 ) were used as a brief measure of dementia staging . from the three options , participants chose the description that best described the patient with dementia still capable of independent living ( mild stage ) , needs some assistance with daily living ( moderate stage ) or needs roundtheclock supervision ( severe stage ) . this brief measure was previously shown to have reasonable agreement with clinical dementia rating scale ( = 0.560.6 ) ( forsell et al . , 1992 ; juva et al . , if predeath grief exists in the asian context ( primary aim ) , it should be measurable on mmcgi with high internal consistency reliability and with convergent and discriminant validities consistent with the original caucasian constructs ( marwit and meuser , 2002 ) . we had four hypotheses regarding convergent validity : mmcgi measures the experience of predeath grief . therefore , it should show stronger correlation with another predeath grief scale , pg12 , than with zbi or cesd.personal sacrifice burden subscale captures the experience of individual losses due to caregiving . it resembles caregiver burden measured by zbi and should correlate stronger with zbi than with pg12 or cesd.worry and felt isolation subscale measures the feelings of losing connection with others . it is a feature of grief reaction and should correlate stronger with pg12 than with zbi or cesd.heartfelt sadness and longing subscale resembles the traditional concepts of grief , that is , one 's intrapersonal reactions to lost relationship . therefore , it should show stronger correlation with another predeath grief scale , pg12 , than with zbi or cesd . it resembles caregiver burden measured by zbi and should correlate stronger with zbi than with pg12 or cesd . it is a feature of grief reaction and should correlate stronger with pg12 than with zbi or cesd . heartfelt sadness and longing subscale resembles the traditional concepts of grief , that is , one 's intrapersonal reactions to lost relationship . we made two hypotheses regarding discriminant validity : heartfelt sadness and longing subscale should correlate poorer with depression scale cesd , consistent with ample evidence demonstrating the difference between grief and depression ( prigerson et al . , 2009).mmcgi should correlate poorly with finances subscale of zbi and positive affect subscale of cesd , because they are distinctly different constructs . heartfelt sadness and longing subscale should correlate poorer with depression scale cesd , consistent with ample evidence demonstrating the difference between grief and depression ( prigerson et al . , 2009 ) . mmcgi should correlate poorly with finances subscale of zbi and positive affect subscale of cesd , because they are distinctly different constructs . first , linear regression was performed , with mmcgi as dependent variable , to identify factors associated with predeath grief . all variables with p 0.075 in univariate regression were entered into multivariate regression , and variables with p > 0.05 in multivariate regression were removed through stepwise backward selections . second , the mean scores of mmcgi and its subscales were compared with those from the original us study ( marwit and meuser , 2002 ) using onesample ttest . we calculated the sample size using bonett and wright 's formulas ( bonett and wright , 2000 , 2015 ) , setting the 95% confidence interval width at 0.2 . referencing to the original study ( marwit and meuser , 2002 ) , we expected the cronbach 's alpha of mmcgi to approximate 0.90 , corresponding to a minimum sample size of 11 ; spearman 's correlation coefficient between mmcgi and pg12 should approximate 0.8 , corresponding to a minimum sample size of 72 . table 1 shows the basic demographics of the participants and the patients with dementia they cared for . basic demographics of the caregivers and the patients with dementia they cared for ( n = 72 ) sd , standard deviation ; mmcgi , marwit meuser caregiver grief inventory . p value derived from univariate linear regression , with mmcgi score as the dependent variable . cronbach 's alpha , which measured the internal consistency of responses , was 0.97 for mmcgi . meanwhile , the cronbach 's alpha was 0.94 for personal sacrifice burden subscale , 0.92 for heartfelt sadness and longing subscale and 0.89 for worry and felt isolation subscale . contrasting among the three scales of pg12 , zbi and cesd ( table 2 ) , mmcgi expectedly correlated stronger with pg12 ( spearman 's = 0.79 , p < 0.001 ) than with zbi or cesd , while personal sacrifice burden subscale correlated stronger with zbi ( = 0.74 , p < 0.001 ) , heartfelt sadness and longing subscale with pg12 ( = 0.76 , p < 0.001 ) and worry and felt isolation subscale with pg12 ( = 0.79 , p < 0.001 ) . spearman 's correlation coefficient between the mmcgi ( total and subscale scores ) and the other scales of related constructa mmcgi , marwit meuser caregiver grief inventory ; pg12 , prolonged grief scale ; zbi , zarit burden interview ; cesd , centre for epidemiologic studies depression scale . p < 0.001 for all the values in the table , after bonferroni adjustment . as hypothesized , heartfelt sadness and longing subscale correlated poorer with cesd ( = 0.58 , p < 0.001 ) . mmcgi also correlated poorly with finances subscale of zbi ( = 0.37 , p = 0.162 ) and positive affect subscale of cesd ( = 0.33 , p = 0.530 ) . table 1 includes the p values derived from univariate regression analysis of factors related to mmcgi . in multivariate linear regression ( table 3 ) , factors associated with higher mmcgi score included caring for patients with severe dementia , spousal relationship , secondary or below education and malay ethnicity . employment and primary caregiver status both had p > 0.05 and were removed from the final model . the final model in multivariate linear regression , with mmcgi score as the dependent variable . mmcgi scores for the respective variables were also shown mmcgi , marwit meuser caregiver grief inventory ; sd , standard deviation ; ci , confidence interval . table 4 compares the mmcgi total and subscale scores between the current study and the original us study ( marwit and meuser , 2002 ) . the worry and felt isolation subscale score was 13.8% higher in this study ( p < 0.001 ) , while the rest of the scores were comparable with those of the usa ( marginal difference of 4.4% to 3.7% ) . comparison of mmcgi scores between the original us study ( n = 166 ) and the current singapore study ( n = 72 ) mmcgi , marwit meuser caregiver grief inventory ; sd , standard deviation ; ci , confidence interval . prior to this study , there were uncertainties about the applicability of predeath grief concept to the asian population . in many ways , this study supported the existence of predeath grief in asia and attests to the universality of human emotions . we showed that predeath grief was measurable in a reliable and valid manner , using an established predeath grief scale of mmcgi . the risk factors of spousal caregivers and advanced dementia reported here were similarly highlighted in a systematic review of the caucasian studies ( chan et al . , 2013 ) . even the mmcgi total and subscale scores were largely comparable with those of the usa ( marwit and meuser , 2002 ) . these results suggest a need for clinicians to be attuned to the predeath grief experience of dementia caregivers even in the noncaucasian populations . this is particularly important because predeath grief in dementia caregivers is commonly disenfranchised ( frank , 2007 ) , and its expression is not generally acceptable in society . unless the clinicians inquire about its presence , dementia caregivers are unlikely to volunteer such feelings and will suffer in silence until they experienced the irrevocable adverse effects of predeath grief . by such time , it may be too late to intervene . asian caregivers showed more worries and felt isolation , and certain ethnicity experienced more predeath grief . to attune to the predeath grief experience of caregivers , clinicians need to be mindful of the disparate presentations of predeath grief in different cultures . in the example of asian caregivers , strong expression of worries and felt isolation may be an indicator of underlying predeath grief . such cultural sensitivity is also necessary to tailor personcentred interventions and to better support the emotional needs of caregivers in multiethnic populations . the influence of culture on predeath grief is an area worth further exploration in future researches . the caregiver risk factors in this study ( spouse , lower education and selected ethnicity ) paralleled the predictors of complicated postdeath grief from a systematic review ( chan et al . , 2013 ) previous studies showed that greater predeath grief is a key predictor of postdeath complicated grief ( blandin and pepin , 2015 ) . identifying shared risk factors between predeath and postdeath grief opens a window of opportunity to target efforts of case finding and early intervention in the predeath setting . such efforts can prevent the development of complicated grief following bereavement ( schulz et al . one of the strengths of this study lies with the location where it was conducted . singapore is a cosmopolitan city in asia and holds a number of major asian cultures , including that of the chinese , indian , malay muslim and eurasian . this environment provided an opportune test bed to answer our research question on the applicability of predeath grief in asia . they alert readers on the likely presence of predeath grief even in the other parts of asia , and serve as an impetus for further researches on predeath grief in asia . in this study , limited proportion of spousal caregivers participated because a number of them could only read in chinese and not in english . we also restricted the number of variables entering multivariate regression so that the analyses remain valid given the sample size . even with these two limitations , we have had reasonable evidence from this study to show that predeath grief do exist among dementia family caregivers in asia . we hope to better represent spousal caregivers and identify even more risk factors in our ongoing study involving chinese mmcgi and larger sample size . in summary , predeath grief is applicable even to the noncaucasian population , and it is detectable with an objective scale like mmcgi . clinicians working with noncaucasian populations need to be attuned to its presence and be sensitive to the influence of culture on its expression key points predeath grief is also applicable to dementia caregivers in a noncaucasian population , and it is detectable with an objective scale like mmcgi.to prevent the adverse outcomes of predeath grief , clinicians working with noncaucasian populations need to be sensitive to its presence and to the influence of culture on its expression.identifying shared risk factors between predeath and postdeath grief allows targeted efforts of case finding and early intervention in the predeath setting , thus preventing complicated grief following bereavement . predeath grief is also applicable to dementia caregivers in a noncaucasian population , and it is detectable with an objective scale like mmcgi.to prevent the adverse outcomes of predeath grief , clinicians working with noncaucasian populations need to be sensitive to its presence and to the influence of culture on its expression.identifying shared risk factors between predeath and postdeath grief allows targeted efforts of case finding and early intervention in the predeath setting , thus preventing complicated grief following bereavement . predeath grief is also applicable to dementia caregivers in a noncaucasian population , and it is detectable with an objective scale like mmcgi . to prevent the adverse outcomes of predeath grief , clinicians working with noncaucasian populations need to be sensitive to its presence and to the influence of culture on its expression . identifying shared risk factors between predeath and postdeath grief allows targeted efforts of case finding and early intervention in the predeath setting , thus preventing complicated grief following bereavement .

objectivepredeath grief is prevalent among dementia family caregivers . when unaddressed , it produces adverse outcomes . with its research primarily conducted in caucasians , its applicability to noncaucasians is uncertain . we explore the existence and the characteristics of predeath grief in a multiethnic asian population using an established predeath grief scale marwit meuser caregiver grief inventory ( mmcgi).methodsseventytwo dementia family caregivers were recruited from a tertiary hospital . existence of predeath grief was shown by its measurability on mmcgi , together with good internal consistency reliability and construct validity . characteristics of predeath grief were explored through multivariate linear regression of mmcgi and by comparing mmcgi scores with those from the original us study using onesample ttest.resultsin the asian context , predeath grief was measurable in a reliable and valid manner . risk factors of predeath grief included caring for patients with severe dementia , spousal relationship and secondary or below education . influence of culture was palpable asians had more worries and felt isolation , and certain ethnicity showed more predeath grief.conclusionspredeath grief is applicable even to the noncaucasian population . it bears much similarity to that in caucasians . yet , its expression is modified by culture . clinicians working with noncaucasian populations need to be sensitive to its presence and to the influence of culture on its expression . 2015 the authors . international journal of geriatric psychiatry published by john wiley & sons , ltd .

systemic lupus erythematosus ( sle ) is a chronic systemic autoimmune disease with considerable heterogeneity in clinical manifestations and disease course . the etiology of sle remains unknown , but evidence suggests that sle results from the interaction of genetic and environmental factors that ultimately promote an abnormal immune response leading to the organ damage . dysregulation of various components of the immune system can be seen at different stages of sle development , but hyperactivity of b cells with excessive production of multiple autoantibodies is perhaps the major immunological stigmata of sle [ 2 , 3 ] . the pathogenic role of b cells in sle is not however strictly linked to the secretion of autoantibodies . b cells can have potent antigen presentation capacity , and they regulate and organize inflammatory responses . indeed , studies in murine models of sle have shown that b cells are critical to the development of disease even when they are unable to secrete autoantibodies [ 5 , 6 ] . moreover , studies in human sle showed that the clinical response obtained by treatment of patients with rituximab , a b cell depleting anti - cd20 antibody , preceded autoantibody decline , thus supporting the autoantibody - independent role of b cells in sle process . a more detailed analysis of molecular mechanisms underlying the sle - associated tissue damaging - immune response revealed that interactions between t and b cells are essential in the development of this disease and that cytokines are key mediators of this interplay . however , because of the redundant functions of cytokines , a major challenge for scientists is to identify key molecule(s ) which can profoundly alter the outcome of sle - associated inflammatory response . here although il-21 was initially considered as a cytokine produced by activated peripheral blood t cells , it is now known that it can be synthesized by a range of differentiated cd4 + t helper ( th ) cells . studies in mice have shown that il-21 is synthesized exclusively by polarized th17 cells , while in human , il-21 can also be made by th1 cells [ 1116 ] . more recently , it has been shown that il-21 is also produced by a subset of cd4 + t cells , which are involved in germinal center ( gc ) formation and function , and denoted t follicular helper cells ( tfh cells ) [ 18 , 19 ] . tfh cells express high levels of the chemokine receptor chemokine ( c x c motif ) receptor 5 ( cxcr5 ) , which allows them to home to and be retained by the lymphoid follicle , where contact with antigen - primed b cells promotes b cell proliferation , isotype switching , and somatic mutation of the ig repertoire [ 20 , 21 ] . . however , these cells can synthesize il-21 if deleted of specific transcription factors , such as t - bet and eomesodermin , and infected with lymphocytic choriomeningitis virus . il-21 activity is mediated by a heterodimeric receptor , formed by the -chain subunit ( shared with il-2 , il-4 , il-7 , il-9 , il-13 , and il-15 receptors ) and its own unique receptor ( designated il-21r ) [ 23 , 24 ] . binding of il-21 to the receptor leads to the activation of the janus - kinase - family proteins ( jak 1 and jak 3 ) and signal transducer and activator of transcription ( stat ) 1 and stat3 and , to a lesser degree , stat4 and stat5 . il-21 also activates members of the mitogen - activated protein kinase ( mapk ) family such as extracellular signal - regulated protein kinases ( erk ) 1/2 in neoplastic cell , epithelial cells , and monocytes [ 25 , 26 ] . strictly linked to the activation of such intracellular pathways is the ability of il-21 to regulate the functional activity of various immune and non - immune cell types . here we highlight some of the biological effects of il-21 on b and t cells , because of the pathogenic role of these cells in sle . for a detailed description of the regulatory functions of il-21 on both immune and non - immune cells , the reader is directed towards recent reviews [ 10 , 23 , 24 , 27 ] . il-21 increases b cell proliferation after activation via the b cell receptor plus t cell - derived costimulatory signals [ 28 , 29 ] . in contrast , il-21 delivers proapoptotic signals to primary b cells and b cells that are activated with anti - igm or toll - like receptors ligands [ 30 , 31 ] . in cultures of cd40-stimulated human b cells , the proliferative effect of il-21 is dependent on stat1 and stat3 activation . in vitro , stimulation of both murine and human b cells with il-21 induces the plasma - cell - associated transcription factor b - lymphocyte - induced maturation protein 1 , now termed pr domain containing 1 , and subsequent plasma cell differentiation as well as the production of isotype - switched immunoglobulins ( i.e. , igg , igm , and iga ) [ 28 , 29 , 33 ] . importantly , the il-21-driven generation of memory b cells and plasma cells relies on stat3 , because il-21 fails to promote the differentiation of ig - secreting cells in cultures of nave b cells isolated from patients with inactivating mutations in stat3 . using a well - established model of b cell differentiation , avery et al . have also shown that the hen egg lysozyme - specific response of il-21r - null b cells is diminished compared with wild - type b cells , as indicated by the marked reduction in the number of antigen - specific plasma cells and production of igm and igg . overall , these results suggest that il-21 and stat3 play a crucial role in initiating and/or maintaining serological immunity by generating antigen - specific effector b cells from nave precursors . by contrast , stat3 does not seem to be necessary for the il-21-driven isotype switching to igg . il-21 enhances the proliferation of anti - cd3-stimulated t cells and acts in concert with other common -chain - dependent cytokines to enhance the growth of cd4 + t cells [ 10 , 24 ] . however , il-21 regulates differently the differentiation and/or maintenance of polarized th cells depending on the species . studies in mice revealed that il-21 can expand either th2 or th17 cell responses and inhibit the synthesis of interferon ( ifn)- [ 11 , 12 , 3436 ] . by contrast , in populations of human peripheral blood or mucosal t cells that are preactivated with anti - cd3 , il-21 enhances the expression / activation of transcription factors that drive th1 cell differentiation ( i.e. , t - bet , stat4 ) [ 14 , 37 ] . il-21 stimulates the proliferation of cd8 + t cells and synergizes with il-15 and il-7 in promoting cd8 + t cell expansion [ 3840 ] . studies in il-21-deficient mice showed that cxcr5 surface expression on cd4 + t cells is greatly reduced after immunization with a t - cell - dependent antigen , and that il-21r expression is significantly higher on cxcr5+cd4 + than on cxcr5cd4 + t cells . adoptive transfer of wild - type cd4 + t cells into il-21r - null recipients followed by immunization rescues gc formation and partially rescues ig production . the fact that il-21 controls the pool of memory b cells and promotes differentiation of b cells into plasma cells suggests that a deregulated il-21 activity may contribute to the development of autoimmune diseases . so , many researchers have evaluated the contribution of il-21 in the pathogenesis of murine models of sle these strains result from a cross between a c57bl/6 female and an sb / le male , and the male offspring of the cross had a 50% mortality rate at 6 months of age . the mice display many of the symptoms common to sle , including lymphadenopathy , splenomegaly , hypergammaglobulinemia , and severe immune complex mediated glomerulonephritis . subsequent studies have verified that the disorder is not gonadal hormonally driven but is y - linked . analysis of multiple genes in splenocytes taken from these mice revealed a marked age - dependent increase in the levels of il-21 mrna as compared to wild - type mice . corresponding to the increase in il-21 mrna , serum levels of il-21 , igg1 , and igg3 were increased in bxsb.b6-yaa+/j mice . importantly , il-21r - deficient bxsb - yaa+/j mice show none of the abnormalities characteristic of sle , thus supporting the key role of il-21 in the accumulation of plasma cells and production of autoantibodies . in this model , the excessive il-21 production did not derive from tfh cells , but rather from an extrafollicular population of icos+ cd4 + t cells . further support to the pathogenic role of il-21 in this model of sle was provided by preclinical studies showing that administration of il-21r / fc , a fusion protein neutralizing il-21 , to bxsb.b6-yaa+ mice results in a decreased production of il-21 , diminished lymphocyte activation , and reduced circulating igg1 levels . proteinuria is also reduced in treated mice , but the therapy does not prevent the symptoms of sle . moreover , follow - up studies showed that the il-21 contribution to sle - like phenotype in bxsb - yaa mice is variable within the time course of disease progression , because blockade of il-21 activity in the early phase is deleterious , whereas later in the time course it is advantageous . the reason why the blocking il-21r / fc regulates differently the pathogenic inflammatory response in bxsb - yaa mice remains unknown . in this context , it is noteworthy that il-21 can exert both inflammatory and anti - inflammatory effects , the latter linked to the induction of il-10 , a counter - regulatory cytokine expressed at high levels both in bxsb - yaa mice and in human sle patients [ 33 , 4547 ] . therefore , blockade of il-21 with il-21r / fc might inhibit il-10 expression , thus exacerbating the severity of sle symptoms in the early phase of the disease . studies in mrl - fas mouse , another model of sle , showed that blockade of il-21 with il-21r / fc significantly reduced proteinuria , lymphadenopathy , skin lesions , circulating autoantibodies , and igg1 and igg2a . in addition , mrl - fas mice treated with anti il-21r / fc showed reduced levels of glomerular igg deposits in the kidney and no thickening in glomerular basement membranes by histological evaluation . il-21r / fc treatment also reduced the number of splenic t lymphocytes and b cells antibodies production . in the mrl - fas mouse , il-21 is primarily made by an extrafollicular population of icos - expressing cd4 + t cells that exhibits reduced expression of p - selectin glycoprotein ligand 1 but is able to produce il-4 and ifn- . evidence for the pathogenic role of il-21 in sle also comes from studies in the sanroque mouse strain , in which a mutation in the ring - type ubiquitin ligase protein family member , roquin , results in the accumulation of excessive numbers of both gc and tfh cells with high levels of icos , excessive il-21 production , and severe sle - like autoimmune phenotype . lupus - like symptoms are dependent on enhanced gc formation as they could be reduced by deletion of even one allele of the bcl6 gene . however , tfh formation in this model seems to be dependent on icos rather than il-21 . a subpopulation of b-1 cells expressing the programmed death ligand 2 ( termed l2pb-1 cells ) has been shown to be enriched for autoreactive immunoglobulin , to be potent in antigen presentation , and to be fully able to facilitate th1 and th17 cell differentiation . l2pb1 cells are found in increased numbers in the periphery of mice with sle and such an increase correlates with anti - dna antibody levels . this well fits with the demonstration that l2pb-1 cells secrete high levels of autoantibodies , such as dsdna - reactive antibodies , and can undergo isotype switch and generate igg1 and igg2b in the presence of il-21 . plasma levels of il-21 are significantly higher in sle patients than in healthy controls , but il-21 levels do not seem to correlate with sle activity . we have recently shown that il-21 mrna expression is enhanced in skin biopsies taken from individuals with sle in comparison to normal controls , even though this increase is less pronounced than that seen in lesional skin of patients with psoriasis . in patients with sle , il-21r is under - repressed on total , nave , and memory b cells , and on plasmablasts , compared to controls . reduced levels of il-21r are associated with nephritis and a high titer of anti - double - stranded dna antibodies in the serum . by contrast , blood t cells of sle patients exhibit normal levels of il-21r . recent genome - wide association ( gwa ) studies have shown that single nucleotide polymorphisms ( snps ) in the chromosome 4q27 region containing il-2 and il-21 are associated with chronic inflammatory disorders , including inflammatory bowel disease ( ibd ) , celiac disease , psoriasis , diabetes , rheumatoid arthritis , asthma , and sle , suggesting a common genetic background for these diseases [ 5662 ] . with regard to sle , a first study genotyped three snps in the il-21 gene in more than 1300 patients with sle and controls . two out of the three snps ( rs907715 and rs2221903 ) were significantly associated with sle . stratification by race revealed that this genetic association was found more in european - american than in african - american sle patients . the fact that both associated snps are located within intronal regions of the il-21 gene suggests however the possibility that they can influence il-21 production . another gwa study analyzed 17 snps in the il-21r gene in 2 independent , ethnically divergent populations ( european and hispanic ) of sle patients and controls . a genetic association between rs3093301 ( a / g ) and sle was found in both the european - derived and hispanic cohorts . moreover , the lupus - risk allele was significantly associated with the presence of malar rash but not other symptoms or signs of sle . the involvement of il-21 in the pathogenic mechanisms causing human sle remains to be demonstrated . however , both the ability of this cytokine to regulate in vitro and in vivo immunological pathways that are relevant in sle and the evidence obtained in murine models of sle suggest that il-21 could play an important role in the production of pathogenic autoantibodies and end - organ damage in this disease . therefore , it is tempting to speculate that inhibitors of il-21 could be useful to attenuate lupus - related clinical manifestations . however , in designing such clinical interventions for blocking il-21 one should take into consideration not only the advantageous effects but also the risk of potential and deleterious consequences for the host . in fact , it is well - known that , at least under specific circumstances , il-21 can also exert anti - inflammatory actions due to its ability to inhibit dendritic cell maturation and stimulate il-10 production [ 45 , 64 ] . in this context , it is also noteworthy that il-21 may stimulate immune responses against tumor cells and promote cd8 + t cell responses against viruses [ 6568 ] . therefore , if so , blockade of il-21 function could attenuate antineoplastic immunity and wake worse the course of viral infections .

systemic lupus erythematosus ( sle ) is an autoimmune disorder characterized by excessive production of a variety of autoantibodies and a wide range of clinical manifestations . pathogenesis of sle is complex and not fully understood . there is however evidence that b and t cells are critical to the development of disease , and that t cell - derived cytokines are involved in the sle - associated inflammatory response . one such cytokine seems to be interleukin ( il)-21 , the latest identified member of the -chain - related cytokine family . il-21 has an important role in the control of the growth , survival , differentiation , and function of both t and b cells , and excessive production of il-21 has been associated with the development of multiple immune - mediated diseases . here we review data supporting the involvement of il-21 in the pathogenesis of sle .

network - driven spindle - like oscillations are a functional hallmark of the developing cerebral cortex . during late prenatal and early postnatal stages of development , spontaneous spindle - like oscillations have been identified as physiological activity patterns in various neocortical areas of different mammalian species [ 14 ] . in humans , so - called delta brushes can be observed in eeg recordings from preterm babies already at gestational week 28 , that is ~12 weeks before normal birth of a full - term neonate ( for review see ) . in ac - filtered eeg recordings , delta brushes are brief rhythmic delta waves ( 0.31.5 hz ) of 50300 v amplitude with a superimposed burst of fast rhythm ( > 8 hz , the brush ) . it has been suggested that delta brushes in human preterm infants correlate with so - called spindle bursts recorded in rodents during the early postnatal period . from a developmental point of view , these early spontaneous activity patterns in developing rodent and human cerebral cortex are probably of similar or even identical origin . rats and mice are altricial - born in a far less mature condition than humans . in rodents the degree of neocortical development at the day of birth ( postnatal day [ p ] 0 ) can be compared to the stage of human cortex between gestational weeks 28 and 32 and the cerebral cortex of a p12p14 rat is comparable to that of the full - term newborn human baby [ 6 , 8 ] . in preterm infants born between gestational weeks 28 and 32 , milh et al . and colonnese et al . recorded eeg signals containing spontaneous and stimulus - evoked delta brushes with oscillatory activity in the frequency range of 8 to 25 hz , suggesting that spontaneous delta brushes may represent a physiological neocortical activity pattern of the human fetus in utero . in the cerebral cortex of rodents , spindle bursts ( beside short gamma oscillations ) constitute the majority of spontaneous activity during the first postnatal week ( figure 1 ) . these spindle bursts resemble in their appearance spindles recorded in the adult brain during sleep . these sleep spindles are one of the hallmarks of human stage 2 sleep for comprehensive overviews the reader is referred to recent reviews by lthi and by mccormick et al . . however , in humans sleep spindles appear 4 to 9 weeks after birth , which is much later than the disappearance of delta brushes around the end of the first postnatal week , thus excluding the hypothesis that delta brushes or spindle bursts gradually develop into sleep spindles . in addition , the frequency profile of spindle bursts and delta brushes displays a rather broad frequency distribution up to 25 hz , whereas sleep spindles present oscillations in a narrower band of ~1015 hz . the present review focuses on spindle burst activity in the cerebral cortex of the developing rat during the first postnatal week and summarizes our current understanding ( i ) on the functional properties of spindle bursts , ( ii ) the mechanisms underlying their generation , ( iii ) the synchronous patterns and cerebral networks associated with spindle bursts , and ( iv ) the physiological and pathophysiological role of spindle bursts during early cortical development . two distinct activity patterns can be observed in the neonatal rat cerebral cortex in vivo : gamma oscillations and spindles bursts ( figures 1 and 2 ) [ 1419 ] . gamma oscillations have a duration of 100 to 300 ms , a frequency of 30 to 40 hz and appear spontaneously every 10 to 30 s ( figures 1 and 2(b ) ) . the properties and functional role of gamma oscillations as well as the mechanisms underlying their generation spindle bursts are characterized by their spindle - like oscillatory appearance , have a duration of 0.5 to 3 s and a frequency in the range of 8 to 30 hz , and occur spontaneously every ~10 s ( figures 1 and 2(a ) ) . as recognized in full - band direct - current ( dc ) coupled recordings , spindle bursts are nested in slow ( delta ) waves , which in infant rats , and preterm human babies [ 22 , 23 ] have been termed spontaneous activity transients ( sats ) . spindle bursts have been described in primary somatosensory cortex ( s1 ) including barrel cortex [ 14 , 1619 ] , in primary visual cortex ( v1 ) [ 10 , 15 ] , in primary motor cortex ( m1 ) , and in prefrontal cortex of anesthetized and awake rats during the first postnatal week . in s1 , spontaneous and stimulus - evoked spindle bursts can be observed as early as p0 [ 17 , 18 ] . in rats , the incidence of spindle bursts declines during the second postnatal week and sporadic spindle bursts are obscured by the ongoing neocortical activity . likewise in humans , delta brushes are replaced by more continuous eeg activity at birth of a full - term infant . this gradual developmental shift from a highly synchronized state of spontaneous activity to a desynchronized state seems to be a fundamental network property of the cerebral cortex during early neonatal stages . spindle bursts synchronize the activity of a local neuronal network of 200 to 400 m in diameter , which resembles the dimension of a whisker - related neocortical column in the immature barrel cortex ( figure 3 ) [ 18 , 25 ] . using a combination of voltage - sensitive dye imaging and high - density multielectrode recordings in p0-p1 rat barrel cortex in vivo , yang et al . could demonstrate that this early spontaneous activity constitutes the later emergence of the whisker - related barrel field map . these data indicate that spontaneous activity patterns at this early age form functional precolumns , supporting the concept of the existence of ontogenetic columns in the radial unit hypothesis . further support for this hypothesis comes from in vivo two - photon calcium imaging in the barrel cortex of both anesthetized and nonanesthetized newborn mice , demonstrating highly synchronous spontaneous burst activity , reminiscent of spindle bursts , in local networks of 100 to 200 m in diameter . these data strongly indicate that early spindle bursts , and probably also gamma oscillations ( for review ) , synchronize early neocortical networks into functional columns at a developmental stage when the upper layers 2/3 have not even been formed ( i.e. , in rats at p0 ) . at this developmental stage , thalamocortical afferents have not reached layer 4 and instead transiently innervate the subplate ( for review [ 28 , 29 ] ) . thalamic afferents form transient glutamatergic synapses with surprisingly mature properties including ampa and nmda receptors [ 3032 ] . in vitro studies in acute brain slice preparations and in intact whole cortical hemisphere preparations have demonstrated that oscillatory network activity in the frequency range of spindle bursts depend on an intact subplate [ 34 , 35 ] . selective removal of the subplate in s1 in vivo causes a significant decline in the occurrence of spontaneous spindle bursts and disturbances in the development of the cortical architecture in the barrel cortex . these data further support the hypothesis that spindle bursts in developing cerebral cortex fulfill an important role in the maturation of the neocortical architecture . in the next section we will discuss the network and molecular mechanisms underlying the generation of spindle bursts in neonatal cerebral cortex . the rodent cerebral cortex develops rapidly during late prenatal and early postnatal stages and at least four different activity patterns may occur sequentially between birth and the end of the first postnatal week ( for review ) . furthermore , the cortex shows a mediolateral and anterior - posterior gradient in development and within the same neocortical area neurons in upper layers 2/3 are ~2 days younger compared to lower cortical layers . many inconsistencies in the literature on the properties of spontaneous activity patterns in newborn rodents and their underlying mechanisms can be explained by the fact that these important developmental differences are often ignored . in addition , 2 days in early rodent cortical development make a large difference , so that the neocortex of a p0 rat ( without layer 2/3 ) can not be compared to that of a p2 rat ( with almost complete lamination ) . already in newborn ( p0-p1 ) rat barrel cortex in vivo , mechanical stimulation of a single whisker elicits in field potential recordings and to some extent detectable also in voltage - sensitive dye imaging ( vsdi ) a sequence of an early gamma oscillation followed by a spindle burst ( figure 3(b ) ) . at this age , the thalamocortical activity reaches the developing cortical network largely via the subplate [ 17 , 31 , 32 ] and is amplified by an intrinsic gap - junction coupled network within the subplate and cortical plate [ 28 , 34 ] . spontaneous and evoked delta brushes can be observed in premature human neonates of 2832 weeks postconceptional age , a developmental stage when the human cerebral cortex resembles that of a newborn rat . impressive examples of large delta brushes are provided in the supplementary eeg videos of milh et al . , demonstrating that a single touch elicits a large oscillatory response in the somatosensory evoked potential ( sep ) recorded above the contralateral parietal cortex . in mature human cortex , seps with smaller amplitudes and shorter durations can be only obtained after averaging of at least 100 epochs . thus , in both species , rats and humans , at a comparable stage of cortical development mechanical stimulation of the sensory periphery elicits in s1 spindle bursts and delta brushes , respectively . simultaneous multielectrode recordings in the barrel cortex and in the ventral posteromedial nucleus ( vpm ) of the somatosensory thalamus of p0-p1 rats in vivo have demonstrated that the majority of spontaneous cortical spindle bursts and also gamma oscillations are not generated within s1 , but rather in subcortical structures or outside of s1 ( figures 4(a ) and 4(b ) ) . at this age a local , functionally defined lesion in the vpm blocks the whisker stimulation - induced cortical responses and also profoundly reduces the spontaneously occurring cortical burst activity , further demonstrating that the majority of the spontaneous spindle bursts in p0-p1 rat barrel cortex are generated in subcortical structures of the whisker - to - barrel cortex pathway ( for further information see ) . silencing the sensory periphery by injection of lidocaine into the whisker pad causes a significant reduction in the occurrence of spontaneous spindle bursts and gamma oscillations by ~50% ( figure 4(c ) ) , indicating that during this developmental period at least half of the spontaneous burst activity in s1 is related to activity in the sensory periphery . a similar peripheral generation of spontaneous burst activity may occur in human preterms and fetuses between gestational weeks 28 and 32 [ 9 , 22 ] . similar to the spindle bursts in s1 , spontaneous spindle bursts in v1 are also largely generated in the sensory periphery . ( for review ) , provide the primary drive for spindle bursts in newborn rat v1 , as demonstrated by simultaneous recordings from the retina and v1 . intraocular injection of forskolin , which augmented retinal waves , increased the occurrence of v1 spindle bursts , and removal of the retina reduced the spindle bursts frequency . as in the somatosensory system , spindle bursts in v1 can be also evoked by stimulation of the sensory periphery . however , since rod- and cone - mediated visual signaling is not functional in rats during the first postnatal week , spindle bursts can not be evoked by light flashes before p8 . at that age , the neocortical response in v1 consists of an early visual evoked response followed by an evoked spindle burst . similar responses could be observed in v1 of preterm infants once photoreceptor mediated light responses occur in the retina ( for review ) . whereas a large amount of experimental data has shown that retinal waves provide the main trigger for the cortical v1 spindles bursts , it is not completely understood which pacemaker drives the spontaneous activity in s1 and how spontaneous activity is generated in the somatosensory periphery . in p3p6 rats spontaneous whisker movements occur during active sleep and are correlated with activation of whisker - related cortical columns in the barrel cortex . in newborn rats the proprioceptive feedback from self - generated myoclonic movements trigger spindle bursts in s1 ( for review ) . spontaneous limb movements of the human fetus during the third trimester of gestation , or those of the preterm infant associated with delta brushes in s1 , are similar to these twitching movements of the neonatal rat and can be also triggered by sensory feedback . kreider and blumberg have demonstrated in 1-week - old rats that the mesopontine region plays a central role in the generation of myoclonic twitching . have shown in newborn rats that spatially confined spindle bursts in s1 are triggered in a somatotopic manner by sensory feedback signals from spontaneous muscle twitches . these spontaneous movements are generated by neuronal networks in the spinal cord , but spindle bursts persisted at a reduced frequency after sensory deafferentation ( spinal cord transection ) in s1 , indicating that spindle burst activity can be also generated in neocortical or thalamocortical circuits during this early period of development . this assumption is also supported by the observation that silencing of the sensory periphery causes only a ~50% reduction in the occurrence of spontaneous spindle bursts ( figure 4(c ) ) . however , since it can not be excluded that the remaining portion of spindle bursts actually conveys activity from adjacent or distant sensory areas ( transmitted via inter- and intrahemispheric connections , see next chapter ) , the outcome of this experiment may underestimate the contribution of the sensory periphery . simultaneous monitoring of forepaw movements , vsdi , and extracellular multielectrode recordings in s1 and m1 of p3p5 rats under light urethane anesthesia have demonstrated that tactile forepaw stimulation triggers spindle bursts in s1 , followed by gamma and spindle bursts in m1 ( figure 5 ) . focal electrical stimulation of corticospinal tract neurons in layer 5 of m1 mimicking physiologically relevant 40 hz gamma or 10 hz spindle burst activity reliably elicited forepaw movements , indicating that m1 cortical spindle bursts are capable of triggering muscle twitches at this age . however , only 23.7% of the spontaneous bursts in m1 triggered forepaw movements and were followed by spindle bursts in s1 ( figures 5(b)(a ) and 5(c ) ) , indicating that only a fraction of m1 activity transients triggers motor responses directly . in 40.7% of the cases , spontaneous movements preceded the burst activity in m1 and s1 ( figures 5(b)(b ) and 5(c ) ) , suggesting that this activity may arise from subcortical regions in the brainstem or spinal cord . the remaining 35.6% of the m1 bursts were unrelated to any movements ( figure 5(c ) ) . the finding that 23.7% of the movements were triggered by m1 bursts as observed by an et al . is in contrast to previous observations , which demonstrated that dissection of neocortical inputs fails to suppress muscle twitches in rat pups . in summary , these data indicate that neocortical spindle bursts in newborn rodents ( and delta brushes in human fetus during the third trimester or in preterms ) are generated by central pattern generator ( cpg ) circuits in spinal cord , brainstem , and motor cortex ( for cpg circuits in mature brain see [ 4244 ] ) . neuropharmacological studies provided insights into the molecular mechanisms underlying the generation and modulation of neocortical spindle bursts . in vivo and in vitro data suggest that gabaergic synapses are not crucial for the generation of spindle bursts or spindle burst - like activity , respectively , but are essential for their spatial confinement to a cortical ( pre- ) column . in contrast , spindle bursts depend on intact glutamatergic synapses including alpha - amino-3-hydroxy-5-methyl-4-isoxazole propionic acid ( ampa ) and n - methyl - d - aspartate ( nmda ) receptors [ 34 , 45 ] . carbachol - induced spindle - like oscillations in p0p3 mouse neocortex in vitro and spindle bursts in p0p2 rat in vivo are blocked or significantly reduced by different gap junction blockers , indicating that electrical synapses are critically involved in the generation of spindle bursts at this neonatal period . however , minlebaev et al . reported for p1p3 rats a significant increase in the occurrence of spontaneous spindle bursts in s1 following application of the gap - junction blocker mefloquine . single - cell recordings revealed additional insights into the mechanisms underlying the generation of spindle bursts . spindle bursts in v1 and s1 are accompanied by a barrage of glutamatergic and gabaergic postsynaptic currents ( pscs ) that are phase - locked to the spindle oscillations ( figure 6 ) . in prefrontal cortex , glutamatergic and gabaergic synaptic inputs to excitatory pyramidal neurons are phase locked to the theta - band component of the spindle , while pscs of inhibitory interneurons are phase locked to the higher beta and gamma frequencies , suggesting that excitatory and inhibitory neurons differentially modulate the distinct components of the spindle bursts . further , the application of cnqx eliminates the glutamatergic pscs and completely blocks the occurrence of spindle bursts , indicating a major causal role of ampa - receptor mediated glutamatergic inputs in the generation of spindle bursts . additional insights have been obtained by experiments in which subplate cells were selectively ablated in s1 shortly after birth . in these animals these in vivo results support previous in vitro studies , in which spindle bursts elicited by cholinergic stimulation were suppressed after removal of the subplate ( see below ) [ 34 , 35 ] . taken together , these data suggest that thalamocortical inputs relayed and amplified by the subplate [ 28 , 29]play an important role in the generation of spindle bursts ( e.g. , [ 17 , 45 ] ) . spindle bursts in the cerebral cortex of newborn rats can be elicited [ 34 , 35 ] and modulated by cholinergic mechanisms . in vitro , spindle burst - like oscillations can be reliably induced by activation of muscarinic acetylcholine receptors , predominantly of the m1 and m5 type . in vivo , spindle bursts in v1 are decreased by ~50% following the application of the muscarinic receptor antagonist atropine . furthermore , blockade of acetylcholine esterase with physostigmine or direct stimulation of the cholinergic basal forebrain nuclei augmented the occurrence v1 spindle bursts , indicating that the cholinergic system facilitates spindle burst activity in developing cerebral cortex . as discussed above , spatially confined spindle bursts in newborn rat cortex synchronize a local neuronal network resembling a neocortical ( pre- ) column ( figure 3 ) . beside these intra - areal synchronization , spindle bursts are also synchronized between different cortical regions within one hemisphere ( intrahemispheric ) . as described above , a tight functional correlation in spontaneous and stimulus - evoked spindle burst activity exists between s1 and m1 cortex ( figure 5 ) . spindle bursts with similar properties as those in v1 and s1 have been also recorded in vivo in the prefrontal cortex of urethane - anesthetized rats older than p2 . the same authors demonstrate that the hippocampus drives this early activity in the prefrontal cortex . beside this intrahemispheric synchronization between different cortical regions , spindle bursts also interact between both hemispheres ( interhemispheric ) . in vivo simultaneous recordings of spontaneous activity in homotopic cortical areas in both hemispheres at the same stereotaxic coordinates and depth have demonstrated that the amount of interhemispheric synchronization in newborn rats is initially rather low and increases during the first postnatal week . this interhemispheric communication of spindle burst activity depends on an intact corpus callosum . in this regard , in unanesthetized newborn rats callosotomy doubled the occurrence of spontaneous spindle bursts , suggesting that the corpus callosum modulates functionally inhibitory interactions between homotopic regions in both hemispheres during the occurrence of spindle burst activity . experiments in p2p15 rats demonstrated that this callosotomy - induced disinhibition is a transient feature of early development that disappears abruptly after p6 . it is not surprising that intra- and interhemispheric interactions of spontaneous activity in the spindle burst frequency range can be also observed in developing human cerebral cortex at early stages . using eeg and functional magnetic resonance imaging ( fmri ) omidvarnia et al . electric resting - state network that shows functional intra- and interhemispheric interactions in the 815 hz frequency range . in summary , several in vitro and in vivo studies have demonstrated that spindle bursts represent elementary states of intra- and interhemispheric synchronization in the very immature cerebral cortex . an increasing amount of experimental and clinical data strongly indicate that spindle bursts play an important role in the physiological development of the cerebral cortex . experimental evidence indicates that spindle bursts may be particularly suited to interfere with early neurodevelopmental processes , and thus disturbances in spindle burst activity may cause long - term structural and functional disorders . at early stages of development spontaneous and sensory - evoked activity patterns influence a variety of developmental processes , such as neurogenesis , apoptosis , neuronal migration , cellular differentiation , network formation , and myelination ( for review , see ) . it is not completely understood how electrical activity controls these different developmental processes and whether distinct activity patterns , such as spindle bursts , play a specific role . however , for the control of apoptotic cell death of immature neurons in vitro and in vivo the essential role of spontaneous network bursts to provide antiapoptotic signals has been demonstrated [ 52 , 56 , 57 ] . for this activity - dependent control of neuronal survival the phosphatidylinositol 3-kinase pathway plays an important role , while the mapk / extracellular signal - regulated kinase or the calcium / calmodulin - dependent protein kinase pathway is not directly involved . since one spontaneous spindle burst is associated at the single neuron level with 510 action potentials and the frequency of spontaneous spindle bursts is ~5 per minute [ 16 , 17 ] , a single neuron discharges with 2550 action potentials per minute . under in vitro conditions this discharge frequency supports neuronal survival of developing neocortical neurons , suggesting that spontaneous spindle bursts in vivo provide an important physiological signal for the control of neuronal survival versus apoptosis in the neonatal cerebral cortex . notably , spindle burst and gamma activity provides an ideal physiological stimulus for the activity - dependent release of bdnf , an important antiapoptotic signal . balkowiec and katz demonstrated for neuronal cultures that 3060 min of electrical burst stimulation ( 50 pulses at 2050 hz at intervals of 20 s ) increased extracellular bdnf levels by 20-fold , whereas stimulation patterns at lower frequency ( albeit producing the same number of extracellular electric shocks ) were ineffective ( for review , see ) . these data indicate that spontaneous spindle bursts represent a physiological trigger for the release of bdnf , which plays an important role in several aspects of development ( for review , see ) . using in vitro and in vivo models it has been recently shown that an experimentally induced inflammation by application of the endotoxin lipopolysaccharide induces rapid ( < 2 h ) alterations in the pattern of spontaneous spindle bursts and gamma oscillations in neonatal rodent cerebral cortex , which subsequently leads to increased apoptotic cell death . these inflammatory effects are specifically initiated by the microglia - derived proinflammatory cytokine tumor necrosis factor alpha and to a lesser extent by the chemokine macrophage inflammatory protein 2 . thus , inflammation causes a fast dysfunction in the pattern of spontaneous burst activity , which subsequently leads to increased apoptotic cell death , most likely by disturbances in the release of survival factors such as brain - derived neurotrophic factor ( bdnf ) acting on neurotrophin tropomyosin - related kinase b / c receptor . furthermore , removal of the subplate massively reduced spindle burst activity and led to a persistent loss of the typical barrel - like whisker representation within layer 4 , indicating that spindle bursts play a role in the development of the neocortical architecture . in summary , these experimental data suggest that any disturbances in the spontaneous activity of the developing cerebral cortex ( including spontaneous spindle bursts ) induce acute dysfunctions , which may cause long - term disorders . however , it remains to be elucidated whether spindle burst in particular can be causally related to neurodevelopmental disturbances . it has been recently shown in extremely preterm infants that the properties of neocortical bursts recorded with eeg and their scaling relationships correlate significantly with later cognitive development . these clinical data suggest that analyses of burst shapes obtained in eeg recordings from preterm and full - term newborn babies may have diagnostic use in neonatal intensive care units and predict the clinical outcome . while it is generally accepted that early electrical activity shapes the maturation of neocortical circuits , it remains an open question whether the specific properties of spindle burst are required or fulfill a distinct role in development . in this regard , it is possible that the spatiotemporal patterns of spindle bursts translate into a local molecular signal which fulfills an important developmental role . in particular as gaba does not seem to be necessary for the generation or maintenance of spindle bursts , and since gaba is essential for neuronal differentiation [ 64 , 65 ] , it is tempting to speculate that spindle bursts control the spatially confined release of gaba in developing local networks . another unresolved issue is that both spindle bursts and gamma oscillations can be observed during the same period of early development . they can be observed in early postnatal rodent brain from the day of birth (; also see ) and occur spontaneously as well as after sensory stimulation . moreover , both can be observed mainly during the critical periods of the primary sensory areas , although spindle bursts probably persist for slightly longer periods . in addition , spindle bursts and early gamma oscillations in newborn cortex are proposed to rely on thalamocortical inputs , in contrast to gamma oscillations in adult cortex which depend on perisomatic gabaergic inhibition . beside their differences in duration , occurrence , and frequency ( see above ) , spindle bursts and gamma oscillations in newborn cortex reveal a number of additional distinctions . in the immature barrel cortex the vast majority ( > 90% ) of spontaneously occurring spindle burst spans several barrel - related columns , whereas the majority ( ~70% ) of spontaneous gamma oscillations are restricted to a single or two barrel related columns . in line with these observations , gamma oscillations evoked by tactile stimulation are also closely related to a single functional column , while evoked spindle bursts span over more than one column . thus , one reason for the coexistence of spindle bursts and gamma oscillations during early postnatal development might be a distinct role in neocortical maturation . gamma bursts may reflect local information processing mostly within a single functional column , thus supporting the maturation of a column related network . in contrast , spindle bursts probably reflect larger local network events and may thus serve to promote the connectivity between neighboring columns . although spindle bursts as well as gamma oscillations are spatially confined to a small network in one neocortical area , it is unclear to what extent and how this activity connects to other cortical and subcortical regions ( e.g. , see [ 18 , 19 ] ) . further , it remains to be studied whether the immature brain shows a spindle burst related resting state and how this network state is altered by sensory activation or by pathophysiological events . finally , it would be most interesting and important to correlate specific patterns of spontaneous activity ( e.g. , delta brush ) recorded by means of full - band direct - current eeg in preterm und full - term human neonates with the acute functional state and with the further development of the child , as impressively done by vanhatalo and colleagues [ 62 , 67 ] . spatially confined spindle bursts and delta brushes represent the most prominent physiological activity patterns in the developing cerebral cortex of newborn rodents and preterm human infants , respectively . spontaneous and stimulus - evoked spindle bursts can be observed in various neocortical areas of different mammalian species and play important roles in the early development of cortical networks . however , it remains to be studied in more detail how exactly spindle bursts influence the maturation of the cortex and how a potential long - term dysfunction due to disturbances in spindle burst activity can be prevented by early intervention . since this important type of brain activity is already present in the human fetus in utero ( either spontaneously occurring or related to sensory inputs from the uterine environment ) , a better understanding of the physiological relevance of spindle burst oscillations is of major clinical relevance .

spontaneous and sensory evoked spindle bursts represent a functional hallmark of the developing cerebral cortex in vitro and in vivo . they have been observed in various neocortical areas of numerous species , including newborn rodents and preterm human infants . spindle bursts are generated in complex neocortical - subcortical circuits involving in many cases the participation of motor brain regions . together with early gamma oscillations , spindle bursts synchronize the activity of a local neuronal network organized in a cortical column . disturbances in spindle burst activity during corticogenesis may contribute to disorders in cortical architecture and in the activity - dependent control of programmed cell death . in this review we discuss ( i ) the functional properties of spindle bursts , ( ii ) the mechanisms underlying their generation , ( iii ) the synchronous patterns and cortical networks associated with spindle bursts , and ( iv ) the physiological and pathophysiological role of spindle bursts during early cortical development .

high - risk vascular surgery patients , including those with thrombotic disorders , present safety and technical challenges for anesthetic management . given the poor tolerance of these patients to hemodynamic perturbations , the safest methods should be considered for surgical anesthesia.1 the factor v leiden ( fvl ) mutation , a single point mutation that confers factor v resistance to protein c degradation , is the most common genetic cause of thrombophilia.2 in this case report , we discuss the successful use of infraclavicular brachial plexus , thoracic paravertebral , and unilateral subarachnoid blocks for a patient with fvl mutation and left axillary artery occlusion . a 58-year - old , 76 kg , 162 cm tall , american society of anesthesiologists ( asa ) physical status ii woman was scheduled for axillo - brachial bypass to treat left axillary artery occlusion ( subclavian steal syndrome ) . the surgeon planned to harvest the patient s left distal saphenous vein for axillo - brachial bypass . the patient had a history of hypertension , chronic obstructive pulmonary disease ( copd ) ( fev1/fvc [ forced expiratory volume in 1 second / forced vital capacity ] ratio : 50% by spirometry ) and fvl mutation . her medications included valsartan 160 mg plus hydrochlorothiazide 25 mg daily , aspirin 300 mg daily , formoterol 12 g plus budesonide 400 g inhaled twice daily , tiotropium bromide 18 g inhaled daily , and theophylline 200 mg twice daily . pre - operative platelet count , bleeding time , prothrombin time , activated partial thromboplastin time ( aptt ) , and international normalized ratio ( inr ) were normal . low molecular weight heparin ( lmwh ) 40 mg was administered subcutaneously 12 hours before the procedure , and her daily copd medications ( formoterol / budesonide inhaler , tiotropium inhaler , and theophylline ) were given on the morning of the procedure . after applying electrocardiography , noninvasive arterial blood pressure , and pulse oximetry monitors , the patient was placed in the sitting position and oxygen was applied via face mask at a rate of 2 l per minute . she was given midazolam 1 mg and fentanyl 25 g for sedation , and 500 ml of crystalloid solution intravenously . the thoracic paravertebral block ( tpvb ) was performed according to the method of eason and wyatt.3 palpation was used to localize the seventh cervical vertebra ( c7 ) and first thoracic vertebra ( t1 ) at the tip of the scapula . spinous processes of t1 through t5 were outlined , and a point was marked 2.5 cm left lateral to each of the spinous processes ( figure 1 ) . skin and subcutaneous tissue were anesthetized along the vertical line with 4 ml lidocaine 1% . a 22-gauge spinal needle was inserted 2.5 cm lateral to the spinous process of t1 , in the direction of t1 . the transverse process was found with the tip of the needle , and then the needle was withdrawn to the skin and redirected caudally to slide under the transverse process . the needle was advanced to the paravertebral space , as identified by loss of resistance to air . after the paravertebral space was identified and aspiration did not produce blood , air , or cerebrospinal fluid , 5 ml of levobupivacaine was injected , and the needle was removed . the patient was placed supine and observed for signs of intravascular or epidural injection and respiratory difficulty . sensation was assessed by using ice - cold and pin - prick tests , repeated at 5-minute intervals . we used comlekci et al s technique for infraclavicular block , in which the highest point of the shoulder is taken as a reference point.4 the infraclavicular region was prepared with povidone - iodine . using a 22-gauge , 100 mm insulated needle and a nerve stimulator ( stimuplexb , braun ag , melsungen , germany ) , we assessed hand responses for n. medianus and n. musculocutaneous at 0.4 ma . a total of 200 mg lidocaine 2% ( 10 ml ) and 150 mg levobupivacain 0.5% ( 30 ml ) was injected slowly with aspirations after every 5 ml ( figure 2 ) . repeated sensory blockade assessments were performed at frequent intervals . ten minutes after completing the tpvb , unilateral spinal block was performed in the leftdown lateral decubitis position . a 25-gauge quincke needle was used to introduce 5 mg of hyperbaric bupivacaine 0.5% for induction at the l3l4 interspace . the left distal saphenous vein was prepared in 15 minutes and the area closed primarily . the patient recovered from brachial and thoracic paravertebral block in 4 hours and spinal block in 2 hours . the pain - free period after the procedure was 7 hours at the axillary artery site and 4 hours at the saphenous vein harvest site . oral medications were started on the second postoperative day , and the patient was discharged on postoperative day 7 without complications . inherited resistance to activated protein c through fvl mutation leads to a thrombophilic state.5 fvl mutation is the most common inherited risk factor for venous thromboembolism , with heterozygous carriers having a 410-fold increased risk of deep vein thrombosis ( dvt ) and homozygous patients having up to a 100-fold increase in dvt risk.6 while planning the patient s anesthesia management , we searched for methods associated with quick recovery , less postoperative pain , less postoperative nausea and vomiting , reduced postoperative analgesic requirements , reduced association with thromboembolic events , and shorter hospital stays . because of the patient s advanced obstructive lung disease , we chose regional anesthesia . this approach was also chosen in this patient with an fvl mutation because regional anesthesia is associated with a lower risk of thrombosis compared to general anesthesia.7 although we chose an infraclavicular block for arm anesthesia , we performed sensorial blockade of the axillary region using a tpvb at the t1 , t2 , and t3 levels.8 tpvb can be performed with the patient sitting , lying on the side , or prone . we chose the sitting position because it provides easy identification of landmarks.9 we believed that unilateral low - dose spinal anesthesia would be sufficient for saphenous vein harvest . hyperbaric bupivacaine at 4 mg and 6 mg doses have been compared and found to be equivalent for unilateral , segmental spinal anesthesia for outpatient knee arthroscopy . the 4 mg dose appears superior to the 6 mg dose because it produces faster spinal anesthesia induction and the patient achieves discharge criteria significantly earlier.10 we used 5 mg of hyperbaric bupivacaine , which proved adequate during saphenous vein harvest . for most patients , lmwh thromboprophylaxis should be discontinued 1012 hours prior to surgery and coagulation status ( aptt ) should be assessed prior to neuraxial blockade.11 although our patient was prone to thrombosis because of her fvl mutation , we discontinued lmwh , as per general recommendations , 12 hours prior to the procedure . performing a combination of regional techniques on one patient is time consuming , may create intraoperative patient discomfort , and may be associated with higher failure rates . these factors may also contribute to an increased risk of technique - specific complications and local anesthetic toxicity.12 however , these regional techniques may be suitable alternatives for patients with thrombophilias , because they may be performed with minimal postoperative pain and early recovery without thromboembolic complications .

anesthetic management of patients with coagulation disorders presents safety and technical challenges . this case describes a 58-year - old woman with factor v leiden mutation who required distal saphenous vein harvest and axillo - brachial bypass to treat axillary artery occlusion . the patient underwent surgery with satisfactory anesthesia using infraclavicular brachial plexus block , thoracic paravertebral block , and unilateral subarachnoid block . these three regional anesthetic interventions were performed in lieu of general anesthesia to minimize risks of thrombotic events , pain , and to decrease recovery time . despite higher failure rates of regional anesthesia , longer time required for procedures , and added discomforts during surgery , the benefits may outweigh risks for selected high - risk patients , including those with factor v leiden mutations .

breast cancer ( bc ) is a prevalent cancer among women worldwide , and it has been estimated that there are 71 cases of bc per year for every 100 000 women in brazil . it is one of the most common causes of death among women , and epidemiological studies indicate that the age of onset is slowly , but steadily , becoming lower . this suggests that there may be changes in environmental factors that are affecting bc risk . the risk factors for bc include early age of menarche , delayed menopause , contraceptive use , hormonal replacement therapy , above - average body mass index , exposure to environmental pollutants , smoking , and alcohol use [ 13 ] . however , it is generally believed that the initiation of bc is a consequence of cumulative genetic damage , which leads to genetic alterations that result in the activation of proto - oncogenes and the inactivation of tumor suppressor genes . a large number of genetic variants that are associated with bc risk have been identified in genes involved in a wide variety of functions , including steroid hormone metabolism , detoxification of environmental carcinogens , dna damage repair , and tumor suppression . as observed in drug and chemical metabolism , there is considerable interindividual variability ( i.e. , polymorphisms ) in the conjugation pathways of both estrogen and catechol estrogens . these person - to - person differences , which are attributed to polymorphisms in the genes encoding for the respective enzymes , may define subpopulations of women with higher lifetime exposure to hormone - dependent growth promotion or to cellular damage from particular estrogens and/or estrogen metabolites . current evidence suggests that the metabolic by - products of estrogens in the body may act as initiators of cellular alterations . estrogen metabolism products such as quinone - catecholestrogen can bind to dna and form dna adducts . the generation of free radicals by metabolic redox cycling between quinone and hydroquinone can damage dna by causing strand breaks , 8-hydroxylation of purines , and lipid hydroperoxide - mediated dna modifications . although still controversial , a number of genes involved in biogenesis ( cyp17 ) , bioavailability ( cyp1b1 and cyp1a1 ) , and degradation ( comt ) of estrogen compounds contain polymorphisms that could affect susceptibility to bc . they are called low penetrance genes ( or sometimes modifier genes ) and are , in this instance , defined as genes in which subtle sequence variations or polymorphisms may be associated with a slightly to moderately increased relative risk for bc . the hypothesis of the present study is that polymorphic variants of the estrogen metabolizing genes cyp17 , cyp1b1 , cyp1a1 , and comt may affect the spontaneous levels of chromosome damage in lymphocytes of bc patients and subsequently modulate bc risk . therefore , the objective of the present work was to correlate the genotypes of polymorphic variants of the above - mentioned genes with the basal levels of chromosome damage in lymphocytes of untreated bc patients and healthy individuals . micronucleus assay was employed to determine the extent of baseline chromosome damage in bc patients and controls , and pcr - rflp was used for genotype analysis . the bc patient group consisted of 62 untreated women diagnosed with in situ or invasive ductal breast carcinoma , ranging in age from 25 to 60 years old ( mean age , 50.5 years old ) and free of any pathology associated with the use of medication that is known to cause dna damage . the control group consisted of 62 women with ages ranging from 25 to 50 years ( mean age , 46.7-years old ) . they were enrolled in the control group after a detailed investigation in order to ensure that they were free from any breast pathology . they came from the same geographical location , their dietary habits were not appreciably different , and they were not occupationally exposed to genotoxic chemicals . none of the subjects reported alcohol consumption , the use of genotoxic medicine , presence of known inherited genetic disorders or chronic diseases or exposure to ionizing or nonionizing radiation , even for diagnostic or therapeutic purposes , for at least one month prior to enrolling in the study . patients and controls enrolled in the present study did not report family history of breast and/or ovarian cancer . this investigation was approved by the national ethics committee ( conep : 1217/2004 ) and was performed in accordance with ethical standards . informed consent of patients and controls was obtained before inclusion in the study and sample collection . samples of venous blood ( 10 ml ) were collected in heparinized and edta vacutainer tubes ( becton dickinson , nj , usa ) by venepuncture under sterile conditions . the samples were coded , immediately protected from direct light , and processed for genotyping and micronucleus assay . genomic dna samples were obtained from blood lymphocytes using a wizard genomic dna purification kit ( promega , madison , wi ) . isolated dna was resuspended in tris - edta buffer ( ph 8.0 ) and stored at 20c until use . the t1931c polymorphism of the cyp17 gene was determined by pcr - rflp with the following primers : sense , 5-caaggtgaagatcagggtag-3 and antisense , 5-gctagggtaagcagcaagag-3. the 145 bp product was digested overnight with 8 u of the restriction enzyme mspa1i . the t allele remained intact , but the c allele was digested into 75 and 70 bp fragments . the v432l polymorphism of the cyp1b1 gene was genotyped with the following primers : sense , 5-tcacttgcttttctctctcc-3 and antisense , 5-aatttcagcttgcctcttg-3. the 650 bp product was digested overnight with 8 u of the restriction enzyme acui . the genotypes of the t3205c polymorphism of the cyp1a1 gene were determined with the following primers : sense , 5-taggagtcttgtctcatgcct-3 and antisense , 5-cagtgaagaggtgtagccgct-3. the 340 bp pcr product was digested for 3 h with 5 u of the restriction enzyme mspi . the c allele was digested into 200 and 140 bp fragments , whereas the t allele remained intact . the genotypes of the v158 m polymorphism of the gene comt were determined with the following primers : sense , 5-tactgtggctactcagctgt-3 and antisense , 5-tgaagctggtgtgaacacct-3. the 114 bp pcr product was digested with 5 u of the restriction enzyme nlaiii . the met allele was digested into fragments of 96 , 54 , 39 , and 27 bp , whereas the val allele was digested into fragments of 54 and 39 bp only . lymphocyte cultures were prepared combining 0.5 ml of isolated lymphocytes in plasma with 5 ml of complete medium containing 78% of rpmi ( sigma - aldrich co. , usa ) , 20% inactivated fetal bovine serum ( gibco - invitrogen , denmark ) , the antibiotics penicillin ( 5 g / ml , sigma - aldrich co. , usa ) and streptomycin ( 10 g / ml , sigma - aldrich co. , usa ) , and 2% phytohemagglutinin ( life technologies , grand island , ny , usa ) to stimulate cell proliferation . cultures were incubated at 37c . after 44 hours of incubation , cytochalasin b ( sigma - aldrich co. , usa ) was added to the cultures to a final concentration of 4 g / ml , according to the method of fenech and morley . one thousand binucleated cells were analyzed per individual and the frequency of binucleated cells micronucleated ( bcmn ) was determined according to the criteria described by fenech . the mann - whitney statistical test was used to compare bcmn between patients and controls . the frequency of bcmn in different genotypes was analyzed with one - way anova and the statistical differences between groups for bc risk was calculated using fisher 's exact test ( two - tailed ) . crude odds ratios ( ors ) were calculated and are given with 95% confidence intervals ( cis ) . table 1 compares and shows the homogeneity of bc patients and control groups according to their smoking habits , menopause status , hormone replacement therapy , and full - term pregnancy . basal level of chromosome damage was measured by micronucleus ( mn ) assay in lymphocytes from 62 bc patients and 62 age - matched controls , as shown in figure 1 . bc patients exhibited higher levels of chromosome damage than controls according to bcmn ( p < .05 ) . the genotype distributions for all cases were in agreement with those predicted by the hardy - weinberg equilibrium . an association between bc occurrence and the c allele of the t1931c polymorphism in the cyp17 gene was observed in the postmenopause group ( or 4.3 ; 95% ci 1.313.7 ) . cyp1b1 v432l polymorphism had no association with bc occurrence in pre- and postmenopause women , however , in the premenopause women we observed increased risk to bc when the c allele of cyp1a1 gene ( t3205c polymorphism ) was present ( or 10.5 ; 95% ci 2.641.7 ) . comt met allele was also associated with bc occurrence in premenopause women ( or 3.2 ; 95% ci 1.18.6 ) . the basal levels of chromosome damage in bc patients and controls among the different genotypes are presented in table 3 . bcmn frequencies were higher in bc than in control group in all genotypes except for val / val of comt gene where differences between patients and controls were not significant ( p = .07 ) . we also evaluated the influence of different genotypes considering the levels of chromosome damage in patient and control groups individually ( figure 2 ) . cyp17 t1931c and cyp1a1 t3205c polymorphisms had no influence in the frequency of bcmn in patients as well as in controls . however , in control group , cyp1b1 432l allele was related to increased frequencies of bcmn ( p = .006 ) . differently , patients with v158 m polymorphism of comt gene presented higher frequencies of bcmn when compared to the wild - type counterparts ( p = .04 ) . finally , we tested the risk to bc in the group of patients and controls that presented frequencies of bcmn higher than the mean ( higher than 18.5 bcmn for patients and higher than 9.5 bcmn for controls ) ( table 4 ) . cyp17 , cyp1b1 , and cyp1a1 polymorphisms did not modify the occurrence of bc , however , comt v158 m polymorphism was more frequent in bc than in control group and resulted in significant or increasing ( or 3.5 ; 95% ci 111.4 ) . dna damage can occur spontaneously or as consequence of exposure to chemical or physical genotoxins . there are modulators that can affect the levels of spontaneous dna damage , including dna repair genes , antioxidant defense genes , and estrogen - metabolizing gene polymorphisms that can lead to the accumulation of genotoxic estrogen subproducts . high serum estrogen levels are thought to be a major risk factor for bc . in vitro and in vivo animal and patient - based studies suggest that estrogens , their metabolic compounds , and the entire biochemical metabolic machinery may play a role in bc carcinogenesis . we can look forward into two different processes by which hormones are related to bc : ( i ) one involves the binding of estradiol to estrogen receptor ( er ) alpha with the stimulation of cell proliferation . errors in dna occurring during replication result in fixed mutations when not well repaired ; ( ii ) the other process results from the formation of genotoxic metabolites of estradiol , which can bind to dna , cause depurination , and also result in mutations . herein , we focused on the second process . therefore , in the present study , the relationship between the snps in four estrogen - metabolizing genes and basal levels of chromosome damage was examined using lymphocytes of untreated bc women and healthy controls once lymphocytes can circulate for years or even decades , accumulating mutations in their dna . results presented here show that the basal levels of chromosome damage detected by mn assay and observed in the control group are in accordance with those previously reported in other control populations of similar age [ 15 , 16 ] . it is well known that mn are formed by the condensation of acentric chromosomal fragments or by whole chromosomes lagging behind during cell division and that this is the only biomarker that allows the evaluation of both clastogenic and aneuploidogenic effects in a vast range of cells , as they are detected in interphase . as we did not observe any statistical difference between smoking and nonsmoking patients or controls ( data not shown ) , we grouped these two subsets together for the analysis of the basal levels of chromosome damage in patients and controls . the investigation of basal levels of chromosome damage in peripheral lymphocytes of untreated cancer subjects has been previously reported . baseline levels of dna damage were significantly higher in bladder cancer patients than in controls , and the frequency of mn in stimulated peripheral blood cells from an untreated leukemia population was significantly greater than that in the control group . similar results were found by lou et al . , who simultaneously investigated both baseline and ionizing radiation - induced ( ir ) genetic damage in peripheral lymphocytes from 36 cancer patients using mn and comet assays . they found that both spontaneous and ir - induced genetic damages were higher in patients than in controls . in the present study , the primary objective was to investigate the relationship between the genotypes of polymorphisms in estrogen - metabolizing genes and the extent of endogenous chromosome damage . although this is not an epidemiological study , the odds ratio for the different genotypes was also examined and displayed interesting findings . the results presented here show that there is a slight significant relationship between the cyp17 polymorphism , and bc risk is postmenopausal women , however , we found no correlation between spontaneous chromosome damage and the t1931c polymorphism of cyp17 gene , which encodes for cytochrome p450c17 , a 17-hydroxylase that catalyzes the conversion of pregnenolone and progesterone to 17-oh - pregnenolone and 17-oh - progesterone , respectively . cytochrome p450c17 also has a 17,20-lyase activity that converts hydroxylase products to dehydroepiandrosterone ( dheas ) and androstenedione , respectively , which can be further converted to estrogens and testosterone . it has been hypothesized that certain genotype variants in the cyp17 gene result in higher hormone levels , leading to an increased risk of bc . however , non - hispanic white women who were heterozygous or homozygous for the variant allele of cyp17 had lower estrone , total testosterone , free testosterone , and dheas concentrations compared to women homozygous for the wild - type allele . chen and pei applied both traditional meta - analysis and bayesian approach to determine the overall effect of cyp17 t1931c polymorphism on risk of bc and detected that carriers of c allele were positively associated with risk of bc in postmenopausal women and were inversely related to bc in pre - menopausal women . the v432l polymorphism of the cyp1b1 gene is located within a catalytic heme - binding domain in exon 3 and has been analyzed in several independent studies that present controversial results . chinese women with the leu / leu genotype at the l432v polymorphism had a higher risk of bc ( or = 2.0 , 95% ; ci = 1.03.7 ) . a positive association was also seen in turkish women but was limited to those with a high body mass index ( or = 2.3 , 95% ; ci = 1.34.2 ) . in contrast , results presented here showed no association between v432l polymorphism and bc occurrence in pre- or postmenopause women ( or 2.5 , 95% ci 0.87.5 and or 0.6 , 95% ci 0.22.1 , resp . ) . a significant inverse association between the val allele and bc risk was previously observed in studies of african american or mixed populations . it is interesting to note that in the control group , carriers of the leu allele exhibited higher levels of dna damage compared to homozygous wild - type individuals while this was not observed in the patients group . we believe that this difference between patients and controls is mostly due to the increased spontaneous levels of chromosome damage in bc which makes it difficult to identify suitable differences in bcmn between polymorphic and wild - type individuals . it has been described that cyp1b1 catalyzes c4 hydroxylation of estradiol ( 4-hydroxyestradiol ( 4-ohe2 ) ) . this metabolite can undergo metabolic redox cycling to generate free radicals such as superoxide and chemically reactive estrogen semiquinone / quinone intermediates , which can form dna adducts [ 27 , 28 ] . anyway , these results suggest further investigation of this polymorphism combined with other low - penetrance gene polymorphisms such as dna repair in the modulation of spontaneous chromosome damage in healthy women . cytochrome p450 1a1 ( cyp1a1 ) is one of the most important phase i enzymes expressed in breast tissue . it catalyzes estrogen 2-hydroxylation , generating the 2-oh estrogen metabolite , which can form dna adducts [ 28 , 29 ] . the 3205tc polymorphism in cyp1a1 is located in the 3 noncoding region and can affect the modulation of enzymatic activity and thereby the susceptibility risk to bc . the results presented here showed that the frequency of this polymorphism was higher in premenopause bc women than in controls and resulted in association to bc risk ( or 10.5 , 95% ci 2.641.7 ) , however , it had no effect on the frequency of bcmn in patients or in control group . the rare ct and cc genotypes were detected in 14 patients and three controls , and it , therefore , resulted in an extensive confidence interval ( ci ) . similar results were observed by taioli et al . that demonstrated an association between this polymorphism and bc cancer risk in african american women ( or = 9.7 95% , ci 2.047.9 ) , and huang et al . nevertheless , the association observed here must be interpreted with caution considering the number of individuals included in the present study and the fact that c allele is rare . the catechol - o - methyltransferase ( comt ) catalyzes the addition of a methyl group to reactive catechol estrogens , converting them into stable methyloxyestrogen conjugates . in patients group , carriers of met allele of the comt gene exhibited higher levels of chromosome damage relative to those homozygous for the wild - type allele . besides , when the analysis considered the association of bc occurrence in individuals that exhibited levels of chromosome damage higher than the mean , met allele was strongly associated with bc occurrence . this was an interesting finding especially if we consider that biomarkers of susceptibility and risk contribute to the identification of high - risk subgroups of the population , independent of whether they are associated with previous exposure or they are involved in a defined pathway or mechanism . it is known that g to a polymorphism at codon 108 in the comt gene leads to a substitution of methionine to valine , resulting in decreased enzymatic activity and increased thermolability [ 34 , 35 ] . as metabolites of catechol estrogens have the potential to induce oxidative dna damage and form dna adducts , the conversion of catechol estrogens into stable conjugates may be important in preventing bc . in a previous study of a taiwanese population , a strong association between the comt polymorphism and bc risk was observed ( or = 3.5 95% , ci 1.1513.3 ) . the correlation between genetic polymorphisms and specific phenotypes was evaluated by synowiec et al . . they correlated a polymorphism in a dna repair gene with oxidative dna damage in 41 bc patients and 48 controls . they demonstrated a strong association between bc occurrence and the c / c genotype of the rad51 - 135g / c polymorphism , the ser / ser genotype of the ogg1-ser326cys polymorphism and the lys / gln genotype of the xpd - lys751gln polymorphism . in contrast , the g / c genotype of the rad-135g / c polymorphism and the lys / lys genotype of the xpd - lys751gln polymorphism exhibited a protective effect against bc . in this study , the risk of bc was calculated in patients and controls that had higher levels of endogenous oxidative dna damage . we previously reported that brazilian untreated bc patients exhibited higher levels of chromosome damage than healthy controls and that xrcc3 polymorphism was weakly associated with the risk to bc in those individuals that presented higher levels of chromosome damage . it is well known that dna damage as detected in peripheral blood represents a surrogate for what would be observed in breast tissue . while the polymorphisms are obviously expressed in both breast tissue and lymphocytes , the level of expression of the genes and in particular , the level of substrate that in our present hypothesis is estradiol , may be and is likely very different within breast tissue and the lymphocytes , however , genome damage in lymphocytes may be correlated with cancer - initiating events in target tissues via a common genetic , dietary , or environmental factor . therefore , the manifestation of effects of the polymorphisms on levels of the reactive quinones may be quite different in breast tissue and lymphocytes , making extrapolation of the findings being reported to implications for bc quite tenuous . as mentioned above , this is not an epidemiological study and although the limited number of participants , we consider that our results point that polymorphisms in the enzymes involved in the metabolism of estradiol / estrone to reactive quinones that cause dna damage may represent low penetrance risk factors susceptible of further detailed investigations . it is unknown whether the healthy controls and patients with bc had equal levels of dna damage before the appearance of clinical symptoms in the patients . it is also unclear whether mn enhancement is a consequence or causative agent of the disease process . however , the followup of those controls who are carriers of met allele of cyp1b1 gene that exhibited higher frequencies of bnmn than wild - type counterparts could help to further answer this question . in conclusion , according to the data presented here both the genetic background of genes involved estrogen metabolism and dna damage in healthy controls and patients can modulate bc risk reflected by higher chromosomal damage in lymphocytes .

in the present study , we investigated the relationship between polymorphisms in the estrogen - metabolizing genes cyp17 , cyp1b1 , cyp1a1 , and comt and genomic instability in the peripheral blood lymphocytes of 62 bc patients and 62 controls considering that increased or prolonged exposure to estrogen can damage the dna molecule and increase the genomic instability process in breast tissue . our data demonstrated increased genomic instability in bc patients and that individuals with higher frequencies of mn exhibited higher risk to bc when belonging val / met genotype of the comt gene . we also observed that cyp17 and cyp1a1 polymorphisms can modify the risk to bc depending on the menopause status . we can conclude that the genetic background in estrogen metabolism pathway can modulate chromosome damage in healthy controls and patients and thereby influence the risk to bc . these findings suggest the importance to ally biomarkers of susceptibility and effects to estimate risk groups .

acetone is the simplest ketone compound . in general , acetone is not considered harmful , and the world health organization has not classified acetone as carcinogenic . however , its prolonged inhalation can not only cause irritation of the mucous membranes , headaches , confusion , and narcotic effects , but lead to coma as well [ 15 ] . for etiological reasons , acetonaemia is classified to be of endogenous and exogenous origin [ 6 , 7 ] . multiple toxicities and physiopathological conditions result in ketosis ( acetonaemia particularly ) , including diabetes mellitus ( dm ) , starvation coupled with physiologic stress , prolonged exercise , during pregnancy , and ethanol toxicity , other alcohol ingestions , drug toxicities , inborn errors of ketone metabolism , alcoholic ketoacidosis , delirium tremens , and hypothermia [ 69 ] . the main symptom of dm is a high blood glucose concentration depending on insulin deficiency . in this case the body can not fully use glucose but could use fatty metabolism instead of glucose for energy . dm , especially diabetes and autoimmune associated diseases , thyroid disease , and diseases that can accompany diabetes ( hypertension , cardiovascular disease , cerebrovascular disease , and renal insufficiency ) can cause pathological changes in most of the tissues , organs , and biological fluids depending on lipotoxicity and glucotoxicity . a lot of medical , chemical , and medicolegal investigations have been carried out with the determination of acetone in blood and other biological fluids [ 2 , 6 , 7 , 10 ] . over the decades , several methods have been used for its determination in biological samples . in the beginning , colorimetric methods were developed and used for the determination of acetone in plasma [ 1113 ] . these methods have common disadvantages such as the lack of specificity and detection limit . in recent decades , gas chromatographs equipped with flame ionization detectors or mass spectrometric detectors have been developed for determination of acetone concentrations in body fluids and in expired air [ 1418 ] . enzymatic methods are more specific but more complex and have long assay times and gas chromatographic methods , although widely used , are applied with difficulty as routine tests . the determination of acetone in the blood is most important in clinical diagnostic laboratory studies . there are three ketone bodies , while the two main ketone bodies are acetoacetate ( acac ) and 3-b - hydroxybutyrate ( 3hb ) , the third ketone body ; acetone ( ac ) is found minimum level . ketone bodies are produced by the liver and used peripherally as an energy source when glucose is not readily available [ 9 , 19 ] . ketone bodies are three water - soluble compounds that are produced as by - products when fatty acids are broken down for energy in the liver and kidney . ketone bodies are produced from acetyl - coa mainly in the mitochondrial matrix of hepatocytes when carbohydrates are so scarce that energy must be obtained from breaking down fatty acids [ 9 , 20 ] . acetone can not be converted back to acetyl - coa , so it is excreted in the urine or exhaled . recently , blood or urine testing kits have been used to test for the presence of acetone in clinical biochemistry laboratories . acetone can also be quantified by sampling the human blood and testing by gas chromatography . described a rapid and simple hplc procedure that can be used for the routine measurement of acetone in biological fluids , such as plasma and urine . according to fujii et al . , it is proposed that liquid chromatography with fluorescence ( lc - fl ) seems to be useful for the determination of acetone in the saliva . in this paper , we present a rapid and simple hplc technique using 2,4-dnph as a derivatizing reagent for quantitative determination and metabolomic research of acetone in biological fluid such as human blood . 2,4-dnph ( sigma - aldrich , 97% ) , acetone ( merck , 99.8% ) , acetonitrile ( sigma - aldrich , 99.8% ) , and methanol ( merck , 99.8% ) were used in this study . for the chromatographic analysis , thermo scientific dionex ultimate 3000 hplc with a thermoacclaim - c18 ( 15 cm 4.6 mm 3 m ) column and uv - vis dad were used . the deionized water was 18.2 mcm ( millipore direct - q3 uv ) and was used throughout the experiments . for the biochemical analyses , cobas 6000 ( roche , germany ) autoanalyzer was used for blood glucose levels . qualitative analysis of total ketones in urine was evaluated by iris iricel 2000 analyzer ( icem velocity ) . in the first stage of our study , following a 12-hour fasting venous blood samples were taken from patients admitted to hospital of the faculty of medicine , canakkale onsekiz mart university . the human blood and urine samples were directly collected into a tube ( without a collection device ) between 08:30 and 11:00 am . clinical biochemistry laboratory blood and urine glucose tests were studied for routine biochemistry using a urine autoanalyzer . the patients were divided into high blood glucose and urine ketone positive subjects ( group 1 ) and high blood glucose and urine ketone negative example subjects ( group 2 ) . the patients with hyperglycemia were 8 females and 7 males ( age : 2187 ; n = 15 ) , while 5 female and 2 male patients had positive urine ketones ( age : 2168 , n = 7 ) and 5 male and 3 female patients had negative urine ketones ( age : 5587 , n = 8) . the blood glucose levels varied between 110 and 320 mg / dl in our patients ( table 1 ) . in the second stage of the study , to determine the probable positive acetone , its quantitative analysis was carried out in biological fluids using the hplc technique . the blood samples were immediately prepared for hplc analysis carried out within 8 hours after sample collection . plasma specimens were deproteinized with acetonitrile ( 1 : 1 , v / v ) ; 2,4-dnph is added to the supernatant ( filtered blood samples ) and treated with acetonitrile ( 2 : 1 , v / v ) to prevent crystallization of the synthesized phenylhydrazone . an aliquot ( 20 microliters ) of the reaction mixture was subjected to hplc at ambient temperature using thermoacclaim - c18 ( 15 cm 4.6 mm 3 m ) column and uv - vis dad with ( methanol / acetonitrile ) water as eluent at a flowrate of 1 ml min and detection at 365 nm [ 2 , 3 ] . the experimental procedures were conducted in accordance with the ethical standards of the helsinki declaration and approved by the canakkale onsekiz mart university human research ethics committee . written informed consent was obtained from all the subjects . the 2,4-dinitrophenylhydrazone standards were prepared by mixing a and b solutions : ( a ) : 0.40 g of 2,4-dnph dissolved in 2.00 ml of h2so4 + 3.00 ml of h2o + 10.0 ml ethanol ; ( b ) : 0.50 g or 1.00 ml of the acetone standard dissolved in 20.0 ml ethanol . after this mixing , a precipitate was formed in each case , isolated through filtration , and dried in vacuum [ 3 , 24 , 25 ] . acetone was added into its 2,4-dnph derivatives by mixing 1.00 ml of a solution containing 200 mg/100 ml of 2,4-dnph with 1.0 ml of h3po4 , and 4.00 ml of the human serum . after 2 h , a 40.0 l aliquot was withdrawn and analyzed by the hplc technique [ 3 , 2427 ] . chromatographic separation was achieved in a thermoacclaim - c18 ( 15 cm 4.6 mm 3 m ) column at uv - vis dad detector ( max 365 nm ) . the injection volume was 20.0 l and the detection was performed at 365 nm . the following gradient was used : ( methanol / acetonitrile ) ( 8 : 2 ) water 60 : 40 ( v / v ) . elution was achieved at retention time ( tr ) 12.10 and flow - rate of 1 ml min . standard calibration curve was prepared with acetone ( 0.5 , 2.5 , 5.0 , 10 , and 20 mmol l ) and 40 l 2,4-dnph in acetonitrile . human samples were prepared by adding 40 l 2,4-dnph and 500 l acetonitrile to 200 l human serum . the calibration curve was constructed by plotting the peak area of the 2,4-dnph derivative of acetone ( y ) versus the concentration of acetone ( x , mmol l ) by linear regression ( n = 4 ) . the equation was found as y = 0.7361x + 0.0877 with a 0.9967 , correlation coefficient ( r ) . the method proposed was validated as described in ich guidelines in parameters of linearity , limit of detection and quantification , accuracy , and precision . the linearity of the method was determined at four concentration levels ranging from 0.5 to 20 mmol l. the calibration curves were constructed by plotting the peak area of the 2,4-dnph derivative of acetone ( y ) versus concentration of acetone ( x ) . the slope , y - intercept , and correlation coefficient were calculated . to check the linearity , the lod was estimated using signal - to - noise ratio of 3 : 1 or ( 3 s / m ) and loq as 10 : 1 or ( 10 s / m ) , at which accuracy and standard deviation were within 20% as per ich [ 28 , 32 , 34 , 35 ] . intraday accuracy and precision were performed for acetone at 5.0 mmol l in replicate ( n = 3 ) . accuracy ( expressed as recovery ) and precision ( expressed as relative standard deviation ) should not deviate by 15% of the nominal concentration . as can be seen from figure 1 , the retention time ( tr ) was obtained as 3.80 min . the most efficient separation of acetone as its 2,4-dnph was obtained with a thermoacclaim c18 column ( 15 cm 4.6 mm 3 m ) at retention time ( tr ) 12.10 min and flowrate of 1 ml min using a ( methanol / acetonitrile ) water elution gradient . no elution problem for acetone as its 2,4-dinitrophenylhydrazone derivative was observed . typical hplc chromatogram of 0.681 mmol l acetone as its 2,4-dnph derivative is given in figure 2 . the hplc chromatogram of 15 mmol l acetone in human blood in the first patient of group 1 is given in figure 3 . in our study , the patients were determined as high blood glucose and urine ketone positive subjects ( group 1 ) and high blood glucose and urine ketone negative example subjects ( group 2 ) . the blood glucose levels , given in table 1 , vary between 110 and 320 mg / dl in our patients , except for sample 7 . many patients in our study have dm disease , except samples numbered 4 , 5 , 11 , 13 , and 15 ( table 1 ) . the calibration plot of peak area against concentration was obtained linear in the range 0.5 to 20 mmol l. the regression equation and correlation coefficient were obtained as y = 0.7361x + 0.0877 with a 0.9967 , correlation coefficient ( r ) . so this value is smaller than 2.2 , and calibration curve is linear . for the human plasma samples , fcritical value for calibration curve at 3 degrees of freedom ( p = 0.05 ) is 3.18 . so , this value is smaller than fcritical value , and obtained values are appropriate . the lod and loq were found as 0.041 and 0.136 mmol l , respectively . the accuracy for acetone in human plasma samples expressed as recovery was found as 98% . for interday assay , the accuracy for acetone in human plasma samples expressed as recovery was found as 96% . 2,4-dnph can be used to qualitatively detect the carbonyl functionality of a ketone such as acetone or aldehyde functional group . a positive test is signaled by a yellow , orange , or red precipitate known as a dinitrophenylhydrazone . if the carbonyl compound is aromatic , then the precipitate will be red ; if aliphatic , then the precipitate will have a more yellow color . the reaction between 2,4-dnph and a ketone such as acetone is shown below:(1)rrco+c6h3no22nhnh2c6h3no22nhncrr+h2othis reaction can be described as a condensation reaction , with two molecules joining together with loss of water . it is also considered an addition - elimination reaction : nucleophilic addition of the -nh2 group to the c = o carbonyl group , followed by the removal of an h2o molecule . 2,4-dnph does not react with other carbonyl - containing functional groups such as carboxylic acids , amides , and esters . for carboxylic acids , amides , and esters , there is resonance associated stability as a lone pair of electrons interacts with the p - orbital of the carbonyl carbon resulting in increased delocalization in the molecule . also with carboxylic acids there is the effect of the compound acting as a base , leaving the resulting carboxylate negatively charged and hence unable to be attacked by this nucleophile [ 36 , 37 ] . in this study , an analytical method was applied for the quantitative determination of acetone in human blood . the determination was carried out using a uv - vis dad detector with hplc . in most of our patients ' blood samples , higher levels of acetone have been measured in the patients who have high level of blood glucose and positive urine ketone ( group 1 ) . the hplc method based on the labeling of acetone with 2,4-dnph seems to offer a rapid , low cost , sensitive , selective , and reproducible methodology for quantification of the acetone level in clinical samples such as human blood . the hplc method described here overcomes many of the problems in the determination of acetone in biological fluids and the preanalytical errors . the volatile ketone is promptly stabilized by conversion into its dnph derivative and rapidly determined without recourse to a solvent extraction step . the method uses very inexpensive reagents . as can be stated by brega et al . , the proposed hplc method can therefore be used to great advantage over current gas chromatographic methods ; it can be used in experiments requiring multiple samples and specific activity determination for the routine measurement of acetone in diabetic patients and in biological monitoring of exposed workers . we also presented acetone as a useful tool for the hplc - based metabolomics investigation of endogenous metabolism and quantitative clinical diagnostic analysis . in the medical and medicolegal practice however , the detection and early identification of acetone could be used as an initial indicator of detection of all these physiopathological conditions and the biological monitoring test and to determine further diagnostic management and timely treatment . determination of acetone levels in blood may be a valid clinical approach in the symptomatic or / and nonsymptomatic cases in the literature for determining treatment strategy and controlling glycemic levels of patients . consequently , the advanced studies which evaluate blood and urine ketone bodies level together should be performed in different physiopathological conditions including clinical and forensic toxicological studies .

using high - performance liquid chromatography ( hplc ) and 2,4-dinitrophenylhydrazine ( 2,4-dnph ) as a derivatizing reagent , an analytical method was developed for the quantitative determination of acetone in human blood . the determination was carried out at 365 nm using an ultraviolet - visible ( uv - vis ) diode array detector ( dad ) . for acetone as its 2,4-dinitrophenylhydrazone derivative , a good separation was achieved with a thermoacclaim c18 column ( 15 cm 4.6 mm 3 m ) at retention time ( tr ) 12.10 min and flowrate of 1 ml min1 using a ( methanol / acetonitrile ) water elution gradient . the methodology is simple , rapid , sensitive , and of low cost , exhibits good reproducibility , and allows the analysis of acetone in biological fluids . a calibration curve was obtained for acetone using its standard solutions in acetonitrile . quantitative analysis of acetone in human blood was successfully carried out using this calibration graph . the applied method was validated in parameters of linearity , limit of detection and quantification , accuracy , and precision . we also present acetone as a useful tool for the hplc - based metabolomic investigation of endogenous metabolism and quantitative clinical diagnostic analysis .

a 38-year - old male patient was referred to asan medical center due to severe shock attributable to contained aortic rupture around the aortic root . the patient had been treated with systemic corticosteroids after being diagnosed with behcet s disease 14 years previously . the patient had previously undergone aortic valve surgery three times in another hospital for the management of aortitis and consequent aortic regurgitation associated with underlying behcet s disease . the first two mechanical aortic valve replacement ( avr ) procedures had been performed 14 years previously , followed by a ross procedure two years after the second operation . when the patient presented to our emergency department , he was in severe shock ( systolic blood pressure , 65 mmhg ) despite maximal intravenous inotropic support ( epinephrine and norepinephrine ) , and exhibited a drowsy mental state . on enhanced computed tomography , a contained aortic rupture was found around the aortic root and the ascending aorta , abutting the sternum . 1 ) . an emergency operation was planned . a transesophageal echocardiography performed in the operating room showed mild aortic regurgitation ( ar ) and severely depressed cardiac function with a left ventricular ( lv ) ejection fraction of 17% despite full inotropic support . massive hemorrhage during the reopening of the sternum and consequent catastrophic events were anticipated ; therefore , deep hypothermic circulatory arrest ( dhca ) was implemented before opening the sternum , using femoral cardiopulmonary bypass ( cpb ) with direct trans - apical lv venting through a left - side mini - thoracotomy ( fig . once the nasopharyngeal temperature cooled to 18c , sternal opening was attempted using an oscillating saw . as expected , a massive hemorrhage occurred from the ruptured aorta while the hematoma was being removed after sternal opening , at which point the cpb was stopped . distal ascending aorta clamping was achieved securely within one minute of dhca , and cpb was restarted . two discrete rupture sites were found in the aorta : one at the distal anastomosis site of the previous ross procedure and the other at the right coronary button site ( fig . 3 ) . after complete adhesiolysis of the mediastinum and the removal of the previously implanted pulmonic autograft , several tiny pseudoaneurysms were identified at the level of the aorto - ventricular junction , and further resection of this area was therefore conducted . dhca was then reimplemented ( 15 minutes ) in order to conduct hemiarch replacement using a 28-mm hemashield vascular graft ( boston scientific , boston , ma , usa ) , which was followed by the reimplementation of cpb and systemic rewarming . during this period , complete root replacement was carried out , using a 27-mm composite mechanical valved conduit ( st . , st paul , mn , usa ) . the circulatory arrest and cardiac ischemic times were 16 minutes and 188 minutes , respectively . the patient failed to be weaned from cpb due to profound lv dysfunction , therefore , cpb was substituted with venous - arterial extracorporeal membrane oxygenation ( ecmo ) . massive hemorrhage from the surgical sites continued despite meticulous hemostatic maneuvers and massive blood product transfusions . the patient was transferred to the intensive care unit ( icu ) with an open sternum . the transfusion of large quantities of blood products for two days resulted in adequate hemostasis . follow - up echocardiography showed a significant recovery of lv function at this point , and the patient was successfully weaned off ecmo after four days of ecmo support , after which a delayed sternal closure was performed . the follow - up computed tomography images and echocardiogram were unremarkable in that neither paravalvular dehiscence nor leakage on the anastomosis site was observed . lv function was found to be normal , with an ejection fraction of 56% without any inotropic support . the patient was discharged on postoperative day 31 without any neurologic sequelae or residual complications . on the echocardiographic follow - up performed six months after the surgery , cardiac function was completely normal , with adequate function of the mechanical valve and no pathologic findings around the aortic root and ascending aorta . the patient has undergone 14 months of follow - up while prescribed an oral corticosteroid medication , and has exhibited no cardiovascular complications . cardiovascular involvement in behcet s disease is a rare but life - threatening condition ; in particular , involvement of the aortic root is known to be the leading cause of death in patients with behcet s disease . surgical treatment of the aortic valve is required in these conditions , however , conventional avr is frequently complicated by prosthetic valve detachment due to the fragility of the aortic tissue and recurrent inflammation . various surgical methods have been suggested to improve the outcomes of the surgical treatment of aortic root diseases associated with behcet s disease [ 46 ] . since observations have revealed that the inflammation in behcet s disease primarily involves the aorto - ventricular junction of the aortic root , the complete resection of the inflammatory tissue in this area has been considered important for preventing the recurrence of the disease . with this in mind , aortic root replacement ( arr ) is considered an ideal surgical option for ar attributable to behcet s disease . the largest series of aortic valve operations in patients with behcet s disease presented 19 patients who underwent 46 aortic valve procedures and demonstrated an 80% reoperation rate after avr alone due to valve dehiscence , whereas excellent results were observed with arr . the authors suggested that arr should be performed as the initial surgical treatment of ar in patients with behcet s disease . ando et al . also reported excellent results of arr procedures in eight patients with behcet s disease ; only one patient died of arrhythmia , while the other seven patients survived through a long follow - up period . of those seven patients , only one patient required reoperation due to valve detachment over the course of a mean follow - up period of 74 months . in agreement with these previous studies , we believe that ar attributable to behcet s disease should be treated with arr as the initial surgical procedure in order to prevent prosthetic valve detachment . in the present case , ar attributable to behcet s disease was initially treated with conventional avr that was followed by a repeated avr procedure and a ross procedure due to recurrent prosthetic valve detachment . the patient ultimately presented with contained aortic rupture with pseudoaneurysm formation around the aortic root and ascending aorta twelve years after the third surgery . since a massive hemorrhage and consequent cardiac arrest were anticipated during the opening of the sternum , we decided to induce dhca with transapical lv venting before opening the sternum . through a left - side mini - thoracotomy , it was possible to achieve lv venting without encountering severe mediastinum adhesion or fatal hemorrhage from the ruptured aorta . this strategy enabled the prompt identification of the ruptured location and the complete resection of the diseased aortic tissue . since this patient had been suffering from cardiac insufficiency attributable to preoperative shock , preventing lv distention injury during dhca was believed to be crucial for minimizing further myocardial damage associated with the surgical procedures . the intensive transfusion of blood products , including platelet concentrate and fresh frozen plasma , was performed after transferring the patient to the icu . serious mediastinal bleeding and a coexisting low cardiac output state requiring ecmo support gradually improved during postoperative icu care after the correction of coagulopathy . we believe that an optimal surgical strategy and dedicated intensive care allowed the patient to recover from a desperate condition . in conclusion , we report the case of a patient with behcet s disease presenting with a contained aortic rupture in the previous surgical sites of the aortic root , who was treated with a sophisticated surgical approach and postoperative care . the long - term outcomes associated with this procedure need to be addressed in further studies .

cardiovascular involvement in cases of behcet s disease is a rare but life - threatening condition , and prosthetic valve detachment is a frequent and serious complication attributable to behcet s disease following the surgical repair of aortic regurgitation . we report the case of a patient with behcet s disease presenting with contained aortic rupture around the aortic root . the patient had previously undergone aortic valve surgery three times due to recurrent prosthetic valve detachment . an emergency operation was performed , consisting of aortic root replacement ( arr ) using a composite valved conduit and the replacement of the hemiarch . arr may be an appropriate surgical option for patients with behcet s disease in order to prevent recurrence of the disease .

colonic necrosis has been described as a rare complication after the administration of kayexalate [ 1 , 2 ] . in this case study , we present a case of calcium polystyrene sulfonate - induced colonic necrosis and perforation to remind clinicians of this rare , but dangerous , toxicity associated with this commonly used medication . a 78-year - old woman with stage 4 chronic kidney disease ( ckd ) due to chronic pyelonephritis , and a right hypoplastic kidney was presented to our emergency department with a 2-day history of abdominal pain . her medical history included epilepsy that was treated with carbamazepine , hypertension and hyperlipidaemia , for which she received carvedilol and atorvastatin . because of the persistent hyperkalaemia , she was treated with calcium polystyrene sulfonate at 30 g / day ( anti - potassium granule ; assos drug , istanbul , turkey ) . as a result of the ckd , she was prescribed calcitriol for a mineral and bone disorder , darbepoetin alpha for anaemia and sodium hydrogen carbonate for acidosis . upon initial physical examination , her abdomen was non - distended with normoactive bowel sounds , but there was diffuse tenderness in her abdomen . significant laboratory values at the time of admission included sodium 138 meq / l , potassium 4.6 meq / l , ph 7.20 , bicarbonate 13 meq / l , blood urea nitrogen 71 mg / dl , creatinine 2.6 mg / dl , leukocytes 15 500/mm , eosinophils 110/mm , haematocrit 32.7% , platelets 221 000/mm and c - reactive protein 10.9 mg / dl . on the second day of hospitalization , the abdominal pain worsened and free air under the diaphragm was found on abdominal radiography . her sigmoid colon was found to be necrotic and perforated . a biopsy was performed and the perforated segment was repaired by primary closure . the surface of the deep ulcer contained necroinflammatory debris and various sized fragments of basophilic crystalloid material with angulated margins on microscopic examination ( figure 1a and b ) . also , there were no features of chronic colitis , including inflammatory bowel disease or chronic ischaemic colitis . ( a ) the basophilic crystalloid material in the necroinflammatory background of the ulcer surface ( haematoxylin and eosin , original magnification , 100 ) . ( b ) the basophilic crystalloid material in the necroinflammatory background of the ulcer surface ( haematoxylin and eosin , original magnification , 400 ) . sodium polystyrene sulfonate can also bind intraluminal calcium , leading to constipation , fecal impaction and subsequent bowel obstruction or perforation . gerstman et al . reported a 0.27% overall incidence , with a higher incidence ( 1.8% ) during the postoperative period . sorbitol enemas , along with experimental evidence suggesting that the necrosis was due to sorbitol rather than the kayexalate in presence of uraemia . extensive transmural necrosis was noted in rats receiving enemas of sorbitol or kayexalate in sorbitol in both the uraemic and non - uraemic groups . as in this case report , renal failure may be an important facilitating factor in the pathogenesis of the necrosis . in contrast to the experimental data , our case was treated with calcium polystyrene sulfonate orally , not rectally . rashid et al . noted that kayexalate in sorbitol given as an enema or orally to treat hyperkalaemia has been reported to induce intestinal necrosis in uraemic patients . they studied clinical and pathologic features of 15 patients and observed kayexalate crystals in tissue specimens from surgical resections and endoscopic biopsies . one possibility is elevated renin levels , commonly seen in renal insufficiency , that predispose the patient to non - occlusive mesenteric ischaemia via angiotensin - mediated vasoconstriction . one gram of kayexalate possesses a theoretical in vitro exchange capacity of 23.1 meq of potassium and in vivo capacity of 1 meq . emmett et al . reported that in vivo potassium - binding capacity may be lower than previously estimated , more on the order of 0.40.8 meq / g of kayexalate resin . in contrast to other minor digestive complications associated with kayexalate treatment , colonic perforation results in significant morbidity and mortality . as a result , potassium exchange resins may , although rarely , induce a colonic perforation , and this diagnosis should be considered in a patient treated as such in case of acute abdomen . the clinicians must be aware of the possible rare and serious complications of potassium exchange resins .

sodium or calcium polystyrene sulfonate ( kayexalate or analog ) is an ion - exchange resin commonly used to treat hyperkalaemia in patients with chronic kidney disease . it is known to cause digestive complications , such as nausea , vomiting and constipation . although rare , colonic necrosis and perforation are very severe complications associated with the medication . in this case report , we present a case of calcium polystyrene sulfonate - induced colonic necrosis and perforation to remind clinicians of this rare , but dangerous , toxicity associated with this commonly used medication .

hiv / aids is spreading rapidly in the society and the major cause is attributed to commercial sex workers sexual behaviour . higher risk of hiv transmission is determined by frequent partner change or higher number of partners ; lack of , low level , or inconsistent condom use ; unprotected anal intercourse ; and presence of certain types of stis , especially genital ulcerative disease , as cofactors . hiv prevalence among sex workers in 2007 was over 30% , and in some cities like kano and abuja , 50% of all brothel - based sex workers are hiv - infected . thus , while hiv prevalence among the general population in nigeria has been declining from its peak of 5.8% in 2001 to 4.1% in 2011 prevalence among brothel - based sex workers has shown no sign of declining . in a recent survey among most - at - risk populations in six states in nigeria , over half of sex workers did not consider themselves at risk of hiv infection . in lagos state , with the highest concentration of sex workers in nigeria , only 16% of brothel - based sex workers felt they were at risk , even though each has on average 34 clients per week . despite their high - risk sexual activity , many sex workers perceive their risk of hiv infection to be low . as in most regions globally , many african individuals support themselves and their dependants through sex work , primarily women . between 1% and 4% of women in several west african capital cities are estimated to be sex workers , with even higher proportions in areas were transport and employment networks increase the demand for paid sex [ 8 , 9 ] . estimated 37% of sub - saharan female sex workers ( fsws ) live with hiv . the risks of hiv transmission experienced by sex workers who inject drugs are related to their individual exposure to factors such as unprotected sex with multiple partners or sharing injecting equipment while injecting drugs . there is evidence from many countries that sex work rates are much higher among female than among male injecting drug users and that female injectors are more likely to have less control over sharing of injecting equipment in a group injection or even with a sexual partner and are more likely to inject after male injecting drug users , thereby increasing their risk for acquiring hiv . the hiv prevalence decreased from 4.3% in 2001 to 1.2% in 2003 and again increased to 2.0% in 2005 and decreased to 1.2% in 2008 [ 12 , 13 ] . factors driving the epidemic in osun state include poverty , multiple sex partners , marital infidelity , high unprotected sexual activities among youths , ignorance , low risk assessment , negative cultural activities such as female circumcision , and migration of people from the high prevalence states that share borders with osun . the sexual behaviour of sex workers according to has been identified as the likely central problem of the transmission of hiv / aids because the business may expose them to the danger of contracting hiv infections if they are ignorant of the use of condom as a protective measure against hiv virus as they engage in sex with multisexual partners in respect of their hiv / aids status . this study therefore investigates sexual practices of female sex workers who inject drugs in osogbo , nigeria . the scope of the study was delimited to sexual behaviour , drug use , awareness of hiv magnitude , and hiv counseling and testing ( hct ) , condom use , and sexually transmitted infection ( sti ) challenges among fsws . osogbo lies on the railway line from lagos to kano and is the trade centre for a farming region . osogbo is the venue of the annual osun - osogbo festival along the river osun . the festival is centered around the sacred grove of the river goddess sun , which is a unesco world heritage site . the population of the state according to the 2006 national population census figures is 3,423,535 , including 1,740,619 ( 50.8% ) men and 1,682,916 ( 49.2% ) women . osun state consists chiefly of the yoruba people , but with variations in dialects ; approximately half of the residents are muslim , with several mosques in major towns such as iwo , ede , osogbo , ikire , and ejigbo . the study population was brothel - based fsws who inject drugs in osogbo , osun state , nigeria . snowball technique was used in selecting a total of 27 fsws who inject drugs from 11 brothels ( urban = 6 brothels ; semiurban = 5 brothels ) in osogbo . this implies that the participants were selected because they are sex workers and also injecting drug users . in - depth interview guide and semistructured questionnaire were developed by the researchers based on the literature reviewed together with input from health promotion specialists at the university of ibadan , nigeria . the questionnaire was used to document female sex workers sociodemographic characteristics and sexual behaviour , while in - depth interview questions focused on drug use , awareness of hiv magnitude and hiv counseling and testing ( hct ) , condom use , and sti challenges . two of the researchers moderated all the interviews , while note - taking was done by other research members to complement the audio recording by the moderator . questionnaire was self - administered before the interview and idis were conducted in a quiet space within their brothels . each interview lasted about 3045 minutesand the discussions were audiotaped with the consent of the participants . the study instruments were reviewed by health promotion experts at the faculty of public health , university of ibadan , and were also pretested among fsws who are also injecting drug users in ibadan which has the same characteristics with the study area to determine how effective the developed instrument would be in collecting appropriate data relevant to the research objectives . a total of four idis and questionnaire each were conducted during the pretest and they were revised based on the issues identified by the team . the quantitative data collected was collated , screened , and entered into computer . the statistical package for social science ( spss ) the analysis was done by reading through the transcribed interviews and listening to the audio records in order to get all major discussions during the interviews and the data were manually analyzed using content analysis technique . approval for the study was sought from the osun state ministry of health . written consent of all the participants was obtained before the interview by giving them informed consent form to fill according to their ability to read and write after explaining the objectives of the study to them . participation in the study was voluntary and there was no criticism of participants who refused to participate or wish to withdraw from the study . no identifier like participants ' name or address was recorded so as to keep the information given by each participant confidential . the age of respondents ranged from 21 to 38 with mean age of 26.2 7.5 . many ( 63.0% ) of the respondents were single , the respondents have been in the business for an average of 5.3 years ( table 1 ) . the age of respondents at first intercourse ranged from 13 to 19 with mean age of 15.7 3.2 . more than half of the respondents ( 55.6% ) reported boyfriend , as first sexual partner , and employer ( 18.5% ) ( figure 1 ) . reasons adduced by respondents for first sexual intercourse included pressure by partner ( 29.6% ) and financial reason ( 25.9% ) ( figure 2 ) . all ( 100.0% ) the respondents ' reported not using a condom at their first intercourse and their reasons included that they did not have one at hand ( 55.6% ) ( figure 3 ) . majority of the respondents reported having a daily average of 4 clients and usually spending at least 30 minutes with the client . the majority ( 88.9% ) of the respondents were current family planning users , and intrauterine device ( iud ) ( 54.2% ) was a major method used by the respondents . all ( 100.0% ) the respondents had ever aborted pregnancy more than once and 66.7% reported using contraceptive when pregnancy happened . less than a third ( 25.9% ) of the respondents had ever experienced abortion complications and these were severe bleeding ( 71.4% ) and internal infection of the abdomen ( 28.6% ) ( table 2 ) . majority of the respondents were aware of the magnitude of hiv in nigeria and also considered hiv as a serious health problem amongst sex workers . a respondent in urban brothel specially saidi consider myself at risk of sexually transmitted diseases including hiv / aids because most times i do n't protect myself when having sex because am not always mentally stable because of the drugs i use . even most times i fight with my clients when i see them because most of them do n't pay me because they know am not normal again because of the drugs have injected , so they use the advantage to get a free sex from me and this is always unprotected sex . i consider myself at risk of sexually transmitted diseases including hiv / aids because most times i do n't protect myself when having sex because am not always mentally stable because of the drugs i use . even most times i fight with my clients when i see them because most of them do n't pay me because they know am not normal again because of the drugs have injected , so they use the advantage to get a free sex from me and this is always unprotected sex . few of the respondents were also of the opinion that other women who are not sex workers are also at risk of hiv as sex workers . a respondent in semi - urban brothel saidi am at risk of hiv as other women who are not into this business because their husbands come here to do business with us , so if we are infected with hiv then we are going to transfer this to their husband and their husband will also transfer to them at home . i am at risk of hiv as other women who are not into this business because their husbands come here to do business with us , so if we are infected with hiv then we are going to transfer this to their husband and their husband will also transfer to them at home . another respondent in urban brothel saidi think we are not even at risk of hiv compared with other women who are not sex workers because i can say no to sex if my client refuse to use condom but married women can not say that to their husband and their husbands are our clients who can easily infect them when infected with this disease from us . i think we are not even at risk of hiv compared with other women who are not sex workers because i can say no to sex if my client refuse to use condom but married women can not say that to their husband and their husbands are our clients who can easily infect them when infected with this disease from us . on the issue of hiv counseling and testing , some of the respondents reported that they had ever tested for hiv and only one of these respondents disclosed to be hiv positive . respondents who had never taken hiv test were also asked for their reasons and their reasons were fear of rejection and discrimination by the community and even among other sex workers if the test is positive . other reasons were inadequate understanding of the importance of hct , fear of high blood pressure , and depression and shock if tested positive . many of the respondents reported use of condom , regular talking of herbs , and good personal hygiene as ways of protecting themselves from hiv . a respondent in urban brothel saidi have been tested twice this year and the results showed that am negative . i have been tested twice this year and the results showed that am negative . another respondent in urban brothel saidi have never been tested because am scared of discrimination even from my colleagues . i have never been tested because am scared of discrimination even from my colleagues . another respondent in semiurban brothel saidi have not been tested in the last 3 years and i engage in unprotected sex intercourse with many of my clients . personally , i do n't think i need to be tested because if am tested positive i will die of hypertension so i think is better i do n't do the test now . i have not been tested in the last 3 years and i engage in unprotected sex intercourse with many of my clients . personally , i do n't think i need to be tested because if am tested positive i will die of hypertension so i think is better i do n't do the test now . cocaine , marijuana , tobacco , and alcohol were reported as the drugs mostly used by the respondents . few of the respondents reported that they were involved in sex work as a means of financing their drug use , while majority reported that they were into sex work before becoming drug users with a major reason to escape from intense burden of their work . other reasons were to become bold , to be confident to negotiate with clients , and to be strong in bed for clients to enjoy their money . a respondent in urban brothel saidi inject cocaine at least twice a week to enable me to be strong and mentally stable . am actually doing this sex work to raise money to finance the drug am taking . i inject cocaine at least twice a week to enable me to be strong and mentally stable . am actually doing this sex work to raise money to finance the drug am taking . another respondent in semiurban brothel saidi inject drugs many times to enable me to be very strong in bed so that my clients can enjoy me . i inject drugs many times to enable me to be very strong in bed so that my clients can enjoy me . all the respondents reported not using a condom at their first intercourse but all had ever used a condom . less than a third reported using a condom with their last client and more than half reported that ninety percent of their regular clients do not like using a condom . nearly all the respondents were willing to have clients not to wear a condom in exchange for accepting more money in return , and the majority of them were aware that using a condom could help prevent hiv transmission . major reason adduced by respondents for inconsistent condom use was mental imbalance effects from drug use . other reasons were as follows : clients objected to use it , it reduces sexual pleasure , and they did not have one at hand . a respondent from urban brothel who disclosed to be hiv positive reported to be inconsistent with condom usage with reason that most of her clients always rejected using a condom and even offer more money for this . the respondent specifically saidi always try my best to enforce my clients to use condom but majority of them always reject this with reason that condom reduce sexual pleasure and i ca n't tell them i am hiv positive because of my previous experience . i always try my best to enforce my clients to use condom but majority of them always reject this with reason that condom reduce sexual pleasure and i ca n't tell them i am hiv positive because of my previous experience . the respondent narrated her previous experience as thusi came from x state to this state to continue this sex work because when i tested positive to hiv , i disclosed this to my friends whom we are doing this business together and within a week the information has spread rapidly to everybody and they started discriminating against me . i suffered a lot during this period as i was not getting any client and life became miserable for me and that was why i travelled down to this state so that i can continue this business as i have nothing else to survive with . am telling you this because you may assist me with treatment and because you have assured me of my confidentiality and if you do n't keep your promise and disclose my status to other sex workers then i will leave this state for another state . i came from x state to this state to continue this sex work because when i tested positive to hiv , i disclosed this to my friends whom we are doing this business together and within a week the information has spread rapidly to everybody and they started discriminating against me . i suffered a lot during this period as i was not getting any client and life became miserable for me and that was why i travelled down to this state so that i can continue this business as i have nothing else to survive with . am telling you this because you may assist me with treatment and because you have assured me of my confidentiality and if you do n't keep your promise and disclose my status to other sex workers then i will leave this state for another state . the respondent further added thati am actually not seeing any sign on my body to show that i am hiv positive only that i easily get tired and that is one of the reasons for my taking drugs and that has been assisting me a lot . i am actually not seeing any sign on my body to show that i am hiv positive only that i easily get tired and that is one of the reasons for my taking drugs and that has been assisting me a lot . questions on sexually transmitted infections ( stis ) were also discussed with the respondents and more than half of the respondents had ever been treated for sti more than once . reported stis among respondents were gonorrhea , syphilis , trichomoniasis , and chlamydia infections . reported signs and symptoms experienced by respondents were pain during sexual act , pus like discharges from vagina , an itch in the vagina region , and pain during urination . a respondent in urban brothel saidi suffered from gonorrhea last year and it was since then have made up my mind not to allow any client to have sex with me without using condom . i suffered from gonorrhea last year and it was since then have made up my mind not to allow any client to have sex with me without using condom . another respondent in semiurban brothel saidhave been infected with gonorrhea and syphilis in this work and i think it was because of my lack of knowledge of these diseases . many times when we attend health talk as sex workers it is always hiv / aids education so many of us do n't really know about other diseases . have been infected with gonorrhea and syphilis in this work and many times when we attend health talk as sex workers it is always hiv / aids education so many of us do n't really know about other diseases . another respondent in semiurban brothel saidi was told last 2 years that i was infected with chlamydia infection when i went for medical check - up due to itches in the vagina region which i do experience then and this was treated for me and i was counseled to always use condom with my client when i disclosed to the matron that am a sex worker . i was told last 2 years that i was infected with chlamydia infection when i went for medical check - up due to itches in the vagina region which i do experience then and this was treated for me and i was counseled to always use condom with my client when i disclosed to the matron that am a sex worker . this is similar to the findings of which also revealed that interviewed fsws have been in the business for an average of 4.1 years . majority of the respondents reported having a daily average of 4 clients . in the russian city of st . petersburg , female injecting drug users who exchange sex for money had a mean of 49.5 sexual partners in the previous 30 days . this is in accord with the findings of who reported that , in the netherlands , a large proportion of sex workers , mainly from latin america and eastern europe , do not use contraceptives other than condoms and some have undergone two or three abortions . this is in accord with the findings of where all the respondents reported sharing needles and injecting equipment . the riskiest activity for hiv infection during injection is frequent sharing of injecting equipment with strangers . the highest infection risk lies in the sharing of needles and syringes , where blood is most likely to collect during injection , especially when jacking back ( injecting into a vein , drawing blood into the syringe , and reinjecting the blood - and - drug mixture ) . even if no blood is visible in either the needle or syringe , hiv may remain trapped in microscopic blood particles . a syringe is a particularly viable atmosphere for hiv within blood , and a contaminated syringe may remain capable of infecting others for up to six weeks . cocaine , marijuana , and heroin were reported as the drugs mostly used by the respondents and also it is also reported that about one third of the idus reported having injected cocaine , heroin , or speedball ( heroin and cocaine ) . all the respondents reported not using a condom at their first intercourse , but all had ever used a condom . less than a third reported using a condom with their last client and more than half reported that ninety percent of their regular clients do not like using a condom . lower levels of condom use have also been shown among those injecting drugs and selling or buying sex in other countries . for example , only 10 percent of injecting drug users from three cities in indonesia , many of whom had multiple - sex worker and other partners , reported using condom . among crack using sex workers in southern brazil , condom use was reported to be lower with clients who paid more , looked clean , and were regulars . among injecting drug user cohorts in vancouver , canada , and in several cities of usa condom use with all types of sexual partners ( clients and casual and primary partners ) is rare or low among those exchanging sex for drugs or money [ 23 , 24 ] . more than half of the respondents had ever been treated for sti more than once . this is in accord with the findings of where more than half of the respondents indicated having sexually transmitted diseases once or more . valdez et al . also reported that 37 percent of respondents reported some history of infection with a sexually transmitted disease since entering sex work . high levels of stis such as syphilis , gonorrhea , chlamydia , and herpes among sex workers have pointed to the urgent need to provide sti prevention and treatment services for this population . respondents have a relatively high number of sexual partners , but this in itself does not necessarily increase their likelihood of becoming infected with hiv ; if they use condoms consistently and correctly , then they will probably be protected no matter how many people they have sex with . some of the respondents reported becoming pregnant even while using family planning and this calls for further investigation to determine the antecedent factors behind this failure . involving sex workers directly in hiv prevention campaigns can raise their self - esteem and empower them , thereby encouraging them to look after their health and to access services that could help them . evidence suggests that sex workers who use / inject drugs face increased risk of sexual hiv transmission because they often have a higher number of clients or sexual partners who are more willing to engage in unprotected sex and have sexual partners who also inject drugs .

female sex workers ( fsws ) who inject drugs have higher risks of hiv infection due to injection drug use and the array of sexual practices employed . this study , therefore , is designed to determine sexual practices of fsws who inject drug in osogbo , nigeria . this study was a cross - sectional descriptive mixed - methods design . twenty - seven fsws who inject drug were selected from 11 brothels by snowball sampling and interviewed with a semistructured questionnaire and in - depth interview guide . the mean age of respondents was 26.2 7.5 . many of the respondents were aware of the magnitude of hiv and some were sex workers first before turning to be drug users . some of the respondents had ever tested for hiv and few had ever been treated for sti more than once . some respondents were willing to have male clients who do not wear a condom in exchange for accepting more money in return . many of the respondents reported use of condom , regular talking of herbs , and good personal hygiene as ways of protecting themselves from hiv . respondents have relatively high numbers of sexual partners . involving sex workers directly in hiv prevention campaigns will encourage them to look after their health and to access services that could help them .

currently , liver transplantation is the only definitive treatment for the end stage liver diseases . however , its widely used was limited by the expensive costs , shortage of donor organs and invasive procedure ( 1 ) . hepatocyte transplantation has emerged as a feasible alternative to liver transplantation in some liver diseases ( 2 ) , but it is limited by organ donors and the low cell quality of available liver tissues ( 3 , 4 ) . although human embryonic stem cells ( hescs ) and umbilical cord blood stem cells ( ucbsc ) could be induced into hepatocyte - like cells in vitro ( 5 ) , their widely used were limited by the low differentiation quality , ethics , and teratoma formation ( 6 ) . recently , the adult stem cells ( ascs ) derived from various tissues , including bone marrow derived mesenchymal stem cells ( bmscs ) , adipose tissue derived mesenchymal stem cells ( adscs ) , and peripheral blood mononuclear cells ( pbmcs ) have been developed as new cell sources contributing to liver regeneration for their high efficiency of hepatogenic differentiation using simple procedures and no ethnic issues ( 710 ) . in the present study , we investigated whether ascs are the ideal seed cells for the liver regeneration from the followings : ascs biology , including isolation , culture , differentiation to hepatocytes , and the further role of ascs in cell therapy and tissue engineering in recent studies , samples of human bone marrow were obtained by lumbar puncture ( lp ) , human adipose tissue were obtained from abdominal subcutaneous adipose tissue of gastric cancer patients or liposuction patients , human peripheral blood were obtained from patients with hbv or healthy adult blood donors , in accordance with the local ethics committee . most laboratory data showed that bmscs could be prepared through the density degree of centrifuge , flow cytometry sorting , and sidewall sieve method ( 1114 ) . side - wall sieve method has become popular one for its simple operation , lower cost , less injury to cell ( 15 ) . adipose tissue was minced with scissors and scalpels into less than 3 mm pieces and isolation of adscs proceeded as previously described ( 16 , 17 ) . generally , human peripheral blood monocytes isolated from donors were isolated by density gradient centrifugation and further purified by adherence separation . in order to obtain more pbscs in shortly time , g / kg / d , administered subcutaneously daily to mobilize pbmcs from bone marrow to peripheral blood ( 18 , 19 ) . then pbmcs were collected by means of aphaeresis . conget et al indicated that the bmscs express many surface agents including cd13 , cd44 , cd29 , cd105 , but did nt express cd1a , cd14 , cd31 , cd34 , cd56 , cd45 ( 20 ) . ascs derived from all three sources displayed no expression of hematopoietic markers ( cd14 , cd34 , cd45 ) , of the stem cell marker cd133 , or the marker for endothelial cells cd144 . more than 90% of mscs derived from the three sources expressed the typical msc marker proteins cd44 , cd73 , cd29 , and cd90 . however , the intensity of expression of cd90 of pbscs was significantly below that of the other tissues . more than 90% of the ascs derived from all three sources expressed hla i ; however , none of the mscs expressed hla ii . cd105 was expressed by a significantly lower percentage of pbscs compared with bm- or ad - scs , whereas , more pb- and bm - scs expressed cd106 than adscs . besides the fact , that they are more heterogeneous ( 23 ) , they reveal a surface antigen marker profile ( 22 , 2426 ) , and differentiation potential similar to bmscs ( 2732 ) . adscs are characterized as cd45 , cd34 + , cd105 + , cd31-(33 ) ( table 1 ) . some studies indicated that functional hepatocytes could be induced from ascs by some cytokines or through coculture with other cell types . however , the potential of the ascs transdifferentiation is generally low . therefore , researchers are keen to explore new methods to induce ascs differentiate into functional hepatocytes in vitro currently . the hepatocyte - like cells from ascs were confirmed from the gene and protein expression . gene expressions were identified by rt - pcr , using the common markers of hepatocytes , including alb , afp , ck18 , ck19 , and cyp3a4 ( 34 ) . protein expressions were usually identified by immunohistochemistry or mmunofluorescence , western blot , from the expression of albumin , ck18 , cyp3a4 , cyp1a1 , cyp2c9 and nadph - p450 ( 3538 ) . to compare the potential of hepatogenic differentiation of the different adult stem cells in vitro , researches indicated adscs have a similar differentiation potential towards the hepatic lineage , similar to bmscs . however , their longer culture period and proliferation capacity differ from the bmscs ( 3941 ) . in recent studies , samples of human bone marrow were obtained by lumbar puncture ( lp ) , human adipose tissue were obtained from abdominal subcutaneous adipose tissue of gastric cancer patients or liposuction patients , human peripheral blood were obtained from patients with hbv or healthy adult blood donors , in accordance with the local ethics committee . most laboratory data showed that bmscs could be prepared through the density degree of centrifuge , flow cytometry sorting , and sidewall sieve method ( 1114 ) . side - wall sieve method has become popular one for its simple operation , lower cost , less injury to cell ( 15 ) . adipose tissue was minced with scissors and scalpels into less than 3 mm pieces and isolation of adscs proceeded as previously described ( 16 , 17 ) . generally , human peripheral blood monocytes isolated from donors were isolated by density gradient centrifugation and further purified by adherence separation . in order to obtain more pbscs in shortly time , pbmcs were mobilized with recombinant g - csf at 510 g / kg / d , administered subcutaneously daily to mobilize pbmcs from bone marrow to peripheral blood ( 18 , 19 ) . conget et al indicated that the bmscs express many surface agents including cd13 , cd44 , cd29 , cd105 , but did nt express cd1a , cd14 , cd31 , cd34 , cd56 , cd45 ( 20 ) . ascs derived from all three sources displayed no expression of hematopoietic markers ( cd14 , cd34 , cd45 ) , of the stem cell marker cd133 , or the marker for endothelial cells cd144 . more than 90% of mscs derived from the three sources expressed the typical msc marker proteins cd44 , cd73 , cd29 , and cd90 . however , the intensity of expression of cd90 of pbscs was significantly below that of the other tissues . more than 90% of the ascs derived from all three sources expressed hla i ; however , none of the mscs expressed hla ii . cd105 was expressed by a significantly lower percentage of pbscs compared with bm- or ad - scs , whereas , more pb- and bm - scs expressed cd106 than adscs . besides the fact , that they are more heterogeneous ( 23 ) , they reveal a surface antigen marker profile ( 22 , 2426 ) , and differentiation potential similar to bmscs ( 2732 ) . adscs are characterized as cd45 , cd34 + , cd105 + , cd31-(33 ) ( table 1 ) . some studies indicated that functional hepatocytes could be induced from ascs by some cytokines or through coculture with other cell types . however , the potential of the ascs transdifferentiation is generally low . therefore , researchers are keen to explore new methods to induce ascs differentiate into functional hepatocytes in vitro currently . the hepatocyte - like cells from ascs were confirmed from the gene and protein expression . gene expressions were identified by rt - pcr , using the common markers of hepatocytes , including alb , afp , ck18 , ck19 , and cyp3a4 ( 34 ) . protein expressions were usually identified by immunohistochemistry or mmunofluorescence , western blot , from the expression of albumin , ck18 , cyp3a4 , cyp1a1 , cyp2c9 and nadph - p450 ( 3538 ) . to compare the potential of hepatogenic differentiation of the different adult stem cells in vitro , researches indicated adscs have a similar differentiation potential towards the hepatic lineage , similar to bmscs . however , their longer culture period and proliferation capacity differ from the bmscs ( 3941 ) . in the present study , we described different ascs could be induced into hepatocyte lineage cells in different culture systems in vitro . it is very safe and easy to acquire the enough ascs , and then induce them into functional hepatocytes in vitro . based on this progress , ascs transplantation might be a novel therapy for the severe liver diseases , and also will be ideal seed cells for liver tissue engineering . however , which ascs are better still needs us to investigate from their preparation , molecular characterization , and functional assay . this paper firstly provides such a concise review focused on ascs biology and differentiates potential , indicating ascs might be an ideal seed cells in cell transplant therapy and tissue engineering . we also believe in the future , some studies will show us the most appropriate ascs for cell transplant or tissue engineering .

adult stem cells ( ascs ) are undifferentiated cells found throughout the body that divide to replenish dying cells and regenerate damaged tissues , which are the powerful sources for cell therapy and tissue engineering . bone marrow - derived mesenchymal stem cells ( bmscs ) , adipose tissue - derived mesenchymal stem cells ( adscs ) , and peripheral blood monocytes ( pbmcs ) are the common ascs , and many studies indicated that ascs isolated from various adult tissues could be induced to hepatocyte - like cells in vitro . however , the isolation , culture protocols , characterization of ascs and hepatocyte - like cells are different . this review aims to describe the isolation and culture procedures for ascs , to summarize the molecular characterization of ascs , to characterize function of hepatocyte - like cells , and to discuss the future role of ascs in cell therapy and tissue engineering .

a 53-year - old woman presented to the emergency department with a 2-week history of abdominal distention and constipation . she had a previous history of having undergone a myomectomy 15 years ago and has been under thyroid hormone replacement therapy for 10 years . seven years ago , she had visited our hospital due to the same symptoms . at the time , abdominopelvic computed tomography revealed markedly dilated entire colon with feces and tapered narrowing in the distal portion ( fig . colonoscopic mucosal biopsy showed mild lymphoplasmacytic infiltrates in the mucosa without muscularis mucosa component . at this visit , laboratory findings were within normal limits except for a slightly decreased tsh level ( 0.48 iu / ml ; normal range , 0.55 to 4.2 ) . serologic study of anti - hu antibody , anti - ri antibody , anti - yo antibody , anti neuromyelitis optica antibody , anti antinuclear antibody , anti thyroid - stimulating hormone receptor antibody , anti - thyroglobulin antibody , and anti - thyroid microsomal antibody were all negative . on abdominopelvic computed tomography , the entire colon was severely dilated and there was no significant difference between the previous and current images . the differential diagnoses included colonic obstruction due to stricture from inflammatory bowel disease or ischemic colitis and a mild form of congenital megacolon . proximal advancement of the scope was not possible due to much fecal material and the patient s pain . after 3 days of conservative management with slow bowel preparation with polyethylene glycol solution , the distended abdomen was decompressed and the symptoms subsided . colon transit time study showed a slow transit constipation pattern . given concern for the possibility of intractable benign luminal stenosis due to adhesive structure or ischemic enteritis there was no evidence of adhesion or mechanical obstruction except for focal physiologic attachment between the visceral and parietal peritoneum . the patient s postoperative course was uncomplicated and the patient s symptoms resolved after the surgery . the wall thickness appeared even without regional differences between the dilated and the narrowed portions . the average number of eosinophils was less than 4 per high power field ( hpf ) . the muscularis mucosa was thickened up to 660 m in the dilated area in contrast to the thin muscularis mucosa in the narrow area ( fig . intermuscular auerbach s myenteric plexuses showed moderate eosinophilic infiltrates up to 22 per hpf ( fig . 3b ) and hypogangliosis with atrophic reduction of the volume of nerve bundle , which became more evident with synaptophysin immunostain ( fig . the atrophic myenteric plexus was accompanied by increased fibrosis and admixed with some cd3 and cd4 dual positive lymphocytes ( fig . immunohistochemical study showed preserved positivity of c - kit and stronger but scantier glial fibrillary acidic protein ( gfap ) reaction in the myenteric plexus when we compared the immunostain results with a comparable descending colon of a 60 year old female patient who received hartmann s operation due to cancer ( fig . there was no significant difference in the severity of the eosinophilic infiltrates in the myenteric plexus between the grossly dilated and narrow portions . however , there was regional difference in the eosinophilic infiltrates between the inner portion and the outer portion of the myenteric plexus . there was no eosinophilic infiltrates in the submucosa and the inner muscle proper in contrast to the increased number of eosinophils , lymphocytes , and histiocytes in the outer muscle layer , subserosa , and serosa . the wall thickness appeared even without regional differences between the dilated and the narrowed portions . the average number of eosinophils was less than 4 per high power field ( hpf ) . the muscularis mucosa was thickened up to 660 m in the dilated area in contrast to the thin muscularis mucosa in the narrow area ( fig . intermuscular auerbach s myenteric plexuses showed moderate eosinophilic infiltrates up to 22 per hpf ( fig . 3b ) and hypogangliosis with atrophic reduction of the volume of nerve bundle , which became more evident with synaptophysin immunostain ( fig . the atrophic myenteric plexus was accompanied by increased fibrosis and admixed with some cd3 and cd4 dual positive lymphocytes ( fig . immunohistochemical study showed preserved positivity of c - kit and stronger but scantier glial fibrillary acidic protein ( gfap ) reaction in the myenteric plexus when we compared the immunostain results with a comparable descending colon of a 60 year old female patient who received hartmann s operation due to cancer ( fig . there was no significant difference in the severity of the eosinophilic infiltrates in the myenteric plexus between the grossly dilated and narrow portions . however , there was regional difference in the eosinophilic infiltrates between the inner portion and the outer portion of the myenteric plexus . there was no eosinophilic infiltrates in the submucosa and the inner muscle proper in contrast to the increased number of eosinophils , lymphocytes , and histiocytes in the outer muscle layer , subserosa , and serosa . digestive motility , an important pathogenic factor of cipo , is a highly coordinated process and depends on smooth muscle contractility and the related pacemaker activity evoked by the interstitial cells of cajal ( iccs ) . both of these mechanisms are finely tuned by the intrinsic enteric nervous system ( ens ) and the extrinsic sympathetic and parasympathetic nerves , independently from the central and peripheral nervous systems . based on histology , cipo can be classified into three major entities depending on the predominant involvement of enteric neurons , iccs , or smooth muscle cells : neuropathies , mesenchymopathies , and myopathies . neuropathy can be classified into two major forms : ( 1 ) inflammatory neuropathies in which a significant inflammatory response is identified within nerve tissue and ( 2 ) degenerative neuropathies characterized by evidence of neurodegenerative aspects in the absence of an identifiable inflammatory response . inflammatory neuropathy is also subdivided into lymphocytic and eosinophilic depending on the prominent infiltrating cells . distinctive findings of eosinophilic neuropathy has mainly been reported in children showing no degenerative pathologic findings such as neuronal loss and no lymphocytic infiltrates . we report a case of eosinophilic myenteric ganglionitis which developed in a middle - aged female patient with the pathologic features of hypogangliosis and cd4-positive lymphocytic infiltrates . however , interleukin 5 ( il-5 ) , a well - known eosinophilic chemotactic factor , has been mentioned in eosinophilic myenteric ganglionitis . although we did not evaluate il-5 , the increased number of cd3 and cd4 dual positive cells may suggest the increased production of il-5 from the infiltrating cd4-positive helper t cells for eosinophilic chemotaxis . a few scattered cd8-positive lymphocytes may be involved in the cytotoxic reaction of the nerve plexus resulting in hypogangliosis . atrophic plexus and cell loss in neuroinflammatory disease were mentioned as the result of the activation of a caspase dependent mechanism ; however , immunoreactivity of bcl-2 in our case showed no definite reduced positivity . enteric glial cells are the main constituent of the ens and the majority of the enteric glial cells are located in the myenteric plexus . recently , the role of enteric glial cells as a neuroprotector and as an immune - modulator in inflammatory processes has been investigated and the myenteric gfap - expressing glial subpopulation was reported to be susceptible to systemic inflammation . in our case , the stronger but scantier expression of gfap seems to be related with the overproduction of gfap by activated enteric glial cells for neuroprotection as a damaged myenteric plexus . the role of the myenteric plexus as an immune - modulator and barrier was evident in the findings of scarce inflammatory reaction in the inner muscle layer in contrast to mild to moderate inflammatory reaction in the outer muscle layer and the subserosa . this regional difference suggests the barrier function of the myenteric plexus to diffusible and systemic immune reactants supplied by the mesenteric bloods through the subserosal and outer muscular layer vessels . there being no difference in the eosinophilic infiltrates in the myenteric plexus between the dilated and narrow areas was also meaningful . similarly , no difference in ens abnormality between the affected and the nonaffected areas in crohn disease has been described . this finding suggests the eosinophilic reaction as a systemic reaction , and the possible discrepancy between the pathologic findings and functional impairment . the pathologic findings such as eosinophilic infiltrates or neuronal loss can be the underlying causes of cipo ; however , the clinical functional impairment is more complex . physiologic abnormality such as disturbed activity of neurotransmitters can be another factor to take into consideration . the increased number of mast cells in the submucosa may be the finding of the crosstalk between mast cells and eosinophils . the thickened muscularis mucosa in the dilated colon can be a compensatory response to prolonged high luminal pressure . the therapeutic effect of surgery is limited since cipo often involves the entire gastrointestinal tract with a progressive nature . as surgical approach can aggravate the underlying condition , it should be reserved for only carefully selected patients . if a confirmed diagnosis of ganglionitis is made through a full - thickness biopsy of the intestinal wall , the patient might benefit from an anti - inflammatory or immunosuppressive therapy before the surgical procedure . however , in the absence of a transmural tissue diagnosis , patients with enteric ganglionitis can not be subject to these nonoperative treatment options . in conclusion , we think this colonic lesion might have begun as a systemic immune reaction showing eosinophilic myenteric ganglionitis and progressed into a chronic inflammatory lesion showing atrophic myenteric plexus , reduced ganglion cells , thickened muscularis mucosa , and subserosal fibrosis . the diagnosis of cipo is mainly clinical , and the histopathology of cipo has frequently been reported as a frustrating experience by pathologists due to limited experience . further combined clinical and histopathological studies are needed in order to enrich the understanding and management cipo .

eosinophilic myenteric ganglionitis is a disorder characterized by infiltration of the auerbach myenteric plexus by eosinophils . as a cause of chronic intestinal pseudo - obstruction ( cipo ) , eosinophilic myenteric ganglionitis has been rarely reported and the majority of the reported cases in the literature were children . we experienced a case of eosinophilic myenteric ganglionitis associated with cipo in a 53-year - old female patient . histologic examination of the resected descending colon showed moderate eosinophilic infiltrates with hypogangliosis in the myenteric plexus . immunohistochemical study revealed increased number of cd4-positive lymphocytes and stronger but scantier glial fibillary acid protein expression in the inflamed myenteric plexus .

metabolic syndrome ( ms ) is a group of cardiovascular ( cv ) risk factors including abdominal obesity , hypertriglyceridemia , low high - density lipoprotein cholesterol ( hdl - c ) , dysglycemia , and high blood pressure ( bp ) . numerous studies have shown that a significant increase in systemic inflammatory markers , such as high sensitivity c - reactive protein ( hscrp ) , interleukin-6 , and tumor necrosis factor- ( tnf- ) , among others , is observed in subjects with ms . high sensitivity c - reactive protein is a good predictor of ms and is strongly associated with abdominal obesity . moreover , this biomarker predicts cv events in healthy people as well as in those with atherosclerotic disease [ 5 , 6 ] . it has been reported that hscrp is elevated in an urban population of santiago , chile , with ms , and there is a clear correlation between ms , hscrp , and subclinical atherosclerosis in this population . thus , the clustering of cv risk factors and inflammation observed in ms increase the risk of atherosclerosis . lipoprotein - associated phospholipase a2 ( lp - pla2 ) is a recently described inflammatory marker . it is an enzyme produced by macrophages that hydrolyzes phospholipids of oxidized low - density lipoprotein ( ldl ) , releasing oxidized fatty acids and lysophosphatidylcholine , which are potent proinflammatory and prooxidative molecules . since oxidized ldl is mostly located in the intima of the artery , the products of lp - pla2 activity are generated mainly in the vascular wall . given that atherosclerosis is an inflammatory disease which begins in the vascular wall , lp - pla2 may have a prominent role in its pathophysiology . both lp - pla2 and hscrp are associated with coronary and cerebrovascular atherosclerotic disease in populations with and without a history of cv events [ 912 ] . furthermore , lp - pla2 is related to cv risk factors , with a strong correlation with ldl , as well as various components of ms ( e.g. , abdominal obesity ) . the vast majority of published studies do not show any association between lp - pla2 and hscrp ; hence , these inflammatory markers have been considered to be independent of each other . the objective of this study was to analyze and compare the levels of lp - pla2 and hscrp as predictors of ms in subjects without atherosclerotic disease . this was a descriptive cross - sectional study in 152 subjects ( 69 women ) , without history of atherosclerotic disease , who were recruited in two preventive cardiology centers of santiago between october 2011 and june 2012 : hospital clnico de la pontificia universidad catlica de chile and hospital de la previsin de carabineros de chile . exclusion criteria were ( a ) history of coronary heart disease and/or carotid or peripheral vascular disease ; ( b ) lipid - lowering therapy ( statins , ezetimibe , fibrates , nicotinic acid , and omega 3 ) ; ( c ) use of oral contraceptives or hormone replacement therapy ; ( d ) chronic intake of anti - inflammatory medicines ( steroidal and nonsteroidal ) ; ( e ) acute and/or chronic inflammatory diseases ; and ( f ) pregnancy . subjects were contacted by telephone and invited to participate by nurses or doctors in charge of the study . after signing the written , informed consent , patients were subjected to clinical data collection and anthropometric and laboratory measurements , all detailed below . during the visit to the centers , subjects were interviewed about demographics , medical history , education , physical activity , family medical history , and intake of medications . weight , height , body mass index ( bmi ) , and hip and waist circumferences were measured . the latter was measured in the midpoint between the last rib and the iliac crest . blood pressure was measured following the recommendations of the seventh joint national committee , with the subject sitting and after 5 minutes of rest . venous blood samples were taken after 12-hour fasting for determining the following biochemical parameters.lp-pla2 : samples for analysis of lp - pla2 activity were frozen and stored at 70c . levels of lp - pla2 activity were determined by enzymatic method ( diadexus , usa ) , cobas 8000 analyzer , c702 module . prior to the analysis , precision and trueness of the enzymatic test were assessed locally , and a calibration curve was constructed , which was sent to the international center for approval . following the approval , the analysis of the subjects ' samples was performed.total cholesterol ( total - c ) : colorimetric enzymatic method , roche diagnostics cobas analyzer cobas 8000 , c702 module.hdl-c : colorimetric homogeneous enzymatic method , roche diagnostics cobas , cobas 8000 analyzer , c702 module.triglycerides : white colorimetric enzymatic method with glycerol , roche diagnostics cobas , cobas 8000 analyzer , c702 module.ldl cholesterol ( ldl - c ) : calculated by friedewald formula.blood glucose : enzymatic method ( hexokinase ) , roche diagnostics cobas , cobas 8000 analyzer , c702 module.creatinine : jaff method , roche diagnostics cobas , cobas 8000 analyzer , c70 module.hscrp : nephelometric method on bn prospec analyzer , siemens ( detection limit 0.16 mg / l).fibrinogen : clauss method in acl top 500 analyzer , instrumentation laboratory . lp - pla2 : samples for analysis of lp - pla2 activity were frozen and stored at 70c . levels of lp - pla2 activity were determined by enzymatic method ( diadexus , usa ) , cobas 8000 analyzer , c702 module . prior to the analysis , precision and trueness of the enzymatic test were assessed locally , and a calibration curve was constructed , which was sent to the international center for approval . following the approval , the analysis of the subjects ' samples was performed . total cholesterol ( total - c ) : colorimetric enzymatic method , roche diagnostics cobas analyzer cobas 8000 , c702 module . hdl - c : colorimetric homogeneous enzymatic method , roche diagnostics cobas , cobas 8000 analyzer , c702 module . triglycerides : white colorimetric enzymatic method with glycerol , roche diagnostics cobas , cobas 8000 analyzer , c702 module . ldl cholesterol ( ldl - c ) : calculated by friedewald formula . blood glucose : enzymatic method ( hexokinase ) , roche diagnostics cobas , cobas 8000 analyzer , c702 module . creatinine : jaff method , roche diagnostics cobas , cobas 8000 analyzer , c70 module . hypertensives were subjects who had a prior diagnosis according to the jnc 7 , with or without pharmacologic therapy , and/or those with an average blood pressure 140/90 mm hg . dyslipidemics were subjects who had ldl - c level 130 mg / dl , hdl - c level < 40 mg / dl in men , < 50 mg / dl in women , or non - hdl - c level 160 mg / dl in the laboratory assessment . subjects were considered diabetics if they had a prior diagnosis , with or without drug treatment , and/or if they had a fasting blood glucose 126 mg / dl during the study , according to the american diabetes association criteria . current smokers were those subjects who smoked daily during the last month , and ex - smokers were those subjects who had at least six consecutive months without smoking . the recent harmonized criteria were used for the diagnosis of ms , which includes a waist circumference 90 cm in men and 80 cm in women for latin american populations . a logistic regression analysis was performed with each biomarker , adjusted for age and gender . smooth receiver operating characteristic ( roc ) curves were constructed with cubic splines for ms ( area under the curve c value = 0.50 implies a predictive value equivalent to chance ) . comparisons of means ( expressed as mean standard deviation [ sd ] ) are based on the analysis of variance and linear regression models . this study included 152 subjects ( 45% women ) with a mean age of 46 11 years . prevalence rate for hypertension , diabetes , dyslipidemia , and current smoking was 30% , 5% , 62% , and 31% , respectively , with no significant differences observed between genders . prevalence of ms was 38% ( 57 subjects ) in the total sample with no significant differences between genders : 42% and 32% in men and women , respectively . men had significantly higher waist circumference ( p < 0.01 ) , systolic and diastolic bp ( p < 0.0001 ) , and creatinine levels ( p < 0.0001 ) than women . mean level of hscrp was 2.1 2.2 mg / l , and there were no significant differences between men and women ( table 2 ) . likewise , there were no significant differences in the prevalence of high levels of hscrp ( defined as > 2 mg / l ) between genders ( 39% in men and 36% in women ) . of note , hscrp was significantly and directly correlated with bmi , waist circumference , blood glucose , systolic bp , non - hdl - c , and fibrinogen , and it was inversely and significantly correlated with hdl - c ( table 3 ) . mean level of lp - pla2 activity was 185 48 nmol / ml / min in the total sample with significant differences between men and women : 201 nmol / ml / min and 166 nmol / ml / min ( p < 0.0001 ) , respectively ( table 2 ) . lp - pla2 was significantly and directly correlated with bmi , waist circumference , diastolic bp , ldl - c , non - hdl - c , and plasma creatinine ; hdl - c was inversely and significantly correlated with lp - pla2 . no significant correlations were found between lp - pla2 and blood glucose , systolic bp , hscrp , and fibrinogen ( table 3 ) . no differences were found between lp - pla2 activity of active smokers and nonsmokers . as shown in table 4 , both lp - pla2 and hscrp were significantly higher in subjects with ms than in those without ms . in subjects with ms , lp - pla2 was 198 45 nmol / ml / min versus 180 48 nmol / ml / min in subjects without ms ( p = 0.03 ) . mg / l in subjects with ms versus 2.2 3.2 mg / dl in those without ms ( p = 0.0001 ) . both biomarkers significantly increased with the number of ms components : lp - pla2 levels increased from 163 nmol / ml / min to 198 nmol / ml / min ( p < 0.01 ) and hscrp increased from 1.5 mg / l to 4.1 mg / l ( p = 0.04 ) when subjects with 0 and 3 components of ms were compared ( figures 1 and 2 ) . the results of the logistic regression analysis for determining ms , which included both biomarkers adjusted for age and gender , were as follows : odds ratio ( or ) for lppla2 : 1.02 ( confidence interval [ ci ] : 1.001.02 , p = 0.03 ) and or for hscrp : 2.5 ( ci : 1.653.80 , p < 0.0001 ) . figure 3 shows the roc curves for both biomarkers adjusted for age and gender : lp - pla2 , c value = 0.66 [ 0.570.74 ] and hscrp , c value = 0.73 [ 0.650.81 ] . according to this analysis , although hscrp shows a better area under the curve , from a statistical point of view , it can not be shown that it is better than lp - pla2 , as the cis of both biomarkers overlap . in this study , we have demonstrated in a population without atherosclerotic disease that levels of lp - pla2 activity and hscrp are elevated in subjects with ms . both biomarkers significantly increased with the number of metabolic risk factors , and both were shown to be independent and statistically significant predictors of ms . the ms corresponds to a clustering of cv and metabolic risk factors and inflammation . in our country , ms prevalence is approximately 30% , and it is occurring at younger ages , probably due to the overweight , obesity , and insulin resistance epidemic . although , the pathophysiologic mechanism involved in ms is not entirely clear , it has been shown that it confers an increased risk for diabetes and atherosclerotic cv disease [ 9 , 15 ] . recent studies have reported that the presence of ms and inflammation ( determined by hscrp ) confers more cv risk than the presence of ms alone [ 16 , 17 ] . high sensitivity c - reactive protein is also a predictor for the development of ms and diabetes . in our country , we have previously reported that subjects with ms and elevated hscrp have more subclinical atherosclerosis and carotid atherosclerotic plaques . therefore , many inflammatory markers related to ms have been investigated in the search of a possible explanation for the elevated cv risk associated with it . the most studied biomarker is hscrp ; however , there are new biomarkers associated with atherosclerotic disease that may be more specific to vascular inflammation . among them is lp - pla2 , an enzyme that acts on oxidized ldl and produces strong inflammatory and oxidative mediators of the intima . epidemiologic and clinical studies have shown an association between atherosclerotic disease and lp - pla2 concentration , in a similar way as reported with hscrp [ 911 , 13 ] . in this study , we have demonstrated that both biomarkers , lp - pla2 and hscrp , predicted ms with a similar power . regarding the association of hscrp with ms , this study confirms the available evidence showing that levels of hscrp are increased in subjects with ms [ 16 , 17 , 20 ] . our results also support the association between hscrp and overall / visceral obesity , hdl - c , non - hdl - c , and glycemia . similar to the findings of ballantyne and colleagues , we demonstrated that hscrp is not associated with lp - pla2 , suggesting that their inflammation pathways are different from a physiologic point of view . conversely , hscrp was related to fibrinogen , also an acute phase protein that has a similar mechanism of production by the liver . hscrp is produced by the liver , primarily by the action of interleukin-6 , a cytokine that increases in the conditions of visceral obesity and insulin resistance . based on this evidence , although hscrp is an important inflammatory marker , it is unspecific . activity levels of lp - pla2 were also significantly higher in subjects with ms in our study . these findings are similar to those from the bruneck study in 2009 . in that study , tsimikas et al . found significant associations between lp - pla2 and ldl - c , non - hdl - c , hdl - c , and homeostasis model assessment insulin resistance ( homa - ir ) , whereas correlations with bmi and waist circumference were weaker . our results are identical to those reported by these authors regarding lipid factors and obesity . however , we did not find any direct correlation with blood glucose , and we did not measure insulin resistance . furthermore , lp - pla2 was not correlated to bp and smoking in our study , which was seen in the bruneck study . lastly , we found no association between lp - pla2 and fibrinogen , which confirms that lp - pla2 inflammatory pathway is not through acute phase reaction . it is important to analyze the relationship between lp - pla2 and ldl - c and non - hdl - c ( atherogenic cholesterol ) . after adjusting for gender and age , our population had an average ldl - c level of 128 mg / dl , which suggests that , even at moderate levels of ldl - c , subjects with ms had elevated activity of this enzyme . this result could be because many subjects with ms have primarily small and dense ldl molecules , which are prone to oxidation and therefore they are substrate for lp - pla2 . the inflammation produced by lp - pla2 is primarily in the vascular level , because lysophosphatidylcholine and oxidized fatty acids , generated after lp - pla2 action over oxidized ldl , induce adhesion molecules and other cytokines in the same vascular wall . thus , it can be hypothesized that , unlike hscrp ( which occurs as a result of inflammation caused by other cytokines from the visceral fat ) , lp - pla2 is directly involved in the inflammatory process , and , subsequently , the atherogenic process of the plaque . thus , lp - pla2 could be a more specific marker of atherosclerotic events in patients with ms . confirmation of this hypothesis must be demonstrated by studies of cv events in patients with ms , who have these biomarkers measured , and with findings adjusted for all the risk factors associated with hscrp and lp - pla2 . in this regard , it should be noted that a meta - analysis evaluating the clinical use of hscrp has shown that this biomarker adds little in the prognosis of cv risk , when adjusting by the risk factors involved in ms . conversely , the fact that lp - pla2 was weakly related to these metabolic factors should preserve its power as a biomarker . the reasons for not having completely cleared lp - pla2 prognosis utility are based primarily on the different techniques used for its measurement , which makes it impossible to compare the results of different studies . in this study , we did not expect to find the same power for hscrp and lp - pla2 as predictors of ms . we expected hscrp to be a better predictor , given its close association with metabolic risk factors included in ms . however , from a statistical point of view ( as demonstrated by the roc curves ) , both had a similar level of prediction . these results suggest that lp - pla2 is related to ms through other pathways that are not fully known and most likely not associated with the metabolic risk factors . we know that lp - pla2 is linked through oxidized ldl , which is elevated in subjects with ms , although it can not be perceived due to the moderate levels of ldl - c observed in the subjects of our study . oxidized ldl , and , for measuring oxidized ldl in the clinic , it required more expensive and sophisticated methods not frequently used in clinical practice . since ms is highly prevalent in our population , our results could provide knowledge about nontraditional risk factors that could help to identify which patients with ms must be treated earlier and more aggressively . this is important , because currently there is a pharmacologic inhibitor of lp - pla2 activity , darapladib , which is being investigated in subjects with atherosclerotic disease . in addition , another study is currently investigating the use of a monoclonal antibody against interleukin-1 for the intervention against high hscrp . if these studies have positive results , the measurement and targeting of these biomarkers might become important components of clinical management of patients . finally , it must be noted that we found no studies in literature that compare lp - pla2 and hscrp as predictors of ms in the general population . our study has some limitations : ( 1 ) it is a cross - sectional study , and hence causality inferences can not be made ; ( 2 ) it was done in a small , nonrandom sample of subjects ; ( 3 ) we did not include homa - ir , which could explain the relationship of insulin resistance with ms and inflammatory markers ; ( 4 ) finally , we only determined lp - pla2 activity instead of mass . this decision was subject to the availability of the laboratory determination in our country . however , currently , it is the most used technique , and it has shown the best correlation with ms . the strengths of the study include the following : ( 1 ) the recruitment of subjects was performed prospectively ; ( 2 ) subjects with lipid - lowering therapy and women with hormone replacement therapy were excluded , among others ; ( 3 ) this study has validated the measurement of lp - pla2 in our country . in conclusion , prospective studies must confirm which of the markers or if both markers are causally related to the atherosclerotic consequences observed in subjects with ms .

high sensitivity c - reactive protein ( hscrp ) is a marker of metabolic syndrome ( ms ) and cardiovascular ( cv ) disease . lipoprotein - associated phospholipase a2 ( lp - pla2 ) also predicts cv disease . there are no reports comparing these markers as predictors of ms . methods . cross - sectional study comparing lp - pla2 and hscrp as predictors of ms in asymptomatic subjects was carried out ; 152 subjects without known atherosclerosis participated . data were collected on demographics , cardiovascular risk factors , anthropometric and biochemical measurements , and hscrp and lp - pla2 activity levels . a logistic regression analysis was performed with each biomarker and receiver operating characteristic ( roc ) curves were constructed for ms . results . mean age was 46 11 years , and 38% of the subjects had ms . mean lp - pla2 activity was 185 48 nmol / ml / min , and mean hscrp was 2.1 2.2 mg / l . subjects with ms had significantly higher levels of lp - pla2 ( p = 0.03 ) and hscrp ( p < 0.0001 ) than those without ms . roc curves showed that both markers predicted ms . conclusion . lp - pla2 and hscrp are elevated in subjects with ms . both biomarkers were independent and significant predictors for ms , emphasizing the role of inflammation in ms . further research is necessary to determine if inflammation predicts a higher risk for cv events in ms subjects .

peripheral arterial aneurysms are rare , and among these , deep femoral arterial ( dfa ) aneurysms are extremely rare [ 13 ] . dfa aneurysms are not associated with any characteristic symptoms , and their early diagnosis is difficult . these aneurysms have a high rate of rupture , and surgery plays an important role in their treatment . however , standard methods have not yet been established because of the rarity of dfa aneurysms [ 36 ] . we describe a case of dfa aneurysm in a patient who was successfully treated with aneurysmectomy and revascularization with an artificial blood vessel graft . a 58-year - old man presented with paralysis and pain in the left leg , and a mass was found in his thigh . because of the growth of the mass and the worsening of his symptoms , the patient visited a neighboring hospital . physical examination revealed a pulsatile mass with a diameter of 8 cm in the left thigh . multidetector computed tomography performed in our hospital revealed a large dfa aneurysm with an intraluminal thrombus ( fig . 1 ) . surgical intervention was planned because of the large size of the aneurysm , the high risk of perforation and the worsening symptoms . figure 1:multidetector computed tomography revealed a large dfa aneurysm ( arrows show aneurysm ) . the common femoral artery , superficial femoral artery , superficial femoral vein and deep femoral vein were recognized and preserved ( fig . 2a ) . after clamping the dfa proximal and distal to the aneurysm , we opened the sac and found a massive thrombus . aneurysmectomy and revascularization of the distal dfa with an artificial blood vessel graft were performed ( fig . pathological examination of the specimen revealed atherosclerosis in the vascular wall and confirmed that it was a true aneurysm . figure 2:(a ) the common femoral artery , superficial femoral artery , superficial femoral vein and deep femoral vein were recognized and preserved . ( b ) aneurysmectomy and revascularization of the distal dfa with an artificial blood vessel graft were performed . ( a ) the common femoral artery , superficial femoral artery , superficial femoral vein and deep femoral vein were recognized and preserved . ( b ) aneurysmectomy and revascularization of the distal dfa with an artificial blood vessel graft were performed . following surgery , a follow - up multidetector computed tomography scan showed good blood flow in the graft , superficial femoral artery and popliteal artery ( fig . figure 3:a follow - up multidetector computed tomography scan showed good blood flow in the graft , superficial femoral artery and popliteal artery . a follow - up multidetector computed tomography scan showed good blood flow in the graft , superficial femoral artery and popliteal artery . although many dfa aneurysms have been associated with other peripheral aneurysms or peripheral occlusive diseases , there was no evidence of aneurysms or occlusive lesions in the other arteries of our patient [ 4 , 7 , 8 ] . therefore , we consider our case of an isolated dfa aneurysm to be extremely rare . there are no characteristic symptoms of dfa aneurysms in the early stage , and their diagnosis is difficult . they are often found accidentally or after their enlargement , when symptoms include pulsating groin swelling , paralysis or pain in the leg , which were the symptoms that our patient experienced . enlarged dfa aneurysms must be treated because of the high risk of rupture , which may cause severe complications , including hemorrhagic shock , ischemia of the foot , thigh embolization , thrombophlebitis and sciatic or femoral neuropraxia [ 1 , 5 , 9 , 10 ] . several treatment options , including simple ligation , revascularization of the distal dfa and bypass grafting to the popliteal artery , exist for dfa aneurysms ; however , standard methods have not been established because of the rarity of dfa aneurysms . in our case , we performed an aneurysmectomy and revascularization to the distal dfa with an artificial blood vessel as the graft . revascularization was performed to avoid thigh ischemia and preserve the collateral circulation if ipsilateral peripheral occlusive disease occurred . we believe that revascularization to the distal dfa should be done whenever it is technically feasible . in previous reports , if the patient must undergo operations such as coronary artery bypass grafting or other peripheral arterial bypass grafting , the saphenous veins will be required for those surgeries . therefore , we did not use the saphenous vein as the graft . in this case , we consider the surgery a success because the patient recovered well from the surgery without complications , his symptoms improved and the blood flow in the leg was preserved . in conclusion , dfa aneurysms are extremely rare . surgery plays an important part in the treatment of this disease , but standard methods have not been established because of its rarity . in our patient , we successfully performed an aneurysmectomy and revascularization to the distal dfa with an artificial blood vessel graft .

a 58-year - old man presented with paralysis and pain in the left leg , and a mass was found in his thigh . because of the growth of the mass and the worsening of his symptoms , the patient visited our hospital . multidetector computed tomography revealed a large deep femoral arterial ( dfa ) aneurysm . surgical intervention was planned because of the large size of the aneurysm , the high risk of perforation and the worsening symptoms . aneurysmectomy and revascularization of the distal dfa with an artificial blood vessel graft were performed . dfa aneurysms are extremely rare . these aneurysms have a high rate of rupture , and surgery plays an important role in their treatment . however , standard methods have not yet been established because of their rarity of dfa aneurysm . we describe a case of dfa aneurysm in a patient who was successfully treated with aneurysmectomy and revascularization with an artificial blood vessel graft .

the endothelial cell dysfunction is observed in myopia and in contact lens wearers [ 2 , 3 ] . the decreasing number of endothelial cells can also be a result of a surgical injury related to the opening of the anterior chamber . many studies have shown that even minor changes in the morphology of the endothelial cells may manifest in the disturbances in the tightness of the endothelial barrier . it has been demonstrated that human corneal endothelial cells have mitotic ability in vitro , but in vivo they do not exit the cell cycle but are arrested in g1 phase . loss of cells is compensated through the expanding and spreading of cells , which over time results in a lack of tightness and corneal oedema . prevention of the corneal endothelium dysfunction , its early detection and immediate treatment are therefore crucial , especially if the problem concerns young patients . noncontact specular microscopy , which evaluates endothelial morphology quickly and easily , can be especially useful with children . there are many reports concerning the analysis of the corneal endothelium in adults with type 1 and type 2 diabetes [ 69 ] ; however , there are no publications concerning the analysis of the cornea in juvenile patients with this disease . the aim of our study was to compare the endothelial cell density and central corneal thickness in diabetic and nondiabetic patients and to evaluate the local and systemic factors which may affect the corneal endothelium in this group . the current study was performed at the department of pediatric ophthalmology and strabismus , medical university of bialystok , poland . for the purpose of this study we examined 123 eyes of 123 patients with type 1 diabetes ( 60 boys and 63 girls ) . the age of diabetic group was 719 years ( mean : 15.34 3.06 years ) . the mean duration of diabetes was 8.02 3.9 years and ranged from 8 months to 16 years . all the diabetic patients were divided into three groups according to diabetes duration : less than 5 years ( 38 patients ) , from 5 to 10 years ( 42 patients ) , and longer than 10 years ( 43 patients ) . 48 persons had bad metabolic control , 37 had moderate metabolic control , and 38 had good metabolic control . at the time of examination , the mean value of hba1c in diabetic patients was 8.02 3.9% ( range 5.5%3.2% ) . ophthalmologic examination in diabetic patients included slit - lamp examination and binocular indirect ophthalmoscopy fundus examination . as controls , the mean age of the control group was 918 years ( mean : 14.58 2.01 years ) . none of the examined patients had history of ocular disease , topical ocular medications , or contact lens wear . data from the right eye of each patient was used in this study . the corneal endothelium density ( ecd ) and central corneal thickness ( cct ) in its central part the aim of this study was to compare ecd and cct in diabetic and nondiabetic patients and to evaluate a correlation between endothelial cell density , central corneal thickness , and local factors ( presence of retinopathy ) and systemic factors ( age , sex , diabetes duration , the level of hba1c , and plasma creatinine level ) . the mann - whitney test for ecd and t - test for cct were used to compare medians in diabetic and control group . the kruskal - wallis test and anova test were used to compare the values of medians in different states of metabolic control . multiple regression analysis was used to analyze the influence of the set of variables for ecd and cct . in the model four continuous variables ( age , diabetes duration , plasma creatinine level , and hba1c ) and two dummy variables ( sex and presence of retinopathy ) were used . in our study the starting model and the final model received by the use of backward elimination of variables are presented . the value of multiple r , r2 , and p value for the global test that r2 is equal to 0 . the normal distribution of the residuals was verified by the use of pearson 's chi square test . the mean endothelial cell density in patients with diabetes was 2435.55 443.43 cells / mm and was significantly lower than in the control group ( 2970.75 270.1 cells / mm ) ( p = 0.0001 ; mann - whitney test ) . ecd values in both groups are presented in figure 1 the mean cct was 0.55 0.03 mm in diabetic group versus 0.53 0.033 mm in control group ( p < 0.0001 ; t - test ) . cct values in both groups are presented in figure 2 in order to determine the systemic and local factors affecting the corneal endothelium in diabetic patients we evaluated the correlation of ecd and cct with the following variables : the patients ' age and sex , duration of diabetes , hba1c level , plasma creatinine level , and presence of diabetic retinopathy . the age of diabetic group was 719 years ( mean : 15.34 3.06 years ) . there was no correlation between ecd and age of diabetic patients ( p value = 0.111 ) . the mean ecd was 2446 488.3 cells / mm in diabetic boys and 2424 394.7 cells / mm in diabetic girls , and these differences were not statistically significant ( p value = 0.99 ) . 38 diabetic patients had good metabolic control ( with hba1c less than 7% ) , 37 subjects had moderate metabolic control ( hba1c from 7% to 8% ) , and 48 persons had poor metabolic control ( hba1c above 8% ) . we did not observed significant differences in ecd in relation to metabolic control ( p value = 0.54 ) . the mean duration of diabetes was 8.02 3.9 years ( ranged from 8 months to 16 years ) . in 38 patients duration of diabetes was shorter than 5 years , 42 persons suffered from diabetes from 5 to 10 years , and 43 persons had diabetes longer than 10 years . the mean ecd values in relation to sex , metabolic control , and duration of diabetes are presented in table 1 . the mean hba1c in diabetic patients was 8.26 1.7% and ranged from 5.5% to 13.2% . there was no significant correlation between ecd and hba1c level ( p value = 0.378 ) . there was no correlation between ecd and plasma creatinine level ( p value = 0.650 ) . we did not observe any correlation between ecd and the presence of diabetic retinopathy ( p value = 0.293 ) . the correlation between ecd and duration of diabetes was statistically significant ( p value < 0.0001 ) ( table 2 ) . the mean cct value in patients with duration of diabetes up to 5 years was 0.539 0.027 mm , with duration of diabetes from 5 to 10 years was 0.551 0.03 mm , and with duration of diabetes over 10 years was 0.558 0.03 mm , and the differences were statistically significant ( p value = 0.0144 ; anova test ) . multiple regression analysis for cct indicated that only duration of diabetes was significantly related to cct . the observed disorders include increased fragility and damage of the corneal endothelium and recurring erosions and increased sensitivity to injuries . experimental research discovered abnormal basement membrane of the endothelium , a decreased number of hemidesmosomes , and a prolonged healing of the cornea and its decreased sensitivity . many studies confirmed that diabetes causes abnormalities in morphology and functioning of corneal endothelium cells . functional disturbances may lead to increased autofluorescence of the cornea and its increased penetrability [ 8 , 9 ] . morphological changes may result in a high variability factor of the endothelial cell surface and decreased percentage of hexagonal cells in corneas in patients with diabetes , using contact specular microscope , when compared to healthy patients [ 811 ] . although morphology of the endothelial cells is interesting in diabetic patients , the limitations of topcon 2000 are obvious ( pleomorphism and polymegathism could not be assessed , and hexagonality was also not available ) . the existing research data encouraged us to examine the corneas in children and adolescents with this chronic illness . we have established that the mean density of corneal endothelium cells in patients with diabetes was reduced in comparison with the control group by 18% . similar results were obtained by roszkowska et al . , who after examining 75 adults with type 1 and type 2 diabetes noted the ecd decreased by 5% in type 2 diabetes , and by 11% in type 1 when compared with healthy persons . lower endothelial cell counts were also proved by sudhir et al . , who examined 1191 adult patients with type 2 diabetes . the mean ecd in their study was 2550 326 cells / mm versus 2634 256 cells / mm in the nondiabetic control group . in our diabetic group ecd was very similar ( 2435.55 443.43 cells / mm ) but ecd in our control group was higher ( 2970.75 270.1 cells / mm ) . we have to remember that our diabetic and non - diabetic subjects were young : the mean age in diabetic group was 15.34 3.06 years versus 14.58 2.01 years in control group . , who did not demonstrate significant changes in mean density of corneal endothelium cells in diabetic subjects , but they only examined patients with type 2 diabetes . we also analyzed the influence of local and systemic factors affecting the density of the corneal endothelium in children and adolescents with type 1 diabetes . in contrast , inoue et al . did not find any ocular and systemic factors that affect the damaging of endothelium in diabetic patients , but it was type 2 diabetes . the authors claim that although they did not show the influence of systemic and ophthalmic factors on the morphology of the corneal endothelium , due to the chronicity of the disease , the relation may not occur during the examination but it may appear some years later . larsson et al . noticed several changes in the endothelium in elderly with diabetes , but the observed anomalies may have been additionally caused by the process of senescence . similarly to our study , lee et al . showed that ecd in adult diabetic patients was significantly lower for diabetes with over 10 years of duration than for diabetes of under 10 years . like other authors , we did not prove the influence of sex , the state of metabolic control , and the presence of diabetic retinopathy on the corneal endothelium [ 11 , 16 , 17 ] . did not detect any relation between the level of glycosylated haemoglobin and the condition of the corneal endothelium . the patients they have examined had similar values of hba1c ( mean 8.2% ) to the mean hba1c level of our patients ( 8.01% ) . they did not detect any influence of diabetes duration ( in contrast to our study ) and the presence of diabetic retinopathy ( similarly to our results ) on the corneal endothelium . in the study of mdis et al . , the hba1c level in adult patients with type 1 diabetes mellitus was inversely correlated with the ecd , but they did not prove such correlation in patients with type 2 diabetes mellitus . the authors concluded that type 1 diabetic corneas are more susceptible to environmental changes than type 2 diabetic corneas . we were unable to demonstrate any correlation between ecd and the presence of diabetic retinopathy . while examining 64 adult patients with diabetes mellitus observed that the severity of diabetic retinopathy was correlated with endothelial cell density , but these correlations were low and the corneal changes were not correlated with glycemic control . inoue et al . reported that the presence of retinopathy ( proliferative also ) as well as laser coagulation in medical history did not affect the density of corneal endothelium cells . recently , the role of growth factors , crp , proinflammatory cytokines , or level of lipids has been emphasised in the pathogenesis of diabetic retinopathy in children [ 20 , 21 ] . their significantly higher blood serum levels have noxious influence on the tiny blood vessels in the retina . specificity of the cornea first of all lack of blood vessels may be one of the reasons , that in many publications concerning with the corneal endothelium in diabetic adults , the analysis of the influence of these factors was not performed [ 11 , 14 , 1719 , 22 ] . shows that the corneal endothelium is a tissue undergoing constant metabolic stress . in normal endothelial cells , circumferential bands of f - actin ( a major component of the cellular cytoskeleton ) help to maintain the regular and functionally efficient , hexagonal shape . kim et al . demonstrated that the corneas of diabetic individuals showed marked irregular f - actin fibers crossing the endothelial cell cytoplasm . they suggested , that these abnormal patterns of f - actin may be the result of constant stress in cell volume regulation in the corneas of diabetic patients . kleinzeller and ziyadeh showed that dissociation of f - actin fibers either chemically or osmotically caused massive cellular swelling . they presumed that this abnormal collocation of f - actin in the endothelium of diabetics may contribute to altered morphology and , in their opinion , the mechanism may be related to sorbitol accumulation within these cells . fujishima and tsubota claim that in molecular pathogenesis of corneal changes significant importance may be given to aldose reductase , the first enzyme of the sorbitol pathway . aldose reductase has been demonstrated immunohistochemically in the corneal endothelium , and the osmotic stress that occurs secondary to sorbitol accumulation could lead to altered endothelial morphology and cell loss . reported that alterations in endothelial morphology resolve within 3 months after the onset of topical aldose reductase inhibitor treatment . the accumulation of advanced glycation end products ( ages ) in the epithelial basement membrane or in descemet 's membrane may play a role in the disorders of diabetic cornea . showed that ages formation on fibronectin and laminin attenuated the attachment and spreading of the corneal endothelial cells . they concluded , that ages formation in descemet 's membrane may be responsible for the corneal endothelial abnormalities in diabetic patients . the next mechanism could decrease na / k - atpase activity , which influences the endothelial pump action and induces the dysfunction of the corneal endothelial cell layer [ 18 , 29 ] . thickness of the cornea indirectly informs about the functioning of the endothelial layer , which plays role as pump , which is responsible for active dehydration of the cornea and also has a barrier function . in our study possible explanations for increased corneal thickness in diabetic patients include ( besides inhibition of the corneal endothelial pump ) an increased endothelial permeability , which result from the metabolic effects of diabetes . another reason could be the increased stromal swelling pressure due to the accumulation of sorbitol or from the glycosylation of corneal collagen . evaluating the condition of the corneal endothelium is important since one of the most frequent reasons of corneal endothelium cells loss is cataract surgery , and cataract , besides diabetic retinopathy , is one of the most common ophthalmic complications of diabetes . the research done by mathew et al . shows that the removal of cataract is exceptionally traumatizing for the endothelium in eyes of diabetic patients . the diabetic endothelium was found to be under greater metabolic stress and had less functional reserve after manual small incision cataract surgery . it can be assumed that for children suffering from diabetes , an eventual development of cataract in the future and the necessity for its surgery may be a factor that significantly increases the risk of dysfunction of corneal endothelium cells . shenoy concluded that evaluation of corneal endothelium in diabetic patients should be part of the protocol for eye care of diabetic patients . the results of this study may support the theory of lower endothelial cell density and thicker cornea in children and adolescents with type 1 diabetes mellitus . duration of diabetes is the factor that affects ecd and cct and observed changes could predispose to corneal dysfunction in the future .

purpose . to evaluate the systemic and local factors that contribute to the damage of endothelial cells in diabetic patients and to compare the endothelial structure of the cornea in diabetic and nondiabetic patients . materials and methods . the endothelial cell density ( ecd ) and central corneal thickness ( cct ) were investigated in 123 eyes of type 1 diabetic patients and in 124 eyes of nondiabetic patients . the mean diabetic patients age was 15.34 3.06 years versus 14.58 2.01 years in the control group . the mean duration of diabetes was 8.02 3.9 years . the corneal endothelium was imaged by the topcon sp-2000p . results . the mean ecd in diabetic eyes was 2435.55 443.43 cells / mm2 and was significantly lower than in control group ( 2970.75 270.1 cells / mm2 ) . the mean cct was 0.55 0.03 mm in diabetic group versus 0.53 0.033 mm in control group . ecd and cct significantly correlated only with duration of diabetes . there was no correlation between ecd and cct and patient age , sex , hba1c level , and plasma creatinine level . conclusions . ecd is decreased and cct is increased in children and adolescents with diabetes mellitus . duration of diabetes is the factor that affects ecd and cct .

the history of total knee arthroplasty ( tka ) designs demonstrates attempts at implants that more closely match patient anatomy for improved treatment results . traditional total knee implant systems were designed to provide components of a wide range of sizes to achieve a proper fit in almost all patients . however , selection of a femoral component of correct size for a particular patient can still be challenging not only because it is common to have a distal femoral anteroposterior ( ap ) dimension going in the middle of usual 3 - 4 mm gap between each sizes of traditional femoral components but also because medial lateral ( ml ) overhang is sometimes encountered even with a traditional femoral component with a precisely matching ap dimension . in sizing of a femoral component , the ap dimension is important to maintain the flexion - extension gap and optimal tension in the extensor mechanism , whereas the ml dimension determines adequate coverage of the resected bone surface , allowing more even stress distribution and smooth tracking of the patellofemoral joint . although there is no evidence on the clinical effect of underhang and overhang of implants , theoretical assumption is possible . if a component is too small ( underhang ) , there will be higher contact stresses on the reduced bone - implant interface , increasing the risk of subsidence and loosening . conversely , if a component is too large ( overhang ) , it may impinge on the surrounding capsular tissues and ligaments , causing pain and limiting the range of motion ( rom ) of the joint . anatomic variations between male and female are well - documented in several anatomic and radiographic studies1,2,3,4,5 ) . in general , females tend to have smaller distal femora and different shapes with a narrower ml dimension for a given ap dimension when compared with males1 ) . in addition , females are thought to have an increased quardriceps angle and a less prominent anterior femoral condyle than males even though controversy still remains6,7 ) . the recent introduction of a gender - specific implant ( gender solutions nexgen high - flex ; zimmer inc . , warsaw , in ) designed specifically for females started the debate that has focused on the effect of gender on the results of tka using traditional implant systems . the gender solutions implant was designed to better accommodate the anatomic differences noted in females with a narrower ml dimension for any given ap dimension . besides , the angle of trochlear groove was increased and the anterior flange thickness and width were reduced to more closely reproduce the native female anatomy . with this background , we designed this present study in order to find out if gender - specific implants reduce the incidence of overhang during operation and there is any clinical or radiological advantage of gender specific implants over traditional implants . we hypothesized : 1 ) the incidence of femoral component overhang would be reduced with the use of gender - specific femoral components and 2 ) clinical and radiological results would show no difference between two types of implants in a short - term follow - up because no clinical disadvantage of overhang has been reported so far . between may 2007 and january 2008 , 136 primary tkas were undertaken in a consecutive series of 92 patients . there were 7 males ( 8 knees ) and 85 females ( 128 knees ) . the criteria for inclusion into the study consisted of female gender , a primary diagnosis of osteoarthritis and a minimum 3-year follow - up . patients with severe bone loss requiring structural bone grafting or metal augmentation were excluded from the present study . their mean age at the time of the operation was 67.9 years ( range , 52 to 80 years ) and their mean body mass index ( bmi ) was 26.8 kg / m ( range , 20.2 to 33.3 kg / m ) . the mean follow - up duration was 41.3 months ( range , 36 to 50 months ) . there were 28 unilateral procedures and 38 bilateral procedures that were performed two weeks apart . we compared the clinical and radiological outcomes of tka using a gender - specific implant ( gender solutions nexgen high - flex ) and a traditional implant ( nexgen legacy posterior stabilized flex [ lps - flex ] , zimmer inc . ) at 3 years after surgery . we also analyzed intraoperative measurements , including the overhang of femoral component and patella tracking , after tka using two different trial components . all the operations were performed by the senior author ( bin ) using the same technique . after eversion of the patella , resection of the femoral condyles was performed to remove a thickness of bone equal to that of the femoral component to be inserted . the femoral cutting block was placed in 3 to 5 of external rotation to the posterior condyles , and this was ascertained by the ap trochlear sulcus . the valgus angle of femoral resection was made using an intramedullary guide , and ap cutting was performed using an anterior referencing system . femoral component sizing was based on the ap dimension of the femur , which was measured with a femoral ap sizer during operation . if a measurement of the ap dimension fell between two sizes , the smaller size femoral component was chosen . using an intramedullary tibial guide , a 10 mm - thick tibial bone resection was performed to obtain a surface that was perpendicular to the shaft of the tibia in the coronal plane , with a 7 posterior slope in the sagittal plane . two different trial femoral components ( gender solutions nexgen high - flex prosthesis and nexgen lps - flex prosthesis ) of the same size were inserted sequentially after femoral and tibial bone cuts . at this moment , overhang of each trial femoral component was examined medially and laterally at three cut surfaces ( anterior , anterior chamfer and distal cut surfaces ) . patellar tracking was also checked with each trial component using the ' no thumb ' test . the rom in which the patella showed congruent tracking was recorded with each trial femoral component and designated as congruent patella tracking rom . based on the coverage of cut surface and patellar tracking , appropriate type of permanent femoral implant was chosen by the surgeon . the patients who received a gender - specific implant ( gender solutions ) were assigned to group i and those with a traditional implant ( nexgen lps - flex ) were to group ii . two days after surgery , drain was removed and continuous passive motion and tolerable weight bearing were started . the entire data were collected prospectively from the beginning of the present study and were compared and analyzed retrospectively . this study was approved by the institutional review board at our hospital and informed consent was waived . presence of overhang at the three femoral cut surfaces and the congruent patella tracking rom of each prosthesis were assessed intraoperatively and compared between groups . postoperatively , the patients were reviewed at six weeks , three months , six months , and annually after surgery . clinical parameters including hospital for special surgery ( hss ) knee score8 ) , flexion contracture , maximal flexion , and complications were evaluated and compared preoperatively and at 3 years after surgery . flexion contracture and maximal flexion were measured using a manual goniometer with the arms aligned along the long axes of the femur and tibia on the lateral side of the knee joint . patients were told to straighten and bend their knee until they felt a slight degree of pain in supine position . all the data at follow - up examinations were recorded by an orthopedic surgeon and compiled by a research assistant who was not a part of the surgical team and had no information about the surgical and radiological findings . radiographic evaluation included standing ap , lateral , and merchant views of both knees at three months , six months , and annually after surgery , which were analyzed using the radiological evaluation system of the knee society9 ) to delineate radiolucency around the components . postoperative patellar tracking was assessed by measuring patella tilt angle and patella displacement as described by gomes et al.10 ) . all the radiographs were analyzed by two of the co - authors ( kim jm and kim sb ) who were blinded to patient information . between may 2007 and january 2008 , 136 primary tkas were undertaken in a consecutive series of 92 patients . there were 7 males ( 8 knees ) and 85 females ( 128 knees ) . the criteria for inclusion into the study consisted of female gender , a primary diagnosis of osteoarthritis and a minimum 3-year follow - up . patients with severe bone loss requiring structural bone grafting or metal augmentation were excluded from the present study . their mean age at the time of the operation was 67.9 years ( range , 52 to 80 years ) and their mean body mass index ( bmi ) was 26.8 kg / m ( range , 20.2 to 33.3 kg / m ) . the mean follow - up duration was 41.3 months ( range , 36 to 50 months ) . there were 28 unilateral procedures and 38 bilateral procedures that were performed two weeks apart . we compared the clinical and radiological outcomes of tka using a gender - specific implant ( gender solutions nexgen high - flex ) and a traditional implant ( nexgen legacy posterior stabilized flex [ lps - flex ] , zimmer inc . ) at 3 years after surgery . we also analyzed intraoperative measurements , including the overhang of femoral component and patella tracking , after tka using two different trial components . all the operations were performed by the senior author ( bin ) using the same technique . after eversion of the patella , resection of the femoral condyles was performed to remove a thickness of bone equal to that of the femoral component to be inserted . the femoral cutting block was placed in 3 to 5 of external rotation to the posterior condyles , and this was ascertained by the ap trochlear sulcus . the valgus angle of femoral resection was made using an intramedullary guide , and ap cutting was performed using an anterior referencing system . femoral component sizing was based on the ap dimension of the femur , which was measured with a femoral ap sizer during operation . if a measurement of the ap dimension fell between two sizes , the smaller size femoral component was chosen . using an intramedullary tibial guide , a 10 mm - thick tibial bone resection was performed to obtain a surface that was perpendicular to the shaft of the tibia in the coronal plane , with a 7 posterior slope in the sagittal plane . two different trial femoral components ( gender solutions nexgen high - flex prosthesis and nexgen lps - flex prosthesis ) of the same size were inserted sequentially after femoral and tibial bone cuts . at this moment , overhang of each trial femoral component was examined medially and laterally at three cut surfaces ( anterior , anterior chamfer and distal cut surfaces ) . patellar tracking was also checked with each trial component using the ' no thumb ' test . the rom in which the patella showed congruent tracking was recorded with each trial femoral component and designated as congruent patella tracking rom . based on the coverage of cut surface and patellar tracking , appropriate type of permanent femoral implant was chosen by the surgeon . the patients who received a gender - specific implant ( gender solutions ) were assigned to group i and those with a traditional implant ( nexgen lps - flex ) were to group ii . two days after surgery , drain was removed and continuous passive motion and tolerable weight bearing were started . the entire data were collected prospectively from the beginning of the present study and were compared and analyzed retrospectively . this study was approved by the institutional review board at our hospital and informed consent was waived . presence of overhang at the three femoral cut surfaces and the congruent patella tracking rom of each prosthesis were assessed intraoperatively and compared between groups . postoperatively , the patients were reviewed at six weeks , three months , six months , and annually after surgery . clinical parameters including hospital for special surgery ( hss ) knee score8 ) , flexion contracture , maximal flexion , and complications were evaluated and compared preoperatively and at 3 years after surgery . flexion contracture and maximal flexion were measured using a manual goniometer with the arms aligned along the long axes of the femur and tibia on the lateral side of the knee joint . patients were told to straighten and bend their knee until they felt a slight degree of pain in supine position . all the data at follow - up examinations were recorded by an orthopedic surgeon and compiled by a research assistant who was not a part of the surgical team and had no information about the surgical and radiological findings . radiographic evaluation included standing ap , lateral , and merchant views of both knees at three months , six months , and annually after surgery , which were analyzed using the radiological evaluation system of the knee society9 ) to delineate radiolucency around the components . postoperative patellar tracking was assessed by measuring patella tilt angle and patella displacement as described by gomes et al.10 ) . all the radiographs were analyzed by two of the co - authors ( kim jm and kim sb ) who were blinded to patient information . the incidence of overhang of the trial femoral component was significantly higher in the knees with the traditional component than those with the gender - specific trial component ( table 1 ) . of the 104 knees , overhang was observed at least in one area in 62 knees with the traditional trial component ( 59.6% ) compared to 26 knees ( 25.0% ) with the gender - specific trial component ( p<0.001 ) . among the six areas where overhang was examined , the lateral anterior cut surface was the area where overhang of the femoral component was most frequent regardless of the type of the trial component . distal medial area was the only exception that did not show statistically significant difference ( p=0.250 ) . sixty - two knees ( 59.6% ) that demonstrated overhang with traditional trial components were replaced with a gender - specific implant ( group i ) and the remaining 42 knees ( 40.4% ) that showed no overhang with traditional trial components were replaced with a traditional implant ( group ii ) . therefore , the incidence of overhang after final implantation was 26/62 knees ( 41.9% ) in group i and no overhang ( 0/42 knees ) was observed in group ii . as a result , 36 knees in group i were able to avoid overhang with use of the gender - specific implants instead of the traditional ones , whereas the remaining 26 knees in group i still showed overhang even with gender - specific implants . ten patients received a gender solutions prosthesis on one side and a lps - flex prosthesis on the other side . the difference of the congruent patella tracking rom between traditional and gender - specific trial components was not significant ( p>0.05 ) ( table 3 ) . hence , there was no case where a gender - specific implant was chosen for better patella tracking . the distribution of the femoral component size ranged from c to e and showed a tendency of selecting larger sizes in the knees with the gender - specific implant ( p=0.045 ) ( table 4 ) . clinical evaluation was performed in all patients including 10 patients who received different implants in bilateral tka . the mean flexion contracture , mean maximal flexion and mean rom improved significantly after operation in both groups . the mean hss score also improved after operation in both groups and the difference between two groups was not significant ( table 5 ) . lateral release was not performed in both groups . at the final follow - up , the mean patella tilt angle and mean patella displacement showed no significant difference between two groups ( table 6 ) . we observed no clinical complications in both groups , including delayed wound healing , deep infection or instability . no indications of component loosening and progressive osteolysis were noted in any zone on the postoperative radiographs . in the present study , the incidence of overhang was reduced by 34.6% in the gender - specific implant group in which overhang was inevitable during trial insertion of the traditional implant . however , clinical disadvantage of overhang was not demonstrated in the short - term follow - up . in 1996 , poilvache et al.4 ) indicated some female femora were narrower than average and the available femoral component with adequate ap dimension might be too broad in these patients . several years later , chin et al.1 ) investigated 200 consecutive knees ( 100 males and 100 females ) during primary tka and reported female femora showed a tendency to have a narrower ml dimension for any given ap dimension than male femora ( ap / ml ratio , 0.82 vs. 0.79 ) . shortly after this study , hitt et al.2 ) performed a well - designed multicenter study enrolling 337 knees ( 128 males and 209 females ) to compare six different contemporary prosthetic systems of traditional design . they reported that all the prostheses tended to present more or less ml overhang in female femora and the tendency was more evident in larger sizes . moreover , they introduced the concept of aspect ratio ( ml / ap ratio ) and demonstrated that the aspect ratio of female femora tended to decrease in larger knees while male femora showed relatively constant aspect ratio throughout the sizes . after this study , the aspect ratio became a standard parameter to describe distal femoral morphology . they also stated manufacturers should consider gender - specific implants or decrease the ml dimension to prevent overhang in females . stimulated by these reports , gender - specific implants were launched and marketed , which lead to the debate as to the relevance of this system6,7,11 ) . proponents of gender - specific implants presented accumulated data on sexual dimorphism of distal femur during decades and possible negative effects of component overhang6,11 ) . on the contrary , opponents pointed out that most recent studies had failed to eliminate or correct for other biases between both sexes such as average height or size of the studied femora and concluded that the unproven use of gender - specific implants may pose ethical or medico - legal dilemmas7 ) . in spite of this , there seems to be no disagreement at least on one point that the reported studies had neither directly demonstrated negative effects of overhang or overstuffing nor investigated actual merits of gender - specific knee arthroplasty with real implants . the paucity of data on negative effects of overstuffing has been taken for granted due to the difficulty of measuring the amount of overhang and controlling multiple possible prognostic factors other than overhang . however , no theoretical advantage was expected with regard to the overhang of femoral implant . thus , we decided to investigate the reduced incidence of overhang in tka using gender - specific implants in asian female patients even though the practical advantage of reduced overhang remained unclear . in this study , the incidence of ml overhang after tka using traditional and gender - specific trial components showed a significant difference ( 59.6% vs. 25.0% ) . in other words , 34.6% of the patients were able to avoid femoral component overhang because gender - specific implants were available . regarding the incidence of 25.0% of overhang even with the gender - specific trial component , these patients would have had greater overhang if it had not been for a gender - specific implant . with respect to the incidence of overhang in six separate areas , only distal medial cut surface did not show significant differences between two groups . this may be attributable to our efforts to lateralize femoral components during operation in order to improve patella tracking regardless of the type of implant . the distribution of selected sizes in each group showed a tendency of higher selection rate of a gender - specific implant in larger sizes ( p=0.045 ) . our data is consistent with those of hitt et al.2 ) that showed a higher aspect ratio in smaller knees and a proportionally lower ratio in larger knees . song et al.12 ) compared clinical and radiologic results of tka using gender - specific implants and conventional unisex design with a minimum follow - up of two years . they found no difference in clinical and radiologic outcomes between two groups and concluded gender - specific implants demonstrated no advantage over standard unisex design . guy et al.13 ) compared 50 males and 50 females to investigate the difference in the morphology of distal femur and the incidence of femoral component overhang . the mean aspect ratio was larger in females ( 1.02 ) than males ( 0.98 ) ( p=0.005 ) . standard implants in tka resulted in a significant increase of overhang in females in terms of both incidence and magnitude than did gender - specific implants . tanavalee et al.14 ) performed a prospective study that compared standard and gender - specific implants with an average follow - up of two years . the selection rate of gender - specific implants was significantly higher in females ( 60.8% ) than males ( 8.2% ) and significantly increased with increasing femoral size . the latter finding was exactly concordant with that in our present study and the selection rate of gender - specific implants in females ( 60.8% ) was very similar to that of our study ( 59.6% ) . they evaluated 122 knees in three subdivided groups ( 42 females with unisex implants , 41 males with unisex implants and 39 females with gender - specific implants ) and concluded that traditional unisex femoral components did not accurately match female anatomy . however , they were not able to demonstrate better radiographic results with gender - specific implants . therefore , the overall conclusion of the study was similar to ours in that traditional femoral components did not seem pertinent to reproduce female anatomy and gender - specific components did not provide a perfect solution , either . asymmetrical component sizes are known to be frequently used in bilateral tka , and the prevalence of which ranges from 6.7% by brown et al.16 ) to 31% by hitt et al.2 ) . in our study , 10 patients had a gender solutions prosthesis in one knee and a lps - flex prosthesis in the contralateral knee . this accounted for 10/38 ( 26.3% ) of all bilateral surgeries in the present study . not only the size but also the shape of both distal femora of a patient may be different from each other according to our data , which alerted us to the risk of the use of the same implant in bilateral tka . we believe the phenomenon of asymmetric component implantation probably originated from the awareness of the fact that anatomy is different from one side to the other . the clinical outcomes of two groups did not present significant differences in terms of motion , performance of patella and hss score in this study . because these parameters are not considered to deteriorate rapidly in short term , long - term observation seems to be necessary to elucidate differences , if any , in clinical outcomes between two groups . one of the limitations of this study is that it is not a true comparative study : implant selection relied on the extent of overhang of each trial femoral component and therefore the true independent clinical outcomes of each implant system were not thoroughly investigated . besides , the minimum 3-year follow - up period may be considered insufficient to determine the longevity of clinical outcomes . however , this study is based on solid evidence from prospective data , which can be regarded as a strength of the study . in the present study , the gender - specific femoral component explicitly reduced the incidence of overhang compared to the traditional femoral component . however , we could not find any correlation between this apparent advantage and more favorable clinical and radiologic outcomes . potential advantages of gender specific implants , such as avoidance of overstuffing in the patellofemoral joint , soft tissue irritation and anterior knee pain , were not identified in this study . there was significant difference in the incidence of overhang of femoral component between the gender - specific implant and the traditional implant . it was observed that 34.6% of the patients were able to avoid femoral component overhang with gender - specific implants . after three years of follow - up , there was no significant difference in clinical and radiological outcomes between two groups . potential advantages of gender specific implants , such as avoidance of overstuffing in the patellofemoral joint , soft tissue irritation and anterior knee pain , should be evaluated in a long - term follow - up study .

purposeto compare the incidence of overhang between two distinct femoral components and whether there is clinical and radiological benefit of gender - specific implants in short - term follow-up.materials and methodsone hundred and four knees in consecutive 66 female patients who underwent primary total knee arthroplasty due to primary osteoarthritis were included in this study . overhang was measured and recorded in every cut surface of femur with both gender - specific and traditional trial femoral components respectively in every patient . then , the knees were divided into two groups according to the type of the permanent femoral component they received . clinical and radiological outcomes were compared between 2 groups at minimum 3 years after operation.resultsmean follow - up duration was 41.3 months ( range , 36 to 50 months ) . sixty two knees ( 59.6% ) showed femoral overhang at least in one area with a traditional trial component , while 26 knees ( 25.0% ) did with a gender - specific trial component ( p<0.001 ) . in terms of range of motion , hospital for special surgery knee score , radiographic result , patella tilt angle and displacement , no significant difference was observed between two groups.conclusionsthe use of gender - specific implants substantially reduced the incidence of femoral overhang but did not demonstrate any clinical , functional or radiologic benefit in short - term follow - up .

congenital heart disease ( chd ) is one of the most common birth defects , with an incidence of nine out of every 1,000 live births.1,2 critical chd ( cchd ) is defined as cardiac lesions that require surgery or cardiac catheterization within the first month ( or within the first year by different definitions ) of life to prevent death or severe end - organ damage.2 although infant mortality has decreased over the past 3 decades for children with all forms of chd , many children are still diagnosed too late to avoid significant morbidity or death.35 delayed diagnosis of cchd is unfortunately all too common , with up to 25% of infants with these defects being missed in newborns when identification is based on clinical symptoms or signs of heart disease even in settings with routine prenatal sonograms.3,68 approximately 40% of these infants with missed diagnoses at birth present in cardiogenic shock at a medical facility and 5% are diagnosed at autopsy.4,5,9 studies in europe and the us have suggested that newborn screening with pulse oximetry testing prior to discharge from the nursery can decrease the number of missed diagnoses by 30%.2,10 in 2011 , pulse oximetry screening for cchd was added to the recommended uniform screening panel by the health and human services secretary.11 in the subsequent years , many states have implemented their own protocols to comply with this recommendation.1215 pulse oximetry has been the mainstay procedure for indirectly detecting hypoxemia in medically ill patients since the 1980s.16,17 the screening of cchd by pulse oximetry involves taking advantage of its ability to detect clinical and more importantly subclinical levels of hypoxemia that should raise suspicion for a cchd . however , pulse oximetry does not readily offer information about decreased stroke volume , which is another physiologic feature of several cchds that could be detectable during the neonatal transition . in this review , we highlight the relevant principles and limitations of pulse oximetry in the context of detecting cchd . the beer lambert bouguer law of physics describes the attenuation of light to the properties of materials through which the light is traveling.16 oxygenated blood absorbs red light at a wavelength of 660 nm , and deoxygenated blood absorbs light in the infrared spectrum at 940 nm.16 computation of oxygen saturation is achieved with the use of calibration algorithms , based on the amount of signals from nonpulsatile ( venous , capillary , bone , and skin ) and pulsatile arterial blood flow , in the red and infrared wavelengths mentioned earlier . a microprocessor removes the continuous signal from the nonpulsatile tissues and vessels , which leaves the pulsatile signal from the arteries to be displayed as a plethysmographic wave form on the pulse oximeter monitor.16 safe use of pulse oximetry requires knowledge of its limitations . there are multiple sources of potential artifacts , which are particularly relevant for neonates and can cause false readings . these include motion artifacts , poor perfusion and cold skin at the site of measurement , irregular rhythms , ambient light , phototherapy or electromagnetic interference , skin pigmentation and jaundice , inappropriate probe positioning ( penumbra effect ) , venous pulsation , intravenous dyes , and presence of abnormal hemoglobin molecules.1618 pulse oximeters recommended for screening for chd should report functional oxygen saturation ( referring to the hemoglobin that is capable of transporting oxygen ) , be motion tolerant , be validated for low perfusion states , and have a 2% root - mean - square accuracy.11 of note , a pulse oximeter s performance is optimized for oxygen saturations ( spo2 ) in the range of 80%100% . the development of the modern pulse oximeter is based on healthy , fit adult individuals who were exposed to different degrees of subambient oxygen with their spo2 being kept between 80% and 100% . therefore , any spo2 < 80% is extrapolated by a computer program.19,20 most new pulse oximeters are able to detect motion and label it as artifact or perform calculations quickly in a way that renders them motion tolerant . the pulse oximeter uses an algorithm to average readings over a period of time . for the most accurate measurement , the average is taken over a shorter period of time , which also increases the delay of the reading and decreases the speed of computation by the machine . for a pulse oximetry measurement to be accurate , the peripheral tissue needs an adequate pulse volume and pressure . in situations such as septic shock , where the extremities are cool and have low perfusion , the pulse oximeter may not reliably assess the oxygen saturation.19,20 competing light sources , such as other machinery , fluorescent lighting , and even cell phones , can overload the semiconductor sensor . in pediatrics , a phenomenon called the penumbra effect occurs.18 pulse oximeters may over - or under - read the spo2 in infants and children because of the small size of their fingers , or the other areas where the pulse oximeter probe is placed , and the different light paths for each wavelength through the peripheral tissue . specific probes for infants and children have been created and should be used to prevent these false measurements . finally , pulse oximetry should not be used in certain circumstances in which the hemoglobin molecule is not completely saturated with oxygen , such as with carbon monoxide poisoning or when methemoglobinemia occurs after administration of certain medications such as antimalarial drugs , nitrates , nitrites , or dyes such as methylene blue . in these instances , pulse oximetry is used in all aspects of newborn care , including resuscitation of newborns in the delivery room to routine monitoring in the operating room . a study by levesque et al21 in 2000 described the normal range of oxygen saturations in term newborns in the first days of life . these investigators evaluated normal oximetry values at sea level , from admission to the newborn nursery to discharge . they also evaluated variables such as sex , gestational age at birth , birth weight , mode of delivery , apgar scores , pre- or postductal site of measurement , and status of the infant at the time of measurement ( sleeping , quiet , and crying ) . pulse oximetry measurements were taken upon admission to the newborn nursery , at 24 hours of life , and at discharge . the overall oxygenation saturation was 97.2%1.6% ( 95% ci 97.1%97.2%).21 they compared the mean values at each time period , which showed a slight increase in oxygen saturation over time for the right hand ( preductal ) and right foot ( postductal ) . rising from statistical significance were only two variables , postnatal age and activity of the infant . postnatal age was found to be statistically significant ( + 0.17% per 24-hour interval from admission to discharge , p=0.0001 ) but not clinically relevant.21 infant s activity was statistically significant with values obtained while the infant was crying , fussy , or awake being lower than the values obtained while sleeping.21 de wahl granelli et al10,22 systematically measured the oxygen saturation in 40,000 newborns and compared the values obtained in children with cchd vs otherwise healthy newborns . in addition , these investigators showed that two of the three infants with cchd were missed by physical examination alone.22 addition of pulse oximetry screening raised the diagnosis rate to 82% . several other studies mostly from europe revealed similar findings.12,2328 these were summarized in a systematic review and meta - analysis of pulse oximetry screening for cchd in the newborn nursery , which included 13 studies with 229,421 infants.28 sensitivity of pulse oximetry was 76.5% ( 95% ci 67.783.5 ) and specificity was 99.9% ( 95% ci 99.799.9 ) for the detection of chd , with the average false - positive rate for these infants being 0.14% ( 95% ci 0.160.33).28 with the average pulse oximetry value being 97.2% during the first days of life for all newborns , pulse oximetry is an excellent tool to evaluate subclinical hypoxemia , that occurs during transitioning physiology of certain chd , such as transposition of the great arteries , truncus arteriosus communis , hypoplastic left heart syndrome , total anomalous pulmonary venous connection , tricuspid atresia , tetralogy of fallot , and pulmonary atresia . these lesions are usually associated with hypoxemia in the newborn period and can cause significant morbidity and mortality if the diagnosis is delayed . in september 2010 , the secretary s advisory committee on heritable disorders in newborns and children ( sachdnc ) considered these seven lesions as primary targets for pulse oximetry screening in the newborn period on the basis of advice from a technical expert panel.2 in 2011 , the sachdnc , in collaboration with the american academy of pediatrics , the american college of cardiology foundation , and the american heart association , convened a work group to outline implementation strategies for pulse oximetry screening in newborns for chd . after reviewing data from existing large studies in sweden and the uk,22,25 the work group proposed a screening protocol based on results of measurements from the right hand ( preductal ) and either foot ( postductal).11 according to the sachdnc protocol , an infant would have a positive ( failing ) screen if at 24 hours of life : 1 ) a pulse oximeter reading was < 90% in either the right hand or either foot . 3 ) a persistent > 3% difference in the right hand and either foot measurement on three measurements each separated by 1 hour . an infant who had 95% in either extremity with 3% difference in the pre- and postductal oxygen saturation would have a negative screen and no further work - up is needed . many states that have initiated pulse oximetry screening have adapted this protocol or a variation there of.12,13,15 kochilas et al14 demonstrated that the sachdnc protocol was the most efficient protocol with the fewest false - positive pulse oximetry screens in the newborn period . data from various studies suggest that this protocol is adequate to detect clinical and subclinical hypoxemia that is associated with the seven primary target lesions based on their almost universal association with at least mild hypoxemia in the neonatal period , when the fetal circulation transitions to postnatal circulation . the protocol should also be effective , although to a lesser degree , to detect the hypoxemia associated with five additional congenital heart lesions that are considered as secondary targets.6,9 these lesions are frequently associated with at least some degree of neonatal hypoxemia and include proximal aortic arch anomalies ( such as interrupted aortic arch or aortic atresia ) , coarctation of the aorta ( coa ) with patent ductus arteriosus , ebstein s anomaly , double outlet right ventricle , and single ventricle lesions . there are additional lesions that are possibly screenable in the neonatal period with the same protocol based on their anatomy and potential for intracardiac or ductal - level shunting . these include aortic stenosis with a patent ductus arteriosus , severe pulmonary stenosis , and complete common atrioventricular canal . finally , there is a category of cardiac lesions that will not have hypoxemia in the newborn period and can be classified as not screenable and include left - sided obstructive lesions such as coa without patent ductus arteriosus and aortic stenosis without a patent ductus arteriosus , ebstein s anomaly without interatrial shunting from right - to - left , and all other lesions that cause left - to - right shunting and valve anomalies that were not included in the previous categories29 ( table 1 ) . although not all chds cause hypoxemia , these types of lesions can still lead to serious complications if not detected early enough to avoid end - organ damage . among them , the most frequent category is comprised of some form of left - sided obstructive lesions for which additional diagnostic strategies may soon become clinically available . one of the most promising ones is the peripheral perfusion index ( ppi ) , which is based on the analysis of the pulse oximetry signal and is displayed on some newer generation pulse oximeters . more specifically , this technique uses the ratio between the pulsatile and nonpulsatile component of the pulse oximetry signal to detect changes in the relative amount of arterial perfusion in the examined site.29,30 as measured , the ppi may be useful in detecting reduction in the arterial circulation at the monitoring site and can be used to detect decreased perfusion in settings of left heart obstructive lesions either globally ( ie , aortic stenosis ) or regionally ( coa ) . important challenges for this technique remain the wide and highly skewed distribution of ppi values in the normal population and sensitivity to environmental factors such as skin temperature . preliminary work with ppi in neonates has established reference values for newborns with cutoff values of < 0.70 for possibly impaired peripheral perfusion and < 0.50 definite hypoperfusion.31 however , further studies with children with various cardiac lesions are needed before incorporating ppi in the screening process for cchd . few studies have shown the cost - effectiveness of pulse oximetry screening in the newborn nursery . two studies published by peterson et al32 estimated that routine screening of newborns would identify an additional 1,189 infants with cchd at their respective birth hospitals that would not have been diagnosed prior to discharge . in addition , they estimated that 20 infant lives would be saved each year by screening and a cost of $40,385 per life - year will be gained.32 the estimated cost for pulse oximetry screening in the newborn nurseries in new jersey was $ 14.09 per newborn , with supplies and labor dividing this cost almost equally.33 peterson et al33 extrapolated that the estimated average cost to screen all newborns in the us regardless of which level of nursery they were in was $ 13.50 per newborn . in the scientific statement from the american heart association and the american academy of pediatrics , mahle et al evaluated the statistical analysis of pulse oximetry screening with data from ten different studies . analysis on the studies with infants who were evaluated after 24 hours of age showed 18 false positives , along with seven false negatives and 51,063 true negatives . with these data , the sensitivity of pulse oximetry was 69.9% and the specificity was 99.9% , with a negative predictive value of 99.9% and a positive predictive value of 47%.2 pulse oximetry is an adequate screening tool , with few false positives and a high negative predictive value . as far as we know at the time of writing this article , there are no sufficient data available on the burden to the health care system with the increase in infant echocardiograms completed due to failed pulse oximetry screening in the nursery . each type of chd affects cardiopulmonary circulation differently , and pulse oximetry is a good screening tool to detect lesions that cause hypoxemia in the first few days of life . chd with impaired perfusion rather than oxygenation will , though , remain undetected by pulse oximetry . the addition of ppi , a derivative of the quantitative analysis of the pulsatile vs nonpulsatile signal of the pulse oximetry , is a promising technique to cover the diagnostic gaps for this population .

congenital heart disease ( chd ) is one of the most common birth defects , with an incidence of nine out of every 1,000 live births . the mortality of infants with chd has decreased over the past 3 decades , but significant morbidity and mortality continue to occur if not diagnosed shortly after birth . pulse oximetry was recommended as a screening tool to detect critical chd in 2011 by the american academy of pediatrics and the american heart association . pulse oximetry is a tool to measure oxygen saturation , and based on the presence of hypoxemia , many cardiac lesions are detected . due to its ease of application to the patient , providing results in a timely manner and without the need for calibrating the sensor probe , pulse oximetry offers many advantages as a screening tool . however , pulse oximetry has also important limitations of which physicians should be aware to be able to assess the significance of the pulse oximetry measurement for a given patient . this review aims to highlight the benefits and shortcomings of pulse oximetry within the context of screening for critical chd and suggests future avenues to cover existing gaps in current practices .

recently , the use of magnetic nanoparticles ( mnps ) in medical applications ( e.g. magnetic fluid hyperthermia ( mfh ) , magnetic drug delivery , magneto - mechanical actuation of cell surface receptors , magnetic gene transfection , and magnetic resonance imaging ( mri ) contrast improvement ) has had an upward trend . the principle of this technique is to destroy cancer cells by heating them up without lethal effects on normal tissues . in comparison with other treatment techniques , this technique is a physical therapy with few side effects and it can be accompanied by other therapies like radiotherapy and chemotherapy to sensitize resistant malignant cells to these therapies . effectiveness of the treatment is dependent on the tumor size and its location in the body . mnps , used as heating mediators , are magnetic materials consisting of one or more inorganic crystals . when they undergo external magnetic field , thermal energy the heating power of mnps depends on the particle size , size distribution , domain state , magnetic anisotropy , the viscosity of the dispersion medium and parameters of the magnetic field , including field strength ( h ) and frequency ( f ) . survival of the malignant cells declines when their temperature reaches 41 to 46 c . in this technique , it is therefore necessary to reach temperatures above 41 c by mnps as the heat sources . tumor cells are more sensitive to heat in comparison to normal cells and consequently these temperatures do not disturb the viability of normal cells . dispersing mnps in a liquid , as a carrier , leads to the production of magnetic fluids . . when fe is substituted by mn , co and zn in m1-xznxfe2o4 ferrites with superior magnetic properties can be produced . several methods are available for synthesizing mnps , but the most popular methods are coprecipitation , thermal decomposition , hydrothermal synthesis , and laser pyrolysis . in this study , we synthesized fe0.5zn0.5fe2o4 ferrite with dextrin coating by the aqueous precipitation method . the samples were characterized by transmission electron microscopy ( tem ) to investigate the size distribution and morphology of the nanostructure . magnetic properties and hysteresis loop of the mnps were studied by vibrating sample magnetometer ( vsm ) . fourier transform infrared ( ft - ir ) was also applied to characterize the structure and purity of the compound . afterwards , we injected these particles intratumorally in mice bearing implanted melanoma cancer to investigate their hyperthermia effect under amf . ferrite nanoparticles ( nps ) were synthesized by the aqueous precipitation route . briefly , individual metal chloride or sulfate , in an appropriate stoichiometric proportions , was dissolved in a dilute nitric acid ( 0.1 mol / l hcl ) solution and heated to 80 c . sodium hydroxide solution ( 4 mol / l ) was prepared separately and heated to 80 c . the hot nitrate precursor solution was then rapidly mixed with magnetic stirring ( final ph of 12.0 ) . then , the solution was cooled and the precipitate was collected by a permanent magnet and washed several times with distilled water to neutralize the supernatant . tem ( zeiss , em10c , 80 kv , germany ) was performed to obtain the morphology and size of the nanostructure . magnetic properties of ferrofluid were determined by drying the fluid and using a vsm ( meghnatis kavir co. , kashan , iran ) at room temperature . surface structure of the sample was identified by using a ft - ir spectroscope ( burker tensor 27 ) . the ft - ir spectrum was acquired in the wavenumber range of 500 - 4000 cm . b16/f10 melanoma cells were purchased from the pasteur institute ( iran ) and cultured in dmem ( dulbecco s modified eagle medium ) medium with 10% fbs ( fetal bovine serum ) and 1% penicillin in a humidified 37c incubator . four- to six - week - old inbred balb / c mice were purchased from the center of comparative and experimental medicine , shiraz university of medical sciences , shiraz , iran . all animals were housed in the laboratory of the center of comparative and experimental medicine under identical conditions . the animals were housed in special cages at a controlled temperature ( 242 c ) with weekly floor exchange . a 12:12 hour light - dark cycle was followed in the above - mentioned animal house center . all animals received care in compliance with the standard animal ethics of iran . to implant solid tumor , 1510 b16/f10 melanoma cells in pbs after approximately one week , the tumors appeared and the mice were followed until their tumors reached an appropriate size . to measure the tumor volume , a caliper was used and the volume was computed using the following formula . where a and b are the largest and smallest diameter of tumor , respectively . after reaching an appropriate tumor size ( about 1 cm ) , hypethermia study was started . in order to generate alternating magnetic field ( amf ) , a horizontal solenoid coil vibrating 100 g at 515 khz ( sine wave pattern ) was used ( shiraz university , school of engineering ) . the solenoid had an inner diameter of 5 cm , a length of 15 cm and 14 turns . it consisted of a water - cooling system to avoid heat induction because of the impedance . a resonant h - bridge zcs inverter with a capacitor bank of 75 nf was employed to derive the coil and inject ac current . capacitor bank was in series with the solenoid coil , which constitutes the resonant tank . the inverter changed dc input voltage to a square wave alternating voltage and was applied to the resonant tank . sinusoidal current flowed through the coil while the frequency of the square wave voltage was adjusted to the resonant frequency of the resonant tank . the injected ac current generated amf whose amplitude was calculated according to the coil characteristics and the magnitude of ac current . where , n : number of turns , l : coil length , 0 : equal to 4*10 and is the magnetic permeability in vacuum , bm : peak value of amf , and i m : peak value of ac current . the peak value of the current was adjusted to 85 a , which was measured using gds-1072 oscilloscope . the maximum input power of the system was 650 w. mice bearing implanted solid tumor in proper conditions were selected and divided into four groups consisting of two mice per group . it included a control group ( group a ) without any treatment administration and a sham treatment group ( group b ) to investigate the effect of magnetic field on the malignant cells . the mice in group b were located in the solenoid coil for 30 min without mnps injection . for evaluating the lethal effects of the fnps on cancerous cells , the mice in group c received only intratumoral injection of fnps . finally , group d was chosen for treatment administration by intratumoral injection of mnps ( 200 l magnetic fluid containing 2 mg fe ) undergoing amf for 30 min . before administrating the experimental treatment , each mouse was anesthetized with intramuscular injection of ketamin / xylazine mix . for histological findings , all treated and non - treated tumors and tissues of the spleen , kidney , and liver were removed 24 hours after the treatment . for this purpose , after dissection , the tissues were removed and each specimen was collected and fixed by immersion in 10% buffered formalin and routinely processed into paraffin . afterwards , sections were cut at 5 m and put in an oven for 45 minutes to remove the extra paraffin from the tissue . after staining with hematoxylin and eosin ( h&e ) , perl s iron staining was carried out to obtain the iron content in groups c and d. finally , each prepared slide was observed under a microscope to distinguish necrosis and inflammation areas and the distribution of mnps through the track of iron . for this purpose , the necrotic surface area was estimated by using graticule ( olympus , japan ) on tumor slides . ferrite nanoparticles ( nps ) were synthesized by the aqueous precipitation route . briefly , individual metal chloride or sulfate , in an appropriate stoichiometric proportions , was dissolved in a dilute nitric acid ( 0.1 mol / l hcl ) solution and heated to 80 c . sodium hydroxide solution ( 4 mol / l ) was prepared separately and heated to 80 c . the hot nitrate precursor solution was then rapidly mixed with magnetic stirring ( final ph of 12.0 ) . then , the solution was cooled and the precipitate was collected by a permanent magnet and washed several times with distilled water to neutralize the supernatant . tem ( zeiss , em10c , 80 kv , germany ) was performed to obtain the morphology and size of the nanostructure . magnetic properties of ferrofluid were determined by drying the fluid and using a vsm ( meghnatis kavir co. , kashan , iran ) at room temperature . surface structure of the sample was identified by using a ft - ir spectroscope ( burker tensor 27 ) . the ft - ir spectrum was acquired in the wavenumber range of 500 - 4000 cm . b16/f10 melanoma cells were purchased from the pasteur institute ( iran ) and cultured in dmem ( dulbecco s modified eagle medium ) medium with 10% fbs ( fetal bovine serum ) and 1% penicillin in a humidified 37c incubator . four- to six - week - old inbred balb / c mice were purchased from the center of comparative and experimental medicine , shiraz university of medical sciences , shiraz , iran . all animals were housed in the laboratory of the center of comparative and experimental medicine under identical conditions . the animals were housed in special cages at a controlled temperature ( 242 c ) with weekly floor exchange . a 12:12 hour light - dark cycle was followed in the above - mentioned animal house center . all animals received care in compliance with the standard animal ethics of iran . to implant solid tumor , 1510 b16/f10 melanoma cells in pbs were injected subcutaneously into the back of the neck of each mouse . after approximately one week , the tumors appeared and the mice were followed until their tumors reached an appropriate size . to measure the tumor volume , a caliper was used and the volume was computed using the following formula . where a and b are the largest and smallest diameter of tumor , respectively . after reaching an appropriate tumor size ( about 1 cm ) , hypethermia study was started . in order to generate alternating magnetic field ( amf ) , a horizontal solenoid coil vibrating 100 g at 515 khz ( sine wave pattern ) was used ( shiraz university , school of engineering ) . the solenoid had an inner diameter of 5 cm , a length of 15 cm and 14 turns . it consisted of a water - cooling system to avoid heat induction because of the impedance . a resonant h - bridge zcs inverter with a capacitor bank of 75 nf was employed to derive the coil and inject ac current . capacitor bank was in series with the solenoid coil , which constitutes the resonant tank . the inverter changed dc input voltage to a square wave alternating voltage and was applied to the resonant tank . sinusoidal current flowed through the coil while the frequency of the square wave voltage was adjusted to the resonant frequency of the resonant tank . the injected ac current generated amf whose amplitude was calculated according to the coil characteristics and the magnitude of ac current . where , n : number of turns , l : coil length , 0 : equal to 4*10 and is the magnetic permeability in vacuum , bm : peak value of amf , and i m : peak value of ac current . the peak value of the current was adjusted to 85 a , which was measured using gds-1072 oscilloscope . mice bearing implanted solid tumor in proper conditions were selected and divided into four groups consisting of two mice per group . it included a control group ( group a ) without any treatment administration and a sham treatment group ( group b ) to investigate the effect of magnetic field on the malignant cells . the mice in group b were located in the solenoid coil for 30 min without mnps injection . for evaluating the lethal effects of the fnps on cancerous cells , the mice in group c received only intratumoral injection of fnps . finally , group d was chosen for treatment administration by intratumoral injection of mnps ( 200 l magnetic fluid containing 2 mg fe ) undergoing amf for 30 min . before administrating the experimental treatment , each mouse was anesthetized with intramuscular injection of ketamin / xylazine mix . for histological findings , all treated and non - treated tumors and tissues of the spleen , kidney , and liver were removed 24 hours after the treatment . for this purpose , after dissection , the tissues were removed and each specimen was collected and fixed by immersion in 10% buffered formalin and routinely processed into paraffin . afterwards , sections were cut at 5 m and put in an oven for 45 minutes to remove the extra paraffin from the tissue . after staining with hematoxylin and eosin ( h&e ) , perl s iron staining was carried out to obtain the iron content in groups c and d. finally , each prepared slide was observed under a microscope to distinguish necrosis and inflammation areas and the distribution of mnps through the track of iron . for this purpose , the necrotic surface area was estimated by using graticule ( olympus , japan ) on tumor slides . magnetic properties of nps were determined by vibrating sample magnetometer ( vsm ) at room temperature . figure 1 depicts the m versus h curve of the dry fe0.5zn0.5fe2o4 ferrite nps with applied field between -8500 oe and 8500 oe at room temperature . as shown in this figure , the particle size , morphology , and agglomeration of mnps can be determined by tem images . m versus h curve of fe0.5zn0.5fe2o4 ferrite ( at room temperature ) indicates the single domain superparamagnetic properties of mnps . tem image of fe0.5zn0.5fe2o4 ferrite shows that the diameter of nanoparticles is around 9 nm . in order to confirm that dextrin was successfully coated on the surface of nps ft - ir spectra of fe0.5zn0.5fe2o4 ferrite shows nps were successfully covered with dextrin . for the in vivo study , the tumor was induced in balb / c mice via the injection of b16/f10 melanoma cells in pbs into the back of the neck of the mice . after approximately one week , the tumors appeared in the animals and the size of tumors was measured with a caliper ( figure 4 ) . figure 5 shows that the mice in groups b and d were located in the center of the solenoid and subjected to amf with 50 a / div current ( figure 6 ) for 30 minutes . mouse bearing implanted tumor was placed in a solenoid ( f=515 khz , h=100 g ) . sinusoidal current flow through the coil could be seen by oscilloscope ( 50 a / div ) . after 24 hours , all mice were sacrificed and each specimen of the tumors was collected and prepared for histological analyses . figure 7 shows stained slides of tumors in four groups . in order to document the presence of fnps between the cells and to investigate their effects on the surrounding medium , perl s iron staining was done.12,13 as shown in figure 7c , iron particles were present in malignant cells . a : group a ( blue arrows show malignant cells ) ; b : group b ; c : iron stained slide of mouse tumor in group c ( yellow arrow shows the presence of fe nanoparticles in malignant cells ) ; d : h&e stained slide of mouse tumor in group d. no significant difference in the necrosis extent was observed ( h&e 400 ) . no trace of fnps was detected in the spleen , kidney , and liver tissues and they had normal tissues , figure 8 . no trace of mnps was observed in the tissues of the liver ( a ) , spleen ( b ) , and kidney ( c ) ( h&e 400 ) . magnetic properties of nps were determined by vibrating sample magnetometer ( vsm ) at room temperature . figure 1 depicts the m versus h curve of the dry fe0.5zn0.5fe2o4 ferrite nps with applied field between -8500 oe and 8500 oe at room temperature . as shown in this figure , the particle size , morphology , and agglomeration of mnps can be determined by tem images . m versus h curve of fe0.5zn0.5fe2o4 ferrite ( at room temperature ) indicates the single domain superparamagnetic properties of mnps . tem image of fe0.5zn0.5fe2o4 ferrite shows that the diameter of nanoparticles is around 9 nm . in order to confirm that dextrin was successfully coated on the surface of nps for the in vivo study , the tumor was induced in balb / c mice via the injection of b16/f10 melanoma cells in pbs into the back of the neck of the mice . after approximately one week , the tumors appeared in the animals and the size of tumors was measured with a caliper ( figure 4 ) . figure 5 shows that the mice in groups b and d were located in the center of the solenoid and subjected to amf with 50 a / div current ( figure 6 ) for 30 minutes . mouse bearing implanted tumor was placed in a solenoid ( f=515 khz , h=100 g ) . sinusoidal current flow through the coil could be seen by oscilloscope ( 50 a / div ) . after 24 hours , all mice were sacrificed and each specimen of the tumors was collected and prepared for histological analyses . figure 7 shows stained slides of tumors in four groups . in order to document the presence of fnps between the cells and to investigate their effects on the surrounding medium , perl s iron staining was done.12,13 as shown in figure 7c , iron particles were present in malignant cells . a : group a ( blue arrows show malignant cells ) ; b : group b ; c : iron stained slide of mouse tumor in group c ( yellow arrow shows the presence of fe nanoparticles in malignant cells ) ; d : h&e stained slide of mouse tumor in group d. no significant difference in the necrosis extent was observed ( h&e 400 ) . no trace of fnps was detected in the spleen , kidney , and liver tissues and they had normal tissues , figure 8 . no trace of mnps was observed in the tissues of the liver ( a ) , spleen ( b ) , and kidney ( c ) ( h&e 400 ) . due to many side effects of common strategies for cancer therapy , these strategies are not considered useful for all kinds and stages of tumors . consequently , researchers are looking for a comprehensive treatment method . parameters that contribute to the induction of heat in this technique are the diameter of nps , anisotropy constant , fluid viscosity , and frequency and amplitude of the magnetic field . in this study , for the characterization of fnps , ft - ir , vsm , tem were done . the absence of hysteresis loop in this curve and non - saturated magnetization at the applied magnetic field , indicated the single domain superparamagnetic properties of mnps . where , m is the molecular weight of the composition ( in gram ) , ms is saturation magnetization , and 5585 is the magnetic factor . by estimating the saturation magnetization through extrapolation ( 30.16 emu / gr ) , nb was calculated to be 1.27 . according to tem image for in vivo study , it is necessary to coat the nps with a polymer . in this study , we used dextrin as a coating for biocompatibility of nps . ft - ir analysis was used to confirm that dextrin was successfully coated on the surface of mnps . in ft - ir spectrum , the peak at 570 cm refers to the characteristic absorption of fe - o bond of magnetic particles . the peaks at 1634.6 cm and 2920.5 cm corresponded to c = o bond and vibration of -ch2- groups , respectively . thus , according to previous studies , fnps were successfully covered with dextrin . in this study , microscopic changes in tumoral cells were investigated 24 hours after treatment among the four mice groups . for this purpose , the degree of necrosis and inflammation ( 400 ) was verified on tumor slides . according to the pathology results , in the tumoral tissue , necrotic cells were easily seen in the central part of all tumors . on tumor slides belonging group c , there was no necrosis cells around nps . thus , it is expected that if fnps are transferred to other organs in intravenous administration , they do not make lethal effects on the normal cells . however , a previous study showed iron / iron oxide core / shell nps with porphyrin coating caused cell damage due to the generation of free radicals and magnetic hyperthermia under the influence of magnetic field . different degrees of necrotic cells could be seen in all specimens , but no significant difference in necrosis extent was observed among the four groups ; thus , this treatment was not powerful enough to remove the cancer cells , which may contribute to particle size . three mechanisms contribute to heat loss in mnps , namely , hysteresis losses , neel and brownian relaxations . bakoglidis et al . investigated the magnetic hyperthermia response of iron oxide nps in the size range of 5 - 18 nm . they divided these nps into three categories ; the superparamagnetic ( spm ) region ( as large as 10 nm ) where heating is mainly because of neel relaxation , the spm - ferromagnetic region ( 10 - 13 nm ) , and the ferromagnetic region ( > 13 nm ) where hysteresis loss mechanism is dominate in heat dissipation . they reported that ferromagnetic materials release heat that is more significant and hysteresis mechanism is more efficient for hyperthermia applications . in addition , jeun et al . reported that the heat efficiency of fe3o4 particles below 9.8 nm under 110 khz magnetic field frequency was insufficient for hyperthermia applications , and the value of specific loss power ( slp ) had an upward trend with an increase in the particle size from 11.5 to 22.5 nm under the same magnetic field . therefore , heat efficiency of fnps with an average diameter of 9 nm under the influence of amf ( 515 khz ) is insufficient for hyperthermia applications . although , ultra - small nps have a lot of potential for use in biomedical applications , it was confirmed that this np could not induce efficient heat for hyperthermia . however , studies show the mnp aggregate to larger cluster after systematic administration and therefore the effective hydrodynamic size of the mnp in the body is different from the initial size of nps . additionally , theoretical study on proton relaxation indicates that the magnetic properties of nps increase significantly upon the enhancement of the particle and the crystallite size . ultra - small nps can be functionalized and guided by an external magnetic field for in vivo studies and after removing the emf , dipoles return to random orientations because of the spm behavior of these nps . unlike ferromagnetic nps however , for the hyperthermia study , spm nps show lower heating efficiency than single domain ferromagnetic nps due to the absence or negligible amounts of hysteresis loss . we also surveyed the presence of nps in other organs such as the spleen , kidney , and liver . no trace of fnps was detected in the spleen , kidney , and liver tissues . thus , mnps remained in the tumor and did not transfer to other organs after intratumoral injection . in systemic administration , large particles ( > 50 nm ) are taken up by the liver cells , but smaller ones generally circulate for a longer period . longer residence time in the blood stream may enhance ( enhanced permeation and retention ) epr effect . therefore , it is expected that in systemic administration , fnps with a diameter of 9 nm are not captured by the liver and accumulated by tumor . a limitation of this investigation was the use of a solenoid coil with one frequency and field strength . the major findings reported here illustrate that fnp ferrofluid coated with dextrin is a good candidate for in vivo approaches without any side effects on body organs . while fe particles were seen in malignant tumor cells , there was no trace of them in other organs . different amounts of necrosis were seen in the tumor tissues of all groups , but no significant difference in the necrosis extent was observed among the groups . therefore , although this np is a good agent for other biomedical applications , like contrast enhancement in mri , it did not induce efficient heat under external magnetic field .

background : nowadays , magnetic nanoparticles ( mnps ) have received much attention because of their enormous potentials in many fields such as magnetic fluid hyperthermia ( mfh ) . the goal of hyperthermia is to increase the temperature of malignant cells to destroy them without any lethal effect on normal tissues . to investigate the effectiveness of cancer therapy by magnetic fluid hyperthermia , fe0.5zn0.5fe2o4 nanoparticles ( fnps ) were used to undergo external magnetic field ( f=515 khz , h=100 g ) in mice bearing implanted tumor.methods:fnps were synthesized via precipitation and characterized using transmission electron microscopy ( tem ) , vibrating sample magnetometer , and fourier transform infrared . for in vivo study , the mice bearing implanted tumor were divided into four groups ( two mice per group ) , namely , control group , amf group , mnps group , and mnps&amf group . after 24 hours , the mice were sacrificed and each tumor specimen was prepared for histological analyses . the necrotic surface area was estimated by using graticule ( olympus , japan ) on tumor slides.results:the mean diameter of fnps was estimated around 9 nm by tem image and m versus h curve indicates that this particle is among superparamagnetic materials . according to histological analyses , no significant difference in necrosis extent was observed among the four groups.conclusion:fnps are biocompatible and have a good size for biomedical applications . however , for mfh approach , larger diameters especially in the range of ferromagnetic particles due to hysteresis loss can induce efficient heat in the target region .

in spite of the relatively high accuracy of endoscopic ultrasound - assisted fine - needle aspiration ( eus - fna ) in diagnosing lymphomas , inadequate sampling by eus - fna often makes it difficult to perform immunohistochemical analysis , thus limiting its application in the classification of lymphoma . natural orifice transluminal endoscopic surgery ( notes ) is a surgical technique by which procedures such as exploration , biopsy , organ resection , and anastomosis can be performed using an endoscope passed through a natural orifice [ such as the mouth , stomach , colon ( or rectum ) , vagina , bladder , or esophagus ] and then entered into the abdominal cavity , mediastinum , or thoracic cavity through an internal incision . the advantages of notes include reduced trauma , faster recovery , absence of scarring , and painlessness , and such procedures have been regarded as third - generation surgery . here we report an eus - assisted retroperitoneal lymph node biopsy performed in a patient who had developed enlarged retroperitoneal lymph nodes with an unknown cause . this procedure was carried out on november 10 , 2014 , after obtaining the approval of the ethics committee and informed consent documents signed by the patient . a 60-year - old male patient was admitted to our hospital complaining of epigastric discomfort , which had persisted for 1 month . a computerized tomography ( ct ) scan suggested the presence of multiple soft - tissue density masses in the patient 's abdominal cavity , the largest of which was 7.7 cm 7.2 cm [ figure 1a and b ] . positron emission tomography - ct ( pet - ct ) showed that these masses had abnormal f - fdg uptake [ figure 2a and b ] . the patient then underwent eus examination ; multiple enlarged retroperitoneal lymph nodes were found between the body of the pancreas and the gastric wall . we then performed eus - fna [ figure 3 ] to obtain a tissue sample for biopsy . pathological examination revealed only a few heterotypic cells [ figure 4 ] . because of the lack of definite pathological evidence , diagnosis of the patient 's condition was extremely difficult . ct scan showing multiple , enlarged soft tissue - density images in the abdominal cavity pet - ct showing the accumulation o f abnormal radioactivity in soft tissue - density images in the abdominal cavity eus - fna of a lymph node eus - fna showing a few heterotypic cells to obtain adequate tissue samples of the enlarged lymph nodes for immunohistochemical analysis , we performed eus - assisted retroperitoneal lymph node biopsy . a standard single - channel gastroscope ( epk - i , pentax , tokyo , japan ) was used throughout the endoscopic procedure ; a linear array ultrasonic endoscope ( eg3830ur ; pentax precision instrument corporation , orangeburg , ny , usa ) was used to evaluate the size of the lymph nodes , their echo characteristics , and localization . a triangle - tip knife and an insulated - tip ( it ) knife ( both from olympus corporation , tokyo , japan ) were used for resection of the gastric wall and enucleation of the lymph node . a pair of hot forceps ( fd-410lr , olympus corporation , tokyo , japan ) was used for gastric wall hemostasis . adequate preoperative communication with the patient and his family was performed and associated issues including the necessity , feasibility , safety , and probable complications of the operation were explained thoroughly . for the procedure , the patient was placed in a supine position and he received standard intravenous anesthesia with propofol . the site nearest to the retroperitoneal lymph nodes in the posterior wall of the gastric body was chosen for puncture . a methylthioninium chloride and saline compound solution was injected into the puncture channel for labeling when the needle was retrieved . the lymph node was also marked by a triangle tip knife with the cautery under eus - guidance . then , the ultrasound transducer was pulled out ; therapeutic gastroscopy with a transparent cap was then performed . a triangle - tip knife was used to incise the full thickness of the gastric wall along the labeled site . the triangle - tip knife was used to separate the tissues surrounding the stomach wall sufficiently until the targeted lymph node capsule was exposed . then , enucleation of the targeted lymph node was performed using an it knife [ figure 5 ] . after tissue samples were obtained , the hot forceps were used to stop the bleeding . the endoscope was retrieved from the stomach and the procedure was completed by closing the incision in the stomach wall using metal clips [ video 1 ] . the results showed : cd3(large cell- ) ; vimentin(+ ) ; pax-5(- ) ; cd15(- ) ; cd20(large cell+ ) ; cd21(+ ) ; ki-67(large cell8%+ ) ; cd30(- ) ; cd68(partly+ ) ; ck(- ) ; mum-1(- ) ; cd10(- ) ; bcl-6(+ ) ; bcl-2(+ ) . the diagnosis was : non - hodgkin lymphoma , germinal center b - cell - like diffuse large b - cell lymphoma [ figure 6 ] . ( a ) the gastroscope entered into the abdominal cavity after incision of the full thickness of the gastric wall . the prelabeled lymph node was found , ( b ) the removed lymph node tissues immunohistochemical staining : cd3(large cell- ) ; vimentin(+ ) ; pax-5(- ) ; cd15(- ) ; cd20(large cell+ ) ; cd21(+ ) ; ki-67(large cell8%+ ) ; cd30(- ) ; cd68(partly+ ) ; ck(- ) ; mum-1(- ) ; cd10(- ) ; bcl-6(+ ) ; bcl-2(+ ) . the diagnosis was non - hodgkin lymphoma , germinal center b - cell - like diffuse large b - cell lymphoma the patient was given standard postoperative treatments and nursing care including ecg monitoring , ceftazidime as prophylaxis against infection , proton pump inhibitors , and nutritional support . the patient 's highest temperature after the procedure was 37.2c and he only felt mild epigastralgia . the blood test results after the procedure were : white blood cell ( wbc ) count 12.8 10/l and percentage of neutrophils 80.3% . the wbc count decreased to 6.5 10/l and the percentage of neutrophils decreased to 61.8% , 4 days after the procedure . the patient gradually returned to a normal diet and normal physical activities by 3 days after the procedure . the patient was then transferred to the department of hematology to undergo further therapy . the r - chop regimen ( pathological evidence is an indispensable part of the diagnosis and differential diagnosis of lymphoma and is significant for the classification of lymphomas . precise pathological classification is critical for the choice of chemotherapeutic regimen in cases of lymphoma . in the early 1990s , eus - fna was first used for tissue biopsy of tumors around the gastrointestinal tract . in spite of its high accuracy , the lack of sufficient tissue material obtained using this technique often renders immunohistochemical staining inconclusive and limits its application in the classification of lymphomas . how to obtain an adequate sample that can be used for immunohistochemical testing is a primary problem in need of a solution . in a study by mohamad et al . , the results of eus - fna of two patients who had suspected lymphoma were negative . however , positive results were obtained by use of eus - guided trucut biopsy ( eus - tcb ) . the authors suggested that the use of eus - tcb with 19 g needle could permit more tissue materials to be obtained in those who had a negative eus - fna result . this minimally invasive procedure can be used for the preoperative diagnoses of some difficult cases . analyzed and compared the efficacies of eus - fna and eus - tcb in the diagnosis and classification of lymphoma . among the 24 included patients , 23 patients underwent eus - fna . only one patient received eus - tcb alone , while the other 22 patients received both eus - fna and eus - tcb . the results showed that the accurate diagnostic rate of eus - guided biopsy was 79% ( 19/24 ) and the accurate rate of eus - guided biopsy for classification was just 66.6% ( 16/24 ) . this indicated that eus - guided biopsy was of limited value in the classification of lymphoma . there have been more than 300 reports of notes clinical applications from around the world . over 50 cases of cholecystostomy and endectomy by transvaginal notes have been performed in germany , and 116 notes surgeries ( including 77 transgastric cholecystotomy cases ) have been reported in brazil . however , there are few case reports regarding the clinical application of notes in china . wang et al . presented a case of laparoscopy - assisted transgastric endoscopic biopsy of a retroperitoneal lymph node . in this case , an endoscopic full - thickness resection ( eftr ) with the aid of laparoscopy was used to extirpate an enlarged retroperitoneal lymph node . pathological and immunohistochemical tests confirmed the diagnosis of retroperitoneal b - cell lymphoma ( diffuse large b - cell lymphoma ) . in recent years , these studies showed that eus was very useful for creating transgastric access and locating the targets . in this study , we successfully used eus - assisted notes to perform enucleation of a retroperitoneal enlarged lymph node without laparoscopic assistance . the use of a minimally invasive procedure not only enables more tissue materials to be obtained but also causes less trauma than a laparoscopy - assisted approach . in this patient , blood test results recovered to normal levels by 4 days after the procedure and the highest postoperative body temperature was just 37.2c . he was also able to return to a normal diet 3 days after the procedure . our experience suggests that this is an alternative and minimally invasive approach for the biopsy of retroperitoneal lymph nodes .

since its introduction in the early 1990s , endoscopic ultrasound - assisted fine - needle aspiration ( eus - fna ) has been used for sampling of extraintestinal mass lesions and peri - intestinal lymphadenopathy . although eus - fna is highly accurate , lymphomas can be challenging to diagnose using eus - fna . we present the case of a 60-year - old male who had experienced upper abdominal discomfort for 1 month . computerized tomography ( ct ) examination revealed multiple soft - tissue shadows located above the pancreatic body . the biggest shadow had a cross - sectional area of 7.7 cm 7.2 cm . positron emission tomography - ct ( pet - ct ) imaging showed increased uptake of 18f - fdg by these soft - tissue shadows . to investigate further , eus was performed and it revealed the presence of multiple hypoechoic round lymph nodes . during the procedure , eus - fna was performed , but only a few dyskaryotic cells were observed by cytological evaluation . eus - assisted retroperitoneoscopy and lymph node biopsy were performed to obtain more tissue for immunohistochemical analysis and subclassification of lymphoma . finally , the patient was diagnosed with non - hodgkin lymphoma , germinal center b - cell - like diffuse large b - cell lymphoma by this technique . eus - assisted transendoscopic retroperitoneal lymph node biopsy is an alternative procedure for the diagnosis of lymphomas .

periodontal diseases are inflammatory conditions of bacterial origin that involve large proportions of inflammatory cells and the sequential activation of different components of the host immune and inflammatory response , aimed at defending the tissues against bacterial aggression , reflecting the essentially protective role of the response . neutrophils protect the host tissues by killing various pathogenic bacteria either by non - oxidative or oxidative means in an intracellular or extracellular environment . non - oxidative killing is mediated by various lysosomal enzymes , peptides and proteins , including lysozyme , bactericidal / permeability - increasing proteins , cationic proteins , defensins and lactoferrin . generation of reactive oxygen species ( superoxide , hydrogen peroxide , hydroxyl radicals and hypochlorous acid and chloramines ) contributes in oxidative killing of the invading microorganisms . numerous researchers have reported that any loss of the neutrophil defense , either due to deficiency in numbers or functions , predisposes an individual to a higher risk of periodontitis . these findings were further corroborated by investigations into various systemic conditions that exhibited impaired peripheral neutrophil number or functions and severe and early form of periodontal disease . these systemic conditions included chediak - higashi syndrome , cyclic neutropenia , leukocyte adhesion deficiency , papillon lefevre syndrome , down 's syndrome , actin dysfunction syndrome and diabetes mellitus , to name a few . however , it has also been demonstrated that the defensive inflammatory host response may lead to increased destruction of host connective tissue and perpetrate the loss of periodontal structures . ample research has demonstrated that in localized aggressive periodontitis ( lap ) , neutrophils are hyperactive and primed and appear to release enhanced levels of oxygen radicals , inflammatory mediators such as cytokines and matrix - degrading enzymes . this hyperactivity and reactivity of neutrophils destroys the adjacent host tissues and contributes to the destructive changes observed in inflammatory periodontitis . the various neutrophil functions investigated for deficiency in lap include chemotaxis , phagocytosis and intracellular killing , leukotriene b4 synthesis , superoxide generation and signal transduction abnormalities . the data from various laboratories in different parts of the world have yielded conflicting results , although most of the laboratories report defective chemotactic and phagocytic function and increased production of reactive oxygen species . there are very few published studies on the prevalence of lap in an indian population , and none on the prevalence of neutrophil functional abnormalities associated with lap in an indian population . the purpose of the present investigation was to examine neutrophil chemotaxis , phagocytosis , intracellular killing and superoxide ion generation in a group of patients with lap compared with healthy subjects in the indian population . in total , 20 patients ( 11 male and nine female ) were selected from the out - patient department of periodontics based on their periodontal conditions . these patients were divided into two groups , one group comprising 11 patients ( five male : six female , mean age group 22 years ) , diagnosed clinically and radiographically by two different investigators as having lap . the other group consisted of nine healthy individuals ( six male : three female , mean age group 21 years ) referred for routine oral prophylaxis procedures without any form of periodontal disease other than mild marginal gingivitis . a standard proforma consisting of demographic information ( name , age , sex , address and occupation ) , medical and past dental history and gingival index of loe and silness ( 1963 ) was recorded for the control group and clinical attachment loss ( cal ) was recorded for the test group [ table 1 ] . laboratory analysis in the form of complete hemogram was performed blind to the clinical diagnosis and showed normal hemoglobin levels , with no other abnormality in total or differential white blood cell ( wbc ) count . bleeding time and clotting time were within normal limits . data on demographics , clinical attachment loss and gingival index of the test and control groups all the subjects included in the study were non - smokers , systemically healthy and did not undergo any form of periodontal therapy for at least 6 months prior to the initiation of the study . a written consent was obtained from all the individuals participating in the study . six milliliters of whole blood was drawn under strict aseptic conditions from the anticubital vein of each patient in two separate vials , one of which contained heparin , and transported to the laboratory . after a total and differential count of wbcs , the blood in the plain vial was incubated at 37c for 2 h and the serum was separated for use in the nbt test for superoxide generation . the blood collected in the vials containing heparin was mixed with an equal quantity of minimum essential medium ( mem containing hank 's balanced salt solution ) and 3 - 5 ml of 6% dextran in 0.15 m sodium chloride ( himedia labs , mumbai , india ) . after this , the supernatant , rich in leukocytes , was collected in test tubes and centrifuged at 5000 rpm for 10 min . the resultant supernatant was discarded and the sediment was gently washed with phosphate - buffered saline and centrifuged at 3000 rpm for 10 min . differential wbc count of the resultant leukocyte - rich cell pellet yielded a neutrophil population of 85 - 87% . this cell pellet was then suspended in mem for use in chemotaxis , phagocytosis and microbicidal assays . the chemotaxis assay assembly consisted of a lower compartment filled with the chemo - attractant casein ( in hank 's balanced salt solution ; himedia labs ) . the upper compartment was made up of a syringe with 5 m pore size calcium acetate filter paper glued at one end and containing the cell suspension . it was placed inside the lower compartment and allowed to stand undisturbed for about 1 h at room temperature . after this , the cell contents in the upper compartment were emptied and the compartment was immersed in 70% methanol such that the glue melted . the filter paper strip was carefully removed , stained with hematoxylene and fixed on a glass slide to observe under the microscope . for the phagocytic assay , candida albicans was grown on sabouraud 's 2% dextrose broth for 48 h at 37c to obtain organisms in the yeast phase only . the candida cells were mixed with the neutrophil - rich cell suspension and kept undisturbed for about 30 min at 37c . the supernatant was then discarded and smears were prepared with the sediment , air dried and stained with giemsa stain . the remaining sediment from the phagocytosis assay was mixed with 2.5% sodium deoxycholate ( himedia labs ) , which lysed the leukocytes but did not damage the candida cells . after about 5 min , 4 ml of 0.01% methylene blue , which stains the ingested candida cells , was added to the tubes and centrifuged at 1500 rpm for 10 min . the supernatant was discarded and wet smears were prepared from the sediment for immediate microscopic observation in a modified neubauer 's chamber . superoxide generation was measured by using the nbt test , which is a qualitative screening test and utilizes stimulated and unstimulated neutrophils for evaluation . escherichia coli ( e. coli ) endotoxin ( himedia labs ) was used to stimulate the cells . briefly , 100 l of whole blood was added to 100 l of nbt ( himedia labs ) and 100 l of e. coli endotoxin . the assembly was incubated at 37c for 20 min and then at room temperature for a further 20 min . the test for all the samples was performed in duplicate and the control was tested simultaneously with addition of mem instead of e. coli endotoxin . in total , 20 patients ( 11 male and nine female ) were selected from the out - patient department of periodontics based on their periodontal conditions . these patients were divided into two groups , one group comprising 11 patients ( five male : six female , mean age group 22 years ) , diagnosed clinically and radiographically by two different investigators as having lap . the other group consisted of nine healthy individuals ( six male : three female , mean age group 21 years ) referred for routine oral prophylaxis procedures without any form of periodontal disease other than mild marginal gingivitis . a standard proforma consisting of demographic information ( name , age , sex , address and occupation ) , medical and past dental history and gingival index of loe and silness ( 1963 ) was recorded for the control group and clinical attachment loss ( cal ) was recorded for the test group [ table 1 ] . laboratory analysis in the form of complete hemogram was performed blind to the clinical diagnosis and showed normal hemoglobin levels , with no other abnormality in total or differential white blood cell ( wbc ) count . bleeding time and clotting time were within normal limits . data on demographics , clinical attachment loss and gingival index of the test and control groups all the subjects included in the study were non - smokers , systemically healthy and did not undergo any form of periodontal therapy for at least 6 months prior to the initiation of the study . a written consent was obtained from all the individuals participating in the study . six milliliters of whole blood was drawn under strict aseptic conditions from the anticubital vein of each patient in two separate vials , one of which contained heparin , and transported to the laboratory . after a total and differential count of wbcs , the blood in the plain vial was incubated at 37c for 2 h and the serum was separated for use in the nbt test for superoxide generation . the blood collected in the vials containing heparin was mixed with an equal quantity of minimum essential medium ( mem containing hank 's balanced salt solution ) and 3 - 5 ml of 6% dextran in 0.15 m sodium chloride ( himedia labs , mumbai , india ) . after this , the supernatant , rich in leukocytes , was collected in test tubes and centrifuged at 5000 rpm for 10 min . the resultant supernatant was discarded and the sediment was gently washed with phosphate - buffered saline and centrifuged at 3000 rpm for 10 min . differential wbc count of the resultant leukocyte - rich cell pellet yielded a neutrophil population of 85 - 87% . this cell pellet was then suspended in mem for use in chemotaxis , phagocytosis and microbicidal assays . the chemotaxis assay assembly consisted of a lower compartment filled with the chemo - attractant casein ( in hank 's balanced salt solution ; himedia labs ) . the upper compartment was made up of a syringe with 5 m pore size calcium acetate filter paper glued at one end and containing the cell suspension . it was placed inside the lower compartment and allowed to stand undisturbed for about 1 h at room temperature . after this , the cell contents in the upper compartment were emptied and the compartment was immersed in 70% methanol such that the glue melted . the filter paper strip was carefully removed , stained with hematoxylene and fixed on a glass slide to observe under the microscope . for the phagocytic assay , candida albicans was grown on sabouraud 's 2% dextrose broth for 48 h at 37c to obtain organisms in the yeast phase only . the candida cells were mixed with the neutrophil - rich cell suspension and kept undisturbed for about 30 min at 37c . the supernatant was then discarded and smears were prepared with the sediment , air dried and stained with giemsa stain . the remaining sediment from the phagocytosis assay was mixed with 2.5% sodium deoxycholate ( himedia labs ) , which lysed the leukocytes but did not damage the candida cells . after about 5 min , 4 ml of 0.01% methylene blue , which stains the ingested candida cells , was added to the tubes and centrifuged at 1500 rpm for 10 min . the supernatant was discarded and wet smears were prepared from the sediment for immediate microscopic observation in a modified neubauer 's chamber . superoxide generation was measured by using the nbt test , which is a qualitative screening test and utilizes stimulated and unstimulated neutrophils for evaluation . escherichia coli ( e. coli ) endotoxin ( himedia labs ) was used to stimulate the cells . briefly , 100 l of whole blood was added to 100 l of nbt ( himedia labs ) and 100 l of e. coli endotoxin . the assembly was incubated at 37c for 20 min and then at room temperature for a further 20 min . the test for all the samples was performed in duplicate and the control was tested simultaneously with addition of mem instead of e. coli endotoxin . the results of the present study demonstrated that lap patients exhibited defective neutrophil function as compared with healthy subject as measured by neutrophil chemotaxis , phagocytosis , microbicidal activity and superoxide generation . chemotaxis assay was performed along with phagocytosis assay , candidacidal assay and nbt test for superoxide generation on the same day and at the same time for each patient and control sample . a non - parametric mann observation and results of neutrophil function test in lap patients as compared with healthy individuals in an indian population the neutrophil chemotactic response to casein was recorded as the mean distance traveled in micrometer ( m ) by the neutrophils toward the chemoattractant . for neutrophils in lap patients , the mean distance traveled was 107.63 m ( sd + 25.98 m ) while that for neutrophils in healthy subjects was 127.44 m ( sd + 10.24 m ) [ graph 1 ] . the difference between the two means was found to be statistically significant ( p < 0.05 ) , indicating that neutrophil chemotaxis was significantly depressed in lap patients as compared with healthy subjects . neutrophil chemotaxis assay the phagocytosis of the candida cells by neutrophils was evaluated and recorded as the mean particle number ( mpn ) of candida cells ingested by neutrophils . the mean mpn in lap patients was 3.18 ( sd + 0.75 ) while that in healthy subjects was 4.61 ( sd + 0.485 ) . the difference between the means of the two groups was found to be statistically highly significant ( p < 0.01 ) , indicating that neutrophil phagocytosis was highly significantly reduced in lap patients as compared with healthy subjects[graph 2 ] . neutrophil phagocytosis assay the intracellular killing of candida cells was recorded as the percentage of candida cells killed after ingestion by neutrophils . the mean percentage of cells killed by neutrophils in lap patients was observed to be 23.27% ( sd + 3.32% ) while that in healthy subjects was 27% ( sd + 2.23% ) . the difference between the two means was statistically significant ( p < 0.05 ) , which can be interpreted as significantly reduced neutrophil microbicidal activity in lap patients as compared with healthy subjects [ graph 3 ] . neutrophil candidacidal assay the superoxide generation from both endotoxin - stimulated and unstimulated cells in both the groups was observed and recorded as % positive non - lymphoid cells . the results demonstrated that the mean % positive non - lymphoid cells in lap patients were 64.72% ( sd + 7.55 ) and that for the healthy subjects was 53.22% ( sd + 8.66 ) . the difference between the two means was found to be statistically significant ( p < 0.01 ) , indicating that superoxide generation by neutrophils in lap patients was highly significantly elevated than in control subjects [ graph 4 ] . the neutrophil chemotactic response to casein was recorded as the mean distance traveled in micrometer ( m ) by the neutrophils toward the chemoattractant . for neutrophils in lap patients , the mean distance traveled was 107.63 m ( sd + 25.98 m ) while that for neutrophils in healthy subjects was 127.44 m ( sd + 10.24 m ) [ graph 1 ] . the difference between the two means was found to be statistically significant ( p < 0.05 ) , indicating that neutrophil chemotaxis was significantly depressed in lap patients as compared with healthy subjects . the phagocytosis of the candida cells by neutrophils was evaluated and recorded as the mean particle number ( mpn ) of candida cells ingested by neutrophils . the mean mpn in lap patients was 3.18 ( sd + 0.75 ) while that in healthy subjects was 4.61 ( sd + 0.485 ) . the difference between the means of the two groups was found to be statistically highly significant ( p < 0.01 ) , indicating that neutrophil phagocytosis was highly significantly reduced in lap patients as compared with healthy subjects[graph 2 ] . the intracellular killing of candida cells was recorded as the percentage of candida cells killed after ingestion by neutrophils . the mean percentage of cells killed by neutrophils in lap patients was observed to be 23.27% ( sd + 3.32% ) while that in healthy subjects was 27% ( sd + 2.23% ) . the difference between the two means was statistically significant ( p < 0.05 ) , which can be interpreted as significantly reduced neutrophil microbicidal activity in lap patients as compared with healthy subjects [ graph 3 ] . the superoxide generation from both endotoxin - stimulated and unstimulated cells in both the groups was observed and recorded as % positive non - lymphoid cells . the results demonstrated that the mean % positive non - lymphoid cells in lap patients were 64.72% ( sd + 7.55 ) and that for the healthy subjects was 53.22% ( sd + 8.66 ) . the difference between the two means was found to be statistically significant ( p < 0.01 ) , indicating that superoxide generation by neutrophils in lap patients was highly significantly elevated than in control subjects [ graph 4 ] the dual role of neutrophils , as defenders and perpetrators , in the periodontal disease process has been well established over the past few years and has recently been reviewed thoroughly . although there are conflicting and contradictory reports of defective neutrophil function from various laboratories around the world , the majority of reports agree that certain neutrophil functions such as adhesion , chemotaxis , phagocytosis and intracellular killing are deficient in periodontitis in general , and in lap in particular . the majority also agree that the neutrophils in lap are in a hyperactive , primed state . this hyperactivity may be attributed to circulating factors , genetic make - up of an individual or environmental effects . these hyperresponsive neutrophils are considered as one of the major reasons for the periodontal tissue destruction seen in lap . the conflicting reports by different laboratories around the world may be attributed to the genetic heterogeneity among the populations studied . the present investigation was aimed to assess neutrophil chemotaxis , phagocytosis , intracellular killing and superoxide generation in lap patients in an indian population . the results of the present study indicate that neutrophils in lap patients exhibit significantly depressed chemotaxis than in healthy subjects . these observations were in conjunction with various earlier reported observations of defective chemotactic function in lap neutrophils . possible mechanisms underlying the reduced chemotactic response have been studied in the past , and include defective calcium influx factor activity , decreased diacylglycerol ( dag ) kinase activity with concomitant increase in protein kinase c ( pkc ) and dag , reduced levels of a surface glycoprotein gp110 and possibly elevated levels of proinflammatory cytokines such as tnf- and il-1 in the serum of lap patients . also , patients with reduced chemotaxis showed a significant reduction in both c5a and fmlp binding sites on the cells , the probable reason being reduction in the reduced receptor density on the cell surface because of the hereditary nature of the disease or aberrant fpr expression . the present study also reports a highly significant decrease in phagocytosis and intracellular killing in lap neutrophils , corroborative of the earlier reports of defective phagocytic function in lap neutrophils . to measure phagocytosis accurately , it is necessary to distinguish between ingested particles trapped intracellularly and surface - bound particles adhering to the plasma membrane . using c. albicans , it is possible to make this distinction by the uptake of trypan blue dye by the extracellular , but not the intracellular , heat - killed candida cells . chemotaxis and phagocytosis are both modulated by a variety of receptors and involve several activation pathways , of which one of the most important is activation of protein kinase c ( pkc ) and regulation of dag and dag kinase . the mechanisms underlying defective neutrophil phagocytosis and intracellular killing , although not as thoroughly studied , have emphasized on intrinsic cellular or cytoskeletal defects of neutrophils , interference of a. actinomycetemcomitans with phagosome and lysosome fusion by ljp neutrophils and suppression of lactoferrin release , reduced phagocytosis of some strains of p. gingivalis and a. actinomycetemcomitans by neutrophils from subsets of aggressive periodontitis patients along with elevation of superoxide production and signal transduction abnormalities such as chronic pkc activation due to elevated levels of dag . also reported that defects in lysosomal - associated membrane protein-2 ( lamp-2 ) result in impaired phagosomal maturation and formation of the phagolysosome in the neutrophils of lamp-2-deficient mice . in general , various investigations into neutrophil functions in lap emphasize that chronic activation of pkc and subsequent downregulation of dag kinase may result in neutrophil functional abnormalities such as decreased chemotaxis , reduced phagocytosis and microbicidal activity . the present study also investigated superoxide generation by neutrophils from lap patients when stimulated by endotoxins . periodontal diseases are largely gram negative infections and , to simulate the local environment in the gingival sulcus , the present study employed the nbt assay that uses e. coli , a gram negative organism that possesses very potent lipopolysaccharide , to induce neutrophil priming . the superoxide generation was observed to be highly significantly elevated in lap individuals . these observations were consistent with those of shapira et al . and hurttia et al . it is hypothesized that reactive forms of oxygen produced in vivo can inactivate protease inhibitors present in biological fluids thus increasing the activity of proteases . besides this makes it possible to presume that such reactive forms of oxygen produced by neutrophils are particularly important factors causing tissue damage . the prevalent observation regarding elevated superoxide generation in lap neutrophils is elevated levels of dag and reduced levels of dag kinase in the neutrophils from lap patients . the hyperactivity and reactivity of peripheral blood neutrophils from periodontitis patients may be a constitutive feature of the cells themselves , or a constitutive characteristic of the host in relation to the elaboration of priming agents into plasma . rates of reactive oxygen species production are important determinants of oxidative stress , a phenomenon that is associated with periodontitis . chapple and matthews also suggested a dual role for neutrophils in the production of oxidative tissue damage , involving a potentially reversible fc-receptor - mediated hyperreactivity and a constitutional hyperactivity relative to baseline oxygen radical release . conflicting results of the present study with observations of some of the earlier studies may be partly attributed to differences in the racial and ethnic background of the individuals studied , differences in methodologies employed to perform the neutrophil function assays and in the interpretation of data obtained from these assays . the limitations of the present study may also include use of c. albicans and e. coli as indicator organisms in neutrophil function assays . although not associated with periodontal diseases , these are most commonly employed in the laboratory for neutrophil assays . the phagocytic response of neutrophils to heat - killed c. albicans may differ from that to commensal organisms such as a. actinomycetemcomitans . similarly , as e. coli is not a commonly isolated organism from periodontal pockets in ap , the response of neutrophils to the e. coli endotoxin challenge may vary from that to commonly isolated organisms such as a. actinomycetemcomitans or p. gingivalis . further studies using organisms associated with periodontal disease for neutrophil function assays are required to more specifically elaborate the neutrophil function in lap . the limitations of the present study included the small sample size , which may not be representative of an indian population , and diagnosis of lap based on clinical and radiographic investigations excluding more conclusive microbiological diagnosis . in conclusion , the findings of the present study support the earlier observations that impaired neutrophil functions such as deficient neutrophil chemotaxis , phagocytosis and intracellular killing and increased superoxide generation are associated with lap , and may also serve as predisposing factors for lap in the indian population . research has indicated a more prominent role of oxidative damage to periodontal tissues due to increased superoxide generation by lap neutrophils . however , further studies with a large sample size are required to ( 1 ) elaborate on more a direct association of microbiological , immunological and genetic factors predisposing to aggressive periodontitis , ( 2 ) ascertain the cause - and - effect relationship of defective host factors such as defective neutrophil functions and aggressive periodontitis in the indian population and ( 3 ) develop therapeutic strategies aimed at reducing this neutrophil - mediated oxidative damage and restore the antibacterial properties of the neutrophils for a healthy periodontal support structure .

background : localized aggressive periodontitis ( lap ) patients exhibit abnormal neutrophil functions to a variety of environmental and host stimuli . the aim of the present study was to evaluate neutrophils chemotaxis , phagocytosis , microbicidal activity and superoxide generation in lap patients of indian origin.materials and methods : eleven lap patients and nine healthy subjects were included in the study . neutrophil chemotaxis was evaluated against an alkali - soluble casein solution using wilkinson 's method . phagocytosis and microbicidal activity assay were performed using candida albicans as an indicator organism . nitrobluetetrazolium ( nbt ) test was used to assess superoxide generation by neutrophils using e. coli endotoxin.results:the chemotactic activity and phagocytic and microbicidal activity were observed to be significantly reduced ( p < 0.01 ) in lap neutrophils . on the contrary , superoxide generation was observed to be significantly increased ( p < 0.01 ) in lap neutrophils compared with healthy individuals.conclusion:the results of the present study suggest that neutrophil functions , namely chemotaxis , phagocytosis and microbicidal activity , are deficient lap patients . however , superoxide generation was significantly increased when stimulated by endotoxins , which may explain the tissue damage seen in lap . these abnormal neutrophil functions may predispose to increased susceptibility for lap . further large - scale studies are required in the indian population to ascertain the cause - and - effect relationship of defective host factors and aggressive periodontitis and to develop treatment strategies for more predictable periodontal treatment outcome .

subjects with chronic insomnia have a higher risk for hypertension [ 13 ] and u - shaped associations have also been demonstrated between sleep duration and hypertension . studies have reported that insomnia is associated with an overall hypersecretion of acth and cortisol , suggesting an activation of the hypothalamic - pituitary - adrenal ( hpa ) axis in these patients [ 58 ] . given the well - established association of hypercortisolemia with significant medical morbidity ( eg , hypertension , metabolic syndrome , osteoporosis ) , insomnia can induce hypertension through an activation of the hypothalamic - pituitary - adrenal ( hpa ) axis . on the other hand , it has also be confirmed that subjects with hypertension are more likely to report emotional disorders such as anxiety , depression , and sleep disorders , which are usually viewed as one of their clinical features , indicating that hypertension is a risk factor for sleep disorders . it is well known that both sleep quality and hypertension are strongly influenced by lifestyle . it has been confirmed that there is a u - shaped association between alcohol consumption and hypertension or chronic insomnia . insomnia is a risk factor for hypertension [ 13 ] , and individuals with hypertension are more likely to have sleep disorders . some behaviors ( eg , exercise , smoking , alcohol consumption ) are risk factors for both sleep disorders and hypertension [ 1315 ] , sleep disorders and hypertension are both risk factors for mortality in the elderly [ 4,1619 ] , and hypertension and sleep disorders are both related to age . from all of these , we can conclude that in very elderly subjects ( aged 90 years or more ) , there is a close association between sleep quality and arterial blood pressure , which may be different from that in he general population of older adults ( aged 60 years or more ) . however , to our knowledge , no population - based study has evaluated the association between sleep quality and arterial blood pressure in the very elderly . in this study we examined the association between sleep quality and arterial blood pressure in the very elderly subjects using data from a sample of chinese nonagenarians and centenarians , the methods were reported previously . in brief , on the basis of the dujiangyan ( located in sichuan province , southwest china ) 2005 census , a cross - sectional study for age - related diseases was conducted in 870 very elderly subjects ( > 90 years ) , which was a part of the project of longevity and aging in dujiangyan ( plad ) . the plad aimed to investigate the relationship between environments , lifestyle , genetics , longevity and age - related diseases . volunteers were examined by trained professional physicians according to basic health criteria and the results were entered into standard forms , especially the questionnaire on lifestyles ( including the pittsburgh sleep quality index ( psqi ) ) . in this analysis , subjects were excluded who were using medicine for treating sleep problems of hypertension , were bedridden , had cancer , a history or clinical evidence of stroke , secondary hypertension , or were receiving anti - hypertensive treatment , had terminal stage of physical disease such as respiratory system disease , cardiovascular disease , kidney disease , dementia or cognitive decline . overall , 21 men and 26 women were not eligible for the study because they had already died or moved away from the area . of the 262 men and 561 women who were interviewed , 46 men and 117 women were excluded for the reasons above . informed consents were obtained from all participants ( as well as their legal proxies ) . the research ethics committee of the sichuan university approved the study . to assure reliability of this information , during the course of interviewing , at least 1 family member , who usually lived with the participant , took part in the interviewing and checked the completed questionnaire for accuracy . the methods were reported previously . sitting or recumbent position , right arm blood pressure ( bp ) was measured twice to the nearest 2 mmhg using a standard mercury sphygmomanometer ( phases i and v of korotkoff ) by trained nurses or physicians . the mean value of 2 measurements was used to calculate systolic bp ( sbp ) and diastolic bp ( dbp ) , and the sbp and dbp were calculated as the mean of right and left arm values in exceptional subjects . the mean of 2 readings was used for classification of bp according to joint national committee ( jnc)vii criteria into normal ( sbp < 120mmhg and dbp < 80mmhg ) , pre - hypertension ( sbp 120139 mmhg and/or dbp 8089 mmhg ) , stage 1 hypertension ( sbp 140159 mmhg and/or dbp 9099 mmhg ) , stage 2 hypertension ( sbp > 160 mmhg and/or dbp > 100 mmhg ) , systolic hypertension was defined as a sbp > 140 mmhg , the isolated systolic hypertension ( ish ) was defined as a sbp > 140 mmhg and a dbp<90mmhg , diastolic hypertension was defined as a dbp > 90 mmhg , and the isolated diastolic hypertension ( idh ) was defined as a sbp < 140 mmhg and a dbp > 90 mmhg . hypertension was defined as a sbp > 140 mmhg and/or a dbp > 90 mmhg and/or receiving anti - hypertensive treatment . subjects with confirmed hypertension and no identified cause of secondary hypertension were diagnosed with essential hypertension . the pittsburgh sleep quality index ( psqi ) the psqi is a questionnaire consisting of 19 items which are coded on a 4-point scale ( 03 ) to obtain 7 subcategories : sleep duration , sleep disturbances , sleep latency , daytime dysfunction , sleep efficiency , sleep satisfaction , and medication use . the sum of all subscores represents the total sleep quality score , ranging between 021 , with higher scores representing lower sleep quality . the individuals were categorized as follows : good sleep quality ( scores between 0 and 5 ) ; fairly good sleep quality ( scores between 6 and 10 ) ; and poor sleep quality ( scores between 11 and 21 ) . psychometric properties have demonstrated good reliability ( internal consistency : 0.89 ; test retest reliability : 0.85 ) and good construct validity for the english language version . the psqi is a widely used tool in research studies and clinical trials , and has been translated into several languages including chinese , spanish , german , and hebrew , with comparable reliability and validity values . internal consistency in the present study was =0.69 . when excluding the medication use subscale due to a low rate of medication users in this sample , internal consistency increased to =0.76 . outcome measures included the total psqi score as well as self - report of sleep latency ( sl ) ( question 2 : how long ( in minutes ) has it usually taken you to fall asleep each night ? ) , and sleep efficiency percentage ( se ) , computed as the ratio between the hours of actual sleep ( question 4 : how many hours of actual sleep did you get at night ? ) and total time in bed ( hours computed based on reported bedtime in question 1 : what time have you usually gone to bed at night ? and reported wake - time in question 3 : what time have you usually gotten up in the morning ? ) , multiplied by 100 . the baseline examination included information on age ( years ) , sex ( male / female ) , smoking habits ( yes or no ) , alcohol consumption ( yes or no ) , tea consumption ( yes or no ) , exercise ( yes or no ) , serum lipid / lipoprotein levels ( including serum triglyceride ( tg ) , total cholesterol ( tc ) , high - density lipoprotein ( hdl ) cholesterol , and low - density lipoprotein ( ldl ) cholesterol ) , fasting blood glucose ( fbg ) , serum uric acid ( sua ) , and body mass index ( bmi ) . the bmi was calculated as body weight in kilograms divided by height in meters squared . serum lipid / lipoprotein levels , fbg and sua were determined by standard laboratory techniques ( performed by a technician in the biochemistry laboratory of sichuan university ) . behaviors of smoking , alcohol consumption , tea consumption , and exercise , which included former and current status , were collected by using a general questionnaire . in the questionnaire , subjects were asked whether they had ever had behaviors of smoking , alcohol consumption , tea consumption , and exercise , and 1 of 3 answers were recorded : never , did in the past , or currently . among those who did , currently or in the past , information was obtained on the average frequency of smoking , using alcohol , drinking tea , and doing exercise , and on the number of years they had engaged in the behavior . the subjects , who did almost everyday during the past year , were classified as those with these habits current , otherwise as without . the subjects , who had done behaviors almost everyday for more than 2 years as of a year before , were classified as those with these habits previously , otherwise as without . alcohol consumption included spirits , liqueurs , wine , sherry , martini , beer , lager , hard cider , stout , and so on . all of the statistical analyses for this study were performed with the spss for windows software package , version11.5 ( spss inc , chicago , illinois , usa ) . baseline characteristics were compared between subjects with and without hypertension , between subjects with and without systolic hypertension , between subjects with and without diastolic hypertension using pearson chi - square or fisher s exact test ( where an expected cell count was < 5 ) for categorical variables and unpaired student s t test for continuous variables . baseline characteristics were also compared among different sleep quality groups using one - way for analysis of variance ( anova ) for continuous variables and pearson chi - square or fisher s exact test ( where an expected cell count was < 5 ) for categorical variables . multiple logistic regression was used to estimate the odds ratio ( or ) and 95% confidence interval ( ci ) of sleep quality as a function of increased risk for hypertension , systolic hypertension or diastolic hypertension . the following items were adjusted : age , sex , serum lipid / lipoprotein , body mass index , blood glucose level , smoking , alcohol consumption , tea consumption , and exercise . a p value < 0.05 was considered to be statistically significant , and all of the p values were 2-sided . the methods were reported previously . in brief , on the basis of the dujiangyan ( located in sichuan province , southwest china ) 2005 census , a cross - sectional study for age - related diseases was conducted in 870 very elderly subjects ( > 90 years ) , which was a part of the project of longevity and aging in dujiangyan ( plad ) . the plad aimed to investigate the relationship between environments , lifestyle , genetics , longevity and age - related diseases . volunteers were examined by trained professional physicians according to basic health criteria and the results were entered into standard forms , especially the questionnaire on lifestyles ( including the pittsburgh sleep quality index ( psqi ) ) . in this analysis , subjects were excluded who were using medicine for treating sleep problems of hypertension , were bedridden , had cancer , a history or clinical evidence of stroke , secondary hypertension , or were receiving anti - hypertensive treatment , had terminal stage of physical disease such as respiratory system disease , cardiovascular disease , kidney disease , dementia or cognitive decline . overall , 21 men and 26 women were not eligible for the study because they had already died or moved away from the area . of the 262 men and 561 women who were interviewed , 46 men and 117 women were excluded for the reasons above . informed consents were obtained from all participants ( as well as their legal proxies ) . the research ethics committee of the sichuan university approved the study . to assure reliability of this information , during the course of interviewing , at least 1 family member , who usually lived with the participant , took part in the interviewing and checked the completed questionnaire for accuracy . the methods were reported previously . sitting or recumbent position , right arm blood pressure ( bp ) was measured twice to the nearest 2 mmhg using a standard mercury sphygmomanometer ( phases i and v of korotkoff ) by trained nurses or physicians . the mean value of 2 measurements was used to calculate systolic bp ( sbp ) and diastolic bp ( dbp ) , and the sbp and dbp were calculated as the mean of right and left arm values in exceptional subjects . the mean of 2 readings was used for classification of bp according to joint national committee ( jnc)vii criteria into normal ( sbp < 120mmhg and dbp < 80mmhg ) , pre - hypertension ( sbp 120139 mmhg and/or dbp 8089 mmhg ) , stage 1 hypertension ( sbp 140159 mmhg and/or dbp 9099 mmhg ) , stage 2 hypertension ( sbp > 160 mmhg and/or dbp > 100 mmhg ) , systolic hypertension was defined as a sbp > 140 mmhg , the isolated systolic hypertension ( ish ) was defined as a sbp > 140 mmhg and a dbp<90mmhg , diastolic hypertension was defined as a dbp > 90 mmhg , and the isolated diastolic hypertension ( idh ) was defined as a sbp < 140 mmhg and a dbp > 90 mmhg . hypertension was defined as a sbp > 140 mmhg and/or a dbp > 90 mmhg and/or receiving anti - hypertensive treatment . subjects with confirmed hypertension and no identified cause of secondary hypertension were diagnosed with essential hypertension . the pittsburgh sleep quality index ( psqi ) the psqi is a questionnaire consisting of 19 items which are coded on a 4-point scale ( 03 ) to obtain 7 subcategories : sleep duration , sleep disturbances , sleep latency , daytime dysfunction , sleep efficiency , sleep satisfaction , and medication use . the sum of all subscores represents the total sleep quality score , ranging between 021 , with higher scores representing lower sleep quality . the individuals were categorized as follows : good sleep quality ( scores between 0 and 5 ) ; fairly good sleep quality ( scores between 6 and 10 ) ; and poor sleep quality ( scores between 11 and 21 ) . psychometric properties have demonstrated good reliability ( internal consistency : 0.89 ; test retest reliability : 0.85 ) and good construct validity for the english language version . the psqi is a widely used tool in research studies and clinical trials , and has been translated into several languages including chinese , spanish , german , and hebrew , with comparable reliability and validity values . when excluding the medication use subscale due to a low rate of medication users in this sample , internal consistency increased to =0.76 . outcome measures included the total psqi score as well as self - report of sleep latency ( sl ) ( question 2 : how long ( in minutes ) has it usually taken you to fall asleep each night ? ) , and sleep efficiency percentage ( se ) , computed as the ratio between the hours of actual sleep ( question 4 : how many hours of actual sleep did you get at night ? ) and total time in bed ( hours computed based on reported bedtime in question 1 : what time have you usually gone to bed at night ? and reported wake - time in question 3 : what time have you usually gotten up in the morning ? ) , multiplied by 100 . the methods were reported previously . sitting or recumbent position , right arm blood pressure ( bp ) was measured twice to the nearest 2 mmhg using a standard mercury sphygmomanometer ( phases i and v of korotkoff ) by trained nurses or physicians . the mean value of 2 measurements was used to calculate systolic bp ( sbp ) and diastolic bp ( dbp ) , and the sbp and dbp were calculated as the mean of right and left arm values in exceptional subjects . the mean of 2 readings was used for classification of bp according to joint national committee ( jnc)vii criteria into normal ( sbp < 120mmhg and dbp < 80mmhg ) , pre - hypertension ( sbp 120139 mmhg and/or dbp 8089 mmhg ) , stage 1 hypertension ( sbp 140159 mmhg and/or dbp 9099 mmhg ) , stage 2 hypertension ( sbp > 160 mmhg and/or dbp > 100 mmhg ) , systolic hypertension was defined as a sbp > 140 mmhg , the isolated systolic hypertension ( ish ) was defined as a sbp > 140 mmhg and a dbp<90mmhg , diastolic hypertension was defined as a dbp > 90 mmhg , and the isolated diastolic hypertension ( idh ) was defined as a sbp < 140 mmhg and a dbp > 90 mmhg . hypertension was defined as a sbp > 140 mmhg and/or a dbp > 90 mmhg and/or receiving anti - hypertensive treatment . subjects with confirmed hypertension and no identified cause of secondary hypertension were diagnosed with essential hypertension . the pittsburgh sleep quality index ( psqi ) was used for the subjective assessment of sleep quality . the psqi is a questionnaire consisting of 19 items which are coded on a 4-point scale ( 03 ) to obtain 7 subcategories : sleep duration , sleep disturbances , sleep latency , daytime dysfunction , sleep efficiency , sleep satisfaction , and medication use . the sum of all subscores represents the total sleep quality score , ranging between 021 , with higher scores representing lower sleep quality . the individuals were categorized as follows : good sleep quality ( scores between 0 and 5 ) ; fairly good sleep quality ( scores between 6 and 10 ) ; and poor sleep quality ( scores between 11 and 21 ) . psychometric properties have demonstrated good reliability ( internal consistency : 0.89 ; test retest reliability : 0.85 ) and good construct validity for the english language version . the psqi is a widely used tool in research studies and clinical trials , and has been translated into several languages including chinese , spanish , german , and hebrew , with comparable reliability and validity values . internal consistency in the present study was =0.69 . when excluding the medication use subscale due to a low rate of medication users in this sample , internal consistency increased to =0.76 . outcome measures included the total psqi score as well as self - report of sleep latency ( sl ) ( question 2 : how long ( in minutes ) has it usually taken you to fall asleep each night ? ) , and sleep efficiency percentage ( se ) , computed as the ratio between the hours of actual sleep ( question 4 : how many hours of actual sleep did you get at night ? ) and total time in bed ( hours computed based on reported bedtime in question 1 : what time have you usually gone to bed at night ? and reported wake - time in question 3 : what time have you usually gotten up in the morning ? ) , multiplied by 100 . the baseline examination included information on age ( years ) , sex ( male / female ) , smoking habits ( yes or no ) , alcohol consumption ( yes or no ) , tea consumption ( yes or no ) , exercise ( yes or no ) , serum lipid / lipoprotein levels ( including serum triglyceride ( tg ) , total cholesterol ( tc ) , high - density lipoprotein ( hdl ) cholesterol , and low - density lipoprotein ( ldl ) cholesterol ) , fasting blood glucose ( fbg ) , serum uric acid ( sua ) , and body mass index ( bmi ) . the bmi was calculated as body weight in kilograms divided by height in meters squared . serum lipid / lipoprotein levels , fbg and sua were determined by standard laboratory techniques ( performed by a technician in the biochemistry laboratory of sichuan university ) . behaviors of smoking , alcohol consumption , tea consumption , and exercise , which included former and current status , were collected by using a general questionnaire . in the questionnaire , subjects were asked whether they had ever had behaviors of smoking , alcohol consumption , tea consumption , and exercise , and 1 of 3 answers were recorded : never , did in the past , or currently . among those who did , currently or in the past , information was obtained on the average frequency of smoking , using alcohol , drinking tea , and doing exercise , and on the number of years they had engaged in the behavior . the subjects , who did almost everyday during the past year , were classified as those with these habits current , otherwise as without . the subjects , who had done behaviors almost everyday for more than 2 years as of a year before , were classified as those with these habits previously , otherwise as without . alcohol consumption included spirits , liqueurs , wine , sherry , martini , beer , lager , hard cider , stout , and so on . all of the statistical analyses for this study were performed with the spss for windows software package , version11.5 ( spss inc , chicago , illinois , usa ) . baseline characteristics were compared between subjects with and without hypertension , between subjects with and without systolic hypertension , between subjects with and without diastolic hypertension using pearson chi - square or fisher s exact test ( where an expected cell count was < 5 ) for categorical variables and unpaired student s t test for continuous variables . baseline characteristics were also compared among different sleep quality groups using one - way for analysis of variance ( anova ) for continuous variables and pearson chi - square or fisher s exact test ( where an expected cell count was < 5 ) for categorical variables . multiple logistic regression was used to estimate the odds ratio ( or ) and 95% confidence interval ( ci ) of sleep quality as a function of increased risk for hypertension , systolic hypertension or diastolic hypertension . the following items were adjusted : age , sex , serum lipid / lipoprotein , body mass index , blood glucose level , smoking , alcohol consumption , tea consumption , and exercise . a p value < 0.05 was considered to be statistically significant , and all of the p values were 2-sided . among the 660 participants , mean age was 93.52 years , 69 were centenarians and 444 were women . there were 150 ( 22.7% ) subjects with normal arterial blood pressure , 133 ( 20.1% ) with pre - hypertension , 216 ( 32.7% ) with stage 1 hypertension , 161(24.5% ) with stage 2 hypertension , 364 ( 55.2% ) with systolic hypertension , and 86 ( 13.0% ) with diastolic hypertension ( tables 1 and 2 ) . there were 385 ( 58.4% ) , 128 ( 19.4% ) and 147 ( 22.2% ) with good , fairly good , and poor sleep quality , respectively . the subjects with good sleep quality were younger in age ( 93.323.32 , 93.953.59 , 93.633.14 , among subjects with good , fairly good , poor sleep quality respectively , p=0.016 ) . the mean sleep quality score was 6.842.15 , the mean sl was 45.8916.72 min , and the mean se was 76.538.78% ( tables 1 and 2 ) . among the different sleep quality groups , none of the differences in levels of sbp , dbp , the prevalence of hypertension , systolic hypertension and diastolic hypertension was significant ( sbp : 140.2123.20 , 138.3522.24 , 140.3722.78 , p=0.703 ; dbp : 72.7712.30 , 72.5010.92 , 72.2312.65 , p=0.889 ; hypertension : 57.66% , 56.25% , 57.14% , p=0.847 ; systolic hypertension : 55.32% , 55.47% , 54.42% , p=0.836 ; diastolic hypertension : 13.50% , 8.59% , 15.64% , p=0.334 among subjects with good , fairly good , and poor quality sleep , respectively ) ( table 1 ) . between subjects with and without hypertension , none of the differences in sleep quality scores , sleep latency , sleep efficiency percentage and prevalence of poor sleep quality was significant ( sleep quality scores : 6.672.59 vs. 6.972.66 , p=0.139 ; sleep latency : 44.1013.17 vs. 47.2318.11 , p=0.235 ; sleep efficiency percentage : 79.1712.64 vs. 74.5614.36 , p=0.473 ; prevalence of poor sleep quality : 22.34% vs. 22.22% , p=0.778 in subjects without and with hypertension , respectively ) . between subjects with and without systolic hypertension , none of the differences in sleep quality scores , sleep latency , sleep efficiency percentage and prevalence of poor sleep quality was significant ( sleep quality scores : 6.712.71 vs. 6.952.48 , p=0.172 ; sleep latency : 44.3513.42 vs. 47.1417.99 , p=0.347 ; sleep efficiency percentage : 79.2111.91 vs. 74.3513.44 , p=0.469 ; prevalence of poor sleep quality : 22.64% vs. 21.98% , p=0.813 in subjects without and with systolic hypertension , respectively ) . between subjects with and without diastolic hypertension , none of the differences in sleep quality scores , sleep latency , sleep efficiency percentage and prevalence of poor sleep quality was significant ( sleep quality scores : 6.773.01 vs. 7.312.67 , p=0.212 ; sleep latency : 45.9913.57 vs. 45.2211.65 , p=0.881 ; sleep efficiency percentage : 76.342.25 vs. 77.792.81 , p=0.786 ; prevalence of poor sleep quality : 26.74% vs. 21.60% , p=0.207 in subjects without and with diastolic hypertension , respectively ) . neither the unadjusted nor the adjusted multiple logistic regression showed that good quality sleep had a function of decreasing the risk for hypertension ( unadjusted odds ratio ( or ) : 0.963 , 95% confidence interval ( ci ) : 0.6541.417 ; adjusted or : 0.981 , 95% ci : 0.7361.328 ) , systolic hypertension ( unadjusted or : 0.968 , 95% ci : 0.6741.497 ; adjusted or : 0.976 , 95% ci : 0.6151.541 ) or diastolic hypertension ( unadjusted or : 0.821 , 95% ci : 0.5431.205 ; adjusted or : 0 . all of these showed that among very elderly subjects there was no association between sleep quality and arterial blood pressure . among the 660 participants , mean age was 93.52 years , 69 were centenarians and 444 were women . there were 150 ( 22.7% ) subjects with normal arterial blood pressure , 133 ( 20.1% ) with pre - hypertension , 216 ( 32.7% ) with stage 1 hypertension , 161(24.5% ) with stage 2 hypertension , 364 ( 55.2% ) with systolic hypertension , and 86 ( 13.0% ) with diastolic hypertension ( tables 1 and 2 ) . there were 385 ( 58.4% ) , 128 ( 19.4% ) and 147 ( 22.2% ) with good , fairly good , and poor sleep quality , respectively . the subjects with good sleep quality were younger in age ( 93.323.32 , 93.953.59 , 93.633.14 , among subjects with good , fairly good , poor sleep quality respectively , p=0.016 ) . the mean sleep quality score was 6.842.15 , the mean sl was 45.8916.72 min , and the mean se was 76.538.78% ( tables 1 and 2 ) . among the different sleep quality groups , none of the differences in levels of sbp , dbp , the prevalence of hypertension , systolic hypertension and diastolic hypertension was significant ( sbp : 140.2123.20 , 138.3522.24 , 140.3722.78 , p=0.703 ; dbp : 72.7712.30 , 72.5010.92 , 72.2312.65 , p=0.889 ; hypertension : 57.66% , 56.25% , 57.14% , p=0.847 ; systolic hypertension : 55.32% , 55.47% , 54.42% , p=0.836 ; diastolic hypertension : 13.50% , 8.59% , 15.64% , p=0.334 among subjects with good , fairly good , and poor quality sleep , respectively ) ( table 1 ) . between subjects with and without hypertension , none of the differences in sleep quality scores , sleep latency , sleep efficiency percentage and prevalence of poor sleep quality was significant ( sleep quality scores : 6.672.59 vs. 6.972.66 , p=0.139 ; sleep latency : 44.1013.17 vs. 47.2318.11 , p=0.235 ; sleep efficiency percentage : 79.1712.64 vs. 74.5614.36 , p=0.473 ; prevalence of poor sleep quality : 22.34% vs. 22.22% , p=0.778 in subjects without and with hypertension , respectively ) . between subjects with and without systolic hypertension , none of the differences in sleep quality scores , sleep latency , sleep efficiency percentage and prevalence of poor sleep quality was significant ( sleep quality scores : 6.712.71 vs. 6.952.48 , p=0.172 ; sleep latency : 44.3513.42 vs. 47.1417.99 , p=0.347 ; sleep efficiency percentage : 79.2111.91 vs. 74.3513.44 , p=0.469 ; prevalence of poor sleep quality : 22.64% vs. 21.98% , p=0.813 in subjects without and with systolic hypertension , respectively ) . between subjects with and without diastolic hypertension , none of the differences in sleep quality scores , sleep latency , sleep efficiency percentage and prevalence of poor sleep quality was significant ( sleep quality scores : 6.773.01 vs. 7.312.67 , p=0.212 ; sleep latency : 45.9913.57 vs. 45.2211.65 , p=0.881 ; sleep efficiency percentage : 76.342.25 vs. 77.792.81 , p=0.786 ; prevalence of poor sleep quality : 26.74% vs. 21.60% , p=0.207 in subjects without and with diastolic hypertension , respectively ) . neither the unadjusted nor the adjusted multiple logistic regression showed that good quality sleep had a function of decreasing the risk for hypertension ( unadjusted odds ratio ( or ) : 0.963 , 95% confidence interval ( ci ) : 0.6541.417 ; adjusted or : 0.981 , 95% ci : 0.7361.328 ) , systolic hypertension ( unadjusted or : 0.968 , 95% ci : 0.6741.497 ; adjusted or : 0.976 , 95% ci : 0.6151.541 ) or diastolic hypertension ( unadjusted or : 0.821 , 95% ci : 0.5431.205 ; adjusted or : 0 . all of these showed that among very elderly subjects there was no association between sleep quality and arterial blood pressure . this study evaluated the association between sleep quality and arterial blood pressure in very elderly subjects . in the present study , arterial blood pressure included systolic blood pressure , diastolic blood pressure , hypertension , systolic hypertension and diastolic hypertension . in the cross - sectional observations , there was no association between sleep quality and arterial blood pressure . several studies have shown that there was a strongly significant association of sleep quality with arterial blood pressure , or with the risk of hypertension among children , adolescents , and adults ( aged 60 years or younger ) . however , many previous studies showed that there was no association of sleep quality with arterial blood pressure in the elderly aged 60 years or older [ 2830 ] . in the previous studies , participants were all less than 90 years of age . in our study , participants were all aged 90108 years . our finding provided the evidence from very elderly subjects and extended this association of sleep quality with arterial blood pressure to very elderly people . combining the results of previous studies and our finding in the present study , we should conclude here that the association between sleep quality and arterial blood pressure changed with age ; that in a population aged less than 60 years it was significant , but that in a population aged more than 60 years ( including general elderly aged 60 years or older and very elderly aged 90 years or older ) it was non - significant . progressive arterial stiffness , as a result of structural and functional changes within the vessel wall , is a feature of aging and may precede the onset of hypertension [ 2631 ] . isolated systolic hypertension , defined as raised sbp but normal dbp , is the predominant form of hypertension in elderly people ( 65 years ) ; systolic - diastolic hypertension is not as common in this population , as dbp stabilizes or even declines with advancing age [ 26,3234 ] . in our study , in very elderly chinese , there were high prevalences of hypertension ( 57.2% ) , systolic hypertension ( 55.2% ) , and isolated systolic hypertension ( 44.2% ) , and low prevalences of diastolic hypertension ( 13.0% ) , systolic - diastolic hypertension ( 11.0% ) and isolated diastolic hypertension ( 2.0% ) . the prevalence of hypertension , systolic hypertension and isolated systolic hypertension in the present study was higher than that in the elderly aged 60 years or older from the previous reports , but the prevalence of diastolic hypertension , systolic - diastolic hypertension and isolated diastolic hypertension were lower than that in the elderly aged 60 years or older from the previous reports . this was consistent with previous findings that with advancing age , progressive arterial stiffness results in sbp increase and dbp decline . these indicate that systolic and diastolic blood pressure has different clinical significance in the elderly , especially in very elderly subjects . considering this , we further independently assessed the association of systolic and diastolic blood pressure with sleep quality when the data were analyzed . all the results showed that there was no association between sleep quality and arterial blood pressure ( including sbp and dbp ) in very elderly subjects ; this strongly confirmed the non - significant association between sleep quality and arterial blood pressure in very elderly subjects . among children , adolescents , and general adults , habitually sleep problems could lead to the development and maintenance of hypertension through elevated sympathetic nervous system activity , waking physical and psychosocial stressors , increased salt retention and changed exposure to raised 24-hour blood pressure and heart rate . extended exposure to these forces could lead to the entrainment of the cardiovascular system to operate at elevated pressure equilibrium through structural adaptations such as arterial and left ventricle hypertrophic remodeling . habitual sleep problems could also contribute to hypertension by disrupting circadian rhythmicity and autonomic balance . dramatic alterations in these parameters in modern industrialized society are theorized to cause the environment sensed by the brain to become metabolically flattened and arrhythmic , disturbing the circadian rhythmicity of blood pressure in susceptible individuals . hypertension is characterized by a disturbance in the circadian rhythmicity of many physiological variables , such as a shifting of the daily blood pressure profile to higher values , an increased prevalence of the non - dipping pattern , increased blood pressure variability , and disturbances in the diurnal rhythm of cardiac output . hypertensive subjects have been shown to have reductions of > 50% in the 3 main neuronal populations of the scn in comparisons between normal subjects and hypertensive subjects who died of myocardial infarction or brain hemorrhage . there was the difference between the elderly and younger adults , children or adolescents , and there was no association of sleep with hypertension in the elderly . the mechanism of the difference is unclear and should be further explored , and might be related with the following . firstly , there was a high mortality rate among the elderly , specifically among the every elderly , and both sleep problem and hypertension were related with high mortality rate . the high mortality in the elderly more likely removed those both with sleep problem and hypertension , and left the one or the other , and removed the association between sleep problem and hypertension . secondly , degradation of arterial wall structure with age resulted in elevated arterial blood pressure , a high risk for hypertension , and insensitivity for sympathetic hormone . in the present study , there was a high prevalence of hypertension ( 57.19% ) in the sample , and provide an evidence for a high prevalence of hypertension in the elderly [ 26,3134 ] . activation of the sympathetic nervous system is an important mechanism that links sleep disorders with combined systolic and diastolic hypertension , which is common in middle - aged hypertensive patients . however , no mechanistic link has been identified between sleep disorders and isolated systolic hypertension that results from age - dependent loss of arterial compliance and accounts for nearly 60% of hypertension in elderly populations [ 3035 ] . thirdly , advanced age is associated with changes in sleep architecture , with increased difficulties in sleep initiation and maintenance . elderly subjects , who are often retired , also have more opportunities to take naps during the day [ 3638 ] . first , 870 subjects aged 90 years or older volunteered for the plad study , and among these 870 volunteers only 660 were included in the statistical analysis . there might be selection biases . because subjects with cancer , a history or clinical evidence of stroke , terminal stage of physical disease such as of respiratory system disease , cardiovascular disease , and kidney disease , usually have symptoms which could lead to insomnia and being bedridden . however , as our sample was from the community , not from hospital or nursing home , the information from them ( excluded from the sample ) might be unable to influence the practical implications of the present study . second , because of the cross - sectional nature of this study , the subjects might change their diets and the conditions related to elevated hypertension and habits of sleep . however , the lifestyles , habits of sleep and food habits of the nonagenarians / centenarians were relatively stable and similar . third , since this was a part of the plad , there might be a survival bias . finally , we did not adjust for other potential confounding factors , such as socio - economic status and family history of sleep disorders . to our knowledge , this is the first study to demonstrate the association between sleep quality and arterial blood pressure in long - lived subjects , but the association was non - significant . this is different from that in children , adolescents , and general adult ( 60 years or younger ) , and is similar with that in the general elderly ( 60 years or more ) . in very elderly chinese , there was high prevalence of hypertension , especially systolic hypertension and isolated systolic hypertension .

summarybackgroundthere is association between sleep quality and arterial blood pressure , but it is still unclear if the association also exists in the very elderly . we examined the individual association between sleep quality and arterial blood pressure among the very elderly.material/methodsthe present study analyzed data from a survey that was conducted on all residents aged 90 years or older in a district with 2,311,709 inhabitants in 2005 . sleep quality was measured using the pittsburgh sleep quality index ( psqi).resultsthe subjects included in the statistical analysis were 216 men and 444 women . there were no significant differences in sleep quality scores , sleep latency , and sleep efficiency percentage and prevalence of poor sleep quality between subjects with and without hypertension . none of the differences in systolic blood pressure , diastolic blood pressure , and prevalence of hypertension , systolic hypertension and diastolic hypertension among subjects with well , fairly and poor sleep quality were significant . multiple logistic regressions showed that unadjusted and adjusted odds ratio ( ors ) of poor sleep quality for increased risk for hypertension were significant.conclusionsamong very elderly subjects , there was no association between sleep quality and arterial blood pressure .

striated muscle cells contain a complex , highly ordered cytoskeleton , mainly composed of interdigitating thick myosin and thin actin filaments . muscle contraction and relaxation occurs by the sliding of myosin filaments past actin filaments under the strict regulation of their accessory proteins , which are responsible for their assembly and maintenance as well as the regulation of contractile activity . myosin - binding protein - c ( mybp - c ) comprises a family of accessory proteins of the thick myosin filaments that encompasses ~2% of the total myofibrillar protein . to date , two major roles have been attributed to mybp - c ; it contributes to the regular organization and stabilization of thick filaments and modulates the formation of cross - bridges between myosin and actin , via direct interactions with both filamentous systems ( as reviewed in [ 3 , 4 ] ) . within the sarcomere , mybp - c localizes to the c - zone , the cross - bridges containing region of the a - band , in 79 transverse stripes that are ~43 nm apart [ 57 ] . three mybp - c isoforms have been identified ; cardiac , slow skeletal , and fast skeletal ( cmybp - c , smybp - c , and fmybp - c ) , encoded by different genes localizing to human chromosomes 11 , 12 , and 19 , respectively [ 8 , 9 ] . the core structure of mybp - c is composed of seven immunoglobulin ( ig ) domains and three fibronectin type iii ( fn - iii ) repeats , numbered from the nh2-terminus as c1c10 ( figure 1 ; ) . a proline / alanine- ( pro / ala- ) rich motif and a conserved linker region , termed mybp - c or m - motif , flank the first ig domain , c1 . cmybp - c possesses three unique features , including an additional ig domain at the extreme nh2-terminus ( termed c0 ) , a 9-residue long insertion within the m - motif containing consensus phosphorylation sites and a 28-amino acid long loop in the middle of domain c5 [ 8 , 11 ] . conversely , smybp - c comprises a subfamily of four alternatively spliced variants ( v ) , v1v4 , differing from one another due to the retention or exclusion of select exons encoding three novel insertions . these are located at the very nh2-terminus within the pro / ala- rich motif , the middle of domain c7 , and the very cooh - terminus of the molecule , following the c10 domain ( figure 1 ; ) . the presence of these insertions may result in different topographies and possibly functions of the smybp - c variants that contain them [ 12 , 13 ] . consistent with this , v1 , which carries the cooh - terminal insertion preferentially localizes to the periphery of the m - band , rather than the c - zone , where it codistributes with its binding partners obscurin and titin ( our unpublished observations ) and plays key roles in the structural integrity of m- and a - bands . the last ig domain ( c10 ) of smybp - c v1 interacts with the second ig domain ( ig2 ) of obscurin . although the presence of the novel cooh - terminal insertion further strengthens this interaction , it is not required for binding . however , this novel motif is both necessary and sufficient to support binding of smybp - c v1 to the last ig domain of titin , m10 , which is a hotspot for mutations associated with tibial muscular dystrophy [ 14 , 15 ] . moreover , our laboratory has recently identified novel phosphorylation sites within the pro / ala- rich motif of the nh2-terminus of smybp - c , specifically ser-59 , located in the novel nh2-terminal insertion and ser-62 are targets of pka , while ser-83 and thr-84 are targets of pkc ; ser-204 is a substrate for both pka and pkc . similar to its cardiac counterpart , phosphorylation of these sites may be essential in regulating the activities of smybp - c . nevertheless , despite the differences in the core structure of the cardiac , slow , and fast mybp - c proteins , they share a ~50% homology and a ~65% identity . although mybp - c was identified over forty years ago as a contaminant of purified myosin , a better understanding of its physiology has emerged in the last two decades , paralleling the discovery of mutations in cmybp - c that result in the development of familial hypertrophic cardiomyopathy ( fhc ; [ 18 , 19 ] ) . consequently , most of our knowledge originates from studies focusing on the cardiac form of the protein . this review will focus on the role of mybp - c in regulating the formation of actomyosin cross - bridges via its direct interaction with both myosin and actin filaments . myosin is a hexameric protein consisting of two heavy chains ( mhc ) and two light chains ( regulatory and essential , rlc and elc , resp . ) . a dimer of heavy chains forms a coiled - coil helix that constitutes the rod or light meromyosin ( lmm ) segment of myosin . toward its nh2-terminus , the helix unwinds and each mhc gives rise to a catalytic head , referred to as subfragment 1 ( s1 ) that has atpase activity and participates in the formation of cross - bridges with filamentous actin [ 21 , 22 ] . the lever arm , referred to as subfragment 2 ( s2 ) , separates the s1 segment from the rod portion , and transduces the chemical energy from the hydrolysis of atp into mechanical movement along the thin filament . a pair of rlc and elc binds in tandem to each head of the s1 segment modulating the speed and force of contraction [ 24 , 25 ] . in addition to myosin light chains and actin , myosin is intimately associated with mybp - c ( table 1 and figure 2 ; ) . the cooh - terminal c10 domain of all three mybp - c isoforms harbors binding sites for the lmm portion of myosin . charged residues r1064 , e1079 , n1083 , r1100 , and r1101 present on the surface of the c10 domain of mybp - c support an electrostatic interaction with residues 15541581 of the rod domain of myosin that has a modest affinity of ~3.5 m [ 28 , 29 ] . moreover , through domains c8c10 , mybp - c interacts with titin , a giant sarcomeric protein spanning half a sarcomere , which is also tightly bound to the thick myosin filament . specifically , mybp - c binds to the first ig domain of an 11-domain superrepeat present in the a - band portion of titin . thus , it has been speculated that the repetitive binding of mybp - c to the a - band portion of titin likely contributes to its periodicity within the c - zone . consistent with these findings , a number of studies have further shown that the cooh - terminal c8c10 domains are necessary and sufficient to target mybp - c to the a - band [ 44 , 45 ] . while binding of the cooh - terminus of mybp - c to the rod domain of myosin may contribute to the maintenance and stability of the thick filament , ( reviewed in [ 3 , 4 ] ) , binding of the nh2-terminus of mybp - c to myosin may mediate contractile regulation a number of biochemical and structural studies have implicated the nh2-terminal portion of cmybp - c , containing domains c0c2 , in binding to the s2 region of myosin . specifically , the m - motif located between the c1 and c2 ig domains has been shown to bind directly to the nh2-terminal 126 residues of the s2 fragment with an affinity of ~4.3 m ; this interaction is abolished by phosphorylation of specific ser residues within the m - motif of cmybp - c via camp - dependent protein kinase [ 4649 ] . similarly , the c2 domain has been also shown to interact with the same 126 residues of the nh2-terminus of the s2 fragment , albeit with considerably lower affinity ( ~1.1 mm ) , compared to the m - motif . interestingly , molecular modeling has suggested that the interaction between the s2 portion of myosin and the c2 domain of cmybp - c is mediated by polar residues . on the other hand , the c1 domain of cmybp - c has been reported to interact with the hinge region between the s1 heads and the s2 fragment , in close proximity to the myosin light chains , while a recent study reported that the first ig domain of cmybp - c , c0 , binds to the rlcs with an affinity of ~3.2 m . in support of this , overexpressed c0 targets to the a - band independently of the rest of the molecule , likely through its interaction with the rlcs . taken together , these findings strongly suggest that cmybp - c may regulate the position and thus proximity of the myosin s1 heads relative to the actin filament , through its interaction with the s2 region and the rlcs , therefore affecting the formation of actomyosin cross - bridges . in addition to the mounting experimental support indicating the intimate association of the nh2-terminus of cmybp - c with the s2 region of myosin , there is also significant evidence supporting its direct interaction with actin thin filaments . early studies have shown that purified full - length cmybp - c binds to filamentous actin ( f - actin ) [ 36 , 50 ] . however , reconstituted thin filaments ( i.e. preincubated with troponin and tropomyosin ) fail to bind purified full length cmybp - c in the presence of edta , a ca chelating agent , but binding is restored upon addition of ca [ 36 , 50 ] . further studies have recently begun to identify the actin - binding domain(s ) of cmybp - c through biochemical and structural approaches . neutron scattering experiments have indicated that recombinant c0c2 binds to f - actin in a repetitive manner , stabilizing it , while biochemical studies have shown that recombinant c0 is capable of directly interacting with f - actin . moreover , bacterially expressed c1-c2 associates with naked f - actin as well as reconstituted thin filaments , likely through the m - motif , exhibiting a binding affinity of ~10 m to naked f - actin [ 40 , 41 ] . this finding has been also substantiated by a recent study that used negative stain electron microscopy and three - dimensional reconstruction to show that bacterially expressed c0c3 is well ordered on actin filaments near subdomain 1 . noticeably , some of the above studies report linear , nonsaturating binding , suggesting the presence of weak electrostatic interactions , as exemplified by the calculated micromolar affinity . consistent with this , these binding reactions depend largely on ph and ionic strength and are regulated by phosphorylation of cmybp - c . thus , an increase in ph or ionic strength significantly decreases the capacity of the aforementioned cmybp - c peptides to bind to f - actin [ 4042 ] . similarly , pretreatment of these recombinant peptides with pka results in a dramatic reduction of their ability to associate with f - actin . importantly , a recent study by rybakova and colleagues investigated the actin - binding capabilities of cmybp - c across its entire length . in support of previously published studies discussed above , the authors found that the nh2-terminal domains of cmybp - c bind to f - actin in a linear , nonsaturating manner , likely mediated by weak electrostatic interactions . however , recombinant full - length cmybp - c , expressed in the baculovirus system , supported a direct and saturating interaction with f - actin with a calculated kd of ~4.3 m . moreover , constructs lacking domains c0c5 exhibited similar binding properties as full - length cmybp - c ; this observation prompted the authors to conclude that the weak actin binding mediated by the nh2-terminus of the molecule does not contribute significantly to the overall affinity of cmybp - c for f - actin . moreover , the authors further showed that the actin binding supported by the cooh - terminal c6c10 domains , unlike the nh2-terminal c0c2 domains , is independent of the regulatory elements , troponin and tropomyosin , of thin filaments , the levels of ca , and the phosphorylation status of the m - motif . although the cooh - terminus of cmybp - c exhibits a higher affinity for f - actin , the minimal binding region and its physiological relevance are still elusive . these open questions are especially important because the cooh - terminal domains c6c10 also harbor binding sites for myosin and titin filaments ( discussed above ) . indeed , space constraints would favor dynamic rather than simultaneous interactions of the cooh - terminus of cmybp - c with the actin , myosin , and titin filaments . however , three - dimensional reconstruction of thick filaments demonstrated that domains c7c10 of cmybp - c run along the length of the myosin rod , suggesting that its interaction with the lmm portion of myosin is not of dynamic nature . collectively these studies suggest the presence of various but weak binding sites for f - actin in the nh2-terminus of cmybp - c as well as the presence of a high affinity , saturating binding site in the cooh - terminus of the molecule . obviously , more work is required before we can obtain a clear understanding of the physiological relevance of these interactions during muscle contraction and relaxation . early work has shown that the skeletal isoforms of mybp - c also interact directly with both thick and thin filaments ; however , their binding is much less characterized ( figure 3 and table 2 ) . similar to cmybp - c , the skeletal isoforms bind to the rod portion of sarcomeric myosin through their c10 domain and to the s2 region through their nh2-terminus . further characterization of the nh2-terminal binding has shown that the first two ig domains of smybp - c ( c1-c2 ) bind to the s2 region with an affinity of ~2.2 m [ 26 , 34 ] . in addition , native skeletal mybp - c ( presumably containing a mixture of the slow and fast isoforms ) coaggregates with f - actin in a ca sensitive manner . it becomes apparent from the above studies that our knowledge on the interaction of slow or fast mybp - c with myosin and actin filaments is limited . the identification of multiple mutations in the cardiac isoform that have been causally linked to the development of fhc is a major contributing factor that has shifted the interest of researchers towards cmybp - c ; however , this has recently changed , as mutations in smybp - c were identified in patients suffering from distal arthrogryposis type 1 ( da1 ) , a disorder characterized by congenital contractures of the hands and feet (; please see below ) . more importantly , the molecular diversity of the skeletal isoforms further complicates the relevant studies as there are at least five different skeletal forms of mybp - c ( one fast and four slow variants ) that share homologous or common sequences , contain novel insertions in the nh2- and cooh - termini , and may coexist in the same muscle , fiber , or even sarcomere . further detailed investigation is , therefore , necessary to evaluate the ability of each isoform to bind to myosin and actin filaments as well as the physiological significance of these interactions . during the last decade , an overwhelming number of mutations ( ~200 ) have been identified in the mybpc3 gene , which encodes the human cardiac mybp - c protein . these include missense , nonsense , deletion / insertion , and frame - shift mutations and have been causally linked to the development of modest and late onset familial hypertrophic cardiomyopathy ( fch ) [ 5658 ] . the majority of the nonsense and frame - shift mutations result in truncated forms of the protein , that lack the cooh - terminus harboring binding sites for lmm , actin and titin , and thus negate the ability of cmybp - c to associate with these filaments and regulate contractile activity . interestingly , these truncated peptides are often undetectable in patient biopsies , possibly due to transcriptional misregulation or rapid degradation . contrary to non - sense and frame shift mutations , missense mutations are generally associated with a less severe cardiomyopathic phenotype and do not affect the structure or stability of the protein although some of them have been suggested to weaken myosin binding . moreover , recent genome wide linkage analysis revealed that mutations in the mybpc1 gene that encodes the human skeletal mybp - c slow protein lead to the development of distal arthrogryposis type 1 , an autosomal dominant disorder characterized by congenital contractures of the hands and feet . two missense mutations have been identified to date , w236r and y856h , which are present in the m - motif and c8 domain , respectively . evaluation of skeletal muscle biopsies obtained from affected individuals revealed that slow twitch fibers were significantly smaller than fast twitch fibers . importantly , the locations of these two mutations indicate possible alterations in the ability of smybp - c to interact with myosin or actin via its nh2-terminus and to associate with the thick and titin filaments through its cooh - terminus . the causal involvement of the mybp - c family of proteins in the development of cardiac and skeletal myopathies , as exemplified by the aforementioned studies , clearly indicates that the members of this multifaceted and complex family are essential components and key regulators of muscle structure and function . through its dynamic interactions with myosin and actin filaments , mybp - c affects the formation and cycling of cross - bridges in three distinct ways : ( i ) by maintaining the normal structure of myosin and actin filaments , ( ii ) by regulating the rate at which myosin and actin interact , and ( iii ) by modulating the atpase activity of myosin . consistent with this , the addition of skeletal mybp - c reduces the critical concentration necessary for myosin polymerization in vitro and results in the formation of longer and more uniform thick filaments [ 61 , 62 ] . in addition , the nh2-terminal region c0c2 of cmybp - c supports actin bundling in vitro , as evidenced by the increased turbidity of f - actin in the solution and the formation of significantly thicker actin filaments , as visualized by electron microscopy . notably , the effect of cmybp - c on actin bundling is dependent on ph and regulated by phosphorylation events mediated by pka . moreover , in vitro motility assays , examining the sliding of actin filaments over myosin heads in the presence of full - length or truncated forms of cmybp - c revealed a significant reduction in their sliding velocity [ 40 , 63 , 64 ] . notably , the degree of reduction was also dependent on the presence of the actin regulatory proteins , troponin , and tropomyosin ; in their presence , there was a lesser effect on the decrease of actin motility over full - length myosin [ 63 , 65 ] , hmm or s1 heads . mybp - c also modulates the atpase activity of myosin , though in an indirect way , via interactions with both myosin and actin filaments supported by its nh2-terminus . in agreement with this , purified smybp - c inhibited the atpase activity of actin - activated myosin ; however , native cmybp - c moderately enhanced the actin - activated atpase activity of myosin [ 66 , 67 ] . surprisingly though , recombinant cmybp - c containing the c0c2 region inhibited the atpase activity of myosin in the presence of actin . the majority of the aforementioned studies have been performed with native proteins obtained from different species or types of muscles , presumably due to ease of purification . however , alyonycheva et al . have convincingly shown that the strength of the interaction between myosin and mybp - c is considerably increased when both proteins are purified from the same source . as such , cmybp - c and skeletal mybp - c bound with higher affinity to cardiac and skeletal myosin , respectively . as our knowledge about the molecular and functional properties of mybp - c broadens , it becomes apparent that through interactions supported by its cooh and nh2 termini , mybp - c contributes to the maintenance of the normal structure of thick filaments , and the regulation of cross - bridges formation and cycling . it has , therefore , been suggested that mybp - c may serve as a linker , whereby its cooh - terminus is necessary for anchoring it to lmm in the a - band , leaving the nh2-terminus available to modulate contractility through dynamic interactions with the head region of myosin and actin . consequently , calaghan and colleagues proposed the tether model to explain how mybp - c modulates the formation of actomyosin cross - bridges . this model suggests that both myosin binding sites on mybp - c act together by limiting the movement of the s1 heads relative to lmm and actin , thereby restricting the formation of actomyosin cross - bridges . phosphorylation of select ser residues within the m - motif of cmybp - c , mediated mainly by pka , regulates the interaction between cmybp - c and myosin by acting as an on - off switch . consequently , when in the on or dephosphorylated state , the nh2-terminus of cmybp - c binds to myosin and limits its interaction with actin , when in the off or phosphorylated state , myosin is free to interact with actin and form cross - bridges . the tether model is centered on the dynamic and highly regulated interaction of mybp - c with myosin and actin filaments . however , it is necessary to understand the orientation of mybp - c within the sarcomere , as well . two proposed models for the topology of mybp - c have been suggested : a collar model in which a trimer of mybp - c molecules wraps around the rod domain of myosin , and an axial model , where the cooh - terminus of mybp - c runs along the length of the rod domain . a stipulation of the collar model is that mybp - c is required to dimerize . in support of this , it has been shown that mybp - c dimerizes through interactions mediated by domains c5 and c7 with c8 and c10 , respectively [ 43 , 72 ] . on the other hand , x - ray diffraction in combination with molecular and computational modeling studies support the axial model , suggesting that the last three domains of mybp - c lay along the length of the myosin rod [ 71 , 73 ] . recently , a study using electron microscopy and three - dimensional reconstructions of cardiac thick filaments further demonstrated that the cooh - terminus of cmybp - c runs along the rod domain surface of thick filaments . although mybp - c dimerization is not essential for the arrangement of mybp - c along the length of the myosin rod , the axial model does not negate its dimerization . nevertheless , in both models the nh2-terminus of mybp - c extends into the interfilament space where it is accessible to bind to the s2 portion of myosin and actin . physiological dynamic interactions of mybp - c with myosin and actin are possible as the distance between thick and thin filaments is ~916 nm and the length of extended mybp - c is ~40 nm . therefore , mybp - c is capable to span the interfilament space , enabling interactions with both actin and myosin filaments . it is not without precedence that regulation of contractility occurs through dynamic interactions with both actin and myosin filaments , as the essential and regulatory light chains bind tightly to myosin , but also interact with actin [ 25 , 76 , 77 ] . additionally , twitchin , a protein similar to titin , characterized in c. elegans as a thick filament regulatory protein , interacts with both myosin and actin in a phosphorylation - dependent manner to modulate cross - bridges formation [ 78 , 79 ] . however , current models ( as described above ) to explain the mechanism of contractile regulation through dynamic binding to myosin and actin are lacking . undoubtedly , more work is necessary in order to understand the precise mechanisms by which mybp - c modulates contractility .

myosin - binding protein - c ( mybp - c ) is a family of accessory proteins of striated muscles that contributes to the assembly and stabilization of thick filaments , and regulates the formation of actomyosin cross - bridges , via direct interactions with both thick myosin and thin actin filaments . three distinct mybp - c isoforms have been characterized ; cardiac , slow skeletal , and fast skeletal . numerous mutations in the gene for cardiac mybp - c ( cmybp - c ) have been associated with familial hypertrophic cardiomyopathy ( fhc ) and have led to increased interest in the regulation and roles of the cardiac isoform . this review will summarize our current knowledge on mybp - c and its role in modulating contractility , focusing on its interactions with both myosin and actin filaments in cardiac and skeletal muscles .

the extraordinary physical and functional properties of dna and rna have led to extensive investigation into their suitability for use in many fields including therapeutics , diagnostics , chemistry , nanotechnology , and molecular computation ( reviewed in refs ( 1 and 2 ) ) . in pursuit of these endeavors , researchers exploit both natural and contrived properties of nucleic acids , including interstrand and intrastrand hybridization , strand displacement , binding of ligands to aptamers , and catalysis by nucleic acid enzymes . in general , nucleic acid enzymes are composed of single strands of either dna ( deoxyribozymes or dnazymes ) or rna ( ribozymes ) that are organized into domains required for enzymatic activity ( catalytic core domains ) and for substrate recognition ( substrate binding domains ) . dnazymes and ribozymes are capable of catalyzing a broad range of chemical reactions including cleavage , ligation , phosphorylation , and deglycoslyation of rna or dna ( reviewed in ref ( 3 ) ) . catalytic nucleic acids , sometimes in combination with aptamers , have been studied extensively for their capacity to provide the basis for biosensors ( reviewed in refs ( 15 ) ) . many strategies use nucleic acid sequences that include an intact catalytic core but have reduced enzymatic potential due to weakened association of the enzyme with its substrate ( reviewed in ref ( 4 ) ) . in such designs , the presence of the target analyte induces a conformation change that increases substrate binding affinity and catalytic activity . for example , the target may disrupt an inhibitory hairpin structure(6 ) or it may bind to the enzyme and substrate , thus strengthening their interaction.(7 ) the disadvantage of this approach lies in the potential for low - level catalytic activity in the absence of the target analyte under low stringency conditions . an alternative and inherently safer strategy involves dividing the catalytic core between multiple oligonucleotides that are capable of combining to form active enzymes . such oligonucleotides have been discovered by extensive re - engineering or in vitro evolution of ribozymes ( reviewed in refs ( 8 and 9 ) ) or by rational design utilizing specific tertiary structures within dnazymes such as guanine quartets(10 ) or stemloops.(11 ) for example , kolpashchikov(11 ) chose the e6 dnazyme for splitting because it contained a nonconserved stemloop that was thought to be required for structural , but not catalytic , function . in this report we describe a general approach for deriving partial catalytic core sequences from nucleic acid enzymes , which can then be incorporated into partial enzymes ( partzymes ) useful for assembly into mnazymes . in contrast to previous studies , our strategy does not necessarily require knowledge of , or exploitation of , predicted or observed structural or functional domains . we demonstrate our approach by engineering multiple partzyme pairs incorporating various sequences derived by splitting catalytic domains from two different nucleic acid enzymes , known as the 1023 and 817 dnazymes.(12 ) while each partzyme is inherently inactive , when they are combined in the presence of an assembly facilitator , they may associate and form a catalytically active , multicomponent nucleic acid enzyme ( mnazyme ) . mnazymes have numerous applications , including use as biosensors , as exemplified below where the target nucleic acids to be detected served as the assembly facilitators . however , assembly facilitators may also be synthetic and/or have multiple components , as shown in examples where mnazymes allowed the creation of molecular switches and cascades . it is the modular nature of mnazymes that imparts the enormous flexibility and functionality , which in turn increases the versatility of nucleic acid enzymes . mnazymes are multicomponent nucleic acid enzymes that are assembled , and therefore catalytically active , only in the presence of an assembly facilitator ( figure 1 ) . these enzymes are composed of multiple partial enzymes , or partzymes , which self - assemble in the presence of assembly facilitators and form active mnazymes that catalytically modify substrates to produce products . figure 1 provides an example of the components of an assembled mnazyme capable of recognizing and cleaving a substrate in the presence of an assembly facilitator . the partzymes have multiple domains including sensor arms that bind to the assembly facilitator or target ; substrate arms that bind the substrate ; and partial catalytic core sequences that , upon assembly , combine to provide a complete catalytic core ( figure 1 ) . mnazymes can be designed to recognize a broad range of input assembly facilitators including , for example , different target nucleic acid sequences . in response to the presence of the assembly facilitator the assembly facilitator may be a target nucleic acid present in a biological or environmental sample . in such cases , the detection of mnazyme activity is indicative of the presence of the target in the sample . this mnazyme was composed of partzymes a and b , each of which contained a substrate arm , a partial catalytic core , and a sensor arm that can bind to an assembly facilitator , such as a target nucleic acid molecule . the assembly facilitator provided the input that directed the assembly of partzymes into an mnazyme with a functional catalytic core capable of cleaving multiple substrates into products thus providing an output . if the substrate is labeled on each side of the cleavage site with a fluorophore ( f ) and a matched quencher ( q ) , then cleavage can be monitored by an increase in fluorescence . the partial catalytic core modules in the following examples were engineered by splitting the catalytic core of the unimolecular dna enzymes known as the 1023 and the 817 dnazymes.(12 ) the general strategy we developed to engineer mnazymes from dnazymes is illustrated in figure 2 . many dnazymes have similar structures : a catalytic core flanked by substrate arms that can be tailored to hybridize to a range of substrates . to engineer a new mnazyme from a dnazyme , positions are identified within the dnazyme catalytic core at which it can be split , so that each partial portion of the catalytic core can be distributed between two oligonucleotides ( candidate partzymes a and b ) . partzymes are designed such that the sequence from 5 to 3 of candidate partzyme a contains ( i ) one sensor arm portion capable of binding to an assembly facilitator molecule , ( ii ) one partial catalytic core portion derived from the dnazyme , and ( iii ) one substrate binding arm portion ; and candidate partzyme b contains ( i ) one substrate binding arm portion capable of binding adjacent to candidate partzyme a on the same substrate sequence , ( ii ) one partial catalytic core portion that contains the remaining bases from the catalytic core of the dnazyme that were not incorporated into the candidate partzyme a , and ( iii ) one sensor arm portion capable of binding to the same assembly facilitator as candidate partzyme a and in a position adjacent to partzyme a ( figure 2 ) . many dnazymes have a catalytic core flanked by substrate binding arms . to engineer mnazymes , sites for splitting the catalytic core of the dnazyme are selected , and then candidate partzymes a and b incorporating sensor arms are designed . candidate partzyme pairs a and b may be tested by mixing them with a substrate and assembly facilitator and then assessing the level of catalytic activity . a series of paired a and b candidate partzymes can be screened to determine whether they exhibit catalytic activity . candidate partzyme pairs are assessed by mixing them with suitable substrates and assembly facilitators and then ascertaining the degree of catalytic activity , if any . once active partzyme pairs have been identified , their partial catalytic cores can be incorporated into a range of new partzymes , which can be tailored for recognition of other assembly facilitators ( e.g. , new targets ) and/or other substrates . the protocol for engineering mnazymes from dnazymes is illustrated in the following example where partzymes , based on the 1023 dnazyme,(12 ) were engineered and used as components for mnazymes , referred to here as type mz1 . the sequence of the complete catalytic core domain of this dnazyme is shown in table 1 . a control 1023 dnazyme ( dz1 ) , which was capable of cleaving a chimeric dna / rna substrate designated sub2i - fb , was synthesized . substrate sub2i - fb was labeled with a fluorophore ( fam ) and quencher ( black hole quencher ) on either side of the cleavage site . the 1023 catalytic core was then split at a number of positions ( table 1 ) and candidate partzyme pairs were synthesized such that candidate partzyme b contained bases from g1 to the split position , and candidate partzyme a contained bases from the split position to a15 . candidate partzymes were designed to hybridize to both an assembly facilitator dna oligonucleotide , af - ro5 , and the substrate , sub2i - fb ( table s1 , supporting information ) . partzyme pairs were incubated in the presence of af - ro5 and sub2i - fb with 50 mm mgcl2 and the change in fluorescence , indicative of cleavage of the substrate , was monitored over time to provide a measurement of kobs ( figure s1 , supporting information ) . partzyme pair core sequences based on the 1023 dnazyme are designated mz1 , with candidate partzyme sequences designated ag . n represents a dna base , and x and y denote the positions with respect to the complete catalytic core of the dnazyme that are split upon incorporation into candidate partzymes a and b , respectively . kobs values were obtained by fitting a hyperbolic function ( by use of prism version 4.0 , graphpad software inc . ) to the fluorescence versus time data ( figure s1 , supporting information ) . the results ( table 1 ; figure s1 , supporting information ) indicated that the most suitable sites for splitting the 1023 catalytic core to generate useful partial core pairs were between c7 and t8 ( mz1c ) and between t8 and a9 ( mz1d ) . partzymes based on splitting the core between a9 and c10 ( mz1e ) showed activity , albeit reduced compared to the mz1c and mz1d splits , and other candidate pairs showed little activity . the kobs values for control 1023 dnazyme and for mz1c and mz1d partzyme designs were comparable . to confirm that the mnazyme cleaves the substrate in a similar fashion to the original dnazyme , figure 3a shows a polyacrylamide gel demonstrating cleavage of the radiolabeled dna / rna chimeric substrate sub2-fb by both a 1023 dnazyme and an mnazyme ( mz2c ) engineered as described above . cleavage of radiolabeled and fluorescent substrates by catalytic nucleic acids and use of mnazymes in the detection of target nucleic acids . ( a ) polyacryamide gel electrophoresis showing uncleaved radiolabeled substrate ( lane 1 ) and cleavage products of this substrate following incubation with a dnazyme ( lane 2 ) and an mnazyme ( lane 3 ) . test reactions contained partzyme a ( ro4aa/1 ) and partzyme b ( ro4ba/1 ) in the presence of af - ro4 or in the absence of an assembly facilitator ( no af ) . control reactions contained partzyme a only with af - ro4 ( control 1 ) ; partzyme b only with af - ro4 ( control 2 ) ; mutated partzyme a with partzyme b and af - ro4 ( control 3 ) ; or partzymes a and b with a noncomplementary oligonucleotide af - ro5 ( control 4 ) . ( c ) use of mz1d mnazymes to determine the limit of detection of a target assembly facilitator by the isothermal direct detection protocol . test reactions contained ro3ad/2-p , ro3bd/2-p , and sub2i - fb , with either various concentrations of target af - ro3 ( 750 , 500 , 250 , 50 , 10 , or 5 pm ) or no assembly facilitator ( no af ) . ( d ) mnazymes were used to monitor the accumulation of amplicons during pcr amplification of the k - ras gene from human genomic dna ( 5-fold dilutions from 100 ng to 32 pg ) . the calibration curve generated from this plot had an r = 0.999 and an efficiency of 94% . to demonstrate the generality of our approach for engineering mnazymes from dnazymes , the process was repeated and additional mnazymes based on variants of the 817 dnazyme(12 ) were produced ; they are designated here as mz2 mnazymes ( tables s2 and s3 , supporting information ) . similar to mz1 mnazymes , the activity of mz2 mnazymes was influenced by the location of the core split ( figure s2 , supporting information ) . to further analyze mnazyme activity , partzymes were synthesized to contain partial catalytic core sequences based on the 817 dnazyme variant 1 ( mz2a ) , along with substrate arms complementary to a substrate ( from ref ( 13 ) ) , referred to here as sub1i - fb . incubation of partzymes a and b in the presence of the complementary assembly facilitator af - ro4 resulted in the formation of active mz2a mnazymes that cleaved sub1i - fb , resulting in an increase in fluorescence over time ( figure 3b ) . control reactions , which lacked either partzyme b ( control 1 ) or partzyme a ( control 2 ) or which lacked the assembly facilitator and had an irrelevant noncomplementary oligonucleotide ( control 4 ) , did not show an increase in fluorescence . this confirmed that the increase in fluorescence was due to catalytic activity of the mnazymes and was dependent on the hybridization and assembly of the partzymes with the assembly facilitator and that the partzymes , either alone or as a pair , had no catalytic activity until they were assembled into mnazymes on the assembly facilitator . to further confirm that the mechanism for generation of fluorescence was cleavage of sub1i - fb by the mnazymes , a c to t mutation was introduced into the partial catalytic core within an mz2a partzyme a. this base is the equivalent of the c nucleotide at position 11 within the complete catalytic core domain and has been reported to be crucial for enzymatic activity of the 817 dnazyme.(14 ) when this mutation was incorporated into partzyme a , the catalytic activity of the mnazyme was abolished ( control 3 , figure 3b ) , consistent with a similar mechanism of catalysis between the dnazyme and mnazymes derived from it . finally , the lack of any activity of the oligonucleotide complexes containing this mutated partzyme confirms that the increase in fluorescence generated by the active mz2a mnazymes is indeed due to cleavage of the substrate and is not due to a beacon-like effect caused by hybridization of the partzyme substrate - binding arms to the substrate . the kinetics of mnazymes were analyzed , with the mz1d core split as an example , and compared to those of the 1023 dnazyme on which the mz1 series was based . both enzymes had identical substrate binding arm sequences and were designed to cleave substrate sub2i - fb . turnover was compared by measuring kcat , that is , the amount of substrate converted to product per unit of time . kinetic studies measured kcat and km values for the mnazyme as approximately 28 min and 95 nm , respectively . in comparison , the kcat and km of the dnazyme were approximately 80 min and 140 nm , respectively . kcat / km was 2.9 10 min m for the mnazyme and 5.6 10 min m for the dnazyme . together these data demonstrate that an mnazyme can function as a true enzyme capable of multiple turnover with only slightly decreased efficiency compared to the dnazyme on which it is based . further , similar to dnazymes , the rate of cleavage by mnazymes was shown to be dependent on many factors including temperature , length of the substrate binding arms , and the constituents and concentration of buffer . the arm length of various substrates was optimized to allow cleavage by mnazymes under various conditions , including those compatible with real - time polymerase chain reaction ( pcr ) . by way of example , measurement of kobs of the substrate sub2i - fb over a broad temperature range showed an optimum at around 50 c ( figure s3 , supporting information ) . in this example the number of bases in the substrate binding arms was 11 and 10 for the a and b partzymes , respectively . analysis of other mnazymes showed that the optimal temperature decreased as the lengths of substrate arms decreased , consistent with faster product dissociation of shorter oligonucleotides at lower temperatures ( data not shown ) . santoro and joyce(12 ) demonstrated that the rate of catalysis of the 1023 dnazyme is also dependent upon the concentration of divalent cations . additional experiments confirmed that the rate of catalysis by this mnazyme was similarly dependent on the concentration of mg . the catalytic rate of this mnazyme increased approximately linearly with increasing concentrations over the range of 0100 mm mg , after which the rate reached a plateau ( data not shown ) . mnazymes can be used as tools for direct detection of target nucleic acids in a protein - free , isothermal , fluorescent format . the limit of detection of such an assay was assessed in a series of three experiments with mz1d partzymes , which were designed to assemble in the presence of the target af - ro3 and cleave sub2i - fb . at 52 c the mnazyme assay could discriminate 5 pm of target assembly facilitator from the background signal ( measured by the absence of target ) ( figure 3c ) . this corresponds to approximately 125 amol or 7.5 10 copies of the target nucleic acid sequence in a 25 l reaction . changes in fluorescence following cleavage of sub2-fb allowed real - time detection and quantification of the k - ras gene from human genomic dna ( figure 3d ) . in the absence of genomic dna , fluorescence did not increase . a standard curve was generated by plotting the log of the initial dna concentration of a serial dilution of genomic dna against the threshold cycle , resulting in a linear plot with a correlation coefficient of 0.999 and a pcr efficiency of 94% . signal was detected in reactions containing as little as 32 pg of genomic dna , corresponding to approximately 10 copies of the target gene , demonstrating the high sensitivity of mnazyme detection coupled with real - time pcr . many variations of the basic mnazyme design , some of which involve using additional component oligonucleotides beyond those illustrated in figure 1 , have been shown to form active mnazymes ( figure 4 ) and provide further functionalities and utility . one possible variation incorporates a partzyme with a truncated sensor arm that requires hybridization of a stabilizer arm adjacent to it on the target to facilitate formation of an active mnazyme ( figure 4a i ) . this type of mnazyme structure can be exploited to discriminate two targets differing by a single base ( figure 5a ) . in the following example , the sensor arm of one partzyme was truncated and contained only five bases , all of which were fully complementary to an assembly facilitator ( af - xdc ) . a second oligonucleotide ( af - xdt ) was identical to af - xdc with the exception that a thymine replaced a cytosine , thus resulting in a single mismatch with respect to the truncated partzyme sensor arm . a stabilizer arm , fully complementary to the adjacent sequence of both af - xdc and af - xdt , was added to the reaction . in the presence of the partzymes , the stabilizer arm , and the fully matched target af - xdc , mnazyme activity resulted in cleavage of the substrate sub2i - fb ( figures 4a i and 5a omission of the stabilizer arm from this reaction resulted in no cleavage , demonstrating this oligonucleotide was essential for formation of active mnazymes incorporating the partzyme with the truncated sensor arm ( figures 4a ii and 5a ii ) . when the partzymes and the stabilizer arm were incubated with the mismatched target af - xdt , no mnazyme activity was evident , consistent with the capacity of mnazymes to discriminate between two sequences differing by only a single base ( figures 4a iii and 5a iii ) . together these data demonstrate that active mnazymes , which incorporate partzymes with truncated sensor arms , are formed only when the stabilizer arm is present and the assembly facilitator is fully matched to the sensor arms . absence of the stabilizer arm and/or the presence of a mismatch in the assembly facilitator within the region that hybridizes to the truncated sensor arm results in an inactive complex . the following abbreviations indicate oligonucleotides in this figure : sa , stabilizer arm ; af , assembly facilitator ; af - mut , mutated assembly facilitator ; afc , assembly facilitator component ; ai , assembly inhibitor ; s , substrate ; pz , partzyme pair ; s1p , cleavage product of substrate 1 . af - mut indicates a sequence that is identical to af with the exception of a single base difference . numbers are used to indicate the presence of more than one of any of the above elements in a particular series of complexes indicated in this figure . for example , the substrate s1 can also function as an ai and in such cases the second function is indicated in brackets . similarly , once s1 is cleaved , one of its products ( s1p ) can function as afc2 . while some oligonucleotide complexes represent active mnazymes ( a i , b i , and c i and ii ) , others are catalytically inactive ( a ii and iii , b ii and iii , and c iii ) . ( a ) detection of a single base difference by use of a partzyme , xdbd/2-p , that has a truncated five - base sensor arm in combination with the stabilizer arm , xdsa - p . reactions also contained xdsa - p in the presence of either ( i ) fully matched target ( af - xdc ) , ( iii ) mismatched target ( af - xdt ) , or ( iv ) no assembly facilitator . an additional control reaction ( ii ) contained fully matched target ( af - xdc ) but lacked the stabilizer arm ( xdsa - p ) . ( b ) demonstration of active mnazymes requiring two assembly facilitator components and inactive complexes incorporating inhibitor sequences . in addition , reactions also contained ( i ) assembly facilitator components afc1-ro1 and afc2-ro1 , ( ii ) no afc2-ro1 , ( iii ) afc1-ro1 and assembly inhibitor ai ( sub6-trb ) , or ( iv ) no assembly facilitator components . reactions contained the components of the initiating mnazyme ( ro5ad/6-p , ro5bd/6-p , and sub6-trb ) and the cascading mnazyme ( ro1ad/2-p , ro1bd/2-p , sub2i - fb , and afc1-ro1 ) . in addition , reactions either contained target af - ro5 ( i , ii ) or lacked target ( iii , iv ) , and fluorescence of either texas red ( i , iii ) or fam ( ii , iv ) was monitored over time . mnazymes can also be designed so that the formation of an active enzyme depends upon the presence of multiple assembly facilitator components ( afc ) , any one of which can be used to switch the enzyme from the on state ( active mnazyme ) to an off state ( inactive complex ) or vice versa . further , inclusion of assembly inhibitor oligonucleotides can result in formation of alternative inactive complexes that incorporate and sequester mnazyme components , including partzymes . structures incorporating two assembly facilitator components , afc1-ro1 and afc2-ro1 , were shown to facilitate active mnazyme formation followed by cleavage of the substrate sub2i - fb and a resultant increase in fluorescence ( figures 4b i and 5b omission of one of the assembly facilitators , afc2-ro1 , from this reaction resulted in inactive complexes ( figures 4b ii and 5b ii ) . an assembly inhibitor oligonucleotide ( ai ) was tested that incorporated one domain equivalent to the sequence of one assembly facilitator component ( afc2-ro1 ) plus a second domain containing noncomplementary bases appended to the termini at the junction with the other assembly facilitator component ( afc1-ro1 ) ( figure 4b iii ) . incubation of the partzymes , one assembly facilitator component ( afc1-ro1 ) , and the assembly inhibitor oligonucleotide ( sub6 ) again produced inactive complexes that were unable to cleave the substrate ( figure 5b iii ) . these alternate structures could function as a molecular switch that is on when afc2-ro1 completes the formation of an active mnazyme and is off when the assembly inhibitor binds in its place , forming an inactive complex . reactions were set up to contain partzymes and a substrate for both an initiating mnazyme ( ro5ad/6-p , ro5bd/6-p and sub6-trb ) and a cascading mnazyme ( ro1ad/2-p , ro1bd/2-p , and sub2i - fb ) plus a first assembly facilitator component , afc1-ro1 , required for the active state for the cascading mnazyme . reactions either contained or lacked the target , af - ro5 . in the presence of the target , the initiating mnazyme was formed , which cleaved the first substrate sub6-trb , resulting in an increase in texas red fluorescence ( figures 4c i and 5c i ) . cleavage of sub6-trb by the initiating mnazyme produces a cleavage product ( s1p ) that is one of two facilitator components required to direct assembly of the cascading mnazyme . the cascading mnazyme then cleaved the second substrate sub2i - fb , causing an increase in the fluorescence of fam ( figures 4c ii and 5c ii ) . as such , an increase in fluorescence of both texas red and fam is indicative of the presence of the target af - ro5 in the reaction . in the absence of target sequence , no significant increase in fluorescence of either texas red or fam was observed over time ( figure 5c iii and iv ) . in its intact state , uncleaved sub6-trb would bind to one of the partzymes of the cascading mnazyme and function as an assembly inhibitor oligonucleotide directing formation of inactive complexes ( schematic in figure 4c iii ) . this paper describes mnazymes , which are a powerful type of nucleic acid enzyme , with increased versatility and functionality that stems from the multiple components that make up these modular enzymes . the specific mnazymes characterized here are dna enzymes that comprise partial enzymes or partzymes , each of which lacks part of the sequence necessary for catalysis . partzymes combine to form catalytically active enzymes only in the presence of one or more assembly facilitators . active mnazymes catalyze substrate modification , thus producing an output signal . when the assembly facilitator is a target nucleic acid , modification of the substrate produces a signal that heralds the presence of the target in the sample . kinetic analysis established that mnazymes are capable of multiple turnover : for each target molecule present , multiple substrate molecules are modified and this results in signal amplification . the kcat values observed for both the dnazyme and mnazyme in this study are on the order of 10-fold higher than the maximum kcat reported for a dnazyme by santoro and joyce.(12 ) several factors , such as the use of higher ph and temperature , may have contributed to this result ; however , the most significant contributor is likely due to differences in the composition of the substrates used in the studies . santoro and joyce(12 ) observed that kcat was highly dependent on the specific base composition of the substrate , and ota et al.(15 ) demonstrated that a specific dnazyme cleaved a dna / rna chimeric substrate with an enhanced rate compared to its all rna counterpart . the substrate used in kinetic studies by santoro and joyce was an in vitro - transcribed rna substrate selected because it corresponded to the start codon within the hiv target . in contrast , our kinetic studies used a dna / rna chimeric substrate that was selected by screening hundreds of different substrate sequences for those that afforded faster turnover rates . a subset of substrates was chosen as the basis for a series of generic substrates , each of which can be incorporated into single or multiplexed mnazymes assays for detection of a broad range of targets . a general strategy for engineering new mnazymes based on dnazymes we demonstrated the generality of this approach by deriving mnazymes from 1023 and 817 dnazymes.(12 ) mnazymes were found to have similar properties to the dnazymes from which they were engineered . for example , these mnazymes were shown to be true enzymes capable of multiple turnover at a rate that is dependent on both the concentration of magnesium ions and the reaction temperature . like protein enzymes , dnazyme catalysis is highly dependent on tertiary structure formation , and factors that impact this even subtly can have a profound influence on the catalytic rate . therefore , it is remarkable that mnazymes have enzyme activity comparable to that of dnazymes when their fundamental differences in structure are considered . this is particularly true for mnazymes based on the 1023 dnazyme , which has no apparent stemloop or other tertiary structures within the catalytic core . such structures could theoretically promote the formation of an active mnazyme catalytic core by supporting association and/or stabilization of partial catalytic domains when the complete dnazyme core has been split between two partzymes . for example , the presence of the structural stemloop within the parent e6 dnazyme most likely contributes to the association of component oligonucleotides that constitute the binary deoxyribozyme developed by kolpashchikov.(11 ) while the enzymes exemplified in our paper all cleave nucleic acid substrates , recent work in our laboratory has extended this repertoire by deriving an mnazyme capable of ligation ( data not shown ) . furthermore , the strategy described here could be applied to derive new mnazymes from either dnazymes or ribozymes , which could have a broad range of catalytic functions . much of the power of mnazymes comes from the modularity imparted by component partzymes . in practice , partzymes can be synthesized to have sensor arms that hybridize to any target nucleic acid of interest and substrate arms that hybridize to any one of a series of generic substrates . examples in this report have used various combinations that incorporated seven partial core sequence pairs , five target binding sequences , and three substrate binding sequences to produce 12 different mnazymes . further , the use of generic substrates as probes provides multiple advantages regardless of the application for which mnazymes are being used . in particular , the use of generic substrates in protocols allowing target detection confers reliable , consistent performance regardless of the target sequence . further , their use reduces assay development time , lowers the cost of goods , and eliminates substrate waste since any excess can be used for the next system . mnazymes have functionalities useful for a range of biotechnical applications , especially molecular diagnostics , where demand is increasing for point - of - care assays and technologies with higher throughput , sensitivity , and specificity . in two demonstrations of utility described here , we reported the use of mnazymes in an isothermal , protein - free assay for direct detection of nucleic acids . the assay detected approximately 125 amol of target in 2 h by fluorescence monitoring in a format compatible with analysis on any general - purpose fluorometer . the ability of this mnazyme to detect a low concentration of target was superior to that reported for the e6 binary deoxyribozyme,(11 ) which only detected 1 nm after 30 h. our mnazyme detection strategy provides a simple , inexpensive protocol suitable for detecting targets present at a high copy number in a point - of - care setting . this isothermal mnazyme protocol could also produce a colorimetric readout if combined with an approach such as that published by lui and lu.(18 ) however , methods that use either dnazymes or split dnazymes to produce a colorimetric readout have the disadvantage that they are not capable of multiplex analysis of more than one target in a single reaction chamber . in alternative detection strategies , mnazymes can be linked to various target amplification technologies including real - time pcr . to date , no binary or split dnazymes have been described as being capable of functioning at the elevated temperatures ( 50 c ) required for pcr . we demonstrated the use of mnazyme real - time pcr to quantify a target gene present in genomic dna and detected as little as 10 copies . the method is highly sensitive and has enhanced specificity compared to other real - time pcr protocols since two partzymes and two pcr primers are required to bind for amplification and detection . a series of generic substrates has been successfully used in multiplex mnazyme real - time pcr assays that simultaneously quantified five rna transcripts.(20 ) furthermore , multiplex mnazyme real - time pcr was also used to follow an rna transcript ( oz1 rna ) and a control transcript in an international phase ii gene therapy clinical trial,(21 ) demonstrating the robustness and utility of the technology in a clinical setting . mnazymes are highly versatile enzymes encompassing a range of structures with variant oligonucleotide composition and structure . for example , mnazymes may incorporate elements such as partzymes with truncated sensor arms and stabilizer arm oligonucleotides , and/or they may require multiple assembly facilitator components for functionality . this further broadens their range of utility to encompass applications such as base - sensitive biosensor elements and computational switches ( reviewed in ref ( 2 ) ) . partzymes with truncated sensor arms , together with stabilizer arms , are particularly useful for detection of sequence variations including mutations and single nucleotide polymorphisms ( snps ) . in this report , detection of a single base change was dependent upon hybridization at 46 c between a five - base truncated sensor arm and a target . it is likely that the truncated sensor arm and the adjacent stabilizer arm synergistically cooperated by coaxial helix stacking to complete the complex . the position within the sensor arm and the specific mismatch both influence the degree of discrimination . many authors have reported that certain mismatches , for example , c - a or g - a , have a greater destabilizing effect on duplexes than others such as g - t . experiments to date indicate these rules are also useful for predicting mismatches between a target assembly facilitator and a partzyme sensor arm that result in inactive complexes . while the experiments reported in this study used an isothermal , direct detection format , we have since used partzymes with truncated arms for mutation and snp detection in an mnazyme real - time pcr ( data not shown ) . in another example , mnazyme structures were formed with the use of multiple assembly facilitator component oligonucleotides , all of which were required for enzyme activity . switch-like effect such that the absence of an assembly facilitator component , and/or the presence of an assembly inhibitor , prevented formation of an active enzyme and resulted in catalytically inactive or off complexes . in contrast , the presence of all abutting assembly facilitator components resulted in catalytically on mnazymes . combining the functions of an mnazyme that is ready to receive input and produce output along with the ability to be turned off and on provides the basis of molecular switches and provides useful components of nucleic acid cascades . these structures can be used for transducing signals , facilitating molecular computation , and/or providing structural elements useful for bioengineering nanoscale machines . our laboratory has exploited mnazyme structures to develop cascade protocols that may be linked to any isothermal mnazyme assay to increase the sensitivity of target detection and/or amplify the signal . the cascade strategy employed here is controlled by a molecular switch where an initiating mnazyme controls the formation of a second cascading mnazyme . the target - dependent assembly of the initiating mnazyme is required to produce an essential component of the cascading mnazyme , and thus the initiating mnazyme is a molecular switch to turn the cascading mnazyme on . while it was useful to label the two substrates with different fluorophores to provide evidence of both stages of the cascade in this example , labeling both substrates with the same fluorophore would be advantageous in an analytical setting , as this would increase the degree of signal amplification afforded for each target molecule . this study demonstrates the power of mnazymes , which are inherently more versatile than the antecedent dnazyme on which they were based . in general , dnazymes or ribozymes recognize and modify a target substrate ; however , the separation of target recognition from substrate modification in the mnazyme design affords increased flexibility and functionality . further , our study discloses a general method for generating new mnazymes from other nucleic acid enzymes . mnazymes use stable nucleic acid components and this will facilitate simple , fast , and cost - effective methods for the detection of targets . mnazymes are a powerful class of enzyme that can be activated by the input of any specific synthetic or biological nucleic acid to produce an amplified output , which can be generic in nature . furthermore , their activity can be switched on or off by the presence or absence of other nucleic acid sequences , and they can be linked in series to produce a molecular cascade or signal transducer . the extraordinary properties of mnazymes confers potential for their integration into diverse devices such as diagnostic biosensors , molecular computers , and/or nanoscale machines . reactions contained a bulk mix consisting of 0.2 m each of partzyme a and partzyme b , pcr buffer ii ( applied biosystems ) , and 25 mm mgcl2 , ( unless stated otherwise ) . in addition , reactions may also have contained a stabilizer , an assembly facilitator or assembly facilitator component(s ) , mutated assembly facilitator , or no assembly facilitator ( no af control reaction ) . the total volume of each reaction was 25 l . reactions were incubated at various , constant temperatures in a smartcycler system thermocycler ( cepheid ) , and fluorescence was monitored over time . a substrate - only reaction was performed to enable subtraction of the background level of fluorescence from the reactions . results in all figures are the averages from replicates and were plotted by use of excel ( version 11 ) . mnazyme activity was monitored by cleavage of a dual - labeled nucleic acid substrate composed of dna ( uppercase ) and rna ( lowercase ) bases . underlined bases indicate the position of a 6-fam ( f ) or texas red ( tr ) moiety on the 5 side of the rna bases and the black hole quencher moiety ( b ) on the 3 side of the rna bases . sub2 was either end - labeled ( sub2-fb , aaggtttcctcguccctgggca ) or internally labeled ( sub2i - fb , aaggtttcctcguccctgggca ) . the substrate used for studies of mz2 mnazymes was sub1i - fb , which was internally labeled ( actcactataggaagagatg ) . partzyme sequences used in these experiments are displayed such that the bases shown underlined form part of the catalytic core of the assembled mnazyme , bases shown in boldface type hybridize with the assembly facilitator , and bases shown in italic type hybridize to the substrate . indicates 3 phosphorylation of the partzyme , which prevents extension when partzymes are used in conjunction with pcr . the assembly facilitators ( af ) af - ro3 ( cagtgacttcacatggggcaatggcaccagcacgggcagcagctggc ) , af - ro4 ( gccattgtcgaacacctgctggatgaccagc ) , and af - ro5 ( gaaggtgtaatccgtctccacagacaaggccaggactcgtttg ) and assembly facilitator component ( afc ) afc1-ro1 ( acttccaggtcgccctgtcttccctgagc ) were homologous to sequence from the human rplp0 gene ( nm_001002 ) . the assembly facilitator af - xdt ( tgccccctcaccctcacgaaggtatacagacagatggacatccagttggtga ) was homologous to the human plxnb1 gene ( nm_002673 ) , and af - xdc ( tgccccctcaccctcacgaaggcatacagacagatggacatccagttggtga ) was homologous to the mutant a5359g(22 ) of the human plxnb1 gene . reactions were performed in triplicate and were monitored in real time at 9 s intervals for 15 min at 55 c by the isothermal direct detection method ( see above ) with the modification of 50 mm mgcl2 . test reactions for mz1 candidate partzyme pairs ( ag series ; see table s1 in supporting information ) each contained 0.2 m af - ro5 or no assembly facilitator . substrate sub2-fb ( 1 m ) was 5 end - labeled in 10 ci [ -p]atp ( perkin - elmer ) , 1 pnk buffer , and 1 unit of polynucleotide kinase ( new england biolabs ) at 37 c for 30 min . the reaction contained 1 immobuffer ( bioline ) , 50 mm mgcl2 , 5 nm labeled sub2-fb , and either 100 nm 1023 deoxyribozyme ( dz1 ) or 100 nm mnazyme [ 100 nm each of ro5ac/2-p ( caaacgagtcctggccttgtcttacaacgagaggaaacctt - p ) , ro5bc/2-p ( tgcccagggaggctagcgtggagacggattacaccttc - p ) , and af - ro5 ] or no enzyme . the reactions were stopped by adding formamide loading dye containing 100 mm ethylenediaminetetraacetic acid ( edta ) . upon completion of the reactions , the uncleaved substrate and cleavage products of the deoxyribozyme and the mnazyme were resolved by electrophoresis on a 16% denaturing polyacrylamide gel . reactions reported in figure 3b were monitored at 36 s intervals for 1 h at 40 c by the isothermal direct detection method ( see above ) with the modifications of 50 mm mgcl2 and 50 mm tris hcl ( ph 9.0 at 25 c ) . test reactions also contained sub1i - fb , ro4aa/1 ( gctggtcatccagcagcggtcgaaatagtgagt ) , and ro4ba/1 ( catctcttctccgagcgtgttcgacaatggc ) in the presence of 0.2 m af - ro4 or in the absence of an assembly facilitator ( no af ) . control reactions included ro4aa/1 with 0.2 m af - ro4 ( no partzyme b , control 1 ) ; ro4ba/1 with 0.2 m af - ro4 ( no partzyme a , control 2 ) ; mutated partzyme a ( ro4aa/1mut gctggtcatccagcagcggttgaaatagtgagt ) with ro4ba/1 and 0.2 m af - ro4 ( control 3 ) ; and ro4aa/1 and ro4ba/1 with 0.2 m af - ro5 ( control 4 ) . three independent experiments were performed , with each reaction done in triplicate and monitored in real time at 18 s intervals for 15 min at 55 c . the isothermal direct detection method ( see above ) was used with the modifications of 50 mm mgcl2 and various concentrations of sub2i - fb ( either 1000 , 750 , 500 , 250 , 200 , 150 , or 100 nm ) . mnazyme test reactions used the mz1d split ( table s1 in supporting information ) and contained 1 m ro5ad/2-p , 1 nm ro5bd/2-p , and 1 m af - ro5 ( equivalent to a maximum concentration of assembled mnazyme of 1 nm ) . the 1023 dnazyme reaction contained 1 nm dz1 . to enable the conversion of fluorescence data into reaction velocities , a standard curve was generated from a range of concentrations ( 1000 , 500 , 250 , 125 , 62.5 , or 31.1 nm ) of totally cleaved substrate . the michaelismenten equation was fitted to the data generated from the enzymatic reactions ( by use of prism version 4.0 , graphpad software inc . ) to derive the enzymatic parameters vmax and km . the kobs values were obtained by fitting a hyperbolic function ( by use of prism version 4.0 , graphpad software inc . ) to the fluorescence versus time data . reactions in figure 3c were performed in duplicate and were monitored at 72 s intervals for 2 h at 52 c by the isothermal direct detection method . all reactions contained ro3ad/2-p ( agctgctgcccgtgctggtgacaacgagaggaaacctt - p ) , ro3bd/2-p ( tgcccagggaggctagctccattgccccatgtgaagtca - p ) , and sub2i - fb . test reactions contained various concentrations of target af - ro3 ( 750 , 500 , 250 , 50 , 10 , or 5 pm ) or no assembly facilitator ( no af ) . human genomic dna extracted from the im9 cell line ( american type culture collection ) was used as template for amplification of the k - ras gene ( nt_009714.16 ) . real - time pcr amplification and quantification of the target sequence the cycling parameters were 95 c for 10 min , followed by 5 cycles of 95 c for 30 s and 55 c for 60 s , and followed by 40 cycles of 95 c for 30 s and 53 c for 60 s. the reactions contained 40 nm forward primer ( 5k - ras tagtgtattaaccttatgtgtgac ) and 200 nm of each reverse primer ( 3k - ras aatgattctgaattagctgtatc ) , k - rasad/2-p ( taaacttgtggtagttggagacaacgagaggaaacctt ) , k - rasbd/2-p ( tgcccagggaggctagctctggtggcgtaggcaagagtgcc ) , and sub2-fb , 8 mm mgcl2 , 10 units of rnasin ( promega ) , 1 immobuffer ( bioline ) , 2 units of immolase ( bioline ) , and either genomic dna template ( 100 ng , 20 ng , 4 ng , 800 pg , 160 pg , or 32 pg ) or no dna ( h2o ) . reactions in figure 5a were performed in duplicate and were monitored at 72 s intervals for 2 h at 46 c by the isothermal direct detection method . all reactions contained xdad/2-p ( actggatgtccatctgtctgacaacgagaggaaacctt - p ) , xdbd/2-p ( tgcccagggaggctagcttatgc - p ) , and sub2i - fb . reactions also contained 0.2 m xdsa - p ( cttcgtgagggtgag - p ) in the presence of either 10 nm af - xdc or 10 nm af - xdt or no assembly facilitator . an additional control reaction contained 10 nm af - xdc but lacked xdsa - p . reactions in figure 5b were performed in duplicate and were monitored at 36 s intervals for 1 h at 47 c by the isothermal direct detection method . all reactions contained ro1ad/2-p ( gctcagggaagacagggcgacctgacaacgagaggaaacctt - p ) , ro1bd/2-p ( tgcccagggaggctagctgaagtcgaggcgtgat ) , and sub2i - fb . in addition , reactions also contained either 0.2 m afc1-ro1 and afc2-ro1 , or 0.2 m afc1-ro1 , or 0.2 m each of afc1-ro1 and assembly inhibitor ai ( sub6-trb ) , or no assembly facilitator components . reactions in figure 5c were performed in duplicate , and fluorescence of both texas red and fam were monitored at 36 s intervals for 1 h at 48 c by the isothermal direct detection method . reactions contained the components of the initiating mnazyme , ro5ad/6-p ( caaacgagtcctggccttgtctacaacgagaggcgtgat - p ) , ro5bd/6-p ( ctgggaggaaggctagctgtggagacggattacaccttc - p ) , and sub6-trb , and the cascading mnazyme ( ro1ad/2-p , ro1bd/2-p , sub2i - fb , and 0.2 m afc1-ro1 ) . in addition , reactions either contained 10 nm target af - ro5 or lacked target .

to increase the versatility and utility of nucleic acid enzymes , we developed multicomponent complexes , known as mnazymes , which produce amplified output signals in response to specific input signals . multiple oligonucleotide partzymes assemble into active mnazymes only in the presence of an input assembly facilitator such as a target nucleic acid . once formed , mnazymes catalytically modify a generic substrate , generating an amplified output signal that heralds the presence of the target while leaving the target intact . we demonstrated several applications including sensitive , isothermal target detection ; discrimination of polymorphisms ; and highly specific monitoring of real - time polymerase chain reaction ( pcr ) . furthermore , we showed their capacity to function as molecular switches and to work in series to create a molecular cascade . the modular nature of mnazymes , together with the separation of input and output functionalities , provides potential for their integration into diverse devices such as diagnostic biosensors , molecular computers , and/or nanoscale machines .

it is not uncommon to see bleeding symptoms in patients in outpatient or hospital - based practice . postpartum hemorrhage complicates pregnancy and accounts for significant morbidity and mortality , particularly in underdeveloped countries.1,2 menorrhagia is a common clinical challenge and is often associated with secondary anemia , excessive fatigue , and a negative effect on health - related quality of life.3 postoperative bleeding is one of the more common complications of surgery . trauma is a leading cause of morbidity and mortality in the younger population.4 while bleeding symptoms may be commonly seen by physicians of multiple specialties , it is unclear how frequently these symptoms belie an underlying undiagnosed congenital or acquired bleeding disorder . in the us , the most common congenital bleeding disorders include von willebrand disease , which affects approximately 1% of the population ( males and females equally),5 and hemophilia a and b combined , which affect approximately 20,000 persons ( essentially all males , with rare exception).6 medications can also affect coagulation or platelet function,7 as can certain herbal supplements.8 trauma and surgery can lead to blood loss , and critical reduction in coagulation factors can lead to additional non - surgical bleeding complications ( coagulopathic bleeding).9 the most ubiquitous method for evaluating coagulation is prothrombin time ( pt)/international normalized ratio ( inr ) and activated partial thromboplastin time ( aptt ) . typically , they are ordered to monitor anticoagulant therapy ( pt / inr for warfarin , aptt for heparin ) , to evaluate coagulation preoperatively , or in response to hemorrhagic symptoms . the pt / inr provides an assessment of the extrinsic ( tissue factor - dependent ) and final common pathways of the coagulation cascade , while the aptt provides an assessment of the intrinsic ( tissue factor - independent ) and final common pathways.10 an example of a potentially life - threatening cause of unexplained recent onset or acute bleeding associated with a prolonged aptt is acquired hemophilia , with an incidence of approximately 1 to 4 per million / year.11 acquired hemophilia primarily affects older adults,12 an ever - growing segment of the population that presents for medical evaluation and care , but may also occur during pregnancy as postpartum hemorrhage or in association with other underlying diseases , including cancer and autoimmune disorders.13 prompt diagnosis is a primary determinant of prognosis in acquired hemophilia14 because initiation of definitive therapy ( ie , hemostatic and immunosuppressive ) is delayed until the diagnosis is made . acquired hemophilia - related bleeding does not respond to the typical management algorithms used for hemorrhaging in a patient and , therefore , is associated with high morbidity and mortality . severe bleeds occur in up to 90% of patients with acquired hemophilia,11 and the reported overall mortality rate in these patients ranges from 8% to 22%.1517 given the rarity of acquired hemophilia , combined with the general lack of familiarity of nonhematologists with this condition , the diagnosis of acquired hemophilia poses a clinical challenge , even in patients presenting with straightforward bleeding and an isolated , prolonged aptt . a survey was conducted of physicians across a number of specialties to identify potential barriers to the effective recognition and management of this rare but important cause of serious bleeding . the survey , based on an actual case found to be the result of acquired hemophilia , focused on participants stepwise evaluation and management of a case patient who presented to the hospital with recent - onset bleeding . the survey also assessed participants history with regard to consulting hematologists , discovering or diagnosing underlying bleeding disorders , and encountering acquired hemophilia in clinical practice . findings pertaining to the interpretation and follow - up of abnormal coagulation studies , enlistment of hematology consultation , and the diagnostic decision process in an actively bleeding case patient were the primary areas assessed in this analysis . results were evaluated across specialties to determine any specialty - specific practice trends that might hinder effective recognition and management of an actively bleeding patient with coagulopathy , including acquired hemophilia . physicians within the specialties of hematology , hematology / oncology , emergency medicine , geriatrics , internal medicine , rheumatology , obstetrics and gynecology , critical care medicine , and general surgery were randomly sampled from the american medical association physician masterfile . physicians who were part of the harris interactive online physician panel were invited via email to participate in the survey , while physicians who were not part of the panel were invited via first class mail . the invitation provided a uniform resource locator address and password for one - time use to log on to the survey site , where invitees first encountered questions to determine eligibility to participate in the survey . eligible physicians were required to be actively practicing , with additional specialty - specific requirements . in particular , hematologists , hematologist / oncologists , general surgeons , and emergency medicine and critical care physicians were required to be practicing at an acute care hospital . for internists and geriatricians , at least 60% of their practice had to consist of patients older than 60 years of age . obstetricians / gynecologists were required to have an active obstetrics practice . upon confirmation of eligibility , participants completed the online survey , the average duration of which was approximately 10 minutes . each specialty was recruited to a final sample of 50 complete responses to ensure a sufficient number of responses to generate reasonable hypotheses about specialists behavior to test in face - to - face interviews . findings from the surveys completed by the physicians specializing in hematology , hematology / oncology , emergency medicine , geriatrics , internal medicine , rheumatology , and critical care medicine are presented herein . general surgeons and obstetrics and gynecology physicians each completed different surveys from the aforementioned specialties ; the findings of those surveys are not included here . survey questions focused on the diagnostic and general therapeutic approach to the management of a case patient ( figure 1 ) whose presentation and clinical course were based on those of a real patient who experienced significant delays in diagnosis despite the involvement of multiple specialists . at each juncture in the case , participants were given lists of actions that included diagnostic tests ( laboratory , radiology , other ) , consultations , and potential treatments addressing bleeding or anemia ( local hemostasis , transfusion ) . since familiarity with the upper limits of normal of the pt / aptt tests was being tested in this study , upper limits of normal were not specified , and aptts were 25% to 30% above the upper limits of normal and set at a value that respective specialties would recognize as abnormal based on their experience with ordering and interpreting this laboratory test . the survey did not specify whether laboratory testing , imaging studies , or consultations in this hypothetical case had to be available immediately or locally , and the assumption was that respondents would answer based on their best medical judgment in an ideal practice situation . rheumatologists were only asked about an initial emergency department / urgent care presentation because it was expected that if a bleeding disorder developed in one of their patients it would present in an outpatient or emergency department / urgent care setting . conversely , critical care specialists started at presentation 2 , since their involvement in a recurrent epistaxis workup would be unlikely . vital signs were adjusted to make it more likely that a potential intensive care unit admission would require their evaluation . since emergency medicine physicians would most likely have ordered an imaging study initially to define the patient s retroperitoneal hematoma , we allowed for the option of a general surgery consultation even though the computed tomography findings were never presented . hematology / oncology respondents were given the option of ordering additional specialty coagulation laboratory tests ( eg , mixing studies ) . participants were additionally questioned about their experiences diagnosing underlying bleeding disorders and , in the case of nonhematologist specialists , consulting hematologist colleagues . conversely , hematology and hematology / oncology specialists were asked about being on the receiving end of consultations , specifically from emergency medicine personnel , and the reasons for those consultations . a total of 302 physicians ( 5051 per specialty ) participated in the case - based survey reported herein . demographics of the specialty groups were consistent : mean age ranged from 45.6 to 50.6 years , and mean years of practice experience ranged from 14.5 to 19.3 . faced with an older adult female patient with recurrent epistaxis , nearly 90% of physicians in each of the surveyed specialties indicated they would have ordered a complete blood count and coagulation studies ( pt / inr and aptt ) as part of the initial evaluation ( figure 2a ) . despite abnormal results of the aptt at 42 seconds ( with an upper limit of normal typically ranging from 35 to 39 seconds , although not specified in the case ) , less than half the physicians in most specialties would have chosen to repeat the coagulation studies as one of the next steps ( figure 2b ) . in contrast , 67% of hematologists would have repeated these studies . less than 45% of surveyed physicians in all nonhematology specialties would have consulted a hematologist after reviewing the initial coagulation study results ( figure 2b ) . rheumatologists were most likely to obtain a consult ( 43% ) , although they were presented with an initial aptt that was more abnormal ( 50 seconds ) than the aptt initially presented to the other specialists . after the patient s second presentation several weeks later with bruising and abdominal / back pain , again nearly 90% or more of respondents ( including critical care specialists , who began their case review at this juncture in the patient s clinical course ) would have ordered both a complete blood and coagulation studies as part of their initial evaluation ( figure 2c ) . when these results revealed a clearly abnormal and markedly changed aptt of 63 seconds , the majority of respondents indicated they still would not have repeated coagulation studies ( figure 2d ) . approximately 75% of internal medicine and geriatrics physicians would have consulted a hematologist at this point , compared with 47% and 50% of emergency medicine and critical care specialists , respectively ( figure 2d ) . sixty percent of hematologists and hematologists / oncologists surveyed would have evaluated the patient s peripheral blood smear at this point . in addition to the aforementioned hematologic laboratory tests , other diagnostic tests and consultations that participants would have recommended from a series of options as part of their evaluation after the patient s second presentation are shown in figure 3 . participants across specialties clearly preferred computed tomography of the abdomen ( 80%84% ) over abdominal ultrasound ( 9%28% ) , upper gastrointestinal endoscopy ( 2%16% ) , or colonoscopy ( 0%16% ) . emergency medicine physicians demonstrated the greatest breadth of testing , with 82% additionally recommending urinalysis and 92% recommending a stool guaiac test . requests for gastroenterology consultations ranged from 10% to 43% and were highest for the internal medicine group , which also had the highest proportion of physicians recommending endoscopy ( 16% each for upper gastrointestinal endoscopy and colonoscopy ) . for the purposes of assessing multiple specialties , the next 12 hours of observation in the case patient s clinical course were described as having occurred in the emergency department . by the end of this observation period , the aptt had further increased to at least twice the upper limit of normal , while the hemoglobin level had decreased . internists and geriatricians were most inclined to repeat the laboratory studies at this juncture ( figure 4 ) . in contrast with previous time points , by this point in the patient s clinical presentation , 73% to 94% of respondents would have consulted a hematologist . the majority of hematologists would have ordered 1:1 mixing studies of patient and normal plasma to rule out coagulation inhibitors ( 97% ) and fibrinogen / fibrin split products testing ( 75% ) in response to the laboratory results obtained after 12 hours of observation , as part of a diagnostic evaluation of the prolonged aptt . emergency medicine physicians were given the additional option of consulting general surgery practitioners ; 2% would have done so after the first set of laboratory results at the second presentation , and 20% would have done so in response to the second set of laboratory results . the percentages of physicians in each specialty who would have recommended admission of this case patient to a general hospital floor or back to a skilled nursing facility or nursing home after initial presentation with subsequent abnormal aptt of 42 seconds are shown in figure 5a . across the majority of specialties , less than 20% of physicians would have recommended overnight admission to a general hospital floor , while more than 50% of physicians in these specialties would have recommended discharge to a skilled nursing facility or a nursing home . in contrast with respondents from other specialties , a slightly higher proportion of hematologists and hematologist / oncologists would have endorsed a higher level of care at this point ; 36% of physicians in this group indicated they would have favored overnight hospital admission over discharge to a skilled nursing facility . the majority of physicians faced with the second presentation would have recommended that the patient be admitted to the hospital . the differences among specialties were most noticeable in allocation to a general hospital floor versus an intensive care unit after review of initial laboratory results or the second set of laboratory results obtained after 12 hours of observation ( figure 5b ) . while approximately 80% or more of physicians in each specialty would have recommended hospital admission in response to both sets of laboratory results , the proportion recommending admission specifically to the intensive care unit increased as the laboratory values deteriorated ( worsening anemia and coagulopathy ) . based on the laboratory results obtained after 12 hours of observation , nearly 50% or more of physicians in each specialty would have recommended admission to the intensive care unit in lieu of a general hospital floor , compared with approximately 40% or less across specialties after the initial laboratory results obtained during the patient s second presentation . the difference was most noticeable for physicians in emergency medicine ( 35%73% ) , the specialty most likely to refer the patient to an inpatient medical / hospitalist or critical care service . more than 85% of emergency medicine and critical care physicians reported having ever discovered an underlying bleeding disorder , while 100% of hematologists and hematologist / oncologists reported having ever diagnosed one ( figure 6a ) . the percentage of physicians in other specialties who had ever discovered an underlying bleeding diathesis ranged from 47% to 65% . among those physicians who had previously diagnosed or discovered a bleeding disorder , 14% to 77% had specifically encountered acquired hemophilia ( figure 6b ) , although this quantitative survey did not assess whether they understood this disorder . hematologists and hematologists / oncologists accounted for the highest percentage in this group , while internal medicine specialists accounted for the lowest percentage . table 2 shows the frequencies with which various specialties had ever consulted a hematologist when they encountered a patient with an abnormal pt / inr and aptt and no history of a bleeding diathesis or medications that affect coagulation . the distribution of reasons for which respondents would have consulted a hematologist is also shown in table 2 . in the majority of specialties ( emergency medicine , geriatrics , and internal medicine ) , the highest percentage of physicians had consulted a hematologist 1 to 2 times for a patient with an unexplained prolonged pt / inr or aptt . in contrast , more than 80% of rheumatologists had consulted a hematologist 3 , 4 , or 5 or more times for the same reason . the highest percentage ( 39% ) of critical care physicians the most common reason for consulting a hematologist was abnormal coagulation study results in the setting of clinical bleeding ( table 2 ) , which was parallel to the findings for the 100 additional surgeons and obstetricians surveyed ( data not shown ) . the most common response among critical care specialists to the open - ended question , what are the circumstances around which you would call for a hematology consult ? was an unexplained abnormal lab or bleeding ( 33 like or similar mentions ) , followed by any unexplained bleeding disorder conversely , the frequency with which hematologists had ever been consulted by an emergency medicine provider for an abnormal pt / inr or aptt in a patient with no history of bleeding diathesis or medications that affect coagulation is outlined in table 2 . the majority ( 57% ) of hematologists had been consulted a minimum of 5 times by their colleagues in emergency medicine . the majority of these consults pertained to significantly abnormal coagulation studies with or without clinical bleeding ( table 2 ) . faced with an older adult female patient with recurrent epistaxis , nearly 90% of physicians in each of the surveyed specialties indicated they would have ordered a complete blood count and coagulation studies ( pt / inr and aptt ) as part of the initial evaluation ( figure 2a ) . despite abnormal results of the aptt at 42 seconds ( with an upper limit of normal typically ranging from 35 to 39 seconds , although not specified in the case ) , less than half the physicians in most specialties would have chosen to repeat the coagulation studies as one of the next steps ( figure 2b ) . in contrast , 67% of hematologists would have repeated these studies . less than 45% of surveyed physicians in all nonhematology specialties would have consulted a hematologist after reviewing the initial coagulation study results ( figure 2b ) . rheumatologists were most likely to obtain a consult ( 43% ) , although they were presented with an initial aptt that was more abnormal ( 50 seconds ) than the aptt initially presented to the other specialists . after the patient s second presentation several weeks later with bruising and abdominal / back pain , again nearly 90% or more of respondents ( including critical care specialists , who began their case review at this juncture in the patient s clinical course ) would have ordered both a complete blood and coagulation studies as part of their initial evaluation ( figure 2c ) . when these results revealed a clearly abnormal and markedly changed aptt of 63 seconds , the majority of respondents indicated they still would not have repeated coagulation studies ( figure 2d ) . approximately 75% of internal medicine and geriatrics physicians would have consulted a hematologist at this point , compared with 47% and 50% of emergency medicine and critical care specialists , respectively ( figure 2d ) . sixty percent of hematologists and hematologists / oncologists surveyed would have evaluated the patient s peripheral blood smear at this point . in addition to the aforementioned hematologic laboratory tests , other diagnostic tests and consultations that participants would have recommended from a series of options as part of their evaluation after the patient s second presentation are shown in figure 3 . participants across specialties clearly preferred computed tomography of the abdomen ( 80%84% ) over abdominal ultrasound ( 9%28% ) , upper gastrointestinal endoscopy ( 2%16% ) , or colonoscopy ( 0%16% ) . emergency medicine physicians demonstrated the greatest breadth of testing , with 82% additionally recommending urinalysis and 92% recommending a stool guaiac test . requests for gastroenterology consultations ranged from 10% to 43% and were highest for the internal medicine group , which also had the highest proportion of physicians recommending endoscopy ( 16% each for upper gastrointestinal endoscopy and colonoscopy ) . for the purposes of assessing multiple specialties , the next 12 hours of observation in the case patient s clinical course were described as having occurred in the emergency department . by the end of this observation period , the aptt had further increased to at least twice the upper limit of normal , while the hemoglobin level had decreased . internists and geriatricians were most inclined to repeat the laboratory studies at this juncture ( figure 4 ) . in contrast with previous time points , by this point in the patient s clinical presentation , 73% to 94% of respondents would have consulted a hematologist . the majority of hematologists would have ordered 1:1 mixing studies of patient and normal plasma to rule out coagulation inhibitors ( 97% ) and fibrinogen / fibrin split products testing ( 75% ) in response to the laboratory results obtained after 12 hours of observation , as part of a diagnostic evaluation of the prolonged aptt . emergency medicine physicians were given the additional option of consulting general surgery practitioners ; 2% would have done so after the first set of laboratory results at the second presentation , and 20% would have done so in response to the second set of laboratory results . the percentages of physicians in each specialty who would have recommended admission of this case patient to a general hospital floor or back to a skilled nursing facility or nursing home after initial presentation with subsequent abnormal aptt of 42 seconds are shown in figure 5a . across the majority of specialties , less than 20% of physicians would have recommended overnight admission to a general hospital floor , while more than 50% of physicians in these specialties would have recommended discharge to a skilled nursing facility or a nursing home . in contrast with respondents from other specialties , a slightly higher proportion of hematologists and hematologist / oncologists would have endorsed a higher level of care at this point ; 36% of physicians in this group indicated they would have favored overnight hospital admission over discharge to a skilled nursing facility . the majority of physicians faced with the second presentation would have recommended that the patient be admitted to the hospital . the differences among specialties were most noticeable in allocation to a general hospital floor versus an intensive care unit after review of initial laboratory results or the second set of laboratory results obtained after 12 hours of observation ( figure 5b ) . while approximately 80% or more of physicians in each specialty would have recommended hospital admission in response to both sets of laboratory results , the proportion recommending admission specifically to the intensive care unit increased as the laboratory values deteriorated ( worsening anemia and coagulopathy ) . based on the laboratory results obtained after 12 hours of observation , nearly 50% or more of physicians in each specialty would have recommended admission to the intensive care unit in lieu of a general hospital floor , compared with approximately 40% or less across specialties after the initial laboratory results obtained during the patient s second presentation . the difference was most noticeable for physicians in emergency medicine ( 35%73% ) , the specialty most likely to refer the patient to an inpatient medical / hospitalist or critical care service . more than 85% of emergency medicine and critical care physicians reported having ever discovered an underlying bleeding disorder , while 100% of hematologists and hematologist / oncologists reported having ever diagnosed one ( figure 6a ) . the percentage of physicians in other specialties who had ever discovered an underlying bleeding diathesis ranged from 47% to 65% . among those physicians who had previously diagnosed or discovered a bleeding disorder , 14% to 77% had specifically encountered acquired hemophilia ( figure 6b ) , although this quantitative survey did not assess whether they understood this disorder . hematologists and hematologists / oncologists accounted for the highest percentage in this group , while internal medicine specialists accounted for the lowest percentage . table 2 shows the frequencies with which various specialties had ever consulted a hematologist when they encountered a patient with an abnormal pt / inr and aptt and no history of a bleeding diathesis or medications that affect coagulation . the distribution of reasons for which respondents would have consulted a hematologist is also shown in table 2 . in the majority of specialties ( emergency medicine , geriatrics , and internal medicine ) , the highest percentage of physicians had consulted a hematologist 1 to 2 times for a patient with an unexplained prolonged pt / inr or aptt . in contrast , more than 80% of rheumatologists had consulted a hematologist 3 , 4 , or 5 or more times for the same reason . the highest percentage ( 39% ) of critical care physicians had consulted a hematologist at least 5 times for such a patient . the most common reason for consulting a hematologist was abnormal coagulation study results in the setting of clinical bleeding ( table 2 ) , which was parallel to the findings for the 100 additional surgeons and obstetricians surveyed ( data not shown ) . the most common response among critical care specialists to the open - ended question , what are the circumstances around which you would call for a hematology consult ? was an unexplained abnormal lab or bleeding ( 33 like or similar mentions ) , followed by any unexplained bleeding disorder conversely , the frequency with which hematologists had ever been consulted by an emergency medicine provider for an abnormal pt / inr or aptt in a patient with no history of bleeding diathesis or medications that affect coagulation is outlined in table 2 . the majority ( 57% ) of hematologists had been consulted a minimum of 5 times by their colleagues in emergency medicine . the majority of these consults pertained to significantly abnormal coagulation studies with or without clinical bleeding ( table 2 ) . bleeding is commonly encountered in outpatient and hospital - based medical practice and can have a wide variety of underlying causes . while bleeding may be relatively common , most acquired and congenital bleeding disorders are uncommon , and some , such as acquired hemophilia , are rare . nevertheless , the consequences of failure to recognize promptly and treat properly a bleeding disorder may be significant.18 in the case of acquired hemophilia , morbidity and mortality rates are particularly high : severe bleeding is experienced by up to 90% of affected patients , and mortality rates are as high as 22%.11 this survey provided a step - wise methodology to tease out specialty - specific patterns of interpretation of clinical data to identify barriers to the diagnosis and treatment of underlying bleeding disorders . the sample size obtained across specialties was sufficient to generalize these findings , at least to the point of identifying specific issues for education and development of clinical decision - making pathways . when presented with a clinical picture that includes a recent history or symptoms of active bleeding , clinicians typically obtain coagulation times , such as the pt / inr and aptt , as part of the initial diagnostic evaluation . proper interpretation of laboratory test results includes recognition of abnormal values and , more important , the potential clinical significance of such results . a common pitfall in the interpretation of coagulation times is failure to appreciate that even mildly abnormal values may represent a serious underlying coagulation deficit . another important observation is to identify how an abnormal laboratory value may have changed over time , which can be facilitated by the ability of electronic medical record systems to display data trends . in the absence of iatrogenic causes , even a mildly elevated pt / inr or aptt may be indicative of true coagulopathy and should not be ignored or dismissed , particularly when there is evidence of bleeding , as was the case with this patient , even at initial presentation . after excluding laboratory error , the differential diagnosis of an isolated prolongation of aptt includes heparin effect , lupus anticoagulant , and deficiency of or antibody against an intrinsic pathway factor ( viii , ix , xi , or xii).19,20 a detailed history , focusing on factors such as heparin exposure,19,20 history of thromboembolism ( lupus anticoagulant),21 and prior personal or family history of bleeding,22 may provide diagnostic clues . laboratory testing should include a 1:1 mixing test of patient plasma with control plasma to determine whether a prolonged aptt is the result of an intrinsic pathway factor deficiency or an inhibitor that continues to block the activity of the intrinsic system even in the presence of control plasma . the majority of inhibitor antibodies identified in this manner will turn out to be lupus anticoagulants . although far less common , this is the same diagnostic pathway that leads to the identification of the antifactor viii antibodies associated with acquired hemophilia.23 unlike acquired hemophilia , lupus anticoagulants typically do not present with bleeding , and the abnormal aptt is due to interference with phospholipid - dependent coagulation reactions . once an acquired antifactor viii antibody is suspected , confirmatory testing includes measuring factor viii activity , which should be significantly reduced , and the bethesda assay,24 which is used to quantitate antifactor viii antibodies inhibitor activity . we found a general lack of appropriate consideration and response to the presenting symptom of bleeding and the prolonged aptt throughout this case study . this is consistent with data from the european acquired haemophilia registry ( each-2 ) , which reported a median delay of 3 days between onset of bleeding symptoms and the diagnosis of acquired hemophilia and a median delay of one day between the first abnormal aptt test in those same patients and the established diagnosis.23 in addition , we found that emergency medicine and critical care physicians were reluctant to consider a bleeding disorder as the primary explanation for this patient s clinical presentation . the disposition of a patient with active hemorrhage and evidence of coagulopathy should be based on several factors , including the patient s current condition and anticipated clinical course , taking into account the presenting vital signs and evolving laboratory findings . at the time of the patient s second presentation , vital signs were notable for mild tachycardia and a pulse pressure at the upper limit of normal , and subsequent laboratory findings indicated a decreasing hemoglobin level and an increasing aptt . these findings alone prompted hospital admission , although the exact location ( general floor versus intensive care unit ) of admission may vary , based on the level of monitoring and nurse - to - patient ratios in a particular hospital . another important variable is the anticipated potential for clinical deterioration , which is based in large part on clinician appreciation of the seriousness of the diagnosis . we found a consistent tendency to consider admission to a general floor bed with the second presentation , even though this was ultimately an unstable , critically ill patient with an undiagnosed bleeding disorder . we also found that the physicians who participated in this survey were reluctant to consult a hematologist as they worked through this case scenario , particularly given that options for additional testing ( liver function , disseminated intravascular coagulation ) were not available to evaluate for common causes of coagulopathy . relative to their reported historical experience with hematology consultations during an average practice experience of approximately 20 years , this survey finding was somewhat surprising . rheumatologists and critical care specialists reported a greater frequency of hematology consultation relative to the other specialties ( table 2 ) . we would expect the emergency medicine physicians to be most likely to consult a hematologist , yet 16% of them reported never having consulted a hematologist . the highest percentage ( 46% ) had only consulted a hematologist 1 or 2 times , and almost one quarter of geriatricians and internists had never consulted a hematologist , even though these specialists would be expected to first encounter patients with undiagnosed bleeding disorders , including acquired hemophilia . one potential reason for not seeking hematology consultation might be the lack of availability of hematology / oncology specialists with expertise in coagulation disorders , including in rural and community hospitals . a limitation of this survey was that one can not interpret the thinking behind the responses of the individual participants . therefore , 31 qualitative 45-minute interviews were conducted subsequent to the quantitative study and focused particularly on critical care ( n = 7 ) , emergency medicine ( n = 6 ) , hematology / oncology ( n = 4 ) , or hematology ( n = 2 ) physicians to understand the reasoning behind their decisions and depth of knowledge ( unpublished data ) . we found that the physicians focus was generally on finding the source ( location ) of bleeding and not on finding the underlying reason for bleeding . this could potentially lead to surgical intervention in the face of an underlying bleeding disorder , with subsequent adverse outcomes . in a series of 67 patients with acquired hemophilia at a single center in bonn , germany , 4 of 5 deaths were the result of surgical intervention for bleeding at outside hospitals in the setting of a delayed diagnosis of acquired hemophilia.13 when queried about their experience encountering and/or diagnosing underlying bleeding disorders , particularly acquired hemophilia , more than 85% of physicians in hematology , hematology / oncology , emergency medicine , and critical care medicine reported having ever discovered or diagnosed an underlying bleeding disorder , compared with 65% , 54% , and 47% of rheumatologists , internists , and geriatricians , respectively . while reports of ever specifically having encountered acquired hemophilia were high , it is unclear from this study whether the participants truly understood the diagnosis . this seems unlikely , given the rarity of acquired hemophilia , relative to the reported frequency of having encountered it . except for hematology and/or oncology ( 77% ) and critical care ( 36% ) specialists , approximately one quarter of surveyed physicians had ever encountered acquired hemophilia . although they accounted for the highest percentage of physicians who had ever encountered this condition , nearly one quarter of hematologists had never encountered acquired hemophilia . subsequent unpublished data from the aforementioned qualitative research further suggest that , compared with hematology practitioners , specialists in hematology / oncology , who likely practice mostly oncology , might be able to identify mixing studies and inhibitors but might not fully understand the underlying pathophysiology that constitutes acquired hemophilia , making it hard for them to recognize the condition . given the survey findings reflecting the infrequency with which most physicians have encountered these conditions , consultation with a hematologist may facilitate the diagnostic evaluation and proper management of a hemorrhaging patient suspected of having an underlying bleeding diathesis , particularly acquired hemophilia . the consulting hematologist can provide specific guidance , leading to the prompt diagnosis and optimal management of an actively bleeding patient with acquired hemophilia , including initiation of immunosuppression , which is usually necessary to eradicate the inhibitor and to prevent additional bleeding episodes . however , this requires a level of familiarity and expertise in treating acquired hemophilia and other rare bleeding disorders that is not often seen outside of an academic hematology practice . this represents yet another barrier to the effective diagnosis and management of this rare yet serious bleeding diathesis . for the hospitalist or intensivist charged with the care of a bleeding patient , immediate stabilization is the initial priority and should take precedence over determination of a specific etiology of bleeding . however , in the presence of underlying coagulopathy , particularly acquired hemophilia or other rare disorders , traditional measures of stabilization may not be as effective as expected . a prolonged pt / inr or aptt in the absence of iatrogenic causes should never be ignored , even with only minimally prolonged values , and determination of the cause of an abnormal coagulation study should carry at least equal weight to looking for the anatomic site of bleeding . any delay in establishing the diagnosis of a bleeding diathesis such as acquired hemophilia can result in significant morbidity or even death . while hospitalists and intensivists should be able to conduct a thorough differential of bleeding and eliminate most common etiologies , consultation with a hematologist ( particularly one with specific expertise in coagulation disorders ) may facilitate the evaluation of coagulopathic patients and subsequent interpretation of diagnostic findings , as well as initiation of appropriate treatment . given the rarity of acquired hemophilia , as exemplified by the findings of this survey , physicians must harbor a high index of suspicion to diagnose this condition promptly in patients who present with recent - onset or acute bleeding . given the high morbidity and mortality the insights from this survey highlights knowledge and practice gaps that could be the focus of targeted educational initiatives , including diagnostic algorithms , to ensure proper and efficient workup of the abnormal laboratory studies that characterize acquired hemophilia .

background : bleeding symptoms commonly seen by multiple physician specialties may belie undiagnosed congenital or acquired bleeding disorders . acquired hemophilia is a potentially life - threatening cause of unexplained acute bleeding manifested by an abnormal activated partial thromboplastin time ( aptt ) that does not correct with 1:1 mixing with normal plasma.methods:practicing physicians ( hematology / oncology , emergency medicine , geriatrics , internal medicine , rheumatology , obstetrics and gynecology , critical care medicine , and general surgery ) completed an online survey based on a hypothetical case scenario.results:excluding surgeons and obstetrician / gynecologist respondents , 302 physicians ( about 50 per specialty ) were presented with an older adult woman complaining of recurrent epistaxis . nearly 90% ordered a complete blood count and coagulation studies ( aptt , prothrombin time [ pt]/international normalized ratio [ inr ] ) . despite a prolonged aptt of 42 seconds , < 50% of nonhematologists would repeat the aptt , and < 45% would consult a hematologist ; emergency medicine physicians were least likely ( 10% ) and rheumatologists were most likely ( 43% ) to consult . after presentation weeks later with bruising and abdominal / back pain , 90% of physicians within each specialty ordered a complete blood count or pt / inr / aptt . despite an aptt of 63 seconds , the majority did not repeat the aptt . at this point , approximately 75% of internal medicine and geriatric physicians indicated they would consult a hematologist , versus 47% in emergency medicine and 50% in critical care . all participants preferred abdominal computed tomography ( 80%84% ) . after 12 hours of additional observation , 73% to 94% of respondents consulted a hematologist . complete blood count revealed anemia and an aptt twice the upper limit of normal ; emergency medicine physicians remained least likely to request a consult.conclusion:determining the cause of an abnormal coagulation study result should carry equal weight as looking for the site of bleeding and could be facilitated by consultation with a hematologist . insight from this survey highlights knowledge and practice gaps that could be the target of focused educational initiatives .

accordingly , the number of reports of laparoscopic pancreaticoduodenectomy ( lap pd ) has gradually increased . although the feasibility and safety of lap pd have been established in institutes particularly experienced in the skilled performance of this technique ( hereafter referred to as experienced institutes ) , the benefit of lap pd beyond conventional surgery has not yet been shown . the management of concomitant abdominal aortic aneurysm ( aaa ) and intra - abdominal malignancy is controversial . three issues must be considered in the development of a treatment technique in such cases . the first is mutual interference because of operative fields that are in close vicinity to one another , resulting in adhesions or collateral injuries . the last is postoperative complications , especially intra - abdominal abscessation with graft infection . in particular , the pancreas is the organ that is most resistant to resolution of these issues because of its anatomical proximity to the aorta and severity of pancreatic fistulae as a postoperative complication . the feasibility and safety of endovascular repair ( evar ) for aaa have been established . in addition , laparoscopic colectomy and evar for aaa were successfully performed in a patient . similarly , lap pd and evar for aaa could have some benefits for patients . however , to the best of our knowledge , concomitant treatment by lap pd and evar has never been reported . a 70-year - old japanese man was referred from vascular surgery for investigation of a pancreatic tumor , which was identified as a cystic tumor of the pancreas head by computed tomography ( ct ) . within 1.5 years he had a previous history of percutaneous coronary intervention for acute myocardial infarction when he was 66 years old and aortic stent grafting for an aaa when he was 68 years old . the aaa was located on the infrarenal aorta with a thrombus of 52 mm ( fig . the height , weight , and body mass index of the patient were 163 cm , 67.3 kg , and 25.3 , respectively . a ct scan showed a cystic tumor of 31-mm diameter in the pancreas head without dilatation of the main pancreatic duct ( fig . 2 ) , but contrast - enhanced endoscopic sonography revealed a nodule among the cyst mucus ( fig . thus , we diagnosed the enlarging tumor as a branched - type ipmn with a nodule and planned to perform a resection . to avoid disturbing the aaa or stimulating a residual aaa , we intended to perform lap pd . open laparoscopy was performed at the umbilicus , and an additional five ports were placed ( fig . the patient had severe visceral steatosis , and the abdominal cavity was filled with omental fat . for mobilization around the ligament of treitz and the fourth portion of the duodenum , an additional port was placed at the middle of the inferior abdomen ( fig . 3 ) . during this procedure , neither duodenal adhesion to the aorta nor other inflammatory changes due to the previously placed stent graft were observed . after mobilization , an upper - middle incision of 15 cm was made , and the pancreas head was excised and removed . a pancreato - jejunal anastomosis was created by hand - suturing between the pancreatic duct and the jejunal mucosa . although the patient required medical therapy for pancreatic fistulae ( grade b according to the international study group on pancreatic fistula [ isgpf ] ) , his postoperative recovery was uneventful . the pathological diagnosis was intraductal papillary mucinous adenoma with small foci of carcinoma in situ ( fig . lap pd after evar for aaa was safely performed with both rigorous preoperative planning and a meticulous operation . although lap pd is one of the most complicated procedures in laparoscopic surgery , its safety and feasibility have been reported in experienced institutions . pancreatic cancer , as a representation of pancreas head tumors , has a poor prognosis . thus , the indications for lap pd in patients with pancreatic cancer are very limited . this case involved an ipmn , and the patient was thus a good candidate for lap pd . there are few operative indications for this type of neoplasia , and reported cases have shown a poor prognosis . when pancreatic cancer and aaa are simultaneously present , pancreatectomy is first recommended , including determination of the stage of the cancer . second , mutual interference between the two conditions is undeniable because of the proximity of the pancreas to the aorta . deiparine advocated division of the retroperitoneal dissection procedure : right - sided dissection for pd and left - sided dissection for abdominal aortic bypass . the last is the severity of the postoperative complications after pancreatectomy . in the present case , laparoscopic dissection of the pancreas head was safely performed without interference of the residual aaa because the axis of the laparoscopic procedure was located apart from the aaa ( fig . moreover , laparoscopic procedures require smaller operative fields using magnified visualization . these are benefits of the laparoscopic approach for patients with aaa . after the resection , an upper abdominal incision was made and reconstruction of the pancreas stump was performed through the incision . this reconstruction involved wirsung anastomosis , which represents the usual manner of standard pd in our institute . total lap pd has been reported in experienced institutes ; however , other reconstruction methods and no reconstruction have also been reported . thus , we performed reconstruction by hand as usual because laparoscopic reconstruction had not replaced hand sewing at that time in our institute . the additional upper incision is adequately located apart from the aaa ; therefore , the reconstruction procedure is safely performed without interference by the aaa . the isgpf grade b pancreatic fistulae healed with medical therapy and without graft infection . the pancreatic tumor was detected during follow - up of the aaa after evar . increased performance of evar for written informed consent was obtained from the patient for publication of this case report and accompanying images . a copy of the written consent is available for review by the editor - in - chief of this journal on request . norihiko ishikawa , mari shimada , and hideki moriyama performed the lap pd with the corresponding author .

introductionmost gastroenterological surgeries , even pancreatic surgery , can now be performed laparoscopically . however , the management of concomitant abdominal aortic aneurysm ( aaa ) and intra - abdominal malignancy is controversial . the performance of endovascular repair ( evar ) for aaa has been increasing ; however , there is no report of laparoscopic pancreaticoduodenectomy after evar.presentation of casea pancreatic tumor was detected during follow - up after evar for aaa . the enlarging tumor was diagnosed as an intraductal papillary mucinous tumor with a nodule . laparoscopic pancreaticoduodenectomy was safely performed . after laparoscopic dissection around the pancreas head , an additional incision was made in the upper abdomen , and pancreatic reconstruction was performed through the incision . in spite of grade b pancreatic fistulae , the patient recovered with medical therapy . the pathological diagnosis was intraductal papillary mucinous adenoma with small foci of carcinoma in situ . the patient has been well with neither recurrence of the tumor nor any cardiovascular events for 18 months.discussionthe management of concomitant malignancy and aaa is challenging , especially in patients with a pancreatic tumor . the reasons for the rarity of treatment include prognosis , anatomical vicinity , and postoperative complications . evar reduces retroperitoneal adhesions . a laparoscopic approach provides a small operative field and decreases mutual interference with aaa . moreover , reconstruction is performed through an upper abdominal incision apart from the aaa . hand - sewing provides more reliable stability of the anastomosis.conclusionthe increasing frequency of performance of evar for aaa and subsequent computed tomography may help to detect malignancy . laparoscopic surgery appears to be a valid approach to malignancy after evar .

vitamin d deficiency was once thought to exclusively affect bone metabolism , but now there is ample evidence of its role in many other conditions including metabolic syndrome , autoimmune diseases , and cancer . vitamin d receptors are recognized to be in numerous extraskeletal tissues , such as pancreas and muscle . a systematic review by pittas and colleagues reported that vitamin d may have a beneficial effect on the action of insulin , either directly or indirectly . several observational studies in adults , including the framingham heart study , have reported an inverse association between vitamin d status and insulin resistance . however , data from children and adolescents do not consistently report this inverse relationship [ 510 ] . even in studies reporting this association in adolescents and children , further , none of the studies in adolescents have studied the effect of puberty on this association . a fall in insulin sensitivity with compensatory increase in insulin secretion has been reported in puberty [ 11 , 12 ] . high prevalence of cardiometabolic risk factors , including insulin resistance ( 64.8% of normal weight children had at least one cardiometabolic abnormality ) , even in healthy young children of normal weight , has been reported in indian children and adolescents . south - asian adolescents , including those with normal bmi , have a higher prevalence of insulin resistance when compared with age and bmi matched european adolescents [ 14 , 15 ] . this ethnic difference in insulin sensitivity was attributed to the higher body fat percentage in south - asian children . an alternative hypothesis to explain the ethnic differences in insulin sensitivity is vitamin d deficiency . despite plenty of sunlight available throughout the year , a high prevalence of vitamin d deficiency has been reported in all age groups including toddlers , school children , pregnant women and their neonates , and adult males and females residing in rural and urban india . vitamin d deficiency has been reported in 8492% children in the age group of 1018 . in view of high prevalence of both , insulin resistance and vitamin d deficiency in asian - indian children and adolescents , we sought to examine the relationship between vitamin d status and insulin resistance . we also sought to determine if puberty affected the relationship of vitamin d status and parameters of insulin resistance and sensitivity in asian - indian children and adolescents . the study was conducted as a prospective observational study after approval from the ethics committee of the all india institute of medical sciences ( aiims ) , new delhi . subjects were selected from the children and adolescents attending the endocrinology outdoor services at aiims for evaluation of obesity . the inclusion criteria included children between the ages of 617 years with obesity as per international obesity taskforce ( iotf ) criteria . the exclusion criteria included subjects with diagnosed diabetes mellitus or taking metformin or any weight reducing drugs , subjects with any known systemic illness or endocrine or metabolic disorders , known to be associated with obesity , or subjects with symptoms to suggest hypothalamic obesity were excluded from the study . blood samples were collected in fasting state ( minimum 9 hours fasting ) , followed by oral glucose tolerance test ( anhydrous glucose = 1.75 gm / kg bodyweight , maximum of 75 gram , dissolved in 250 ml of water , ingested over 5 minutes ) with sample collection at 60 and 120 minutes after glucose ingestion . height was measured with holtain 's stadiometer ( holtain inc . , crymych , pembs . waist circumference was measured ( nonelastic measuring tape ) at the end of normal expiration at the midpoint between the iliac crest and the lower edge of ribs in the midaxillary line . blood pressure ( bp ) was measured in the right upper limb in sitting position with a mercury sphygmomanometer after 5-minute rest with an appropriate size cuff . total body fat was measured with dual energy x - ray absorptiometry ( dxa , hologic qdr 4500 with pediatric software , hologic inc . , fat mass index ( fmi , fat mass in kg / height in meter ) and fat - free mass index ( ffmi , lean body mass+ bmc)/(height in meter ) were calculated . bone mineral area , content ( bmc ) and areal density ( abmd ) at the femoral neck , lumbar spine ( l1 - 5 ) , and forearm were measured , using a hologic qdr 4500a fan beam dxa machine . hologic spine phantom ( hologic spine phantom number 21373 ) was scanned daily before subject evaluation . the measured phantom bone mineral density was stable throughout the study period at 0.9150.945 gm / cm . similarly , whole body phantom ( hologic wb number 1252 ) was also scanned before subject evaluation and remained stable during study period . the in vivo precision error for adults was 0.62% for femoral neck abmd , 0.65% for lumbar spine abmd , and 0.77% for forearm abmd . however , in view of additional radiation exposure , the reproducibility of these scans was not assessed among children . pubertal stage was assessed by a single endocrinologist , based on breast stage and pubic hair development in girls and genitalia development in boys . b - mode ultrasonography was used to measure cimt ( 7.510 mhz probe , philips envisor ultrasound machine ) using standard protocol . insulin resistance ( ir ) was calculated by computer - based model called homeostasis assessment model ( homa - ir ) utilizing fasting blood glucose and fasting serum insulin levels .the whole body insulin sensitivity index ( wbisi ) or matsuda index was calculated by the formula suggested by matsuda and defronzo .area under curve ( auc ) was calculated for blood glucose ( 0 , 60 , and 120 minutes ) and serum insulin ( 0 , 60 , and 120 minutes ) by using trapezoidal rule . insulin resistance ( ir ) was calculated by computer - based model called homeostasis assessment model ( homa - ir ) utilizing fasting blood glucose and fasting serum insulin levels . the whole body insulin sensitivity index ( wbisi ) or matsuda index was calculated by the formula suggested by matsuda and defronzo . area under curve ( auc ) was calculated for blood glucose ( 0 , 60 , and 120 minutes ) and serum insulin ( 0 , 60 , and 120 minutes ) by using trapezoidal rule . complete blood counts , liver function tests , renal function test , serum calcium ( corrected calcium with serum albumin ) , phosphate , alkaline phosphatase , uric acid , and blood glucose were measured in all subjects with an automated chemistry analyzer ( roche hitachi 912 chemistry analyzer , gmi inc . glycosylated hemoglobin ( hba1c ) was measured in whole blood using ion - exchange high performance liquid chromatography ( bio - rad laboratories inc . , ca , us ) . serum insulin was measured on an autoanalyzer ( roche elecsys 2010 ) using electrochemiluminometric assay . serum total cholesterol ( tc ) , triglyceride ( tg ) , and hdl cholesterol ( hdl - c ) levels were estimated directly with automated analyzer , while ldl cholesterol ( ldl - c ) was estimated by using the friedewald equation . vitamin d deficiency was defined as a serum 25(oh)d levels less than 20 ng / ml . this was further subdivided to severe , moderate , and mild vitamin d deficiency if serum 25(oh)d levels were < 5 ng / ml , 5<10 ng / ml , and 10<20 ng / ml respectively . statistical analysis was carried out using spss ( spss version 11.5 ; spss inc . , continuous variables not showing normal distribution were treated with appropriate log transformations before any parametric analysis . student 's t - test was used to determine statistical difference between subjects with serum 25(oh)d < 10 ng / ml or more . pearson 's correlation was used to assess association between continuous variables and partial correlations were used to determine relationship after adjusting for relevant covariates . stepwise linear regression analysis was used with serum 25(oh)d as dependent variable in the model . values for serum 25(oh)d , serum insulin , homa - ir , and matsuda isi were not normally distributed ; therefore , they were logarithmically transformed which yielded normal distribution . a total of 62 subjects participated in this cross - sectional study from june 2008 to may 2009 . the mean age of subjects was 13.0 3 years ( 35 boys ; 27 girls ) . assessment of pubertal status by tanner 's method showed 19 subjects in stage 1 , 11 in stage 2 , 6 in stage 3 , 3 in stage 4 , and 23 subjects in puberty stage 5 . no significant difference was observed between boys and girls except higher whr in boys than girls ( 0.93 0.05 versus 0.89 0.05 ; p = 0.013 ) . all study subjects were classified as vitamin d deficient ( mean sd 8.5 4.2 ng / ml ; maximum minimum : 3.919.2 ng / ml , median = 6.9 ng / ml ) . severe vitamin d deficiency ( < 5 ng / ml ) was seen in 11 subjects ( 17.7% ) while 30 subjects had serum 25(oh)d levels between 5<10 ng / ml ( 48.3% ) . serum 25(oh)d equal to or more than 10 but less than 20 ng / ml was present in 21 subjects ( 33.8% ) . pearson correlation coefficient analyses showed significant inverse relationship between serum 25(oh)d and total body fat percentage ( r = 0.31 , p = 0.01 ) and serum 25(oh)d and fmi ( r = 0.33 , p = 0.01 , figure 1 ) . partial correlation analysis showed persistence of significant correlation between serum 25(oh)d and total body fat percentage ( r = 0.37 , p = 0.005 ) and between serum 25(oh)d and fmi ( r = 0.36 , p = 0.006 ) even after adjustment for age , sex , pubertal stage , and bmi . although blood glucose and serum insulin levels showed inverse relationship trends with serum 25(oh)d , but these were not statistically significant ( data not shown ) . similarly , no statistically significant correlation was seen between serum 25(oh)d and parameters of insulin sensitivity or resistance . fmi showed positive correlation with homa - ir ( r = 0.26 ; p = 0.04 ) , auc insulin ( r = 0.41 ; p = 0.001 ) , auc glucose ( r = 0.31 ; p = 0.018 ) , and negative correlation with wbisi ( r = 0.42 ; r = 0.001 ) . however , total body fat did not show any correlation with parameters of glucose and insulin parameters . on the basis of serum 25(oh)d , subjects were divided into two groups , group a with serum 25(oh)d level < 10 ng / ml ( n = 41 ) and group b with serum 25(oh)d level 10 ng / ml ( n = 21 ) ( an arbitrary cutoff to compare subjects with mild vitamin d deficiency with moderate and severe vitamin d deficiency , 25 ) . no statistical significant difference was seen in blood glucose , serum insulin , and auc for both , glucose and insulin between group 1 and group 2 . trends for higher insulin resistance ( homa - ir ) and lower insulin sensitivity ( wbisi ) were seen in subjects with lower serum 25(oh)d concentrations but not statistically significant . total body fat percentage and fmi were significantly higher in group 1 in comparison to group 2 ( p = 0.015 and 0.007 , resp . ) . however , no significant difference was seen in waist and hip circumference and waist hip ratio . similarly , ffmi , lipid parameters , or thyroid profile were also not different between the two groups . group 1 ( prepubertal group ; tanner stage 1 ) had 19 subjects , group 2 ( peripubertal group ; tanner stage 2 , 3 , and 4 ) had 20 , while group 3 ( postpubertal group ; tanner stage 5 ) had 23 subjects . no significant correlation was seen between vitamin d status and any other parameters including total body fat percentage , fmi and parameters of insulin resistance and sensitivity in the prepubertal ( group 1 ) or peripubertal groups ( group 2 ) . partial correlations after adjusting for age , sex , and bmi were also nonsignificant for both groups ( data not shown ) . in postpubertal subjects ( group 3 , n = 23 ) , a significant inverse correlation was seen between serum 25(oh)d and homa - ir ( r = 0.41 , p = 0.03 , figure 2 ) . this association between serum 25(oh)d and homa - ir persisted even after control for age , sex , and bmi ( r = 0.505 , p = 0.023 ) . positive trends of association between serum 25(oh)d and wbisi were also seen , but this was not significant ( r = 0.29 , p = 0.17 ) , even after adjusting for age , sex , and bmi ( r = 0.35 , p = 0.13 ) . serum 25(oh)d showed significant inverse correlation with total body fat percentage ( r = 0.58 , p = 0.003 ) and fmi ( r = 0.49 , p = 0.016 ) which improved after adjusting for age sex and bmi ( r = 0.63 , p = 0.002 for total body fat percentage ; r = 0.582 , p = 0.007 for fmi ) . serum 25(oh)d also showed significant inverse relationship with fasting serum insulin levels ( r = 0.44 , p = 0.034 ) which improved after adjusting for age , sex , and bmi ( r = 0.53 , p = 0.016 ) . when prepubertal subjects ( group 1 ) were compared with the prepubescent subjects ( group 2 ) , no significant difference was seen in any of the parameters including blood glucose , serum insulin , homa - ir , and wbisi . similarly , when prepubertal subjects were compared with all other subjects ( subjects in group 2 and 3 combined ) , significantly higher insulin sensitivity ( wbisi , mean sd 6.07 5.7 versus 3.42 2.7 , p < 0.01 ) and lower insulin resistance ( homa - ir , mean sd 3.68 2.7 versus 5.22 4.0 , p = 0.04 ) were seen in prepubertal subjects . this is the first study examining the relationship between serum 25(oh)d levels and parameters of insulin resistance in obese asian - indian children . our study confirms a significant inverse relationship between serum 25(oh)d levels and body fat indices as reported in other studies in children [ 6 , 10 ] . we demonstrated trends for that higher serum 25(oh)d levels are associated with better insulin sensitivity and less insulin resistance though this correlation did not reach statistical significance . one of the main reasons for the absence of significance could be the universal presence of vitamin d deficiency ( < 20 ng / ml ) resulting in very narrow range of serum 25(oh)d . we also show , for the first time , that the association between serum 25(oh)d and insulin resistance and sensitivity is influenced by pubertal staging . when subjects were stratified by pubertal stage , only subjects in tanner stage 5 exhibited a significant correlation between serum 25(oh)d and homa - ir , while prepubertal subjects did not show statistically significant correlation . high prevalence of vitamin d deficiency has been reported across the age range in pediatric , adolescent , and adult in obese individuals [ 5 , 6 , 2631 ] . this may be just reflection of very high prevalence of vitamin d deficiency in asian - indians [ 16 , 17 ] . however , high prevalence of vitamin d deficiency has been reported in obese children and adolescents from populations where vitamin d deficiency is not that prevalent [ 5 , 6 , 10 ] . reis et al . , in a study of 3577 adolescents in the age group of 1219 years , found significantly lower serum 25(oh)d levels in adolescents with bmi > 95th centile in comparison to subjects with bmi < 95th centile ( p < 0.001 ) . it has been postulated that increased sequestration of vitamin d in fat tissues , leading to decreased vitamin d bioavailability , low dietary vitamin d intake due to poor nutritional habits , and minimal sun exposure due to sedentary indoor lifestyle are important factors associated with high prevalence in the obese population [ 29 , 32 ] . it has also been suggested that vitamin d deficiency is a cause of common obesity and can account for the secular trends in the prevalence of obesity and for individual differences in its onset and severity . the inverse relationship between serum 25(oh)d and insulin resistance in adults have been reported in most studies , though some have failed to find this association . in comparison , reports in children and adolescents have shown conflicting results . delvin et al . , in a study of 1745 french - canadian children , reported modest but significant negative associations between serum 25(oh)d and homa - ir . each 10-nmol / l ( 4 ng / ml ) increase in serum 25(oh)d was associated with lower glycemia and homa - ir . did not find significant correlation between serum 25(oh)d levels and insulin , homa - ir and homa - b % . in another study based on nhanes data of 3577 adolescents from 20012004 showed that serum 25(oh)d levels were inversely associated with plasma glucose concentration ( p = 0.01 ) . lenders et al . , in a study of 58 obese adolescents , did not found any correlation between insulin indices and serum 25(oh)d in both , adjusted and unadjusted models . in our study , we did not find a statistically significant association between serum 25(oh)d and parameters of insulin sensitivity and resistance . however , trends for higher blood glucose and serum insulin levels were seen in subjects with low serum 25(oh)d level . no significant difference was seen in insulin resistance and sensitivity between group 1 and group 2 . this is in contrast to the observations made by ashraf et al . who reported in 51 african - american obese female adolescents that higher serum 25(oh)d concentrations were associated with significantly higher wbisi ( p = 0.018 ) but no difference in homa - ir . these differences may be due to marked difference in severity of obesity as mean bmi of study subjects was 43.3 9.9 in study by ashraf et al . , while it was 29.3 9.8 kg / sqm in our study subjects . studies in adult population have shown that parameters of insulin sensitivity and insulin resistance as calculated by hyperinsulinemic euglycemic clamp study ( m value ) correlated better with serum 25(oh)d levels than indirect parameters like homa - ir . studies with direct measures of insulin sensitivity like clamp studies are required to further investigate these issues in pediatric and adolescent population . the phenomenon of pubertal insulin resistance has been well described in cross - sectional as well as longitudinal studies . a longitudinal study of insulin resistance in american children showed that during puberty , insulin sensitivity decreased by 50% , with a compensatory increase in plasma insulin which is independent of the changes in body fat . these changes are thought to be mediated by interaction of various hormones during puberty including increased growth hormone secretion . no studies have yet reported the relationship between serum 25(oh)d concentrations and insulin sensitivity and resistance in pediatric and adolescent population and the potential influence of puberty except one . lenders et al . reported no significant association between serum 25(oh)d and insulin indices , even after adjusting for tanner stage . however , our study , for the first time , shows that the relationship between serum 25(oh)d concentrations and insulin resistance is influenced by puberty . when subjects with all stages of puberty were analyzed , no significant correlation was found , but when subjects in tanner stage 5 were analyzed alone , significant inverse correlation was seen between serum 25(oh)d concentrations and homa - ir ( r = 0.41 , p = 0.03 ) . this association persisted / improved after controlling for age , sex , and bmi ( r = 0.505 , p = 0.023 ) but disappeared when subjects were controlled for fmi ( r = 0.27 , p = 0.24 ) . most of the evidence of an association between adiposity and vitamin d status comes from adult studies [ 4 , 33 ] . a recently published study of 382 healthy subjects aged 621 years with dxa scans showed that serum 25(oh)d concentrations were more likely to be lower in those with greater bmi z - scores and fm , but not ffm . when this association was adjusted for possible covariates in the model , it became nonsignificant . . showed that obese adolescents with and without vitamin d deficiency differed according to fm and fm percentage , but not lean mass and this relation of fm percentage to serum 25(oh)d remained significant after potential confounding variables were adjusted for . showed that vitamin d deficiency was associated with higher visceral adipose tissue in white and greater subcutaneous adipose tissue in black american children and adolescents . in agreement with these studies , our findings indicate that body fat indices ( total body fat percentage and fmi ) are inversely correlated with serum 25(oh)d level . this correlation persisted even after adjustment for possible covariates like age , sex , and bmi . the differences in study findings may be explained in part by differences in study design and underlying sample characteristics like bmi and variation in serum 25(oh)d concentrations . the major strengths of our study are the use of robust measures of insulin sensitivity and resistance based on ogtt rather than fasting sample and all our study subjects belonged to one ethnic group . we have used dxa for assessment of body fat as opposed to several studies which have used bmi as a surrogate for body fat . the limitations of our study included , small sample size , presence of vitamin d deficiency in all study subjects which gave us very narrow range of serum 25(oh)d concentrations thereby limiting comparisons . absence of pth measurement was another important limitation , since it has been implicated in pathogenesis of insulin resistance and metabolic syndrome . another important limitation was inability of dxa technique to differentiate between subcutaneous and visceral adipose tissue . in conclusion , our study demonstrates a significant inverse relationship in obese asian - indian children between body fat indices and serum 25(oh)d concentrations which persists even after adjustment of covariates . the association between serum 25(oh)d and parameters of insulin sensitivity and resistance is influenced by puberty as a significant association was found between these two variables in postpubertal subjects ( tanner stage 5 ) only which remained significant even after adjustment for covariates . as association is not equal to causation , future longitudinal research studies are required in determining role of puberty on association between serum 25(oh)d and insulin resistance in both , obese as well as subjects with normal bmi .

to study the effect of puberty on the relationship between serum 25-hydroxyvitamin d ( 25(oh)d ) and parameters of insulin kinetics in obese asian - indian children . material and methods . the study population included 62 obese asian - indian children and adolescents in the age group of 617 years . blood glucose , serum insulin , and serum 25(oh)d were measured . total body fat was measured by dual energy x - ray absorptiometry . indices of insulin resistance ( homa - ir , auc for insulin ) and sensitivity ( wbisi ) were calculated after oral glucose tolerance test . result . a total of 62 subjects ( 35 boys ; mean age = 13.0 3 years ; bmi = 29.3 4.8 kg / sq m ; 19 subjects in tanner stage 1 , 11 in stage 2 , 6 in stage 3 , 3 in stage 4 , and 23 subjects in tanner stage 5 ) were studied . all study subjects were vitamin d deficient with a mean serum 25(oh)d of 8.5 4.2 ng / ml . no significant relationship was observed between serum 25(oh)d and parameters of insulin kinetics in prepubertal children . however , a significant inverse correlation was seen between serum 25(oh)d and homair ( r = 0.41 , p = 0.03 ) in postpubertal subjects . conclusion . the relationship between vitamin d status and parameters of insulin kinetics are affected by puberty .

transurethral resection of bladder tumor ( turbt ) is well - established modality for treating non - muscle - invasive bladder cancer . furthermore , it is essential for evaluating the pathological depth of bladder tumors in resected specimens . despite improvements in endoscopic instruments and imaging systems , there are well - recognized complications associated with turbt , such as bladder perforation , irrigation fluid absorption , bleeding , infection , damage to the ureteric orifices , obturator nerve stimulation , and tumor cell implantation . among these , bladder perforation is one of the most frequent complications of turbt and is sometimes associated with obturator reflex . several researchers have reported that the risk of obturator nerve stimulation is from 10.6% to 11% . several advances in instruments and techniques , including modifications of resectoscope design and snare resection , have been proposed to remove pedunculated bladder tumors . however , difficulties remain in safely collecting adequate specimens for flat tumors . we introduce an easy technique for superficial bladder cancer that uses a standard monopolar resectoscope loop . in this study , we demonstrate the easy resection technique for superficial bladder tumors that we have termed the " grasp and bite " turbt technique and compare it with conventional turbt . this was a retrospective study in the department of urology of chonnam national university hospital ( gwangju , korea ) between january 2012 and april 2013 . this study included a total of 29 men and 6 women who had bladder tumors with superficial lesions and who underwent turbt . patients with a huge unresectable mass , an invasive tumor shown during preoperative imaging , or an irradiated bladder were excluded . group 1 patients were treated by use of the conventional turbt technique , and group 2 patients underwent the grasp and bite technique . conventional turbt was performed in 2012 and grasp and bite turbt was performed in 2013 . this study was approved by the institutional review board of chonnam national university hospital ( irb no . transurethral resection was performed with a monopolar resectoscope system ( 24-fr karl storz , tuttlingen , germany ) . for the grasp and bite technique , the operator initially defined the tumor morphology and safe margin . in small and flat lesions , the tumor and surrounding mucosal lesions were positioned between the resection loop and the end portion of the resectoscope sheath . we call this the " grasp " step ( fig . 1 ; video clip , supplementary material ) . using the grasping technique , the tumor lesions were lifted up . in the " bite " step , the surgeon maintained a tight hold and resected the tumor while grasping . a cystoscopic view and schematic illustrations 1 . for resection during the bite step , the movement of the electrode loop was straight backward in a linear direction . resection was performed carefully so as not to cause bladder perforation without excessive bladder wall distention . the resection site was carefully inspected for bleeding and coagulation . in bulky or pedunculated tumors , after removal of the bulging mass , the base lesion and surrounding mucosa were resected carefully by using grasp and bite turbt as mentioned earlier . specimens were measured by hematoxylin and eosin ( h&e ) staining and immunohistochemical smoothelin stain to determine the muscularis mucosae . independent t - tests were used to compare the mean values of two independent parametric continuous variables . the mann - whitney test was used to compare the median values of two nonparametric continuous variables . this was a retrospective study in the department of urology of chonnam national university hospital ( gwangju , korea ) between january 2012 and april 2013 . this study included a total of 29 men and 6 women who had bladder tumors with superficial lesions and who underwent turbt . patients with a huge unresectable mass , an invasive tumor shown during preoperative imaging , or an irradiated bladder were excluded . group 1 patients were treated by use of the conventional turbt technique , and group 2 patients underwent the grasp and bite technique . conventional turbt was performed in 2012 and grasp and bite turbt was performed in 2013 . this study was approved by the institutional review board of chonnam national university hospital ( irb no . transurethral resection was performed with a monopolar resectoscope system ( 24-fr karl storz , tuttlingen , germany ) . for the grasp and bite technique , the operator initially defined the tumor morphology and safe margin . in small and flat lesions , the tumor and surrounding mucosal lesions were positioned between the resection loop and the end portion of the resectoscope sheath . we call this the " grasp " step ( fig . 1 ; video clip , supplementary material ) . using the grasping technique , the surgeon maintained a tight hold and resected the tumor while grasping . a cystoscopic view and schematic illustrations are shown in fig . 1 . for resection during the bite step , the movement of the electrode loop was straight backward in a linear direction . resection was performed carefully so as not to cause bladder perforation without excessive bladder wall distention . the resection site was carefully inspected for bleeding and coagulation . in bulky or pedunculated tumors , after removal of the bulging mass , the base lesion and surrounding mucosa were resected carefully by using grasp and bite turbt as mentioned earlier . specimens were measured by hematoxylin and eosin ( h&e ) staining and immunohistochemical smoothelin stain to determine the muscularis mucosae . independent t - tests were used to compare the mean values of two independent parametric continuous variables . the mann - whitney test was used to compare the median values of two nonparametric continuous variables . statistical analyses were performed with ibm spss statistics ver . 20.0 ( ibm co. , armonk , ny , usa ) . from january to april 2012 , 16 patients were enrolled who had undergone conventional turbt . from january to april 2013 , the grasp and bite technique was applied to 19 patients with superficial bladder tumors . the two groups had comparable demographic and intraoperative data , including patient age , tumor size , patient gender , multiplicity , tumor morphology , and tumor location ( table 1 ) . the mean operative time was similar in the conventional and grasp and bite groups ( 35 minutes vs. 35 minutes , respectively ) . the mean duration of irrigation , duration of catheterization , and hospital stay were not significantly different between the groups . there were no cases of bladder perforation , excessive obturator reflex , or persistent hematuria in either group . 2 ) . there was no significant difference in recurrence rates between the conventional and grasp and bite turbt groups during the 14-month follow - up ( 43.8% vs. 31.6% , p=0.458 ) . the purpose of turbt is complete removal of all visible tumors for curative intent and acquisition of adequate tissue for pathologic evaluation . also , tumor recurrence after turbt is relatively high . for non - muscle - invasive bladder cancer , the probability of recurrence after 1 year ranges from 15% to 70% . many obstacles can arise during the procedure , such as bladder perforation , bleeding , obturator reflex , and damage to the ureteral orifices . to overcome these difficulties , many surgical techniques and innovative devices for example , endoscopic snare resection of a bladder tumor can be used to remove pedunculated tumors . although the snare method offers the advantage of en block resection , smaller and sessile tumors are not suitable for this technique . new devices such as water jet hydrodissection and the thulium : yttrium aluminium garnet laser may be applicable for en block resection of superficial tumors . however , further studies and development of these techniques are necessary to determine efficacy and oncologic outcomes . variable methods of tumor resection have been developed and used in association with tumor size and location . the side - to - side bidirectional lateral rotating handgrip and loop allows excellent precision and control over the loop under direct vision . loop modification , the so - called " runner , " can push the bladder wall away during resection , thereby widening the view and decreasing the risk of perforation . we wanted a technique in which we could use a conventional resectoscope and loop without accessory equipment . when using a wire - loop resectoscope , the tumor must be resected thoroughly to obtain adequate muscle tissue . the movement of the loop is somewhat fast and deep to avoid charring of the surrounding mucosa and bladder perforation . direct pressure to the bladder wall may allow deeper tissue to protrude into the bladder wall and then be easily removed by use of the resectoscope . instead of the curvilinear movement of the resectoscope end according to the curved bladder surface , the easy linear motion can make a curved resection with grasping . using our " grasp and bite " technique , beginners can easily perform turbt safely . often , small and sessile tumors are missed and severely damaged during resection and cauterization . the grasp and bite technique , the protruding tumor can be removed f irst by the conventional method , and then the stalk and base lesion can be removed by the grasp and bite technique . , the grasp and bite technique could have difficulties in a posterior or posterolateral located tumor because of its moving mechanism . in our series , two posterior and four posterolateral tumors were successfully accessed by use of suprapubic pressure and nearly emptying the bladder . similar to conventional turbt , it was also difficult to access posterior tumors with the grasp and bite method . in our consecutive cases , general anesthesia was preferred in the conventional and grasp and bite groups ( 81.3% vs 57.3% , p=0.138 ) . also , tumor locations did not differ significantly between the two groups ( p=0.253 ) . we postulated that the grasping method , which lifts the tissue away from the obturator nerve and tightly holds the grasped tissue , may eventually weaken the reflex . considering this small population , however , thus , there could have been a selection bias . also , the study population was small , and thus we were unable to perform statistical analysis of the oncologic outcome as recurrence - free survival . this was a preliminary study to develop a surgical technique performed by an experienced surgeon . larger prospective studies with a long follow - up period will be necessary to show the oncologic advantage of this technique . to determine the feasibility of the technique for inexperienced surgeons , future study will address a teaching protocol comparing the conventional and grasp and bite methods . investigation of tissue adequacy of proper muscle and effect of coagulation will be our next subject of study . our study demonstrated that grasp and bite turbt is a safe and effective method for removing superficial bladder tumors . this technique has merit for flat tumors and can also be used for pedunculated and poorly accessible tumors . the grasp and bite method may be a shortcut for overcoming the difficulties of turbt . an accompanying video can be found in the ' urology in motion ' section of the journal homepage ( www.kjurology.org ) . the supplementary data can also be accessed by scanning a qr code located on the fig . 1 of this article , or be available on youtube ( http://youtu.be/2xjdgspzvy4 ) .

purposetransurethral resection of bladder tumor ( turbt ) can be a challenging procedure for an inexperienced surgeon . we suggest an easy technique for turbt , which we have named the " grasp and bite " technique . we describe this technique and compare its effectiveness and safety with that of conventional turbt.materials and methodsmonopolar turbt ( 24-fr karl storz ) was performed in 35 patients who had superficial bladder tumors . after defining the tumor margin , the tumor and surrounding mucosa were grasped by use of a loop electrode and resectoscope sheath . with tight grasping , linear moving resection was performed . the patients ' demographic , intraoperative , and postoperative data were analyzed between the conventional and grasp and bite turbt groups.resultsof 35 patients , 16 patients underwent conventional turbt ( group 1 ) , and the other 19 patients underwent grasp and bite turbt ( group 2 ) . both groups were similar in age , tumor multiplicity , size , anesthesia method , and location . grasp and bite turbt could be performed as safely and effectively as conventional turbt . there were no significant differences in irrigation duration , urethral catheterization , postoperative hemoglobin drop , or length of hospital stay . no significant side effects such as bladder perforation , severe obturator reflex , or persistent bleeding occurred . there were no significant pathological differences between specimens according to the type of resection technique.conclusionsthe grasp and bite turbt technique was feasible for superficial bladder tumors . it may be a good tool for inexperienced surgeons owing to its convenient and easy manner .

clinicians rely on patients recall of their symptoms to adjust therapies and monitor responses , but the accuracy of patients recall , particularly between clinic visits , is poor.1 patients recall recent symptoms more clearly than earlier , more remote symptoms , and more severe symptoms more clearly than mild symptoms.2 recognition of day - to - day variability in symptoms is also important for revealing important trends ( improvement or worsening ) , but measuring such variability is difficult without close tracking of symptoms . daily symptom variation is important because it may obscure the ability to detect signal ( of improvement ) from the noise ( of random daily variation ) . taken together , these challenges highlight the problems of symptom recall as the basis for making treatment recommendations , and either altering or escalating therapeutic interventions.1,3 inflammatory bowel disease ( ibd ) causes chronic inflammation of the gastrointestinal tract , often resulting in severe symptoms . despite optimal treatment , over 40 percent of patients continue to have symptoms that reduce their quality of life ( qol ) such as persistent fatigue,4,5 abdominal bloating , or fecal urgency.6 careful attention to a patient s symptomatic response to therapies using paper symptom logs is often inadequate or too cumbersome to provide sufficient evidence to guide treatment.7 improved methods are needed to ease symptom data collection and to facilitate conducting individual ( n - of-1 ) therapeutic trials to improve symptoms and qol . currently , 78 percent of teenage patients carry mobile devices capable of easily collecting and transmitting symptom data on a daily basis.8 there is growing interest among patients ( e.g. , the quantified self community)5,9,10 and physicians1115 in leveraging mobile technologies for improving patient outcomes . according to a recent study by the pew research center , over 40 percent of americans who have a chronic health condition track their symptoms or health indicators such as headaches , blood sugar , blood pressure , etc.5 but there is little evidence of the impact that frequent monitoring of self - reported data has on patient qol or other health outcomes.16 while some limited progress has been made in integrating formalized patient - reported outcome ( pro ) measures into clinical practice , these early efforts provide little evidence to guide the effective integration of pros into real - world patient care setting.16,17 efforts at implementation are made more difficult by technical limitations ( e.g. , many care centers lack electronic tools to collect pros , and completing repeated online surveys are not sustainable for most patients over time ) . most published studies utilizing pros in real - world patient care settings have demonstrated that the pros have little or no impact on patient outcomes.16 this may be due to a lack of validated tools to monitor patient symptoms or qol reliably and prospectively in real time.16 standardized qol indicators for general assessment have not been studied widely for assessing patients in clinical practice . the patient - reported outcomes measurement information system ( promis ) survey instruments may be appropriate for this purpose , but promis surveys have been validated only in population studies.18,19 promis tools have not been studied in individual patients or over short periods , and have not been broadly incorporated into routine patient care . as part of an ongoing study exploring innovations in health care , we evaluated a prototype tool to determine if continuous symptom tracking aids in improving patients and physicians understanding of symptom patterns and if tracking can help patients and physicians work together to better target treatments.20,21 we also attempted to determine if promis measures can be used in individual patients to track qol over short periods , as opposed to populations of patients for which they were developed . we report , here , important learning from a small number of patients who completed prototype testing of this tool . this study was conducted as part of the ongoing work of the collaborative chronic care network ( c3n ) project that is developing innovations to facilitate more continuous , collaborative relationships between patients and families with chronic illness and their physicians . the tools described here emerged from a structured design process involving patients , families , clinicians , and innovators that entails testing novel concepts in small - scale prototypes and larger pilot studies prior to deployment.21 this manuscript describes the prototype testing of the novel concept . we took advantage of the collaborative network of patients , physicians , and researchers who participate in the improvecarenow network to engage participants.21 we recruited adolescent patients with ibd who were engaged in their care and were interested in further improving their symptoms and qol . each patient ( along with a parent if under 18 years old ) met with his or her pediatric gastroenterologist and a researcher at the time of study enrollment to discuss the patient s medical history and to plan the details of the patient s individual study . the researcher and patient agreed upon a means of assessing symptoms by selecting from a list of preassembled measures . they were allowed to create their own symptom measure if none was available to fit their needs . we used a daily , automated text - message service to prompt patients to submit symptom scores via text message . we did not limit the number of questions that could be asked per day , but suggested each patient select no more than three to four . at study enrollment , the questions were delivered each day at this prespecified time , and did not vary from day to day or with responses to prior questions . we also used the promis survey instruments to measure fatigue and pain interference across all patients.22,23 these short surveys have been extensively validated in a wide range of chronic medical conditions and children.18,19,24 the promis surveys query information on specific outcomes for the preceding seven days . the results were converted to t - scores , where a score of 50 represents the mean score of the general pediatric calibration sample ( standard deviation 10).25 t - scores greater than 50 represent worse fatigue or pain interference . we deployed the appropriate promis survey weekly via a web - based interface to each patient . we compared individual patients promis survey results with their average symptom scores from the same period . we used individual - level , statistical process control methods for time - sequence data to monitor the patient s daily symptom data and weekly promis survey data as they were collected over time.26 we established each patient s individual baseline pattern of symptom variation , or common cause variation . symptom data points that fell outside three sigma control limits were noted as special cause variation , or variation outside stable baseline variability.26,27 these data , along with calculated control limits , were graphed on a microsoft excel spreadsheet for visual representation of data for patient and physician review . for prototype testing , we used a heavily manual process including creating microsoft excel graphs by hand and sharing data with patients and providers via email that was possible for only a small number of patients . the purpose of this type of manual prototype was to inform more robust pilot testing facilitated by automated tools being developed by the research team . patients were given access to their own data and were provided with graphical displays of their data at each meeting . physicians and patients jointly reviewed the data on a regular basis in prearranged consultations ( in person or by phone , according to each family and physician s preferences ) . these meetings were opportunities for patients and physicians to learn from patterns of data and to make treatment decisions if they felt this was appropriate . this study and the consent procedures were approved by the institutional review boards of both the university of michigan health system ( hum00051651 ) and cincinnati children s hospital medical center ( 20103380 ) . all adult participants provided written informed consent to participate in this study . written informed consent was obtained from parents of minors and written assent from minors themselves for participation in this study . patients tracked symptoms in order to monitor for changes in disease status , to generate hypotheses as to what makes their symptoms better or worse , or as part of a specific n - of-1 study . we present three patients who tracked their symptoms , prior to implementation of n - of-1 testing , and for whom symptom patterns led to important revelations . a 19 year old with longstanding indeterminate colitis underwent proctocolectomy with ileal pouch anal anastomosis at the age of 10 years . she had frequent nocturnal stools that woke her up to six times per night and caused fatigue contributing to her poor qol . she had been treated every eight weeks with long - term infliximab therapy by intravenous infusion . in collaboration with her physician , she decided to track her total number of daily stools and the number of stools waking her from sleep each night . she historically awoke to have a bowel movement from three to six times per night on average . during her baseline observation period , there was a span of nine consecutive nights with fewer episodes of nocturnal stools ( fig . 1 ) . however , she failed to recognize this nine - night interval of improved nighttime waking for stools despite the shift in symptoms that qualified as special cause variation.26 this shift occurred immediately following an infusion of infliximab , which led her treating physician to conclude that infliximab may have played a role in her transiently improved stool pattern . however , this postinfusion improvement was not reproduced after her subsequent four infliximab infusions , during which time her daily symptoms were continuing to be recorded . she did experience reproducible improvement in her stools with repeated courses of antibiotics that were prescribed for non - ibd related symptoms by her primary care physician ( previously illustrated in kaplan et al.20 ) . during those times when she experienced decreased nighttime bowel movements , she also felt less fatigued ( fig . 2 ) . upon reviewing these data she confirmed that she felt less fatigued and more energetic . the patient and her physician both felt that the graphical representation of her symptom data aided in their visualization and understanding of the treatment effects . both the patient s mother and her physician were interested in trying different probiotics to determine if one would be more effective than others for managing her fecal urgency . over the course of repeated periods on and off different probiotics,20 her fecal urgency gradually improved ( fig . 3 , green dashed ovals ) , though there was no discernable difference during periods with or without the use of probiotics.20 the reduction in fecal urgency over time was reflected in improved promis fatigue score ( fig . after three months of monitoring , the patient developed an exacerbation of the disease ( fig . after he was diagnosed with crohn s disease , his running pace decreased and had not returned to his pre - illness baseline , and he was interested in assessing his running potential after starting treatment with biological therapy . at his enrollment in this study , his baseline promis fatigue t - score was 35.7 , indicating that he was less fatigued than the average healthy individual . he received his first infliximab infusion on may 25 , 2011 , with induction regimen completed by july 6 , 2011 , ( usually consisting of three infusions spread over six weeks ) . his number of daily bowel movements decreased within four days of completing the induction regimen , and attained a steady state along with a special cause ( fig . his running pace also improved significantly from a mean 8.4 to 7.6 minutes / mile , indicated by a special cause shift as well . a 19 year old with longstanding indeterminate colitis underwent proctocolectomy with ileal pouch anal anastomosis at the age of 10 years . she had frequent nocturnal stools that woke her up to six times per night and caused fatigue contributing to her poor qol . she had been treated every eight weeks with long - term infliximab therapy by intravenous infusion . in collaboration with her physician , she decided to track her total number of daily stools and the number of stools waking her from sleep each night . she historically awoke to have a bowel movement from three to six times per night on average . during her baseline observation period , there was a span of nine consecutive nights with fewer episodes of nocturnal stools ( fig . 1 ) . however , she failed to recognize this nine - night interval of improved nighttime waking for stools despite the shift in symptoms that qualified as special cause variation.26 this shift occurred immediately following an infusion of infliximab , which led her treating physician to conclude that infliximab may have played a role in her transiently improved stool pattern . however , this postinfusion improvement was not reproduced after her subsequent four infliximab infusions , during which time her daily symptoms were continuing to be recorded . she did experience reproducible improvement in her stools with repeated courses of antibiotics that were prescribed for non - ibd related symptoms by her primary care physician ( previously illustrated in kaplan et al.20 ) . during those times when she experienced decreased nighttime bowel movements , she also felt less fatigued ( fig . 2 ) . upon reviewing these data she confirmed that she felt less fatigued and more energetic . the patient and her physician both felt that the graphical representation of her symptom data aided in their visualization and understanding of the treatment effects . both the patient s mother and her physician were interested in trying different probiotics to determine if one would be more effective than others for managing her fecal urgency . over the course of repeated periods on and off different probiotics,20 her fecal urgency gradually improved ( fig . 3 , green dashed ovals ) , though there was no discernable difference during periods with or without the use of probiotics.20 the reduction in fecal urgency over time was reflected in improved promis fatigue score ( fig . after three months of monitoring , the patient developed an exacerbation of the disease ( fig . after he was diagnosed with crohn s disease , his running pace decreased and had not returned to his pre - illness baseline , and he was interested in assessing his running potential after starting treatment with biological therapy . at his enrollment in this study , his baseline promis fatigue t - score was 35.7 , indicating that he was less fatigued than the average healthy individual . he received his first infliximab infusion on may 25 , 2011 , with induction regimen completed by july 6 , 2011 , ( usually consisting of three infusions spread over six weeks ) . his number of daily bowel movements decreased within four days of completing the induction regimen , and attained a steady state along with a special cause ( fig . his running pace also improved significantly from a mean 8.4 to 7.6 minutes / mile , indicated by a special cause shift as well . we found that monitoring daily symptom data detects the daily variation in patients symptoms . establishing a patient s background pattern of symptom variation can serve as a baseline from which to make subsequent comparisons when making changes in diet or medications . significant ( special cause ) deviations in symptoms from the baseline pattern are also associated with variation in promis instrument assessments . our data from these patients suggest that the promis measures of fatigue and pain , in particular , are responsive to changes in patient symptoms over short periods and on the individual level . these findings lend face validity to daily remote monitoring of patient symptoms , and pave the way for broader use of the promis instruments for use in patient - centered improvement research . using standard promis instruments , in addition to more customized patient symptoms , may enable greater learning across patients because measures are well validated and standardized in pediatric populations . we also found that knowledge about daily symptom variation and promis assessments of key outcomes may be clinically relevant . improving patients and physicians understanding of symptom patterns for the purpose of tailoring treatments to the patients needs is essential for understanding response to attempted therapies . it is also essential to measure symptoms that are important to patients in order to help patients achieve good qol even if other traditional measures show minimal symptoms . this work is important and expands upon the very few previous studies of the impact of daily monitoring on outcomes for chronic diseases.16 the most widely documented evidence is for asthma in which daily monitoring is associated with improved asthma control.28 there is also evidence that daily monitoring via mobile phone of adults with heart failure results in improved qol , improved self - care,12 and in one study decreased mortality.29 similarly , in insulin - dependent diabetes mellitus , daily monitoring leads to improved glucose control , more symptom - free days , and decreased perceived disease - related anxiety.13 despite limited evidence of benefit from mobile symptom tracking in some studies , the majority of studies do not demonstrate a measurable improvement in patient outcomes.16 one significant issue that has not previously been addressed is the day - to - day variability of patient symptoms . as we ve seen in our study , there is substantial daily variability in symptoms , which may obscure the detection of improvements , when they occur . the work reported here builds on previous studies by developing tools that enable daily symptom tracking to better understand and detect changes in patient symptoms . understanding the nuances of symptom variation at baseline , and in response to therapeutic challenges is critical for targeting therapies for improvement and for determining the individual efficacy of therapeutic changes . comparing daily symptom data with weekly promis surveys provides internal consistency of the pro measures collected for each patient . together , the collection of these pro data from patients facilitates real - time feedback to patients and their physicians about the effectiveness of therapies . these methods offer substantial improvements over routine clinical practice and over prior mobile health ( mhealth ) studies lacking such feedback . these were highly engaged patients who volunteered for this study ( as is the case in many studies ) . it is possible that daily or weekly symptom measurement may not be feasible in less - engaged patients . however since 75 percent of young people have mobile phones and 88 percent of them send and respond to numerous text messages daily ( typical 14 to 17 year olds send and receive 100 text messages per day),30 it remains to be determined how important engagement with care is to these assessments . there is also some concern that daily monitoring may lead to increased attention to symptoms and worsening of perceived physical health.31 however others have demonstrated that patients with chronic constipation who monitored daily symptoms had no worse outcomes than those monitored weekly in a prospective randomized clinical trial.32 others found improved outcomes with daily monitoring for irritable bowel syndrome , asthma , and heart failure.12,13,33 it will be important to determine if certain patients may benefit more from these methods and if others should avoid these methods to minimize attention to symptoms . note that a patient with longstanding daily symptoms may have a significant improvement in symptoms without being aware of such changes ( as was seen with patient 1 ) . conversely , perceived improvement may not actually represent real improvement , but may be misattributed background variation which , when graphically evaluated , represents common cause variation ( as seen with patient 2 ) . this was seen with the initial improvement in nocturnal stools following infliximab infusion ( patient 1 ) , which was not reproduced after subsequent infusions . it is tempting to infer a causal relationship between two events because of their close proximity in timing , even if the association is coincidental . the accuracy of the interpretation of associations has direct implications for how most physicians and patients routinely test therapies by ad hoc trial and error . it is only with careful monitoring of outcomes , and with replication ( through experimentation ) , that we can distinguish between causal relationships or associations.34,35 this study also highlights the importance of tracking the symptoms that patients feel are most important to them . the example of patient 3 , whose gastrointestinal symptoms and fatigue were not severe , illustrates this additional benefit of our individualized , patient - centered approach . by closely monitoring the symptom that was most important to the patient ( his running pace ) , he and his physician were able to determine when a significant improvement occurred in the outcome that was of greatest importance to the patient . our findings in this early study highlight the difficulty in assessing treatment efficacy based on casual assessment of symptom relief for an individual patient . these findings are of great importance to improving the physician s ability to adequately understand their patient s disease activity and the symptoms contributing to qol for the purpose of tailoring treatments to the needs of the patient . this applies when new therapies are initiated and also during changing , modifying , weaning off , or withdrawing therapies ( as could be the case with steroid weaning for ibd treatment ) . these new methods offer the potential to support more collaborative decision - making and to improve clinicians and patients abilities to identify the right treatments both through informal learning and planned experimentation with individual patient therapeutic ( n - of-1 ) trials . we must have a clear understanding of the effects of an intervention ( medical , dietary , behavioral , etc . ) on the patient s symptoms . only with a clear understanding of symptom variation , and an ability to discern a meaningful change in symptom pattern from the background variation , can we fully understand the effects of therapies on symptoms and know with certainty whether or not a therapy improves symptoms and qol . the new methods described here are the building blocks for transforming the doctor - patient relationship to become more collaborative and to provide improved evidence for understanding individual patient treatment efficacy for better treatment individualization . based on the success of daily tracking using customized and standardized measures ( promis measures ) , and the utility of graphical display of symptoms using statistical process control , we are developing more advanced technology ( web platform and mobile app ) to enable tracking and real - time visualization of this data for the patient and provider . we are also examining ways to better support patients and providers in using pro data to enable formal n - of-1 studies of pediatric patients with ibd . n - of-1 studies are well established as a method to improve patient symptoms . however , until now these methods have been limited in their scope of use . we are currently expanding these methods that we have described here to develop the means of conducting n - of-1 studies concurrently with larger numbers of patients who may have different complaints and different treatments . these methods have the potential to benefit patients broadly and to improve patient - physician engagement and communication across a wide range of clinical settings . clinicians and patients should consider the use of mobile daily symptom data collection to improve care and outcomes . daily symptom tracking can facilitate improved informal learning as well as rigorous n - of-1 experimental studies to objectively assess individual treatment efficacy and individualize patient care.34,35 prototype studies have provided proof of concept so that these methods can be further expanded to enable more robust learning designed to improve symptoms and qol for pediatric patients with ibd and others with differing chronic health conditions .

introduction : improving symptoms for patients with chronic illness is difficult due to poor recall and imprecise assessments of therapeutic response to inform treatment decisions . daily variation in symptoms may obscure subtle improvement or lead to erroneous associations between symptom changes and alteration in medication or dietary regimens . this may lead to mistaken impressions of treatment efficacy ( or inefficacy ) . mobile health technologies that collect daily patient reported outcome ( pro ) data have the potential to improve care by providing more detailed information for clinical decision - making in practice and may facilitate conducting single subject ( n - of-1 ) trials.methods:interrupted time series to prototype mobile health enabled data collection for three patients . we recruited pediatric patients with established inflammatory bowel disease who had persistent symptoms . based on their self - identified most troubling symptoms , patients were sent customized , daily - automated text messages to assess the extent of their symptoms . standardized , pro measurement information system ( promis ) surveys were deployed weekly . individual statistical process control charts were used to assess variation . patients met with physicians regularly to interpret their data jointly.results:we report the experience of 3 patients with inflammatory bowel disease , each with different symptoms . daily symptom monitoring uncovered important patterns , some of which even patients were unaware before reviewing their symptom data . important associations were found between symptom variation and changes in medications and diet . promis survey results assessed longitudinally accurately reflected changes in patient symptoms.conclusions:we demonstrated how pros can be implemented in practice . monitoring and analyzing daily symptom data , using both customized and standard pros , has the potential to detect meaningful variation in symptom patterns , which can inform clinical decision - making or can facilitate conducting formal n - of-1 trials to further improve outcomes .

autoimmune hepatitis ( aih ) is an immune - mediated chronic liver disease characterized by hepatocellular inflammation , necrosis , and fibrosis , which can progress to cirrhosis and fulminant hepatic failure if left untreated . aih has a female predominance occurring in all ages and races , and both the median and mean age of initial disease presentation are in the forties , similar to many other autoimmune diseases . diagnostic criteria are based on demonstration of characteristic autoantibodies , elevated immunoglobulins , and histological features of hepatitis in the absence of viral liver disease . standard treatment of aih includes corticosteroids alone or in combination with azathioprine ( aza ) that aims at the normalization of transaminase and immunoglobulin g ( igg ) levels in serum which correlate with histological disease activity . it is reported that although majority of the patients respond well to the standard therapies , about 20% of patients do not due to either intolerance or refractory disease . in treatment failure , alternative immunosuppressive and biological agents including mycophenolate mofetil ( mmf ) , tacrolimus , cyclosporine and anti tnf antibodies should be considered . the patient we report here is a 48-year old woman who was diagnosed with autoimmune hepatitis and was treated with standard therapy corticosteroids and aza . however , she could not tolerate the side effect of aza induced pancytopenia and skin rash . mmf in combination with corticosteroid was opted as second - line treatment and the patient was able to achieve remission with these treatments . a 48 year - old woman was admitted to department of hepatology due to fatigue and jaundice which lasted for approximately 10 days . she had history of heavy drinking ( 60 g of alcohol every day ) and herbal tea intake ( solomons seal tea twice a week ) for recent 6 months . on physical examination , icteric sclera was observed but there was neither hepatomegaly nor splenomegaly . upon admission , the laboratory findings were as follows : alanine aminotransferase ( alt ) level 472 iu / l , aspartate aminotransferase ( ast ) level 556 iu / l , alkaline phosphatase ( alp ) 123 iu / l , total bilirubin 3.44 mg / dl , direct bilirubin 1.91 mg / dl and prothrombin time internal normalisation ratio ( inr ) 1.05 . viral markers including hepatitis a antibody immunoglobulin m ( hav ab igm ) , hepatitis b surface antigen ( hbsag ) and hepatitis c antibody ( hcv ab ) were negative . since the patient was a middle aged woman , further assessments to rule out autoimmune hepatitis were also performed . anti - smooth muscle antibody ( sma ) and antinuclear antibody ( ana ) were positive with the ratio of 1:640 for ana and igg was increased to 1,940 mg / dl . liver biopsy was performed and the histologic findings revealed mild portal inflammation with lymphoplasmacytic , neutrophilic and eosinophilic infiltration along with minimal interface activity ( fig . 1 ) . as the histologic findings were not typical for autoimmune hepatitis along with spontaneous slow decrease in ast ( 48 iu / l ) and total bilirubin ( 0.64 mg / dl ) levels the patient was followed up as out - patient without treatment . follow up ana was positive with the ratio of 1:1,280 and igg level was further elevated to 2,420 mg / dl ( fig . 2a , b ) . based on the lab findings and fluctuation of ast and alt levels , diagnosis of autoimmune hepatitis type she was initially prescribed with prednisolone 30 mg which was tapered to 5 mg in in 4 months and was maintained in combination with aza 25 mg daily . the patient showed treatment response with decrease in serum ast and alt levels along with decrease in total igg levels after 3 months of treatment ( fig . after 3 months of maintenance treatment , aza - related side effect , pancytopenia and alopecia developed . hemoglobin level decreased to 9.6 g / dl , white blood cell count decreased to 2,150 mm/l ( absolute neutrophil count 377 mm/l ) and platelet count decreased to 82,000 mm/l ( fig . 2c ) . furthermore , hair loss occurred 2 months after treatment with aza which aggravated until the patient had to wear a wig . therefore , aza was discontinued after 5 months of treatment and was maintained on 5 mg of prednisolone daily . after discontinuation of aza , complete blood count returned to normal range within 1 month while hair growth returned to normal 2 months later . although the symptoms subsided after quitting aza , there were wax and wane in ast and alt levels after 27 months of steroid monotherapy , exacerbation of aih occurred . the steroid dose was escalated to 30 mg again in combination with 50 mg of azathioprine . unfortunately , after 20 days of combination treatment , aza - related side effect occurred again , especially severe alopecia was noted , unabling the patient to maintain on standard treatment . as remission was not achieved with first - line treatment , steroid monotherapy was maintained for another 2 months which was decreased to 15 mg during that duration and mmf 1 g per day was added thereafter . after administration of mmf , her symptoms had subsided and normalization of liver enzymes and igg level was achieved . the patient is still on 5 mg of steroid and 500 mg of mmf and is stable for 12 months after the acute exacerbation of aih . autoimmune hepatitis ( aih ) was first reported in 1951 as a fluctuating persistent hepatitis of young woman with marked elevations of serum immunoglobulins . the median and mean age of initial disease presentation are in the forties and the etiology of aih is unknown , but it carries all features of an autoimmune disease : genetic predisposition , spontaneous disease fluctuations , circulating autoantibodies , auto - reactive t cells , inflammatory infiltrations in liver , and a good response to immunosuppressive agents [ 1,5,7 - 9 ] . aih can be diagnosed when compatible clinical signs and symptoms , laboratory abnormalities ( increased serum ast or alt , and total igg ) , serological ( autoantibodies ) , and histological ( interface hepatitis , lymphocytic and lymphoplasmacytic infiltrates in portal tracts and extending in the lobule , hepatic rosette formation , or chronic hepatitis with lymphocytic infiltration ) findings are present ; and other conditions that can cause chronic hepatitis , including viral , metabolic , cholestatic , hereditary , and drug - induced diseases , have been excluded . studies reported that when aih is left untreated , as many as 40% of the patients with severe disease die within 6 months and of the remaining 40% develop cirrhosis . therefore , when clinical , laboratory or histologic features of active liver inflammation is present , immunosuppressive treatment should be initiated . standard treatment includes corticosteroids alone or in combination with aza that aims at the normalization of transaminase and igg levels in serum which correlate with histological disease activity . in adults with aih , standard treatment can be started with prednisolone ( starting with 30 mg daily and tapering down to 10 mg daily within 4 weeks ) in combination with azathioprine ( 50 mg daily or 1 - 2 mg / kg body weight ) or prednisolone alone ( starting with 40 - 60 mg daily and tapering down to 20 mg daily within 4 weeks ) . although most of the patients respond very well to immunosuppressive treatment , 20% of patients do not respond to or are intolerant to standard therapy . the overall frequency of aza - related side effect is 10% , which can be improved after dose reduction or discontinuation . aza is a non - selective immunosuppressant that acts by inhibition of several enzymes involved in purine synthesis and this contributes to side effects including bone marrow suppression , nausea , rash , alopecia , arthralgias , neoplastic and malabsorption . the frequency of cytopenia in azathioprine treated patients with autoimmune hepatitis is 46% , and the occurrence of severe hematologic abnormalities is 6% . thiopurine s - methyltransferase ( tpmt ) is part of a cascade of enzymes responsible for the metabolism of thioprine drugs including aza , 6-mercaptopurine ( 6-mp ) and 6 thioguanine ( 6-tg ) . some studies reported that activity of tpmt could be used to predict those who would respond to treatment and those who would show hematologic side effects as the patients with deficient tpmt activity are at severe risk of developing bone marrow suppression [ 15 - 17 ] . however , tpmt testing is not routinely performed as it is time consuming and not widely available in the past 15 years alternative immunosuppressive and biological agents for those who can not tolerate standard treatment were evaluated . unfortunately , the treatment options for patients who failed to respond to standard therapy is still limited . mmf , an ester pro - drug of mycophenolic acid which acts as a noncompetitive inhibitor of inosine monophosphate dehydrogenase , the rate - limiting enzyme involved in the de nevo synthesis of purines were studied as an alternative to standard treatment . according to the aasld practice guidelines , mmf or cyclosporine have had the most empiric use as alternative medications and mmf ( 2 g daily orally ) is the most promising current agent where improvement of hepatitis can be expected in 39 - 84% of patients . several studies suggested mmf as an alternative treatment for those who are either refractory or intolerant to steroid and azathioprine combination therapy . study by zachou et al . prospectively studied the efficacy and safety of mmf in treatment nave aih patients where 88% of 59 patients responded to the treatment and even allowed rapid steroid tapering with eventually withdrawal of steroid . another study by sharzehi et al . based on retrospective cohort including 90 patients showed that those who were unable to continue corticosteroid and azathioprine well tolerated mmf with 88% of patients maintaining complete remission . these studies initiated mmf at a dose of 1.5 - 2 g / day in the former while 1g / day in the latter study . the reported side effect was gastrointestinal symptoms including nausea , vomiting and diarrhea , rash and hair loss but no evidence of bone marrow suppression was observed . our patient did not experience any of the mentioned side effects after 10 months of mmf treatment . although the second - line therapy with mmf seems promising , the response rates varied according to reason for stopping aza treatment where patients with aza intolerance had higher response rates to mmf than those who had shown insufficient response to aza ( 43% vs. 25% ) . in our case , pancytopenia and alopecia occurred after 3 months of standard treatment ( corticosteroid and aza ) . unfortunately after 27 months of corticosteroid monotherapy , hepatitis flare recurred and was stabilized with addition of aza but again the same side effect , pancytopenia and alopecia developed . like other studies , alternative treatment with mmf was considered as a good option and we expect better treatment response for our patient as she had shown sufficient response to aza .

autoimmune hepatitis ( aih ) is an immune - mediated chronic liver disease characterized by hepatocellular inflammation , necrosis , and fibrosis , which can progress to cirrhosis and fulminant hepatic failure . the standard treatment for aih includes corticosteroids alone or in combination with azathioprine . although most patients achieve remission using the standard regimen , some patients do not respond due to either drug intolerance or refractory disease ; in such cases alternative immunosuppressive agents should be explored . the second - line therapies are cyclophilin inhibitors such as cyclosporine a or tacrolimus , and nowadays mycophenolate mofetil ( mmf ) is widely used if azathioprine - based therapies are not tolerated . although these are recommended as an alternative to the first - line regimen , there is insufficient evidence for the efficacy of second - line therapies , with the evidence based mainly on expert opinion . therefore , we report an aih patient receiving the standard regimen in whom remission did not occur due to side effects to azathioprine , but was successfully treated with mmf in combination with corticosteroids as an alternative to the standard regimen .

the deleterious impact of hydrosalpinx on fertility is best exemplified by studies showing a 50% reduction in ivf pregnancy rates in their presence ( 1 ) . chlamydia infection and pelvic inflammatory disease are associated with an increased risk of both tubal infertility and ectopic pregnancy , with the association proportionate to the number of infections(2 ) . various theories have been proposed to explain the observation of lower ivf pregnancy rates in the setting of hydrosalpinx , to include mechanical effects , embryo and gametotoxicity , decreased expression of key implantation molecules , and/or a direct effect on the endometrium leading to intrauterine fluid formation(3 ) . drainage of hydrosalpingeal fluid into the endometrial cavity may exert a mechanical effect by washing the transferred embryo from the cavity . cultured epithelial cells isolated from hydrosalpinx affected tubes produce a fluid which is hostile to both spermatozoa and early mouse embryo development(4 ) . multiple studies have demonstrated deficiency in endometrial markers of embryo receptivity in the presence of hydrosalpinx . il-2 , t lymphocytes , cd3 + , cd8 + , cd4 + , alpha v beta 3 integrin , lif , mmps and hoxa10 have all been shown to be decreased in the endometrium from women with versus without hydrosalpinx(5 - 8 ) though molecular inflammatory changes in the setting of hydrosalpinx are documented , a hysteroscopic endometrial phenotype in the presence of hydrosalpinx has not been well characterized . eighteen months prior to presentation , she was diagnosed with a chlamydial infection by cervical culture and treated with a single dose azithromycin . a negative chlamydia culture was confirmed four months prior to presentation . as part of her initial infertility evaluation at our center , a hysterosalpingogram ( hsg ) was performed in a window of doxycycline prophylaxis which demonstrated bilateral hydrosalpinges , with the right tubal diameter measuring greater than the left and bilateral tubal occlusion ( figure 1 ) . hysterosalpingogram demonstrating bilateral distal tubal occlusion with hydrosalpinges staged combined endoscopy was performed in the operating room . hysteroscopy revealed endometrial inflammation as evidenced by diffuse hyperemia and mucosal oedema ( figure 2a ) . laparoscopic findings were significant for a right sided hydrosalpinx measuring over 2 centimeters in diameter and dense adhesions of the bowel to the left hydrosalpinx and ovary . she underwent right salpingectomy and left tubal occlusion at the visible isthmic region via clip placement . she experienced an unremarkable postoperative course and was scheduled for in vitro fertilization ( ivf ) treatment . hysteroscopic appearance of endometrial cavity immediately pre - salpingectomy ( a ) and six months after interruption of communication with hydrosalpinges ( b ) . for each series , images from left to right depict right cornual , fundal and left cornual regions , respectively . six months later , the patient underwent hysteroscopic endometrial cavity evaluation in preparation for ivf . at hysteroscopy , the patient subsequently underwent ivf with transfer of a single blastocyst culminating in the delivery of a healthy infant at term . given the importance of tubal patency in non - ivf treatment and the detrimental impact of tubal pathology such as hydrosalpinx , oviductal evaluation represents an important part of the initial female infertility work up . given the trend among women to present for infertility care later in the reproductive lifespan , patients undergoing ivf may not have had prior tubal evaluation via either of the standard modalities . transvaginal ultrasound in the detection of hydrosalpinx has been reported , albeit with user - dependent sensitivity . the present case suggests that an inflamed endometrial appearance may be a hysteroscopic sign of a communicating hydrosalpinx requiring dedicated tubal study prior to initiating fertility treatment as depicted in figure 2 , the endometrial surface appears erythematous with friable and tortuous vascularity in the setting of a hydrosalpinx , and normal appearing after surgical interruption of hydrosalpingeal drainage . subsequent ivf treatment and transfer of a single blastocyst resulted in an intrauterine pregnancy , with subsequent term delivery , indicative of functional embryo receptivity on a molecular level , although no molecular evidence is available for confirmation . in view of these findings , we propose an inflamed appearance at hysteroscopy for endometrial cavity screening should prompt a dedicated study to rule out hydrosalpinx prior to proceeding with ivf treatment . to date , such a hysteroscopic endometrial phenotype in the presence of hydrosalpinx has not been described .

we report the hysteroscopic findings in a 22 year old nulligravid patient with bilateral communicating hydrosalpinges . the inflamed hyperemic endometrial cavity encountered preoperatively normalized at second look hysteroscopy six months after bilateral tubal interruption . the patient underwent successful ivf with culmination in a singleton , live birth . we propose that an inflamed appearance at hysteroscopy , done for endometrial cavity screening , should prompt a dedicated study to rule out hydrosalpinx prior to proceeding with ivf treatment . to date , such a hysteroscopic endometrial phenotype in the presence of hydrosalpinx has not been well characterized .

chromosomal abnormalities affect about 0.5% of living newborns , and are associated with congenital malformation , cognitive defects , learning disabilities , seizures , etc . cytogenetic techniques can diagnose chromosomal abnormalities , and investigate the possible etiology of birth defects . it is important to know the clinical data of chromosome abnormalities in order to explore the corresponding relationships between the phenotypes and certain chromosome abnormalities , and increase the evidences of initial clinical indications of these types of disorders in different ages . furthermore , the cytogenetic outcomes can guide medical professionals the optimal treatment , social function training , and predicting the possible prognosis . our tertiary care referral center previously reported the results of cytogenetic survey from 1996 to 2010 , which allowed us to closely gain insight into the incidence and distribution of the cytogenetic abnormalities in outpatient children suspected with congenital disorders . the purpose of the present study was to collect data among children who were suspected with chromosomal disorders from january 1 , 2011 to march 31 , 2014 in the children 's hospital , zhejiang university , and tried to establish and update our previous database of common chromosomal anomalies that could be useful for genetic counseling and reminding the medical professionals which kind of patients should be transferred to genetic analysis . we collected children who were suspected with chromosomal disorders from january 1 , 2011 to march 31 , 2014 since this study was an update to the previous report by the same team in the children 's hospital , zhejiang university . the informed consents were obtained from children 's parents / guardians or other legally authorized representatives before the chromosome analysis preparation , including clinical interview of the medical histories and blood sample collections . the clinical features were recorded and the blood sample were collected , and then the blood samples were sent to the medical biology and genetic department laboratory for cytogenetic analysis at zhejiang dian diagnostics , which is an independent third - party medical diagnostic service institution . according to the reasons for referral for cytogenetic analysis , we divided them into 4 groups : group 1 , who presented with specific clinical stigmata ( such as up slanting palpebral fissure , prominent epicantic folds , micrognathia , etc . ) ; group 2 , who had speech or motor developmental delay , or both , or learning disabilities ; group 3 , who presented with congenital genitourinary defects ( including ambiguous genitalia , abnormality of male external genitalia , concealed penis , cryptorchidism , shield chest , widely spaced nipples and amenorrhoea , etc . ) ; and group 4 ( miscellaneous group , including obesity , congenital heart diseases , primary seizures and other indications not listed in the above three groups ) . for those who presented with both specific clinical stigmata and genitourinary defects we would put them into 1 group according to the main complains of their main problems . for routine cytogenetic analysis , 0.5 to 1.0 ml peripheral blood samples were collected from the patients and stored into heparinized test tubes . the karyotypes were determined by g - banding using trypsin and giemsa ( gtg ) . at least 30 cells were routinely analyzed ; in cases of mosaicism , this number was increased to approximately 100 metaphases . the karyotypic descriptions were reported according to the international system for human cytogenetic nomenclature recommendations ( iscn , 1995 ) . the percentage of abnormal cases in each group and the distribution of the numerical and structural abnormalities were determined . we used the chi - squared test to evaluate the detection rates and types of chromosomal anomalies among groups according to different classification criteria . we collected children who were suspected with chromosomal disorders from january 1 , 2011 to march 31 , 2014 since this study was an update to the previous report by the same team in the children 's hospital , zhejiang university . the informed consents were obtained from children 's parents / guardians or other legally authorized representatives before the chromosome analysis preparation , including clinical interview of the medical histories and blood sample collections . the clinical features were recorded and the blood sample were collected , and then the blood samples were sent to the medical biology and genetic department laboratory for cytogenetic analysis at zhejiang dian diagnostics , which is an independent third - party medical diagnostic service institution . according to the reasons for referral for cytogenetic analysis , we divided them into 4 groups : group 1 , who presented with specific clinical stigmata ( such as up slanting palpebral fissure , prominent epicantic folds , micrognathia , etc . ) ; group 2 , who had speech or motor developmental delay , or both , or learning disabilities ; group 3 , who presented with congenital genitourinary defects ( including ambiguous genitalia , abnormality of male external genitalia , concealed penis , cryptorchidism , shield chest , widely spaced nipples and amenorrhoea , etc . ) ; and group 4 ( miscellaneous group , including obesity , congenital heart diseases , primary seizures and other indications not listed in the above three groups ) . for those who presented with both specific clinical stigmata and genitourinary defects we would put them into 1 group according to the main complains of their main problems . for routine cytogenetic analysis , 0.5 to 1.0 ml peripheral blood samples were collected from the patients and stored into heparinized test tubes . the karyotypes were determined by g - banding using trypsin and giemsa ( gtg ) . at least 30 cells were routinely analyzed ; in cases of mosaicism , this number was increased to approximately 100 metaphases . the karyotypic descriptions were reported according to the international system for human cytogenetic nomenclature recommendations ( iscn , 1995 ) . the percentage of abnormal cases in each group and the distribution of the numerical and structural abnormalities were determined . we used the chi - squared test to evaluate the detection rates and types of chromosomal anomalies among groups according to different classification criteria . there were totally 4129 children referred to cytogenetic analysis from january 1 , 2011 to march 31 , 2014 , including 1857 boys and 2272 girls . the average age was 51.7 months , median age was 33 months , and age ranged from 1 day to 18 years and 11 months old . the ratios between cases referred for cytogenetic analyses and total outpatient visits were 1:1607 ( 1036/1,665,048 ) in 2011 , 1:1364 ( 1328/1,812,521 ) in 2012 , 1:1318 ( 1448/1,908,152 ) in 2013 , and 1:1329 ( 317/421,532 ) in first quarter of 2014 , respectively . there was no statistical difference between the referral ratios in these years by chi - squared test ( = 0.03 , p = 0.99 ) . but compared to previous report , the referral ratios were higher than that in 2010 ( = 448 , p < 0.001 ) . there were 769 children who had chromosome abnormalities , accounting for 18.62% of all referral cases . the detection rates of abnormalities were 19.66% ( 365/1857 ) for boys and 17.78% ( 404/2272 ) for girls the detection rate of autosomal anomalies was higher in boys ( 16.6% vs 12.2% , = 16.6 , p < 0.001 ) , but the detection rate of sex - linked chromosomal anomalies was higher in girls than that in boys ( 5.6% vs 3.0% , = 17.2 , p < 0.001 ) . rate of chromosome abnormalities in boys and girls in different age groups according to referral causes , there were 4 groups set . the percentages of chromosome abnormalities for groups 1 , 2 , 3 , and 4 were 59.1% , 10.6% , 7.9% , and 13.1% , respectively . in group 1 , in which children presented with specific clinical stigmata , the detection rate of chromosome abnormalities was highest . while for group 3 , in which children presented with congenital genitourinary defects , the detection rate of chromosome abnormalities was lowest ( fig . totally , 359 cases were detected with down syndrome , 76.9% ( 276 cases ) of which were found before their age of 1 year old , and 90.5% ( 325 cases ) were found before their ages of 3 years old . there were 81 children with turner syndrome ( ts ) , 76.5% of which ( 62/81 ) were diagnosed after 6 years old . almost half of the children with ts had the karyotypes of mosaicism ( table 2 ) . types of chromosomal abnormalities according to age when children were transferred to do cytogenetic analysis , the detection rates were higher in neonates and infants than that in others ( fig . further analysis showed that most of chromosome abnormalities were trisomy 21 , accounting for almost half of all the abnormalities ( fig . , we presented the cytogenetic results of 769 cases in detail ( table 2 ) . four hundred two ( 19.5% ) were confirmed by cytogenetic analysis and their results were consistent with primary diagnosis . in the other 2070 children with unknown causes , 326 ( 15.7% ) this investigation showed the total detection rate of chromosome abnormalities was 18.62% in the cases suspected with congenital disorders , which was lower than that in the previous report undertaken by the same study group from 1996 to 2010 . in recent 2 decades , it could only indicate the occurrence rate of 1 disease in the group of suspected with 1 type of disease in 1 center . the data from these patients were compared with that from our previous published group of children ( 19962010 ) from the same children 's hospital . we found that the ratios between cases referred for cytogenetic analyses and total outpatient visits were increased from 2011 to 2014 . compared to the previous report , these present ratios were higher than that before 2006 , and equal to that post-2007 . this result indicated that the professionals had more awareness of transferring the children with clinical manifestations of unknown causes to do cytogenetic analysis . the most notable feature of these types of disorders is diagnostic difficulty since they present diverse clinical pictures . physical examination , radiographic studies , and histologic investigation may prove to be equivocal . the data from table 1 showed that the detection rate for the children < 1 year old was higher than that for other older age groups . , we should take it into consideration that the percentage of trisomy 21 was 76.9% in total chromosome abnormalities in 0 to 1 year group , and 90.5% in 0 to 3 years group . these data indicated that professionals had the experiences to identify this common syndrome , even when patients were very young . however , for the most common sex - linked chromosome abnormality of ts , only 5 children were diagnosed in 1 year old , accounting for 6.2% of total diagnosed cases ( more than 2/3 of them ( 31/81 ) were identified until children had more symptoms in their adolescent stages . this indicates the clinical doctors were not familiar with ts , especially for those who were very young . if we found the patients in their early stages , we could treat them timely , which would greatly improve their prognosis , including final stature and sexual development . therefore , it was significant to do more training with the diverse features of these type of chromosome disorders , which would contribute to finding them early , and being treated timely . if we compared the detection rate according to the reasons for referral for cytogenetic analysis , we found that the group 1 which presented with specific clinical stigmata had highest detection rate of 59.1% . among them , 91.9% ( 330/359 ) of down syndromes , 25.0% of tss were found . we speculated that , as mentioned above , our professionals were familiar with down syndrome , but were not with ts . we should take the factor of detection resolution into consideration that might lead to low detection rate . normal development of the genitourinary ( gu ) tract is a complex process that frequently goes abnormal . in male children , the most frequent congenital gu anomalies are cryptorchidism ( 14% ) , hypospadias ( 1% ) , and micropenis ( 0.35% ) . current evidences suggested that monogenetic changes contributed to the congenital genitourinary defects , which could be detected at dna levels or fluorescence in situ hybridization ( fish ) or array comparative genomic hybridization ( acgh ) rather than chromosomal analysis . the professionals could transfer these children to do next - generation sequencing or microarray and promote the detection rate . most of them were transferred to do cytogenetic analysis because they were found with abnormal appearance of the external genitalia , or without second sexual features in their ages of more than 12 years . thus , the percentages of sex - linked chromosome abnormalities in children older than 6 years were higher than those younger than 6 years ( 10.9% vs 52.7% ) . presently , most professional organizations have recommended more higher resolution tests for children suspected with chromosome abnormalities , as acgh testing . but the acgh testing is not popularized among general hospitals in china , and which is more expensive . secondly , we did not establish an interaction between a specific type of chromosome abnormality and clinical features . actually , because of the miscellaneous information , the clear relationship is not established . as an alternative designation , we divided these clinical features into 4 groups to define an extensive association of clinical features with chromosome abnormalities . this study demonstrated the detection rates of chromosome abnormalities in children who were suspected with chromosomal disorders . there were 769 children who had chromosome abnormalities , accounting for 18.62% of all referral cases . among the affected , the percentage of sex - linked chromosomal abnormalities in all was 23.8% . combined with previous report , we established a database of common chromosomal anomalies and the clinical features and remind the medical professionals what kind of patients should be transferred to genetic analysis .

abstractthis study aimed to investigate the detection rate of chromosome abnormalities in children suspected with congenital disorders in 1 single center , identify any differences according to different classification criteria , and try to enlighten the medical professionals what clinical features should be transferred for cytogenetic analysis.from january 1 , 2011 to march 31 , 2014 , children who were suspected with chromosomal disorders were included . all the cytogenetic analyses were performed in the medical biology and genetic department laboratory in zhejiang dian diagnostics . we evaluated the variants of clinical indications , and incidence and types of chromosomal abnormalities among groups.during the study period , 4129 samples were collected and analyzed . among them , 769 children were detected with chromosome abnormalities , accounting for 18.62% of all referral cases . the ratio of sex - linked chromosomal abnormalities to autosomal ones was 1:3.2 . the detection rates were 19.66% ( 365/1857 ) for boys and 17.78% ( 404/2272 ) for girls . most of trisomy 21 were found before the age of 1 year old , while most of children with turner syndrome were found after 6 years old . the group presenting with specific clinical stigmata had highest detection rate of 59.1%.we demonstrated the detection rates of chromosome abnormalities in children who were suspected with chromosomal disorders . combined with previous report , we established a database of common chromosomal anomalies and the clinical features that could be useful for genetic counseling and remind the medical professionals what kind of patients should be transferred to genetic analysis .

posterior urethral valve ( puv ) is the commonest cause of renal impairment in boys during early childhood . despite a systematic approach in deciding choice of therapy in each case , renal failure antenatal diagnosis of hydronephrosis is possible since 1980s but sensitivity and specificity of ultrasound for antenatal diagnosis of puv remains low . specific diagnosis is required as puv is associated with a poorer prognosis as compared to other causes of hydronephrosis which are diagnosed antenatally , such as pelvi - ureteric junction obstruction ( pujo ) and vesico - ureteric reflux ( vur ) . antenatal diagnosis helps in parental counseling and considering options for antenatal intervention if the diagnosis could be made with precision & foetuses with puv and poorer prognosis could be identified antenatally . renal damage in puv occurs as a result of activation of renin - angiotensin system pathway , in which renin is an early marker . therefore , the levels of renin must be raised early in those cases with puv who progress to renal damage . this study was designed to study the role of cord blood plasma renin activity ( pra ) and ultrasonography in antenatal diagnosis and prognostication in puv . this was a prospective observational study conducted over a period of 1.5 years between january 2013 to june 2014 . patients with parental refusal to consent and disappearance of hydronephrosis on 3 trimester ultrasound were excluded . ethical clearance was obtained and patients were enrolled from obstetrics and gynecology outpatient department ( opd ) . all the patients were followed up with serial ultrasounds till delivery and severity of hydronephrosis was assessed using the society of fetal urology ( sfu ) grading [ table 1 ] . in patients with bilateral hydronephrosis , two milliliter of cord blood was collected for pra estimation at the time of cord clamping in ethylenediaminetetraacetic acid ( edta ) . ultrasonography kidney ureter , bladder ( usg kub ) to confirm hydronephrosis and baseline renal function tests ( rft ) were done at 48 hours of birth . diagnosis of puv was confirmed by micturating cystourethrogram ( mcu ) done soon after birth . accordingly , patients were divided into two groups : puv and those with hydronephrosis due to vesico - ureteric reflux and pelvi - ureteric junction obstruction ( non - puv ) . patients diagnosed as puv were managed according to our current clinical practice and published protocol . all puv patients were followed up with mcu , glomerular filtration rate ( gfr ) and tc-99 m dimercaptosuccinic acid ( dmsa ) scan at 1 and 6 months post - operatively . puv patients were divided into two groups based on gfr values at 6 months post - operatively those with a gfr < 60 and those with a gfr 60 ml / min/1.73 m bsa . society of fetal urology ( sfu ) grading of hydronephrosis blood sample collected from peripheral vein was immediately transported to laboratory in an ice pack . upon arrival in the laboratory , pra was measured by radioimmunoassay of generated angiotensin - i using diagnostics biochem canada inc ( dbc ) enzyme - linked immunosorbent assay ( elisa ) kit ( can - ra-4600 ) and values expressed as ng the records of all patients were studied regarding gestational age at detection of hydronephrosis , surgical intervention , antenatal ultrasound findings , cord blood and follow - up pra values , vur , and renal cortical scarring on dmsa . data analysis was done using ibm statistical package for the social sciences ( spss ) statistics 20 ( ibm , armonk , new york , united states ) . values were reported as number ( % ) or mean standard deviation ( range ) as appropriate , unless otherwise reported . blood sample collected from peripheral vein was immediately transported to laboratory in an ice pack . upon arrival in the laboratory , pra was measured by radioimmunoassay of generated angiotensin - i using diagnostics biochem canada inc ( dbc ) enzyme - linked immunosorbent assay ( elisa ) kit ( can - ra-4600 ) and values expressed as ng / ml / hr . the records of all patients were studied regarding gestational age at detection of hydronephrosis , surgical intervention , antenatal ultrasound findings , cord blood and follow - up pra values , vur , and renal cortical scarring on dmsa . data analysis was done using ibm statistical package for the social sciences ( spss ) statistics 20 ( ibm , armonk , new york , united states ) . values were reported as number ( % ) or mean standard deviation ( range ) as appropriate , unless otherwise reported subsequently , 21 of them were excluded as hydronephrosis disappeared in the third trimester scan . the data of remaining 25 patients ( 10 puv and 15 non - puv ) were analyzed . in the non - puv group , 9 ( 60% ) had pujo including two with bilateral pathology while 6 patients had non - obstructive hydronephrosis . the mean gestational age at detection of hydronephrosis was 154.6 15.9 days ( range : 132 - 191 days ) . the mean age was 150.9 12.8 days ( range : 134 - 167 days ) and 157.1 17.6 days ( range : 132 - 191 day ) in puv and non - puv patients , respectively ( p = 0.349 ) . overall , 19 ( 76% ) patients had bilateral and 6 ( 24% ) had unilateral hydronephrosis on initial ultrasound . all 10 puv patients had bilateral hydronephrosis . in the non - puv group , 9 ( 60% ) had bilateral while 6 ( 40% ) had unilateral hydronephrosis . the distribution of grades of hydronephrosis was not significantly different among these two groups ( p = 0.669 ) [ table 2 ] . grades of hydronephrosis on initial ultrasound ( n = 25 , p = 0.669 ) the keyhole sign was seen in 6/25 patients on initial antenatal ultrasound . it was observed in 1/15 ( 6.7% ) non - puv patients and 5/10 ( 50% ) puv patients ( p = 0.023 ) . the sensitivity and specificity of keyhole sign for diagnosing puv were 50% and 93.33% , respectively . the mean amniotic fluid index ( afi ) on initial ultrasound was 12.3 3.4 cm ( range : 4.6 - 19.8 cm ) . it was 10.1 2.8 cm ( range : 4.6 - 16.2 cm ) and 13.8 2.9 cm ( range : 9.0 - 19.8 cm ) in puv and non - puv group , respectively ( p = 0.005 ) [ table 3 ] . oligohydramnios was observed in only one ( 10% ) patient in the puv group and none in the non - puv group on initial ultrasound . amniotic fluid index on initial ultrasound ( n = 25 ) the mean cord blood pra value for controls and patients were 3.95 1.57 ng / ml / hr ( range : 2.26 - 8.00 ng / ml / hr ) and 10.71 5.57 ng / ml / hr ( range : 1.51 - 20.71 mean pra was 7.08 3.28 ng / ml / hr ( range : 1.51 - 12.58 ng / ml / hr ) and 16.15 3.36 ng / ml / hr ( range : 11.05 - 20.71 ng / ml / hr ) for non - puv and puv group , respectively [ figure 1 ] . the difference in mean cord blood pra values between the controls and non - puv group ( p = 0.004 ) , puv and control groups ( p < 0.0001 ) , and puv and non - puv groups ( p < mean pra was10.47 3.26 ng / ml / hr ( range : 6.32 - 16.08 there was a significant fall in pra in all patients of puv post valve ablation ( p < 0.0001 ) . mean cord blood plasma renin activity ( pra ) values in various groups , noh : non - obstrcutive hydronephrosis , all pra values are expressed as ng / ml / hr mean plasma renin activity ( pra ) values in posterior urethral valve ( puv ) patients : pre- ( cord blood ) and post - valve ablation , all pra values are expressed as ng / ml / hr baseline serum creatinine ( at 48 hours of birth ) was within normal limits in 2/10 ( 20% ) puv patients while it was raised in 8/10 ( 80% ) patients . the mean baseline creatinine was 1.33 0.96 ( range : 0.30 - 3.20 mg / dl ) . vesicoureteric reflux was seen in 9 ( 90% ) of 10 puv patients at the time of diagnosis . renal cortical scan ( dmsa ) done 1 month post ablation showed presence of scars in 6 ( 60% ) out of 10 puv patients . it was unilateral in 4 ( 66.6% ) and bilateral in 2 ( 33.3% ) patients . at 6 months post ablation , scars were unilateral in 6 ( 60% ) , bilateral in 2 ( 20% ) , and absent in 2 ( 20% ) patients . the mean gfr was 37.3 18.21 ml / min/1.73 m ( range : 12 - 70 ml / min/1.73 m ) and 61.0 24.44 ml / min/1.73 m ( range : 29 - 89 ml / min/1.73 m ) at 1 and 6 month post valve ablation . four patients had gfr < 60 while 6 had gfr 60 ml / min/1.73 m bsa at last follow - up . mean cord blood pra among puv patients with gfr < 60 and those with gfr > 60ml / min/1.73 m bsa was 19.73 0.81 ng / ml / hr ( range : 18.72 - 20.71 ng / ml / hr ) and 13.77 1.73 ng / ml / hr ( range : 11.05 - 15.57 ng / ml / hr ) , respectively ( p < 0.0001 ) [ table 4 ] . correlation between glomerular filtration rate ( gfr ) and plasma renin activity ( pra ) in posterior urethral valve ( puv ) patients ( n = 10 ) gestational age at detection of hydronephrosis , bladder wall thickness , amnitotic fluid index , and presence of cortical cysts did not significantly correlate with renal impairment among puv patients . posterior urethral valve is one of the most serious congenital urinary tract anomalies that can lead to deleterious effect on future bladder and renal function . despite improvement in survival as many as 25 - 60% of these patients may have significant impairment in renal function in long - term follow - up . the gold standard for post - natal diagnosis is micturating cystourethrography , while pre - natal diagnosis is dependent on routine screening ultrasonography which has a low specificity and is operator dependent . despite the ability to identify features of bladder outlet obstruction early in fetal development , there is no consensus on how to incorporate early detection into current screening protocols . in centers where antenatal salvage therapies in the form of amnio - infusion and vesico - amniotic shunts are being used , the correct diagnosis of puv would improve the efficacy of these procedures with respect to case selection . with our current screening strategy , mortality and long term morbidity from puv will likely remain unchanged until it is possible to intervene prior to the onset of irreversible renal damage . new biologic markers will allow for more effective diagnosis and intervention at earlier stages of fetal development . bajpai et al . , first documented the activation of renin angiotensin system ( ras ) using pra , in patients with posterior urethral valves . they found that mean pra in patients with renal damage was significantly higher as compared to patients with normal renal function . in the present study , cord blood pra was raised in all patients of puv , whereas no other parameter showed such consistency . cord blood pra was raised in 2/3 of the patients with pujo and none of the patients with non - obstructive hydronephrosis . also , the levels were significantly higher in cases of puv than those without puv ( p < 0.0001 ) . keyhole sign was seen on initial antenatal ultrasound in higher proportion of puv patients ( 50% versus 6.7% ) ( p = 0.023 ) . the sensitivity of this sign for diagnosis of puv was 50% and specificity was 93.3% . these values were higher compared to those reported in a series published by bernardes et al . oligohydramnios did not significantly correlate with diagnosis of puv ( p = 0.40 ) . on the other hand , mean afi values were significantly lower in patients with puv when compared to non - puv patients . renal function deterioration has been linked to age at presentation , gfr , prenatal diagnosis , renal dysplasia , vur , renal scarring , nadir creatinine during 1 year of life , upper tract obstruction , bladder dysfunction , and urinary tract infection ( uti ) . in the recent decades , the choice of therapy in puv could be easily discerned by a step - wise approach using the step - ladder protocol . endoscopic valve ablation has become the mainstay of treatment for puv . in the present study , age at presentation has been suggested as a predictor of renal function in children with puv . prenatal diagnosis was initially thought to improve the outcome , but earlier studies failed to show that the long - term outcome in prenatally detected puv patients is better than symptomatic patients detected postnatally . hutton et al . , reported that antenatal detection before 24 weeks of gestation predicted a poorer prognosis than later detection , with more than 50% of the earlier diagnosed group dead or in renal failure within 4 years of follow - up . in the present study , there was no significant difference in gestational age at detection among patients who ultimately developed renal impairment and those who did not . the decline in renal function after valve ablation is accompanied by activation of ras reflected in a gradual rise in pra . in an earlier study , fall in gfr , high grade vur , scars , and raised serum creatinine were not consistent in detecting renal damage in puv patients but plasma renin activity was found to be high in all such patients . it has been shown that increase in plasma renin activity ( pra ) precedes all the other presently accepted criteria of renal damage . in a recent study of 58 patients with puv , mean pra was high in all but four patients and the fall in gfr was preceded by a rise in pra by a significant interval , implying that ras is activated much earlier before the fall in gfr becomes evident . therapy with angiotensin converting enzyme - inhibitors stabilizes and then improves renal function , thereby , retarding the pace of renal damage . in the present study mean cord blood pra values were higher in patients who had gfr < 60 compared to those with a gfr 60ml / min/1.73 m bsa ( p < 0.0001 ) . this study highlights the diagnostic significance of cord blood pra in infants with puv . in this pilot study , we have noted that utrasound parameters are not very useful in the antenatal diagnosis of puv and measurement of pra in the fetuses may have the potential to differentiate poor from better prognosis puv patients . on antenatal ultrasound keyhole sign , bladder cycling , oligoamnios , and cortical cysts are not consistent findings in posterior urethral valves . in congenital hydronephrosis cord blood pra is significantly higher in cases with puv than those without puv and falls significantly after valve ablation .

aims : study on prognostic significance of antenatal ultrasonography and renin angiotensin system activation in predicting disease severity in posterior urethral valves.materials and methods : antenatally diagnosed hydronephrosis patients were included . postnatally , they were divided into two groups , posterior urethral valve ( puv ) and non - puv . the studied parameters were : gestational age at detection , surgical intervention , ultrasound findings , cord blood and follow up plasma renin activity ( pra ) values , vesico - ureteric reflux ( vur ) , renal scars , and glomerular filtration rate ( gfr).results : a total of 25 patients were included , 10 puv and 15 non - puv . all infants with puv underwent primary valve incision . gfr was less than 60 ml / min/1.73 m2 body surface area in 4 patients at last follow - up . keyhole sign , oligoamnios , absent bladder cycling , and cortical cysts were not consistent findings on antenatal ultrasound in puv . cord blood pra was significantly higher ( p < 0.0001 ) in puv compared to non - puv patients . gestational age at detection of hydronephrosis , cortical cysts , bladder wall thickness , and amniotic fluid index were not significantly correlated with gfr while pra could differentiate between poor and better prognosis cases with puv.conclusions:ultrasound was neither uniformly useful in diagnosing puv antenatally , nor differentiating it from cases with non - puv hydronephrosis . in congenital hydronephrosis , cord blood pra was significantly higher in cases with puv compared to non - puv cases and fell significantly after valve ablation . cord blood pra could distinguish between poor and better prognosis cases with puv .

where applicable , please follow the ontology for biomedical investigations : http://obi-ontology.org/page/main_pageimage 1organism / cell line / tissuenih3t3 mouse fibroblasts and transformed nih3t3-k - rassexnasequencer or array typemouse genechip mogene-1_0 -st - v1 arrays ( affymetrix)data formatraw data : cel files , normalized data : soft , miniml , txtexperimental factorsimmortalized nih3t3 mouse fibroblasts vs. transformed nih3t3-k - ras mouse fibroblasts cultured at 1 mm glucose and daily treated with 10 m forskolinexperimental featurestranscriptome profiling of genes modulated by pka in immortalized and transformed mouse fibroblasts . cells were cultured in medium with 1 mm glucose and then treated with fsk . the samples for the microarray analysis were collected at 72 h of culture in low glucose after daily treatments with fsk.consentdata are publicly available the immortalized nih3t3 and the transformed nih3t3-k - ras cell lines were routinely maintained in dmem medium ( life technologies ) with 10% new born calf serum ( life technologies ) , 25 mm glucose and other supplements as described previously , . all cells were cultured at 37 c in 5% co2 . to perform the array experiments after 16 h , the cells were washed twice with phosphate - buffered saline and incubated in medium ( time 0 ) supplemented with 1 mm glucose instead of 25 mm ( fig . hence pka activation , the cells were daily treated with 10 m forskolin ( fsk ) ( sigma - aldrich ) starting from 24 h after medium replacement . fsk - treated and untreated ( dmso ) cells were then collected for array analyses at 72 h of culture in low glucose medium , which corresponds to 48 h of fsk - treatment ( fig . the basal and the fsk - induced camp levels as well as the pka activation in both cell lines are described in , . cells were lysed in trizol ( invitrogen , carlsbad , ca ) and total rna was further purified with the rneasy kit ( qiagen , hilden , germany ) following manufacturer instructions . rna quality and integrity was determined by an agilent 2100 bioanalyzer ( agilent technologies , santa clara , ca ) on rna nano chips . single strand biotinylated cdna was generated as follows : 100 ng of total rna were subjected to 2 cycles of cdna synthesis with the ambion wt expression kit ( life technologies ) . the first cycle - first strand synthesis is performed using an engineered set of random primers that exclude rrna - matching sequences and include the t7 promoter sequences . after second - strand synthesis , the resulting cdna is in vitro transcribed with the t7 rna polymerase to generate a crna . this crna is subjected to a second cycle - first strand synthesis in the presence of dutp in a fixed ratio relative to dttp . single strand cdna is then purified and fragmented with a mixture of uracil dna glycosylase and apurinic / apirimidinic endonuclease 1 ( affymetrix ) in correspondence of incorporated dutps . dna fragments are then terminally labeled by terminal deoxynucleotidyl transferase ( affymetrix ) with biotin . the biotinylated dna was hybridized to the mouse genechip mogene-1_0 -st - v1 arrays ( affymetrix ) , containing almost 23000 genes selected from mus musculus genome databases refseq , ensembl and genbank . chips were washed and scanned on the affymetrix complete genechip instrument system , generating digitized image data ( dat ) files . data where then normalized using a global normalization approach with the affymetrix gene expression console software , generating the .cel files microarray analysis was performed on biological triplicate samples . labeling of cdna and hybridization to mouse mogene-1_0 -st - v1 arrays were performed at the irgs , biogem , ariano irpino ( av ) , italy . the samples included ( see table 1 ) : 1 ) gsm1666885 , immortalized nih3t3 mouse fibroblasts replicate 1 , 2 ) gsm1666886 , immortalized nih3t3 mouse fibroblasts replicate 2 , 3 ) gsm1666887 , immortalized nih3t3 mouse fibroblasts replicate 3 , 4 ) gsm1666888 , nih3t3-k - ras transformed mouse fibroblasts replicate 1 , 5 ) gsm1666889 , nih3t3-k - ras transformed mouse fibroblasts replicate 2 , 6 ) gsm1666890 , nih3t3-k - ras transformed mouse fibroblasts replicate 3 , 7 ) gsm1666891 , immortalized nih3t3 mouse fibroblasts treated with fsk replicate 1 , and 8) gsm1666892 , immortalized nih3t3 mouse fibroblasts treated with fsk replicate 2 , 9 ) gsm1666893 , immortalized nih3t3 mouse fibroblasts treated with fsk replicate 3 , 10 ) gsm1666894 , nih3t3-k - ras transformed mouse fibroblasts treated with fsk replicate 1 , 11 ) gsm1666895 , nih3t3-k - ras transformed mouse fibroblasts treated with fsk replicate 2 , 12 ) gsm1666896 , nih3t3-k - ras transformed mouse fibroblasts treated with fsk replicate 3 . all samples were normalized through the robust multi - array average ( rma ) method with background correction and quantile normalization at core genes level as implemented by the r / bioconductor package differential expression analysis was performed using a linear model approach with least - square fitting and false discovery rate correction as provided by the r / bioconductor package two differential expression tests ( contrasts ) were performed : untreated normal cells vs. fsk - treated normal cells - denoted as nf / n - and untreated transformed cells vs. fsk - treated transformed cells - denoted as tf / t . only the probe of each gene having the minimum average p - value in the two contrasts has been kept . there were 496 genes ( probes ) with absolute log fold - change at least 1 for contrast nf / n and 208 genes for contrast tf / t . to gain a better understanding of the biological implications of these expression profiles , we performed a comprehensive gene set analysis of the differential expression results using the implementation provided by the r / bioconductor package the analysis used as gene sets the 202 kegg pathways classified as metabolic or signaling as downloaded on jan . the pathways contain 6885 unique genes of which 6010 are included in the analysis dataset ( approx . piano provides the implementation of several gene set analysis tests and of a consensus methodology to summarize the results obtained with the different tests . this approach should be less sensitive to possible hidden biases of the single tests and allows , for example , the integration of sources of gene expression data with other types of high throughput data like proteomic data obtained by 2-dige . the following tests have been performed : a)meanb)medianc)sumd)maxmeane)gene set enrichment analysis ( gsea)f)parametric analysis of gene set enrichment ( page)g)reporter featuresh)wilcoxon rank - sum testi)tail strength . gene set enrichment analysis ( gsea ) parametric analysis of gene set enrichment ( page ) wilcoxon rank - sum test tests ( a)(f ) used t - values computed in the differential expression analysis while tests ( g)(i ) used the corresponding p - values ( and log fold changes , for devising the directionality ) . tests ( f ) , ( g ) , ( h ) computed gene set statistics significance via the theoretical null distribution , while the remaining ones via gene ( re)sampling . each test may return distinct p - values for each gene set ( i.e. , pathway ) in the following directionality classes : distinct - directional ( up or down ) , indicated as dist(up ) and dist(down ) , which tests if the expression of the gene set as a whole is regulated in a distinct direction ( either up or down ) . by definition , a gene set can not be significant in both classes as regulations in opposite directions will cancel out the effect.mixed-directional ( up or down ) , indicated as mix(up ) and mix(down ) , which tests if a significant subset of its genes are coordinately up- or down - regulated . a gene set can be significant in both classes.non-directional , indicated as nondir , which tests if the expression of the gene set as a whole is significantly perturbed , discarding any information about directionality . distinct - directional ( up or down ) , indicated as dist(up ) and dist(down ) , which tests if the expression of the gene set as a whole is regulated in a distinct direction ( either up or down ) . by definition , a gene set can not be significant in both classes as regulations in opposite directions will cancel out the effect . mixed - directional ( up or down ) , indicated as mix(up ) and mix(down ) , which tests if a significant subset of its genes are coordinately up- or down - regulated . non - directional , indicated as nondir , which tests if the expression of the gene set as a whole is significantly perturbed , discarding any information about directionality . a total of 35 p - value vectors over the 45 possible combinations for each contrast have been obtained since not all tests returned results for all the directionality classes . the combined analysis of the p - values obtained by each gene set in the various directionality classes provides a clear picture of the ( possible ) regulation of that gene set . consistency of the results has been empirically verified by computing the pairwise correlation between the p - value vectors . 2 depicts the correlation matrices of the p - value vectors of contrast nf / n on the left and of contrast directionality classes of the p - value vectors are indicated by the annotation bars on the top and on the left of the two plots , while letters refer to the different gene set analysis tests as per the previous list . according to the plots , p - value vectors of the same directionality class ( obtained with different tests ) have strong correlation ( see , for example , the class dist(dn ) ) , p - value vectors of similar classes have a non - negligible correlation ( see , for example , mix(dn ) and dist(dn ) ) and p - value vectors of opposite classes are inversely correlated ( see , for example , dist(dn ) and dist(up ) ) . further confirmation is provided by the dendograms depicted on the sides , showing that tests of the same directionality class cluster together . the only exception is test ( i ) tail strength for the distinct down - directional class on both contrasts . despite that , the p - values of the gene sets on these classes still appear to be in accordance ( albeit less correlated ) with the rest of the results and represent only a small perturbation that the final consensus analysis should easily accommodate . principal component analysis ( pca ) of the same set of vectors provides a second empirical support to the consistency of the results computed by the different tests . 3 depicts each p - value vector ( represented as the corresponding letter , see the list above ) for contrast nf / n ( left ) and contrast tf / t ( right ) in the space of its first three principal components ( x- and y - axes and size ) . ideally , each directionality class ( represented by colors ) should cluster together in the resulting space . the most evident outliers are those computed by test ( i ) for the distinct down - directional class , that are placed near the vectors of the mixed down - directional class . piano computes the overall consensus rank by first ranking each gene set in each directionality class for each method according to the predicted p - value . these ranks are then aggregated according to the borda rule ( a single - winner election method ) . 4 depicts the aggregated ranks obtained by the gene sets that ranked in the first 5 positions in at least one directionality class . directionality classes are represented as columns while numbers and colors represent the position of each gene set ( row ) in the aggregated rank of that column for both contrast nf / n ( left block ) and contrast tf / t ( right block ) . in general , a pathway can be considered significantly regulated in a particular directionality class if it ranks in the first positions ( albeit a general cut - off does not exist ) . as expected , pathways ranking high ( i.e. , small numbers ) in a directionality class ( see , for example , ribosome biogenesis in eukaryotes ) are ranked low ( large numbers ) in the opposite directionality class . such a representation provides a clear and concise comparison of the regulation of pathways in the two contrasts , highlighting common regulation of pathways in normal and transformed cells , like ribosome biogenesis in eukaryotes , down - regulated in both nf / n and tf / t , and specific pathway regulations , different for the two cell lines , like the up - regulation of pathway steroid biosynthesis detected in tf / t but not in nf / n . the immortalized nih3t3 and the transformed nih3t3-k - ras cell lines were routinely maintained in dmem medium ( life technologies ) with 10% new born calf serum ( life technologies ) , 25 mm glucose and other supplements as described previously , . all cells were cultured at 37 c in 5% co2 . to perform the array experiments after 16 h , the cells were washed twice with phosphate - buffered saline and incubated in medium ( time 0 ) supplemented with 1 mm glucose instead of 25 mm ( fig . hence pka activation , the cells were daily treated with 10 m forskolin ( fsk ) ( sigma - aldrich ) starting from 24 h after medium replacement . fsk - treated and untreated ( dmso ) cells were then collected for array analyses at 72 h of culture in low glucose medium , which corresponds to 48 h of fsk - treatment ( fig . the basal and the fsk - induced camp levels as well as the pka activation in both cell lines are described in , . cells were lysed in trizol ( invitrogen , carlsbad , ca ) and total rna was further purified with the rneasy kit ( qiagen , hilden , germany ) following manufacturer instructions . rna quality and integrity was determined by an agilent 2100 bioanalyzer ( agilent technologies , santa clara , ca ) on rna nano chips . single strand biotinylated cdna was generated as follows : 100 ng of total rna were subjected to 2 cycles of cdna synthesis with the ambion wt expression kit ( life technologies ) . the first cycle - first strand synthesis is performed using an engineered set of random primers that exclude rrna - matching sequences and include the t7 promoter sequences . after second - strand synthesis , the resulting cdna is in vitro transcribed with the t7 rna polymerase to generate a crna . this crna is subjected to a second cycle - first strand synthesis in the presence of dutp in a fixed ratio relative to dttp . single strand cdna is then purified and fragmented with a mixture of uracil dna glycosylase and apurinic / apirimidinic endonuclease 1 ( affymetrix ) in correspondence of incorporated dutps . dna fragments are then terminally labeled by terminal deoxynucleotidyl transferase ( affymetrix ) with biotin . the biotinylated dna was hybridized to the mouse genechip mogene-1_0 -st - v1 arrays ( affymetrix ) , containing almost 23000 genes selected from mus musculus genome databases refseq , ensembl and genbank . chips were washed and scanned on the affymetrix complete genechip instrument system , generating digitized image data ( dat ) files . data where then normalized using a global normalization approach with the affymetrix gene expression console software , generating the .cel files labeling of cdna and hybridization to mouse mogene-1_0 -st - v1 arrays were performed at the irgs , biogem , ariano irpino ( av ) , italy . the samples included ( see table 1 ) : 1 ) gsm1666885 , immortalized nih3t3 mouse fibroblasts replicate 1 , 2 ) gsm1666886 , immortalized nih3t3 mouse fibroblasts replicate 2 , 3 ) gsm1666887 , immortalized nih3t3 mouse fibroblasts replicate 3 , 4 ) gsm1666888 , nih3t3-k - ras transformed mouse fibroblasts replicate 1 , 5 ) gsm1666889 , nih3t3-k - ras transformed mouse fibroblasts replicate 2 , 6 ) gsm1666890 , nih3t3-k - ras transformed mouse fibroblasts replicate 3 , 7 ) gsm1666891 , immortalized nih3t3 mouse fibroblasts treated with fsk replicate 1 , and 8) gsm1666892 , immortalized nih3t3 mouse fibroblasts treated with fsk replicate 2 , 9 ) gsm1666893 , immortalized nih3t3 mouse fibroblasts treated with fsk replicate 3 , 10 ) gsm1666894 , nih3t3-k - ras transformed mouse fibroblasts treated with fsk replicate 1 , 11 ) gsm1666895 , nih3t3-k - ras transformed mouse fibroblasts treated with fsk replicate 2 , 12 ) gsm1666896 , nih3t3-k - ras transformed mouse fibroblasts treated with fsk replicate 3 . all samples were normalized through the robust multi - array average ( rma ) method with background correction and quantile normalization at core genes level as implemented by the r / bioconductor package differential expression analysis was performed using a linear model approach with least - square fitting and false discovery rate correction as provided by the r / bioconductor package two differential expression tests ( contrasts ) were performed : untreated normal cells vs. fsk - treated normal cells - denoted as nf / n - and untreated transformed cells vs. fsk - treated transformed cells - denoted as tf / t . only the probe of each gene having the minimum average p - value in the two contrasts has been kept . there were 496 genes ( probes ) with absolute log fold - change at least 1 for contrast nf / n and 208 genes for contrast tf / t . to gain a better understanding of the biological implications of these expression profiles , we performed a comprehensive gene set analysis of the differential expression results using the implementation provided by the r / bioconductor package the analysis used as gene sets the 202 kegg pathways classified as metabolic or signaling as downloaded on jan . the pathways contain 6885 unique genes of which 6010 are included in the analysis dataset ( approx . package piano provides the implementation of several gene set analysis tests and of a consensus methodology to summarize the results obtained with the different tests . this approach should be less sensitive to possible hidden biases of the single tests and allows , for example , the integration of sources of gene expression data with other types of high throughput data like proteomic data obtained by 2-dige . the following tests have been performed : a)meanb)medianc)sumd)maxmeane)gene set enrichment analysis ( gsea)f)parametric analysis of gene set enrichment ( page)g)reporter featuresh)wilcoxon rank - sum testi)tail strength . gene set enrichment analysis ( gsea ) parametric analysis of gene set enrichment ( page ) wilcoxon rank - sum test tests ( a)(f ) used t - values computed in the differential expression analysis while tests ( g)(i ) used the corresponding p - values ( and log fold changes , for devising the directionality ) . tests ( f ) , ( g ) , ( h ) computed gene set statistics significance via the theoretical null distribution , while the remaining ones via gene ( re)sampling . each test may return distinct p - values for each gene set ( i.e. , pathway ) in the following directionality classes : distinct - directional ( up or down ) , indicated as dist(up ) and dist(down ) , which tests if the expression of the gene set as a whole is regulated in a distinct direction ( either up or down ) . by definition , a gene set can not be significant in both classes as regulations in opposite directions will cancel out the effect.mixed-directional ( up or down ) , indicated as mix(up ) and mix(down ) , which tests if a significant subset of its genes are coordinately up- or down - regulated . a gene set can be significant in both classes.non-directional , indicated as nondir , which tests if the expression of the gene set as a whole is significantly perturbed , discarding any information about directionality . distinct - directional ( up or down ) , indicated as dist(up ) and dist(down ) , which tests if the expression of the gene set as a whole is regulated in a distinct direction ( either up or down ) . by definition , a gene set can not be significant in both classes as regulations in opposite directions will cancel out the effect . mixed - directional ( up or down ) , indicated as mix(up ) and mix(down ) , which tests if a significant subset of its genes are coordinately up- or down - regulated . non - directional , indicated as nondir , which tests if the expression of the gene set as a whole is significantly perturbed , discarding any information about directionality . a total of 35 p - value vectors over the 45 possible combinations for each contrast have been obtained since not all tests returned results for all the directionality classes . the combined analysis of the p - values obtained by each gene set in the various directionality classes provides a clear picture of the ( possible ) regulation of that gene set . consistency of the results has been empirically verified by computing the pairwise correlation between the p - value vectors . 2 depicts the correlation matrices of the p - value vectors of contrast nf / n on the left and of contrast tf / t on the right . directionality classes of the p - value vectors are indicated by the annotation bars on the top and on the left of the two plots , while letters refer to the different gene set analysis tests as per the previous list . according to the plots , p - value vectors of the same directionality class ( obtained with different tests ) have strong correlation ( see , for example , the class dist(dn ) ) , p - value vectors of similar classes have a non - negligible correlation ( see , for example , mix(dn ) and dist(dn ) ) and p - value vectors of opposite classes are inversely correlated ( see , for example , dist(dn ) and dist(up ) ) . further confirmation is provided by the dendograms depicted on the sides , showing that tests of the same directionality class cluster together . the only exception is test ( i ) tail strength for the distinct down - directional class on both contrasts . despite that , the p - values of the gene sets on these classes still appear to be in accordance ( albeit less correlated ) with the rest of the results and represent only a small perturbation that the final consensus analysis should easily accommodate . principal component analysis ( pca ) of the same set of vectors provides a second empirical support to the consistency of the results computed by the different tests . 3 depicts each p - value vector ( represented as the corresponding letter , see the list above ) for contrast nf / n ( left ) and contrast tf / t ( right ) in the space of its first three principal components ( x- and y - axes and size ) . ideally , each directionality class ( represented by colors ) should cluster together in the resulting space . the most evident outliers are those computed by test ( i ) for the distinct down - directional class , that are placed near the vectors of the mixed down - directional class . this confirms the observation made before for fig . 2 and , again , this should not represent an issue for the consensus analysis . piano computes the overall consensus rank by first ranking each gene set in each directionality class for each method according to the predicted p - value . these ranks are then aggregated according to the borda rule ( a single - winner election method ) . 4 depicts the aggregated ranks obtained by the gene sets that ranked in the first 5 positions in at least one directionality class . directionality classes are represented as columns while numbers and colors represent the position of each gene set ( row ) in the aggregated rank of that column for both contrast nf / n ( left block ) and contrast tf / t ( right block ) . in general , a pathway can be considered significantly regulated in a particular directionality class if it ranks in the first positions ( albeit a general cut - off does not exist ) . as expected , pathways ranking high ( i.e. , small numbers ) in a directionality class ( see , for example , ribosome biogenesis in eukaryotes ) are ranked low ( large numbers ) in the opposite directionality class . such a representation provides a clear and concise comparison of the regulation of pathways in the two contrasts , highlighting common regulation of pathways in normal and transformed cells , like ribosome biogenesis in eukaryotes , down - regulated in both nf / n and tf / t , and specific pathway regulations , different for the two cell lines , like the up - regulation of pathway steroid biosynthesis detected in tf / t but not in nf / n . the microarray data and the piano computational approach here presented , have allowed to the identification of significant pro - survival processes , otherwise hidden , regulated by pka in glucose deprivation . a part from our approach , these data could be further used to uncover other mechanisms of cancer resistance mediated by pka , whose role in cancer progression has assumed great relevance in recent years , , .

forskolin ( fsk ) induces activation of protein kinase a ( pka ) . this activation protects specifically some cancer cells from death induced by glucose starvation . cell effects upon fsk treatment prompted us to investigate in detail the physiological role of pka in the activation of pro - survival mechanisms in glucose starvation . in this regard we performed a microarray analysis of normal nih3t3 and transformed nih3t3-k - ras mouse fibroblasts cultured at 1 mm glucose and daily treated or not with 10 m fsk until 72 h of growth , when the samples were collected . the microarray is deposited into gene expression omnibus under series gse68266 . the microarray data revealed that the activation of pka regulates the expression of genes involved in metabolic , stress - response and pro - survival processes , like glutamine metabolism , autophagy and unfolded protein response , preventing cancer cell death in glucose starvation . altogether these findings suggest that pka activation , by inducing a complex transcriptional program , leads to cancer survival in nutrient stress , a typical feature of developing tumor . these transcriptional data , identifying this important role of pka , will be useful to identify novel target in cancer therapy .

epilepsy is the second most prevalent neurological disorder ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 1\ ) \end{document}% prevalence ) affecting approximately 67 million people worldwide with up to 75% from developing countries . eeg is important in developing countries for various clinical uses , including evaluation of suitability for epilepsy surgery and treatment of status epilepticus . epilepsy diagnosis is complicated by the fact that electrical potentials recorded from scalp electroencephalogram ( eeg ) have poor sensitivity to localized epileptiform activity , especially high frequency oscillations ( hfos ) which now attract a lot of attention as a promising marker of epileptogenesis and ictogenesis . misdiagnosis of epilepsy is very common in patients of all ages and occurs in up to 50% of the patients , . the hfos occupy the gamma ( 30100 hz ) , ripple ( 100200 hz ) and fast ripple ( 200500 hz ) bands along with delta , theta , alpha and beta bands making up the remainder of the spectral content of eeg . appearance of fast eeg rhythms at the onset of seizures in humans and animal models of epilepsy has been described in many studies and first reported in humans by fisher et al . and allen et al . . fast rhythms are especially evident in intracranial recordings in patients considered for surgical treatment of their drug - resistant epilepsy . in recent years , there has been a surge in the number of publications on hfos and their role in epilepsy ( for reviews , see , . numerous studies on animal models and epilepsy patients have revealed that : 1 ) hfos are one of the most common early manifestations of seizures recorded within minutes before seizure onset ; 2 ) hfos appear to be the most likely eeg correlate of a seizure onset zone ( soz ) ; and 3 ) the removal of hfo - generating areas correlates with good surgical outcome , , , . thus , hfos appear to be an excellent marker for the epileptogenic zone but their recording from scalp with conventional disc electrodes is difficult . eeg has been a very useful research and clinical instrument for almost a century since its first human use by hans berger in 1920s . the distance to the brain and the relatively large electrical impedance of the skull significantly reduce small electrical signals generated by individual neurons . to be detectable at the scalp , the signal should be produced by a relatively large population of neurons acting more or less synchronously at scales of at least several centimeters . only in this case does the sum of individual neuronal signals become large enough to be detected at the scalp . synchronous activity within neuronal assemblies is now considered an important mechanism of information processing , and this synchrony is expressed through neuronal oscillations occurring at different frequencies spanning several orders of magnitude from infraslow ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 0.01\ ) \end{document } hz ) up to ultrafast ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 1000\ ) \end{document } hz ) ( for a comprehensive review of brain oscillations , see . there is an intricate relationship between temporal and spatial scales of brain oscillations in that higher frequency oscillations ( e.g. , high gamma band > 60 hz ) usually occur at smaller distances between sources and involve smaller neuronal populations . this is because more precise synchronization dictated by shorter oscillatory periods can be achieved only through shorter neuronal connections with shorter propagation times or non - synaptic communication such as ephaptic interactions and gap junctions . long distance connections have longer propagation times and therefore synchronization at larger distances is less precise and preferentially occurs at lower frequencies ( e.g. , within delta , theta , alpha and beta bands ) . as a result , high frequency oscillations > 60 hz usually reflect activity within well localized neuronal assemblies at spatial scales of a few mm , which are not typically visible from the scalp . thus , conventional eeg has lower sensitivity to high frequency components of brain activity , . conventional scalp eeg is also contaminated by noise represented by non - brain electrical activity such as ocular artifacts , scalp muscle potentials , and the electrocardiogram . additional noise is occasionally produced by abnormally large electrode impedance , electrode movement , amplifier drifts , etc . the noise can be as large as , or even larger than , the brain potential of interest and represents a significant problem for electroencephalographers and neurologists . this noise shows high spatial coherence due to smearing effects of the head volume conductor . conventional disc eeg recordings also have reference electrode problems . a common average reference and concentric electrodes have been proposed to resolve the reference electrode problems . however , in the common average reference recordings , it is possible that components present in most of the electrodes but absent or minimal in the electrode of interest may appear as ghost potentials . thus , there is a strong need to develop new types of electrodes beyond conventional disc electrodes . to overcome the poor signal - to - noise ratio and reference problems of the disc electrodes besio et al . have developed the tripolar concentric ring electrode ( tcre ) , a transformative electrode configuration . the tcre consists of three electrode elements - outer ring , middle ring , and the central disc ( fig . 1 , b ) . it is distinctively different from the disc electrode which has a single element ( fig . the novelty of the tcre lies in a new principle of recording electrical activity of the brain from the scalp through its three closely spaced elements . it can provide three separate signals from the three electrode elements used to record two bipolar differential signals for the tripolar laplacian derivation first described in as a weighted sum { 16*(m - d)-(o - d ) } where o , m , and d are the potentials on the outer ring , middle ring , and central disc , respectively . the tripolar signal is a ' hardware realization ' of the laplacian i.e. , the second spatial derivative of the scalp signal . unlike other software methods ( ' laplacian montages ' in conventional eeg systems ) which approximate the laplacian by taking differences between disc electrodes placed , at best , > 1 cm apart , the tcre performs the laplacian automatically and takes bipolar differences of the surface potentials from closely spaced ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 1\ ) \end{document } mm ) concentric electrode elements with our custom preamplifier . with the electrode elements so close to each other , globally originating sources such as eye blinks , muscle or motion - related artifacts contribute nearly equally and are attenuated sharply when bipolar differences are performed by the preamplifier . compared with disc signals , eeg recorded with the tcre ( teeg as described by ) has about a 4-fold ( 374% ) increase in signal - to - noise ratio ( snr ) and less than one - tenth ( 8.27% ) the mutual information between signals recorded from two adjacent tcres ( which suggests a significant attenuation of volume conductance effects and , as a result , higher spatial resolution ) , . the tcre also has strong attenuation of various artifacts , 100 db at one radius from the electrode . figure 1.conventional disc electrode ( a ) and tripolar concentric ring electrode ( b ) . conventional disc electrode ( a ) and tripolar concentric ring electrode ( b ) . the current study presents preliminary data from patients with epilepsy in whom teeg was recorded in parallel with clinical conventional scalp eeg . the goal is to demonstrate that tripolar electrodes may provide a unique opportunity to record hfos from scalp and thus improve diagnosis of epilepsy and localization of the seizure onset and the irritative zones . their implementation into clinical practice may eventually help determine where to place and reduce the number of intracranial grids and depth electrodes to be implanted during presurgical evaluation of patients . patients for this study were recruited from the national institute of neurology and neurosurgery ( ninn ; ten patients ) and rhode island hospital ( rih ; two patients ) . patients were referred by the epilepsy clinic in each institution with the diagnosis of drug resistant epilepsy using the international league against epilepsy criteria . diagnosis of epilepsy and epileptic seizures was based on the international classification of seizures 1981 and epileptic syndromes 1989 . ictal and non - ictal recordings were obtained ; for non - ictal recordings lateralization and localization of the irritative zone ( iz ) was determined whereas in ictal recordings the soz or iz was identified with the appearance of behavioral changes associated with focal low voltage , fast activity , flattening or slow wave interruption . to verify that tcres can be used to record physiological signals we recorded from three healthy subjects , one at stanford school of medicine and two at the university of rhode island ( uri ) placing conventional disc electrodes over the occipital lobe ( at the o1 and o2 locations of the 1020 international electrode system with reference at a1 and ground electrode placed on the right collar bone ) . two tcres were placed right next to the conventional electrodes at the o1 and o2 locations ( o1 ' and o2 ' positions , respectively ) . the tcre eeg signals ( teeg ) were first preconditioned with our custom made preamplifiers ( gain \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(=20\ ) \end{document } ) and then passed to the bipolar inputs of the ( stanford - eeg-1200 , nihon kohden corporation , tokyo , japan ; uri - grass technologies aura ltm-64 , grass technologies , west warwick , ri ) clinical eeg recording system . all signals were recorded with 1 to 120 hz band - width at 500 s / s and stored to the hard drive for post processing . the subjects were asked to close and open their eyes repeatedly to record the presence or absence of alpha rhythms , respectively . the study 's recording protocol was designed in such a way as to avoid any interference with clinical eeg recording and evaluation . at both the ninn and rih during the attachment of clinical conventional disc electrodes ( referred to as ' eeg electrodes ' or ' eeg signals ' in the subsequent text ) , the patient 's scalp was cleaned with nuprep and then eeg electrodes were placed at the 1020 international electrode system locations using ten-20 paste . collodion was also used to help hold all electrodes in place at the ninn . to obtain teeg recordings in parallel to the clinical eeg , the tcres were placed just behind the disc electrodes in locations close to the 1010 sites and attached to the scalp with ten-20 paste ( fig . the ground was placed on the forehead ( rih , ninn ) and the reference electrode was placed on the forehead at rih and on the oz location at ninn . clinical eeg was recorded with the comet as40 system ( grass technologies , west warwick , ri ) and stored separately for further clinical evaluation . the eeg sampling rate was 200 samples per second ( s / s ) and the low - pass filter was 70 hz ( ninn and rih ) . the teeg data were pre - amplified with the gain equal to either 6 ( for eight patients ) or 100 ( for four patients ) and amplified and digitized with an aura ltm-64 system ( grass technologies , west warwick , ri ) at different sampling frequencies for different patients . for four patients the data were filtered 1100 hz and digitized at 200 s / s , another six were filtered 1200 hz and digitized at 400 s / s and for the remaining two patients the data were filtered 1250 hz and 1500 hz respectively and digitized at 1600 s / s . the 60 hz notch filter was used for all patients . the recording sessions at the ninn usually lasted for six hours , from around 7 am to 1 pm . for the two patients at rih , the recording was stopped shortly after the patient had a seizure ( 130 minutes total ) and for the other the recording lasted 66 minutes . the ninn recording protocol included requests to patients to be sleep deprived the night before coming for a video - eeg monitoring and all patients signed an additional consent form as antiepileptic drugs dosage was reduced by half the previous day of the recording . recorded data were reviewed by board certified neurologists and seizure onset time and duration were determined for each seizure . seizure onset time was defined as the beginning of the first observable seizure pattern in either eeg or teeg . note : t7/p7 and t8/p8 are the same as t3/t5 and t4/t6 in 1020 nomenclature . the blue rings are for standard 1020 electrode locations . the 105 montage with tcres ( red ) placed near the 1020 locations . note : t7/p7 and t8/p8 are the same as t3/t5 and t4/t6 in 1020 nomenclature . the data were stored on the hard drive and exported from twin ( grass technologies , west warwick , ri ) into ascii text files that were then imported to matlab ( mathworks , natick , ma ) and converted to .mat files . for the teeg , next we used a modified version of the algorithm reported by gardner et al . for detection of hfos combined with the visual inspection to rule out high frequency artifacts . the algorithm performed a continuous short - time fourier transform to calculate the power within a particular frequency band over consecutive and half - overlapping one - second epochs using a hamming tapering window . as a result simple thresholding was also used to find events which had power significantly higher than the interictal background eeg . then the time periods when the hfo power was exceeding its average plus two standard deviations were found . frequency bands starting at high gamma band ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 60\ ) \end{document } hz ) and up to the upper bound of the band - pass filter that varied for individual patients were searched for hfos . we selected segments up to an hour prior to the onset of the seizures and generated spectrograms to follow the temporal dynamics of pre - seizure hfos . for the patients who had epileptiform activity only but no seizures we calculated spectrograms on 10 minute long segments centered on the periods of epileptiform activity . for the patients who did not have either epileptiform activity or seizures we performed spectral analysis on multiple 10 minute long segments at various times during the recording . to assess the relationship between teeg channels containing hfos preceding seizures and the soz the ratio between the number of teeg channels with hfos in the soz and the total number of teeg channels with hfos was calculated for each patient whose seizure was recorded . we also calculated the ratio between the total number of teeg channels with hfos and the total number of teeg channels to assess how widespread the hfos were for each patient . both ratios were averaged for the five patients with seizures included in this study . only the channels containing hfos preceding the seizures were assessed . soz or iz was determined for each patient independently by three epileptologists ( iemj , jng , and rsf ) based on eeg data and videos only . the epileptologists did not have access to teeg data and were not aware of the hfo detection results . in one patient the third soz was determined by the location of resection by the surgeon . a disjunctive ( logical or ) that is all channels determined as part of the soz ( iz ) by either one of the epileptologists were considered to be a part of the soz ( iz ) . first , we demonstrate the suitability of tcres to provide a sound eeg signal sensitive to physiological rhythms such as alpha activity as well as their insensitivity to myogenic activity which often corrupts scalp eeg recordings especially in the high frequency range > 30 hz . if the tcre attenuates volume conductance effects and has a greater sensitivity to local sources , how good is it for recording global brain rhythms such as alpha activity ? figure 3 , ( a / c ) shows the signals that were recorded with eyes closed / open and their corresponding power spectral densities are presented in fig . 3 , ( b / d ) . the alpha waves were present in teeg ( top trace , blue ) coinciding with the corresponding alpha rhythm recorded by conventional disc electrodes ( middle and bottom traces , red and green respectively ) ( fig . the alpha waves were blocked in both teeg and eeg when the eyes were open ( fig . 3 , c , d ) . thus , this result demonstrates the ability of tcres to record physiological rhythms . figure 3.global physiological rhythms such as alpha activity observable with eyes closed can be recorded by tcres . panel a : the top trace ( blue ) is teeg from a tcre placed next to the conventional disc electrode at o1 . the middle trace ( red ) both the top and middle traces are recorded with respect to the reference on the right collar bone ( rcb ) . the bottom trace ( green ) is from the o1 electrode with respect to the a1 reference electrode . panel b : the power spectral densities for the signals of panel a. panels c and d are the signals and power spectral densities when the eyes are open . it is evident that the alpha waves are present in both teeg and eeg records , specifically between 4 and 5 seconds ( panel a ) , which is confirmed by the spectra displaying a peak just below 10 hz ( panel b ) but are not seen in panels c and d. global physiological rhythms such as alpha activity observable with eyes closed can be recorded by tcres . panel a : the top trace ( blue ) is teeg from a tcre placed next to the conventional disc electrode at o1 . both the top and middle traces are recorded with respect to the reference on the right collar bone ( rcb ) . the bottom trace ( green ) is from the o1 electrode with respect to the a1 reference electrode . panel b : the power spectral densities for the signals of panel a. panels c and d are the signals and power spectral densities when the eyes are open . it is evident that the alpha waves are present in both teeg and eeg records , specifically between 4 and 5 seconds ( panel a ) , which is confirmed by the spectra displaying a peak just below 10 hz ( panel b ) but are not seen in panels c and d. a relative insensitivity of tcres to myogenic activity is demonstrated by teeg and eeg records obtained from the same subject during head movements ( fig . the bottom trace ( green ) it is evident that from about 3 to 6 seconds there is a high - frequency contamination in the bipolar disc recordings that is significantly attenuated in the teeg ( top trace , blue ) . this high - frequency interference coincided with head movements and is most likely caused by muscle artifacts . 4 shows the power spectral densities for the traces in panel a. the teeg power ( blue ) has the lowest power in the higher frequencies range where the muscle artifact is prominent in the records from the conventional bipolar disc signals . from our experience , such attenuation of myogenic artifacts panel a : the top trace ( blue ) is teeg from a tcre placed next to the conventional disc electrode at o1 . both the top and middle traces are recorded with respect to the reference on the right collar bone ( rcb ) . the bottom trace ( green ) is from the o1 disc electrode with respect to the a1 reference electrode . conventional eeg is heavily corrupted by muscle artifacts which are evident from approximately 3 to 6 seconds of the record ( middle [ red ] and bottom [ green ] traces ) coinciding with head movements by the subject . these artifacts are nearly unnoticeable in the teeg ( top trace [ blue ] ) . panel b : the power spectral densities for the signals shown in panel a. the power spectrum for the teeg ( blue ) has much less high frequency power ( > 30 hz ) due to myogenic activity . panel a : the top trace ( blue ) is teeg from a tcre placed next to the conventional disc electrode at o1 . both the top and middle traces are recorded with respect to the reference on the right collar bone ( rcb ) . the bottom trace ( green ) is from the o1 disc electrode with respect to the a1 reference electrode . conventional eeg is heavily corrupted by muscle artifacts which are evident from approximately 3 to 6 seconds of the record ( middle [ red ] and bottom [ green ] traces ) coinciding with head movements by the subject . these artifacts are nearly unnoticeable in the teeg ( top trace [ blue ] ) . panel b : the power spectral densities for the signals shown in panel a. the power spectrum for the teeg ( blue ) has much less high frequency power ( > 30 hz ) due to myogenic activity . we separated the patients into 3 groups : ( 1 ) patients who had clinical seizures ( n\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(=5\ ) \end{document } ) , ( 2 ) patients with epileptiform activity but no seizures ( n\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(=3\ ) \end{document } ) , and ( 3 ) patients with neither epileptiform activity nor seizures ( n\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(=4\ ) \end{document } ) . simultaneously recorded eeg and teeg and the corresponding spectrograms from representative patients are shown in fig . 5 and 6 . 5 the patient was a 42-year - old woman with seizure onset at 9 years . she had been diagnosed with right temporal lobe epilepsy and had undergone a right temporal lobectomy but seizures persisted . she was on clonazepam ( cnz ) , lamotrigine ( ltg ) and phenytoin ( pht ) at the time of the study . in figure 5 conventional eeg data are on panels a , b , e , f , and i and teeg data are on panels c , d , g , h , and j. the conventional eeg data were obtained from the bipolar montage ( channel f8-f4 ) . this patient had a generalized seizure ( onset at approximately 610 seconds in the time scale of panels a - d in fig . the seizure activity is evident by large increases in signal amplitude and power at all frequencies . panels e , f , g , and h are eleven - second eeg and teeg segments ( marked by the black line in panel c ) shown at higher sequential resolution . note the series of high gamma - band hfos between approximately 60 to 80 hz ( highlighted by ellipse in panel c ) occurring about every two seconds which are clearly evident in the teeg but not eeg , starting approximately 10 min prior to the generalized seizure activity ( compare panels a and c as well as e and g ) . with further zoom in panels i and j showing two - second segments , one can see a high frequency burst in the teeg ( black horizontal line in the panel j ) which is not present in the eeg ( panel i ) . conventional eeg ( bipolar montage , channel f8-f4 , panels b , f and i , 170 hz , 200 s / s ) , teeg ( from a tripolar electrode placed right behind the f8 position ( f8 ' ) , panels d , h and j , 1100 hz , 200 s / s , ) and their spectrograms ( panels a , e and panels c , g for eeg and teeg , respectively ) starting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 10\ ) \end{document } min before and continuing during a generalized seizure with onset marked with the dashed vertical black line . panels e - h : eleven - second eeg and teeg segments ( marked by the black line in panel c ) are shown at higher temporal resolution . note a series of high gamma - band bursts hfos at 6080 hz ( highlighted by ellipse in c ) occurring about every 2 s , which are clearly seen in the teeg spectrogram only . panels i , j : further zoom - in of two - second segment shows one hfo in teeg ( black horizontal line in the panel j ) while this hfo is absent in eeg ( panel i ) . figure 6.panel b shows 12 minutes of bipolar eeg from fp2-f4 ( 170 hz , 200 s / s ) . panel e shows 30 seconds of eeg from panel b at the onset of the generalized seizure ( dashed line ) . note the high gamma - band burst hfos just prior to the partial seizure ( highlighted by ellipse in panel c ) . conventional eeg ( bipolar montage , channel f8-f4 , panels b , f and i , 170 hz , 200 s / s ) , teeg ( from a tripolar electrode placed right behind the f8 position ( f8 ' ) , panels d , h and j , 1100 hz , 200 s / s , ) and their spectrograms ( panels a , e and panels c , g for eeg and teeg , respectively ) starting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 10\ ) \end{document } min before and continuing during a generalized seizure with onset marked with the dashed vertical black line . panels e - h : eleven - second eeg and teeg segments ( marked by the black line in panel c ) are shown at higher temporal resolution . note a series of high gamma - band bursts hfos at 6080 hz ( highlighted by ellipse in c ) occurring about every 2 s , which are clearly seen in the teeg spectrogram only . panels i , j : further zoom - in of two - second segment shows one hfo in teeg ( black horizontal line in the panel j ) while this hfo is absent in eeg ( panel i ) . panel b shows 12 minutes of bipolar eeg from fp2-f4 ( 170 hz , 200 s / s ) . panel e shows 30 seconds of eeg from panel b at the onset of the generalized seizure ( dashed line ) . note the high gamma - band burst hfos just prior to the partial seizure ( highlighted by ellipse in panel c ) . this patient was a 45 year old male , with onset of epilepsy at 22 years . his seizures consisted of simple partial seizures followed by complex partial with or without secondarily generalization . he was on carbamazepine ( cbz ) and ltg at the time of the recording . his magnetic resonance imaging showed left mesial temporal sclerosis and a right frontal venous angioma . panel d shows teeg recorded from location fp2 ' directly behind the fp2 disc electrode . , we also found gamma band bursts ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 70\ ) \end{document } hz ) in the teeg , approximately three minutes before the seizures . 6(c ) highlights the high gamma - band burst hfos in the teeg at location fp2 ' approximately 4 min prior to the partial seizure . the hfos are present throughout the pre - seizure spectrogram but became more consolidated around 70 hz about 3 min prior to the seizure . it is also important that the teeg during the tonic seizure was less contaminated with muscle and movement artifacts than the eeg ( compare panels e and f ) . also note the higher power in eeg ( compare panels a and c ) from approx . 400 to 650 s when the patient was still anxious / disoriented and moving while recovering from the seizure . during that same period overall , hfos preceding seizures were present in teeg data from all five patients whose seizures were recorded . for these five patients , out of all the teeg channels that contained hfos an average of 78.2% were also in the soz ( iz ) ( patient minimum : 42.9% ; maximum : 100% ) . the average percentage of the total number of teeg channels recorded that contained hfos is equal to 35.5% ( patient minimum : 5.3% ; maximum : 73.7% ) . figure 7 portrays the relationship between the clinical soz ( iz ) and hfo - containing channels for two patients that we present the spectrograms for in fig . 5 ( panel a ) and fig . 6 ( panel b ) respectively . figure 7.panel a shows the relationship between the clinical seizure onset zone ( soz ) and hfo - containing channels for the patient from fig . panel b shows the relationship between the clinical irritative zone ( iz ) and hfo - containing channels for the patient from fig . soz or iz was determined for each patient independently by three epileptologists ( iemj , jng , and rsf ) based on eeg data and videos only . panel a shows the relationship between the clinical seizure onset zone ( soz ) and hfo - containing channels for the patient from fig . panel b shows the relationship between the clinical irritative zone ( iz ) and hfo - containing channels for the patient from fig . soz or iz was determined for each patient independently by three epileptologists ( iemj , jng , and rsf ) based on eeg data and videos only . it has been shown that eeg is frequently contaminated by artifacts originating from various sources such as scalp muscles , eye blinks , eye movements , or patient movement . state what is intuitively obvious ; artifacts obscuring the eeg at the time of seizure onset can greatly hinder the interpretation of the recorded seizures . during a tonic or tonic - clonic seizure , muscle activity is very prominent and is reflected in the eeg by random , high frequency signals . . showed that when eegs were moderately contaminated with muscle artifacts blind source separation techniques based on canonical correlation provides signals that are more reliable for source estimation than raw eeg . they also found that in cases of severe muscle contamination of the eeg or when the muscle artifact had different spatial information than the spatial information of the epileptiform event , the blind source separation techniques based on canonical correlation did not work well . in general , a major reason why eeg has not reached its full potential in epilepsy diagnosis is due to artifact contamination . we have found that the tcre and the teeg automatically attenuate myogenic activity and movement artifacts . in contrast to the typical examples of artifact contaminated conventional bipolar eeg , the teeg is much less contaminated by the muscle activity . it should be noted that conventional approaches to remove muscle artifacts by digital signal processing ( such as filtering ) lead to significant loss of information since the gamma band activity and hfos in general are within the bandwidth of the electromyogram ( emg ) . in other words , removing the emg will inevitably remove high frequency components of the brain activity , which is not a desirable result . the teeg , although not as obvious , may also be contaminated with emg ( fig . 4 , a , top blue trace ) but from a much more local source . the emg from more distant sources have nearly equal contributions on the elements of the tcre and are cancelled out when the potentials on the elements are subtracted from each other with bipolar differences . in this study , we show , in a limited number of patients , that high - power hfo activity is detected at specific locations on the scalp surface in the teeg records of patients with epilepsy . this hfo activity was apparent prior to seizures . although there may have been hfo activity at various times in the teeg during the recordings the bandwidth of the hfo activity narrowed sharply and the power increased just prior to the seizure . we did not see the hfo activity in the conventional eeg , even when using bipolar montages which somewhat attenuates global artifacts . in some instances this may partially be due to the low - pass filter used to record the eeg . the eeg low - pass filter was set to 70 hz and this cutoff frequency may have partially filtered out gamma band activity . however , the gamma band activity detected in the teeg was often well below 70 hz , in one case as low as 63 hz , and in these cases the gamma band activity was still undetected in the conventional eeg . when a low - pass digital filter was applied to the teeg at 70 hz the hfos were still present in the teeg for patients whose hfos were below 70 hz . we do not know what the upper limit is for detecting hfos on the scalp . in rats we have found an increase in power up to 300 hz during ptz - induced seizure electrographic activity recorded with teeg . in two patients having seizures for whom the data were filtered 1250 hz and 1500 hz respectively and digitized at 1600 s / s we have detected hfo preceding seizures in gamma , ripple , and fast ripple bands at frequencies up to 425 hz . a potential disadvantage of recording with tcre is deemphasis of non - artifactual signals with wide spatial distribution , for example , spike - waves . we intend to develop the circuitry to record both teeg and an eeg approximation from the same sensor in the near future . despite a limited sample size of patients having seizures in this study ( 5 patients ) we were able to investigate a possible correlation between the clinically determined soz ( iz ) and the location of hfos detected by tcres . hfos preceding seizures were present in teeg data from all five patients whose seizures were recorded in an average of 35.5% of the patient 's teeg channels . out of those channels containing hfos an average of 78.2% were within soz ( iz ) determined independently by three epileptologists based on eeg data and videos . in all five patients who had recorded seizures , at least one channel in the soz exhibited hfos ( in one out of five patients hfos were present in a single teeg channel ) . in two patients no specific soz was determined but rather an iz . for those two patients hfos were present in 7 and 4 teeg channels respectively out of 20 ( 35% ) and 19 ( 21% ) channels total respectively which is on par with the population average of 35.5% . examples of the relationship between the soz ( iz ) and hfo - containing channels are presented in fig . the third epileptologist ( jng ) mentioned 4 hz activity over central leads but no clear electrographic onset . soz definitions from all three epileptologists ( iemj , jng , and rsf ) overlapped for two channels ( fp2 and f4 ) over the right frontal lobe . these preliminary results support that teeg is capable of detecting hfos from the scalp , which are not usually seen in conventional eeg records . further studies with more patients are needed to more accurately correlate locations of ( presumably abnormal ) hfo activity with clinically determined soz as well as the resected tissue in surgical cases . due to the improved signal quality of teeg we hope that in the future we will be able to use teeg to detect seizures and trigger stimulation to attenuate seizures . we have shown that transcranial focal electrical stimulation ( tfs ) via tcres in rat models was effective in reducing penicillin - induced myoclonic jerks , , pilocarpine - induced status epilepticus ( an extreme form of continuing seizures ) and in a third model , we applied tfs in rats treated by pentylenetetrazole ( ptz ) . tfs significantly reduced ptz - induced hypersynchrony at the beta and gamma frequencies , most recently we developed an automatic non - invasive seizure control system based on tfs and tested it successfully on rats .

epilepsy is the second most prevalent neurological disorder ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 1\ ) \end{document}% prevalence ) affecting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 67\ ) \end{document } million people worldwide with up to 75% from developing countries . the conventional electroencephalogram is plagued with artifacts from movements , muscles , and other sources . tripolar concentric ring electrodes automatically attenuate muscle artifacts and provide improved signal quality . we performed basic experiments in healthy humans to show that tripolar concentric ring electrodes can indeed record the physiological alpha waves while eyes are closed . we then conducted concurrent recordings with conventional disc electrodes and tripolar concentric ring electrodes from patients with epilepsy . we found that we could detect high frequency oscillations , a marker for early seizure development and epileptogenic zone , on the scalp surface that appeared to become more narrow - band just prior to seizures . high frequency oscillations preceding seizures were present in an average of 35.5% of tripolar concentric ring electrode data channels for all the patients with epilepsy whose seizures were recorded and absent in the corresponding conventional disc electrode data . an average of 78.2% of channels that contained high frequency oscillations were within the seizure onset or irritative zones determined independently by three epileptologists based on conventional disc electrode data and videos .

alternative rna splicing , export and translational regulation all contribute to cancer formation and progression ( venables , 2006 ) . these are dependent on the complex secondary structure of the 3 utr of the mrna recognised by binding proteins such as t - cell intracellular antigen-1 ( tia-1 ) ( eberhardt et al . , tia-1 contains three domains with rna recognition motifs ( rrm1 , rrm2 and rrm3 ) , the latter two of which have been shown to be important for rna binding and selectivity ( bauer et al . , 2012 ) ( cruz - gallardo et al . , 2014 ) . it has been shown to be alternatively spliced in exon 5 to form two isoforms ( a and b , supplementary figure 1a ) with b being the most active in its splicing activity , but tia-1 also acts as a stress induced translational inhibitor , localising to stress granules with polya rna ( izquierdo and valcarcel , 2007 ) . tia-1 alters both co - transcriptional and post - transcriptional rna processing and binds vegf - a rna ( suswam et al . , 2005 ) , the alternative splicing of which influences the angiogenic capability of colorectal carcinomas ( crc ) ( ferrara et al . , 1991 ) . vegf - a exists as multiple alternatively spliced isoforms . exons 6 and 7 endow heparin binding ( houck et al . , 1991 ) , whereas exon 8 determines angiogenicity ( harper and bates , 2008 ) . exon 8 has two splice acceptor sites , a proximal one , and a distal one , 66 bases downstream . the stop codon for the two isoforms lies the same number of bases downstream from the respective splice sites , resulting in two families of isoforms of the same amino acid number but with differing six amino acid sequence at the end , which alter the angiogenicity of the protein . proximal splice site ( pss ) selection in exon 8 results in the angiogenic family ( vegf - axxx where xxx is the number of amino acid e.g. vegf - a165 ) , but distal splicing ( dss ) results in vegf - axxxb , anti - angiogenic proteins of the same size ( e.g. vegf - a165b ) ( bates et al . , 2002 ) . a variable but specific upregulation of the angiogenic isoforms is seen in colorectal cancer ( varey et al . , 2008 ) , but although the mechanisms underlying this splice specificity are being elucidated in epithelial cells ( amin et al . , 2011 ; nowak et al . , 2008 ) and lung cancer ( merdzhanova et al . , 2010 ) , isoform specific control of expression , particularly in colon cancer is still not known . hek293 , colonic carcinoma cell lines ht29 , hct116 , hac7 , sw480 , osteosarcoma saos-2 and u2os , the cervical cancer cell line , hela , prostate cancer cells , pc3 and lncap ( atcc ; rockville , md ) ls174 t ( ecacc , salisbury , uk ) , adenoma cell lines aa / c1 and rg / c2 , the in vitro transformed adenoma cell line aa / c1/sb/10 , referred to as 10c cells ) and breast cancer mda - mb231 ( caliper , perkinelmer , usa ) , human haemangioma endothelial cells ( esc and stem cells ( hsc , a kind gift from joyce bischoff at harvard university ) , myeloma rpmi-8226 , and cardiac myocytes ( hcm , from promocell ) were maintained as described previously ( williams et al . , 1990 ) or according to manufacturer 's instructions cancer cell lines were validated by str profiling ( identicell , aarhus , denmark ) . an initial study was undertaken on six frozen paired colon samples ( normal and cancerous ) from partial colon resection . additional rna was extracted from 40 formalin fixed , paraffin embedded ( ffpe ) samples of normal and tumour tissues . these were taken from 22 male , 18 female patients , mean age 69 years , 28 with colon carcinoma , 18 with rectal carcinoma and 1 with carcinoma of the rectosigmoid junction . an antibody to stia-1 was generated by abgent europe using a klh - conjugated vslkngq ncpg peptide . for access cell experiments used at least 3 independent biological replicates ( independent experiments ) , with each western blot , elisa and rt - pcr carried out on independently replicated samples . for western blot , and rt - pcr where gels are shown , each is indicative of a representative of 3 experiments , and quantitated as per the figures . protein samples from homogenised tissue ( 100 g ) and whole cell extract ( 50 g ) were prepared using standard protocols . protein lysates were resolved on sds page , and probed with a mouse monoclonal anti - cox-2 antibody ( sc-19999 ; santa cruz ) , a / b - tubulin ( sigma ) anti - vegf - a165b igg ( a56/1 ; r&d systems ) , rabbit polyclonal anti - tia-1 antibody ( sc-28237 ; santa cruz , detects full length and stia-1 isoforms ) , or sc-166246 c10 mouse monoclonal n - terminal ; santa cruz and rabbit anti - vegf - a igg ( a-20 , sc-120 ; santa cruz ) , goat polyclonal laminin ( santa cruz ) and mouse monoclonal lamin ( cell signalling ) antibodies . proteins were detected using chemiluminescence and analysed by nih image , or by fluorescently labelled secondary antibody imaging and imaged using a licor odyssey , and quantified using licor imaging software . elisas were carried out as described previously ( varey et al . , 2008 ) . the vegf - a165b elisa uses an antibody that can detect all vegf - axxxb isoforms but no vegf - axxx isoforms , but its affinity to all of the different vegf - axxxb isoforms is not known . we have assumed that it is the same as vegf - a165b and therefore calculated the levels making this assumption . for immunoprecipitation , 50 l of protein a or protein g ( depending on capture antibody ) magnetic beads ( millipore ) was washed with 500 l pbs containing 0.1% tween 20 . after removing the buffer , the beads were resuspended in 100 l of pbs/0.1% tween , capture antibody added to the beads and incubated at room temperature for 10 min before adding the protein samples . the protein samples and antibody were incubated at 28 c overnight with continuous mixing , washed with pbs / tween0.1% before adding sample buffer . for spectrometry , after running the samples on page , the gel was fixed in methanol : acetic acid ( 50%:7% ) for 30 min and stained with sypro ruby ( molecular probes ) overnight . the gel was de - stained in methanol : acetic acid ( 10%:7% ) , washed and imaged . the band was excised and subjected to in - gel tryptic digestion using a progest automated digestion unit ( digilab uk ) . the resulting peptides were fractionated using a dionex ultimate 3000 nanohplc system in line with an ltq - orbitrap velos mass spectrometer ( thermo scientific ) as described in the supplementary material . raw data files were processed and quantified using proteome discoverer software v1.2 ( thermo scientific ) and searched against the swissprot human database using the mascot search engine ( matrix science ) with parameters as described in the supplementary material . total rnas from tissue ( 200 mg ) and cells ( 10 ) was extracted using the rneasy kit ( qiagen ) and quantified . rna was extracted from 25 m thick sections of the ffpe samples by rneasy ffpe kit ( qiagen ) according to the manufacturer 's protocol . standard rt - pcr protocols were used . for cdna synthesis form ffpe samples random primer or gene specific primers were used . extracted dna was cloned into the p - gem t - easy plasmid ( promega ) and sequenced using standard synthetic primers complementary to the vector dna at the junction of the cloning site ( t7 , sp6 ) and a ptarget sequencing primer 5-ttacgccaagttatttaggtgaca-3 by the dundee sequencing service ( dundee university ) . 2002 ) . in brief , 5 10 cells were harvested , cross linked with 1%formaldehyde at room temperature for 10 min and quenched with 0.25 m glycine . protein was extracted , and 100 g of pre - cleared protein incubated with 3 g tia-1 antibody immobilized using seize primary immunoprecipitation kit ( pierce biotechnology ) and immunoprecipitates isolated by centrifugation . total rna was extracted from immunoprecipitates and subjected to rt - pcr as described . the vegf - a sequence from 35 bp upstream of exon 8a to 35 bp downstream of exon 8b was inserted into the adml - ms2 plasmid as previously described ( nowak et al . , 2008 ) . nuclear protein ( 75 g ) from hek 293 or aa / c1/sb/10 ( 10c ) cells were prepared and added to the ms2-mbp fusion protein / vegf - rna mix in 0.5 mm atp , 6.4 mm mgcl2 , 20 mm creatine phosphate for 1 h at 30 c . proteins bound to the ms2-mbp / vegf - ms2 rna complex were affinity selected on amylose beads and immunoblot analysis was performed on 30 g of protein samples using tia-1 antibody and -tubulin antibodies as previously described ( nowak et al . , 2008 ) . the cdna for fltia-1was generated by rt - pcr amplification using pfu turbo dna polymerase ( stratagene ) and primer tia-1-forward and tia-1-reverse from rna from the rg / c2 adenoma cell line . the 1239 bp fragment was cloned in p - gem t - easy vector and sequenced in both directions . the cdna for both variants ( 13-exon tia-1ncbi nm_022173.2 and 12-exon tia-1ncbi nm_022037.2 ) were obtained using these primers . for in vitro expression , the cdna were subcloned into mammalian expression vectors , ptarget ( promega ) and pcdna3 according to the manufacturer 's instructions . two custom sirnas targeting stia-1 ( sirna1 : cagaattgccctggctaacta and sirna2 : gccctggctaa ctacaagcta ) and control sirna ( lopez de silanes et al . , 2005 ) were assessed . two colon cancer cell lines were transfected with mixture of the 2 sirnas ( 50 nm ) using hiperfect transfection reagents ( qiagen ) . transfection : ls174 t and 10c cells were electroporated with 2 g cdna of ptarget - fltia-1 cdna in ptarget and pmaxgfp vectors ptarget vector or pmaxgfp with an amaxa nucleofector system using a basic epithelial cell transfection kit ( vpi-1005 , amaxa gmbh ) . transfectants were analysed 24 h post nucleofection by trypan blue staining and fluorescence microscopy for gfp control plasmid . transfected cells were selected using g418 ( 500 g / ml ) for 4 weeks . the cells were 4% formaldehyde fixed ( 15 min ) and incubated in 1%bsa/10% normal goat serum in tbs - tween ( 0.1% ) for 1 h. the cells were then incubated with the antibody ( ab40693 rabbit polyclonal c - terminal , or sc-166246 c10 mouse monoclonal n - terminal , 5 g / ml ) overnight at + 4 c . the secondary antibody ( red ) was alexa fluor donkey anti - rabbit and ( green ) alexa fluor 488 , used at a 1/500 dilution for 1 h. vector shield hoescht was used to stain the cell nuclei ( blue ) . for confocal imaging cells were imaged using a 60 oil immersion objective with a numerical aperture ( na ) of 1.45 cell clumps were selected at random and a z stack of 25 images taken for each clump of cells . the central slice of each of these stacks was selected and used for the analysis of the ratio of staining between the nucleus ( hoescht ) and cytoplasmic regions of the cell . in nih image , the tia-1 positive area and the hoescht positive area were determined by tracing and nuclear area subtracted from the tia-1 positive area and expressed as a proportion of the tia-1 positive area . the vegf - a full length 3utr was cloned downstream of the thymidine kinase promoter and the firefly luciferase orf ( construct luc - vegf165 ) , ( from jean - jacques feige and nadia cherradi , cea grenoble ) . this was modified using the quick - change ii xl site - directed mutagenesis kit ( stratagene ) to delete the first 66 nucleotides ( luc - vegf - a165b ) of exon 8 . the plasmid prl - tk ( promega ) encoding renilla luciferase was used as an internal control . luciferase activities were measured with the dual - luciferase reporter assay system ( promega ) on a victor 3 1420 multilabel counter luminometer ( perkinelmer ) . vegf - a mrna decay was measured in 10c cells after transfection with tia-1 and treatment with 10 mol / l actinomycin d. cells were harvested every hour over a 4-h period , and vegf - a isoform mrna levels analysed by rt - pcr . experiments were undertaken under home office license after review by the university of bristol ethical review group . 2 10 untransfected , fltia-1 , vegf - a165 , doubly or vector transfected cells in 0.2 ml pbs were injected subcutaneously in the dorsum of 6 balb / c nude mice ( from university of bristol colony ) for each group . mice were treated bi weekly with mouse igg ( sigma ) , or an anti - mouse vegf - a neutralising antibody ( liang et al . sample size was calculated a priori using g - power on the basis of being able to have 80% power to detect a mean sd 2 0.5 fold change in size and vascular density . 5 m thick de - waxed , re - hydrated sections were microwaved in 0.01 m sodium citrate , ph 6.0 for 15 min at 800 w. sections were incubated with vegf - a receptor 2 ( rabbit mab 2479 cell signalling ) or cd31 antibodies at 1:100 overnight at 4 c in a humidity chamber and after application of secondary antibodies and appropriate enzymatic process ( vector dab kit ) , vessel number counted in 10 randomly selected areas for each tumour ( n = 6 ) . results were analysed using prism software , given as mean sem unless otherwise stated and compared using one - way anova or t tests for comparison of means . p < 0.05 was considered significant . power analysis on non - significant results were carried out to ensure power of at least 80% ( using g - power ) . in five carcinoma ( ls174 t , hct116 , sw480 , ht29 , hca7 ) and an adenocarcinoma line ( 10c ) tia-1 rna , but not two other rna binding proteins , ttp and hur , was lower than in adenoma lines rg / c2 , and aa / c1 ( figure s1a , b ) , amplified using primers to detect full length ( fltia-1 ) . expression of the 4243 kda tia-1 protein was similar across cell lines , but in all carcinoma and the 10c cells , an additional band at 16 kda was seen ( figure 1a , figure s1c ) suggesting the presence of a truncated tia-1 protein . amplification of tia-1 mrna sequence from the 10c cells using primers that flank exons 6 and 7 revealed an additional product only in carcinoma and 10c cells ( figure 1b , figure s1d ) , due to the insertion of a 66bp sequence ( figure 1c ) from the intron between exons 6 and 7 of the tia-1 gene ( figure 1e ) . this was also present in braf v600e + ve a375 melanoma cells ( figure s1e ) . the 66base sequence is flanked by a classical consensus splice sequence ( ag gu ) and has a 73% cu sequence immediately upstream , suggesting a cassette exon undergoing alternative splicing . this sequence results in a unique 11 amino acid sequence and a premature stop codon , which should generate a 144 amino acid truncated tia-1 peptide of 15.8 kda ( figure 1d ) , with only rna recognition motif ( rrm)-1 and part of the rrm 2 , consistent with the short band ( 16 kda ) detected by the anti - tia-1 antibody in figure 1a . sequencing of tia-1 genomic dna from 10c cells showed no mutations in the region around either the 5 or 3 acceptor or donor sites . this mrna is identical to a splicing event described as exon 6a when tia-1 itself was over - expressed in a cancer cell line ( le guiner et al . , 2001 ) , but its endogenous expression has not previously been demonstrated . a blast search of this sequence showed identity in 66 ests , all consistent with it being this tia-1 isoform . a rabbit antibody raised to the unique peptide sequence of stia-1 detected the protein in 10c cells ( figure s1f ) . immunoprecipitation of 10c protein ( figure s1 g ) and immunoblotting for tia-1 showed both short and full length tia-1 . ip with a fltia-1 antibody and ib with stia-1 demonstrated that the protein detected with this antibody was tia-1 in ls174 t cells as well . ( figure s1h ) . both protein ( figure 1e top image ) and rna expression ( figure 1e bottom image ) of stia-1 was seen in sarcoma ( saos ) , hela , prostate cancer ( pc3 and lncap ) , and mda - mb-231 breast cancer cells , but not osteosarcoma ( u2os ) , haemangioma endothelial ( a differentiated phenotype ) , haemangioma stem ( hsc ) , human embryonic kidney ( hek ) , or rpmi-8226 leukaemia cells or human cardiac myocytes ( hcm ) . of the 18 cell lines in which stia-1 was investigated , only 2 of the 8 that lacked stia-1 have ras mutations or over - expression , whereas 8 of the 10 that did express stia-1 had ras over - expression or activating mutations , and the other two are braf mutants , ( ht29 and a375 cells ) . in both ls174 t and 10c cells the ras inhibitor farnesyl thiosalysilic acid ( fta ) significantly inhibited splicing to stia-1 in a dose dependent manner ( figure 1f ) . downstream signalling through mek and pi3k was required for this splicing in both cell lines ( figure s2a ) . ras is active in aac1 and rgc2 cell lines ( stia-1 negative ) indicating that it is necessary , but not sufficient to induce splicing of tia-1 . ras inhibition ( figure 2a , figure s2b d ) and mek and pi3k inhibition ( figure s2e ) also switched splice form expression from vegf - a165a to vegf - a165b in both 10c and ls174 t cell types . vegf - a165b protein expression was confirmed by immunoprecipitation and peptide sequencing ( figure s3a and b ) . these results suggest that hyperactivated ras signalling also acts to repress vegf - axxxb splicing , so we investigated a possible link between vegf and tia-1 . isoform specific sirna knockdown of stia-1 ( figure s3c g ) , reduced vegf - a165a mrna in both 10c and ls174 t and increased vegf - a165b mrna in 10c cells but made no difference in aa / c1 cells ( figure 2b ) . vegf - a165b but not vegf - a165 protein was significantly increased by sirna to stia-1 in ls174 t cells ( p = 0.013 ) , whereas only a significant reduction in the vegf - a165a isoform expression was seen in 10c cells ( p = 0.03 , figure 2c ) . thus there was a significant switch in the ratio of isoforms in both cell types towards a less angiogenic phenotype after knockdown of stia-1 ( figure 2d , p < 0.01 anova ) . conversely , vegf - a165b expression was rescued by fltia-1 expression in 10c and ls174 t cells measured by rt - pcr ( figure 3e ) , immunoblot ( figure 2f ) and elisa ( figure 2 g ) , in 10c cells to 90% vegf - a165b ( p < 0.001 compared with ctrl ) , similar to aa / c1 cells and in ls174 t from 5 1.8% to 12 1.5% of total vegf - a ( p < 0.01 ) . the reverse experiment ( over - expression of stia-1 ) resulted in lethality of unknown cause . fltia-1 also reduced cyclooxygenase-2 protein expression ( figure s3h ) , a known tia-1 target . rna immunoprecipitation ( rip ) of tia-1 showed that , whereas 10c , aa / c1 and rg / c1 all expressed both vegf - a165 and vegf - a165b , immunoprecipitation with an anti - tia-1 antibody pulled down only vegf - a165 in aa / c1 and rg / c1 ( cells expressing only fltia-1 ) , but no vegf - a mrna in stia-1 expressing cell lines ( 10c , hca7 and ls174 t , figure 2h ) . in 10c and ls174 t cells , transfection with fltia-1 restored tia-1 binding to vegf - a165a mrna , as evidenced by pull down of vegf - a165 mrna , compared with untransfected cells ( ctrl , figure 2i ) . the binding site of tia-1 to vegf - a mrna was demonstrated , in hek cell nuclear extract using an ms2-mbp binding assay ( nowak et al . , 2008 ) to reside in the vegf - a exon 8a sequence and 35 bases of intron 7 ( figure 2j ) . nuclear extract from 10c cells , while containing tia-1 ( as seen in the flow through unbound eluate in the lower panel ) , was unable to bind to vegf - a mrna sequences ( figure 2j ) . fltia-1 has been shown to function both as a translational repressor ( piecyk et al . , 2000 ) and an alternative splicing factor ( barron and lou , 2012 ; del gatto - konczak et al . , 2000 ) . expression of fltia-1 did not affect the half - lives of vegf - a165b and vegf - a165 mrna in 10c cells , determined by investigating mrna decay rates in cells treated with the transcriptional inhibitor actinomycin d ( actd ) ( figure 3a and b ) . although it did increase the amount of vegf - a165b at the start of the experiment , figure 3c shows that there was no difference in the half - life . there was no significant difference in expression of a luciferase reporter containing the 3utrs of vegf - a165a or vegf - a165b ( figure 3d ) in either 10c or ls174 t cells ( figure 3e ) , but transfection with fltia-1 resulted in significantly increased luciferase activity with the vegf - a165b 3utr compared with the vegf - a165a 3utr ( figure 3f ) indicating preferential translation of vegf - a165b by tia-1 as well as inhibiting splicing of vegf - a165 . rrm 2 and 3 are required for 3 translational repression ( bauer et al . , 2012 ) , and this is lacking in stia-1 , which only contains the full length rrm1 , which can not by itself bind to rna ( dember et al . , 1996 ) but is required for fltia-1 binding to specific sequences ( e.g. u rich sequences ) ( bauer et al . , 2012 ) , thus conferring specificity . thus , while stia-1 prevents fltia-1 mediated repression of splicing of the pro - angiogenic isoforms of vegf - a , the increased level of vegf - a165b mrna and protein induced by over - expression of fltia-1 in colon cells suggest that it also acts to selectively represses translation of the exon 8a containing isoforms . this may explain why the effects of stia-1 sirna knockdown on rna and protein were subtly different in the two cell types ( in 10c it increased vegf - a165b rna but not protein , and vice versa in ls174 t cells ) . nuclear / cytoplasmic separation of proteins ( figure s4a ) showed that in aac1 cells , tia-1 is found as the full length form but only in the nucleus , whereas in ls174 t and 10c cells a substantial proportion of all tia-1 isoforms and products were expressed in the cytoplasm , including stia-1 ( figure 3 g ) . this was confirmed by transfection of cells with full length tia-1 fused to gfp ( figure 3h ) , and by immunofluorescent staining with antibodies to the n terminus of tia-1 ( detects all isoforms , figure s4b ) . stia-1 knockdown , surprisingly , resulted in enhanced cytoplasmic staining for tia-1 ( figure 3i ) . this was confirmed in fltia-1-gfp over - expressing 10c cells , with stia-1 knockdown ( figure s4c ) . n - terminal staining ( which would detect stia-1 ) was reduced , consistent with a reduction in only the stia-1 component ( figure s4c ) . nuclear localisation of gfp - fltia-1 was reduced by stia-1 knockdown ( figure 3j ) . stia-1 therefore alters localisation of the fltia-1 within the cell , thereby preventing it from binding vegf - a mrna . the increased not decreased cytoplasmic tia-1 resulting from reduced of stia-1 in k - ras mutant cells suggests that the gfp - fltia-1 fusion protein localisation is stia-1 dependent . thus stia-1 expression in k - ras mutant cells may act by preventing fltia-1 nuclear trafficking ( to and from the nucleus ) , preventing it from interacting with other potential regulators of splicing and/or translation . this in turn alters the expression of vegf - a isoforms and results in a more angiogenic phenotype of the tumours . co - immunoprecipitation indicated that it is possible that the two isoforms could complex together ( figure s1 the localisation experiments indicate that in carcinoma cells fltia-1 excess either by over - expression with gfp , by knockdown of stia-1 or both results in cytoplasmic fltia-1 and/or vegf - a165b expression , whereas in adenoma cells , where k - ras is not over - expressed , fltia-1 is nuclear . stia-1 and vegf - a165b rna were detected in freshly isolated human colorectal cancer samples ( figure 4a ) confirmed by sequencing . quantification showed both a significant increase in stia-1 expression in cancer ( figure 4b ) and a significantly lower vegf - a165b : vegf - a165 ratio in the tumours than the controls ( figure 4c ) . in histological samples , fltia-1 rna ( figure 4d ) was found only in 13 of 31 tumours ( 42% ) but 22 of 30 normal tissues ( 73% , p = 0.02 , fisher 's exact test ) samples ( figure 4e ) . the proportion of tumours that were k - ras mutant was significantly higher ( p < 0.01 ) in those that had no fltia-1 , and k - ras mutant tumours rarely ( 2/13 cases ) had fltia-1 detected ( figure 4f ) . quantification showed that stia-1 exceeded fltia-1 in 20 of 31 tumours ( 65% ) but in only 9 of 30 normal tissues ( 30% , p = 0.01 , figure 4 g ) . moreover , stia-1 was present in 14 of the 15 dukes ' c ( 93% ) , but only 9 of the 16 dukes ' b ( 56% , p < 0.01 , figure 4h ) . there was a significantly higher microvascular density ( figure 4i ) in samples in which stia-1 but not fltia-1 was found ( figure 4j ) . over - expression of fltia-1 inhibited tumour growth in nude mice ( figure 5a ) , resulting in significantly smaller tumours at 18 and 21 days after injection ( figure 5b ) . this reduction was rescued by vegf - a165 cdna expression i.e. vegf - a that was not under the control of alternative splicing ( figure 5b ) . tumours from fltia-1 transfected cells had reduced tumour vessel density ( figure 5c and d , p < 0.01 ) . mice with ls174 t tumours expressing fltia-1 treated with an anti - vegf - a antibody ( liang et al . , 2006 ) grew significantly faster compared with igg ( figure 5e ) , at the same rate as wild type tumours treated with anti - vegf - a treatment ( figure 5f ) , but still more slowly than untreated wild type tumours ( figure 5 g ) . here we identify a novel k - ras dependent endogenous splice variant of tia-1 , short tia-1 ( stia-1 ) in crc patients and cancer cells , which controls vegf - a isoform expression . this stia-1 had been identified as being capable of being induced by over - expression of recombinant mouse tia-1 in human hek cells ( le guiner et al . , 2001 ) . this is the first demonstration that this splice variant may be present in human cancers , and that it may regulate vegf splice form expression . stia-1 , containing only one rna recognition motif ( rrm ) , prevented binding of full length tia-1 to vegf - a165 mrna , blocking both its angiogenic vegf splice site repression and its promotion of translation of anti - angiogenic vegf - a165b . it has previously been shown that rrm2 and 3 are required for binding of tia-1 to rna ( bauer et al . , 2012 ) , and interestingly , rrm3 is required for binding to c rich stretches ( cruz - gallardo et al . , 2014 ) . moreover , inclusion of exon 5 in tia-1 results in a higher splice activity , indicating that the activity of the protein can be altered by alternative splicing ( izquierdo and valcarcel , 2007 ) . as both of these domains are disrupted in stia , it is likely that stia-1 does not act by directly binding to the vegf rna and preventing fltia-1 from exerting its effects , and the direct mechanism of repression of distal splicing is not known . as stia-1 did co - immunoprecipitate with fltia-1 it is possible that stia-1 acts by heterodimerising with fltia-1 and preventing it from repressing splicing and/or translation of the pro - angiogenic isoform , vegf - a165a . cancer cells over - expressing fltia-1 formed smaller , less vascular tumours than those expressing stia-1 , and were more resistant to anti - vegf antibodies such as bevacizumab . these results indicate that k - ras mediated splicing of an rna binding protein may regulate isoform specific expression of vegf , providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti - angiogenic therapy . this opens up the question as to what is the downstream mechanism through which k - ras regulates tia-1 splicing . there is no clear evidence as to how k - ras activation can induce splicing switches . splicing is regulated by the complex interaction of splicing factor proteins that can be phosphorylated , localise to the nucleus and take part in a complicated interaction with other rna binding proteins and auxillary factors that control splice site location . the mechanisms through which tia-1 is spliced is not known , and none of the known splicing factors have yet been identified as controlling exon 6a splicing . interestingly , over - expression of human fltia-1 did not induce stia-1 splicing in cancer cells , in contrast to that shown by mouse flyia-1 in human hek cells ( le guiner et al . , 2001 ) . the finding that over - expression of vegf - a165 can reverse the effect of fltia-1 expression on tumour growth inhibition suggests that the effect was vegf splicing dependent , through anti - angiogenic activity , or through direct effects on the cells low vegf - a165b levels predict bevacizumab response in crc ( bates et al . , 2012 ) , but , bevacizumab provides significant clinical benefit in patients irrespective of k - ras mutations ( hurwitz et al . , 2009 ) . although patients with k - ras mutations did do better on bevacizumab compared with controls ( 9.3 compared with 5.5 months , an increase of 3.8 months ) , they did not perform as well as patients with wild type k - ras ( 13.5 versus 7.4 , an increase of 6.1 months ) . it would therefore be interesting to know how patients with k - ras mutations , in which stia-1 is expressed , responded . it is not yet clear whether stia-1 predicts survival , or can drive angiogenesis by reducing the anti - angiogenic vegf - a165b splice variants , or by increasing the vegf - a165 variant . these vegf - a165b variants have been shown to be down regulated so far in melanoma ( pritchard - jones et al . , 2007 ) , colon ( varey et al . , 2008 ) , prostate ( woolard et al . , 2004 ) , and kidney ( bates et al . , 2002 ) cancers , but the mechanism of regulation of these variants in cancer is still poorly described ( amin et al . , 2011 ; nowak et al . , 2010 , 2008 ) . the loose relationship between stia-1 and vegf - a165b down regulation in patients suggests a complex interaction , just as for alternative splicing of p73 and vegf - a165b isoforms , also demonstrated in colon cancer ( diaz et al . , 2008 ) . this is supported by the findings that ras inhibition in 10c cells was more effective at lower doses than in ls174 t cells , and that the rna and protein levels of vegf - a165b in the presence or absence of shtia-1 are not directly correlated . the effect of tia-1 on rna stability , as shown in figure 3 indicates that tia-1 is likely to act in a pleiotropic manner on the amount of vegf isoforms present acting by altering translation specifically for one set of isoforms , by regulating splicing at the proximal splice site , or , most likely , by changing both splicing and translation . binding of tia-1 to the vegf rna was shown , but it is also possible that other vascular growth factors are regulated by tia-1 , although these have not yet been published . in summary , we show that alternative splicing of tia-1 in human colon cancer may regulate the balance of the pro- and anti - angiogenic vegf - a isoforms , and control tumour growth and angiogenesis in animal models . this suggests that tia-1 can act as a splicing and translational regulator of vegf - a and alternative splicing of tia-1 may lead to enhanced angiogenesis and tumour growth in colorectal cancer , and resistance to anti - vegf - a therapy . mhz , ema , cha designed and carried out experimental procedures , ed , ks , xy , kh , as , od 's , kbb carried out experimental procedures , acw , djk , ahjs , rsm , mrl , and sjh contributed to the design and secured funding for the experimental work , ahrv contributed to design and interpretation of the experiments , dob designed the experiments , secured funding , and wrote the paper .

the angiogenic capability of colorectal carcinomas ( crc ) , and their susceptibility to anti - angiogenic therapy , is determined by expression of vascular endothelial growth factor ( vegf ) isoforms . the intracellular protein t - cell intracellular antigen ( tia-1 ) alters post - transcriptional rna processing and binds vegf - a mrna . we therefore tested the hypothesis that tia-1 could regulate vegf - a isoform expression in colorectal cancers . tia-1 and vegf - a isoform expression was measured in colorectal cancers and cell lines . we discovered that an endogenous splice variant of tia-1 encoding a truncated protein , short tia-1 ( stia-1 ) was expressed in crc tissues and invasive k - ras mutant colon cancer cells and tissues but not in adenoma cell lines . stia-1 was more highly expressed in crc than in normal tissues and increased with tumour stage . knockdown of stia-1 or over - expression of full length tia-1 ( fltia-1 ) induced expression of the anti - angiogenic vegf isoform vegf - a165b . whereas fltia-1 selectively bound vegf - a165 mrna and increased translation of vegf - a165b , stia-1 prevented this binding . in nude mice , xenografted colon cancer cells over - expressing fltia-1 formed smaller , less vascular tumours than those expressing stia-1 , but fltia-1 expression inhibited the effect of anti - vegf antibodies . these results indicate that alternative splicing of an rna binding protein can regulate isoform specific expression of vegf providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti - angiogenic therapy .

the lack of hepatic g6pase was obtained by a specific deletion of g6pc exon 3 in the liver using a cre - lox strategy in mice , which was recently described by mutel et al . male adult ( 68 weeks old ) b6.g6pc.sa and c57bl/6j mice ( charles rivers laboratories ) were injected intraperitoneally once daily with 100 l of tamoxifen ( 10 mg / ml ) on 5 consecutive days , to obtain l - g6pc and l - g6pc ( wt ) mice , respectively ( 14 ) . mice were housed in the animal house of lyon 1 university , in controlled temperature ( 22c ) conditions , with a 12-h light12-h - dark cycle . the specific hepatic deletion of g6pc exon 3 in the l - g6pc mice was always verified by pcr on purified liver genomic dna after euthanasia , as described by mutel et al . all procedures were performed in accordance with the guidelines established by the european convention for the protection of laboratory animals . insulin , glucagon , and corticosterone concentrations were determined with mouse elisa kits from chrystal chem , biovendor , and neogen , respectively . catecholamines and amino acids levels were determined by high - performance liquid chromatography ( centre hospitalier universitaire , lyon ) . -hydroxybutyrate was measured using optium ketone test strips with optium xceed sensors ( abbott diabetes care , u.k . ) . blood glucose was determined with an accu - chek go glucometer ( roche diagnostics ) during fasting experiments . hepatic glycogen determinations were carried out as described by keppler and decker ( 15 ) . a catheter was inserted into the right jugular vein under anesthesia , and mice were allowed to recover for 46 days . after 6 h or 24 h of fasting , mice were infused with a bolus ( 92.5 kbq ) of [ 3-h ] glucose . we then administered [ 3-h ] glucose at a rate of 6.3 kbq / min . , we checked that a steady state of plasma [ 3-h ] glucose - specific activity reached from 30 min of infusion ( data not shown ) . plasma glucose concentration did not vary during the infusion time . at a steady state of glycemia and [ 3-h ] glucose - specific activity ( between 60 to 90 min ) , egp was calculated as ra using the simplified equation of steele , by dividing the [ 3-h ] glucose infusion rate by the [ 3-h ] glucose - specific activity ( 16 ) . l - alanine and l - glutamine tolerance tests were performed after 6 h or 24 h of fasting . mice were injected intraperitoneally with l - alanine or l - glutamine ( 2 g / kg bw ) . blood glucose levels were determined from the tail vein at 0 , 15 , 30 , 45 , 60 , and 120 min after injection . for glucagon challenge , wt mice were injected intraperitoneally with 0.9% nacl or glucagon ( glucagen , novo nordisk ) at a dose of 200 ng / g of weight . kidneys and intestine were rapidly removed and fixed in formaldehyde for chromatin immunoprecipitation ( chip ) experiments , as described previously ( 17 ) . for gcgr antagonist experiments , l - g6pc mice were dosed by gavage with 50 mg / kg of gcgr antagonist l168,049 ( glucagon receptor antagonist ii , calbiochem ) in 5% peg-400/5% tween-80/90% h2o or vehicle , as described previously ( 18 ) . the liver and kidneys were rapidly removed , with tongs chilled previously in liquid n2 , and frozen . g6pase and pepck - c activities were assayed at maximal velocity , as described previously ( 19 ) . immunoblotting was carried out using purified anti - rat g6pc ( 20 ) , anti - pepck ( santa cruz biotechnology ) , and antiglutaminase ( kindly provided by dr . reverse transcription and real - time pcr were performed using sequence - specific primers ( supplementary table 1 ) , with ribosomal protein ml19 transcript ( rpl19 ) as a reference . chromatin obtained was then sheared with the enzymatic shearing kit ( active motif ) , yielding chromatin fragments of 500200 bp in size . each immunoprecipitation was performed with about 10 g of chromatin using the chip - it express kit ( active motif ) . chromatin complexes were immunoprecipitated for 1618 h at 4c while rotating with either phospho - creb ( ps133 ) antibody ( epitomics ) , glucocorticoid receptor antibody ( sc-1004 ; santa cruz biotechnology ) , or gfp antibody ( santa cruz ) as a negative control . after dna purification , quantitative pcr amplification was performed using primers specific for the camp or glucocorticoid response units of pck1 and g6pc promoters ( supplementary table 1 ) . the lack of hepatic g6pase was obtained by a specific deletion of g6pc exon 3 in the liver using a cre - lox strategy in mice , which was recently described by mutel et al . male adult ( 68 weeks old ) b6.g6pc.sa and c57bl/6j mice ( charles rivers laboratories ) were injected intraperitoneally once daily with 100 l of tamoxifen ( 10 mg / ml ) on 5 consecutive days , to obtain l - g6pc and l - g6pc ( wt ) mice , respectively ( 14 ) . mice were housed in the animal house of lyon 1 university , in controlled temperature ( 22c ) conditions , with a 12-h light12-h - dark cycle . the specific hepatic deletion of g6pc exon 3 in the l - g6pc mice was always verified by pcr on purified liver genomic dna after euthanasia , as described by mutel et al . all procedures were performed in accordance with the guidelines established by the european convention for the protection of laboratory animals . insulin , glucagon , and corticosterone concentrations were determined with mouse elisa kits from chrystal chem , biovendor , and neogen , respectively . catecholamines and amino acids levels were determined by high - performance liquid chromatography ( centre hospitalier universitaire , lyon ) . -hydroxybutyrate was measured using optium ketone test strips with optium xceed sensors ( abbott diabetes care , u.k . ) . blood glucose was determined with an accu - chek go glucometer ( roche diagnostics ) during fasting experiments . hepatic glycogen determinations were carried out as described by keppler and decker ( 15 ) . a catheter was inserted into the right jugular vein under anesthesia , and mice were allowed to recover for 46 days . after 6 h or 24 h of fasting , mice were infused with a bolus ( 92.5 kbq ) of [ 3-h ] glucose . we then administered [ 3-h ] glucose at a rate of 6.3 kbq / min . , we checked that a steady state of plasma [ 3-h ] glucose - specific activity reached from 30 min of infusion ( data not shown ) . plasma glucose concentration did not vary during the infusion time . at a steady state of glycemia and [ 3-h ] glucose - specific activity ( between 60 to 90 min ) , egp was calculated as ra using the simplified equation of steele , by dividing the [ 3-h ] glucose infusion rate by the [ 3-h ] glucose - specific activity ( 16 ) . l - alanine and l - glutamine tolerance tests were performed after 6 h or 24 h of fasting . mice were injected intraperitoneally with l - alanine or l - glutamine ( 2 g / kg bw ) . blood glucose levels were determined from the tail vein at 0 , 15 , 30 , 45 , 60 , and 120 min after injection . for glucagon challenge , wt mice were injected intraperitoneally with 0.9% nacl or glucagon ( glucagen , novo nordisk ) at a dose of 200 ng / g of weight . kidneys and intestine were rapidly removed and fixed in formaldehyde for chromatin immunoprecipitation ( chip ) experiments , as described previously ( 17 ) . for gcgr antagonist experiments , l - g6pc mice were dosed by gavage with 50 mg / kg of gcgr antagonist l168,049 ( glucagon receptor antagonist ii , calbiochem ) in 5% peg-400/5% tween-80/90% h2o or vehicle , as described previously ( 18 ) . immediately after gavage , mice were fasted and killed 6 h later . the liver and kidneys were rapidly removed , with tongs chilled previously in liquid n2 , and frozen . g6pase and pepck - c activities were assayed at maximal velocity , as described previously ( 19 ) . immunoblotting was carried out using purified anti - rat g6pc ( 20 ) , anti - pepck ( santa cruz biotechnology ) , and antiglutaminase ( kindly provided by dr . reverse transcription and real - time pcr were performed using sequence - specific primers ( supplementary table 1 ) , with ribosomal protein ml19 transcript ( rpl19 ) as a reference . chromatin obtained was then sheared with the enzymatic shearing kit ( active motif ) , yielding chromatin fragments of 500200 bp in size . each immunoprecipitation was performed with about 10 g of chromatin using the chip - it express kit ( active motif ) . chromatin complexes were immunoprecipitated for 1618 h at 4c while rotating with either phospho - creb ( ps133 ) antibody ( epitomics ) , glucocorticoid receptor antibody ( sc-1004 ; santa cruz biotechnology ) , or gfp antibody ( santa cruz ) as a negative control . after dna purification , quantitative pcr amplification was performed using primers specific for the camp or glucocorticoid response units of pck1 and g6pc promoters ( supplementary table 1 ) . g6pase activity was disrupted specifically in the liver by temporal and tissue - specific deletion of the g6pc gene based on a cre - lox strategy ( 14 ) . in brief , transgenic b6.g6pc mice were crossed with transgenic b6.sa mice ( 21 ) to generate b6.g6pc.sa mice , expressing inducible cre specifically in the liver . the treatment of adult b6.g6pc.sa mice with tamoxifen induced the excision of g6pc exon 3 , specifically in the liver ( 14 ) , leading to a complete lack of hepatic g6pase activity ( fig . this finding is consistent with these mice being unable to mobilize their glycogen stocks during fasting ( fig . 1c ) , leading to a hepatomegaly ( table 1 ) . in wt mice , indeed , glycogen stock was about 40% of the fed level at 6 h of fasting and was completely exhausted after 12 h of fasting ( 0.6 mg / g of liver , n = 4 ) . although mice with the total knock - out of g6pc die after weaning , these l - g6pc mice are viable . moreover , all l - g6pc mice survived prolonged fasting ( 45 h ) , although hepatic g6pase activity was still undetectable throughout the fasting period ( fig . because hepatic glucose production is thought to be critical for glucose homeostasis during fasting periods , plasma glucose concentration and egp were determined in l - g6pc and wt mice during prolonged fasting . l - g6pc mice displayed the same blood glucose level as control mice in the fed state ( fig . consistent with the role of hepatic glycogenolysis as an important pathway of glucose production in the early postabsorptive state , egp levels were found to be lower in 6 h fasted l - g6pc animals than in control animals ( fig . this was correlated with a lower blood glucose in 6 h fasted l - g6pc mice ( fig . g6pase activities ( a ) and glycogen content ( b ) were assayed in the liver of l - g6pc ( black bar ) and wt ( white bar ) mice in the fed state ( 0 h ) or in fasted states ( 6 h , 24 h , and 40 h ; n = 5 to 6 mice per group ) . c : blood glucose concentrations were determined afterward for 0 , 6 , 16 , 24 , 30 , 40 , and 45 h fasting in l - g6pc and wt mice . d : egp was determined in conscious l - g6pc ( black bar ) and wt ( white bar ) mice fasted for 6 h or 24 h ( n = 6 mice per group ) . data were obtained 5 weeks after gene deletion and are expressed as means sem . , significantly different with respect to the value in fed state in each group ( p < 0.01 ) . and , significantly different with respect to the value at 6 h of fasting in each group ( p < 0.05 and p < 0.01 , respectively ) . body and liver weights and plasmatic parameters of 6 h fasted l - g6pc mice values are expressed as means sem . data were determined 5 weeks after gene invalidation from l - g6pc and wt l - g6pc mice upon 6 h of fasting . values significantly different from wt ( * p < 0.05 and * * p < 0.01 ) are indicated ( mann - whitney test ) . concomitant with this drop in blood glucose observed at 6 h fasting , ketogenesis was rapidly induced in l - g6pc animals . at 6 h fasting , the amounts of ketone bodies ( -hydroxybutyrate ) in l - g6pc mice were fivefold greater compared with the control mice that were able to produce high levels of glucose by inducing glycogenolysis ( supplementary fig these results were accounted for by fast induction of the mitochondrial 3-hydroxy-3-methylglutaryl - coa synthase ( hmgcs2 ) expression in 6 h fasted l - g6pc mice ( supplementary fig . it is important to recall here that ketone bodies provide an alternative form of cellular energy fuel in postabsorptive situations characterized by low glucose availability . this is true notably for the brain , which may derive up to 70% of its energy from ketone bodies ( 6 ) , and for the kidney and intestine , the two alternative gluconeogenic organs , which derive 50% of their energy from ketone bodies in postabsorptive state ( 22,23 ) . it therefore seems unlikely that l - g6pc mice lacked energy fuels for these essential organs . it is noteworthy that differences in egp between l - g6pc and control animals were no longer observed after longer fasting periods , and that l - g6pc mice controlled blood glucose effectively during long fasting periods ( fig . we even observed a significant increase in blood glucose between 6 h and 16 h fasting , such that the blood glucose concentration of l - g6pc mice eventually reached that of control mice after 30 h fasting ( fig . by contrast , blood glucose concentration decreased steadily during fasting in control mice , eventually reaching a plateau at 100 mg / dl after 24 h of fasting ( fig . it was hypothesized that l - g6pc mice maintained their glycemia through rapid induction of extrahepatic gluconeogenesis , compensating for lack of glucose production by the liver . it is noteworthy that blood glucose was the same in l - g6pc and control mice in the fed state , although glucose injection was necessary in the total g6pc knockout mice to maintain blood glucose ( 24 ) . to explain this difference , we assessed the expression of gluconeogenic genes in the kidneys and intestine of fed animals . renal g6pc mrna levels of fed l - g6pc mice were about 1.7-fold higher than those in control mice ( fig . finally , specific g6pase activity in the kidney was 40% higher in l - g6pc than in control mice ( fig . 2c ) . on the contrary , no modification of g6pc and pepck - c expression was observed in the intestine of l - g6pc compared with control mice ( fig . only g6pase was upregulated in the kidneys of l - g6pc mice in the fed state , reflecting constitutive induction of the gluconeogenic pathway in the kidneys of l - g6pc compared with wt mice . expression of main gluconeogenic enzymes in the kidneys ( a c ) and the intestine ( d f ) of fed l - g6pc mice . a and d : level of g6pc and b and e : western blot and quantification analysis for g6pc and pepck - c proteins . c and f : specific g6pase and pepck - c activity of homozygous l - g6pc ( black bar ) and wt ( white bar ) mice . data were obtained 5 weeks after gene deletion in fed mice ( n = 6 mice per group ) and are expressed as the mean sem . values significantly different from wt ( * p < 0.05 , * * p < 0.01 ) are indicated . after 6 h fasting , the expression of the genes encoding major regulatory enzymes of extrahepatic gluconeogenesis [ g6pase , pepck - c , and glutaminase ] was dramatically induced in both the kidneys and intestine of l - g6pc mice . in the kidneys , amounts of g6pc mrna and protein were about twice those in the control ( fig . 3a and b ) , and specific g6pase activity was 36% higher than in control mice ( fig . renal pepck - c expression was increased concomitantly , resulting in a threefold increase of pck1 mrna and protein and specific pepck - c activity more than twice that of l - g6pc mice ( fig . there was also an induction of glutaminase in the kidneys of l - g6pc mice ( fig . a similar pattern of induction of gluconeogenic gene expression was observed for the three enzymes in the intestine of l - g6pc mice ( fig . thus , the expression of gluconeogenic genes was rapidly induced in both the kidneys and intestine of l - g6pc mice in postabsorptive state . expression of main gluconeogenic enzymes in the kidneys ( a c ) and the intestine ( d f ) of 6 h fasted l - g6pc mice . a and d : level of g6pc and pck1 mrna expressed as a ratio relative to rpl19 mrna level . b and e : western blot analysis and quantification for g6pc , pepck - c , and glutaminase proteins . c and d : specific g6pase and pepck - c activity of homozygous l - g6pc ( black bar ) and wt ( white bar ) mice . ( n = 6 mice per group ) , and are expressed as means sem . values significantly different from wt ( * p < 0.05 , * * p < 0.01 ) are indicated . these results strongly suggested that the l - g6pc mouse rapidly upregulated gluconeogenesis in the kidneys and intestine once food glucose was lacking . it is noteworthy that there is clear - cut organ substrate selectivity for the liver , kidney , and intestine gluconeogenesis ( 9 ) . whereas gluconeogenesis from glutamine only takes place in the kidneys and intestine , gluconeogenesis from alanine and lactate is the key pathway in the liver ( 4,9,25 ) . to substantiate the view that postabsorptive or fasting l - g6pc mice were able to regulate plasma glucose from extrahepatic gluconeogenesis in the absence of hepatic gluconeogenesis , we performed tolerance tests from alanine ( exclusive hepatic substrate ) and glutamine ( exclusive extrahepatic substrate ) . after 6 h of fasting , there was a marked increase in plasma glucose upon glutamine injection in l - g6pc mice , whereas only a marginal increase was observed in wt mice ( fig . 4a ) . on the contrary , l - g6pc mice were unable to increase their plasma glucose in response to alanine injection ( fig . surprisingly , there was no increase in plasma glucose in wt mice injected with alanine ; this might be a result of the preponderant role of hepatic glycogenolysis in egp and the nonactivation of gluconeogenesis at 6 h fasting in normal mice ( see fig . wt mice dramatically increased plasma glucose in response to alanine injection after 24 h of fasting , a time where glycogen stores were exhausted and liver gluconeogenesis was enhanced ( fig . l - g6pc mice were still unable to convert the injection of alanine in increased plasma glucose ( fig . 4c ) . also in line with the absence of liver gluconeogenesis in l - g6pc mice , plasma alanine and lactate concentrations were 5060% higher than in wt mice ( table 1 ) . on the other hand , the plasma concentration of glutamine was slightly lower in l - g6pc mice than in wt mice , which was consistent with an increased utilization of glutamine ( table 1 ) . taken together , these data strongly suggested that l - g6pc mice regulated plasma glucose from extrahepatic gluconeogenesis exclusively . after 6 h ( a and b ) or 24 h ( c ) of fasting , l - g6pc ( black circles ) and wt ( open squares ) mice were injected with glutamine ( a ) or alanine ( b and c ) . blood glucose levels were measured every 15 min for 2 h. data were obtained 5 weeks after tamoxifen treatment . percent values relative to time 0 were expressed as means sem ( n = 6 mice / group ) . values significantly different from wt ( * p < 0.01 ) ; significantly different with respect to the value before alanine or glutamine injection ( p < 0.01 ) . we then investigated the mechanisms underlying expression changes of gluconeogenic genes in both the kidneys and intestine in the absence of g6pc in the liver . first , we analyzed the hormonal status of l - g6pc mice in both fed and 6 h fasting states . in mammals , sophisticated hormonal control by insulin , glucagon , glucocorticoids , and cathecholamines maintains blood glucose within narrow limits , orchestrating the uptake and production of glucose . in both the fed state and after 6 h fasting , glucagon levels were higher in l - g6pc mice than in control mice ( fig . , l - g6pc mice exhibited a lower insulin level than that of control mice after 6 h fasting ( fig . 5b ) . this resulted in a twofold ( fed state ) to threefold ( 6 h fasted state ) increase in the glucagon - to - insulin ratio in l - g6pc mice in comparison with that of the control mice ( fig . moreover , corticosterone levels were markedly increased in l - g6pc mice compared with the control mice after 6 h fasting ( table 1 ) , but not in the fed state ( 63.0 10.8 ng / ml in l - g6pc mice vs. 65.0 14.0 ng / ml in wt mice ) . however , the epinephrine and norepinephrine plasma concentrations were similar in both l - g6pc and control mice ( table 1 ) . this is in agreement with the observation that a hypoglycemic state around 60 mg / dl is mild and does not represent a stressed condition from the metabolic viewpoint , especially in the light of the concomitant increased availability of ketone bodies ( see above ) . glucagon regulates gluconeogenic gene expression in the kidneys and intestine of l - g6pc mice . plasma glucagon ( a ) , plasma insulin ( b ) , and glucagon - to - insulin ratio ( c ) of fed or 6 h fasted l - g6pc ( black bars ) and control ( white bars ) mice are shown . d : rt - pcr analysis of the expression of glucagon receptor in the liver , kidney , and intestine of control mice fasted for 6 h. e - g : p - creb binding to the g6pc or pck1 promoter was analyzed by chip assay from kidneys or intestine of fed mice ( e ) or 6 h fasted mice ( f and g ) . h : level of pck1 mrna expressed as a ratio relative to rpl19 mrna level in the liver of l - g6pc mice treated or not with gcgr antagonist l-168,049 ( black bars ) and wt mice ( white bars ) fasted for 6 h. i and j : level of g6pc mrna expressed as a ratio relative to rpl19 mrna level in the kidneys ( i ) and intestine ( j ) of l - g6pc mice treated or not with gcgr antagonist l-168,049 ( black bars ) and wt mice ( white bars ) fasted for 6 h. data were obtained 5 weeks after gene deletion ( n = 6 mice per group ) and are expressed as means sem . values significantly different from wt ( * p < 0.05 , * * p < 0.01 ) and untreated l - g6pc mice ( $ p < 0.05 , $ $ p < 0.01 ) are indicated . , significantly different fed state ( p < 0.01 ) . in the light of these findings , glucagon could be a major candidate responsible for the induced expression of gluconeogenic genes in the kidneys and intestine of l - g6pc mice . glucagon receptors ( gcgr ) are known to be present in the liver , but gcgr are also expressed in the kidneys , notably in the proximal tubules and , to a lesser extent , in the intestine ( 2628 ) . we confirmed these data in l - g6pc mice where gcgr mrna was substantially expressed in the kidneys ( 10 times less than in the liver , however ) and more weakly in the intestine ( about 500 times less than in the liver ) of both control and l - g6pc mice ( fig . 5d ) . to date , the specific role of gcgr in these organs is still unexplained . however , two previous articles suggested a possible role of glucagon in the kidney cortex and intestine : 1 ) glucagon stimulated camp production in a kidney cortical cell line ( 29 ) and 2 ) glucagon infusion caused a net release of glucose into portal blood in dog ( 30 ) . the gcgr is coupled to gs proteins , stimulating adenylate cyclase and increasing intracellular camp levels ( 26 ) . this activates protein kinase a ( pka ) , which phosphorylates camp - response element binding protein ( creb ) . the phosphorylated form of creb ( p - creb ) induces the transcription of g6pc in the kidneys and intestine , via binding to the gene promoter ( 17 ) . we then examined whether p - creb could bind to the g6pc and pck1 promoters by performing a chip assay . on the fed state , p - creb was bound to the g6pc promoter in the kidneys of the fed l - g6pc mice , but not to that of the wt mice ( fig . p - creb was bound neither to the g6pc promoter nor to the pck1 promoter in the intestine of fed l - g6pc or wt mice ( not shown ) . after 6 h fasting , p - creb was bound to the renal g6pc promoter , but still not to the pck1 promoter of l - g6pc mice ( fig . it is noteworthy that in the intestine of 6 h fasted l - g6pc mice , p - creb was bound to both g6pc and pck1 promoters ( fig . unequivocally that the binding of p - creb on g6pc promoter in the intestine and kidney of l - g6pc mice was dependent on the increase of plasmatic glucagon , we administered orally a glucagon receptor antagonist to l - g6pc mice ( l-168,049 , 50 mg / kg body ) ( 18 ) . l-168,049 inhibits the fixation of glucagon on gcgr and glucagon - stimulated camp synthesis in cho cells expressing human gcgr ( 31 ) . in the liver of l - g6pc mice , pck1 mrna expression was decreased by half 6 h after the administration of gcgr antagonist ( fig . 5h ) , demonstrating the efficiency of the suppression of glucagon signaling . in agreement with the role of glucagon in the induction of extrahepatic gluconeogenesis , the administration of the antagonist prevented the increase of the g6pc expression in both the kidneys and intestine of 6 h these results strongly suggested that the increase of glucagon levels could account for the induction of g6pc expression in the kidneys and intestine of l - g6pc mice . an intriguing observation , however , is that the g6pc gene was induced in the kidneys but not in the intestine in fed l - g6pc mice ( figs . 2 and 5 ) . this might be dependent on differential sensitivity to glucagon in both organs , possibly related to the higher expression level of gcgr in the kidneys ( see above ) . although this reasoning may appear speculative , this phenomenon might to some extent be related to the fact that the kidney rapidly become predominant in participating in egp during fasting ( 8) , whereas the participation of the intestine is weaker and is induced later in the same situation ( 5 ) . in agreement with this rationale , the binding of p - creb to the renal g6pc promoter occurred from 6 h of fasting in wt mice ( fig . 5f ) . to increment our findings and confirm that the extrahepatic glucagon effects observed herein were not an adaptation specific to l - g6pc mice but could also take place in wt , we carried out a glucagon challenge in fed wt mice . after 30 min of glucagon administration , the plasma glucagon rose to the level of 6 h fasted l - g6pc mice ( figs . consistent with the results obtained in l - g6pc mice , p - creb was firmly bound to the g6pc promoter in both tissues ( fig . moreover , in response to glucagon injection , p - creb bound to the pck1 promoter only in the intestine ( fig . 6b ) , as already noted in l - g6pc mice ( fig . 5 ) . in conclusion , these results show definitively for the first time that g6pc gene expression is regulated by glucagon not only in the liver , but also in the kidneys and intestine , under physiological conditions . moreover , glucagon can also regulate pck1 in the intestine , but not in the kidneys . it must be noted that the increased concentration of corticosterone might also play a role in the induction of extrahepatic gluconeogenesis . glucocorticoids activate gluconeogenesis via the binding of glucocorticoid receptors ( grs ) to glucocorticoid response element on both g6pc and pck1 promoters . these regulations have been essentially documented in the liver up to now ( for review see ) . using chip experiments , we showed that grs were bound to g6pc and pck1 promoters in both the kidney and intestine of 6 h fasted l - g6pc and wt mice ( supplementary fig . the recruitment of grs to pck1 promoter was markedly increased in both the kidney ( about threefold compared with wt ) and intestine ( sixfold compared with wt ) of l - g6pc mice ( supplementary fig . 2a and b ) . there was also a robust recruitment of grs to the g6pc promoter in the intestine of l - g6pc mice compared with wt mice ( about eightfold compared with wt ; supplementary fig . these data suggested that glucocorticoids concurred in the induction of g6pc and pck1 gene expression in the kidney and intestine of l - g6pc mice . this does not exclude that they could act in synergy with p - creb to stimulate both g6pc and pck1 gene transcription in the kidney and intestine , as it was observed previously in the liver ( 33,34 ) . a : plasma glucagon of wt mice injected with saline solution ( white bar ) or glucagon ( black bar ) . b : p - creb binding to g6pc or pck1 promoter was analyzed by chip assay from the kidneys and intestine , 30 min after the injection of saline solution or glucagon in wt mice . in view of these results , another mechanism should be involved in the induction of pepck - c expression in the kidneys of 6 h fasted l - g6pc mice . despite a high glucagon - to - insulin ratio the transcription of pck1 gene is known to be controlled by metabolic acidosis in the kidneys , whereas it does not respond to changes in ph in the liver ( 35,36 ) . it is noteworthy that the plasmatic levels of free fatty acids , ketone bodies , and lactate were higher in l - g6pc mice than in control mice , after 6 h fasting ( table 1 , supplementary fig . consistently , urinary ph of 6 h fasted l - g6pc mice was more acidic compared with that of 6 h fasted control mice ( fig . both l - g6pc and wt mice presented the same neutral urinary ph , ranging from 6.5 to 7.0 . to test whether acidosis is the mechanism by which the pck1 gene was induced in l - g6pc mice , we counteracted acidosis by the addition of 0.28 m nahco3 in the drinking water of l - g6pc mice for 3 days ( 37 ) . the urinary ph of 6 h fasted l - g6pc mice increased after treatment with nahco3 and was not different from that of control mice ( fig . this led to a specific normalization of pck1 expression at the mrna and protein levels in the kidneys , but not in the intestine , of 6 h fasted l - g6pc mice treated with nahco3 ( fig . g ) . on the contrary , g6pase expression was not affected in the kidneys and in the intestine after nahco3 treatment ( fig . g ) , confirming that g6pc expression is not regulated by acidosis ( 35 ) . these data also strongly suggested that pck1 expression is not regulated by acidosis in the intestine . a : follow - up of urinary ph of l - g6pc mice ( black bars ) , l - g6pc mice treated with 0.28 mol / l nahco3 in drinking water ( gray bars ) , and wt ( l - g6pc mice , white bars ) on the fed or postabsorptive state . b and c : expression levels of mrna encoding g6pc or pck1 gene in the kidneys ( b ) or in the intestine ( c ) of 6 h fasted mice . d g : western blot quantification and enzyme activity assays of g6pase and pepck determined in the kidneys ( d and f ) or in the intestine of 6 h fasted mice ( e and g ) . data were obtained 5 weeks after gene deletion and are expressed as mean sem . values significantly different from wt ( * p < 0.05 ; * * p < 0.01 ) , from the fed state ( $ $ p < 0.01 ) , and from l - g6pc without nahco3 treatment ( p < 0.01 ) . the liver has always been considered the major source of egp until now . in contradiction with this dogma , we here report that , after a transient drop in plasma glucose as a result of incapacity to mobilize glycogen stores , the absence of hepatic glucose production has no major effect on the control of fasting plasma glucose . instead , the early induction of gluconeogenesis in the kidneys and intestine occurs , permitting sustentation of egp and blood glucose right from the start of fasting periods . this perfectly matches what has been observed during the anhepatic phase of liver transplantation in humans ( 38,39 ) . our data also emphasize that an essential function of the liver is the rapid tuning of blood glucose during nutritional transitions , via the handling of glycogen stores . however , the first major finding of this study is that the liver is not an irreplaceable source of endogenous glucose in the absence of food glucose . similarly , glucagon , which is well known to stimulate hepatic glucose release via activation of glycogenolysis and gluconeogenic gene expression , has up to now been considered as only targeting the liver . in opposition to this other dogma , the second major finding here is that glucagon also plays a key role in the transcriptional regulation of renal and intestinal gluconeogenic genes . this may account for the basal induction of renal g6pc in the absence of hepatic glucose production and for the rapid induction of intestinal g6pc and pck1 once food is lacking . either fasting or type 2 diabetes is characterized by increased glucagon secretion in humans , which is believed to exert a key role in the augmented egp of diabetes ( 40 ) . in these two situations , moreover , the renal glucose production could play a crucial role in the counterregulation of insulin - induced hypoglycemia in humans , a situation of increased glucagon and cortisol secretions ( 41,42 ) . at least , the important role of the kidney evidenced here might also explain why patients with renal failure are prone to hypoglycemia ( 44 ) . in conclusion , our study provides a definitive quantitative estimate of the capacity of extrahepatic gluconeogenesis to sustain fasting egp , regardless of the contribution of the liver . it also extends the regulatory role of glucagon to the control of gluconeogenesis in the kidneys and intestine . this leads us to conclude that the current dogma relating to the relative role of the liver vis - - vis extrahepatic gluconeogenic organs in glucose homeostasis should be reconsidered . g6pase activity was disrupted specifically in the liver by temporal and tissue - specific deletion of the g6pc gene based on a cre - lox strategy ( 14 ) . in brief , transgenic b6.g6pc mice were crossed with transgenic b6.sa mice ( 21 ) to generate b6.g6pc.sa mice , expressing inducible cre specifically in the liver . the treatment of adult b6.g6pc.sa mice with tamoxifen induced the excision of g6pc exon 3 , specifically in the liver ( 14 ) , leading to a complete lack of hepatic g6pase activity ( fig . this finding is consistent with these mice being unable to mobilize their glycogen stocks during fasting ( fig . 1c ) , leading to a hepatomegaly ( table 1 ) . in wt mice , indeed , glycogen stock was about 40% of the fed level at 6 h of fasting and was completely exhausted after 12 h of fasting ( 0.6 mg / g of liver , n = 4 ) . although mice with the total knock - out of g6pc die after weaning , these l - g6pc mice are viable . moreover , all l - g6pc mice survived prolonged fasting ( 45 h ) , although hepatic g6pase activity was still undetectable throughout the fasting period ( fig . because hepatic glucose production is thought to be critical for glucose homeostasis during fasting periods , plasma glucose concentration and egp were determined in l - g6pc and wt mice during prolonged fasting . l - g6pc mice displayed the same blood glucose level as control mice in the fed state ( fig . consistent with the role of hepatic glycogenolysis as an important pathway of glucose production in the early postabsorptive state , egp levels were found to be lower in 6 h fasted l - g6pc animals than in control animals ( fig . this was correlated with a lower blood glucose in 6 h fasted l - g6pc mice ( fig . g6pase activities ( a ) and glycogen content ( b ) were assayed in the liver of l - g6pc ( black bar ) and wt ( white bar ) mice in the fed state ( 0 h ) or in fasted states ( 6 h , 24 h , and 40 h ; n = 5 to 6 mice per group ) . c : blood glucose concentrations were determined afterward for 0 , 6 , 16 , 24 , 30 , 40 , and 45 h fasting in l - g6pc and wt mice . d : egp was determined in conscious l - g6pc ( black bar ) and wt ( white bar ) mice fasted for 6 h or 24 h ( n = 6 mice per group ) . data were obtained 5 weeks after gene deletion and are expressed as means sem . , significantly different with respect to the value in fed state in each group ( p < 0.01 ) . and , significantly different with respect to the value at 6 h of fasting in each group ( p < 0.05 and p < 0.01 , respectively ) . body and liver weights and plasmatic parameters of 6 h fasted l - g6pc mice values are expressed as means sem . data were determined 5 weeks after gene invalidation from l - g6pc and wt l - g6pc mice upon 6 h of fasting . values significantly different from wt ( * p < 0.05 and * * p < 0.01 ) are indicated ( mann - whitney test ) . concomitant with this drop in blood glucose observed at 6 h fasting , ketogenesis was rapidly induced in l - g6pc animals . at 6 h fasting , the amounts of ketone bodies ( -hydroxybutyrate ) in l - g6pc mice were fivefold greater compared with the control mice that were able to produce high levels of glucose by inducing glycogenolysis ( supplementary fig these results were accounted for by fast induction of the mitochondrial 3-hydroxy-3-methylglutaryl - coa synthase ( hmgcs2 ) expression in 6 h fasted l - g6pc mice ( supplementary fig . it is important to recall here that ketone bodies provide an alternative form of cellular energy fuel in postabsorptive situations characterized by low glucose availability . this is true notably for the brain , which may derive up to 70% of its energy from ketone bodies ( 6 ) , and for the kidney and intestine , the two alternative gluconeogenic organs , which derive 50% of their energy from ketone bodies in postabsorptive state ( 22,23 ) . it therefore seems unlikely that l - g6pc mice lacked energy fuels for these essential organs . it is noteworthy that differences in egp between l - g6pc and control animals were no longer observed after longer fasting periods , and that l - g6pc mice controlled blood glucose effectively during long fasting periods ( fig . we even observed a significant increase in blood glucose between 6 h and 16 h fasting , such that the blood glucose concentration of l - g6pc mice eventually reached that of control mice after 30 h fasting ( fig . by contrast , blood glucose concentration decreased steadily during fasting in control mice , eventually reaching a plateau at 100 mg / dl after 24 h of fasting ( fig . it was hypothesized that l - g6pc mice maintained their glycemia through rapid induction of extrahepatic gluconeogenesis , compensating for lack of glucose production by the liver . it is noteworthy that blood glucose was the same in l - g6pc and control mice in the fed state , although glucose injection was necessary in the total g6pc knockout mice to maintain blood glucose ( 24 ) . to explain this difference , we assessed the expression of gluconeogenic genes in the kidneys and intestine of fed animals . renal g6pc mrna levels of fed l - g6pc mice were about 1.7-fold higher than those in control mice ( fig . finally , specific g6pase activity in the kidney was 40% higher in l - g6pc than in control mice ( fig . 2c ) . on the contrary , renal pck1 expression and pepck activity were not modified ( fig . no modification of g6pc and pepck - c expression was observed in the intestine of l - g6pc compared with control mice ( fig . thus , only g6pase was upregulated in the kidneys of l - g6pc mice in the fed state , reflecting constitutive induction of the gluconeogenic pathway in the kidneys of l - g6pc compared with wt mice . expression of main gluconeogenic enzymes in the kidneys ( a c ) and the intestine ( d f ) of fed l - g6pc mice . a and d : level of g6pc and pck1 b and e : western blot and quantification analysis for g6pc and pepck - c proteins . c and f : specific g6pase and pepck - c activity of homozygous l - g6pc ( black bar ) and wt ( white bar ) mice . data were obtained 5 weeks after gene deletion in fed mice ( n = 6 mice per group ) and are expressed as the mean sem . values significantly different from wt ( * p < 0.05 , * * p < 0.01 ) are indicated . after 6 h fasting , the expression of the genes encoding major regulatory enzymes of extrahepatic gluconeogenesis [ g6pase , pepck - c , and glutaminase ] was dramatically induced in both the kidneys and intestine of l - g6pc mice . in the kidneys , amounts of g6pc mrna and protein were about twice those in the control ( fig . 3a and b ) , and specific g6pase activity was 36% higher than in control mice ( fig . renal pepck - c expression was increased concomitantly , resulting in a threefold increase of pck1 mrna and protein and specific pepck - c activity more than twice that of l - g6pc mice ( fig . c ) . there was also an induction of glutaminase in the kidneys of l - g6pc mice ( fig . a similar pattern of induction of gluconeogenic gene expression was observed for the three enzymes in the intestine of l - g6pc mice ( fig . thus , the expression of gluconeogenic genes was rapidly induced in both the kidneys and intestine of l - g6pc mice in postabsorptive state . expression of main gluconeogenic enzymes in the kidneys ( a c ) and the intestine ( d f ) of 6 h fasted l - g6pc mice . a and d : level of g6pc and pck1 mrna expressed as a ratio relative to rpl19 mrna level . b and e : western blot analysis and quantification for g6pc , pepck - c , and glutaminase proteins . c and d : specific g6pase and pepck - c activity of homozygous l - g6pc ( black bar ) and wt ( white bar ) mice . ( n = 6 mice per group ) , and are expressed as means sem . values significantly different from wt ( * p < 0.05 , * * p < 0.01 ) are indicated . these results strongly suggested that the l - g6pc mouse rapidly upregulated gluconeogenesis in the kidneys and intestine once food glucose was lacking . it is noteworthy that there is clear - cut organ substrate selectivity for the liver , kidney , and intestine gluconeogenesis ( 9 ) . whereas gluconeogenesis from glutamine only takes place in the kidneys and intestine , gluconeogenesis from alanine and lactate is the key pathway in the liver ( 4,9,25 ) . to substantiate the view that postabsorptive or fasting l - g6pc mice were able to regulate plasma glucose from extrahepatic gluconeogenesis in the absence of hepatic gluconeogenesis , we performed tolerance tests from alanine ( exclusive hepatic substrate ) and glutamine ( exclusive extrahepatic substrate ) . after 6 h of fasting , there was a marked increase in plasma glucose upon glutamine injection in l - g6pc mice , whereas only a marginal increase was observed in wt mice ( fig . l - g6pc mice were unable to increase their plasma glucose in response to alanine injection ( fig . surprisingly , there was no increase in plasma glucose in wt mice injected with alanine ; this might be a result of the preponderant role of hepatic glycogenolysis in egp and the nonactivation of gluconeogenesis at 6 h fasting in normal mice ( see fig . wt mice dramatically increased plasma glucose in response to alanine injection after 24 h of fasting , a time where glycogen stores were exhausted and liver gluconeogenesis was enhanced ( fig . 4c ) . on the contrary , l - g6pc mice were still unable to convert the injection of alanine in increased plasma glucose ( fig . 4c ) . also in line with the absence of liver gluconeogenesis in l - g6pc mice , plasma alanine and lactate concentrations were 5060% higher than in wt mice ( table 1 ) . on the other hand , the plasma concentration of glutamine was slightly lower in l - g6pc mice than in wt mice , which was consistent with an increased utilization of glutamine ( table 1 ) . taken together , these data strongly suggested that l - g6pc mice regulated plasma glucose from extrahepatic gluconeogenesis exclusively . after 6 h ( a and b ) or 24 h ( c ) of fasting , l - g6pc ( black circles ) and wt ( open squares ) mice were injected with glutamine ( a ) or alanine ( b and c ) . blood glucose levels were measured every 15 min for 2 h. data were obtained 5 weeks after tamoxifen treatment . percent values relative to time 0 were expressed as means sem ( n = 6 mice / group ) . values significantly different from wt ( * p < 0.01 ) ; significantly different with respect to the value before alanine or glutamine injection ( p < 0.01 ) . we then investigated the mechanisms underlying expression changes of gluconeogenic genes in both the kidneys and intestine in the absence of g6pc in the liver . first , we analyzed the hormonal status of l - g6pc mice in both fed and 6 h fasting states . in mammals , sophisticated hormonal control by insulin , glucagon , glucocorticoids , and cathecholamines maintains blood glucose within narrow limits , orchestrating the uptake and production of glucose . in both the fed state and after 6 h fasting , glucagon levels were higher in l - g6pc mice than in control mice ( fig . , l - g6pc mice exhibited a lower insulin level than that of control mice after 6 h fasting ( fig . this resulted in a twofold ( fed state ) to threefold ( 6 h fasted state ) increase in the glucagon - to - insulin ratio in l - g6pc mice in comparison with that of the control mice ( fig . moreover , corticosterone levels were markedly increased in l - g6pc mice compared with the control mice after 6 h fasting ( table 1 ) , but not in the fed state ( 63.0 10.8 ng / ml in l - g6pc mice vs. 65.0 14.0 ng / ml in wt mice ) . however , the epinephrine and norepinephrine plasma concentrations were similar in both l - g6pc and control mice ( table 1 ) . this is in agreement with the observation that a hypoglycemic state around 60 mg / dl is mild and does not represent a stressed condition from the metabolic viewpoint , especially in the light of the concomitant increased availability of ketone bodies ( see above ) . glucagon regulates gluconeogenic gene expression in the kidneys and intestine of l - g6pc mice . plasma glucagon ( a ) , plasma insulin ( b ) , and glucagon - to - insulin ratio ( c ) of fed or 6 h fasted l - g6pc ( black bars ) and control ( white bars ) mice are shown . d : rt - pcr analysis of the expression of glucagon receptor in the liver , kidney , and intestine of control mice fasted for 6 h. e - g : p - creb binding to the g6pc or pck1 promoter was analyzed by chip assay from kidneys or intestine of fed mice ( e ) or 6 h fasted mice ( f and g ) . h : level of pck1 mrna expressed as a ratio relative to rpl19 mrna level in the liver of l - g6pc mice treated or not with gcgr antagonist l-168,049 ( black bars ) and wt mice ( white bars ) fasted for 6 h. i and j : level of g6pc mrna expressed as a ratio relative to rpl19 mrna level in the kidneys ( i ) and intestine ( j ) of l - g6pc mice treated or not with gcgr antagonist l-168,049 ( black bars ) and wt mice ( white bars ) fasted for 6 h. data were obtained 5 weeks after gene deletion ( n = 6 mice per group ) and are expressed as means sem . values significantly different from wt ( * p < 0.05 , * * p < 0.01 ) and untreated l - g6pc mice ( $ p < 0.05 , $ $ p < 0.01 ) are indicated . , significantly different fed state ( p < 0.01 ) . in the light of these findings , glucagon could be a major candidate responsible for the induced expression of gluconeogenic genes in the kidneys and intestine of l - g6pc mice . glucagon receptors ( gcgr ) are known to be present in the liver , but gcgr are also expressed in the kidneys , notably in the proximal tubules and , to a lesser extent , in the intestine ( 2628 ) . we confirmed these data in l - g6pc mice where gcgr mrna was substantially expressed in the kidneys ( 10 times less than in the liver , however ) and more weakly in the intestine ( about 500 times less than in the liver ) of both control and l - g6pc mice ( fig . 5d ) . to date , the specific role of gcgr in these organs is still unexplained . however , two previous articles suggested a possible role of glucagon in the kidney cortex and intestine : 1 ) glucagon stimulated camp production in a kidney cortical cell line ( 29 ) and 2 ) glucagon infusion caused a net release of glucose into portal blood in dog ( 30 ) . the gcgr is coupled to gs proteins , stimulating adenylate cyclase and increasing intracellular camp levels ( 26 ) . this activates protein kinase a ( pka ) , which phosphorylates camp - response element binding protein ( creb ) . the phosphorylated form of creb ( p - creb ) induces the transcription of g6pc in the kidneys and intestine , via binding to the gene promoter ( 17 ) . we then examined whether p - creb could bind to the g6pc and pck1 promoters by performing a chip assay . on the fed state , p - creb was bound to the g6pc promoter in the kidneys of the fed l - g6pc mice , but not to that of the wt mice ( fig . p - creb was bound neither to the g6pc promoter nor to the pck1 promoter in the intestine of fed l - g6pc or wt mice ( not shown ) . after 6 h fasting , p - creb was bound to the renal g6pc promoter , but still not to the pck1 promoter of l - g6pc mice ( fig . it is noteworthy that in the intestine of 6 h fasted l - g6pc mice , p - creb was bound to both g6pc and pck1 promoters ( fig . unequivocally that the binding of p - creb on g6pc promoter in the intestine and kidney of l - g6pc mice was dependent on the increase of plasmatic glucagon , we administered orally a glucagon receptor antagonist to l - g6pc mice ( l-168,049 , 50 mg / kg body ) ( 18 ) . l-168,049 inhibits the fixation of glucagon on gcgr and glucagon - stimulated camp synthesis in cho cells expressing human gcgr ( 31 ) . in the liver of l - g6pc mice , pck1 mrna expression was decreased by half 6 h after the administration of gcgr antagonist ( fig . 5h ) , demonstrating the efficiency of the suppression of glucagon signaling . in agreement with the role of glucagon in the induction of extrahepatic gluconeogenesis , the administration of the antagonist prevented the increase of the g6pc expression in both the kidneys and intestine of 6 h these results strongly suggested that the increase of glucagon levels could account for the induction of g6pc expression in the kidneys and intestine of l - g6pc mice . an intriguing observation , however , is that the g6pc gene was induced in the kidneys but not in the intestine in fed l - g6pc mice ( figs . 2 and 5 ) . this might be dependent on differential sensitivity to glucagon in both organs , possibly related to the higher expression level of gcgr in the kidneys ( see above ) . although this reasoning may appear speculative , this phenomenon might to some extent be related to the fact that the kidney rapidly become predominant in participating in egp during fasting ( 8) , whereas the participation of the intestine is weaker and is induced later in the same situation ( 5 ) . in agreement with this rationale , the binding of p - creb to the renal g6pc promoter occurred from 6 h of fasting in wt mice ( fig . 5f ) . to increment our findings and confirm that the extrahepatic glucagon effects observed herein were not an adaptation specific to l - g6pc mice but could also take place in wt , we carried out a glucagon challenge in fed wt mice . after 30 min of glucagon administration , the plasma glucagon rose to the level of 6 h fasted l - g6pc mice ( figs . consistent with the results obtained in l - g6pc mice , p - creb was firmly bound to the g6pc promoter in both tissues ( fig . moreover , in response to glucagon injection , p - creb bound to the pck1 promoter only in the intestine ( fig . 6b ) , as already noted in l - g6pc mice ( fig . 5 ) . in conclusion , these results show definitively for the first time that g6pc gene expression is regulated by glucagon not only in the liver , but also in the kidneys and intestine , under physiological conditions . moreover , glucagon can also regulate pck1 in the intestine , but not in the kidneys . it must be noted that the increased concentration of corticosterone might also play a role in the induction of extrahepatic gluconeogenesis . glucocorticoids activate gluconeogenesis via the binding of glucocorticoid receptors ( grs ) to glucocorticoid response element on both g6pc and pck1 promoters . these regulations have been essentially documented in the liver up to now ( for review see ) . using chip experiments , we showed that grs were bound to g6pc and pck1 promoters in both the kidney and intestine of 6 h fasted l - g6pc and wt mice ( supplementary fig . the recruitment of grs to pck1 promoter was markedly increased in both the kidney ( about threefold compared with wt ) and intestine ( sixfold compared with wt ) of l - g6pc mice ( supplementary fig . there was also a robust recruitment of grs to the g6pc promoter in the intestine of l - g6pc mice compared with wt mice ( about eightfold compared with wt ; supplementary fig . these data suggested that glucocorticoids concurred in the induction of g6pc and pck1 gene expression in the kidney and intestine of l - g6pc mice . this does not exclude that they could act in synergy with p - creb to stimulate both g6pc and pck1 gene transcription in the kidney and intestine , as it was observed previously in the liver ( 33,34 ) . a : plasma glucagon of wt mice injected with saline solution ( white bar ) or glucagon ( black bar ) . b : p - creb binding to g6pc or pck1 promoter was analyzed by chip assay from the kidneys and intestine , 30 min after the injection of saline solution or glucagon in wt mice . in view of these results , another mechanism should be involved in the induction of pepck - c expression in the kidneys of 6 h fasted l - g6pc mice . despite a high glucagon - to - insulin ratio the transcription of pck1 gene is known to be controlled by metabolic acidosis in the kidneys , whereas it does not respond to changes in ph in the liver ( 35,36 ) . it is noteworthy that the plasmatic levels of free fatty acids , ketone bodies , and lactate were higher in l - g6pc mice than in control mice , after 6 h fasting ( table 1 , supplementary fig . consistently , urinary ph of 6 h fasted l - g6pc mice was more acidic compared with that of 6 h fasted control mice ( fig . both l - g6pc and wt mice presented the same neutral urinary ph , ranging from 6.5 to 7.0 . to test whether acidosis is the mechanism by which the pck1 gene was induced in l - g6pc mice , we counteracted acidosis by the addition of 0.28 m nahco3 in the drinking water of l - g6pc mice for 3 days ( 37 ) . the urinary ph of 6 h fasted l - g6pc mice increased after treatment with nahco3 and was not different from that of control mice ( fig . this led to a specific normalization of pck1 expression at the mrna and protein levels in the kidneys , but not in the intestine , of 6 h fasted l - g6pc mice treated with nahco3 ( fig . g6pase expression was not affected in the kidneys and in the intestine after nahco3 treatment ( fig . g ) , confirming that g6pc expression is not regulated by acidosis ( 35 ) . these data also strongly suggested that pck1 expression is not regulated by acidosis in the intestine . a : follow - up of urinary ph of l - g6pc mice ( black bars ) , l - g6pc mice treated with 0.28 mol / l nahco3 in drinking water ( gray bars ) , and wt ( l - g6pc mice , white bars ) on the fed or postabsorptive state . b and c : expression levels of mrna encoding g6pc or pck1 gene in the kidneys ( b ) or in the intestine ( c ) of 6 h fasted mice . g : western blot quantification and enzyme activity assays of g6pase and pepck determined in the kidneys ( d and f ) or in the intestine of 6 h fasted mice ( e and g ) . data were obtained 5 weeks after gene deletion and are expressed as mean sem . values significantly different from wt ( * p < 0.05 ; * * p < 0.01 ) , from the fed state ( $ $ p < 0.01 ) , and from l - g6pc without nahco3 treatment ( p < 0.01 ) . the liver has always been considered the major source of egp until now . in contradiction with this dogma , we here report that , after a transient drop in plasma glucose as a result of incapacity to mobilize glycogen stores , the absence of hepatic glucose production has no major effect on the control of fasting plasma glucose . instead , the early induction of gluconeogenesis in the kidneys and intestine occurs , permitting sustentation of egp and blood glucose right from the start of fasting periods . this perfectly matches what has been observed during the anhepatic phase of liver transplantation in humans ( 38,39 ) . our data also emphasize that an essential function of the liver is the rapid tuning of blood glucose during nutritional transitions , via the handling of glycogen stores . however , the first major finding of this study is that the liver is not an irreplaceable source of endogenous glucose in the absence of food glucose . similarly , glucagon , which is well known to stimulate hepatic glucose release via activation of glycogenolysis and gluconeogenic gene expression , has up to now been considered as only targeting the liver . in opposition to this other dogma , the second major finding here is that glucagon also plays a key role in the transcriptional regulation of renal and intestinal gluconeogenic genes . this may account for the basal induction of renal g6pc in the absence of hepatic glucose production and for the rapid induction of intestinal g6pc and pck1 once food is lacking . either fasting or type 2 diabetes is characterized by increased glucagon secretion in humans , which is believed to exert a key role in the augmented egp of diabetes ( 40 ) . in these two situations , , the renal glucose production could play a crucial role in the counterregulation of insulin - induced hypoglycemia in humans , a situation of increased glucagon and cortisol secretions ( 41,42 ) . at least , the important role of the kidney evidenced here might also explain why patients with renal failure are prone to hypoglycemia ( 44 ) . in conclusion , our study provides a definitive quantitative estimate of the capacity of extrahepatic gluconeogenesis to sustain fasting egp , regardless of the contribution of the liver . it also extends the regulatory role of glucagon to the control of gluconeogenesis in the kidneys and intestine . this leads us to conclude that the current dogma relating to the relative role of the liver vis - - vis extrahepatic gluconeogenic organs in glucose homeostasis should be reconsidered .

objectivesince the pioneering work of claude bernard , the scientific community has considered the liver to be the major source of endogenous glucose production in all postabsorptive situations . nevertheless , the kidneys and intestine can also produce glucose in blood , particularly during fasting and under protein feeding . the aim of this study was to better define the importance of the three gluconeogenic organs in glucose homeostasis.research design and methodswe investigated blood glucose regulation during fasting in a mouse model of inducible liver - specific deletion of the glucose-6-phosphatase gene ( l - g6pc/ mice ) , encoding a mandatory enzyme for glucose production . furthermore , we characterized molecular mechanisms underlying expression changes of gluconeogenic genes ( g6pc , pck1 , and glutaminase ) in both the kidneys and intestine.resultswe show that the absence of hepatic glucose release had no major effect on the control of fasting plasma glucose concentration . instead , compensatory induction of gluconeogenesis occurred in the kidneys and intestine , driven by glucagon , glucocorticoids , and acidosis . moreover , the extrahepatic action of glucagon took place in wild - type mice.conclusionsour study provides a definitive quantitative estimate of the capacity of extrahepatic gluconeogenesis to sustain fasting endogenous glucose production under the control of glucagon , regardless of the contribution of the liver . thus , the current dogma relating to the respective role of the liver and of extrahepatic gluconeogenic organs in glucose homeostasis requires re - examination .

mankind is still struggling against the parasites , plasmodium is considered as cause of malaria since ancient times and is the leading cause of mortality worldwide , infecting approximately 3.3 billion people were at risk globally in 2011 ( 1 - 3 ) . both treatment and control of malaria are hampered by the spread of resistance to common antimalarial drugs , especially against p. falciparum ( 6 , 7 ) . in pakistan , malaria threatens millions of people , due to poor conditions ; and it remains endemic in most parts of country ( 8) . five different species of plasmodium : p. falciparum , p. vivax , p. malariae and p. ovale , p. knowlesi cause human malaria ( 9 , 10 ) . two species of plasmodium have reported in pakistan : p. vivax ( 75% ) and p. falciparum ( 25% ) ( 8) . as there is no vaccine available for malaria and current treatments suffer from several limitations ( 11 ) , hence the emphasis falls on accurate diagnosis of malaria to provide novel drugs to treat different types of malaria , especially for p. falciparum the most fatal infection ( 12 - 15 ) . polymerase chain reaction ( pcr ) has proved to be an efficient , sensitive and specific method for diagnosis of mixed infections , low parasitaemia and species detection for malaria ( 10 , 13 , 16 - 20 ) . pcr has the potential to overcome all the limitations of the traditional diagnostic method , but their high cost limits their clinical implication for malaria diagnosis ( 3 , 21 ) . the present study was aimed to determine the epidemiology of malaria by comparison of microscopy and nested pcr . genus - specific and species - specific primers for 18s rrna gene of plasmodium species were used for two plasmodium species ( p. falciparum and p.vivax ) infection by nested pcr . whole blood ( 5 ml ) was drawn by sterilized syringes and collected in edta vacutainer tubes . negative controls blood was obtained from students of department of zoology , university of the punjab , lahore . the blood smears were stained with 1% giemsa stain in phosphate - buffered saline ( ph 7.0 ) and examined under the microscope at a magnification of 1,000x for the presence of malaria parasites . dna was extracted from 200 l of edta blood using qiaamp dna blood mini kit ( qiagen , germany ) according to given protocol . the extracted dna was stored at -20 c. the purified dna templates were used for amplification of 18s rrna gene using primers ( table 1 ) , as described by ( 16 ) , in nested pcr . all the oligonucleotides were prepared from cemb ( center for excellence in molecular biology ) , lahore . the pcr master mix for 50 l reaction was prepared by mixing 5.0 l of 10x pcr buffer ( 500 mm kcl , 100 mm tris - hcl [ ph 8.8 at 25c ] ) ( fermentas , eu ) , 4.0 l of 10 m deoxyribonucleoside triphosphate ( dntps ) ( fermentas , eu ) , 5.0 l of 2.5 mm mgcl2 ( fermentas , eu ) , 1.5 l of each primer ( 10 m ) , 0.5 l taq dna polymerase ( 1 u/l ; fermentas , eu ) and 22.5 l nuclease - free water . fifty l reactions using 40 l of master mix and 10 l of dna template were performed . the reaction conditions used for pcr1 were : hold at 95 c ( 10 min ) ; 35 cycles of : denaturation at 94 c ( 1 min ) , annealing at 60 c ( 2 min ) , and extension at 72 c ( 2 min ) ; hold for final extension at 72 c ( 10 min ) and hold for indefinite period at 4 c . the sensitivity , specificity , and efficacy of nested pcr were calculated by using these formulas , respectively : [ true positives / ( true positives + false negatives ) 100% ] ; [ true negatives / ( true negatives + false positives ) 100% ] ; and [ 1 ( false negatives + false positives / total ) 100 ] . whole blood ( 5 ml ) was drawn by sterilized syringes and collected in edta vacutainer tubes . negative controls blood was obtained from students of department of zoology , university of the punjab , lahore . the blood smears were stained with 1% giemsa stain in phosphate - buffered saline ( ph 7.0 ) and examined under the microscope at a magnification of 1,000x for the presence of malaria parasites . dna was extracted from 200 l of edta blood using qiaamp dna blood mini kit ( qiagen , germany ) according to given protocol . the purified dna templates were used for amplification of 18s rrna gene using primers ( table 1 ) , as described by ( 16 ) , in nested pcr . all the oligonucleotides were prepared from cemb ( center for excellence in molecular biology ) , lahore . the pcr master mix for 50 l reaction was prepared by mixing 5.0 l of 10x pcr buffer ( 500 mm kcl , 100 mm tris - hcl [ ph 8.8 at 25c ] ) ( fermentas , eu ) , 4.0 l of 10 m deoxyribonucleoside triphosphate ( dntps ) ( fermentas , eu ) , 5.0 l of 2.5 mm mgcl2 ( fermentas , eu ) , 1.5 l of each primer ( 10 m ) , 0.5 l taq dna polymerase ( 1 u/l ; fermentas , eu ) and 22.5 l nuclease - free water . fifty l reactions using 40 l of master mix and 10 l of dna template were performed . the reaction conditions used for pcr1 were : hold at 95 c ( 10 min ) ; 35 cycles of : denaturation at 94 c ( 1 min ) , annealing at 60 c ( 2 min ) , and extension at 72 c ( 2 min ) ; hold for final extension at 72 c ( 10 min ) and hold for indefinite period at 4 c . the sensitivity , specificity , and efficacy of nested pcr were calculated by using these formulas , respectively : [ true positives / ( true positives + false negatives ) 100% ] ; [ true negatives / ( true negatives + false positives ) 100% ] ; and [ 1 ( false negatives + false positives / total ) 100 ] . microscopy is a conventional method for detection of malaria , but pcr has been developed for the rapid and correct diagnosis of malaria . from total of 100 clinically positive samples , 60 patients were negative and 40 patients were positive for malaria by microscopy ; whereas by nested pcr , 41 specimens were positive and 59 specimens were negative for plasmodium spp . the comparison of malaria epidemiology in south punjab observed by clinical symptoms , microscopy , and nested pcr in present study was presented in table 2 . according to table 3 , one specimen was found to be infected by p.vivax by microscopy but it was confirmed to be negative for plasmodium spp . by nested pcr . 1 mixed infection ( p. falciparum and p. vivax ) was diagnosed by microscopy ; rather nested pcr determined mixed infections ( p. falciparum and p. vivax ) in 10 specimens . plasmodium was detected in 41% samples by nested pcr as compared to 40% by microscopy . species identification by nested pcr was done for all plasmodium positive samples ( 41% ) . out of which 15% were having p. falciparum infection , 16 were having p. vivax infection , and 10 were mixed infections ( p. falciparum and p. vivax ) . figure 1 is showing nested pcr result of three samples with p. falciparum , p. vivax and mixed infections . incorrect speciation of p. falciparum and p. vivax was resolved by nested pcr in 8 samples ; p. falciparum was identified in 4 clinically and microscopically positive specimens that showed p. vivax infection by nested pcr ; and 4 p. vivax positive specimens were shown to be p. falciparum infection by nested pcr . all concordant results for parasite identification were resolved by nested pcr ; nested pcr was able to determine plasmodium dna in 2 specimens that were depicted to be negative for malaria by microscopy . the sensitivity , specificity , and efficacy of nested pcr were calculated to be 95% , 98% , and 97% , respectively . the specificity and sensitivity of nested pcr , calculated from present study was better than that of microscopy . malaria is a life - threatening infection impacting most of the developed countries of the world . the who recommended method and the gold standard for routine laboratory diagnosis of malaria is microscopy , despite its decreased sensitivity and specificity in situations of low parasite density and mixed infections . compared to microscopy , molecular methods ( pcr ) has achieved much higher detection sensitivities and specificities , especially in cases of low parasitaemia or mixed infections and differential diagnosis of plasmodium species ( 17 , 21 - 23 ) . they are more important due to the automation of the process and have objective of reading the results by machines . this makes them a valuable option for large - scale epidemiological studies ( 24 ) . especially , nested pcr has proven to be a sensitive method for diagnosis of all species of plasmodium and has expected to exceed the sensitivity of microscopic examination ( 25 ) . moreover , nested pcr has appeared to be effective in correcting wrong diagnosis , identified as plasmodium species by microscopist . this was obvious in the present study with the misdiagnosed plasmodium negative specimens ( 1% ) . the non concordant smear positive / pcr negative cases can also be attributed to either degradation of parasite dna or low parasitemia combined with degradation of parasite dna ( 26 ) . in certain cases , parasite morphology is damaged due to exposition to prophylactic medication or auto - medication , making malaria diagnosis by microscopy difficult ( 27 ) , which may lead to the death of the patient by improper medication . it was found that 8 specimens were microscopically misdiagnosed as p. falciparum or p. vivax infection and they were diagnosed correctly by pcr . it is suggested that it can be due to chemoprophylactic effect on the shapes of the parasites ( 22 ) . in 2% of samples , the parasite could not be determined by microscopy ; by pcr , the parasite was detected even in a very low quantity . it has been found that the pcr assay is usually effective in detecting malarial mixed infections than microscopy ( 16 , 21 , 23 ) but not in all situations ( 28 ) . infected samples were confirmed to be mixed infection ( p. falciparum and p. vivax ) by the pcr but only 2.4% was identified by microscopy . in case of mixed infections , it was suggested that one species has the ability to dominate over other species ; as a result , one may be overlooked in microscopic examination ( 29 ) . in present study , 6 samples , microscopically diagnosed p. vivax infection were determined as mixed infection ( p. falciparum and p. vivax ) by pcr and 3 samples microscopically detected p. falciparum , were depicted as mixed infection ( p. falciparum and p. vivax ) by pcr . it is clear from the present study that p. vivax have higher tendency to dominate over p. falciparum . detection of mixed infection may be of clinical importance because interactions between different species simultaneously infecting the same individual could result in significant changes in the course of the infection and disease . it may also be helpful in the effective treatment of malaria because the treatment of malaria depends on the correct diagnosis of the species ( 15 ) . as p. falciparum and p. vivax have developed resistance against specific drugs and there are many drugs which are effective for p. vivax but not for p. falciparum . for example , mefloquine is an effective drug for treatment of p. vivax malaria but not effective on p. falciparum malaria . pcr detected a high number of mixed infections in the samples analyzed , but its routine clinical use for diagnosing malaria is still under consideration because of its high cost and resource requirements ( 18 , 21 ) . the high prevalence of p. vivax ( 39% ) may lead to serious complications like cerebral malaria but the comparatively less prevalent ( 36.6% ) p. falcip - arum also poses a significant health hazard . compared to microscopy , the nested - pcr is a rapid , sensitive , and specific method for the detection of malaria . the primary goal of the present study was to assess the value of a pcr - based method for the routine diagnosis of malaria at species level and study was conducted to evaluate the epidemiology of malaria in south punjab . although the number of samples used here are small , but the high degree of both sensitivity and specificity is encouraging . larger studies of both p. falciparum and p. vivax malaria in endemic regions will enhance the generalizability of the present findings .

backgroundmalaria is well known for its fatalities worldwide , plasmodium vivax and the plasmodium falciparum are the two important species of malaria reported from pakistan and creating lots of morbidities across the country.methodstudy was conducted to determine the surveillance of malaria in south punjab by microscopy and polymerase chain reaction ( pcr).resultsamples out of 100 patients were found positive for malarial parasites . one patient was found with mixed infection , whereas p. falciparum and p. vivax infections were detected in 17 and 22 patients , respectively . in nested pcr , genus - specific primers for plasmodium species . in round 1 and species - specific primers for p. falciparum and p. vivax in round 2 were used . by the application of pcr 41% were found to be infected by plasmodium spp . among plasmodium positive patients : mixed , p. falciparum and p. vivax infection were detected in 10 , 15 and 16 patients , respectively . thirty nine microscopically positive patients confirmed to have plasmodium spp . one negative by pcr , 2 microscopically negative patients had shown plasmodium spp . infection ( p. falciparum and p. vivax ) by pcr . in total samples , p. falciparum , p. vivax and mixed infection accounted for 36.6% , 39.0% and 24.3% , respectively.conclusionmicroscopy was found deficient for interpretation of mixed infections , low parasitaemia , and species specific diagnosis . the sensitivity , specificity and efficacy of nested pcr was calculated 95% , 98% and 97% , respectively , showing pcr as a more effective and efficient diagnostic tool for malaria .