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this study was approved by the institutional review board (irb) of chonbuk national university hospital. informed consent was obtained from all patients who underwent surgery according to the helsinki declaration. to examine the location and expression of four suggested csc markers, we collected 91 surgical specimens of formalin - fixed, paraffin - embedded hccs resected in the department of pathology, chonbuk national university hospital, between january 2011 and december 2013. among these patients, nine underwent transarterial chemoembolization, which resulted in near total necrosis of the tissue. the remaining 82 patients were analyzed in our study. in each case, clinicopathologic findings, including age, gender, etiology, background liver disease, ascites, serological data including serum albumin level and -fetoprotein (afp) level, microvessel invasion, intrahepatic metastasis, histologic grade, and follow - up data, were obtained from hospital records. tumors were staged according to the criteria of the 2010 american joint committee on cancer (ajcc) tnm classification. follow - up period was defined as the period from the date of initial surgery to the date of either last follow - up or death. for immunohistochemical (ihc) staining, 10% formalin - fixed, paraffin - embedded tissue sections of representative areas of tumor were prepared into 4-m - thick tissue samples. ihc staining was performed using a fully automated ihc system with the bond polymer refine detection kit (leica bond, newcastle upon tyne, uk) according to the manufacturer s instructions. the sources of antibodies used in this study and conditions of the procedure are listed in table 1. the immunoreactivity of the specimens was interpreted according to the intensity of staining and proportion of positive cells. the intensity of cytoplasmic and membranous staining was graded into four levels : no immunostaining (0), weak (1), moderate (2), and intense (3). the proportion of positive cells was scored as follows : 0 (none), 1 (1%), 2 (2%10%), 3 (11%33%), 4 (34%66%), and 5 (67%100%). the sum index was obtained by totaling the scores of intensity and proportion of staining. if the final score was equal to or greater than 4, the immunoreactivity was considered positive ; otherwise, the immunoreactivity was considered negative. for negative controls, sections were treated in the same way, except that they were incubated with tris - buffered saline instead of primary antibodies. associations between the clinicopathological factors and expression of antibodies were tested using a chi - square test. univariate and multivariate cox proportional regression analyses for overall survival and relapse - free survival were performed. this study was approved by the institutional review board (irb) of chonbuk national university hospital. informed consent was obtained from all patients who underwent surgery according to the helsinki declaration. to examine the location and expression of four suggested csc markers, we collected 91 surgical specimens of formalin - fixed, paraffin - embedded hccs resected in the department of pathology, chonbuk national university hospital, between january 2011 and december 2013. among these patients, nine underwent transarterial chemoembolization, which resulted in near total necrosis of the tissue. the remaining 82 patients were analyzed in our study. in each case, clinicopathologic findings, including age, gender, etiology, background liver disease, ascites, serological data including serum albumin level and -fetoprotein (afp) level, microvessel invasion, intrahepatic metastasis, histologic grade, and follow - up data, were obtained from hospital records. tumors were staged according to the criteria of the 2010 american joint committee on cancer (ajcc) tnm classification. follow - up period was defined as the period from the date of initial surgery to the date of either last follow - up or death. for immunohistochemical (ihc) staining, 10% formalin - fixed, paraffin - embedded tissue sections of representative areas of tumor were prepared into 4-m - thick tissue samples. ihc staining was performed using a fully automated ihc system with the bond polymer refine detection kit (leica bond, newcastle upon tyne, uk) according to the manufacturer s instructions. the sources of antibodies used in this study and conditions of the procedure are listed in table 1. the immunoreactivity of the specimens was interpreted according to the intensity of staining and proportion of positive cells. the intensity of cytoplasmic and membranous staining was graded into four levels : no immunostaining (0), weak (1), moderate (2), and intense (3). the proportion of positive cells was scored as follows : 0 (none), 1 (1%), 2 (2%10%), 3 (11%33%), 4 (34%66%), and 5 (67%100%). the sum index was obtained by totaling the scores of intensity and proportion of staining. if the final score was equal to or greater than 4, the immunoreactivity was considered positive ; otherwise, the immunoreactivity was considered negative. for negative controls, sections were treated in the same way, except that they were incubated with tris - buffered saline instead of primary antibodies. associations between the clinicopathological factors and expression of antibodies were tested using a chi - square test. univariate and multivariate cox proportional regression analyses for overall survival and relapse - free survival were performed. a p - value of <.05 was considered statistically significant. the 82 patients included 72 males and 10 females with a mean age of 57 years (range, 26 to 77 years). hcc formation was attributed to the presence of hepatitis b virus (hbv) surface antigen in 64 patients, alcohol - related complication in seven patients, presence of anti hepatitis c virus antibody in four patients, and unknown etiology in seven patients. the surrounding liver tissue showed cirrhosis in 56% of the patients (46/82) and chronic hepatitis with varying degrees of fibrosis in the remaining 44% of the patients (36/82). in cirrhotic livers, all four examined markers showed intense immunoreactivity in proliferating reactive bile ductules. these ductules are thought to originate from hepatic progenitor cells and thus were considered as an internal positive control. 1). in 82 hcc samples, 46 were epcam - positive (56%) and five were k19-positive (6%). of the 82 hccs, only two cases showed cd133 and cd56 expression, respectively. analysis of the relationships between expression of k19, cd133, and cd56 and clinicopathologic features was not meaningful, since the number of cases with k19, cd133, and cd56 expression was very small. however, epcam expression significantly correlated with higher histologic grade (p=.006), elevated afp level (p=.001), and microvessel invasion of the tumor cells (p=.030) (table 2). additionally, epcam expression was strongly associated with k19 expression (p=.041). in 82 patients with hcc, follow - up intervals ranged from 0.1 to 69 months (mean of 23 months). the mean overall survival time of patients with epcam - expressing hcc was 57.23.7 months, while that of epcam - negative hcc was 62.02.9 months. the mean relapse - free survival time of patients with epcam - expressing hcc was 40.55.9 months, while that of patients with epcam - negative hcc was 53.85.0 months. however, these results were not statistically significant. in univariate cox regression analysis, t category significantly correlated with poor patient overall survival (p=.003). additionally, t category, preoperative serum afp level, and vascular invasion were associated with decreased relapse - free survival (p=.019, p=.048, and p=.011, respectively). multivariate analysis revealed that t category and vascular invasion were independent indicators of relapse (p=.011 and p=.007, respectively). the 82 patients included 72 males and 10 females with a mean age of 57 years (range, 26 to 77 years). hcc formation was attributed to the presence of hepatitis b virus (hbv) surface antigen in 64 patients, alcohol - related complication in seven patients, presence of anti hepatitis c virus antibody in four patients, and unknown etiology in seven patients. the surrounding liver tissue showed cirrhosis in 56% of the patients (46/82) and chronic hepatitis with varying degrees of fibrosis in the remaining 44% of the patients (36/82). in cirrhotic livers, all four examined markers showed intense immunoreactivity in proliferating reactive bile ductules. these ductules are thought to originate from hepatic progenitor cells and thus were considered as an internal positive control. 1). in 82 hcc samples, 46 were epcam - positive (56%) and five were k19-positive (6%). of the 82 hccs, only two cases showed cd133 and cd56 expression, respectively. analysis of the relationships between expression of k19, cd133, and cd56 and clinicopathologic features was not meaningful, since the number of cases with k19, cd133, and cd56 expression was very small. however, epcam expression significantly correlated with higher histologic grade (p=.006), elevated afp level (p=.001), and microvessel invasion of the tumor cells (p=.030) (table 2). in 82 patients with hcc, follow - up intervals ranged from 0.1 to 69 months (mean of 23 months). the mean overall survival time of patients with epcam - expressing hcc was 57.23.7 months, while that of epcam - negative hcc was 62.02.9 months. the mean relapse - free survival time of patients with epcam - expressing hcc was 40.55.9 months, while that of patients with epcam - negative hcc was 53.85.0 months. cox regression analysis, t category significantly correlated with poor patient overall survival (p=.003). additionally, t category, preoperative serum afp level, and vascular invasion were associated with decreased relapse - free survival (p=.019, p=.048, and p=.011, respectively). multivariate analysis revealed that t category and vascular invasion were independent indicators of relapse (p=.011 and p=.007, respectively). carcinogenesis is currently explained using two models, one involving a traditional stochastic onset and a second based on the role of cscs. according to the csc model, only a small population of tumor cells has the ability to divide and repopulate within the tumor. cscs are responsible for tumor initiation, maintenance, growth, metastasis, and relapse after therapy [3 - 6 ]. the expression of several proposed csc markers in hcc has been reported and might prove to be useful for predicting the prognosis of hcc patients [11,14 - 16 ]. however, little is known about the relationships between the ihc expression of csc markers in paraffin - embedded tissue sections and clinicopathologic factors or clinical outcomes of hcc. epcam is a transmembrane intercellular protein that was initially posed as a homophilic cell adhesion molecule and is highly up - regulated in most human epithelial cancers, including hcc [14 - 16,18 ]. epcam has been validated as a marker of stem cells in the liver, and epcam - positive hcc is likely to have originated from hepatic stem cells. in the present study, we observed that epcam expression significantly correlated with tumor progression factors of hcc, such as elevated afp level, vascular invasion, and high tumor grade. these findings are in agreement with previous studies showing that expression level of epcam correlates with de - differentiation and vascular invasion and is associated with the high afp level in hcc. the prognosis of patients with epcam - negative hcc is considered to be better than that of those with epcam - positive hcc [11,14 - 16,20,21 ]. although epcam expression was not found to be an independent predictor of survival in patients with hcc in this study, epcam expression was found to be associated with well - known unfavorable prognostic factors in hcc. thus, the use of epcam expression as an unfavorable prognostic factor of hcc is reasonable. furthermore, gene expression profile study has demonstrated that epcam - positive and afp - positive hccs have more aggressive behavior and poor clinical outcome, whereas epcam - negative and afp - negative hccs have good prognosis. based on these observations, epcam has attracted considerable attention as a possible therapeutic target for patients with hcc. taken together, our findings suggest that epcam is a critical player in facilitating vascular invasion of tumor cells, leading to de - differentiation in hcc with high serum afp level, and might be useful for predicting the prognosis of hcc patients. in this study, a proportion of hccs (56%) expressed epcam, and epcam expression was associated with k19 expression. these findings are in agreement with previous reports showing that the expression of epcam and k19 was positively correlated. the proportion of tissues expressing epcam in this study was a little higher than in earlier series, which reported epcam in 15.9%48.7% of hccs [15 - 17,20 - 23 ]. kimura. have demonstrated that epcam expression was observed more often in hcc patients with hbv than in those with other etiologies, and the immunoreactivity of epcam has been found in up to 78% of hcc patients with hbv. the high proportion of hcc patients with hbv (74%) examined in this study might be a possible explanation for the higher epcam - positive rate. it has been hypothesized that the transformation of hepatic stem / progenitor cells (maturation arrest theory) underlies the occurrence of hcc expressing csc markers. the expression of csc markers in hcc can develop as an acquisition of progenitor cell features during the de - differentiation of cancer cells (de - differentiation theory). kim and park have proposed that hcc expressing csc markers should be designated as hcc with stemness - related marker expression in order to avoid the implication that these hccs originated from hepatic stem / progenitor cells. only two of 82 hcc specimens showed positive staining for cd133 and cd56, suggesting that cd133 and cd56 expression is rare in paraffin - embedded hcc tissues. it has been reported that 0%88% of human hcc tissue samples express cd133, and the expression of which is negatively correlated with presence of hbv. the reason for this high discrepancy of positive rate of cd133 and cd56 in hcc is unclear. in our study, we employed the bond polymer ihc staining method to avoid endogenous biotin contamination, and all four examined markers always showed strong immunoreactivity in reactive bile ductules, which were considered as internal positive controls. the discrepancy in the expression rates of cd133 and cd56 is not easily explained but might be related to the different criteria for positivity, the quality of tissue samples analyzed, and the unique antibodies and immunostaining methods used in different studies. our negative results in analysis of cd133 and cd56 through the use of specific antibodies in an established automated ihc system does not exclude the presence of these markers and can be linked to the different etiologies according to geographic background. additional investigations with a larger population of hcc and strict criteria for positive immunoreactivity are necessary to determine the expression and clinical implication of these markers. in conclusion, we showed that the epcam expression in paraffin - embedded tissue sections of hcc is associated with tumor progression factors and might be useful for predicting the prognosis of hcc patients. the high proportion of epcam expression in hccs and its associations with unfavorable prognostic factors of hcc provide a basis for further investigation of anti - epcam targeted therapy.	background : increasing evidence has shown that tumor initiation and growth are nourished by a small subpopulation of cancer stem cells (cscs) within the tumor mass. cscs are posited to be responsible for tumor maintenance, growth, distant metastasis, and relapse after curative operation. we examined the expression of csc markers in paraffin - embedded tissue sections of hepatocellular carcinoma (hcc) and correlated the results with clinicopathologic characteristics.methods:immunohistochemical staining for the markers believed to be expressed in the cscs, including epithelial cell adhesion molecule (epcam), keratin 19 (k19), cd133, and cd56, was performed in 82 hcc specimens.results:epcam expression was observed in 56% of the hccs (46/82) and k19 in 6% (5/82). epcam expression in hcc significantly correlated with elevated -fetoprotein level, microvessel invasion of tumor cells, and high histologic grade. in addition, epcam expression significantly correlated with k19 expression. the overall survival and relapsefree survival rates in patients with epcam - expressing hcc were relatively lower than those in patients with epcam - negative hcc. all but two of the 82 hccs were negative for cd133 and cd56, respectively.conclusions:our results suggest that hccs expressing epcam are associated with unfavorable prognostic factors and have a more aggressive clinical course than those not expressing epcam. further, the expression of either cd133 or cd56 in paraffin - embedded hcc tissues appears to be rare.
respiration is a portal of entry for not only atmospheric gases, but also for harmful particulate pervasive in the environment. the pulmonary epithelium is therefore continually exposed to microorganisms, but remains sterile under normal physiologic conditions. this remarkable phenomenon is a testament to the innate immune defenses that provide a silent mode of broad immune protection. the importance of the innate immune system in protecting the lungs from infection is clearly illustrated in the pathologic condition that arises in cystic fibrosis (cf) (mucoviscidosis), which severely damages the pulmonary innate immune defenses. cystic fibrosis is the most common lethal genetic disorder affecting the caucasian population, with an incidence of 1 in 2,500 births. cf is caused by an autosomal recessive mutation in the cystic fibrosis transmembrane conductance regulator (cftr) gene within chromosome seven. this mutation results in the functional defect in the cyclic adenosine monophosphate stimulated pulmonary chloride pump causing abnormal ion transport in epithelial cells [4, 5 ]. cf is therefore a disease of ion transport across the epithelium, affecting fluid secretion in exocrine glands and the epithelium of the respiratory, reproductive, and gastrointestinal tracts. although cf causes a multitude of pathophysiologic effects, the most significant effect is the impaired ciliary clearance that results in the accumulation of mucus in the lung, creating a haven for bacteria. moreover, the dehydrating conditions in the lung caused by the elevated levels of sodium chloride in the airway secretions severely weaken the host pulmonary innate defenses. the initial acute pulmonary infection of the cf lung is typically a result of colonization by haemophilus influenzae and staphylococcus aureus, while the ensuing chronic infection is caused by pseudomonas aeruginosa [7, 8 ]. the chronic infection in the lungs of cf patients caused by p. aeruginosa is responsible for the high rate of morbidity and mortality associated with this genetic disease. pseudomonas aeruginosa is a ubiquitous, antibiotic resistant, gram - negative opportunistic bacterium. at 6.3 million base pairs, this large genome provides the genetic machinery that enables p. aeruginosa to readily undergo significant genetic and phenotypic transformations in response to environmental changes, contributing to its versatility and antibiotic resistance potential. although p. aeruginosa is pervasive in the environment, it only causes infection in immunodeficient hosts, e.g., cf patients, patients with acquired immunodeficiency syndrome, burn victims, etc. among the many clinical manifestations of p. aeruginosa infection, p. aeruginosa s opportunistic mode of infection is most known in the chronic pulmonary infection that is the hallmark of cf. once acquired, p. aeruginosa almost always colonizes the lungs of cf patients for life. the innate immune system provides the first line of defense against microorganisms pervasive in the environment. unlike the adaptive immune system, innate immunity is non - specific, lacks memory, and is not influenced by previous exposure. antimicrobial peptides (amps) are cationic endogenous antibiotic proteins expressed throughout the epithelium that are effectors of the innate immune system. amps exert antimicrobial activity in a concentration - dependent manner, making their expression a critical factor in host defense. the amphiphathic nature of amps contributes to their effectiveness at interacting with hydrophobic and anionic components of the bacterial membrane. cathelicidins, -defensins, -defensins, and -defensins are among the major classes of human amps. beta - defensins are at the interface between the adaptive and innate immune systems ; beta - defensins exhibit chemotactic function towards immature dendritic cells, memory t cells expressing the chemokine receptor ccr6, neutrophils primed with tumor necrosis factor (tnf)-, and mast cells [17, 18 ]. individual beta - defensins have specific antimicrobial activity. among the various types of defensin amps, only the expression of human beta - defensin-2 (hbd-2) and human beta - defensin-3 (hbd-3) is increased following stimulation by pro - inflammatory cytokines ; all other defensin amps are continuously expressed. however, although the expression of hbd-2 and hbd-3 can be stimulated by pro - inflammatory cytokines, e.g., tnf-, interleukin (il)-1, il-17, and il-22, these antimicrobial peptides are still expressed in unstimulated cells in basal amounts [20, 21 ]. an additional difference between these two amps that are induced by humoral stimulation is that hbd-2 primarily targets gram - negative bacteria, such as p. aeruginosa, while hbd-3 exerts broad bacteriostatic activity against both gram - positive and gram - negative bacteria. however, hbd-2 is most concentrated in the epithelia of the lung, tonsils, and trachea, and therefore plays a critical role in the prevention of pulmonary infection [23, 24 ]. the inducible properties of hbd-2 suggest it plays a significant role in innate immune defense. human beta - defensin-2 is a cationic, 41 amino acid, 4 kda, amp intricately involved in the innate immune response of vertebrates that works synergistically with other antimicrobial molecules, such as lactoferrin and lysozyme [24, 25 ]. like other beta - defensins, hbd-2 is a monomeric protein containing six conserved cysteine residues forming three core disulfide bonds. the initial contact between hbd-2 and invading microorganisms is an electrostatic amphipathic attraction between the cationic amp and the negatively charged phospholipid groups of the bacterium s phospholipid bilayer [27, 28 ]. following initial electrostatic attraction, hbd-2 exerts its antimicrobial effects through insertion within the phospholipid bilayer disrupting the membrane integrity of the invading bacteria resulting in the collapse of membrane potential and death of the invading pathogen. nuclear magnetic resonance (nmr) analysis of the crystal structures of hbd-2 suggests that the formation of a hbd-2 octamer is a prerequisite to the binding of the bacteria cell surface and subsequent increases in membrane permeability. a common theme in pathogen host interactions is the selection against virulence factors required for the establishment of infection, as the stage the infection shifts from acute to chronic. genetic variants are selected that promote long - term survivability and clonal expansion, while variants that no longer provide a survival advantage are selected against. in the cf lung, p. aeruginosa undergoes significant genetic and phenotypic transformations in response to changes in the pulmonary milieu. aeruginosa mutates to a mucoid, flagella - deficient phenotype over the course of chronic pulmonary infection [31, 32 ]. the changes in the expression of p. aeruginosa virulence factors affect the expression of hbd-2 in the pulmonary epithelium that weakens the innate immune defense of the lung. flagellum is a structure common to most gram - negative bacteria derived from flagellin monomers that confers motility, promotes adhesion, and consequently is a significant bacterial virulence factor. flagellum is a bacterial ligand that is detected by toll - like receptor (tlr) 5. the activation of tlr5 by flagellum initiates an inflammatory response that includes the up - regulation of hbd-2 via a nuclear factor (nf)-b dependent pathway in airway epithelial cells. the loss of flagella expression during the transition to the mucoid phenotype allows p. aeruginosa to evade the antimicrobial activity of hbd-2 through decreased tlr5 stimulation, contributing to p.aeruginosas pathogenesis in the cf lung [21, 35 ]. although p. aeruginosa isolates from the chronic stage of pulmonary infection are flagella - deficient, other virulence factors, which are tlr agonists and stimulate hbd-2 expression, remain expressed. for example, lipopolysaccharide (lps) is an endotoxin attached to the outer membrane of gram - negative bacteria that is an agonist of tlr 4. although lps expression does not decrease as pulmonary infection shifts from the acute to chronic stage, the cellular responsiveness to lps decreases. a study involving the exposure of airway epithelial cells to a regime of two discrete bacterial infections demonstrated reduced tlr responsiveness in the second bacterial challenge due to down - regulation of the irak1 signaling protein, which is involved in nf-b activation. irak1 phosphorylation leads to the activation of nf-b and ap-1, which are two transcription factors that induce the up - regulation of il-8 and hbd-2 in airway epithelial cells. although this in vitro model only measured the production of il-8, not hbd-2, these results provide a mechanistic explanation for the reduced levels of hbd-2 expression in the chronic stage of pulmonary infection in cf patients. furthermore, the reduced expression of hbd-2 in the lung in advanced chronic pulmonary infection (owing to decreased tlr responsiveness) provides further insight as to why p. aeruginosa only colonizes the lung post-s.aureus and h. influenzae infection. moreover, this underscores the potential influence of hbd-2 in the progression of chronic pulmonary infection in cf patients. the down - regulation of tlr4 expression in the airway epithelia in response to acute infection may result in reduced hbd-2 expression, promoting p. aeruginosa colonization. inflammation is a protective tissue response to infection or injury. in the context of the cf lung, exposure of the airway epithelium to p. aeruginosa induces the expression of the potent neutrophil chemokine il-8, initiating neutrophil infiltration. however, in the cf lung the abnormal accumulation and persistence of neutrophils produces an inflammatory response that severely damages the lung [43, 44 ]. the quorum - sensing controlled production of rhamnolipid by p. aeruginosa induces rapid necrotic killing of invading neutrophils, which explains why the neutrophils do not significantly contribute to the elimination of p. aeruginosa in the cf lung [4547 ]. in the cf lung, infiltrating neutrophils and most p. aeruginosa strains secrete elastase a serine protease that exerts diverse biological effects that contribute significantly to the progression of pulmonary cf disease [48, 49 ]. elastase is a potent protease that exerts antimicrobial activity against most gram - negative bacteria, but not against p. aeruginosa. the viability and morphology of p. aeruginosa remains unaltered even when exposed to neutrophil elastase (ne) concentrations as high as 25 m, which is commonly present in the cf lung. after a short life span cathepsins are cysteine proteases secreted by macrophages that are involved in the remodeling of the extracellular matrix. pulmonary macrophage influx occurs in response to the elevated levels of apoptotic neutrophils in the lungs of cf patients resulting in cathepsin secretion into the bronchoalveolar fluid (baf) of the cf lung [51, 53 ]. beta - defensins have a conserved core structure of three disulfide bridges, which are susceptible to proteolytic cleavage by cathepsins present in the baf. specifically, cathepsins b, l, and s have been found to cleave the disulfide bonds of hbd-2 and hbd-3 resulting in their degradation and loss of antimicrobial activity. in addition to the high concentrations of cathepsins in the baf of the cf lung, the low ph of the cf baf promotes optimal enzymatic activity for cathepsin proteolytic activity ; most cathepsins have optimal proteolytic function in acidic ph and lose their proteolytic properties at physiologic ph. the baf of cf patients is acidic because of impaired bicarbonate transport across the pulmonary epithelium caused by the cftr mutation. furthermore, the elevated [cl ] present in the baf resulting from the functional cftr defect reduces the efficacy of hbd-2 due to the reduced electrostatic interaction between the cationic hbd-2 peptide and the anionic resting membrane potential of invading microorganisms. the overexpression of cathepsins during chronic pulmonary infection may cause increased degradation of hbd-2, promoting bacterial colonization and infection. the innate immune system provides the first line of defense against microorganisms pervasive in the environment. unlike the adaptive immune system, innate immunity is non - specific, lacks memory, and is not influenced by previous exposure. antimicrobial peptides (amps) are cationic endogenous antibiotic proteins expressed throughout the epithelium that are effectors of the innate immune system. amps exert antimicrobial activity in a concentration - dependent manner, making their expression a critical factor in host defense. the amphiphathic nature of amps contributes to their effectiveness at interacting with hydrophobic and anionic components of the bacterial membrane. cathelicidins, -defensins, -defensins, and -defensins are among the major classes of human amps. beta - defensins are at the interface between the adaptive and innate immune systems ; beta - defensins exhibit chemotactic function towards immature dendritic cells, memory t cells expressing the chemokine receptor ccr6, neutrophils primed with tumor necrosis factor (tnf)-, and mast cells [17, 18 ]. individual beta - defensins have specific antimicrobial activity. among the various types of defensin amps, only the expression of human beta - defensin-2 (hbd-2) and human beta - defensin-3 (hbd-3) is increased following stimulation by pro - inflammatory cytokines ; all other defensin amps are continuously expressed. however, although the expression of hbd-2 and hbd-3 can be stimulated by pro - inflammatory cytokines, e.g., tnf-, interleukin (il)-1, il-17, and il-22, these antimicrobial peptides are still expressed in unstimulated cells in basal amounts [20, 21 ]. an additional difference between these two amps that are induced by humoral stimulation is that hbd-2 primarily targets gram - negative bacteria, such as p. aeruginosa, while hbd-3 exerts broad bacteriostatic activity against both gram - positive and gram - negative bacteria. however, hbd-2 is most concentrated in the epithelia of the lung, tonsils, and trachea, and therefore plays a critical role in the prevention of pulmonary infection [23, 24 ]. the inducible properties of hbd-2 suggest it plays a significant role in innate immune defense. human beta - defensin-2 is a cationic, 41 amino acid, 4 kda, amp intricately involved in the innate immune response of vertebrates that works synergistically with other antimicrobial molecules, such as lactoferrin and lysozyme [24, 25 ]. like other beta - defensins, hbd-2 is a monomeric protein containing six conserved cysteine residues forming three core disulfide bonds. the initial contact between hbd-2 and invading microorganisms is an electrostatic amphipathic attraction between the cationic amp and the negatively charged phospholipid groups of the bacterium s phospholipid bilayer [27, 28 ]. following initial electrostatic attraction, hbd-2 exerts its antimicrobial effects through insertion within the phospholipid bilayer disrupting the membrane integrity of the invading bacteria resulting in the collapse of membrane potential and death of the invading pathogen. nuclear magnetic resonance (nmr) analysis of the crystal structures of hbd-2 suggests that the formation of a hbd-2 octamer is a prerequisite to the binding of the bacteria cell surface and subsequent increases in membrane permeability. host interactions is the selection against virulence factors required for the establishment of infection, as the stage the infection shifts from acute to chronic. genetic variants are selected that promote long - term survivability and clonal expansion, while variants that no longer provide a survival advantage are selected against. in the cf lung, p. aeruginosa undergoes significant genetic and phenotypic transformations in response to changes in the pulmonary milieu. p. aeruginosa mutates to a mucoid, flagella - deficient phenotype over the course of chronic pulmonary infection [31, 32 ]. the changes in the expression of p. aeruginosa virulence factors affect the expression of hbd-2 in the pulmonary epithelium that weakens the innate immune defense of the lung. flagellum is a structure common to most gram - negative bacteria derived from flagellin monomers that confers motility, promotes adhesion, and consequently is a significant bacterial virulence factor. flagellum is a bacterial ligand that is detected by toll - like receptor (tlr) 5. the activation of tlr5 by flagellum initiates an inflammatory response that includes the up - regulation of hbd-2 via a nuclear factor (nf)-b dependent pathway in airway epithelial cells. the loss of flagella expression during the transition to the mucoid phenotype allows p. aeruginosa to evade the antimicrobial activity of hbd-2 through decreased tlr5 stimulation, contributing to p.aeruginosas pathogenesis in the cf lung [21, 35 ]. although p. aeruginosa isolates from the chronic stage of pulmonary infection are flagella - deficient, other virulence factors, which are tlr agonists and stimulate hbd-2 expression, remain expressed. for example, lipopolysaccharide (lps) is an endotoxin attached to the outer membrane of gram - negative bacteria that is an agonist of tlr 4. although lps expression does not decrease as pulmonary infection shifts from the acute to chronic stage, the cellular responsiveness to lps decreases. a study involving the exposure of airway epithelial cells to a regime of two discrete bacterial infections demonstrated reduced tlr responsiveness in the second bacterial challenge due to down - regulation of the irak1 signaling protein, which is involved in nf-b activation. irak1 phosphorylation leads to the activation of nf-b and ap-1, which are two transcription factors that induce the up - regulation of il-8 and hbd-2 in airway epithelial cells. although this in vitro model only measured the production of il-8, not hbd-2, these results provide a mechanistic explanation for the reduced levels of hbd-2 expression in the chronic stage of pulmonary infection in cf patients. furthermore, the reduced expression of hbd-2 in the lung in advanced chronic pulmonary infection (owing to decreased tlr responsiveness) provides further insight as to why p. aeruginosa only colonizes the lung post-s.aureus and h. influenzae infection. moreover, this underscores the potential influence of hbd-2 in the progression of chronic pulmonary infection in cf patients. the down - regulation of tlr4 expression in the airway epithelia in response to acute infection may result in reduced hbd-2 expression, promoting p. aeruginosa colonization. inflammation is a protective tissue response to infection or injury. in the context of the cf lung, exposure of the airway epithelium to p. aeruginosa induces the expression of the potent neutrophil chemokine il-8, initiating neutrophil infiltration. however, in the cf lung the abnormal accumulation and persistence of neutrophils produces an inflammatory response that severely damages the lung [43, 44 ]. the quorum - sensing controlled production of rhamnolipid by p. aeruginosa induces rapid necrotic killing of invading neutrophils, which explains why the neutrophils do not significantly contribute to the elimination of p. aeruginosa in the cf lung [4547 ]. in the cf lung, infiltrating neutrophils and most p. aeruginosa strains secrete elastase a serine protease that exerts diverse biological effects that contribute significantly to the progression of pulmonary cf disease [48, 49 ]. elastase is a potent protease that exerts antimicrobial activity against most gram - negative bacteria, but not against p. aeruginosa. the viability and morphology of p. aeruginosa remains unaltered even when exposed to neutrophil elastase (ne) concentrations as high as 25 m, which is commonly present in the cf lung. after a short life span cathepsins are cysteine proteases secreted by macrophages that are involved in the remodeling of the extracellular matrix. pulmonary macrophage influx occurs in response to the elevated levels of apoptotic neutrophils in the lungs of cf patients resulting in cathepsin secretion into the bronchoalveolar fluid (baf) of the cf lung [51, 53 ]. beta - defensins have a conserved core structure of three disulfide bridges, which are susceptible to proteolytic cleavage by cathepsins present in the baf. specifically, cathepsins b, l, and s have been found to cleave the disulfide bonds of hbd-2 and hbd-3 resulting in their degradation and loss of antimicrobial activity. in addition to the high concentrations of cathepsins in the baf of the cf lung, the low ph of the cf baf promotes optimal enzymatic activity for cathepsin proteolytic activity ; most cathepsins have optimal proteolytic function in acidic ph and lose their proteolytic properties at physiologic ph. the baf of cf patients is acidic because of impaired bicarbonate transport across the pulmonary epithelium caused by the cftr mutation. furthermore, the elevated [cl ] present in the baf resulting from the functional cftr defect reduces the efficacy of hbd-2 due to the reduced electrostatic interaction between the cationic hbd-2 peptide and the anionic resting membrane potential of invading microorganisms. the overexpression of cathepsins during chronic pulmonary infection may cause increased degradation of hbd-2, promoting bacterial colonization and infection. many factors contribute to the pathogenesis of p. aeruginosa in the lungs of cf patients (fig. 1). it is becoming increasingly evident that the regulation of hbd-2 expression and degradation has profound implications in pulmonary infections. hbd-2 is an indicator of inflammation and an essential component of the innate immune system. the regulation of hbd-2 activity, expression, and prevention of degradation (cathepsin inhibitors) are potential therapeutic options for cf patients that may decrease the high rates of morbidity and mortality associated with this common genetic disease.fig. 1compromised hbd-2 function in the cf lung promotes chronic pulmonary infection by the opportunistic pathogen p. aeruginosa compromised hbd-2 function in the cf lung promotes chronic pulmonary infection by the opportunistic pathogen p. aeruginosa	cystic fibrosis (cf) is the most common genetic disease affecting the caucasian population. chronic pseudomonas aeruginosa pulmonary infection is the major cause of morbidity and mortality in cf patients. human beta - defensin-2 (hbd-2) is an inducible pulmonary antimicrobial peptide that exerts bacteriostatic activity in a concentration - dependent manner. the decreased expression and compromised function of hbd-2 contributes to the pathogenesis of p. aeruginosa infection in the cf lung. the purpose of this review is to outline the significance of hbd-2 in p. aeruginosa chronic pulmonary infection in cf patients.
disparity in the distribution of health resources is considered as a major health policy issue in both developed and developing countries (1). the availability of health care resources is one of the main criteria to measure the efficiency and equality in the health system (2). there are significant correlation between higher health resources such as beds, medical supplier and lower mortality rate and higher life expectancy (3). inequitable distribution of health care services is a major barrier for promotion health status and improving health system delivery in any country. inequality in the distribution of health resources is high in both developed and developing countries. however this inequality is both between countries and within countries (4, 5). the iranian health care system is characterised by a public - private mix of health care provision. the public sector provides a considerable part of delivery health services ; whereas the private sector accounts for only 10% of hospitals bed, 7.4% of health care centers and 27.5% of rehabilitation facilities (6). but it is important to note that there are a positive linkage between quantity of health resources and their distribution and health status of people. so investigate the status of distribution health human resources (number of physician, dentist, nurses, pharmacist and others staff) and health physical resources (number of pharmacy, hospital beds, laboratory, rehabilitation centers and etc.) the previous studies conducted in iran and others countries have focused on equality in distribution of health status, health care and health human resources (5, 712) ; while a few studies have focused on equality of distribution in health physical resources such as pharmacy, rehabilitation centers and laboratory (13, 14). in the past decade, the quantity of health resources such as pharmacy, health house and beds hospitals has increased substantially in the public health sector in iran. based on data from the statistical center of iran, the number of pharmacies has increased from 6022 in 2001 to 8644 in 2011 and the number of hospital beds has risen from 93762 in 2001 to 116987 in 2011 (15) in addition, how the resources are distributed is another important issue because the distribution of health care resources and their access is considered as one of the factors affecting on the health of people. there is a few information on the geographic distribution of physical resources in the health sector across provinces of iran and this issue that how they are distributed less investigated in the previous studies. also, at the time of this study, we could not find any study published in the literature related to the distribution of pharmacy, rehabilitation, laboratory and radiology centers in iran and the main focus of previous studies conducted in iran had been on hospital beds and health house (12, 16, 17). the aim of this study was to investigate the inequality in geographic distribution of physical resources in the public health sector across the provinces in iran in 2001 and 2011. the iran country consists of 31 provinces, based on the census 2011, with a population of around 75149669 of people, which is located in the eastern mediterranean region with an area of 1,648,000 km. similar to other studies (5, 13, 14, 1719), the inequality measures (the gini coefficient (gc), index of dissimilarity (i d) and gaswirth index of disparity (gid)) were used to examine the disparity in the distribution of health physical resources in iran. the gc is used commonly for measuring inequality in the distribution of health care resources. it is varies between 1 (perfect inequality) and 0 (perfect equality). as similar to the other studies (12, 20) in this study we used the following formula (21). g=1i=0k1(yi+1+yi)+(xi+1xi) where : g : gini indexyi : cumulative percentage of health variable in the ith provincexi : cumulative percentage of the population (ranked by variables) in the ith provincek : total number of provinces. cumulative percentage of health variable in the ith province cumulative percentage of the population (ranked by variables) in the ith province total number of provinces. the index of dissimilarity represents the percentage of total health variables which would need to be redistributed across provinces to achieve a situation of perfect equality (22)., the index of dissimilarity is calculated with following formula : id=12i=1n\|xipxih\| where : id : index of dissimilarityxip : ith province s population sharexih : ith province s health variable sharen : total number of provinces. index of dissimilarity ith province s population share ith province s health variable share total number of provinces. this index estimates how many new health variable should be added to the entire population to reach to the level received by referenced group (23). this index is calculated in two steps : first, the fraction of the entire population which is underserved relative to the reference groups is calculated as follow : u=i=1k1i(prefpi) where : i : ith province s population share;pref;:the maximum number of health variables in the province of reference group;pi : number of health variables in the province. ith province s population share ; the maximum number of health variables in the province of reference group ; number of health variables in the province. then, the gid is calculated by gid = up ; where p : the average of health variable in entire population (provinces of iran). in the study, each province was considered as a unit analysis and the 30 provinces included in the study (data for alborz province are not available and, then, exclude from the analysis). data on number of health house (hh), hospital s beds, pharmacy, laboratory, rehabilitation centers and radiology centers were obtained from the statistical centre of iran and ministry of health and medical education for years 2001 and 2011 (15). furthermore, in another sensitivity analysis, tehran province was excluded from the analysis to examine inequality in the distribution of physical health resources across the remaining provinces. the reason for this was that tehran has special situation as the capital of the country and the more resources are concentrated in tehran. it is similar to the study conducted by kiadaliri. on distribution of dentists in iran (20). the iran country consists of 31 provinces, based on the census 2011, with a population of around 75149669 of people, which is located in the eastern mediterranean region with an area of 1,648,000 km. similar to other studies (5, 13, 14, 1719), the inequality measures (the gini coefficient (gc), index of dissimilarity (i d) and gaswirth index of disparity (gid)) were used to examine the disparity in the distribution of health physical resources in iran. the gc is used commonly for measuring inequality in the distribution of health care resources. it is varies between 1 (perfect inequality) and 0 (perfect equality). as similar to the other studies (12, 20) in this study we used the following formula (21). g=1i=0k1(yi+1+yi)+(xi+1xi) where : g : gini indexyi : cumulative percentage of health variable in the ith provincexi : cumulative percentage of the population (ranked by variables) in the ith provincek : total number of provinces. cumulative percentage of health variable in the ith province cumulative percentage of the population (ranked by variables) in the ith province total number of provinces. the index of dissimilarity represents the percentage of total health variables which would need to be redistributed across provinces to achieve a situation of perfect equality (22)., the index of dissimilarity is calculated with following formula : id=12i=1n\|xipxih\| where : id : index of dissimilarityxip : ith province s population sharexih : ith province s health variable sharen : total number of provinces. index of dissimilarity ith province s population share ith province s health variable share total number of provinces. this index estimates how many new health variable should be added to the entire population to reach to the level received by referenced group (23). this index is calculated in two steps : first, the fraction of the entire population which is underserved relative to the reference groups is calculated as follow : u=i=1k1i(prefpi) where : i : ith province s population share;pref;:the maximum number of health variables in the province of reference group;pi : number of health variables in the province. ith province s population share ; the maximum number of health variables in the province of reference group ; number of health variables in the province. then, the gid is calculated by gid = up ; where p : the average of health variable in entire population (provinces of iran). in the study, each province was considered as a unit analysis and the 30 provinces included in the study (data for alborz province are not available and, then, exclude from the analysis). data on number of health house (hh), hospital s beds, pharmacy, laboratory, rehabilitation centers and radiology centers were obtained from the statistical centre of iran and ministry of health and medical education for years 2001 and 2011 (15). furthermore, in another sensitivity analysis, tehran province was excluded from the analysis to examine inequality in the distribution of physical health resources across the remaining provinces. the reason for this was that tehran has special situation as the capital of the country and the more resources are concentrated in tehran. it is similar to the study conducted by kiadaliri. on distribution of dentists in iran the average of beds has increased from 14.3 per 10,000 populations in 2001 to 16 in 2011 and the average of radiology centers has increased from 0.27 in 2001 to 0.36 in 2011. the average of physical resources per 10,000 populations in public health sector in iran for the years 2001 and 2011 is shown in table 1. average health physical resources per 10,000 populations in the iran in 2001 and 2011 the gc for beds was 0.158 in 2001 and 0.13 in 2011. also, the gc for radiology centers was 0.19 in 2001 and 0.16 in 2011. the study showed the mean gc for all health physical resources in 2001 with and without tehran was 0.178 and 0.158, respectively. also, the mean gc for all health physical resources in 2011 with and without tehran was 0.163 and 0.155, respectively. the gc of health resources across the provinces in iran 2001 and 2011 is shown in table 2. gini coefficient for physical resources in the health public sector in iran in 2001 and 2011 the i d and gid for physical resources across the provinces of iran are shown in tables 3 and 4, respectively. the highest and lowest value of i d in 2001 and 2011 were belonged to the rehabilitation centers (18%) and health houses (8%), respectively. the mean i d for all physical health resources in 2001(2011) with and without tehran was 13 % (13 %) and 12 % (11 %), respectively. index of dissimilarity for physical health resources across the provinces in iran in 2001 and 2011 b : sample excluding tehran ; x : the value of index of dissimilarity ; y : redistributed based on index of dissimilarity gaswirth index of disparity for physical health resources across the provinces in iran in 2001 and 2011 b : sample excluding tehran ; x : the value of gaswirth index of disparity ; y : increased based on gaswirth index of disparity. also, the number of beds and pharmacy which should be added to current beds and pharmacy in 2001 was 23440 and 7226 and in 2011 was 23397 and 7797, respectively. furthermore, the mean gid for all physical health resources in 2001(2011) with and without tehran was 1.5 (1.2) and 1.4 (0.98), respectively. inequality in the distribution of health care resources is one of the main barriers for access to healthcare and improving health status of population throughout the world. in addition, both the quantity of health resources and their distribution is affecting on overall level of health of a population. in this study, we evaluate inequality in the distribution of physical health resources in the public sector by three practical indexes of gini coefficient, index of dissimilarity and gaswirth index of disparity. the results showed although the number of health resources in 2001 had increased in comparison with 2011, but inequality in their distributions was increased. the numbers of laboratories (11%), rehabilitations (30%) and pharmacies (126%) in 2011 have increased in comparison with 2001, while inequality in the distribution of these resources had increased according to the gini coefficient although it is slightly. the gini coefficient for laboratories and pharmacies was 0.2 and 0.13 in 2001, while in 2011 it was 0.21 and 0.14 respectively. similar to our study, ameryoun (16) founded the gini coefficient for icu and post icu were 0.17 and 0.15. also, a study (14) found that the gc for beds of hospital was 0.2 and it was higher in compared to our findings. the main reason for this differences is that in our study only public sector data is used while in nishiura study, both beds in public and private sector were considered. the gini coefficient for radiology centers in our study was 0.19 in 2001 and 0.16 in 2011. da he. revealed that the gini coefficient for cts was 0.14 and 0.15 in 2006 and 2009 in china, respectively (24). the current study showed when excluding the tehran province from total sample, the value of gini coefficient was reduced and the equity in distribution of health resources was increased. this indicates that the physical health resources per 10,000 populations in tehran province is more than the others provinces. for example, the number of beds per 10,000 populations was 2 times more in tehran (with the highest per capita beds) compared to ilam (with the lowest per capita beds) which is consisted with others studies conducted in iran (16, 20). results of a study conducted by kiadaliri showed that the number of dentist per 100,000 populations in tehran (the highest density) was 11 time higher in compared to khorasan razavi (the lowest density) in 2009 (20). besides, based on the gc results, our finding indicated that the distribution of hh in 2011 has decreased in compared to 2001. based on the i d results, if government wants to increase equity in the distribution of physical health resources in the country, near 1 out of 10 beds, 0.9 out of 10 pharmacy, 1.2 out of 10 laboratory centers, 1.8 out of 10 rehabilitation centers, 1.3 out of 10 radiology centers and 0.8 out of 10 hh should be redistributed. also, according to the i d results, the main redistribution should be happen from tehran, esfahan and fars provinces to other provinces specially ilam, kurdestan and khorasan razavi. kiadaliri. demonstrated that 3 out of 10 dentist should be redistributed from the over served provinces to the under - served provinces. in addition, they concluded that 31583 dentists in iran are needed to reach the entire population to the level received by best groups (tehran province) (20). the result also showed that if the government wants to reach the level of all the provinces to the level of tehran (reference group), about 0.2 per 10 beds current, 0.9 per 10 pharmacy, 2.5 per 10 laboratory centers, 2.5 per 10 rehabilitation centers, 2 per 10 radiology centers and 0.09 per 10 hh should be added to current physical resources. the current study showed that when tehran province is excluded from the sample, the inequality in distribution of physical health resources has decreased. our results can help to managers and planners in health system in iran for planning in order to decreasing inequality in distribution of health physical resources. in addition, the current study provided a holistic view about situation of equality in the distribution of physical health resources especially in pharmacies, laboratories and rehabilitation centers, for health policy makers. finally, we hope the results of the current study could help health policy - makers for improving the delivery of health care and promotion of health status because one of the main factors which affect the health status of people is physical accessibility to health care resource. first, the data used in this study was obtained from the iran statistical yearbooks, so potential measurement error in the data registry is possible. second, the inequality measurement may be underestimated because we considered only public health sector. this study is the first national study which assessed inequality in the distribution of physical resources such as pharmacy, laboratory and radiology centers. this study showed that inequality in the distribution of physical resources across the provinces in iran is slight and the mean value of gc for all of physical health resources is less than 0.2. also, it is recommended that the results of this study to be considered in decision making on the distribution of physical resources across the provinces in iran. 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.	background : one of the major health policy issues, in the both developed and developing countries, is the equality in the distribution of health resources. the aim of this study was to investigate the disparity in the distribution of health physical resources across the provinces of iran in 2001 and 2011.methods:this was a cross - sectional retrospective study which investigated inequality in the distribution of health physical resources by three indexes of gini coefficient, gaswirth index and index of dissimilarity. the data on provinces were obtained from the yearbook statistics and ministry of health, and medical education. the excel software was used to calculated indexes.results:the finding showed the mean gini coefficient for all variables was 0.178 in 2001 and 0.158 in 2011. besides, the mean gaswirth index and index of dissimilarity were 11.5 and 1.5% in 2001 and 11 and 1.4% in 2011, respectively.conclusion:there was slightly inequality in distribution of physical health resources in iran. according to the results of three indexes, this study showed when tehran province excluding from total sample, the inequality was decreased.
newcastle disease (nd) and infectious bursal disease (ibd) are the two most dreaded viral diseases of poultry in nigeria as they cause severe economic losses in domestic and wild bird populations resulting from illness, reduced egg production, immunosuppression, and death following infection with pathogenic strains of their respective causative viruses. despite efforts to prevent and control them over the years, circulation of the causative virus among free - roaming and wild birds has been reported as one of the factors responsible for the sporadic outbreaks of nd and ibd among free - roaming village chickens as well as commercial poultry flocks [1, 2 ]. newcastle disease (nd) is an acute, highly contagious, rapidly spreading viral disease affecting birds of all ages and is characterized in chickens by respiratory, circulatory, gastrointestinal, and nervous signs. the clinical signs seen in infected birds vary widely and are dependent on viral factors like pathogenicity (which depends on virulence and tropism of the virus), host factors (species, age, and immune status), concurrent infections, route of exposure, duration and magnitude of the infection dose, and external factors such as social and environmental stress. according to docherty and friend, it is capable of infecting over 230 species from more than one - half of the 50 orders of birds. these include domestic poultry [7, 8 ] and wild birds such as house sparrows, hawks, crows, double - breasted cormorants, and waterfowls [913 ]. wild birds constitute a natural reservoir of low - virulence viruses, while poultry are the main reservoir of virulent strains. the most virulent form of nd virus (ndv) causes up to 100 percent mortality in affected flocks. on the other hand, infectious bursal disease (ibd) is a highly contagious immunosuppressive viral infection of chicks (36 weeks old) causing severe economic and production losses worldwide. although turkeys, ducks, guinea fowls, and ostriches may be infected, clinical disease occurs solely in chickens. however, serological evidence of the infection has been reported in free - living wild birds such as cordon bleu and village weaver, wild water birds, antarctic penguins, cattle egrets, and wild turkeys and cranes. moreover, ibdv antibodies were detectable in the sera of sedentary and migratory wild bird species in japan, suggesting that they play a key role in the natural history of ibd while the virus was isolated from wild birds in korea. although several studies have been conducted on francolins in south africa [2325 ], sparse information exists on the west african - based double - spurred francolin, francolinus bicalcaratus (synonym : pternistis bicalcaratus), which is a gamebird in the pheasant family phasianidae of the order galliformes. it is a resident breeder in tropical west africa and feeds on insects, vegetable matter, and seeds. according to keith., seven afrotropical francolin species including the double - spurred francolin have been found in nigeria where they are widely consumed as bush meat. in a comparative biochemical study of meat quality and digestive enzymes, their meat was reported to be tastier, juicier, more palatable, and richer in protein than domestic chicken meat. based on these desirable qualities of francolin meat, it is needful to consider them as an alternative source of affordable animal protein to the ever - increasing nigerian human population. moreover, although they are found mostly in the wild, mbinkar. noted that the species is considered a good candidate for future domestication due to the universal acceptability of its meat and its adaptability to anthropogenically altered environments, which may be occasioned by extensive bush burning and intensive grazing of grasslands. therefore, since studies on their biology and ecology, which can provide a basis for their eventual domestication, have been conducted [2831 ], there is a need to also investigate the infections to which they are susceptible. apart from few studies which showed that francolins are affected by diseases such as marek 's disease, coccidiosis, toxoplasmosis, and bacterial sinusitis, there is sparse information on viral diseases such as nd and ibd in francolins. this study was therefore designed to investigate the presence of ndv and ibdv antibodies in free - living double - spurred francolins caught in the wild and sold at a popular live - bird market (lbm) located in shasha, ibadan, southwest nigeria. the study was conducted in ibadan (latitude 7 23 n and longitude 3 56 e), the capital city of oyo state, southwest nigeria. this region is the core of the nigerian poultry industry with ibadan being a major city from where poultry (day - old chicks, broilers, and point - of - lay pullets) and poultry inputs (drugs, vaccines, and feed ingredients) are distributed to other parts of the country. the city also has some lbms, of which the one located at shasha is popular as it is a sales point for diverse bird species brought by peasant farmers from adjoining rural communities and traders from the northern part of the country that transport cattle, sheep, and goats to southwest nigeria. blood samples collected via the jugular vein between april and august 2012 from 56 double - spurred francolins at shasha live - bird market were poured into sterile sample bottles without anticoagulant and allowed to clot at room temperature. although larger sample population of spurred francolins existed in the market and prior consultations were made with the traders on the importance of the project, the number of birds available for bleeding was restricted to 56 in view of the refusal of traders to have their birds bled. the 56 sera were screened for antibodies to ndv and ibdv using the haemagglutination inhibition (hi) test and agar gel precipitation test (agpt) as described by durojaiye and adene and hirai. positive control nd and ibd sera were obtained from the national veterinary research institute, vom, nigeria. antibodies to ndv and ibdv were also detected and quantified in 42 francolin sera (the remaining 14 sera had been exhausted) using commercial nd and ibd enzyme - linked immunosorbent assay (elisa) kits (proflok plus, synbiotics corporation, kansas city, usa), respectively, according to the manufacturer 's instructions. positive and normal control nd and ibd sera were used to validate the tests. as specified by the kit manufacturer, a serum dilution of 1 : 50 was used and optical density (od) values were read at 405 nm with an elx800 universal microplate reader (bio - tek, vermont, usa). valid ndv or ibdv elisa results were obtained when the average od value of the normal control serum was less than 0.250 and the corrected positive control value range was between 0.250 and 0.900. data obtained were analysed with column statistics using graphpad prism version 5.0 (graphpad software, san diego, ca, usa) and p values < 0.05 were considered significant. prevalence of ndv antibodies in the tested francolin sera was 25.0% (14/56) and 35.7% (15/42) using the hi and elisa tests, respectively (table 1). the hi antibody titers ranged from 1 : 2 to 1 : 32 while mean ndv antibody titer obtained with the elisa was 3206 (95% ci : 12615152). only 3 (5.4%) of the tested sera were positive for ibdv antibodies with the agpt while 24 (57.1%) were positive using elisa (table 2). mean ibdv antibody titer obtained with the elisa was 5735 (95% ci : 29198550) and there was a 50% agreement between the agpt and elisa. using the elisa, 28.6% (12/42) of the tested sera had antibodies to both ndv and ibdv. this study investigated the presence of ndv and ibdv antibodies in free - living double - spurred francolins caught in the wild and sold at shasha lbm, which is a trading centre where different avian species including indigenous chickens, pigeons, guinea fowls, turkeys, and francolins are sold in ibadan, oyo state, southwest nigeria. to our knowledge, there is no information available on viral diseases of double - spurred francolins which have been suggested as a good candidate for domestication in order to meet the animal protein needs of the nigerian populace. therefore, this first report on the detection of ndv- and ibdv - specific igy antibodies in the sera of free - living francolins in nigeria is an indication of previous exposure of these birds to the two viruses. since they were not routinely vaccinated, it is possible that the birds acquired their nd and/or ibd seropositive status through exposure to other infected wild or domestic birds that were shedding the viruses. the seropositivity detected could be due to circulating nd and ibd vaccine viruses which the birds might have contracted through interaction with vaccinated free - range birds or even through operating occasionally around commercial poultry farms.. noted that wild birds in the pet or exotic bird trade have the potential to transmit parasites, bacteria, and viruses which may or may not be pathogenic in their normal host but pose threats when introduced to new geographic locations and new host species. in this study, we observed that wild and domestic bird species were kept by the traders in the same cages and this is consistent with a previous report that nd, for example, is spread by contact between birds and exacerbated by birds being mixed together in rural markets. as the francolins were usually kept at the lbms for about 3 - 4 weeks before being sold, it is likely that they acquired the viruses from infected domestic or wild bird species with which they were kept in the same cages. moreover, the francolins tested in this study were all apparently healthy except for one that showed signs of torticollis at the time of sample collection (figure 1). therefore, the detection of ndv and ibdv antibodies in their sera suggests that they were subclinically infected and could serve as reservoirs shedding the viruses into the environment. previous studies have implicated wild birds as possible reservoirs / vectors of these viruses for domestic poultry [16, 21, 39 ]. moreover, kim. suggested wild - type virus transmission between wild and domestic birds as the origin of the similarity of ndv strains found in wild birds and domestic birds in lbms. it is noteworthy that 28.6% (12/42) of the sera tested by the elisa technique had high titres of both ndv and ibdv antibodies, which is an indication that the birds were coinfected with the two viruses. additionally, the detection of a higher proportion of positive samples by the elisa technique compared to the hi test and agpt shows that it is more sensitive than the latter two tests for detecting ndv- and ibdv - specific antibodies, respectively, in francolin sera. this is consistent with the reports of bell. and marquardt. who also found that the elisa was more sensitive than the hi test and agpt for detection of antibodies to ndv and ibdv, respectively. this study has shown that free - living double - spurred francolins are susceptible to infection with ndv and ibdv and can serve as reservoirs of these viruses, thus acting as a means of transmission to domestic poultry. therefore, if they are to be domesticated for intensive rearing as an alternative source of animal protein, a vaccination programme which includes nd and ibd vaccinations should be developed and implemented to protect them from clinical disease. in addition, the detection of ndv and ibdv antibodies in francolins sold at lbms where they have close interaction with commercial and village chickens and other wild birds warrants continuous surveillance for these diseases because of increased concerns that low - virulence wild bird viruses could become more virulent in domestic bird populations. further studies to isolate the two viruses from francolins and determine their level of pathogenicity should be conducted.	the double - spurred francolin francolinus bicalcaratus has been identified as a good candidate for future domestication due to the universal acceptability of its meat and its adaptability to anthropogenically altered environments. therefore, in investigating the diseases to which they are susceptible, serum samples from 56 francolins in a major live - bird market (lbm) in ibadan, southwestern nigeria, were screened for antibodies against newcastle disease (nd) and infectious bursal disease (ibd) viruses. haemagglutination inhibition (hi) test and enzyme - linked immunosorbent assay (elisa) revealed 25.0% and 35.7% prevalence of nd virus (ndv) antibodies, respectively, while 5.4% and 57.1% prevalence of ibd virus (ibdv) antibodies was detected by agar gel precipitation test (agpt) and elisa, respectively. this first report on the occurrence of ndv and ibdv antibodies in apparently healthy, unvaccinated double - spurred francolins from a lbm suggests that they were subclinically infected with either field or vaccine viruses and could thus serve as possible reservoirs of these viruses to domestic poultry. furthermore, if they are to be domesticated for intensive rearing, a vaccination plan including nd and ibd should be developed and implemented.
in april 2003 the directors of the human genome project (hgp), an international scientific research project coordinated by the united states department of energy and the national institutes of health national human genome research institute, announced that the first draft of the map of the human genome had been completed. it was anticipated that mapping the complete set of dna would revolutionize health care and lay the groundwork for the development of clinical markers with predictive capabilities and, thereby, shift the disease - treatment trajectory and lead to preventive interventions, tailored treatments, and averted deaths (figure 1). likewise, it was anticipated that the map would lead to a better understanding of the causes of cardiac disease, cancer, diabetes, alzheimer 's disease, mental disorders, and other common and rare diseases ; the development of diagnostic tests to detect errant genes ; the development of new classes of medicines based on gene sequence and protein structure function ; and the development of therapies which use genes in treating genetic and acquired diseases [2, 3 ]. for population groups known to experience excess disease - related morbidity, and mortality it was believed that the ability to use genetics to predict, prevent, detect, and more effectively treat disease held tremendous promise. ten years after completion of the map, hgp leaders report several accomplishments of the hgp. they report the identification of approximately 1,800 disease genes [4, 5 ], the development of more than 2,000 genetic tests for various human diseases / conditions, and the development and ongoing testing of more than 350 biotechnology - based genetic products. yet, in a more subdued voice, they note that the anticipated clinical benefit from the hgp has yet to unfold. several public and private entities across the country have been established and have begun collecting and storing tissue specimens for genetic testing to support and expand the program of study initiated by the hgp. according to reports authored by respected scientists in the field of genetics, in 1999 close to 300 million tissue samples most of which were collected during routine clinical and surgical procedures were stored in public health departments, blood banks, pathology archives, and researchers ' laboratories within the united states [710 ]. it has been estimated that the number of tissue samples collected since that time has increased by more than 20 million a year. many in the scientific, medical, and advocacy arena, concerned about the excess disease - related morbidity and mortality and health disparities experienced by african americans and other racial / ethnic minority populations, believe that the inclusion of biological specimens (and other health - related data) from african american and other racial / ethnic minority populations is essential [1214 ]. however, an ever increasing number of reports allude to the limited inclusion of biological specimens (and other health - related data) from ethnic / racial minorities in biorepositories [1523 ]. the manner and degree to which african americans have been involved in medical research a population often cited as being unduly burdened by disease have long been expressed as a concern by leaders in the scientific community. while several efforts have been undertaken to identify factors inhibiting the participation of african americans in health - related research, few efforts have been undertaken to have highlight factors associated with the engagement of african americans in health - related research and gene testing [15, 2230 ]. a focused study of factors presumed to be associated with the participation of african americans in health - related genetic research was therefore proposed. the study was conducted by a team of nurse scientists, health educators, clinicians, and biostatisticians using principles of community engagement and community - based research [31, 32 ]. the study was designed to assess factors associated with the engagement of african americans in health - related research among a targeted group of african - american men and women. more specifically, the study was designed to assess the influence of knowledge about genetics ; beliefs regarding benefits and risks of gene testing ; perceptions regarding the utility of genetic testing ; and the involvement of health care providers in decisions regarding participation in health - related genetic research and their willingness to participate in health - related research that involved gene analysis. the organizing framework designed for the study included constructs deemed to be essential to informed decision - making and engagement in health - related genetic research [4, 3336 ] (figure 2). in applying the framework in this study, the independent process variables were knowledge about genetics ; beliefs regarding the benefits and risks of gene testing ; perceptions regarding the utility of genetic testing ; and involvement of health care providers in health care decisions. more specifically, the organizing framework hypothesized the influence of knowledge about genetics ; beliefs regarding benefits and risks of gene testing ; perceptions regarding the utility of genetic testing ; and directive and nondirective involvement of health care providers on decisions made relative to participation in health - related genetics research and willingness to participate in health - related genetic research. an exploratory cross - sectional study design was used to examine the influence of knowledge about genetics ; beliefs regarding benefits and risks of gene testing ; perceptions regarding the utility of genetic testing ; and the involvement of health care providers on decisions made relative to participation in health - related genetics research and willingness to participate in health - related genetic research among a targeted group of african american men and women. a nonprobability sample of african american men and women who resided in a large, densely populated, economically, socially, and culturally diverse urban community in the midwest was recruited to the study. prospective participants who were 18 years of age or older ; able to communicate in english ; willing to complete a questionnaire about gene testing and genetic research ; and able and willing to consent to participate in the study were invited to participate in the study by members of the research team. the investigative team worked in collaboration with the black health coalition of wisconsin (bhcw) to recruit study participants. four community leaders, identified by the coalition director, were hired to and served as facilitators and recruiters for the study. after completing an online module on the protection of human subjects, each study facilitator was provided an overview of the study protocol and an overview of the procedures and methods used for data collection. flyers and announcements explaining the project and providing contact information were posted by the study facilitators at community centers, heath centers, social service centers, and other public venues frequented by diverse groups of african american men and women. individuals expressing an interest in participating in the study were contacted by a study facilitator. after which, a meeting was arranged to further discuss the purpose of the study, to describe the procedures to be used in gathering study data, and to obtain their written consent to participate in the study. to facilitate the collection of the study data, the questionnaire was administered by the study facilitators. prospective participants were informed that completion of the study questionnaire was voluntary and that receipt of services and/or support at the recruitment sites was not contingent on their participation. prospective participants were informed that no names or personal identifiers would be requested or recorded. included in the questionnaire were quantitative measures relevant to involvement of providers in health care decisions and decisions made by regarding participation in health related genetic research. quantitative measures developed, validated and used in national cohort studies to assess genetics knowledge, beliefs regarding the merits and risks of genetic testing, and perceptions regarding the utility of genetic testing [3742 ] were also included. eleven true - false items were used to assess knowledge of genetics principles and implications ; two items were used in which participants were asked to describe how much they felt they knew about genetics ; and four forced - choice items were used to assess knowledge of ongoing health - related genetic studies being conducted within the region. the items included statements suggesting the benefits, anticipated consequences, and negative impact of gene testing in which participants were asked to describe their beliefs using a 5-point likert scale (1 = strongly disagree to 5 = strongly agree). items inferring the importance of gene testing for risk assessment, screening, early detection, and treatment were included. twenty items were used to assess involvement of health care providers in decision making regarding gene testing. the items included statements in which the participants were asked to characterize the extent to which they discuss health concerns with providers, the extent to which their provider understand their background, needs, concerns and values, and the medical judgments made by the providers on their behalf using a 4-point likert scale (1 = strongly disagree to 4 = strongly agree). also an item in which participants were asked if they believed their provider would recommend genetic testing if he or she believed it would cause them harm was included. (v) willingness to participate in health - related research that includes gene analysis. twenty one items were used to assess willingness to participate in health - related research that included gene analysis. items in which participants were asked if they had ever participated in medical research were included. if participants reported that they had never participated in medical research, they were queried as about their willingness to participate and their willingness to provide personal, social, occupational and medical information, and biological specimens for genetic analysis. seventeen items were included within the questionnaire to elicit data reflective of the participant 's gender, age, education, marital status, employment status, income, personal or family history of a chronic disease / condition, previous involvement in health - related research, insurance status, primary source of health care, and perceived health status. content validity and appropriateness of the questionnaire for use among the targeted population were assessed by the study investigators prior to initiation of the study. descriptive and inferential statistics, computed using spss - pc version 20 (spss inc., chicago, il), were used to analyze the study data. descriptive statistics (including frequency, percentages, measures of central tendency, and measures of variability) were used to describe the characteristics of the study sample. inferential statistics (including cross tabulations and chi - square analyses) were used to identify factors associated with willingness to participate in health - related genetic research. the research protocol of the study was reviewed and approved by the institutional review board for the protection of human subjects at the university of wisconsin milwaukee. of the 212 study participants, 45.8% (n = 97) were men and 54.2% (n = 115) were women (table 1). the mean age of the study participants was 43.04 years (sd = 6.14 ; range 1995). the majority of the study participants were single (62.3%, n = 132) ; employed full or part time (50.5%, n = 107) ; and had attended or completed college (60.8%, n = 129). sixty four percent (n = 135) reported incomes of $ 29,999 or less and 80.7% percent (n = 171) reported that they were insured. when asked to describe their health status, 73.6% (n = 156) described their health status as good, very good, or excellent, and 43.4% (n = 92) reported a history of a chronic disease / condition. ninety four percent (n = 192) of the african american men and women involved in the study reported that they knew little about genetics. however, data suggested that most were aware that healthy parents could bear a child with a hereditary disease ; were aware of the implications of consanguinity and late parity on the expression of heritable disease / conditions and birth outcomes ; and were aware of the influence of the environment on multifactorial genetic disorders (e.g., asthma, congestive heart disease, diabetes) (table 2). in addition, when questioned about tests used for genetic analysis, most indicated that they were aware of tests used in newborn screening. study participants had varied beliefs about the benefits, consequences, and risks associated with genetic testing (table 2). gene testing is currently used in the health care arena for carrier screening, preimplantation genetic diagnosis, prenatal diagnostic testing, newborn screening, presymptomatic testing for predicting adult - onset disorders, presymptomatic testing for estimating the risk of developing adult - onset disorders, and confirmatory diagnosis. most study participants reported that they believed that genetic testing would lead to improved treatments and improve health outcomes. yet, several study participants expressed concerns about adverse consequences that could result from the diagnosis of a genetically - linked condition / disease (e.g., potential breech of their privacy, emotional trauma, stigma, and discrimination). fifty seven percent (n = 121) of the study participants expressed beliefs and concerns that the diagnosis of a genetically - linked condition would not remain confidential. fifty two percent (n = 111) expressed beliefs and concerns about the effect of a genetically - liked condition on their family. twenty two percent (n = 47) of the study participants expressed beliefs and concerns that they would not be able to emotionally handle the diagnosis of a genetically - linked condition. thirteen percent (n = 28) reported that if they were found to carry a genetically - linked condition others would view them negatively and 8.0% (n = 17) reported that it would cause them to feel ashamed. most study participants perceived that the results of gene testing would be useful to providers attempting to make patient care decisions and to individuals attempting to improve their overall health status (table 2). when questioned about the utility of gene testing 93.9% (n = 199) reported that they believed that gene testing would be useful to providers when assessing patient 's health risks ; 93.9% (n = 199) reported that they believed that gene testing would help providers in their attempts to validate (or rule out) the presence of indolent disease ; and 90.1% (n = 191) of the study participants reported that they believed that gene testing would aid providers in their attempts to provide personalized treatments and health care. similarly, 93.4% (n = 198) of the study participants reported the belief that the results of gene testing would help them make decisions about how to live a healthier life. the merits of directive and nondirective involvement of health - care providers in decision making about gene testing have been widely reported in the literature [33, 35, 36, 4446 ]. nondirective involvement of health - care providers (relative to gene testing) implies that the patient is given relevant information about a genetic test by the health care provider and is left to make his or her own choice about testing. directive involvement of health care providers (relative to gene testing) implies that the health - care provider reviews relevant information about a genetic test and makes the decision for the patient about testing and the patient concurs. most health care providers and genetic counselors when discussing gene testing, in an effort to distance gene testing from any association with eugenics, tend to be more inclined toward nondirective approaches. yet, while there has been much debate about whether any discussion with patients is completely nondirective, research suggests that most clients, attempting to make decision about genetic testing and genetics related research expect information, advice and help in making decisions [35, 36 ]. review of the study data revealed that the majority of the participants valued their health - care provider 's knowledge and advice when making health decisions. seventy percent (n = 148) of the study participants reported that they completely trusted their provider 's judgment about their medical care ; 89.6% (n = 190) reported that the medical information they received from providers was accurate and up - to - date, and 81.1% (n = 172) reported that they always tried to follow the provider 's advice. when probed further, 67.5% (n = 143) reported that their providers put their medical needs above costs and all other considerations and 89.6% (n = 190) reported that they trusted that their provider would refer them for specialized testing if there was a need. twenty percent (n = 43) of the study participants reported that they had been previously involved in a health - related research study. among those who indicated that they had not been previously involved in a health - related research study, fifty one percent (n = 85) reported that they would be willing to complete detailed questionnaires about their family health history, social history, occupational history, psychological, and/or emotional health. fifty three percent (n = 70) reported that they would be willing to allow researchers to collect environmental samples from their home. fifty two percent (n = 82) reported that they would be willing to provide researchers biological specimens (e.g., tissue, blood, saliva, hair, nail clippings) for genetic analysis. fifty four percent (n = 70) reported that they would be willing to participate even if the study were longitudinal and required the collection of data over several years. study findings implicate the impact of knowledge, perceptions, and beliefs on the willingness of african american men and women to participate in health - related genetics research (table 3). participants with higher levels of knowledge about genetics and heritable diseases ; with understanding of the benefits, risks, and utility of genetic testing ; and who had previous involvement in a health - related research study were more likely to report a willingness to engage in health - related genetic research than study participants with lesser knowledge about genetics, those with lesser knowledge, and those who had not previously been involved in a health - related research study. participants reporting higher levels of trust in their provider 's knowledge and judgment and participants reporting that their providers listened well to their concerns about their health and well - being were more likely to report a willingness to engage in health - related research than those who did not. in addition, as might be expected, participants reporting that they had been informed and offered the opportunity to participate in a study were more likely to express a willingness to engage in a health - related research study than those who were not. the use of nonprobability sampling and self - reported measures used in this exploratory study limit the generalizability of the findings. yet, in spite of these constraints, the findings warrant careful consideration. increasing the involvement of minority participants in health - related research accounts of events that have negatively impacted the engagement of african americans have been widely reported in the scientific literature. also are codes, guidelines, procedures, and regulations are reported to prevent these and other unethical behaviors conducted in the name of science [47, 48 ]. on june 10, 1993, the nih revitalization act of 1993, pl 103 - 4 was signed into law. the legislation directed the national institutes of health to establish guidelines for inclusion of women and minority groups and their subpopulations in nih - funded clinical research, unless a clear and compelling rationale and justification that inclusion is inappropriate with respect to the health of the subjects or the purpose of the research. despite the enactment of legislation specific to the inclusion of minorities in health - related research and the adoption of codes, guidelines, procedures, and regulations to prevent unethical behaviors in research and protect the rights and well - being of persons involved, the contribution of specimens to biorepositories by african americans and the participation of african americans in health - related genetic research are limited. this study was designed to assess factors deemed to be essential to the engagement of african americans in health - related research that involves gene testing. more specifically, the study was designed to assess the influence of knowledge about genetics ; beliefs regarding benefits and risks of gene testing ; perceptions regarding the utility of genetic testing ; and the involvement of health care providers on decisions regarding participation in health - related genetic research. the results of this study support hypotheses proposed in the organizing framework relative to influence of knowledge about genetics ; beliefs regarding benefits and risks of gene testing ; perceptions regarding the utility of genetic testing ; and the involvement of health care providers on decisions relative to participation in health - related genetics research and the willingness to engage in health - related genetic research. as hypothesized, study findings revealed that knowledge about genetic research, perceptions regarding the utility of genetic testing, beliefs regarding the benefits and risks of genetic testing, and previous involvement in research are associated with participant 's willingness to engage in health - related genetic research. study participants reporting higher levels of trust and engagement with providers were more likely to report a willingness to engage in health related research than those who did not. the most surprising were the findings that the vast majority of the african american men and women involved in the study reported support of health - related genetic research ; however, most indicated that they had never been asked. this study is unique in that it attempted to highlight factors associated with the participation of african americans in health - related research as well the processes, outcomes, and principles of informed decision making. the findings suggest the need for research that further examines factors presumed to be associated with the involvement of african americans in health - related genetic research. the most important appears to be the need for research that explores the manner and extent to which african americans are informed about the benefits, risks, and utility of genetic testing ; research that explores the manner and extent to which african americans are informed and afforded the opportunity to participate in health - related genetic research ; and research that explores the manner and extent to which the decisions of african american men and women relative to participation in health - related genetic research (as assured during the consenting process) are supported. the findings also suggest that the need for strategies to better inform african americans of opportunities to contribute health information and biological specimens to biorepositories and to better engage researchers and health care providers in efforts to inform, recruit, and support african american men and women willing to participate in health - related genetic research. without the design of interventions to better inform, recruit, engage, and support the decisions of african americans (and persons representing other minority population groups) willing to participate in health - related genetic research it would be reasonable to anticipate that current trends relative to their involvement will remain unchanged.	the involvement of african americans in research has long been expressed as a concern by the scientific community. while efforts have been undertaken to identify factors inhibiting the participation of african americans in health - related research, few efforts have been undertaken to have highlight factors associated with their engagement of health - related research. an exploratory study of factors presumed to be associated with participation in health - related research was conducted among a nonprobability sample of african americans (n = 212) from a large urban community in the midwest. the study was guided by a framework that hypothesized the influence of knowledge, beliefs, and perceptions about genetics and the involvement of providers in decision - making on willingness to participate in health - related genetic research. the results revealed that knowledge, beliefs, and perceptions about genetics and the involvement of providers were associated with willingness to engage in health - related genetic research (p <.05). the most interesting, however, was that 88.7% of the participants who had not previously been involved in a health - related study who expressed a willingness to participate reported that they had never been asked. study findings suggest the need for research that further examines factors associated with the involvement of african americans in health - related genetic research.
double crowns have not only been used to retain implant - supported overdentures (iods) in the mandible and the maxilla with positive long - term prognoses and high implant and denture survival rates, but they were also applied as attachments for residual natural teeht.123 a number of modifications for the fabrication of implant - supported double - crowns have been described and used in clinical studies, including frictional parallel - sided (telescopic) or conical crown designs mainly fabricated from cast noble alloys.45678 furthermore, telescopic crowns with a clearance fit fabricated from noble and non - precious alloys have been used as attachments for iods9101112 as well as prefabricated double - crown systems.13 the use of all - ceramic materials for the fabrication of double - crown attachments was first described in 2000.14 this so - called ceramo - galvanic double - crown (cgdc) is based on a conical crown design. it consists of a tapered all - ceramic primary crown and a secondary crown made from galvano - formed gold that are luted to a reinforcing cast denture framework (fig. it was assumed that replacing the cast metal by ceramics and electroplated gold would improve the wear resistance compared with conventional cast double crowns, thus leading to a more constant and predictable retentive force of the removable denture.14 the concept of the cgdc was clinically evaluated on a short - term basis with 32 patients wearing 33 dentures supported by natural teeth or implants. during this initial phase, the primary crowns were fabricated mainly from leucite - reinforced glass - ceramics (empress 1, ivoclarvivadent, schaan, liechtenstein). this material exhibited increased fracture rates and therefore was later replaced with yttriastabilized zirconia polycrystals (y - tzp or zirconia).15 subsequently, conical crowns with zirconia primary crowns and electroplated copings as female parts have been evaluated in several in vitro studies.161718 the cgdcs demonstrated clinically acceptable mean retentive forces and reduced excursive retentive force development compared with cast double crown systems.1718 the retentive force was influenced by the abutment height and the taper. it was concluded that zirconia primary crowns with a sufficient height and 2 taper can serve as an alternative to gold alloy primary crowns.16 y - tzp has been clinically evaluated as a framework material for crowns and fixed partial dentures (fpds) in numerous clinical trials with promising results.1920 nevertheless, to the best of our knowledge, the treatment concept of cgdcs for the attachment of iods has only been described in several case reports.21222324 long - term clinical evaluations are needed. the present retrospective clinical study evaluates patients with solely implant - supported overdentures in the maxilla and the mandible. the patients were restored with double - crown - retained iods with zirconia primary crowns and electro - formed secondary crowns (cgdcs) in a private dental practice. this investigation sought to determine the survival rates of implants / abutments / primary crowns and dentures as well as biological and technical complication rates over a mean observational time of > 5 years.252627 this retrospective clinical evaluation was conducted in a private practice (hanau, germany). the study is based on the analysis of primary patient data as well as the evaluation of clinical results from iods that were rigidly retained by double crowns with zirconia primary crowns and electroplated secondary crowns. the study was reviewed by the ethics committee of the georg - august - university, gttingen, germany, and authorized by this board (application no. 4/7/13). recommendations to strengthen the reporting of observational studies in epidemiology (strobe) were followed. 28 patients provided with double crown - retained iods between july 2003, and july 2010 and who attended a maintenance program including supportive post - implant hygiene therapy (sit) were identified. these patients were contacted during their respective annual maintenance appointments and were suggested to participate in the clinical study after having been briefed in writing about its aims and course. patients who submitted written informed consent were included if they met the following inclusion criteria : surgical and prosthetic treatment was received in the study practice the same implant design (ankylos, dentsply implants gmbh, mannheim, germany), and an identical prosthetic concept (ceramo - galvanic - double crowns according to p. weigl) were used inclusion of a minimum of 4 and maximum of 8 implants per restoration regular supportive post - implant hygiene therapy (sit) (at least annually) a functional period of > 3 years with the final restorations in place full medical history available, which includes the identification of these potential risk factors : medication (immune suppression and bisphosphonate), cardiovascular disease, rheumatoid arthritis, diabetes, smoking habits these exclusion criteria were applied : patients who did not completely fulfill the inclusion criteria non - compliance in supportive post - implant hygiene therapy (sit) (minimum 1/year) use of other implant systems than the ankylos system application of other attachments than ceramo - galvanic double crowns (cgdc) restorative planning aimed at a quadrangular support with a minimum of 4 and a maximum of 8 implants per iod. surgery was performed under local anesthesia according to the manufacturer 's protocol. the same experienced clinician (sr) performed all clinical procedures. antibiotics were administered 1 hour before and 1 week after surgery (amoxicillin 1000 31/d or clindamycin 300 31/d). wound control occurred 7 days (suture removal) and 21 - 28 days after surgery. prosthodontic treatment was performed according to the technique proposed by p. weigl and encompasses the following steps : 1. impressions were obtained on the implant level using screw - retained impression copings, a custom impression tray, and a polyether material (impregum soft, 3 m espe gmbh, seefeld, germany). during the same appointment, 2. zirconia primary crowns were fabricated on titanium abutments (ankylos balance posterior, dentsply implants gmbh, mannheim, germany) with various heights and angulations using a cam system (cercon smart ceramics, degudent gmbh, hanau, germany). the crowns were milled with a 2 taper, and the minimum thickness was 0.5 mm. the ceramic surface was sprayed with a thin conductive silver layer, and the copings were placed in an electroplating device (solaris, degudent gmbh, hanau, germany). processing time as well as the current for electroplating was adjusted in order to guarantee a minimum thickness of 0.2 mm for the gold coping. a reinforcing denture framework was cast in one piece from a cocr alloy, including complete coverage of the secondary crowns. between the denture framework and the secondary coping, after definitive placement of the titanium abutments (torque : 15 ncm), the primary crowns were luted adhesively onto the abutments using an auto - curing composite cement (agc cem, wieland dental+technik gmbh, pforzheim, germany) (fig. the denture framework was then luted with the secondary crown intraorally using the same auto - curing composite (fig. 4. the passively fitting framework was than used as a basis for a definitive bite registration. a total impression encompassing the joint secondary crowns and the denture framework was obtained using a custom tray and a polyvinylsiloxane material (aquasil monophase, dentsply detrey gmbh, konstanz, germany). the framework was completely covered with denture base resin, and resin - based denture teeth were added (fig. 2c). the patients were then scheduled for a supportive post - implant hygiene therapy visit (sit). in at least one prophylaxis appointment per year, the patient compliance was assessed. during these sections, the following data were evaluated : the periodontal and peri - implant tissue status using the quigley - hein plaque index (qhi) ; measurement of peri - implant probing depths (ppds) with a millimeter - scaled periodontal probe (pcp 15, hu - friedy manufacturing company, llc, chicago, il, usa) at 4 sites per implant (mesio - buccal, disto - buccal, mesio - oral, and disto - oral), any bleeding on probing was documented (bop ; 30 seconds following probing) ; and radiographs for implants with positive bop and a ppd 5 mm using the long - cone parallel technique). patient motivation was reinforced during the follow - up sessions, and all subjects were repeatedly instructed on in - home plaque control. all implants as well as the teeth involved were then professionally cleaned with polishing paste and a rubber cup. if periimplant mucositis was diagnosed, a special ultrasonic tip was used for scaling around the implants (kavo sonicflex implant, kavo dental gmbh, biberach, germany). these areas were then instrumented manually prior to the subgingival application of chlorhexidine gel (corsodyl 1% dental gel, glaxosmithkline gmbh, hamburg, germany).2930 the patients included in this retrospective clinical study were evaluated according to the following parameters by using patient records : age at the concluding examination, gender, smoking habits, medical history, anatomical implant position (fdi system), number of implants, implant loss until data acquisition, time of denture insertion, opposing dentition, and functional period. an experienced dentist who did not place the implants performed the clinical examination of the patients during the last sit appointment. he evaluated these technical and biological complications of teeth / implants as well as the removable dentures : material fractures, screw loosening, retention loss and/or defects of the retention elements, relines, peri - implantitis. radiographs that measured the extent of peri - implant bone loss referenced to baseline radiographs (prosthetic delivery) were taken in order to confirm the diagnosis of peri - implantitis for implants with positive bop and a ppd 5 mm. intraoral radiographs were obtained using a parallel technique, they served to assess the peri - implant bone level and were evaluated by an experienced dentist (dz).1112 the clinical procedures for the sit and the methods for data collection have been applied in previous studies by the same working group11122930 the maintenance of either an implant or a prosthetic reconstruction in the mouth was defined as survival, independent of biological and/or technical complications.27 the period between the time of placement and the last follow - up appointment or, for failures, the appointment scheduled to address the failure as documented in the patient 's file, were defined as survival time.2627 the criteria proposed by albrektsson.25 were the basis for the calculation of cumulative implant success rates. all technical complications that were related to either the overdenture or the implant abutment (e.g. abutment fracture, loosening of the abutment screw, fracture of the denture material (base / teeth), retention loss, attachment defects) were documented. treatments occurring per restoration per year (t / r / y) and treatments occurring per patient per year (t / p / y)6111229 formed the basis for the calculation of incidence rates for technical complications peri - implant mucositis was recorded for implants with ppd > 4 mm, and bop. the following final points were used as diagnostic criteria for peri - implantitis : ppd 5 mm, positive bop / suppuration, radiographic bone loss with a distance of at least 3.5 mm between implant shoulder and bone level.111230 as the sample size is small, no significant statistical analysis of the potential factors that influence the treatment results was possible. for this reason, 14 of this lot (63.1 6.8 years / range : 52.1 - 75.7) received a final clinical follow - up examination between september 2013 and june 2014. the dropout rate was 17.6% (1 patient declined further participation due to severe illness, and 2 patients died). of the included patients, 7 were female (50%), and 7 were male. the medical histories revealed that 2 patients (14.3%) suffered from diabetes type 2, 5 (35.7%) patients were afflicted with a cardiovascular disease. the mean implant follow - up period was determined at 6.2 2.3 years (minimum 3.5 years, maximum 10.5 years). the 14 patients included in the study received a total of 18 cgdc - retained prostheses (4 patients received restorations in both, the maxilla and mandible). eleven cgdc - retained prosthesis were placed in the mandible, and 7 were placed in the maxilla. the mean prosthetic follow - up period was 5.9 2.2 years (range : 3.2 - 9.9 years). a total of 86 implants with a morse taper connection (ankylos, dentsply implants, mannheim, germany) was inserted : 38 implants (44.2%) were placed in the maxilla, whereas 48 implants (55.8%) were inserted in the mandible (table 2). the mean length was 12.2 1.7 mm (range : 9.5 - 14 mm). due to the absence of osseointegration, one implant (upper molar region) had to be removed at the time of second - stage surgery. none of the remaining implants failed after functional loading (cumulative implant survival rate : 98.8% after 6.2 2.3 years). a total of 85 implants were provided with cgdc according to the protocol proposed by p. weigl. the number of implants included in one restoration ranged between 4 and 8 (mean : 4.7 1.1). peri - implant mucositis (positive bop and ppd > 4 mm) was observed in 40 implants (47.1%) and 10 patients (71.4%). the mean radiographic bone loss was 0.7 0.6 mm (range 0 - 3.6 mm). one of the 85 functionally loaded implants exhibited clinical symptoms of peri - implantitis according to the selected criteria (radiographic bone loss of 3.5 mm, a ppd 5 mm, and positive bop). this resulted in an implant - based peri - implantitis rate of 1.2% (patient - based peri - implantitis : 7.1%). this implant was rated as a radiographic failure. according to the criteria defined by albrektsson.,25 this led to a cumulative implant success rate of 97.7% (table 3). one out of the 85 titanium abutments fractured after a functional period of 5.43 years and required replacement. the cumulative abutment survival rate was 98.2% after a mean observational time of 5.9 2.2 years (range : 3.2 - 9.9 years). none of the zirconia primary crowns fractured, this led to a cumulative primary crown survival rate of 100%. each of the 14 included patients received one or two removable telescopic dentures supported by 4 - 8 implants and attached by cgdcs. the opposing dentition of 3 patients (21.4%) was restored with a fixed reconstruction ; 3 patients (21.4%) wore full dentures, and 2 patients (14.3%) wore tooth - supported, removable dentures. the opposing jaws of 2 patients (14.3%) were restored with iods supported by milled bars, and 4 patients (28.6%) received iods supported by cgdcs in both jaws (table 1). all dentures were found functional at the time of investigation. after a mean observational period of 5.9 2.2 years (range : 3.2 - 9.9 years) a total of 15 technical complications were registered for 8 out of the 18 restorations (44.4%) in 8 different patients. five of these complications were related to the cgdc (33.3%) and 10 (66.6%) to the removable dentures. tooth fractures were the most frequent complication (n=6) and required the replacement of 1 to 3 denture teeth. one removable denture exhibited 3 different denture teeth fractures during the entire observational period of 5.1 years. three other removable dentures required realignment, and one denture required a repair of the fractured part of the denture base material. apart from the above mentioned loss of retention, for all removable restorations, a sufficient retention force was maintained over the entire observational period, and clinical interventions to improve the retention characteristics were not necessary in any of these cases. in total, incidence rates of 0.139 treatments per denture per year (t / d / y) and 0.179 treatments per patient per year (t / p / y) were recorded. table 4 provides information on both, the technical complications as well as the required prosthetic maintenance. this practice - based retrospective study presents clinical data for the survival / success of cgdc - supported iods in 14 patients restored with a total of 85 implants after a mean observational period of 5.9 years. the csr of the implants was documented with 98.5%, 100% of the prosthetic reconstructions remained in function during this time. the observed rates for technical and biological complications were low, the findings did not endanger the continued functioning of the iods in any way. for the evaluation of the present study, it had to be considered that it is based on a small patient lot only. a control group with other types of attachments (e.g., conventional double crowns or ball attachments) that matched the study group was not available. however, notwithstanding these limitations, the study population exclusively consisted of patients with implants and prosthetic restorations with an identical design. only implants with a morse taper connection (ankylos, dentsply implants gmbh, mannheim, germany) and double crowns manufactured according to the cgdc technique were included. this is an important aspect of the present study because most of the existing clinical data with double crown - retained iods were collected in university settings, covering mean observational times of less than 5 years.23456789 the results of the present study revealed a cumulative survival rate of 98.8% for the implants (cumulative success rate according to albrektsson.25 : 97.7%) after a mean observational time of 6.2 2.3 years. after a mean observational period of 5.9 2.2 years this finding meets the survival rates documented for double crown - retained implant - borne overdentures. the published data on implant - supported double crown - retained overdentures in edentulous jaws revealed survival rates of 97% - 100% for implants, and 93% - 100% for overdentures for mean observational periods of up to five years.23678 a specific aspect of the present retrospective clinical evaluation is the use of the ceramo - galvanic double crown. during the entire observational period, none of the zirconia primary crowns fractured. to date, zirconia has been evaluated exclusively as a framework material for crowns and fixed partial dentures. it has demonstrated a high reliability and low fracture rate for functional periods of up to 10 years for these indications.1920 due to the fact that the iods are rigidly retained by the cgdcs, high mechanical loads are generated by the distal extensions. based on the findings of the present study, no increased fracture rate of the unveneered zirconia was detected if used as primary crowns to rigidly attached iods. this presents a new finding not yet reported by clinical investigations. during evaluation of the approaches and concepts used for restorative treatment, the rate of required prosthetic maintenance was seriously accounted for.123 in the literature, few studies detail the technical complications linked to implant - supported removable dentures using various types of attachments (bars, ball attachments, and double crowns). the requirements for maintenance varied between 0.222 and 4.03 treatments per patient per year (t / p / y).1671112 preferably, the results of the present study should be compared with studies that also report technical complication rates for iods retained by double - crowns. a prospective study that assessed 4 interforaminal implants in the mandible over 3 years of follow - up, the need for prosthetic maintenance was documented at 0.41 t / p / y with bar structures compared to 0.45 t / p / y for telescopic restorations6. in a practice - based retrospective evaluation of double crown - retained overdentures after a minimum observational time of 10 years, a technical complication of 0.333 treatments / patient / years was reported.11 another retrospective study that evaluated the technical complication rate of maxillary iods also using ankylos implants and double crown attachments documented that all dentures remained in function (prosthetic survival rate:100%) after a mean observational time of > 5 years.12 technical maintenance procedures were required at a rate of 0.222 t / p / y. this result meets the findings of the present study with a technical complication rate of 0.178 t / p / y. these findings reveal that cgdc - retained iods require a comparatively low maintenance, all the more when considering that about 66% of the appointments were purely prosthesis - related (e.g. fractures of prosthetic teeth, relinings). similar to other studies with solely implant - supported overdentures, technical complications applying to the removable restoration parts (fractures of resin components) were the most frequent type of technical complication.16781112 it is a limitation in the prosthetic design evaluated in the present study that the primary crowns were cemented adhesively to the abutments. in case of an abutment screw loosening, thus the preparation of an access cavity is needed to retighten the abutment screw. in several in vitro investigations, the retentive forces of cgdcs have been evaluated.161718 from these investigations, it was concluded that cgdcs have sufficient retentive properties and are not susceptible to excessive wear. none of the 18 re - examined dentures displayed insufficient retention, and no interventions to improve retention were necessary during the entire observational period. the good retentive characteristics in the present study are potentially explained by two design aspects of the restorations : 1. it was possible for all 85 cgdcs to maintain a minimum height of at least 5 mm. the latter appears to be important, as in vitro investigations have demonstrated that the height of the primary crown significantly affects the retention force generated by cgdcs.16 under these preconditions, no increased risk for technical complications of cgdcs compared with metal - based double crowns was detected for a mid - term clinical evaluation with a mean observational time of > 5 years. in addition to technical complications, the clinical performance of implant - supported restorations can be challenged by biological complications, e.g. peri - implantitis. based on the published results, within 5 - 10 years after implant insertion, peri - implantitis occurs in 10% of implants and 20% of patients. nevertheless, the individually reported data showed a significant variation26 which is due to differences in the study populations, observational times, level of maintenance measures, and the application of different criteria for the definition of peri - implant mucositis and peri - implantitis.27 apart from smoking habits and history of peri - implantitis, inadequate oral hygiene is one of the risk factors linked to peri - implantitis.303132 however, information on biological complications, especially with iods, remains sparse.23 in the present study, 47.1% of the implants in 10 out of 14 patients (71.4%) exhibited clinical symptoms of peri - implant mucositis (bop + ppd > 4 mm). considering the selected criteria (crestal bone loss 3.5 mm, ppd 5 mm, positive bop), one of the implants demonstrated symptoms of peri - implantitis (implant - based prevalence : 1.2%, patient - based prevalence : 7.1%). these findings should preferably be matched with results evaluated with a comparable implant design and prosthetic treatment concept. in another practice - based study of 20 patients restored with overdentures supported by four implants (using the same implant design as in the present case series) and double - crown attachments, an implant- and patient - based peri - implantitis rate of 10% was detected after a mean observational time of 5.6 years.12 the same group also reported results for double crown - retained iods placed in non - smoking patients after a mean observational period of 14.1 years. this study documents a patient - based peri - implantitis rate of 9%.11 the results of the present study match these findings. based on these findings for double crown - retained iods, peri - implantitis occurs in 7 to 10% of patients for mean observational periods of 5 - 14 years, which is somewhat lower than the expected rate of 20% for observational periods of 5 - 10 years. this is of great importance as the positive effect of supportive post - implant hygiene therapy on minimizing the risk for peri - implantitis has been demonstrated in various clinical studies.303132 the design of the prosthesis itself might be another factor that could help to reduce the risk for peri - implantitis. double crown - retained iods offer good cleaning access in oral hygiene homecare procedures, thus reducing the risk for hyperplasia and peri - implantitis. this hypothesis was supported by the findings of clinical trials comparing iods that are bar- or telescopic crown - retained46 ; the two studies determined significantly more hyperplasia and plaque accumulation and hyperplasia for bar attachments. another possible reason for the fairly low peri - implantitis rates can be observed in the patient selection in the three cited studies. all three studies included either only non - smokers or a small number of smokers (5 years. technical complications mainly related to the removable parts should be anticipated during the functional period without further affecting the function of the iods. for patients restored with cgdc - retained iods who regularly attend a supportive post - implant hygiene therapy, biological complications (peri - implantitis) additional studies covering a larger sample and higher evidence levels (rcts) are necessary to validate the results of this study and to compare this treatment concept with alternative treatment concepts.	purposethis retrospective study aims at the evaluation of implant - supported overdentures (iods) supported by ceramo - galvanic double crowns (cgdcs : zirconia primary crowns + galvano - formed secondary crown).materials and methodsin a private practice, 14 patients were restored with 18 iods (mandible : 11, maxilla : 7) retained by cgdcs on 4 - 8 implants and annually evaluated for technical and/or biological failures / complications.resultsone of the 86 inserted implants failed during the healing period (cumulative survival rate (csr) implants : 98.8%). during the prosthetic functional period (mean : 5.9 2.2 years), 1 implant demonstrated an abutment fracture (csr - abutments : 98.2%), and one case of peri - implantitis was detected. all iods remained in function (csr - denture : 100%). a total of 15 technical complications required interventions to maintain function (technical complication rate : 0.178 treatments / patients / year).conclusionconsidering the small sample size, the use of cgdcs for the attachment of iods is possible without an increased risk of technical complications. however, for a final evaluation, results from a larger cohort are required.
during the last decades, cation exchange (ce) reactions have emerged as a new strategy for the fabrication of nanomaterials via postsynthetic chemical modification. at the nanoscale, ce reactions were applied and studied mostly on semiconductor ii vi, iii v, and iv vi compounds. in this method, cations of a presynthesized parent nanocrystal (nc) can be partially or completely replaced by new guest cations with preservation of its size, shape, and, in some cases, even crystal structure. a major characteristic of such selective transformation is the overall preservation of the anion sublattice of ncs owing to the usually much larger size of anions, relative to cations, and thus their lower mobility in the lattice. depending on the extent of ce, doped, alloyed or heterostructured ncs and completely exchanged ncs can be prepared by varying the ratio between host and guest cations. it was also shown that ce enables the synthesis of metastable nc structures, as well as specific architectures that are hardly accessible via a direct synthesis route, such as dot - in - rods znse / zns and pbse / pbs ncs. moreover, ce reactions had already been successfully employed in bioassays. despite ce at the nanoscale having been studied for more than one decade, only a few works have addressed the fundamental mechanisms of this process, as ce reactions have been used mainly as a means for synthesizing nanomaterials. one of the most exploited classes of materials toward ce is represented by copper chalcogenide ncs, one reason being the large number of copper vacancies that these compounds can sustain, which translates in an efficient ion exchange process mediated by vacancy diffusion. in this work, we have carefully investigated room temperature ce in copper selenide (cu2xse) ncs involving two divalent cations (zn and cd) in the presence of trioctylphosphine (top) as a promoter of the exchange, with the aim of elucidating the effect of the density of copper vacancies in the starting cu2xse ncs on the rate of exchange. note that, in both cases, the entering cations have stable oxidation states (+ 2), and additionally, they form phases (znse, cdse) that are in principle immiscible with cu2xse. this simplifies the analysis as we do not expect the occurrence of redox reactions or the pervasive formation of ternary alloys. the only remarkable difference between the two cations is that zn has an ionic radius (0.6) comparable to that of cu (0.6), while cd is larger (0.78) ; however, both zn and cd adopt a tetrahedral coordination with the se anion sublattice. in both cases discussed here, partial ce led essentially to janus - like nc heterostructures represented by a znse (or cdse) domain sharing a close - to - flat interface with the remaining cu2se portion, while core shell geometries were never observed. such mutual arrangement of domains in the heterostructures helps minimizing the interfacial energy and is supportive of an exchange mechanism in which ions are mobile enough to attain this stable configuration. a commonly observed trend was that the exchange was faster and could easily reach completion when performed on heavily substoichiometric cu2xse ncs, that is, ncs that initially presented a high density of cu vacancies, which points to vacancy diffusion as one of the main drivers of exchange. our strategy of starting from ncs with a high density of cu vacancies as templates should give therefore access to a wide range of nc materials under mild conditions (room temperature). we found that raman spectroscopy can easily discriminate between a sample of heavily substoichiometric ncs and a sample of closer to stoichiometric ncs, as the former presents a peak ascribable to a se se vibrational mode, which is instead absent in the latter. this allowed us to monitor the stoichiometry of any remaining cu2xse domains / ncs in partially exchanged samples. in all reactions tested, we always found that the stoichiometry of the nonexchanged domains was closer to cu2se, even when starting from nc samples with many cu vacancies. nevertheless, heavily substoichiometric ncs remained more reactive than the closer to stoichiometric ncs even at longer reaction times (several hours). the re - establishment of the cu2se stoichiometry should be mainly due to the diffusion of cu ions from the exchanged domains. however, if this was the only possible mechanism operative, we would expect a quick equilibration in reactivity between samples which initially differed in number of cu vacancies. we actually found that an important side reaction was the preferential extraction of se atoms from cu2xse ncs to the solution phase, operated by top, which was more efficient in heavily cu - deficient cu2xse ncs. while, over time, this extraction tended to reduce the density of cu vacancies in the nonexchanged cu2xse domains, it also involved the possibility for se ions to diffuse through the cu2xse lattice (in addition to cu ions). another notable finding was that, at higher temperatures (150 c here), the heavily substoichiometric cu2xse ncs were quickly converted to close to stoichiometric cu2se, mainly by fast extraction of se atoms by top. therefore, both cu2xse and cu2se ncs behaved similarly with respect to cation exchange, and the overall exchange efficiency was considerably lower than at room temperature. copper(ii) acetylacetonate (cu(acac)2, 97%), zinc nitrate hexahydrate (zn(no3)26h2o, 99%), zinc acetate (zn(oac)2, 99.99%), cadmium nitrate tetrahydrate (cd(no3)24h2o, 99%), 1-dodecanethiol (ddt, 98%), oleylamine (olam, 70%), octylamine (octam, 99%), oleic acid (olac, technical grade, 90%), tetrachloroethylene (tce, 99%), anhydrous methanol, toluene, and tetrahydrofuran were purchased from sigma - aldrich. cadmium oxide (cdo, 99.999%) and tri - n - octylphosphine (top, 97%) were purchased from strem. the synthesis of cu2xse ncs was performed using a standard schlenk line technique, similarly to the synthesis of cu2xseys1y nanoplatelets already reported by us. first, a se - precursor was prepared according to the previously reported method. se powder (10 mm ; 0.79 g) was mixed with 5 ml of ddt and 5 ml of olam, and the mixture was subsequently degassed under vacuum at 50 c for 1 h. se powder completely dissolved through the reduction by ddt, forming a brown stock solution of alkylammonium selenide, which was cooled to room temperature and stored in a n2 filled glovebox. in a typical nc synthesis, 524 mg of cu(acac)2 (2 mmol) was mixed with 6 ml of ddt and 19 ml of olam in a 100 ml three - neck round - bottom flask, and the mixture was degassed under vacuum (pressure 10 torr) and vigorous stirring at 60 c for 1 h. the flask was then filled with nitrogen and quickly heated to 220 c (45 min to reach the temperature). at this temperature, a mixture of 2 ml of the se - precursor (2 mm of se), prepared as described above, with 3 ml of ddt was swiftly injected from a syringe into the flask leading to a sudden color change from orange to greenish - brown. the reaction mixture was kept at 220 c for 4 min and then cooled to room temperature. to purify the cu2xse ncs, half of the prepared crude solution (15 ml) was centrifuged under inert gas atmosphere. the precipitate was washed twice by dissolution in 3 ml of toluene with subsequent addition of 1 ml of methanol (as a nonsolvent) and centrifugation. the cu2xse ncs were dissolved in 3 ml of toluene and stored in a glovebox. the other half (15 ml) of the reaction mixture obtained as described above was used for the reduction of cu2xse ncs toward close - to - stoichiometric cu2se ncs. for this, 524 mg of cu(acac)2 (2 mmol) was mixed with 3 ml of ddt, 6 ml of olam, and 9 ml of ode in a three - neck round - bottom flask, and the resulting solution was degassed under vacuum (pressure 10 torr) and vigorous stirring at 60 c for 1 h. then, the flask was filled with nitrogen and the temperature was raised to 120 c to dissolve cu(acac)2. thereafter, the temperature was lowered to 100 c and 5 ml of top was injected followed by subsequent addition of 15 ml of the crude reaction cu2xse nc mixture. the resulting mixture was maintained 20 min at 100 c with subsequent cooling to room temperature. the purification of the reduced ncs was performed in the same way as for the cu2xse nc sample. in a typical ce reaction, a certain amount of a 0.1 m methanol stock solution of a guest cation precursor (zn(no3)26h2o, or cd(no3)24h2o) was diluted with 0.51 ml of methanol and mixed with 1 ml of tetrahydrofuran, 0.5 ml of top, and 0.2 ml of cu2xse (cu2se) ncs in toluene (cu content = 0.050.06 mm). thereafter, the ncs were precipitated by centrifugation of the reaction mixture and washed by addition of 12 ml of toluene with subsequent sonication and centrifugation in order to remove organic residues. the precipitate obtained was washed twice in a similar way (with 12 ml of methanol) to remove the excess of cation salts. finally, the ncs were dispersed in 0.51 ml of toluene and stored in a glovebox. in the case of top treatment, the same procedure, except for the addition of the guest cation precursor, was followed. for quantitative experiments, ce reactions (cu cd and cu zn) were also performed at 150 c using a standard schlenk line technique. in the case of cu cd exchange, first, the cd - precursor was prepared by degassing a mixture of 38.4 mg of cdo with 0.4 ml of olac and 12 ml of ode in a three - neck round - bottom flask under vacuum (pressure of 10 torr) and under vigorous stirring at 60 c for 1 h. afterward, the flask was filled with nitrogen, heated to 250 c and kept at this temperature until complete dissolution of cdo (1520 min). then, the temperature was lowered to 150 c and, a mixture of 1 ml of the nc suspension in toluene (cu = 0.3 mm) with 2 ml of top was injected. some 1.52 ml samples of the reaction mixture were collected at 1, 2, 5, and 10 min after the injection, while keeping the mixture at 150 c. for cu zn exchange, a mixture of 1 ml of the ncs in toluene (cu = 0.3 mm), 0.3 ml of the zn - precursor (prepared by dissolving 10 mm of zn(oac)2 in 5 ml of olam and 5 ml of octam previously degassed), corresponding to a cu / zn ratio of 1, and 2 ml of top was injected in previously degassed ode (12 ml) at 150 c under inert gas. the mixture was kept stirring at 150 c with sampling at 1, 2, 5, and 10 min after the injection. the samples of the reaction mixtures were centrifuged, and the nc precipitates were washed 23 times by redispersion in 1 ml of toluene, addition of approximately 0.5 ml of methanol and subsequent centrifugation. the top treatment of the ncs was performed following the same procedure, but without addition of the guest cation precursor. samples were prepared by dropping diluted nc suspensions onto carbon coated 200 mesh copper grids for conventional tem analyses, with subsequent evaporation of the solvent. conventional tem imaging was done on a jeol jem-1011 microscope equipped with a thermionic gun (w filament) operating at 100 kv accelerating voltage. high - resolution tem (hrtem), energy - filtered tem (eftem) and energy - dispersive x - ray spectroscopy (eds) analyses were performed on a jeol jem-2200fs microscope equipped with a schottky emitter working at 200 kv, a ceos spherical aberration corrector of the objective lens allowing for a spatial resolution of 0.9, and an in - column imaging filter (-type). eds mapping and compositional quantification of the ncs was determined in scanning tem (stem)-high angle annular dark field (haadf) imaging mode, using a bruker quantax 400 system with a 60 mm xflash 6 t silicon drift detector. eds quantification was carried out using the cliff - lorimer ratio method for the cu k, se k, cd l and zn k peaks, and elemental maps were obtained by integrating the corresponding peaks in the eds spectra. for hrtem and eds chemical analyses, nc solutions were drop - cast onto ultrathin carbon - coated au grids and the experiments were carried out using an analytical double tilt be holder in order to minimize background and spurious signals. in case of partial cd exchange, better spatially resolved elemental maps were obtained by eftem (three - windows method), due to the favorable energy position of the m45 ionization edge of cd and the l23 ionization edge of cu (slit width : 30 ev for cd m45, 50 ev for cu l23). no comparably clear results were obtained by eftem mapping of zn. the geometric phase analysis (gpa) tool for gatan digital micrograph written by c. t. koch and v. b. zdl was applied to hrtem images for direct visualization of lattice constant variation in domains with different composition within individual ncs. the diffractometer was equipped with a cu source and a gbel mirror to obtain a parallel beam and to suppress cu k radiation (1.392). the samples were prepared by drop casting concentrated nc dispersions onto a zero background silicon substrate. absorbance spectra of ncs dispersed in tce were measured in 1 cm path length quartz cuvettes using a varian cary 5000 uv vis - nir spectrophotometer. these were performed under nitrogen atmosphere in a closed chamber (by linkam) in order to avoid laser - induced oxidation phenomena. spectra were collected using a renishaw invia microraman system exciting the samples at 633 nm by means of a 50 objective with a nominal power ranging from 0.5 to 5 mw and acquisition time from 10 to 100 s. inductively coupled plasma optical emission spectroscopy (icp - oes) analysis, performed on an icap 6000 spectrometer (thermoscientific), was used to quantify the composition of the ncs. the samples were digested in aqua regia (hcl / hno3 = 3/1 (v / v)) prior to measurements. in this work, we have developed a method for the synthesis of heavily p - doped cu2xse ncs that does not require any additional postsynthetic oxidizing treatment. they were prepared similarly to a synthesis of cu2xseys1y nanoplatelets, previously reported by us, with the only difference being the ratio between cu- and se - precursors of 1/1 (used here), as compared to 2/1 of our previous work. in the presence of an excess of se, copper selectively reacts with selenium (instead of ddt) and ultimately yields copper selenide ncs with nearly spherical shapes and diameters around 15 nm (see figure 1a). stem - eds analysis of individual ncs revealed a deviation of their composition from particle to particle : the cu / se ratio ranged from 1.28 to 1.50, giving an average cu1.42se0.97s0.03 composition. at the same time, the icp analysis of several samples from different batches revealed an overall cu1.60se0.93s0.07 composition (that is, icp gave a higher cu / se ratio than eds). the minor inclusion of sulfur in these ncs comes primarily from the passivating ddt molecules, although we can not exclude a partial inclusion of sulfur in the nc due to decomposition of some of the ddt molecules during the synthesis. the as - prepared ncs exhibited an intense localized surface plasmon resonance (lspr) with absorption maximum at 1100 nm (see the red spectrum in figure 1d), similar to that observed by our group on oxidized cu2xse ncs, which is attributed to the collective oscillation of holes. this is indicative of the presence of a significant number of copper vacancies in the as - prepared ncs. tem images of initial (a) and reduced cu2xse (b) ncs with corresponding xrd patterns (c) (red, before reduction ; black, after reduction). the experimental patterns are compared to database powder diffraction files of cubic cu7.16se4 (pdf card # 01 - 071 - 4325) and cu2se berzelianite (01 - 071 - 4843). optical absorption spectra of cu2xse ncs before (red) and after reduction (black) (d) and corresponding raman spectra (e). from these cu2xse ncs, we could prepare closer to stoichiometric ncs by in situ incorporation of cu ions. as seen from figure 1a c, after the reduction treatment the ncs preserved their size, shape and crystal structure (cubic berzelianite). filling of vacancies in the reduced sample with cu ions led to a slight dilation of the lattice unit cell (evidenced by a shift of the diffraction peaks in xrd to smaller bragg angles, figure 1c) and to the complete damping of the lspr in the nir (figure 1d). the increase in cu content in the reduced ncs was also proven by raman analysis and by compositional analysis (both eds and icp). the raman spectrum of the as - synthesized (that is, oxidized or vacant) cu2xse ncs (x = 0.58) evidenced a feature at 260 cm that can be attributed to a se se vibrational mode, whereas the spectrum of the reduced cu2se nc sample did not exhibit any remarkable feature, as already reported in the literature and as also observed by us in cu2xse and cu2xseys1y ncs with x < 0.2. the presence / absence of this raman - active se se vibrational mode was a useful tool to discriminate between stoichiometric and nonstoichiometric ncs in all the experiments that follow. according to the eds analysis of individual reduced ncs, the cu / se ratio varied from 1.63 to 2.09 (1.79 on average), with an average cu1.65se0.92s0.08 composition (see supporting information table si1 for details). again, icp analysis indicated instead a higher cu / se ratio (2.1, overall composition cu1.68se0.8s0.2). despite these differences in chemical quantification between icp and eds that affected all samples, both techniques estimated a 1020% increase in cu content going from the oxidized to the reduced sample. for the sake of simplicity, in the following we will refer to the initial, oxidized ncs as all these reactions involve the use of top as a necessary chemical favoring the ce reaction, since practically no ce was observed without it. the common justification is that top, as a soft base, promotes the extraction of the soft acid cu. on the other hand, it is also known that top can partially extract chalcogenide atoms from metal dichalcogenide ncs and transform them to metal chalcogenides (in some cases even at temperatures as low as 65 c). therefore, before starting the various experiments, we decided to test the stability of both cu2xse and cu2se ncs against top under the same conditions at which the ce reactions were carried out (and which will be discussed later). the ncs were incubated at room temperature overnight, after which they were precipitated by addition of methanol followed by centrifugation and were rinsed several times with methanol. xrd patterns were acquired on the ncs, while the presence of cu and se in the supernatant was quantified by icp. especially for the cu2xse sample, i.e., the one with high density of cu vacancies, the top treatment caused a considerable dilation of the unit cell, as can be seen in the xrd patterns of figure 2a, which corroborates a variation in the composition toward cu2se (that is, filling of the vacancies with cu). also, additional peaks, compatible with cu2se bellidoite (see bulk patterns in figure 2c), appeared in the top treated sample. this is a tetragonal phase in which the unit cell can be viewed as built from a stacking of 2 2 2 berzelianite cells along the three crystallographic directions, and slightly stretched (a bit more along c than along a and b). this expansion accommodates the larger number of cu atoms of cu2se bellidoite compared to cu2xse berzelianite. indeed, whereas the lattice parameter of berzelianite is 5.69, those of bellidoite are 11.52 (a, b) and 11.74 (c), both larger than 5.69 2 = 11.24. we conclude that a fraction of ncs remained in the berzelianite phase but with a stoichiometry closer to cu2se, while a fraction of ncs evolved to tetragonal bellidoite cu2se. less drastic changes in xrd peak positions were seen instead for the cu2se sample (figure 2b), although also in this case a fraction of ncs underwent a transition to cu2se bellidoite. xrd patterns of oxidized (a) and reduced (b) copper selenide ncs before and after the top treatment. the experimental patterns are compared to database powder diffraction files of tetragonal bellidoite cu2se (00 - 029 - 0575), cubic cu7.16se4 (01 - 071 - 4325), and cu2se (01 - 071 - 4843) (c). data from xrd were then compared with the results of elemental analysis (by icp) on the supernatant collected after precipitation of the ncs, which revealed the presence of both copper and selenium, a sign that the ncs were partially etched by top. etching did not change the average size and size distribution of the particles in an appreciable way (supporting information figure si1). taking into account 18.5% loss of se in vacant particles and their initial cu7.16se4 and final cu2se cubic phases, we calculated their size reduction from 15 to 14.1 nm, which apparently is within the error of the nc size estimation presented in supporting information figure si1. for the cu2se sample, the relative amounts of cu and se in solution were such that their ratio was 1.85:1 (essentially the same as in the ncs, see supporting information table si2), indicating roughly equal leakage of cu and se from the ncs. for the cu2xse sample instead many more se atoms than cu atoms were extracted (this time the cu : se ratio in solution was 0.45, that is, 1:2.2). the preferential extraction of se by top from the cu2xse ncs explains their evolution toward cu2se composition found by xrd, since the remaining unetched portions of the ncs became then enriched in cu. the same top treatment, at 150 c, also led to cu enrichment of the nc composition, by extraction of se by top, in both cu2xse and cu2se ncs. the notable difference, with respect to the room temperature case, was that this time the se extraction, and therefore filling of cu vacancies, was completed already within 1 min of the reaction (see supporting information table si3), and further heating during the following 30 min did not induce any significant change in the composition of the ncs, both for the initial cu2xse and cu2se samples. we will come back to the influence of top on ce later in this work. the as - prepared cu2xse ncs and the reduced cu2se ncs underwent partial ce reactions. for the cu cd case, we tested various ratios of added cd ions to cu ions in the ncs, at room temperature : from 1:20 (0.05) to 1:1 (see supporting information table si4). since there will be a replacement by one cd ion every two cu ions, even if all the cd ions added are taken up by the ncs, we expect that the exchange will be closer to completion only for the 1:2 (0.5) and 1:1 cases. as can be seen from the eftem maps of figure 3a, b, a typical product of such reactions contained janus - like particles of separated cu- and cd - containing domains, suggesting the formation of cdse in the exchanged regions (in line with the immiscibility of cu2se and cdse seen in the bulk) and indicating that ce started at one location of the particles and from there it propagated through the nc. we rationalize the formation of janus particles by considering that cations with low coordination with the se sublattice, such as the cd ions discussed here and the zn ions, which will be analyzed shortly, should have higher diffusivity in the cdse and znse phases, respectively, than in the cu2xse phase, in analogy to the findings of ha. for cu cd (zn) ce reactions in cu2s ncs. therefore, ion replacement can be initiated and propagated in a way that a partially exchanged structure is the result of various steps that have eventually led to a relatively stable configuration. such events are likely to be initial ion diffusion throughout the nc lattice, preferential exchange with the cu cations in the most energetically favored locations, and subsequent growth of the cdse and znse domains by a constant supply of the guest cations through the corresponding guest phase, in a way that the overall exchanged domain (cdse or znse) of a nc can minimize its interfacial area with the remaining nonexchanged (cu2xse) domain. cu2se ncs obtained by partial ce at room temperature from vacant cu2xse ncs (a), with eftem mapping of cu (red) and cd (green) (b). note that se maps are not shown in the images, as no appreciable variation is observed over individual ncs. cu2se nc with corresponding mean dilation map as obtained by gpa (d) and fft (e). the experimental patterns are compared to database powder diffraction files of tetragonal bellidoite and cubic alpha cu2se (pdf cards 00 - 029 - 0575 and 00 - 101 - 0581, respectively), cu7.16se4 (01 - 071 - 4325), and cdse (01 - 088 - 2346). the red vertical dotted line represents the position at which the se se vibrational mode in cu2xse should be observed. diagram displaying the evolution of the cd / cu ratio in the heavily substoichiometric (cu2xse, red) and close to stoichiometric (cu2se, black) ncs over the time of the cu cd ce reaction (h). since the lattice parameter of cubic cdse is larger than that of cu2se (see supporting information table si5), we could monitor the evolution of the reactions both by hrtem and xrd. a typical hrtem image of a janus particle (figure 3c) revealed matching of lattice parameters with cubic cdse (a = 6.1) and cu2se (a = 5.8) in the respective domains, confirmed by 4.5(0.9)% mean dilation in the cdse domain relative to the cu2se one as obtained by gpa (see figure 3d). the fast fourier transform (fft) pattern presented in figure 3e evidences the epitaxial orientation between the two phases, where red and green arrows point at spots corresponding to cu2se and cdse phases, respectively. figure 3f reports xrd patterns of partially exchanged ncs, starting from cu2xse ncs, and characterized by different exchange yields. already by incorporation of a small amount of cd, the structure of the unexchanged cu2xse ncs transformed to tetragonal cu2se bellidoite phase. two concurrent effects, both causing the decrease in the density of cu vacancies in the nonexchanged domains, may be responsible for this transformation to bellidoite : the preferential se etching operated by top on ncs (as discussed earlier), and the likely diffusion of the cu ions expelled from the exchanged domains toward the not yet exchanged cu2xse ncs. further exchange led to the increase of the cubic cdse peaks and to the fading of cu2se ones. raman spectroscopy allowed us to make additional conclusions on structure and composition. a typical raman spectrum of a sample of cdse janus ncs obtained from cu2xse ncs (figure 3 g, red spectrum) exhibited a sharp peak at 207 cm, which can be assigned to the longitudinal optical phonon mode of cdse and its overtone at 414 cm. no other peaks were present, so that we could exclude the presence of alloyed phases, as well as any nonstoichiometric cu2xse (as we saw no peak at 260 cm), which suggests that the unexchanged copper selenide domains / ncs had a low density of cu vacancies. it is noteworthy that basically the same type of spectrum was recorded on partially exchanged samples starting from cu2se ncs (figure 3 g, black spectrum). it appears that both partial exchange reactions, the one on cu2xse ncs and the one on cu2se ncs, converged essentially to the same type of heterostructures. on the other hand, an important outcome of these experiments was that ce was more efficient when done on cu2xse ncs than on cu2se ones. we compared, for example, the kinetics of the cu cd exchange on cu2xse and cu2se ncs by monitoring two parallel reactions starting from a ratio of cd ions to cu ions (feed ratio) equal to 0.5 (1:2). the evolution of the actual cd : cu ratio in the ncs over the reaction time is summarized in figure 3h (additional data are reported in supporting information table si6 and figure si2). as follows from the results, the cu2xse ncs underwent ce much faster than the cu2se ones : a roughly 10-fold difference between the cd : cu ratios in the two samples (histograms of figure 3h) at the beginning of the reaction developed to a 30-fold difference after 3 h. after 7 h, the cd / cu ratio in the initial cu2xse ncs had reached 0.45, while in the sample prepared from the initial cu2se ncs it was only 0.015. these experiments demonstrate the impact of copper vacancies on the kinetics of ce. in fully stoichiometric compounds, cations occupying regular sites in the crystal lattice can move to an interstitial site leaving a vacancy behind. such interstitial - vacancy pairs (known as frenkel pairs) can move through the solid by hopping from site to site. this process is limited by solid - state diffusion within a nc, as shown by groeneveld. for zn cd ce. for ncs with a large number of vacancies acting as carriers of both host and guest cations, this diffusion should proceed much faster. on the other hand, we verified that, even for the cu2xse ncs, the exchange rate does slow down over time. this can be seen from figure si2 of the supporting information, which reports the experimental cd / cu ratio in cu2xse ncs over several hours (red markers). the cd / cu ratio followed a linear trend, corresponding to a growth rate of the volume fraction of cdse that follows a c/(1 + ct) dependence over time t (with c equal to a constant, see discussion in the supporting information), that is, the exchange rate slowed down over time, despite our experiments still having a considerable amount of cd ions and of available top in solution. a linear growth of the volume fraction of cdse, on the other hand, would correspond to a time evolution of the cd / cu ratio that is proportional to t/(1 kt) with k equal to a constant (see supporting information), which is steeper than linear, especially at later times. one reason for such a slowdown in the growth rate can be the filling of cu vacancies, although other parameters might play an important role and will require further scrutiny. however, a general consideration that should hold is that the ease of formation of cu vacancies in copper selenide will make the exchange rate in this material always higher than a rate determined by frenkel defect diffusion or by an interface - controlled reaction. further increase of the initial cd : cu ratio up to 1, i.e., by adding double excess of cd precursor relative to cu (considering this reaction as 2cu cd), led to practically complete exchange of copper ions in the initial substoichiometric cu2xse ncs, with a resulting cd : cu ratio of 373, whereas in the initial close to stoichiometric cu2se particles, this ratio was only 22 (as a reminder, the reactions were run overnight, see supporting information table si4). these results again clearly indicate the difference between the two samples and represent an important guideline when exploiting ce reactions as a means to prepare materials in which the amount of impurity atoms has to be minimized. also, in analogy with the case of blank top treatment discussed above, we always found se in significant amounts in supernatant solutions at the end of the reactions. the loss of se was independent from the cd : cu feed ratio and was more pronounced for the cu2xse ncs than for cu2se ncs. by quantifying the content of selenium in ncs and in the solution, we estimated that the cu2xse ncs had lost around 30% of se, while the se loss for the cu2se ncs was half of that value, around 15%. this observation suggests that ce goes through at least partial etching of the ncs, which again is more pronounced in ncs with higher density of cu vacancies. also, in line with previous works on ce, we note that top is an important ingredient : without top, even starting from a cd : cu feed ratio equal to 1, basically no exchange took place (the cd : cu ratios were 0.013 starting from cu2xse ncs and 0.003 starting from cu2se ncs). similar to the case of the room temperature cu cd partial ce discussed above, the reaction at a higher temperature (150 c) yielded cdse however, differently from the room temperature reactions, at 150 c we did not observe a remarkable difference in the ce kinetics between the cu2xse and the cu2se samples (see supporting information table si7 and figure si4), as both samples exchanged with a similar rate (which was even slightly higher for the initial close to stoichiometric cu2se ncs). this can be attributed to a quick filling of the cu vacancies by rapid extraction of se atoms by top. one additional potential reason for similarity in reactivities for the two samples is that, at 150 c, the extracted cu ions may start competing with the cd ions (or with the zn ions, as we shall see briefly) that are still in the solution phase for entry in the ncs (re - entry in the case of cu), which should further contribute to a fast reduction of the density of cu vacancies in the ncs. in a first series of experiments, the ratio of zn ions added to cu ions in the ncs was set to 1:4 (0.25). here, as in the cd case figure 4a, b reports haadf - stem images and superimposed cu and zn stem - eds compositional maps over groups of ncs, after exchange on the cu2xse (panel a) and cu2se (panel b) ncs. in both samples, all particles exhibited a janus structure, in line with the results on cd discussed earlier. unfortunately, for this system neither hrtem (figure 4c) nor xrd (figure 4f) could confirm that the exchanged domain was pure znse, that the nonexchanged domain had remained copper selenide (cu2xse or cu2se), and that no partial ternary alloy compounds had formed (however never reported for the bulk), due to the low mismatch between cubic znse and cu2xse (see supporting information table si5). for example, in the hrtem image of the janus particle reported in figure 4c, no variation of the lattice parameter (a = 5.8) is appreciated throughout the nc. raman spectroscopy, on the other hand, was more informative (figure 4 g). for the znse cu2se ncs samples prepared from cu2se ncs, as well as for the znse ncs prepared from cu2xse, the raman spectra exhibited only one band peaked at 240 cm, which can be interpreted as the longitudinal optical phonon mode of znse. no other peaks were present, not even the one at 260 cm of the initial cu2xse ncs, which would fall in the region marked by the red dashed line in figure 4 g. this indicates that the unexchanged copper selenide domains, in both samples, had compositions close to cu2se. it also excludes the formation of alloys, again in line with the immiscibility of cu2se and znse observed in the bulk. haadf - stem images and corresponding stem - eds cu (red) and zn (green) elemental maps of znse cu2se ncs obtained from oxidized cu2xse (a) and reduced cu2se (b) ncs by partial ce at room temperature. note that se maps are not shown in the images, as selenium was homogeneously distributed over individual particles. cu2se janus - type nc (c) with corresponding fft (d) and stem - eds map (e). the experimental patterns are compared to database powder diffraction files of cubic cu2se (pdf card 01 - 088 - 2043), and znse (01 - 071 - 5978). cu2se ncs derived from cu2se and cu2xse ncs (g). here again, the red vertical dotted line marks the position at which the se se vibrational mode in cu2xse should be observed. diagrams displaying dependence of the zn / cu ratio in the vacant (red) and reduced (black) ncs on the zn / c feed ratio in the cu zn ce reaction (h). as in the cd case, the exchange with zn was more efficient on ncs that had initially a large number of cu vacancies (see supporting information table si6). figure 4h reports zn : cd ratios in the ncs (as measured by icp) for both cu2xse and cu2se ncs when working with a feed ratio of zn : cu of 1:4 and 1:1, at room temperature. again, it is especially attractive that almost full exchange (zn : cu ratio of 67) at room temperature was possible for the cu2xse ncs by employing only double excess of zn ions relative to cu (we remind that one zn ion replaces two cu ions), while, for the initial cu2se sample, the exchange under the same conditions yielded ncs that contained still a considerable fraction of cu (zn : cu ratio was around 5). overall, cd and zn ions behaved quite similarly at room temperature, in terms of exchange kinetics and of their dependence on initial density of cu vacancies, as well as in terms of structure and composition of intermediate exchange products. when the cu zn exchange was carried out at 150 c, we did not observe any significant difference between cu2xse and cu2se nc samples, similar to the high temperature cu cd ce reactions discussed above (see supporting information table si9 and figure si5) : after 10 min of reaction, the zn : cu ratio had reached 0.33 in the case of cu2xse and 0.28 in the case of cu2se ncs. again, this implies that copper vacancies were quickly filled, since already after 1 min of the reaction the zn ions had replaced approximately 30% and 20% of cu ions in cu2xse and cu2se ncs, respectively, after which the exchange slowed down considerably (see supporting information table si9). also, the structure of the resulting particles was similar to that of room temperature exchange products, i.e., janus znse cu2se dimers, without detectable formation of ternary cu zn se alloy phases. overall, by comparing the cu zn and cu cd exchange reactions at 150 c, we can conclude that, under the same experimental conditions, cd ions were more reactive toward the ncs than were the zn ions. it appears that, under these conditions, ce will be favored thermodynamically by a higher bond strength of the newly forming phase, which in our case is cdse, since the bond dissociation energies (enthalpy changes) of cd se, cu se, and zn se bonds are 310, 293, and 136 kj / mol, respectively. the advantage of room temperature ce that needs to be emphasized here is that even a cd(zn)/cu feed ratio equal to 1 is sufficient to achieve almost complete exchange. at the same conditions, but at 150 c, for example only 35% of copper ions are replaced by zn ions. the latter results are in line with published works, in which quantitative exchange of cu ions by zn at high temperatures was made possible only by employing a large excess of zn ions. the major conclusion of this work is that cu vacancies play a key role in cation exchange reactions involving copper selenide ncs, as their presence accelerates the exchange process. therefore, the use of ncs with a high density of cu vacancies, as done in this work, can simplify cation exchange reactions and make them more practical, for example, by significantly reducing the ratio between host and guest cations and by working under mild conditions, for example, at room temperature. moreover, room temperature conditions were found advantageous compared to higher (150 c) temperature conditions, owing to the preservation of copper vacancies over time, which resulted in a much more efficient exchange on substoichiometric cu2xse ncs. also, since top acts as both complexing agent for cu ions and for se (in the form of se - top), it is conceivable that its role as an enhancer of cation exchange is more multifaceted than previously thought. we additionally believe that partial exchange processes investigated in this work can be applied to other copper chalcogenide ncs yielding janus - like structures. moreover, by subjecting such synthesized cu2x zn(cd)x dimers to a further exchange, it should be possible to selectively convert the unexchanged cu2x domain to yet another material, thus giving accessibility to a wide range of heterostructures.	we have investigated cation exchange reactions in copper selenide nanocrystals using two different divalent ions as guest cations (zn2 + and cd2 +) and comparing the reactivity of close to stoichiometric (that is, cu2se) nanocrystals with that of nonstoichiometric (cu2xse) nanocrystals, to gain insights into the mechanism of cation exchange at the nanoscale. we have found that the presence of a large density of copper vacancies significantly accelerated the exchange process at room temperature and corroborated vacancy diffusion as one of the main drivers in these reactions. partially exchanged samples exhibited janus - like heterostructures made of immiscible domains sharing epitaxial interfaces. no alloy or core shell structures were observed. the role of phosphines, like tri - n - octylphosphine, in these reactions, is multifaceted : besides acting as selective solvating ligands for cu+ ions exiting the nanoparticles during exchange, they also enable anion diffusion, by extracting an appreciable amount of selenium to the solution phase, which may further promote the exchange process. in reactions run at a higher temperature (150 c), copper vacancies were quickly eliminated from the nanocrystals and major differences in cu stoichiometries, as well as in reactivities, between the initial cu2se and cu2xse samples were rapidly smoothed out. these experiments indicate that cation exchange, under the specific conditions of this work, is more efficient at room temperature than at higher temperature.
the construction of specific surface architectures via controlled self - assembly is a key goal in the design of nanoscale molecular electronic devices. in this respect, particularly, attractive candidates are guanine (g) and its derivatives, which are unique among the dna constituents for their propensity to form quadruplex structures, known to be stabilized through hoogsteen and watson - crick base pairing [1, 2 ]. moreover, guanine has a low ionization potential due to which it plays a key role in electrical conductivity of dna - based materials. various research groups have recently reported on the evolution of the art of making surface deposited 1-dimensional (1d) structures known as guanosine wires (g - wires) [312 ]. however, despite the promising experimental results, basic processes responsible for wire formation are still far from being fully understood. two main steps are fundamental to drive the topic from a laboratory curiosity towards the technologically relevant g - wire engineering : (a) achieving control of self - folding and self - stimulated end - to - end fusion of the macromolecular quadruplexes in solution as a function of their molecular composition (i.e., base sequence) and solution conditions, and (b) understanding the effect of surface interactions on material deposition from solution phase onto the solid substrate. in this work, we report on how particular modifications of the base sequence of a g - rich oligonucleotide, which are supposed to affect the folding geometry of the quadruplex, affect its ability to form g - wires in solution phase. the idea of wire formation is based on the previously established finding that gc sticky ends may be used to link quadruplexes into longer 1d aggregates. a series of oligonucleotide sequences with one or two gc ends and central sequences of different length were designed for this purpose (see table 1). the folding topology [14, 15 ] has been previously investigated, and quadruplex formation has been recently studied by mergny.. in these studies, length and nature of propeller loops were studied, but end - to - end fusion of the quadruplexes was not considered. here, we focused on end - to - end fusion of these quadruplex folds. polyacrilamide gel electrophoresis (page) is a conventional tool to differentiate between macromolecular objects of different length. during migration through an alternative noninvasive method for determining the size of macromolecular objects is dynamic light scattering (dls). this method was extensively applied to study dna molecules over a wide range of sizes [1721 ]. specifically, it has been used to characterize g - quartet stacking in solutions of single guanosine molecules [2226 ], and for studying formation of g - quadruplexes [2730 ]. protozanova and macgregor compared the use of dls and page on sequences with long terminal tracks. these tracks were based on sequences such as d(a15g15) and d(a13g15tc), which form frayed wires with a g - quadruplex stem and nonguanine portions reaching out as single - stranded arms. we investigated the formation and dimensions of the supramolecular objects formed in aqueous solution of specially designed g - rich dna sequences (table 1) by combining dls and page measurements. we studied the effects of temperature, oligonucleotide concentration, method of preparation, and base sequence to explore the basic principles of g - wire formation. oligonucleotides (see table 1) were ordered from eurogentec (belgium) as 40 nm desalted syntheses and reconstituted in water. dialysis was performed at a concentration of 100 m dna in the presence of 100 mm nacl buffered with 10 mm napi at ph 6.8, then diluted in same concentration of buffer or h2o to specified concentrations immediately prior to measurements. where heat treatment is specified, oligonucleotides at 100 m dna in presence of 100 mm nacl, 10 mm napi (ph 6.8) were heated to 93c for ten minutes and left to cool in heating block to room temperature prior to experiment. for dls measurements, the oligonucleotides with a 0.4 mm concentration were used. for the concentration dependence studies gel electrophoresis experiments were performed on 15% native bis / acrylamide gels, utilising 1x tbe running buffer supplemented with 5 mm nabo2 to retain folded architectures. oligonucleotide samples were prepared to a final concentration of 2 m dna in presence of 100 mm nacl buffered with 10 mm napi at ph 6.8 with 5% sucrose to facilitate sample loading. gels were run at 120 v and 4c for a maximum of 2 hours and stained with 1x sybr gold (invitrogen, paisley, uk) in 1x tbe buffer. in order to compare migration rates between different oligonucleotide samples, a generuler ultra low range (10200 bps) dna ladder (fermentas, york, uk) was used. dynamic light scattering experiments (dls) were performed using a digital correlator (alv - laser vertriebgesellschaft), a goniometer, and a photomultiplier detector. the light source was a frequency doubled nd : yag laser with a wavelength of 532 nm. the incident laser beam was linearly polarized in the direction perpendicular to the scattering plane. the scattered light of the same polarization was detected at scattering angles 50 130. the capillary containing the sample had an inner diameter of 5 mm and was immersed in an index matching bath with a diameter of 10 cm to minimize stray light from the outer capillary wall. we measured the autocorrelation function g2(t) = i(0)i(t)/i of the average scattered light intensity i. most of the measurements were performed in a mixed regime, in which the intensity autocorrelation function is related to the field correlation function g1(t) by the relation (1)g2(t)=1 + 2(1jd)jdg1(t)+jd2g12(t), with jd being the ratio between the intensity of the light that is scattered inelastically and the total scattered intensity. the field correlation function g1(t) for systems with a polydisperse size distribution can be expressed by stretch exponential functions (2)g1(t)=iaiexp (ti)i, where ai is the amplitude of the ith relaxation mode and the stretch exponent lies in the range 0 i 1. the average relaxation time of the ith relaxation mode is given by (3)i=ii(1i), where (1/i) is the gamma function. the parameter i is a measure of the width of the distribution of relaxation times. very narrow distributions correspond to i ~ 1, while smaller values of i indicate broader distributions. the characteristic relaxation times of the observed dynamic modes were obtained by fitting the measured autocorrelation curves g2(t) to (1) and (2). according to the polarized scattering (so called vv scattering) detected in our experiments, the translational diffusion coefficients were calculated as (4)di=1iq2, where q is the scattering wave vector given as q = (4n/)sin (/2) with n = 1.33 being the solution refractive index, the laser wavelength and the scattering angle. in most cases, several correlation curves were measured for every solution and averaged values of the fitting parameters were taken for further consideration. from the translational diffusion coefficients, the corresponding hydrodynamic radius is calculated as (5)rh = kbt6d, with kb being the boltzmann constant, t the absolute temperature, and the solvent viscosity. for dilute solutions of rod - like particles the hydrodynamic theory of tirado and garcia de la torre [3436 ] can be applied as long as the ratio of length to diameter p = l / d is in the range 2 p 30. the translational diffusion coefficient in this theory is given by (6)d = kbt3l(ln p+), where is the end - effect correction term given as = 0.312 + 0.565/p 0.100/p. knowing the diameter of the studied rod - like assembly, thus the length of the objects can be estimated. the sequences were first investigated by page to check for folding of oligonucleotides into higher order architectures. samples prepared by heat treatment show a lesser degree of macromolecular assembly as compared to dialysed samples for all investigated oligonucleotides (figure 1). the band with the highest mobility in each lane corresponds to a bistranded monomeric unit. the shortest dialysed sequences (sequences 2 and 3) show only few albeit sharp bands pointing to the formation of smaller discrete stable species and a very low degree of macromolecular self - assembly. the longer sequences 1, 5 and 6, on the other hand, indicate possible stepwise assembly into large aggregates. in all investigated samples, we detected a slow diffusive mode with correlation times in the range of 110 ms (at = 90) (figures 2 and 3). such a mode corresponds to translational motion of large globular aggregates with hydrodynamic radii in the range of micrometers. they are usually attributed to the presence of loose multichain associates formed due to electrostatic interactions, but their nature is still not quite resolved [20, 37, 38 ]. as these aggregates are to our opinion not connected with g - wire formation, they will not be further considered. in all heat treated samples, only the above mentioned slow mode exists, so they will not be further discussed. solely the slow mode is observed also in the dialysed sequence 2 (figure 3(a)), which is the only sequence containing adenine base. in dialysed sequence 3, an additional fast diffusive mode can be faintly resolved at large scattering angles (> 90). the corresponding diffusion coefficient calculated from (4) has a value of df = 3.0 0.3 10 m / s (table 2), which indicates fast translational motion of very small scattering objects, most probably single oligonucleotides. in dialysed sequence 4, the fast mode is more profound and can be clearly resolved (figure 3(b)). the dls autocorrelation functions of dialysed sequences containing gggg repeats (sequences 1, 5, and 6) all show two relaxation modes. the angular dependence of the inverse relaxation time of the fast mode (figure 4) reveals quadratic dispersion as given by (4). the resultant diffusion coefficients df obtained for solution concentration of 0.4 mm are listed in table 2. the obtained values suggest that the fast mode is most probably associated with translational motion of oligonucleotides assembled into g - quadruplex structures. the dialysed sequence 4 at 0.4 mm dna was also used to investigate the temperature dependence of the fast diffusive mode (figure 5). a relative amplitude of the fast mode decreased with increasing temperature and for t > 338 k the fast mode could not be resolved anymore. to reveal possible temperature - induced structural changes of the scattering objects, the values of diffusion coefficient df obtained at different temperatures were divided by temperature t and multiplied by the corresponding solvent viscosity (t). the resulting values of df(t)/t as a function of temperature are shown in figure 5. one can notice that they remain constant throughout the investigated temperature range, which indicates that no structural modification of the scattering objects (quadruplexes) takes place. the effect of oligonucleotide concentration on the value of df was investigated for dialyzed sequence 1 (figure 6). although it appears that the values decrease with increasing concentration, no precise conclusion can be drawn due to the large uncertainty of the measured data points. at low solution concentrations and short relaxation times however, for dna concentrations below 1 mm, which was the case in all our measurements, the effect of electrostatic interactions on solution dynamics is usually negligible and so the solution can be considered as infinitely dilute. both, page and dls analysis, reveal that the method of sample preparation is essential for the level of self - assembly in solution. we observe formation of slower migrating species for all oligonucleotides in page for the dialysis treated samples. sequences 1, 2, 5, and 6 appear to form larger assemblies, whilst sequences 3 and 4 do not. the most striking results are for sequences 2 and 6, where one or two species of lower molecular weight were formed in the annealing treatment versus numerous larger species through dialysis. page comparison between sequences 2 and 3 shows that there are sequence effects in the kinetics of formation for the same topology. when comparing dialysis, a band appearing at nominal 15 bp in sequence 3 appears only very faintly in sequence 2, while a faster, and a slower, migrating bands appear quite distinctively in both. the effect of sticky ends is also shown when comparing sequences 1 and 6. while between nominal 10 and 35 bp they have the same number of bands appearing in approximately the same positions, their intensities vary substantially. for these particular sequences, the difference in the annealing treatment is greater, with sequence 6 showing a very strong fast migrating band at nominal 16 bp. in contrast, the fastest migrating band for seq1 is at nominal 12 bp. in general, and as expected, it is apparent that the greater the oligonucleotide length the longer the species formed. indeed, it is apparent for the longest oligonucleotide sequence (sequence 5) that the macromolecular dna objects break into smaller units in a less discrete manner than for the other sequences. the smear for low migrating dna in sequence 5 may thus be due to steric hindrance by the pore size of the gel. for the shortest of the dialysed sequences, sequences 2 and 3, the absence of any measurable fast dls mode for the former and the observation of a faint extremely fast mode for the latter, can be explained only by the presence of very small scattering objects, most probably single oligonucleotide molecules. this finding is not consistent with page results (figure 1), which reveal additional slower diffusing species resulting in sharp bands, probably signifying multimers. similar discrete bands are seen also in the picture of page for dialysed sequence 4. for this sequence, the diffusion coefficient of the fast dls mode gives quadruplex length of 8.8 0.8 nm (table 2), which is considerably larger than the length of a single oligonucleotide strand (about 4.8 nm). the two dialysed intermediate - length sequences (sequences 1 and 6) both exhibit the same page band attributed to fast migrating monomers and a strong band at about 35 bps (accordingly to the reference duplex dna ladder). the main difference between the two oligonucleotides is in the formation of two intermediate bands for sequence 6, while sequence 1 forms no intermediate species. interestingly, the two strong bands (at 35 bps) very well agree with the quadruplex length l estimated from the dls measurements, which is 5.0 0.4 nm for sequence 6 and 6.1 0.5 nm for sequence 1, respectively. the smaller apparent length of sequence 6 can be explained by the presence of the intermediate species contributing to the scattering. the diffusion coefficient measured for sequence 1 (df = 1.06 0.037) is in very good agreement with the values obtained for similar folded structures by hydrodynamic modelling. in page, the majority of the material is so slowly migrating through the gel, that just a broad smearing is observed. dls measurements, on the other hand, reveal the presence of supramolecular assemblies with the length of 11.0 0.2 nm, which is only slightly larger than the length of a single strand. in contrast to page, dls also reveals a relatively narrow distribution of aggregate lengths. this is seen from the dls stretch exponent factor for the fast mode, which is in the range 0.9 100 m, this is not consistent with what we intuitively know, that is, higher concentrations tend to favour multimer formation. another aspect arises from the slow dls mode, which signifies that in addition to wire - like assemblies there are also some other aggregate types present in solution. these aggregates, most probably loose multipolyion associates, might correspond to the slow diffusing objects observed in page. dls experiments bring also information on temperature and concentration dependence of the self - assembly. the temperature dependence (figure 5) shows that up to t = 65c the length of the self - assembled objects remains constant. nevertheless, the amplitude of the fast dls mode strongly decreases by increasing temperature, while the overall scattering intensity is only slightly reduced. this signifies that the slow mode, which mainly contributes to the scattering intensity, is not much affected by heating. but relatively to it, the scattering intensity related to the fast mode becomes more and more weak. this can be explained by temperature - driven dissociation of the self - assembled structures of well - defined size into much smaller single oligonucleotides, which are practically invisible by dls. dependence of the fast dls mode on the concentration of the solution (figure 6) also supports the idea of formation of the self - assembled structures with well - defined length, which are not affected by modifications of the solution concentration. on the contrary to wire - like assemblies formed from single guanosine molecules (gmp), it seems that formation of supramolecular assemblies of g - rich oligonucleotides, does not exhibit any critical solution concentration, at which the assembling would become profound. a series of g - rich oligonucleotides was studied by page and dls to investigate the formation and dimensions of g - quadruplexes. both methods show that thermal annealing induces much less macromolecular self - assembly than the dialysis method. this demonstrates that not only the base sequence, but also the folding kinetics play an important role in the self - assembly process. on one hand, this makes the phenomenon very complex, but on the other hand it provides a possibility for fine tuning of the assembling features via external stimuli. further studies are needed to find the source of the differences and how they can be modulated. page and dls show the best agreement on quadruplex dimensions for sequences of intermediate length (sequences 1, 4 and 6). the last band of sequence 4 coincides with the dls signal arising from aggregates of a 9 nm length. the two similar sequences 1 and 6 give also aggregates of similar length 56 nm and these agree with a strong page band observed for both sequences. for the shortest sequences 2 and 3, page suggest the formation of multimers not detected by dls. the only dls signal comes from very fast diffusing objects with effective dimensions below 1 nm pointing to single oligonucleotides. for the largest sequence, on the other hand, page suggests very long aggregates giving a broad smeared band at the beginning of the lane. in dls instead, a well defined fast mode is attributed to a species with the length of about 11 nm. because we are able to assess intermediate lengths, we are currently investigating the mechanism of self - assembly of these wires by combining both methods. thus, the combination provides valuable information on the g - quadruplex formation towards control of its length.	the formation and dimensions of g - wires by different short g - rich dna sequences in solution were investigated by dynamic light scattering (dls) and polyacrilamide gel electrophoresis (page). to explore the basic principles of wire formation, we studied the effects of base sequence, method of preparation, temperature, and oligonucleotide concentration. both dls and page show that thermal annealing induces much less macromolecular self - assembly than dialysis. the degree of assembly and consequently length of g - wires (5 - 6 nm) are well resolved by both methods for dna sequences with intermediate length, while some discrepancies appear for the shortest and longest sequences. as expected, the longest dna sequence gives the longest macromolecular aggregates with a length of about 11 nm as estimated by dls. the quadruplex topologies show no concentration dependence in the investigated dna concentration range (0.1 mm0.4 mm) and no structural change upon heating.
alloantigen - specific t cells and b cells cause acute cellular and humeral rejection through the recognition of graft antigen by highly evolved immune receptors. these receptors, t cell receptors and immunoglobulins, are capable of recognizing an immense variety of antigens due to their numerous encoding genes and due to the process of somatic rearrangement of their encoding dna. the immense diversity of the cell receptors also predicates that for a novel antigen, only a limited pool of lymphocytes will have specificity towards that antigen. consequently, in order to conduct an effective immune response, intense expansion of antigen - specific lymphocytes is required. because this expansion may take several days, a more immediate defense system is also required to address microbial invasions that are capable of rapid progression. the innate immune system has come to the forefront of immunological research with the discovery of toll - like receptors (tlrs) (reviewed in [1, 2 ]) along with the appreciation that the context in which the antigen is recognized is critical for promoting the immune response. tlrs are pattern recognition receptors (prrs) that are expressed on both nonlymphoid and lymphoid tissues, especially antigen- presenting cells such as dendritic cells and macrophages. their ligation initiates intracellular signal transduction cascades that lead to nf-b activation and the upregulation of the adhesion molecules, costimulatory molecules and cytokines that are essential to immune activation [4, 5 ]. characterization of the ligands and function of the various tlrs has revealed that the innate pathways are critical to the development of a robust adaptive immune response [13, 69 ]. unlike the immensely variable antigen recognition epitopes of t cell receptors and antibodies, tlrs have a fixed genomic structure and are capable of binding a limited repertoire of ligands. some of the resultant lack of variability is overcome by the presence of multiple receptor types ; for example, there are currently 13 tlrs identified in mice and humans. despite their limited antigen recognition capability, their conservation between evolutionarily distant species hints that they may bind molecules that are indispensible to microbes such that they can not be mutated or ablated. the benefit of the tlr fixed receptor structure is that a large number of innate immune cells can recognize a pathogen and respond immediately. tlrs have been identified with affinities for molecules associated with infection and tissue injury. however, their ability to recognize pathogen - associated molecular patterns (pamps) is best described. some tlrs (tlr1, 2, 4, 5 and 6) are located on the outer cell membrane and recognize microbial molecules derived from bacteria, fungus, and parasites (reviewed by akira., 2006) for example, tlr2 recognizes bacterial peptidoglycan, fungal phospholipomannan, and trypanosomal tgpi - mutin and tlr4 recognizes bacterial lipopolysaccharide (lps), fungal mannan, and trypanosomal glycoinositol phospholipids. other tlrs (tlr3, 7, 8 and, 9) are located within the in the endosomal / lysosomal compartment and bind bacteria- and virus - derived nucleic acids. for example, tlr3 binds viral double - stranded rna, tlr7 and tlr8 bind viral single - stranded rna, and tlr9 binds bacterial and viral double - stranded dna. tlrs share homology with the type i transmembrane toll receptor discovered in the fruit fly (drosophila melanogaster) initially identified for its role in controlling dorsal - ventral polarity during embryogenesis. it was later discovered that toll also induces production of antimicrobial peptides in response to fungal infection in adult fruit flies. considering the role of the toll receptor in development as well as primitive innate immunity, it is not surprising that tlrs have endogenous ligands in addition to microbial ligands. endogenous tlr ligands arising from tissue damage are termed damage - associated molecular patterns or damps, and they are becoming increasingly recognized for their role in immune regulation (figure 1) [1214 ]. examples include heat - shock protein 60 (hsp60), hsp70, surfactant protein a, -defensin 2 high - mobility - group box 1 protein (hmgb1), and extracellular matrix molecules such as hyaluronan, fibronectin, and and heparan sulfate [16, 17 ]. some controversy exists with regards to potential contamination of damps with pamps (e.g., lps) resulting in false positive results of tlr stimulation [1820 ]. this is especially relevant to protein stimulators that have been synthesized from bacterial recombinant technology. nonetheless, there is accumulating evidence for the role of damps in shaping the overall immune response especially when derived from stressed, injured, or necrotic cells [21, 22 ]. while multiple tlrs exist, they share common intracellular signaling pathways. these include myeloid differentiation primary response protein (myd88), through which all tlrs signal with the exception of tlr3, which utilizes trif (toll / il-1r domain containing adaptor inducing ifn-). signal transduction pathways though both myd88 and trif have been described for tlr4. these pathways converge with the activation of nfb, which results in the transcription of multiple immune stimulatory genes involved in immune cell development, maturation and, cytokine production and proliferation [23, 24 ]. several recent studies have highlighted the importance of the innate immune system in allograft rejection in mouse and human transplantation. in a minor antigen mismatch model of graft rejection using hy - mismatched skin grafts, myd88 knockout recipients showed transplant survival > 100 days whereas wild - type recipients rejected skin allografts at a median of 16 days. interestingly, selective deletion of tlr2 alone (tlr2 knockout hosts) only slightly prolonged skin allograft survival, and deletion of tlr4 (tlr4 knockout hosts) did not prolong skin allograft survival. these results imply that other myd88-dependent pathways contribute to alloimmunity (e.g., il-1) or that significant redundancy in the signaling pathways exists. in contrast, myd88 deficiency does not prolong survival of fully mismatched allogeneic skin and heart transplants. however, in both the minor and major mismatch experiments, myd88 deficiency leads to a defect in th1- dependent alloimmunity [25, 26 ]. this suggests that the myd88 pathways skew the immune response towards th1- type immunity and is sufficient to mediate allograft rejection when only minor antigen mismatches are present. however, this effect is outweighed by the stronger immunologic stimuli of a full antigen mismatch. polymorphisms of tlr4 at asp299gly and thr399ile cause endotoxin hyporesponsiveness, and these mutations are relatively common in the human population. kidney transplant recipients who carry these tlr4 polymorphisms have been noted to have fewer episodes of acute rejection. this same tlr4 polymorphisms decrease the incidence of acute allograft rejection when present in lung transplant donors, but not recipients. a trend toward reduced acute graft - versus- host disease following bone marrow transplantation is noted when either the bone marrow donor or recipient carries these polymorphisms, but the effect is greater when the recipient carries the tlr4 mutation. interestingly, hepatitis c - infected liver transplant recipients with the tlr4 asp299gly mutation are found to have significantly worse long - term graft outcomes than recipients lacking this mutation. overall, these studies provide clinical substantiation of the experimentally observed importance of tlr4 in graft rejection. tissue injury during the pre- and posttransplant periods may result from a multitude of mechanisms, and these factors contribute to end - organ damage and affect allograft survival (figure 2). immune activation in the donor organ during the pretransplant period begins with brain death and the neuropathology associated with brainstem herniation. as the medulla becomes ischemic, vagal activity ceases, resulting in massive sympathetic outflow and high levels of catecholamines. in addition to affecting cardiac function by stimulating tachycardia, hypertension, and dysrhythmia, catecholamine release results in peripheral vasoconstriction that can contribute to end - organ ischemia. following the resolution of the catecholamine storm, the sympathetic tone is lost, resulting in vasodilatation and reperfusion injury to tissue followed by hypotension that can again cause organ hypoperfusion. brain death also results in an outpouring of inflammatory cytokines, including il-6 that has been shown to correlate with worse recipient outcomes in the setting of kidney transplantation. in addition to the release of a multitude of cytokines, acute brain injury has also been shown to up - regulate endogenous innate immune activators hsp70 and hmgb1, as well as to cause the release of fibrinogen fragments. interestingly, while inflammation derived from acute brain injury has been shown to be dependent on myd88, it has been shown to be independent of tlr2 and tlr4, the receptors identified for the majority of damps. potentially, alternative pathways of myd88-mediated tlr signaling act to transduce these inflammatory signals. the effect of adverse proinflammatory and neurophysiologic events on the donor organ quality that arise from brain death has led to the initiation of several studies investigating preprocurement donor cytoprotective therapies. for example, dopamine pretreatment has been observed to protect rat kidney allografts from cold ischemic injury [38, 39 ], potentially by augmenting the expression of the heat - shock protein, heme - oxygenase-1 [40, 41 ]. recently, in a randomized controlled study of human kidney transplantation, donor pretreatment with dopamine significantly improved early graft function. donor pretreatment with intraperitoneal glutamine in the rat kidney transplant model diminishes early structural damage due to prolonged preservation reperfusion injury. preconditioning with oral vitamin e has also been noted to improve post - ischemic recovery of systolic function following rat cardiac transplantation. necrotic cell death that can result from cold ischemia, ischemia - reperfusion injury, and surgical trauma has been shown to elicit a strong inflammatory response. for example, necrotic or damaged cells release hmgb1, a chromatin - binding protein that can act as an endogenous activator of tlr4 and cell signaling mediated by myd88 when released extracellularly [46, 47 ]. hmgb1 can be secreted by activated monocytes and macrophages and is passively released during cellular necrosis. hmgb1 is a potent promoter of inflammation that results in the release of cytokines and chemokines that promote inflammatory tissue damage [47, 48 ]. surgical trauma to the donor organ incurred during procurement and to the donor and recipient during the transplantation procedure can release damps and activate the immune system. however, probably the most significant contributor to either organ or end - organ injury during transplantation is the ischemic injury caused by cold storage followed by the warm reperfusion at the time of engraftment. the association between rejection and increased duration of cold ischemia has been well established. for example, fingolimod (fty720), a sphingosine-1-phosphate receptor agonist that interferes with lymphocyte trafficking, provides tubular epithelial protection in the presence of severe preservation - reperfusion injury in a rat kidney transplant model. reperfusion of ischemic organs results in activation of inflammatory pathways and complement cascades that increase graft immunogenicity [5153 ]. murine models of ischemia - reperfusion injury provide evidence for the role of tlrs in mediating reperfusion injury. in mouse models of kidney reperfusion injury, both tlr2 and tlr4 expressions are increased amongst renal epithelial cells. similarly, in models of myocardial ischemia - reperfusion injury, mice deficient in tlr4 develop smaller areas of myocardial infarction when compared to injury in wild - type mice. tlr4 has also been shown to be a key factor in liver ischemia - reperfusion injury. following transplantation, the liver remains susceptible to additional sources of innate immune activation from infection. microorganisms, either from bacteria translocation across injured bile duct epithelium or from post - operative infections in the form of bilomas, abscesses, wound infections, and viral infections, may initiate inflammatory cascades that adversely affect the allograft survival. inflammatory responses due to systemic viral infections such as cytomegalovirus (cmv), herpes viruses, adenovirus, and polyomaviruses ; moreover viral infections within the transplanted organ such as hepatitis b and hepatitis c liver infections, adenovirus heart infection, and cmv graft infections [58, 64, 65 ] have been associated with adverse clinical outcomes. in experimental models, systemic viral infections are also known to result in allograft rejection and disruption of immunoregulation [6670 ]. the activation of innate immune pathways is likely important in directing the initial activation of the allograft rejection response, but they also may disrupt established immune tolerance. stimulation of tlr receptors with lps or cpg dna has been shown to abrogate transplant tolerogenic regimens in both skin and heart transplant models [71, 72 ]. while the mechanism of the break in tolerance is not well understood, it may be caused by the failure of graft - reactive cd8 t cells to undergo apoptosis, the blockade of treg function, or the accumulation of tregs at the graft site. although multiple pathways by which damps may activate innate immune responses have been discovered, additional pathways await discovery. for example, programmed cell death through apoptosis may release different immune mediators than cells that died from stress or injury. evaluation of lysates of otherwise healthy cells made necrotic by dounce - lysis or freeze thaw cycles, demonstrates that they contain factors that are able to induce nf-b via tlr2 present on fibroblasts and macrophages. the activating agent must be present in healthy cells at the time of lysis and does not require de - novo synthesis. this demonstrates that not all dead cells are equally stimulatory and that genetic programs may exist to sequester damps when programmed cell death occurs. evaluation of a different cell fraction may also illuminate a larger variety of damps. for example, mitochondria are intracellular organelles with bacterial origins and have recently been discovered to harbor damps. cells made necrotic through freeze - thaw cycling release mitochondrial n - formyl peptides that stimulate il-8 production by monocytes. similar to bacteria, mitochondrial dna is rich in cpg dinucleotides which are the ligand for tlr9 in monocytes. for example, uric acid has been recently shown to elicit an acute inflammatory response to sterile cell death in mice. new research has implicated many damps to be molecules that increase the efficacy of pamps rather than being true tlr ligands themselves. further research into the precise mechanism of damp - tlr binding needs to be done to confirm which damps are ligands and which only facilitate the binding of true ligands. it also remains unclear whether receptor competition exists when multiple damps are present simultaneously, as would be expected at sites of injury. similarly, there may exist competition between damps and pamps for tlrs. alternatively, damp- and pamp - mediated cell signaling may synergize. additional pathways of molecular signaling that contribute to the propagation of innate immune signals need to be further investigated. for example, several micrornas (mirnas) have been found to regulate the innate response to pathogens. mirnas are a recently described family of small, noncoding rnas that regulate gene expression by interfering with protein translation and by targeting messenger rna for degradation. already there is evidence that mirna-146 serves as a negative feedback inhibitor of tlr signal transduction and that mirna-125b and mirna-155 regulate the stimulation of tnf- production by the innate immune system. the role of non - tlr innate receptor families in the regulation of the immune response is also just beginning to be uncovered. for example, the nod - lrr and card - helicase proteins, which comprise a huge family of receptors involved in pathogen recognition [80, 81 ], have only recently been defined. unlike tlrs, which are imbedded in cell surface or lysosomal - endosomal membranes, these receptors are cytosolic and recognize pathogen - associated molecules within the cytosol. like tlrs they can produce an inflammatory response driven by nf-b thus, resulting in immune activation. one member of the nlr family, nlrp3 (nlr family, pyrin domain containing 3) has been found to be involved with the sterile inflammatory response caused by necrotic cells and in the stimulation of il-1 secretion triggered by cholesterol crystals. we need to further our understanding of the innate immune pathways that contribute to the alloimmune response leading to acute, as well as chronic, graft rejection. these studies need to look at the contributions of both exogenous and endogenous innate immune stimulants and how these two sources of ligands may function in synergistic activation pathways. also, some ligands may function as competitive inhibitors, and their role in immune suppression could provide a novel route of immunosuppression. finally, targeting the innate pathways can be instituted at multiple timepoints in the transplant setting : in the donor beginning with brain death, during procurement, cold storage, reperfusion, immediately postoperatively, or in the late postoperative period in the setting of infection or chronic rejection. as our understanding of the immune systems grows, the mechanisms by which effective allograft rejection responses are initiated become increasingly complex. the role of allogeneic t cells and b cells in precipitating rejection has been well established ; however, more recent investigations have highlighted the way in which innate immune responses may skew or direct adaptive immunity. although evolutionarily primitive, these receptors appear to propagate innate immune activation and to facilitate activation of adaptive immunity in ways that are only presently being elucidated. in the case of allograft immunity, initiation of innate immune signals through damps and pamps can activate potent immune stimulatory pathways that increase allograft vulnerability to the host immune system. strategies for successful modulation of these signals will likely improve allograft outcomes and allow for the minimization of systemic immunosuppressive therapies.	studies of the immune mechanisms of allograft rejection have predominantly focused on the adaptive immune system that includes t cells and b cells. recent investigations into the innate immune system, which recognizes foreign antigens through more evolutionarily primitive pathways, have demonstrated a critical role of the innate immune system in the regulation of the adaptive immune system. innate immunity has been extensively studied in its role as the host 's first - line defense against microbial pathogens ; however, it is becoming increasingly recognized for its ability to also recognize host - derived molecules that result from tissue damage. the capacity of endogenous damage signals acting through the innate immune system to lower immune thresholds and promote immune recognition and rejection of transplant grafts is only beginning to be appreciated. an improved understanding of these pathways may reveal novel therapeutic targets to decrease graft alloreactivity and increase graft longevity.
the focus on dental erosive wear by both the public and dental health professionals appears to be increasing. this may be due to a decrease in caries levels in industrialized countries and/or to high prevalence of erosion lesions documented in recent epidemiological studies [24 ]. the diagnosis and the potential treatment needs of patients with dental erosive lesions are considered to be a significant challenge to clinicians. it is known that dental erosive wear is a multifactorial condition, and that it is important to evaluate the potential factors which could lead to identification of persons at risk. in order to assess and reveal possible predisposing factors, a record of patient 's dietary intake should be registered as part of a comprehensive case history involving general health, habits, saliva flow rates and buffer capacity. a study by lussi and schaffner showed that active lesions will progress when no adequate preventive measures are implemented. it is therefore important to detect the condition as early as possible and dentists should be trained to register even the first signs of the condition. this becomes especially important when taking into account the trend that patients often do not seek treatment until the lesions have reached such an advanced stage that restorative therapy is needed. furthermore, the clinical appearance of the eroded tooth surfaces is the most essential sign since there are no diagnostic tools available for early, quantitative detection. the clinical examination of the teeth should be performed systematically using a simple and accurate scoring system. the scientific literature shows that there has been an increasing focus on the aetiology, prevalence, and treatment options for dental erosive lesions. however, a survey among general dental practitioners from 2003 showed that a large proportion of dentists advised their patients about the erosive wear only occasionally or rarely. of the 1686 12 year olds included in that study, less than 10% could recall their dentist mentioning the condition. recent studies have also identified that an inadequate level of information about dental erosive wear was given by dental practitioners to adolescents / adults in possible risk groups [10, 11 ]. furthermore, another recent study showed that the knowledge and awareness about dental erosion among dental students, faculty members and patients in a brazilian dental school were poor. therefore, the aim of the present study was to perform a survey on public dental practitioners asking them about their experiences, awareness, knowledge of diagnosis, and choice of treatment options for dental erosive wear in 18- to 30-year - old patients. a precoded questionnaire was sent electronically to all dentists (n = 1262) employed in the public dental health service (pdhs) in norway in april 2011, using the internet - based software program questback. information was collected on the respondents ' sex, age, home county, and type of dental practice, and to which extent the respondents were involved in the diagnosis and treatment of dental erosive wear. in one part the dentists were asked how they register and document dental erosive wear in their patients aged 1830 years. they were also asked questions relating to their experience of erosive lesions, such as their estimation of the distribution and prevalence of erosive lesions among these patients. the second part of the questionnaire involved a patient case, where the dentists were asked to record their choice of treatment including general patient advice and/or type of restoration. a brief patient history as well as colour clinical photographs of labial and palatal surfaces of upper anterior and molar teeth with differing severity of erosive lesions was provided (figure 1). patient case a 28-year - old woman who had an eating disorder with vomiting as a teenager, but is now healthy. a 28-year - old woman who had an eating disorder with vomiting as a teenager, but is now healthy. question onewhat type of advice would you give this patient (you can make more than one choice) ? (1) information about dietary and drinking habits ; (2) information about brushing technique / habits ; (3) recommend rinsing with fluoride ; (4) recommend rinsing with chlorhexidine ; (5) recommend fluoride tablets ; (6) refer to specialist, faculty clinic, or other dentist ; (7) recommend specific toothpaste or rinse. what type of advice would you give this patient (you can make more than one choice) ? (1) information about dietary and drinking habits ; (2) information about brushing technique / habits ; (3) recommend rinsing with fluoride ; (4) recommend rinsing with chlorhexidine ; (5) recommend fluoride tablets ; (6) refer to specialist, faculty clinic, or other dentist ; (7) recommend specific toothpaste or rinse. question twohow would you treat the teeth in the maxillary anterior and posterior regions and the mandibular posterior teeth (you can make more than one choice) ? (1) no treatment ; (2) treat locally with fluoride solution (e.g., 2% naf, duraphat, fluor protector) ; (3) apply bonding material ; (4) apply flow composite ; (5) restore with glass ionomer cement ; (6) restore with composite filling material ; (7) restore with compomer ; (8) restore with ceramic laminate / facet / inlay / onlay ; (9) restore with crown. how would you treat the teeth in the maxillary anterior and posterior regions and the mandibular posterior teeth (you can make more than one choice) ? (1) no treatment ; (2) treat locally with fluoride solution (e.g., 2% naf, duraphat, fluor protector) ; (3) apply bonding material ; (4) apply flow composite ; (5) restore with glass ionomer cement ; (6) restore with composite filling material ; (7) restore with compomer ; (8) restore with ceramic laminate / facet / inlay / onlay ; (9) restore with crown. the questback program was configured to automatically send reminders to all participants who had not replied after 4, 7, and 12 weeks. the data was processed using spss version 18.0.3 (statistical package for the social sciences ; spss inc.,, usa) and statistical evaluation was carried out by means of descriptive statistics. as the upper 1st molars were most commonly suggested for operative treatment by the dentists, multivariate logistic regression analyses were performed to test the association between operative treatment of these teeth (dependent variable) and the following independent variables : dentists ' age, gender, practice location, and choice to refer. adjusted results were obtained by including all the selected variables in one regression analysis. in the adjusted analysis the result for each variable was adjusted for all the other variables. results were reported using odds ratio (or) with 95% confidence interval (ci) and p value (p). the probability level for statistical significance a total of 1262 dentists were invited to participate in the study, and 783 dentists had responded after three reminders. respondents who stated that they did not normally work with patients having dental erosive wear (n = 78) were excluded from the statistical analyses. a response rate of 60% was calculated according to standard definitions of the american association for public opinion research. the respondents ranged in age from 25 to 72 years (mean 43.3 years, sd 13 years) and consisted of 65% females and 35% males. almost all respondents (n = 695, 98.6%) registered dental erosive lesions in their patient 's charts. of these, 50% used a specific scoring system : 31% used a two - graded scoring system (enamel dentine) and 14% used a more detailed system, while 5% did not report details on their scoring system. erosive lesions were registered at the surface level by 58% of the respondents, at the tooth level by 15%, and at the individual level by 26%, while 1% did not answer the question. documentation with clinical photographs was reported as being often performed by 9% of the dentists, sometimes by 51%, and never by 40%. the corresponding values for documentation with study models were 5% (often), 60% (sometimes), and 35% (never). the dentists reported that the erosive lesions were most often located on the palatal surfaces in upper anterior teeth (79%), followed by first mandibular molars (74%) and first maxillary molars (32%). according to 36% of dentists there were no gender differences in the prevalence of erosive lesions. while 40% of the dentists reported that erosive lesions were more common in males than in females, only 4% of the dentists stated that more females than males had erosive lesions, and 20% were unsure. of the dentists with more than 1015 years clinical experience (n = 404), the majority (81%) reported a higher prevalence of erosive lesions today compared with 1015 years ago. eight percent of the dentists did not have the impression of seeing more erosive lesions today, while 11% were unsure. half of the dentists (52%) did not think that young adults (aged 1830 years) with dental erosive lesions had more caries than those without erosive lesions. higher caries prevalence amongst patients with erosive lesions was estimated by 19% of dentists ; 19% stated no differences and 10% were unsure. most of the dentists (77%) reported that they usually found a probable cause of the erosive lesions, 22% occasionally, and only 1% reported that they seldom found a probable cause. the most common causes given were consumption of carbonated beverages (97%), followed by acidic juices (72%), fruits (46%), sport drinks (24%), and acidic diet (20%). reflux and eating disorders with vomiting were reported as more uncommon causes (8%) by the dentists. when patients were registered as having erosive lesions, 21% of the dentists always recorded a dietary history. a quarter of the dentists (24%) reported that they often recorded a dietary history, 36% only occasionally, and 19% reported that they never recorded dietary history in patients registered with erosive lesions. the type of dietary questionnaire used was highly variable ; 51 dentists (9%) used a precoded questionnaire, 31% asked the patient to record all they consumed during a certain period of time (amount, time of day, etc.), while the remaining 60% used different techniques, such as oral interviews and describing text in the dental chart. more than half of the respondents (57%) estimated that their patients with erosive lesions had a normal amount of saliva, while 37% did not know. only a small percentage of dentists estimated that their erosion patients had less than normal saliva flow (4%) and 2% thought they had more than normal amounts of saliva. only two dentists (1%) reported that they always measured saliva secretion, 1% reported measuring saliva flow often, 25% only occasionally, while 73% reported that they never measured saliva secretion in patients with dental erosive lesions. most of the dentists (78%) treated their patients with dental erosive wear themselves, while 9% referred the patients to another dentist or specialist / university faculty clinic. eight percent of all the male dentists would refer all patients with erosive lesions to another dentist or specialist / university faculty clinic, compared with 10% of the female dentists. the mean age was 44.6 years (males) compared with 43.3 years (females) among the dentists who treated their patients themselves. significantly more dentists living in the eastern part of norway (12%) referred patients compared with dentists living in the northern (7%) and western (7%) parts of norway (p = 0.03). a previously calibrated examiner scored the severity of the erosive lesions on the surface level based on the clinical photos using a tested scoring system for erosive lesions (vede system). enamel lesions (grade 2) were registered on central incisors and upper 2nd molars, while dentine lesions (grades 3, 4, and 5) were seen on lateral incisors, all 1st molars and lower 2nd molars, indicating a case with severe erosive lesions (figure 1).. the majority of dentists (88.2%) would give the patient information about good dietary and drinking habits and recommend that the patient should rinse with fluoride (87.1% of the dentists). more than half the dentists (58.4%) would give information about tooth - brushing technique and/or brushing habits. forty - four percent of the dentists chose to place a filling, and 18.8% chose prosthodontic treatment for these teeth. the lower 2nd molars were the most likely teeth to receive either no treatment other than advice (35.3%) or local treatment with fluoride solution / use of a bonding material (40.6%). these teeth were also least likely to be restored with a filling (20.1%). there were no obvious differences between the treatments chosen for the different upper incisor teeth, and most dentists would choose to restore them with a filling. an odds ratio of 1.5 indicated that for upper 1st molars, the female dentists suggested operative treatment significantly more often than the male dentists. even though there are few comparable studies, this survey tends to suggest that the majority of dentists are aware of the problem of dental erosion and are relatively up to date regarding the clinical recording and diagnosis of dental erosive wear. the major points to note are that the documentation of erosive dental wear was not standardized, and little or no priority was given to dietary and salivary analyses. despite this, and more encouragingly, the majority of dentists reported that they were confident of finding the cause of the erosive wear and also in treating their own patients that need reparative therapy due to dental erosive wear. the questionnaire used in this study was aimed at dentists working in the norwegian pdhs who were currently involved in the diagnosis and treatment of dental erosion in their clinical practice. in general, these dentists see a large proportion of adolescents and young adults, and based on recent prevalence studies this patient group may be considered as being at high risk for dental erosion [3, 4, 15, 16 ]. importantly, responding dentists who reported that they did not normally work clinically with erosive dental wear were excluded from the study. measures were taken to ensure a high response rate in accordance with a systematic review of questionnaires. the questionnaire was styled in a personal manner, and a realistic and illustrative case with background information was presented early in the questionnaire, as was proven successful in a previous questionnaire - study on dental caries. in order to emphasize the importance of a reply, when sending reminders to the dentists, respondents were informed of the response rate so far for the questionnaire. unfortunately, as this questionnaire was anonymous, no information could be retrieved about the nonresponders, and a meaningful nonresponse analysis could not be performed. another limitation of this study is that such questionnaires are based on the respondents ' ability to recall. furthermore, given that the specificity and sensitivity of the dentists ' diagnosis of erosive wear were not assessed, the accuracy of the data can not be quality controlled. recent prevalence studies have shown that erosive tooth wear is a common problem in current dental practice and is a challenge for the practitioner [3, 4, 12, 15, 16 ]. in the present survey, the majority of dentists participating in this study reported a higher prevalence of erosion lesions today, compared with 1015 years ago. even though lesion due to the combination of erosion and abrasion / attrition is a common phenomenon, one can expect that the presence of mainly erosive wear lesions exceeded the number of erosion / abrasion / attrition lesions in the age group included in this study. to be able to record the presence and severity, as well as the progression of erosive lesions, use of a grading system the majority of the dentists reported that they used a specific scoring system registered at the tooth surface level. in most cases this was the registration system included in the electronic patient journal system. approximately one quarter of the dentists registered the erosions at the patient level only, which does not allow for specific followup of lesion progression. regarding the oral distribution of the erosive lesions, the most commonly affected tooth surfaces were the palatal surfaces of the upper incisors and the occlusal surfaces of the lower 1st molars. these results are in accordance with many previous studies in adolescents [10, 21, 22 ]. even though approximately one - third of dentists had the impression that there were no differences between the sexes, 40% reported more dental erosive wear in males. several studies have reported a higher prevalence of dental erosion in male adolescents [2, 4, 15, 22 ]. in a recent study the consumption of soft drinks and other acidic beverages was found to be related to both age and gender. one could expect that a higher intake of such beverages (often containing sugars), typically consumed between meals, could also lead to more dental caries. while half the respondents in the present study thought that patients with dental erosion did not have more dental caries, about one fifth of the dentists reported a higher incidence of caries in these patients, and about the same percentage reported that patients with dental erosion had the same amount of caries as patients without dental erosion. results from other studies regarding presence of dental erosive wear and caries experience are also ambiguous. some studies have demonstrated a significantly higher caries experience among children with high prevalence of dental erosion [24, 25 ], whereas others could not verify this association [26, 27 ]. in order to identify possible aetiological factors in patients presenting with dental erosion, a dietary history is extremely important, as behavioural factors such as eating and drinking habits play a key role in the pathogenesis. only half of the dentists in this study reported either occasionally or never, recording a dietary history in patients with dental erosion, and only a small proportion used a precoded questionnaire. this is a worrying finding, as the effective prevention of progression of erosive lesions often requires clear, individualized dietary advice. apart from behavioural factors, biological patient factors also play an important role in the pathogenesis of dental erosion. it is well known that a high salivary flow rate favours the prevention or minimization of initial acid attack. in the present study the majority of dentists reported that they did not measure saliva secretion in their dental erosion patients. this reflected the estimate by more than half the dentists that their patients had normal amounts of saliva. however, more than one - third of the dentists reported that they did not know about the saliva status of their dental erosion patients, possibly indicating a lack of knowledge about the importance of saliva in the aetiology of dental erosive wear. however, if the large majority of patients seen by these dentists are adolescents or young adults, the percentage of patients presenting with subnormal levels of saliva secretion is not expected to be very high. in the present study, approximately two - thirds of the dentists reported that they usually found a probable cause of the dental erosion in their patients. nearly all dentists reported that high consumption of carbonated soft drinks was the most likely cause. acidic drinks are thought to be one of the most important factors leading to dental erosion, especially when taking into account that the consumption of such drinks has increased greatly over the last decades [30, 31 ]. norway is no exception, with a yearly mean consumption of well over 100 liter per person, placing them in the top five among the european countries. frequent intake of fruit and/or fruit juices was also commonly reported by the dentists to be causative factors in their patients. the risk of developing erosive tooth wear was found to be 37-times higher in persons with a greater than twice daily intake of citrus fruits than in persons eating fruit less often. dental personnel may in many cases be the first health workers to observe signs of eating disorders. in the present questionnaire dentists regarded reflux and eating disorders with vomiting as relatively uncommon causes of dental erosion in their patients. while no difference was reported in the prevalence of dental erosion between young icelandic adults and patients with gastroesophageal reflux disease (gerd), a more recent study found that patients with eating disorders (mean age 21 years) were at 8.5-times higher risk of having dental erosion. thorough documentation of the patient case is a standard part of following the progression of erosive lesions and treating patients with erosive tooth wear. for patients where the degree of damage suggests the need for extensive rehabilitation, insurance companies or social security systems usually expect solid documentation. in the present study, the majority of dentists only occasionally documented their patient cases by taking clinical photographs or making study models. surprisingly, more than a third of the dentists never used this type of documentation. this may be related to the possible mild severity of the patient cases or the fact that they are not aware of the importance of documenting such wear. a 3-year longitudinal study on the dynamics of tooth erosion in adolescents concluded that the condition progressed steadily in children with dental erosion. at the same time, there are reports that according to patients, dentists do not appear to give enough information about this problem. in a recent study examining the status of the dentition in professional norwegian wine tasters, seven of the nine wine tasters registered with dental erosive wear had not been informed by their dentist / dental hygienist about the presence of these lesions. in another study examining dental erosion in a group of individuals training regularly at a training studio, 82% of the physically active young adults with erosive wear who recently had been to their dentist / dental hygienist claimed they had not been informed about the presence of these lesions. furthermore, in the study on dental students, faculty members, and patients in a brazilian dental school, hermont and coworkers have recently shown that knowledge and awareness about dental erosion are poor, and they concluded that there was a need for better understanding and communication. maybe it is still true that many dentists typically ignore or overlook the very early stages of erosive tooth wear, dismissing tooth surface loss as a normal and inevitable occurrence of daily living, and thus not worth spending time on any specific interventive activity, as was suggested a few years ago. in the present survey most of the dentists reported that they perform the reparative treatment of their own patients with dental erosion, and only nine percent referred the patients to another dentist, a specialist or a university faculty clinic. there were no significant age or gender differences with regard to this aspect, an observation that was made in a similar clinical decision - making questionnaire among prosthodontists. this indicates that most of the general dentists in this study feel competent in their technical skills. however, with respect to treatment options, significantly more of the female dentists who responded to the present questionnaire chose to restore the upper 1st molars. regarding referral patterns, not surprisingly a greater proportion of dentists in the eastern, more populated part of norway, where there are many specialists, referred their patients for restorative treatment, compared with those located in the northern and western parts of norway, where there are few specialists. the patient case included in the current study prior registration of the erosive damage by one of the authors indicated that the patient had a combination of mild enamel and severe dentine lesions. the results of the questionnaire suggest that the responding dentists had few problems registering this difference. the 1st upper molars were regarded as the teeth with the greatest need for a restoration, probably because in this patient the palatal cusp was almost completely worn away. however, also the lateral incisors, upper 2nd molars, and central incisors were recommended to be filled by over a third of the dentists. surprisingly, even though the lateral incisors and lower 1st molars had exposed dentine, more than a quarter of the dentists suggested that no restorative treatment was necessary. in contrast, about two - thirds of the dentists would restore enamel lesions on central incisors and 2nd molars using resin composites. this inconsistency could be a result of the difficulty for dentists in differentiating between more severe enamel lesions and early dentine lesions, as discussed in the literature. another consideration could be related to dentists ' focus on the tooth level rather than considering the patient case as a whole. in a similar questionnaire - based study, for a patient case involving palatal erosion in maxillary incisors without loss of the incisal edges, most specialists chose the prevention option that involved covering the worn surfaces with a dentine bonding agent and prescription of fluoride mouthwash. restorative management in the form of direct composites was most commonly chosen by uk dentists while the non - uk prosthodontists were more divided, half of them choosing to leave the teeth untreated, about one - third choosing composites, and almost 10% choosing to crown the teeth. in a recent study performed on dentists in the dental practice - based research network, direct resin - based composites were the most common type of restorations placed in noncarious tooth defects. although the patient group in that study was older than the average age of patients seen by the dentists in the present study, the choice of resin composites is a common factor. several authors have published strategies for the restorative treatment of patients with dental erosive tooth wear. some clinicians have suggested that adequate function and aesthetics can be achieved when dental erosion lesions are restored using a combination of bonded ceramic overlays to reestablish vertical dimension and composite resin to restore the worn palatal and incisal surfaces of the upper anterior teeth. recently a new classification system (ace classification : anterior clinical erosion), strictly related to the clinical observation of the status of eroded anterior maxillary teeth affected, was proposed. minimally invasive techniques with composite were recommended to restore the palatal surfaces, while the facial surfaces were recommended to be treated with ceramic veneers when necessary. the authors concluded that the question of whether it is preferable to start earlier with a lighter, less invasive rehabilitation or later with a highly aggressive but eventually more resistant one is still open for debate. in all these studies it would appear that direct composite restorations are considered to be the main approach in less severe cases. despite the limitations of gathering information using a survey of this type, the results tend to suggest that the responding dentists are relatively up to date regarding the clinical recording and diagnosis of dental erosive wear, although dietary and salivary analyses were not given priority. furthermore, although documentation was not standardized, the majority of dentists reported that they felt confident of finding the cause of the erosive wear and of treating their patients themselves. as dental erosion appears to be increasing in prevalence, one would expect that newly trained dental health personnel will need to be even more aware of the necessity of careful and early diagnosis of this problem and that they will be even better equipped to face the future clinical challenges related to dental erosive wear.	this study aimed to investigate dentists ' general experience, knowledge about diagnosis, and treatment of dental erosive wear in young adults. a questionnaire was sent to 1262 norwegian public dental health - employed dentists. the response rate was 60%. results indicated that most dentists recorded erosive wear, half of them used a specific scoring system, and half registered lesions at the tooth surface level. lesions were reported most often on palatal surfaces of upper anterior teeth (79% of dentists), on occlusal surfaces of lower 1st molars (74%), and on upper 1st molars (32%). half the dentists used clinical photographs for documentation and 60% made study models. while 40% reported more erosive lesions in males, 36% reported no gender differences. high intake of carbonated beverages and acidic juices were reported as the most common cause by 97% and 72% of the dentists, respectively. only 21% of dentists recorded the patient 's dietary history, and 73% never measured saliva secretion. the majority (78%) of the dentists treated patients with erosive wear themselves. in general, the survey suggests that the dentists are relatively up to date regarding the clinical recording, diagnosis, and treatment of dental erosive wear. however, dietary and salivary analyses were not given priority, and early, preventive treatment was lacking.
the clinical benefits associated with adoptive immunotherapy of some mabs have established the clinical pertinence of several antigens as immune therapeutic targets. for some therapeutic antibodies such as the anti - cd20 rituximab or the anti - her2 trastuzumab, cell - mediated immunity (antibody - dependent cellular cytotoxicity (adcc)) has been recognized as one of the mechanisms responsible for their clinical efficiency [1, 2 ]. cellular subsets capable of mediating adcc include neutrophils, monocytes / macrophages, and a subset of natural killer (nk) cells. as an example of a strategy to improve patient adcc potential, we have shown in the context of breast cancer patients that their nk population could be amplified in vitro up to 425-fold using a straightforward culture procedure. yet, because of technical limitations associated with the clinical use of nk cells (poor recovery after freezing and thawing, poor in vitro expansion compared to t cells) and the numerous approaches that have become available to engineer lymphocytes (see for review), we have also considered the possibility of arming t cells with a receptor that would enable them to mediate adcc. in the above study, we have shown that, after transduction with fcriiia / fcri (referred to as cd16/) receptor fusion gene, cd4 + and cd8 + cytotoxic t lymphocytes displayed a stable expression of the cd16/ receptor at their surface and mediated adcc. thus, associating a therapeutic mab and an adoptive transfer of cd16/ transduced t cells could combine the advantages associated with the functional potential of cytotoxic lymphocytes and recognition of the target cells unrestricted by the major histocompatibility complex. another way to reach the same objective is to redirect t cells with the so - called chimeric t cell receptor (car) or t bodies, a strategy pioneered more than two decades ago by the team of eshhar [68 ], which has recently shown impressive antitumor effects in patients with hematologic diseases (for a review see gill and june). these are fusion proteins between single chain variable fragments (scfv) from a monoclonal antibody and an intracellular signaling domain such as cd3 or fcri. the above two strategies will be referred to in the text as the adcc approach (a treatment relying on an mab + an adoptive transfer of cd16 armed t cells) and the car approach (the adoptive transfer of car armed t cells). both strategies have the same fundamental advantage, which is the combination of the cellular immune potential of t cells with recognition of a target cell unrestricted by the mhc. in particular, in terms of safety, as highlighted by morgan. in their in - depth analysis of a serious adverse event following the administration of t cells transduced with a car against her2, a major difference between mab administration and adoptive transfer of car engineered t cells is that while ab is cleared by the body, car t cells can continuously produce effector cytokines and can expand in cell numbers following antigen stimulation. this may be a real advantage or a real danger, depending on the tumor restriction of the antigen targeted. provided that the tumor antigen targeted is appropriately restricted, the main advantage of the car approach would be its efficiency. on the other hand, the adcc approach allows the search of a correct dosage simply by changing the dose of the mab. the aim of the present study was to directly compare the car and adcc approaches with a clinically relevant target antigen. to this end, as a first step in designing a model, we equipped the same cytotoxic lymphocyte line (the human nk cell line nk-92) with either a fcriiia / fcri receptor (referred to as nk-92) or an anti - her2/fcri car receptor (referred to as nk-92) and compared their efficiency in killing her2 positive tumor target cells in vitro and in vivo. while in vitro comparison between these two effectors has highlighted an advantage in the car approach in terms of cytotoxic potency, the in vivo experiments performed in nsg mice have not enabled a car / adcc comparison but instead have revealed an off - target interaction which blocked the potential antitumoral efficacy of the car modified lymphocytes. the cd16/ chimeric cdna comprises the leader (s) and the two extracellular domains (ec1 and ec2) of human cd16 and two amino acids (aa) of the extracellular domain of the human fcri (pro4-gln5), as well as the intact transmembrane (tm) and intracellular (ic) domains (figure 1(a)). the trastuzumab - based car contains the vl and vh from the mab (ab4d5 - 8), separated by a linker, the human ch2-ch3 igg2 as a spacer, and the same signaling domain as that of cd16 (figure 1(a)). after transduction (see section 4), 41% of the nk-92 expressed cd16 and 36% expressed the car. after immunomagnetic purification using anti - cd16 and anti - human igg2a - fc - specific mab (see section 4), essentially pure populations of nk-92 and nk-92 were obtained (figure 1(b)). first, we showed that the spontaneous cytotoxic activity (tested against k562, e / t ratio : 30/1) of the untransduced and the transduced nk-92 was not significantly modified by the retroviral transduction (figure 2(a)). next, adcc activity of the nk-92 against the bt474 cell line was tested in the presence of increasing concentration of trastuzumab (as in the example shown in figure 2(b)) and demonstrated a plateau (close to 50% specific lysis) at 10 g / ml (e / t ratio : 30/1). note that a background lysis was observed for nk-92 + trastuzumab (compared to nk-92 + rituximab), likely due to background expression of cd16 by the nk-92 cell line. for all other in vitro experiments, trastuzumab was used at a concentration of 10 g / ml. next, we compared the efficiency of target cell lyses induced either after direct recognition of the her2 ag by the nk-92 alone or after indirect recognition by the nk-92 in the presence of trastuzumab. to this end, target cells (the her2 negative mda - mb-468 and the her2 positive bt474) were coated or not with trastuzumab, and the untransduced nk-92 was used as a negative control. cytotoxic activities of nk-92 and nk-92 against trastuzumab coated or not coated mda - mb-468 and bt474 are summarized in figure 2(c) (at an effector - to - target ratio of 30 : 1). neither nk-92 nor nk-92 presented a significant level of cytotoxicity against the her2 negative mda - mb-468 cell line (figure 2(c), left panel). when tested against the bt474, nk-92 showed a high level of cytotoxic activity (101 3% at 30 : 1). killing of bt474 was also observed by nk-92 in the presence of trastuzumab, although this occurred at a level below that observed by nk-92 (46 8% at the same effector - to - target ratio of 30 : 1). in addition, the preincubation of the bt474 in the presence of trastuzumab drastically reduced the killing by nk-92 (figure 2(c), right panel). to confirm the differences in cytotoxic performance between nk-92 and nk-92, further comparison was performed against 4 different her2 positive cell lines : bt474, bt474 scid (a subclone of bt474 used for the in vivo experiments), mcf7, and mda - mb-231 (figure 3). these data showed that, in vitro, in these experimental conditions, based upon 4 hr cytotoxicity assays, the direct pathway of killing (by nk-92) was always more efficient than the indirect pathway (by nk-92) (figures 3(a), 3(b), 3(c), and 3(d)). having shown that nk-92 were more effective in vitro than nk-92 against several her2 positive tumor cells, we developed a mouse xenograft model to test whether such difference could also be evidenced in vivo. six - week - old female nsg mice were injected subcutaneously in the left side with 5 10 bt474 cells, and when the tumor reached a volume of 50 mm, the mice received the indicated treatment (all mice were sacrificed before the tumor volume reached 2500 mm). we first tested the capacity of ip injection of nk-92 and trastuzumab to control the growth of a subcutaneous established nk-92 resistant bt474 carcinoma (figure 4(a)). in the absence of addition of effector cells, weekly injections of trastuzumab (15 mg / kg) alone when 5 10 nk-92 cells were used in combination with trastuzumab (15 mg / kg) injected 24 h prior to nk cells, complete tumor regression was observed. note that tumor regression began after a single dose but that complete regression required 4 injections once per week of 5 10 nk-92 cells (figure 4(a)). to confirm that adcc was the mechanism responsible for tumor regression, experiments were repeated with the untransduced nk-92 or with nk-92 in the presence of an irrelevant antibody (rituximab, directed against cd20, an ag not present at the surface of bt474). as the result of a mechanism of tumor destruction due to adcc, neither the untransduced nk-92 in the presence of trastuzumab nor nk-92 in the presence of rituximab were able to control the growth of the bt474 tumor (figures 4(b) and 4(c), resp.). in contrast to the in vitro situation, where nk-92 has demonstrated a higher efficacy of bt474 killing compared to nk-92 + trastuzumab, no tumor regression was observed even after 4 injections with nk-92 (figure 4(d)). before investigating in more detail the possible reasons for the discrepancy between in vitro and in vivo results, we first controlled the injection route and reproduced the experiment. with nk-92 iv instead of ip injection, we observed the same inefficacy (data not shown). to investigate the unexpected total lack of in vivo efficacy of nk-92, we compared the dissemination abilities of nk-92 and nk-92 in the nsg mouse model. for this purpose, 2 10 nk-92, nk-92, or nk-92 cells were injected into nsg mice bearing bt474 tumors (tumor size ranging between 50 and 100 mm). twenty - four hours later, mouse blood, liver, tumor, lung, and spleen were collected, and the percentages of cd45 positive cells were analyzed by facs analysis using specific ab. as shown in figures 5(b), 5(c), and 5(d), no major differences between nk-92, nk-92, and nk-92 spreading were found within blood, spleen, and liver, suggesting no major differences in their ability to recirculate within the nsg mice after ip injection. however, whereas nk-92 and nk-92 can be found in tumors even at a low but significant ratio, no nk-92 could be found inside the tumor (figure 5(a)). we then analyzed whether preinjection of either trastuzumab, competing for her2 binding with car - her2, or cetuximab, recognizing her1 also expressed at the surface of the bt474 cells without interfering with car - her2 binding, could induce a chemoattractive signal for nk cells inside the tumor. as shown in figure 5(a), opsonization of tumors with specific ab does not attract nk-92 effectors. to confirm these data, we injected 3 10 csfe labelled nk-92, nk-92, or nk-92 cells into nsg mice bearing bt474 tumors (tumor size ranging between 50 and 100 mm) and collected the tumors 72 hours later. we then performed ihc on frozen sections by direct reading of cfse labelled nk effectors and visualization of the tumor cell nuclei by counterstaining with dapi. as shown in figure 6(a), nk-92 and nk-92 could be found in different parts of the tumors, either as a cluster or as single cells. preinjection of trastuzumab did not affect the nk-92 frequency within the tumor (figure 6(a), panels 2 and 3). in support of the previous facs analysis, nk-92 effectors could never be found within the tumors. we observed that where present at all, most nk-92 were blocked at the edge of the tumor in the form of clusters (figure 6(b), right panel) whereas nk-92 could be found everywhere inside the tumor (figure 6(b), left panel). suspecting a possible interaction between mice macrophages and nk-92, we counterstained our frozen ihc sections with anti - iba1 antibodies specific to mouse macrophages (wako). as shown in figure 6(c) right panel, a yellow merge staining always located at the edge of the tumor with nk-92 was observed, suggesting that mice macrophages (in red) could aggregate with nk-92 effectors (in green). this was never observed inside the tumor, or with nk-92 for which isolated macrophages are distributed everywhere inside the tumor (figure 6(c), left panel, red arrow). taken together, these data suggest that mice macrophages aggregating nk-92 might have prevented them from infiltrating inside the tumor, explaining the lack of efficacy for these type of effectors. as shown in figure 5(a), 24 h after injection, nk-92 and nk-92 are present within the tumor, but not nk-92. this was observed whether or not the mice were pretreated with trastuzumab. because pretreatment of the mice with trastuzumab should have blocked recognition of cross - reactive mice antigens (if any), the interaction between mice macrophages and the car was unlikely to be due to the specific vh - vl part of the car. in contrast, although nsg mice lack t cells, b cells, and natural killer cells, they nevertheless still have neutrophils, monocyte / macrophages, and dendritic cells. and although these cells are functionally defective because of the nod / shilt genetic background, they nevertheless bear fc receptors (as shown in figure 7(a), the cd16 - 32 staining of the cd11b+/gr-1 + myeloid nsg splenocytes). and mice fc receptors can cross - react with the human fc (see section 3). to directly address the possibility of interaction between nsg fc - bearing cells and the nk-92, nsg splenocytes were cocultured with nk-92, nk-92, or nk-92 at a 5 : 1 ratio. after 18 hours, nk-92 cell activation was analyzed by quantifying human ifn in supernatant by elisa. as shown in figure 7(b), coincubation of nsg splenocytes with nk-92, nk-92, or nk-92 led to detectable production of ifn only by nk-92. this production level is far from being insignificant as it corresponds to half of the ifn level produced by nk-92 after coincubation with the her2 bt474 tumor cells (9.3 ng / ml and 18.4 ng / ml, resp.). taken together, these results strongly suggest that nk-92 not only have been physically prevented from penetrating the tumor due to being trapped by the surrounding macrophages but also were functionally activated by this off - target interaction. we compared in the present study two constructs allowing us to implement two strategies relying on an adoptive transfer of cytotoxic lymphocytes to improve the targeting of her2 positive tumors. in the first, the lymphocytes are equipped with a human cd16 receptor (fcriiia / fcri fusion protein) to permit adcc in the presence of trastuzumab. in the second, we used a first generation car encompassing scfv derived from trastuzumab and a ch2-ch3 higg2 spacer between the scfv and the transmembrane domain. in both constructs, the signaling domain was the fcri. after transduction with cd16 and car receptor genes, the human nk cell line nk-92 displayed stable cell surface expression of cd16 and car receptors. specific cytotoxic activity against her2 positive target cells was demonstrated in both cases, and when we compared their potency against four her2 positive target cell lines, nk-92 always performed better than nk-92. to our knowledge, only two studies boissel., using the two different therapeutic monoclonal anti - cd20 mabs rituximab and ofatumumab, demonstrated that the cytotoxic activity of nk-92 cells expressing cd20-targeting first - generation car against primary cll cells was superior to the adcc by nk-92 in the presence of anti - cd20 monoclonal antibodies. and tassev., in their detailed description of the retargeting of nk-92 cells using an hla - a2-restricted ebna3c - specific chimeric receptor (a tcr - like antibody), also directly compared the two systems and conclude that the car mediated approach proved far more effective at killing target cells compared with adcc. to what extent can these observations be generalized ? for adcc, the outcome of effector / target cell interactions will depend on the number of cd16 receptors, their affinity with the antibody used to recognize the target, the number of target antigens, nonspecific interactions between the target and effector (e.g., lfa-1/icam1), time, concentration of abs in the liquid phase, and so forth. similarly for car recognition, car density, car affinity, and antigen expression level will all influence the outcome of the interaction. in addition, for car, other variables will also be important, such as the choice of transducing chain, or the addition of an accessory signal (such as the 4 - 1bb). in the present study as well as in the study undertaken by boissel. and tassev., because the effector was a clone cell line, the only difference in every effector / target interaction was the way target cells were recognized : either by cd16 + ab or by the corresponding car. thus, these three studies suggest that the ranking car > adcc remains true (i) against different target antigens (and in particular cd20 and her2), (ii) with scfv of different affinities (rituximab and ofatumumab have an affinity with the cd20 antigen of approximately 5.45 and 4.76 nm, resp., while the (ebna clone 315) scfv has an affinity of 291 nm with the hla - a2/ebna complex), (iii) with different signaling domains, that is, cd3 or fcri, and (iv) with or without the costimulatory signaling domain 4 - 1bb. we then attempted to implement a xenogeneic model to continue the comparison in vivo. in nsg mice, we found that ip injection of nk-92 and trastuzumab induced total regression of established subcutaneous bt474 tumors (at least until day 40). but in contrast to already published results, nk-92 that we designed had no antitumor activity, in vivo in nsg. further analysis demonstrated that, in contrast to nk-92, nk-92 did not reach the tumor and were aggregated at the periphery of the tumor by the mice macrophages. in addition, in vitro coincubation of nk-92 but not nk-92 in the presence of nsg splenocytes led to activation and ifn- production. an important difference between nk-92 and nk-92, as well as between nk-92 used in the present study and that used by schnfeld. for nk-92, we used a ch2-ch3 igg2 hinge, no hinge was used for the nk-92, and in their nk-92 (also against her2) schnfeld. used the cd8. such spacer is sometimes required in the design of a car, for the following reasons. beyond the variables cited previously, t bodies by their inventor e. eschar, can be seen as a sort of tcr having the specificity of an antibody. the tcr recognizes mhc - peptide complexes ; these are a unique class of antigens which are characteristic of the exposition at the target cell surface and their physical accessibility to the tcr is homogeneous. the size of a tcr molecule is thus naturally adapted to sense the peptide / mhc complex exposed in the target cell. in the same way, the size of the cd16 is naturally adapted to catch the fc of the ab bound to the target cell surface, though in this case with more variability than for the tcr, since the exposition of the fc will depend on the position of the epitope recognized. in nature, this difficulty is solved by the flexibility of the ab, which may derive from specific characteristics of both the fab and the fc region (for a review, see). consequently, while the steric constraints associated with the target cell recognition by a lymphocyte equipped with a tcr or a cd16 receptor are essentially natural, this is not the case for a car, whose ability to catch the ag and transmit the signal will rely in part on the characteristics of the car hinge region. indeed, the distance of the epitope to the target cell surface, as well as the flexibility and length of the car hinge region, matters in regard to car recognition. in line with the spatial constraints cited above, it has been shown that a protruding hinge region is needed for efficient activation of lymphocytes armed with a car recognizing a membrane - proximal epitope, whereas lymphocytes equipped with a car specific for a membrane - distal epitope can be efficiently engaged without extracellular spacer element [16, 17 ]. as many others before us [1722 ], we used an igg hinge region because at first glance it presented many advantages. first, in line with spatial constraints, the igg fc domain can provide both flexibility and the possibility of different lengths by adapting the number of ch2 or ch3 molecules ; for clinical applications, it lacks immunogenicity ; and for detection, it allows the use of anti - fc reagents. in addition, we made the choice to use the human igg2 as a spacer domain in our car design because of its lower affinity for the human fcrs. furthermore, replacement of the igg1 ch2 sequences with those of igg2 was shown to eliminate in vitro the activation of car t cells by human fcr - bearing cells and simultaneous cross - activation of cytokine production by innate immune cells. clearly, this precaution was not enough. cars are designed to be used in humans and their study in xenogeneic models may reveal specific traps. in particular, the receptors for the fc domain of igg, fcrs, are quite dissimilar in binding abilities and expression pattern between human and mouse. mice have three activating fcrs (mfcri, mfcriii, and mfcriv) and one inhibitory fcriib. in nsg mice, neutrophils and monocytes constitute most of the remaining detectable mouse immune cells. dendritic cells and macrophages are also present in the nsg mouse and although they are functionally defective because of alleles in the nod / shilt genetic background, they express activating fcrs. dendritic cells express fcri and macrophages express all the fcrs, and even though they are not functional, they could nevertheless interact with the spacer derived from human iggfc. moreover, human igg2 can bind to the murine fcriib and fcriii and induce a potent adcc with mouse nk cells and mouse polymorphonuclear leukocytes. finally, the absence of antitumoral activity of nk-92 with the human full - length igg2 fc derived spacer observed in the present study supports and extends the results obtained using cd19-car designed with a full - length human igg4 fc spacer, which failed to eradicate raji tumors in nsg mice unless the entire ch2 domain responsible for fcr binding was removed. in conclusion, our results extend the number of target antigens for which the car approach performs better than the adcc approach in vitro in terms of cytotoxic activity. moreover, after the recent work of hudecek., we provide additional evidence stressing the potential dramatic effect in vivo of the spacer domain of cars, even those devoid of intrinsic signaling capacity. nk-92, the human nk cell line (atcc, rockville, md), was grown in rpmi 1640 culture medium (gibco, cergy pontoise, france) supplemented with 10% fbs (paa laboratories, les mureaux, france), 100 iu / ml il-2 (proleukin) (chiron corporation, emeryville, us), 2 mm l - glutamine (gibco), penicillin (100 iu / ml), and streptomycin (0.1 g / ml) (gibco). epstein - barr b - lymphoblastoid cell lines (blcls) were derived from donor peripheral - blood mononuclear cells (pbmcs) by in vitro infection with ebv - containing culture supernatant from the marmoset b95 - 8 cell line (atcc) in the presence of 1 g / ml cyclosporin - a. the her2 negative mda - mb-468 and the her2 positive bt-474, mda - mb-231, and mcf7 breast cancer cell lines were obtained from atcc. cell lines were cultured in complete medium consisting of dmem (sigma aldrich, st. quentin fallavier, france), 10% heat - inactivated foetal calf serum, 2 mm glutamine (sigma aldrich), 100 u / ml penicillin, and 10 g / ml streptomycin (sigma aldrich). expression of the trastuzumab- (4d5-) based car construction against her2 and of the human cd16 (fcriiia / fcri) on the surface of nk-92 was determined by direct immunofluorescence using anti - cd16 (clone 3g8) and anti - human igg2a - fc that recognize the ch1-ch2 spacer of the car (clone hp6002), respectively. for staining 0.1 10 cells (untransduced nk-92, nk-92, and nk-92) were incubated for 15 minutes at room temperature at the indicated mab concentrations diluted with pbs supplemented with 0.1% human albumin in a final volume of 30 l. after staining, plates were centrifuged, the supernatant was discarded by flicking, and wells were washed twice with 200 l ice - cold pbs. negative controls were set up in the presence of a control isotype in case of direct staining or in the absence of first abs for indirect staining. in case of indirect staining, cells were washed after the first incubation and the second ab was used at saturating concentration. two million phoenix - ampho cells were seeded into 10 cm diameter dishes 24 h prior to transfection. the transfection was performed with 15 g pmx / cd16 or pmx / car plasmid dna using cacl2 precipitation (invitrogen). the conditioned medium was collected 48 h after transfection, filtered through 0.45 m pore - size filters, and kept at 80c until use. the viral titer was determined by the transduction of jurkat t cells (1 10 cells per well in 6-well plates) with serial dilutions of virus and analyzed for cd16 or car expression 4 days after infection. the nk-92 cell line was resuspended in rpmi 1640 culture medium supplemented with 10% fbs and 100 iu / ml of recombinant il-2, seeded at 1 10 cells in 1 ml per well into 6-well plates, and exposed to 2 2 ml of retroviral supernatant by spinoculation (2400 g, 1.5 h, and 32c) in the presence of 4 g / ml polybrene (sigma, st. mock (nontransduced) controls were performed in parallel, by which the supernatant of untransfected packaging cells was added to the nk-92 cell line. the transduction efficiencies were assessed 5 days later by flow cytometry after staining the cd16 and the car with a pe - conjugated mouse anti - cd16 (clone 3g8) or a pe - conjugated mouse anti - human igg2a - fc (clone hp6002), respectively. after transduction, nk-92 and nk-92 cells were stained with mouse anti - cd16 (clone 3g8) and anti - human igg2a - fc (clone hp6002), respectively, and immunoselected using anti - mouse - igg coated beads (dynabeads m-450, dynal as, oslo, norway), according to the supplier 's instructions. the target cells were labelled with 100 ci cr for 1 h at 37c, washed four times with culture medium, and plated at the indicated effector - to - target cell ratios in 96-well flat - bottom plates. the indicated mab was incubated with the target cells for 20 mins at room temperature before the addition of effector cells. after 4 h incubation at 37c, 25 l of supernatant was removed from each well and mixed with 100 l scintillation fluid, and cr activity was counted in a scintillation counter (microbeta, perkin elmer, courtaboeuf, france). the results are expressed as the percentage of lysis, which is calculated according to the following equation : (experimental release spontaneous release)/(maximal release spontaneous release) 100, where the experimental release represents mean counts per minute (cpm) of target cells in the presence of effector cells, spontaneous release represents the mean cpm of the target cells incubated without effector cells, and maximal release represents the mean cpm of the target cells incubated with 1% triton x-100 (sigma). nod / scid - il2r (nsg - jax) mice were bred and maintained under pathogen - free conditions at the floralis jean roget institute (ujf grenoble, france) in sterile intraventilated cages. mice were acclimated for 1 week before experimental use in centre lon brard animal facilities (anican). all animal experiments were performed in compliance with french government guidelines and inserm standards for experimental animal studies (agreement b69 - 388 - 0202). they were approved by the local ethics committee of centre lon brard, ens, pbes, and p4 laboratory (cecapp, lyon, france). six - week - old female nod / scid - il2r mice were injected subcutaneously in the left side with 5 10 bt474 cells. when tumors reached a minimal volume of 50 mm, mice were individually identified and randomly assigned to the control or treated groups (5 to 15 mice per group) and treatments were initiated. next, tumor growth was monitored twice a week by measuring two perpendicular diameters with calipers. tumor volume (v) was calculated using the following equation : v = (a b)/2, where a is the width of the tumor (small diameter) and b the length (large diameter), both in millimeters. nonirradiated nk-92, nk-92, or nk-92 effector cells (5 10 each) were injected with ip once a week for 4 weeks. trastuzumab (15 mg / kg) or rituximab control antibody (15 mg / kg) was given ip 24 hours before injection of nk-92 or nk-92. mice were sacrificed before the tumor volume reached 2500 mm. each tumor was dissected and either fixed in formol and processed for histopathological examination or used for rna extraction. frozen excision of bt474 tumors was analyzed by immunohistochemistry on 5 m tissue sections using monoclonal anti - human cd45 (2d1, dako) or iba1 (019 - 19741, wako) antibodies or direct cfse visualization. suspensions were stained with an antibody cocktail and then analyzed by flow cytometry for expression of cd11b (mac-1) versus gr-1 (ly-6 g) and cd11b versus cd16/32 (fcrriii / fcrrii). over 80% of splenocytes were positive for cd16/32 (see figure 7, 1/3 experiments). nk-92 (nk-92, nk-92, or nk-92) and splenocytes or the her2 positive cell line bt474 were cocultured at an effector ratio of 5 : 1 and 2 : 1, respectively, in 96-well round bottom culture plates in rpmi 1640 supplemented with 10% fcs and il2 (100 iu / ml). after 18 h, supernatants were collected and human ifn- content was estimated using an elisa kit (affymetrix, e - biosciences).	the present work was designed to compare two mechanisms of cellular recognition based on ab specificity : firstly, when the anti - her2 mab trastuzumab bridges target cells and cytotoxic lymphocytes armed with a fc receptor (adcc) and, secondly, when her2 positive target cells are directly recognized by cytotoxic lymphocytes armed with a chimeric antigen receptor (car). to compare these two mechanisms, we used the same cellular effector (nk-92) and the same signaling domain (fcri). the nk-92 cytotoxic cell line was transfected with either a fcriiia - fcri (nk-92cd16) or a trastuzumab - based scfv - fcri chimeric receptor (nk-92car). in vitro, the cytotoxic activity against her2 positive target cells after indirect recognition by nk-92cd16 was always inferior to that observed after direct recognition by nk-92car. in contrast, and somehow unexpectedly, in vivo, adoptive transfer of nk-92cd16 + trastuzumab but not of nk-92car induced tumor regression. analysis of the in vivo xenogeneic system suggested that the human ch2-ch3 igg2 used as a spacer in our construct was able to interact with the fcr present at the cell surface of the few nsg - fcr+ remaining immune cells. this interaction, leading to blockage of the nk-92car in the periphery of the engrafted tumor cells, stresses the critical role of the composition of the spacer domain.
coronary artery disease (cad or atherosclerotic heart disease) is the end result of the accumulation of atheromatous plaques within the walls of the coronary arteries that supply the myocardium (the muscle of the heart) with oxygen and nutrients. although the symptoms and signs of cad are noted in the advanced state of disease, most individuals with cad show no evidence of disease for decades as the disease progresses before the first onset of symptoms, often a " sudden " heart attack, finally arises. after decades of progression, some of these atheromatous plaques may rupture and (along with the activation of the blood clotting system) start limiting blood flow to the heart muscle. the disease is the most common cause of sudden death, and is also the most common reason for death of men and women over 20 years of age. according to present trends in the united states, half of healthy 40-year - old men will develop cad in the future, and one in three healthy 40-year - old women. a family history of early cad is one of the less important predictors of cad. screening for cad includes evaluating high - density and low - density lipoprotein (ldl) (cholesterol) levels and triglyceride levels. despite much press, most of the alternative risk factors including homocysteine, c - reactive protein, lipoprotein (a), coronary calcium, and more sophisticated lipid analysis have added little if any additional value to the conventional risk factors of smoking, diabetes, and hypertension. ldl particles can transport cholesterol into the artery wall, retained there by arterial proteoglycans and attract macrophages which engulf the ldl particles and start the formation of plaques, increased levels are associated with atherosclerosis. over time, vulnerable plaques rupture, activate blood clotting, and produce arterial stenosis, which if severe enough results in heart attack, stroke, and peripheral vascular disease symptoms and major debilitating events. in healthy individuals, hdl particles are able to remove cholesterol from atheroma within arteries and transport it back to the liver for excretion or reutilization, which is the main reason why the cholesterol is carried within hdl particles. very low - density lipoprotein, high - density lipoprotein (hdl), ldl are all the vehicles for peroxidation, it is the ldl which gets the maximum load. oxidatively modified ldl (ox - ldl) leads to generation of foam cells, main factor of atherosclerosis. ox - ldl level gives better idea of atherogenic potential of an individual ; quantitation of malondialdehyde (mda) by thiobarbituric acid method is one of the commonly utilized indices of lipid peroxidation. the aim of the present study was to investigate the relationship and degree of association between increased oxidative stress and abnormal lipid profile in cad cases and comparing the same with healthy controls. a total of 42 immediately survived myocardial infarction outpatients with cad of both sex in the age group of 41 to 74 years (mean sd = 61.71429 8.75210), attending cardiology department at mamata medical college hospital, khammam, were included in the study. a total of 33 age- (mean sd = 59.24242 9.57219) and sex - matched healthy controls were included in the study. persons with diabetes mellitus, renal disease, and smokers were excluded from the study. oxidative stress was assessed by measuring mda, and dyslipidemia was assessed by measuring lipid profile parameters in all the subjects. thiobarbituric acid reactive substances was estimated in plasma, described by k. satoh using mda as reference standard, expressed as nmol / ml of plasma. total cholesterol (chod - pap - method),[912 ] -hdl (hdl precipitating method),[1315 ] ldl (friedwald formula), and triacylglycerol (tag) (gpo - pap method)[912 ] were measured on merck microlab 200 semiautoanalyzer in all the subjects. student t - test unpaired was used to compare the means between cases and controls at 5% level of significance. equivalent nonparametric tests like rank sums test (unpaired samples) and mann - whitney u test (unpaired samples) were also used to compare the means between cases and controls at 5% level of significance. correlation analysis was calculated using pearson 's correlation coefficient and the nonparametric equivalent spearman 's rank correlation coefficient at 95% confidence of interval. student t - test unpaired was used to compare the means between cases and controls at 5% level of significance. equivalent nonparametric tests like rank sums test (unpaired samples) and mann - whitney u test (unpaired samples) were also used to compare the means between cases and controls at 5% level of significance. correlation analysis was calculated using pearson 's correlation coefficient and the nonparametric equivalent spearman 's rank correlation coefficient at 95% confidence of interval. in the present study, oxidative stress was assessed by measuring mda and dyslipidemia was assessed by measuring lipid profile parameters in 42 immediately survived myocardial infarction outpatients with cad and 33 healthy controls. mda was significantly increased in cases (mean sd = 6.61429 0.61891) than controls (mean sd = 3.07030 0.44168) as p = 0.0000001. total cholesterol was significantly high in cases (mean sd = 259.90476 12.04020) than controls (mean sd = 177.48485 11.11340) as p = 0.0000001. hdl cholesterol was significantly decreased in cases (mean sd = 34.80952 3.95239) than controls (mean sd = 43.45455 6.16487) as p = 0.0000001. ldl cholesterol was significantly high in cases (mean sd = 147.52381 20.53817) than controls (mean sd = 107.09091 6.10514) as p = 0.0000001. finally, triacylglyceride was significantly high in cases (mean sd = 218.90476 17.65912) than controls (mean sd = 130.48482 13.02910) as p = 0.0000001 as shown in our table 1. showing the statistical analysis of data of mda and lipid profile parameters correlation analysis was calculated using pearson 's correlation coefficient and nonparametric equivalent spearman 's rank correlation coefficient. at 5% level of significance, assuming that at least one of the variable is normally distributed, calculation of pearson 's correlation coefficient showed a statistically highly insignificant positive correlation between mda and total cholesterol (r = 0.258) as p = 0.098346, between mda and ldl cholesterol (r = 0.199) as p = 0.205302, and between mda and hdl cholesterol (r = 0.134) as p = 0.399128. significant negative correlation was observed between mda and triacylglyceride (r = -0.314) as p = 0.042863 which was statistically significant as shown in our table 2. calculation of correlation analysis by nonparametric equivalent spearman 's rank correlation coefficient also showed the same result as pearson 's correlation coefficient analysis, as shown in table 2. correlation analysis was also represented graphically by scatter diagram. in all the scatter figures 14, mda values are taken on horizontal axis and lipid profile parameters values are taken on vertical axis. in all the scatter figures 1,2,3, the points are scattered randomly around the arbitrary central linear line suggesting no statistically significant linear relationship between increased oxidative stress and abnormal lipid profile. no linear relationship was observed between mda and total cholesterol as shown in our scatter diagram one. similarly, no linear relationship was observed between mda and hdl cholesterol as shown in our scatter diagram two. furthermore no linear relationship was observed between mda and ldl cholesterol as shown figure 3. in contrast to this, we have observed a statistically significant negative relationship between mda and tag, as shown in figure 4. no linear relationship between mda and total cholesterol no linear relationship between mda and hdl cholesterol no linear relationship between mda and ldl cholesterol negative linear relationship between mda and tag our study showed a significant increase in the oxidative stress in cad cases than controls. similarly, hdl less than 35 mg / dl is a known independent high - risk factor for arteriosclerosis. but in our study, both pearson 's correlation coefficient analysis and nonparametric equivalent spearman 's rank correlation coefficient analysis showed a statistically highly insignificant correlation between mda level and each lipid profile parameter in cad cases. furthermore, the scatter diagrams between mda and each lipid profile parameter also showed no correlation. in our correlation coefficient analysis study, we could not establish a statistically significant linear relationship between increased oxidative stress and abnormal lipid profile parameters, except tag which showed a significant negative correlation. this clearly suggests that increased oxidative stress and abnormal lipid profile are two independent risk factors in the pathomechanism of atherogenesis.	background : coronary artery disease (cad) is the most common cause of sudden death, none and death of men and women over 20 years of age. the aim of the study was to know if there is any linear correlation between oxidants and abnormal lipid profile parameters in cad.materials and methods : the present study includes 42 known cad cases (age = 4175 years) and 33 age- and sex - matched healthy controls. malondialdehyde (mda), total cholesterol, high - density lipoprotein (hdl) cholesterol, and triacylglyceride were measured and low - density lipoprotein (ldl) cholesterol was calculated in both cases and controls, respectively.results:mda was significantly increased in cases than controls (p = 0.0000001). total cholesterol was high in cases than controls (p = 0.0000001). hdl cholesterol was significantly decreased in cases than controls (p = 0.0000001). ldl cholesterol was high in cases than controls (p = 0.0000001). triacylglyceride was high in cases than controls (p = 0.0000001). insignificant positive correlation were observed between mda and total cholesterol (r = 0.258), between mda and ldl cholesterol (r = 0.199), and between mda and hdl cholesterol (r = 0.134). negative correlation was observed between mda and triacylglyceride (r = -0.314).conclusion : increased oxidative stress and abnormal lipid profile were observed in cad cases. our study showed that statistically significant linear relationship could not be established between increased oxidative stress and abnormal lipid profile parameters, suggesting that increased oxidative stress and abnormal lipid profile are two independent risk factors in the pathomechanism of atherogenesis.
many methods of drug development have passed through fashion, from high - throughput screening, to targeted molecular design and natural product selection. a technique that has been re - invented at regular intervals (usually with a new name) is one that ironically also re - invents the drugs in question. drug re - profiling, (also known as therapeutic switching, repurposing or drug life cycle management) is the re - development of a drug for a use that is an alternative disease or patient population than that for which it was originally developed. for a drug to have a biological effect, it is almost guaranteed to have more biological effects than the developers intend, whether caused by off - target action, whereby the drug hits multiple types of biological sites, which is more common in older drugs ; or those side effects that are inevitable due to the action of the drug, such as initial light - headedness being caused by a drug designed to reduce blood pressure. by harnessing either of these classes of serendipitous drug action, a new application for the drug may be developed. the cost of bringing a drug from the early discovery stages through to the market currently stands at an estimated average of $ 2.6 bn. this cost incorporates even more than a simple total of the numbers of patients that go through the multiple phases of clinical trials, the animals that must be tested on during the preclinical stage and the initial screening, design and development stages ; they must also include a factor of the number of failures of drug candidates throughout this process. statistics for failure can range in estimate from 90% to 99+% depending on the source or stage of development considered, with the majority of these being due to toxicity found prior to (and more rarely during) clinical trials or a lack of relevant bioavailability found during early clinical trials, where the drug is found to not be absorbed and present in the appropriate body part to treat the disease. in drug reprofiling these risks all but disappear, with drug metabolism and pharmacokinetics already established in a reincarnation of a previously developed drug, and the decision of route of administration and formulation can be made in a much more informed manner. the failure risk of lack of efficacy in the treatment of a disease remains during clinical trials, but leapfrogging the early stages, and minimising these risks highlights the biggest advantages to the technique, demonstrating lower risks, costs and time of development. when you already know the pharmacokinetics, safety, tolerability, contraindications and special populations of a drug under development, half the patient information sheet is already written. if it were all that simple though, surely everyone would be doing it, so let us also consider the challenges, and a few of the solutions with worked examples. off - label prescriptions of drugs may be frowned upon by regulators, but impossible to stop by gps in practice particularly where the label is geographically constrained, for example bupropion has an fda (us) label as a smoking cessation aid and an anti - depressant ; yet in the uk has a label only for smoking cessation, leading to temptation by uk physicians to prescribe it off label for depression - related diseases. this act of prescription is not illegal, if a physician feels that a patient will benefit from an off - label prescription and a labelled drug is not available to that patient, then this is an acceptable use. it is not, however, allowed for a company to promote the use of their licensed drugs to be used in any disease state or patient population for which it does not have full regulatory approval. this legality of non - promotion of off - label uses of drugs has frequently been enforced, with highly visible lawsuits against companies such as j&j for the actions of their sales representatives in recommending risperidone prescription for geriatric patients, but why would a pharmaceutical company carry out the trials to register an alternate use if the drug is already off - patent and available at a fraction of the price through a generics company ? a challenge for the re - development of a drug into a new disease use, therefore, needs a differentiating feature to secure both the intellectual property prescription to allow premium pricing but also to prevent off - label use of the original product which will have limited or no remaining patent coverage. the latter may be achieved simply by using a geographical switch, for example or by using a drug that is no longer in regular use, such as thalidomide which has moved from being withdrawn an anti - emetic after the teratogenic properties were discovered, to be reborn as first as a treatment of leprosy and later yet again as an anti - cancer treatment of multiple myeloma courtesy of celgene s re - development program. an even lower risk strategy is demonstrated by the re - licensing of paroxetine from long - standing use as the anti - depressant treatment, paxil, to be re - launched in 2013 as brisdelle, a treatment of menopause - related hot flashes with differentiation provided merely by a decrease in dose from 20 mg to 7.5 mg, below the minimum tablet size previously available, as well as below the level for which side effects were commonly observed. intellectual property protection, in the traditional form, of a patent has been clarified in recent years, with the european patent office defining second medical use patents as those quoting a known medicament to be used for the treatment of a disease in a sufficiently inventive way. the us patent office allows consideration of methods of treatment to cover a comparable, although slightly differently worded coverage of marketing of a drug development program delineated by disease rather than solely by drug structure. although this does not prevent off - label uses, with astute alterations to either the formulation or dosing level / schedule, this protection is sufficient that the prescription of the new branded treatment will be preferred over the generic. for example, by the use of a delayed release formulation, or an entire switch in the route of administration. an anti - histamine cream to treat an insect bite may be possible to affect with a crushed oral anti - histamine tablet in a plain cream, but why would the prescriber not opt for the pre - formulated, dermal cream. reformulation can be explored in much greater detail as a strategy for reprofiling ; in fact, it is the answer to many of the criticisms of the entire paradigm of recycling old drugs for new uses the true way of adding bling to a development process ! arguments against reprofiling include the automatic expectation of side effects in the form of the effects of the original use, lack of composition of matter patent protection, off - label prescription risk and overcoming any prejudices or prior reputations of the drug. our development program val401 provides an example of the combination of reprofiling with reformulation above, and as such we explore the details below to provide an outline of a successful reprofiling process. val401 is a formulation of risperidone in a lipid - filled capsule currently in clinical trials for the treatment of non - small cell lung cancer. risperidone has a history of clinical use as an anti - psychotic, being developed originally as an oral tablet for use as a once or twice a day treatment, formulations have extended to include oral disintegrating tablets and extended release intramuscular injections. risperidone has a chequered and very public history. as one of the first of the group known as atypical anti - psychotics, it was first approved for use for the chronic treatment of schizophrenia and bipolar disorder in 1993, revolutionising treatment for many patients, who had received no new treatment options for many years. the licensed uses have expanded across the decades of international clinical use, and it became the first anti - psychotic to be specifically prescribed for adolescent schizophrenia, and is now used also to treat autism - related aggression in children. however, the developers, janssen, have come under intense scrutiny as they faced accusations of under - reporting some side effects, with gynecomastia in pubescent boys being of particular distress. off - label use has also been widely evident, with literature reports of risperidone being used in late stage cancer patients to treat chemotherapy - induced delirium and nausea ; and in teenage girls to treat anorexia. in fact, all anti - psychotics (both typical and atypical) carry a black box warning against the treatment of delirium in geriatric patients after a meta - analysis demonstrated an increase in the incidence of death in treated patients. on this matter janssen have defended a number of lawsuits for off - label marketing, where sales representatives were accused of promoting use in the geriatric population in nursing homes. in spite of this sometimes negative publicity, risperidone remained an ideal choice for a reprofiling project. despite a host of known dopamine and serotonin receptor interactions, the extent of the clinical anti - psychotic effect of risperidone is not fully explained, by comparison to other antipsychotic drugs. therefore, when the opportunity presented to look for other biological causes of effect, we were intrigued to discover that risperidone inhibits an enzyme known as hsd10 (hydroxysteroid dehydrogenase type 10). this is a redox enzyme, involved in many intracellular reduction and oxidation reactions with substrates including steroids, fatty acids and lipids. in our experiments, although there is no published evidence that this inhibition contributes to the anti - psychotic properties of the drug, there are many literature references to hsd10 as a putative, but as yet unexploited, target for anti - cancer treatments. it is interesting to note that hsd10 is overexpressed in a number of adenocarcinoma cell lines, and in our experiments val401 is selectively effective at targeting these cells. this data provided sufficient evidence to investigate risperidone as a reprofiling candidate, of an off - target example, however, initial results using the drug directly against relevant cell lines in vitro provided insufficient therapeutic effect. formulation screening with lipids remedied this lack, and val401 was confirmed as a specific reformulation of lipidic risperidone, with preclinical studies demonstrating safety, pharmacology and anti - cancer efficacy sufficient to build a package of data including the clinical data available on conventional risperidone. val401 is now entering the clinical stage of development by moving straight to a phase 2 clinical trial for the treatment of end stage non - small cell lung adenocarcinoma patients, entering with the dose level and schedule intended in the eventual market label. the pre - knowledge of side effects, contraindications, tolerability and special indications allowed an exemption from phase 1 trials to be effected, and the ready availability, as well as pre - knowledge of formulation and stability parameters allowed an accelerated cmc program. the conversion of this active ingredient from anti - psychotic use to use in terminally ill cancer patients, allows an acceptance of the side effect profile. as a general rule, the more severe the disease, the greater side effect burden can be tolerated ; but in this case the argument is even greater. the greatest side effect from chronic risperidone use is reported as being an increase in appetite leading to a significant gain in weight in the patients, as late stage lung cancer patients are usually suffering from the wasting of cachexia, this side effect is seen as an advantage ; while the primary pharmacology is not perceived as being an additional hurdle, as the modulation of dopamine and serotonin levels is expected to provide an amelioration of potential instability in the patients. the pubescent male gynecomastia is of course completely irrelevant in this patient population, as the late stage lung adenocarcinoma population includes very few if any pubescent boys. this provides an example whereby the side effects and original desired effects are either acceptable in the patient population, or in fact irrelevant to the patient. at the beginning of this reprofiling project as the drug was only just coming off patent for the original composition of matter patent, this was judged ideal, as the active ingredient was available as a generic, but the development was recent enough to ensure that development was carried out to modern standards. the reformulation required to achieve the anti - cancer activity of risperidone provided opportunity for patent protection of the formulation and process surrounding this, as well as for the method of treatment utility patents. in fact we have been granted us patent protection on both these elements, and international protection is pending, demonstrating the strength of the program strategy [16, 31 ]. our other current clinical program (val201) is a good demonstration of how a drug life cycle management can be built in from the very beginning of the program. in this example it is a function of the mechanism of action, the inhibition of the docking of estragon and androgen receptors to the sh3 domain of src kinase. this inhibition is believe to be the primary mode of action for the use of val201 in the treatment of prostate cancer, for which it is currently in clinical trials as a sub - cutaneous injection ; but even before this trial is complete, investigations are underway to reformulate val201 api into a newly designated drug product (imp) val301 into an implantable device or an oral formulation for use in the treatment of endometriosis. in this case we will be able to project the safety and tolerability data from the men in the prostate cancer clinical trial to short - cut the phase 1 clinical trial, and use the pharmacokinetic data to provide an indication of the level of dosing needed in the women enrolled in the endometriosis trial. this package of information will allow a fast - tracked discovery process to design a trial that co - ordinates the level of drug being release from the implant into the patient s bloodstream with a measure of disease modifying effect such as a reduction in pain or lowering of endometrial lesions. this therapeutic switch from prostate cancer to endometriosis provides a life cycle management that will extend the patent protection over val201, include an element of reformulation to produce the implant, which may also ultimately be also used in cancer patients, and potentially provide an accelerated solution to a very much unmet medical need. by developing both disease indications nearly in parallel, in this case we are able to assist both programs by transfer of real - time information about patient responses, formulations and opportunities. off - label prescriptions of drugs may be frowned upon by regulators, but impossible to stop by gps in practice particularly where the label is geographically constrained, for example bupropion has an fda (us) label as a smoking cessation aid and an anti - depressant ; yet in the uk has a label only for smoking cessation, leading to temptation by uk physicians to prescribe it off label for depression - related diseases. this act of prescription is not illegal, if a physician feels that a patient will benefit from an off - label prescription and a labelled drug is not available to that patient, then this is an acceptable use. it is not, however, allowed for a company to promote the use of their licensed drugs to be used in any disease state or patient population for which it does not have full regulatory approval. this legality of non - promotion of off - label uses of drugs has frequently been enforced, with highly visible lawsuits against companies such as j&j for the actions of their sales representatives in recommending risperidone prescription for geriatric patients, but why would a pharmaceutical company carry out the trials to register an alternate use if the drug is already off - patent and available at a fraction of the price through a generics company ? a challenge for the re - development of a drug into a new disease use, therefore, needs a differentiating feature to secure both the intellectual property prescription to allow premium pricing but also to prevent off - label use of the original product which will have limited or no remaining patent coverage. the latter may be achieved simply by using a geographical switch, for example or by using a drug that is no longer in regular use, such as thalidomide which has moved from being withdrawn an anti - emetic after the teratogenic properties were discovered, to be reborn as first as a treatment of leprosy and later yet again as an anti - cancer treatment of multiple myeloma courtesy of celgene s re - development program. an even lower risk strategy is demonstrated by the re - licensing of paroxetine from long - standing use as the anti - depressant treatment, paxil, to be re - launched in 2013 as brisdelle, a treatment of menopause - related hot flashes with differentiation provided merely by a decrease in dose from 20 mg to 7.5 mg, below the minimum tablet size previously available, as well as below the level for which side effects were commonly observed. intellectual property protection, in the traditional form, of a patent has been clarified in recent years, with the european patent office defining second medical use patents as those quoting a known medicament to be used for the treatment of a disease in a sufficiently inventive way. the us patent office allows consideration of methods of treatment to cover a comparable, although slightly differently worded coverage of marketing of a drug development program delineated by disease rather than solely by drug structure. although this does not prevent off - label uses, with astute alterations to either the formulation or dosing level / schedule, this protection is sufficient that the prescription of the new branded treatment will be preferred over the generic. for example, by the use of a delayed release formulation, or an entire switch in the route of administration. an anti - histamine cream to treat an insect bite may be possible to affect with a crushed oral anti - histamine tablet in a plain cream, but why would the prescriber not opt for the pre - formulated, dermal cream. reformulation can be explored in much greater detail as a strategy for reprofiling ; in fact, it is the answer to many of the criticisms of the entire paradigm of recycling old drugs for new uses the true way of adding bling to a development process ! arguments against reprofiling include the automatic expectation of side effects in the form of the effects of the original use, lack of composition of matter patent protection, off - label prescription risk and overcoming any prejudices or prior reputations of the drug. our development program val401 provides an example of the combination of reprofiling with reformulation above, and as such we explore the details below to provide an outline of a successful reprofiling process. val401 is a formulation of risperidone in a lipid - filled capsule currently in clinical trials for the treatment of non - small cell lung cancer. risperidone has a history of clinical use as an anti - psychotic, being developed originally as an oral tablet for use as a once or twice a day treatment, formulations have extended to include oral disintegrating tablets and extended release intramuscular injections. as one of the first of the group known as atypical anti - psychotics, it was first approved for use for the chronic treatment of schizophrenia and bipolar disorder in 1993, revolutionising treatment for many patients, who had received no new treatment options for many years. the licensed uses have expanded across the decades of international clinical use, and it became the first anti - psychotic to be specifically prescribed for adolescent schizophrenia, and is now used also to treat autism - related aggression in children. however, the developers, janssen, have come under intense scrutiny as they faced accusations of under - reporting some side effects, with gynecomastia in pubescent boys being of particular distress. off - label use has also been widely evident, with literature reports of risperidone being used in late stage cancer patients to treat chemotherapy - induced delirium and nausea ; and in teenage girls to treat anorexia. in fact, all anti - psychotics (both typical and atypical) carry a black box warning against the treatment of delirium in geriatric patients after a meta - analysis demonstrated an increase in the incidence of death in treated patients. on this matter janssen have defended a number of lawsuits for off - label marketing, where sales representatives were accused of promoting use in the geriatric population in nursing homes. in spite of this sometimes negative publicity, risperidone remained an ideal choice for a reprofiling project. despite a host of known dopamine and serotonin receptor interactions, the extent of the clinical anti - psychotic effect of risperidone is not fully explained, by comparison to other antipsychotic drugs. therefore, when the opportunity presented to look for other biological causes of effect, we were intrigued to discover that risperidone inhibits an enzyme known as hsd10 (hydroxysteroid dehydrogenase type 10). this is a redox enzyme, involved in many intracellular reduction and oxidation reactions with substrates including steroids, fatty acids and lipids. in our experiments, although there is no published evidence that this inhibition contributes to the anti - psychotic properties of the drug, there are many literature references to hsd10 as a putative, but as yet unexploited, target for anti - cancer treatments. it is interesting to note that hsd10 is overexpressed in a number of adenocarcinoma cell lines, and in our experiments val401 is selectively effective at targeting these cells. this data provided sufficient evidence to investigate risperidone as a reprofiling candidate, of an off - target example, however, initial results using the drug directly against relevant cell lines in vitro provided insufficient therapeutic effect. formulation screening with lipids remedied this lack, and val401 was confirmed as a specific reformulation of lipidic risperidone, with preclinical studies demonstrating safety, pharmacology and anti - cancer efficacy sufficient to build a package of data including the clinical data available on conventional risperidone. val401 is now entering the clinical stage of development by moving straight to a phase 2 clinical trial for the treatment of end stage non - small cell lung adenocarcinoma patients, entering with the dose level and schedule intended in the eventual market label. the pre - knowledge of side effects, contraindications, tolerability and special indications allowed an exemption from phase 1 trials to be effected, and the ready availability, as well as pre - knowledge of formulation and stability parameters allowed an accelerated cmc program. the conversion of this active ingredient from anti - psychotic use to use in terminally ill cancer patients, allows an acceptance of the side effect profile. as a general rule, the more severe the disease, the greater side effect burden can be tolerated ; but in this case the argument is even greater. the greatest side effect from chronic risperidone use is reported as being an increase in appetite leading to a significant gain in weight in the patients, as late stage lung cancer patients are usually suffering from the wasting of cachexia, this side effect is seen as an advantage ; while the primary pharmacology is not perceived as being an additional hurdle, as the modulation of dopamine and serotonin levels is expected to provide an amelioration of potential instability in the patients. the pubescent male gynecomastia is of course completely irrelevant in this patient population, as the late stage lung adenocarcinoma population includes very few if any pubescent boys. this provides an example whereby the side effects and original desired effects are either acceptable in the patient population, or in fact irrelevant to the patient. at the beginning of this reprofiling project as the drug was only just coming off patent for the original composition of matter patent, this was judged ideal, as the active ingredient was available as a generic, but the development was recent enough to ensure that development was carried out to modern standards. the reformulation required to achieve the anti - cancer activity of risperidone provided opportunity for patent protection of the formulation and process surrounding this, as well as for the method of treatment utility patents. in fact we have been granted us patent protection on both these elements, and international protection is pending, demonstrating the strength of the program strategy [16, 31 ]. our other current clinical program (val201) is a good demonstration of how a drug life cycle management can be built in from the very beginning of the program. in this example it is a function of the mechanism of action, the inhibition of the docking of estragon and androgen receptors to the sh3 domain of src kinase. this inhibition is believe to be the primary mode of action for the use of val201 in the treatment of prostate cancer, for which it is currently in clinical trials as a sub - cutaneous injection ; but even before this trial is complete, investigations are underway to reformulate val201 api into a newly designated drug product (imp) val301 into an implantable device or an oral formulation for use in the treatment of endometriosis. in this case we will be able to project the safety and tolerability data from the men in the prostate cancer clinical trial to short - cut the phase 1 clinical trial, and use the pharmacokinetic data to provide an indication of the level of dosing needed in the women enrolled in the endometriosis trial. this package of information will allow a fast - tracked discovery process to design a trial that co - ordinates the level of drug being release from the implant into the patient s bloodstream with a measure of disease modifying effect such as a reduction in pain or lowering of endometrial lesions. this therapeutic switch from prostate cancer to endometriosis provides a life cycle management that will extend the patent protection over val201, include an element of reformulation to produce the implant, which may also ultimately be also used in cancer patients, and potentially provide an accelerated solution to a very much unmet medical need. by developing both disease indications nearly in parallel, in this case we are able to assist both programs by transfer of real - time information about patient responses, formulations and opportunities. therefore, in summary, we present reprofiling as an economic and environmentally friendly alternative to traditional drug development. there is the recycling of out dated solutions of the problems into the shiny new tools in the physician s toolkit. although frequently out of fashion, this process of checking the fundamental science beyond compounds should be included in the to - do list of any company, when looking forward to expand the clinical pipelines. too often reprofiling is used only in an emergency to shift a candidate that has failed efficacy trials in a cancer indication to an alternative cancerous body - part ; whereas we believe an understanding of the overall biological action of a drug can allow access to entirely new opportunities for use. with ever increasing understanding of genomics, epi - genetics and network pharmacology we have confidence that the ability to harness the power of old compounds will become ever more valuable. the two examples described above within the valirx portfolio are ideal examples of the strategy in practice, and demonstrate how a forward - thinking company can maximize opportunities from a basic science understanding ; it is the authors opinion that the acceptability of this approach will continue to increase in trend. sjd and gsm are employed by valirx plc and its subsidiaries and have been involved in the direct project management of the val201, val301 and val401 programs discussed herein.	drug development has moved along way forward from the days of with doctors peddling cauldrons of herbs and spices, however, the process can still miss opportunities for full exploitation of a drug s potential. drug reprofiling provides a chance for an established or a forgotten drug to move into a new area of therapy, whether related to the known effects or in a completely new area. in an era of environmental awareness and spiraling costs for traditional drug development, a strategy to squeeze every benefit out of drugs with known safety, tolerability and pharmacological parameters must be a strategically sound desire. we explore examples of success in reprofiling, draw comparisons between techniques, and finally provide two examples from the valirx plc development pipeline currently undergoing the process.
traditional risk factors include age, gender, bp, total cholesterol, high - density lipoprotein, smoking, diabetes mellitus, family history of cardiovascular disease, microalbuminuria, or gfr<60 these new risk factors include homocysteine, hscrp, fibrinogen, triglyceride, hba1c, lipoprotein a, plasma myeloperoxidase, red cell glutathione peroxidase 1, ethnic origin, socioeconomic status, and use of antihypertensive drugs. a test may have different levels of efficacy in determining short- or long - term risks. it also needs to be easily applicable, have a high predictive value in cvr assessment, and be accurately reproducible. the main aim in determining cvr is to predict cardiovascular endpoints that may emerge within a certain time period. there are differences between endpoints in the various cvr prediction modalities used for this purpose, however. initially cvr estimates were made for the next 5 years, and cv events and mortality were defined as endpoints. in light of subsequent developments, cardiovascular, cerebrovascular, peripheral vascular events, and factors included in initial risk scoring were age, gender, systolic bp, smoking, and diabetes mellitus, while in later years, factors such as hdl, family history, triglycerides, and hba1c levels began to be used in different scoring predictors. global cvr calculation considers the cumulative impact of all factors beyond the simple effect of each factor alone. in the majority of the predictive tools, the proportional hazards technique is used, based on semi - parametric cox or parametric weibull methods. the first is the concept of discrimination, the power to distinguish whether or not a clinical endpoint will result. the second important concept is calibration, the measurement of the extent to which predicted results match the clinical outcomes. another concept is reclassification, which measures the effect of an additional factor on a clinical endpoint when a new risk factor is added to a risk calculation technique. the most frequently employed are framingham, score, procam, reynolds, qrisk, assign, aric, progetto cuore, and personal heart 2007 (table 1). risk calculation is performed with the determination of factors included in the calculation in light of these studies and of the strength of these factors. in some cvr calculations, charts showing risk factors and what the total risk will be are used, while in others scores are given for each factor, if present, and risk is calculated on the basis of the total score. today, no matter what the intermediate stages of the calculation technique are employed, data being entered into an electronic environment have accelerated not only the obtaining of results, but also the calculation speed, as well as improving the ease of application. it must not be forgotten, however, that there are differences in calculation techniques and that population differences can affect evaluation techniques and risk assessment. the first and widely used globally cvr assessment method is the framingham risk score (table 1). this is an assessment modality based on a study performed on a population in the usa. in later years, the number and type of risk factors have been updated. electrocardiography and left ventricle hypertrophy were used in the first two versions, though these parameters are not employed in later versions. depending on the population in which the study was performed, however, adjustments are necessary if it is to be performed in other populations. the score calculation technique is a system based on 12 prospective studies in 11 countries in europe. score is an evaluation system that does not include diabetes mellitus (dm) as a risk assessment determinant, although dm is an important risk factor of cardiovascular events. another important difference between it and other evaluation methods is that score includes fatal cardiovascular events as an endpoint (table 1). total cholesterol / hdl ratio was used in the first version, while in the new risk assessment, and parallel to the new dyslipidemia guidelines, hdl has been added as a separate variable. the procam assessment method is a system based on research in germany, mainly involving industrial workers. in the first version, gender difference was not used in risk assessment. one important difference is that family history was first included in the assessment parameters in this calculation method, in contrast to other systems (table 1). one important feature of this system is the use for the first time of antihypertensive therapy as a determining factor in risk assessment. the possibility of initial risk changing with treatment in subsequent years needs to be borne in mind. apart from aric, antihypertensive use is also employed as a determining factor in the final version of framingham, progetto cuore 2004, shs 2006, and qrisk. in fact, lipid - reduction therapy and anti - platelet therapy may clearly also be separate factors in risk determination in addition to antihypertensive therapy. however, only reynolds uses lipid - reduction therapy as a risk determination factor (table 1). use of platelet - inhibiting drugs is still not used in risk assessment in any system. the assign 2007 and qrisk scoring systems the most important feature of these two assessment systems is that socioeconomic status was used as a risk determinant for the first time. younger age may lead to the masking of true risk despite the presence of many significant risk factors. in contrast, the risk calculation error in the elderly may be an overestimation of absolute risk because of advanced age. a significant problem in addition to overestimation in risk assessment is that this can lead to associated overmedication. adjustments in terms of ethnicity data therefore need to be made in risk assessment methods. on the other hand, consideration of variation in socioeconomic status that has emerged as a risk determinant in recent years will reduce assessment errors. finally, it should not be forgotten that there might be parameters that vary throughout a patient 's life that that need to be updated at intervals. cvr assessment should be performed in all hypertensive patients, the results shared with the patient, and modifiable risk factors effectively treated. risk assessment methods being based on the population in which the patient lives and the inclusion of factors such as ethnicity variations, socioeconomic status, and medication use will contribute to the improvement of risk assessments.	several calculation modalities are used today for cardiovascular risk assessment. cardiovascular risk assessment should be performed in all hypertensive patients. risk assessment methods being based on the population in which the patient lives and the inclusion of factors such as ethnicity variations, socioeconomic status, and medication use will contribute to improvements in risk assessments. the results should be shared with the patient, and modifiable risk factors must be effectively treated.
in september of 1977, gruentzig performed the first coronary angioplasty as a nonsurgical method for coronary artery revascularization on a 40-year - old patient in zurich, switzerland. the angioplasty in fact induces a controlled injury to the coronary vessel and has two major limitations - acute vessel closure (6%-8%) and restenosis (30%-50%). the pathophysiology of acute vessel closure after angioplasty involves denudation of the endothelium of the coronary artery followed by rapid accumulation of fibrin and platelets, disruption of the atheromatous plaque with intimal dissection and medial tearing, and elastic recoil. restenosis involves smooth muscle proliferation and neointimal hyperplasia [2, 3 ]. in an attempt to overcome these problems, bare metal stents (bms) were introduced into clinical practice in 1986. bms are metallic scaffolds deployed within a diseased coronary artery segment to optimize the lumen integrity by tacking dissection flaps against the vessel wall and providing mechanical lumen patency. two large clinical trials, the belgium netherlands stent arterial revascularization therapies study (benestent) and the north american stent restenosis study (stress) [3, 4 ], showed bms significantly decrease the incidence of target - lesion revascularization from 25%-35% with percutaneous coronary angioplasty (ptca) to 10%-15% with stenting. the success in treatment of acute vessel closure came with a price - increased rates of acute (24h) and subacute (24h to 30 days) stent thrombosis, which was addressed with aggressive anticoagulation attempts. the benestent and the stress study reported subacute stent thrombosis of 3.5% and 3.4% respectively despite the complex anticoagulation regimens used (dextran, aspirin, dipyridamole, heparin, and warfarin) [3, 4 ]. the introduction of intravascular ultrasound, high pressure balloons during stent deployment, and the establishment of dual antiplatelet therapy (dapt) after stent placement contributed to the decrease of bms thrombosis (currently 1.2%) [5, 6 ]. the usefulness of a dual antiplatelet therapy was demonstrated by the pci - cure study in which 2658 patients with acute coronary syndrome (acs) underwent percutaneous coronary intervention (pci). patients were randomly assigned to one - year treatment with clopidogrel and aspirin (asa) or placebo and asa. in this study, an overall 31% reduction (p = 0.002) of cardiovascular mortality or myocardial infarction (mi) rate the difference between both groups appears during the first 3 months, and stays constant or slightly increases up to 12 months. the results of the pci - cure study provided the basis for recommending the institution of dapt of clopidogrel and asa for patients presenting with acs, including the patients treated with stents. the purpose of this review is to provide an overview of the changing culture of coronary artery stenting, in addition to discussing perioperative management strategies and controversies surrounding coronary stents and antiplatelet therapy. a comprehensive literature search of medline was conducted using as keywords : antiplatelet therapy, non - coronary surgery, drug - eluting stents, and stent thrombosis. over 250 relevant articles were found, 88 of which we have cited and discussed in this article based on their specific relevance to perioperative management, perioperative bleeding, and perioperative thrombosis in patients with drug eluting stents (des) on dapt presenting for invasive procedures, mechanisms of stent thrombosis. currently, there are three categories of antiplatelet agents in use : acetylsalicylic acid (asa), platelet p2y12 receptor inhibitors (clopidogrel, prasugrel, ticagrelor), and platelet gpiib - iiia inhibitors (eptifibatide, tirofiban, abciximab). asa is recommended for primary prevention only for diabetic patients with risk of cardiovascular disease. when used for secondary prevention, asa is a lifelong therapy. in their meta - analyses of 50,279 patients for secondary prevention for coronary artery disease, biondi - zoccai. showed that the cardiac complication rate was three times higher after asa withdrawal and increased even more in patients with coronary stents [8, 12 ]. there was, on average, a 10.6-day period between withdrawal from asa and thrombotic events (8.5 days for coronary symptoms). p2y12 inhibitors include the thienopyridines, clopidogrel and prasugrel, and the cyclopentyl triazolopyrimidine ticagrelor. clopidogrel, a pro - drug, is metabolized to active metabolite in the liver in a two - step process by cyp3a4/3a5 and cyp2b6/1a2/2c9/2c19 esterases. clopidogrel decreases the risk of mi in unstable angina by 18% and the risk of coronary stent thrombosis by 30%. addition of clopidogrel to asa decreases the relative risk in the combined end point of cardiovascular death, mi, or stroke by 20%. prasugrel, also a pro - drug, converts to active metabolite more rapidly, in only one step (cyp3a4 dependent). the metabolite achieves 2.2 times higher level than that of clopidogrel. in phase iii clinical trials (trilogy - acs), prasugrel, when compared to clopidogrel, produced a statistically significant reduction of 19% in the primary endpoint of cardiovascular death, nonfatal mi, or nonfatal stroke in the ua / nstemi population (p=0.0004). a 34% decline was observed in urgent target vessel revascularization (p 2.5 mm to < 3.5 mm (< 3.7 mm for paclitaxel stents). clinical trials investigating the restenosis of stents found a 74% reduction of restenosis at 4 years of implantation. as a result, the use of des significantly increased (up to 85% of all stents placed) in the u.s. and the des were used off label in high - risk populations (diabetics, patients with acs, low ejection fraction, or renal failure), high - risk lesions (bifurcating lesions, long ones, small vessel lesions, in stent lesions, multiple lesions, left main disease, saphenous vein graft lesions), or other conditions that were excluded from the initial trials. stent thrombosis and des in 2003, 290 cases of subacute stent thrombosis occurring after sirolimus des implantation were reported to the fda along with a 20% mortality rate. the goal of the basel stent kosten effektivitts trial - late thrombotic events (basket - late) study was to determine the true incidence of late stent thrombosis, mi, and death in 746 patients randomized to receive des or bms. patients on dapt for 6 months without any adverse cardiac events had clopidogrel stopped and were followed for an additional 12 months. results showed the following : late stent thrombosis - related events (death and mi) occurred two to three times more frequently in patients with des than those with bms;late stent thrombosis carried a four times higher risk of cardiac death / mi (p < 0.00010);late stent thrombosis and its complications occurred up to 1 year after clopidogrel discontinuation. late stent thrombosis - related events (death and mi) occurred two to three times more frequently in patients with des than those with bms ; late stent thrombosis carried a four times higher risk of cardiac death / mi (p < 0.00010) ; late stent thrombosis and its complications occurred up to 1 year after clopidogrel discontinuation. authors concluded that while des use in 100 patients avoids five target lesion revascularization events at 6 months, it unfortunately leads to 3.3 late (within 18 months) deaths or mi. at the 55world congress of cardiology, two meta - analyses were presented, showing a significant increase in the rate of total mortality and q wave mi in des compared to bms at after 12 months and up to 3 years [40, 41 ]. experimental models of des demonstrate incomplete healing, fibrin deposition, and inflammatory cells, indicating a hypersensitivity reaction [2, 42 ] while bms demonstrate complete endothelialization at 28 days. sirolimus and paclitaxel have shown to impair endothelial function both within the stent and in the distal coronary artery, leading to delayed arterial healing of the stent itself, as well as enhancing the risk for distal arterial ischemia and coronary occlusion. in addition, they enhance expression of the endothelial tissue factor, which creates a prothrombotic state. despite equal stenosis severity and follow - up duration, patients with des, compared to bms, more frequently have collaterals insufficient to prevent ischemia during occlusion. the most powerful histological predictor of stent thrombosis has been incomplete endothelial coverage of the stent. several studies and registries have identified clinical predictors for delayed endothelial coverage and thrombosis of des. acs, left ventricular ejection fraction < 30%, treatment of bifurcating lesions, renal insufficiency, and diabetes have shown to be strong predictors of stent thrombosis [41, 45,46,47,48,49,50 ]. the concerns about late stent thrombosis resulted in an emergency fda advisory panel meeting in december 2006, which reassured that des in the studied on - label settings appear safe and efficacious, but warned that data regarding safety and efficacy in off - label situations is not available and will likely not match results seen in the lower - risk on - label settings. multiple registry studies have since shown that off - label des use is indeed associated with a roughly two- to threefold increase in clinical adverse events, including stent thrombosis, compared with on - label use. despite these findings, it is clear that des remain superior to bms even in high - risk off - label situations. recently, a comprehensive meta - analysis of almost 10,000 randomized controlled trial patients and over 180,000 observational study patients confirmed the overall benefit of des over bms in both on - label and off - label populations. evidence showed trends to reduced (randomized trials) or significantly reduced (observational studies) death and mi, and dramatic significant reductions in target vessel revascularization (regardless of study type). antiplatelet therapy and des controversy surrounding the des late thrombosis issue creates a controversy about the length of antiplatelet therapy in patients with des. the initial recommendations made by the fda / american college of cardiology (acc)/american heart association (aha)/society for cardiovascular angiography interventions (scai) and the stent manufacturers were completely arbitrary. they advised patients to remain on dapt for a minimum of 3 months after the implantation of sirolimus des and 6 months after paclitaxel des followed by life - long asa therapy. in 2005, a focused update of the acc / aha / scai pci guidelines recommended that all patients who receive a des should be given clopidogrel for at least 12 months in the absence of an increased risk of bleeding. with the growing number of publications concerning the safety of des, the fda published a scientific advisory in january of 2007, endorsed by 5 major professional societies : aha / acc / scai, the american college of surgeons (acs), and the american dental association (ada). once again, the importance of 12 months dapt after placement of des and life - long asa therapy was emphasized. once further, in the 2011 accf / aha / scai guidelines for pci, the role of dapt for prevention of thrombosis in patients with stents was strongly re - enforced with class ib recommendation for treatment with p2y12 inhibitor for at least 12 months after pci with des in addition to indefinite therapy with asa and class ii b recommendation for consideration of dapt beyond 12 months. dapt trial, which is currently ongoing, compares 12 versus 30 months of dapt among 15 000 patients treated with des. this trial is powered to assess the primary efficacy endpoints of differences in stent - thrombosis rates and major adverse cardiovascular / cerebrovascular events (macce), with a primary safety endpoint of major bleeding. currently, around 60% of patients undergoing pci receive des and are placed on dapt for at least one year. it is suggested that 5% of these patients will require non - cardiac surgery during this time, posing a unique challenge during the perioperative period. des management - non - cardiac surgery the decision to stop antiplatelet medications before invasive procedures in order to decrease the risk of bleeding may expose patients with des to increased risk for stent thrombosis, mi, and cardiac death. conversely, continuing antiplatelet therapy in order to prevent stent thrombosis may expose patients to increased risk of bleeding and need for transfusions during invasive procedures. current literature on the use of antiplatelet agents in surgery reports the average surgical blood loss increase by asa is approximately 2.5% - 20%. in non - cardiac surgery, a meta analysis of 474 studies on the impact of low dose asa on surgical blood loss showed that asa alone increases the average intraoperative hemorrhagic risk by a factor of 1.5, but does not increase mortality and morbidity. possible exceptions may be intracranial neurosurgery and transurethral prostatectomy where asa has been a contributing factor to fatal outcomes [12, 59 ]. the major side effect of clopidogrel is increased risk of spontaneous hemorrhage by 38% (incidence 1%-2%). in non - cardiac surgery increased bleeding has been described in transbronchial biopsy and pacemaker and defibrillator implantation [60, 61 ]. prasugrel is a 10 times more potent platelet inhibitor than clopidogrel ; however, it is associated with a statistically significant increase in non - cabg (coronary artery bypass grafting) major bleeding (2.4% vs. 1.8%, p=0.03) and fatal bleeding (0.4% vs. 0.1%, p=0.002) compared to clopidogrel. although ticagrelor is a reversible p2y12 inhibitor, the plato trial showed higher incidence of non - cabg related bleeding (rr 1.18) and significantly increased incidence of fatal intracranial bleeding (rr 10.95). with dapt, the bleeding time increases three- to fourfold over asa alone, and surgical blood loss increases by an average of 30% to 50%. significant bleeding in patients on dapt undergoing different non - cardiac surgical procedures has been described. however, other case reports and series have not found such association [57, 67,68,69 ]. some surgical procedures are associated with significant mortality and morbidity if bleeding is encountered, such as intracranial or spine surgery, where even a small amount of bleeding can cause brain or cord compression and irreversible brain or cord damage. the surgical bleeding and transfusion rates in the rest of the surgical procedures, although increased by 50%, were not associated with an increase in mortality and morbidity. risk of perioperative thrombosis the incidence of major coronary adverse events after pci is estimated around 4% to 5%, and 20% to 45% of these events can result in death [56, 70 ]. patients are most vulnerable immediately after a pci because the stenotic lesion is transformed into an unstable area due to the rupture of its endothelial covering. when undergoing surgery during this early period, the rate of mortality (30%) and morbidity (20 - 40%) is 5 to 10 times higher than matched patients undergoing the same operation under maximal medical therapy or after appropriate delay [68, 71 ]. premature discontinuation of antiplatelet therapy has been found to be the strongest predictor of stent thrombosis, carrying significant risk of mortality and morbidity even when the discontinuation is not associated with surgery [38, 48, 72 ]. as long as des have not endothelialized, the patient is absolutely dependent on the antiplatelet medications for stent thrombosis prevention. surgery itself produces a prothrombotic and proinflammatory state that increases the risk of stent thrombosis. the stress response to surgery includes sympathetic activation and cytokine release that promotes shear stress on arterial plaques, enhanced vascular reactivity conductive of vasospasm, increased platelet activation, and increased hypercoagulability [73, 74 ]. perioperative management currently, there is no definite standard of care for the perioperative management of patients with coronary stents, though deep understanding of the mechanisms of des and the indications for antiplatelet therapy could ensure patient safety and optimize outcome. a survey conducted on 374 interventional cardiologists found that although there is agreement among interventional cardiologists on the optimum delay for surgery after stenting, on the need for bms or balloon angioplasty alone if early noncardiac surgery is needed, and on treatment of perioperative thrombosis, there is significant inconsistency on the optimum antiplatelet therapy for patients who need surgery early after stent implantation. if the physicians most intimately involved with the management of patients with des do not agree on how to manage antiplatelet therapy perioperatively, we may speculate the chance of having consensus among anesthesiologists, surgeons, internists, and other interventional physicians on this topic is minimal. it is likely that the full implications of stopping antiplatelet therapy are not fully appreciated, and a reasonable fear of bleeding predominates. different algorithms for perioperative antiplatelet therapy management in patients with stents have been suggested [75, 77,78,79,80,81 ], but there are no prospective studies evaluating those algorithms. table 2 summarizes recent recommendations on the decision to proceed with antiplatelet therapy and/or surgical intervention, taking into account bleeding risk. until such studies are conducted and evidence - based guidelines are established, every institution should have well - publicized policies and guidelines to manage these patients. patients with stents, especially des, should be identified early in the preoperative work - up. each case should be managed on an individual basis and the risk and consequences of stent thrombosis should be weighed against those of perioperative bleeding. des = drug eluting stents ; asa = aspirin ; dapt = dual antiplatelet therapy. according to the 2011 accf / aha / scai guidelines, even before considering stent implantation, patients should be evaluated for possibility of surgery in the following 12 months and should not be treated with des if such possibility exists. percutaneous angioplasty or bms, requiring a minimum of 4 to 6 weeks of antiplatelet therapy, or medical management only if the surgery can not be delayed, should be considered instead. routine prophylactic coronary revascularization should not be performed in patients with stable coronary artery disease before non - cardiac surgery because of possible significant harm to the patient [55, 56 ]. once patients present for surgery with a stent in place, consideration should be given to the electiveness of the surgical procedure. elective surgery should not be performedwithin 4 to 6 weeks of stent placement or within 12 months of des placement in patients who s antiplatelet therapy will need to be discontinued perioperatively [55, 56 ]. assessed the perioperative outcome of patients undergoing non - cardiac surgery after coronary stent implantation in a single center registry. after multivariable analysis, the predictor of primary endpoint, defined as perioperative occurrence of major adverse events, was the time interval between stenting and surgery with a statistically significant increase in the number of events when surgery was performed within 6 weeks of bms placement. there was no difference in the major bleeding between the groups with different antiplatelet regimens. once again, these results supported the aha / acc recommendations on timing of non - cardiac surgery after stent implantation. if a patient with des presents for an elective procedure more than one year after the stent placement and still on dapt, the management of the antiplatelet medications will depend on the bleeding risk of the specific surgical procedure and on the complexities of the des (thrombotic risk). grouped the surgical procedures into three groups according to their bleeding risk : low bleeding risk surgical procedures that usually do not require blood transfusion, such as peripheral, plastic, and minor general surgery ; biopsies ; minor orthopedic procedures ; minor ent (ear - nose - throat) procedures ; endoscopies ; anterior chamber of the eye surgeries ; or dental extractions and dental surgery.intermediate bleeding risk surgical procedures that frequently require blood transfusions. examples can be visceral surgery ; cardiovascular surgery ; major orthopedic, ent, and reconstructive surgery ; or endoscopic urology.high bleeding risk possible bleeding in a closed space such as intracranial neurosurgical procedures ; spinal canal surgery ; or posterior chamber of the eye surgery. low bleeding risk surgical procedures that usually do not require blood transfusion, such as peripheral, plastic, and minor general surgery ; biopsies ; minor orthopedic procedures ; minor ent (ear - nose - throat) procedures ; endoscopies ; anterior chamber of the eye surgeries ; or dental extractions and dental surgery. examples can be visceral surgery ; cardiovascular surgery ; major orthopedic, ent, and reconstructive surgery ; or endoscopic urology. high bleeding risk possible bleeding in a closed space such as intracranial neurosurgical procedures ; spinal canal surgery ; or posterior chamber of the eye surgery. there is a growing agreement that dapt needs to be continued indefinitely in patients with complex stent placing procedures, such as stenting of bifurcating lesions, left main stents, overlapping stents, stents within stents, small vessel stents, multiple stents, saphenous vein graft stents, chronically occluded stents, or in patients with co - morbidities, such as diabetes, low ejection fraction, end stage renal disease, malignancies, advanced age, or with resistance to antiplatelet medications and a history of stent occlusion / thrombosis. for surgical procedures with low risk for bleeding intermediate - bleeding - risk surgical procedures should be approached on a case - by - case basis. in patients with complex stent procedures and co - morbidities, the dapt should be continued despite the risk for increased bleeding. in all other patients, clopidogrel and ticagrelor can be stopped 5 days before surgery and prasugrel 7 days before surgery, while asa should be continued. dapt should be restarted as soon as possible postoperatively (ideally within 24 hours) with a loading dose of 300 mg - 600 mg clopidogrel, or prasugrel 60 mg, or ticagrelor 180 mg [57, 83 ]. patients with 12 months completed of dapt and low risk for thrombosis of the coronary artery stents can stop clopidogrel and ticagrelor 5 days prior to surgery and prasugrel 7 days before surgery while continuing asa. asa discontinuation even more than one year after des placement may lead to stent thrombosis [83, 84 ]. for patients with coronary stents with high risk for thrombosis presenting for high - risk bleeding surgical procedures, bridge therapy has been suggested. use of short - acting gp iib / iiia inhibitors, such as tirofiban or eptifibatide, has been proposed as a bridge between the time of the thienopyridine discontinuation and surgery. either medication is given as infusion and requires patient admission to the hospital 3 days after the discontinuation of thienopyridines. infusion is stopped 4 to 6 hours prior to surgical procedure and restarted as soon as possible after the surgery upon agreement between cardiology and surgery [85, 86 ]. usually, heparin infusion commences, though there is no evidence supporting that heparin offers efficient protection in high - risk coronary situations. nonsteroidal anti - inflammatory drugs (nsaids), such as ibuprofen, inhibit cox-1, as asa does. since action is reversible and platelet function is completely recovered within 24 hours of their discontinuation, nsaids are suggested as an alternative to asa. however, all of these bridging techniques are controversial, with little data published to support use, and are associated with an increased cost. cangrelor, an investigational parenteral, reversible, direct p2y12 platelet inhibitor with its extremely short (5 to 9 minutes) half - life, may present an alternative for bridge therapy in the near future. in the recently published results from the bridge (maintenance of platelet inhibition with cangrelor) trial, cangrelor was effective at maintaining platelet inhibition in patients on thienopyridines who required bypass surgery. this prospective, randomized, double - blind, placebo - controlled, multicenter trial evaluated 210 patients on thienopyridine therapy awaiting cabg. the thienopyridine was stopped and patients were randomized to treatment with cangrelor or placebo for at least 48 hours which then was stopped 1 to 6 hours prior to surgery. the cangrelor group had low levels of platelet reactivity throughout the treatment period compared to patients on placebo. excessive cabg related bleeding occurred in 11.8% of patients on cangrelor vs 10.4% in patients on placebo. there was no difference in major bleeding prior to cabg, although minor bleeding was slightly higher in the cangrelor group. in surgical procedures where even a small postoperative hemorrhage can have disastrous consequences, such as intracranial surgery, spinal surgery in the medullary canal, and surgery of the posterior chamber of the eye, both p2y12 inhibitors and asa may need to be discontinued 7 days prior to surgery. management of patients with stents in need of urgent surgical procedures depends on the time interval between des placement and the surgical procedure. if the time is less than one year (or less than one month for bms), then the dapt has to be continued throughout, except for surgeries in enclosed spaces. less invasive surgical techniques and alternative treatments should be considered in such patients. if patients present for an urgent surgical procedure more than 12 months after the des placement, the same paths as elective surgical procedures should be followed. future developments of des management current efforts are directed towards creating reversible short - acting platelet inhibitors that can be used in the perioperative period, creating des with more predictable endothelialization, and developing point - of - care tests of platelet function that can help the perioperative management of patients on antiplatelet therapy. even though both the champion platform study and the champion pci trial, which compared clopidogrel and cangrelor, were terminated early for the lack of efficacy end points, cangrelor may prove valuable in the perioperative period as a bridge therapy as shown by the results of the bridge trial. although this path still involves hospital admission ahead of planned surgery and requires iv infusion which will be associated with increased cost, cangrelor may provide better platelet inhibition than that of the currently proposed combination of gpiib / iiia inhibitors and heparin. a new generation des was introduced in 2008 : zotarolimus des system, which uses phosphorylchlorine - base, biocompatible polymer and the everolimus des, approved in july 2008. recently, the results of the isar - test trial were published showing that a polymer - free dual - drug sirolimus- and probucol - eluting stents are non - inferior to the second generation zotarolimus - eluting stent. new platforms (cobalt - chromium and platinum - chromium), new delivery systems, new polymers allowing better biocompatibility and/or flexibility and will attempt to decrease the rate of late stent thrombosis and expand stent indications. in february 2012, the fda approved the first des for use in patients with diabetes based on the results of the resolute trial - the resolute integrity zotarolimus stent. several days later, based on the results of the horizons - ami trial, the fda approved the first des for treatment of acute myocardial infarction - the ion paclitaxel eluting stent. alternatively to the traditional bms and des, a simple chemical coating may be effective in preventing thrombotic events, shortening the duration of required antiplatelet therapy, and allowing for safe perioperative discontinuation of antiplatelet agents. coatings such as titanium - nitride - oxide, dimethyl sulfoxide, cd34 antibodies and stents containing an integrilin - binding cyclic arg - gly - asp peptide are being explored. in recent years, there has also been a significant effort in developing point - of - care tests for assessment of the effects of the antiplatelet drugs for guidance of the perioperative management of patients on antiplatelet therapy. the gold standard is still light transmittance aggregometry, but the test requires significant sample preparation, special equipment, and trained personnel and is difficult to use in everyday practice. plateletworks, verifynow, and thromboelastography platelet mapping are some current available tests that are not as reliable as the light transmittance aggregometry and still require special equipment and trained personnel. understanding the risks of stopping antiplatelet therapy by the healthcare professionals is of paramount importance. use of a multidisciplinary team approach, including the interventional cardiologist, anesthesiologist, and surgeon, to guide perioperative management is imperative. invasive procedures on patients with des, at high risk for stent thrombosis, and dapt discontinued should be performed in centers with around - the - clock invasive cardiology services as the primary option. however, smaller institutions may not have such coverage, and should not be fully excluded from performing invasive procedures due to the overall high volume of stent implantation. outpatient surgery on such patients should not be performed, and ideally, patients should be kept in the hospital for at least 48 hours for cardiac monitoring or until antiplatelet medications are restarted.	significant advancements in percutaneous treatment of coronary artery disease have been achieved with the introduction of bare metal stents. they have two major drawbacks : acute / subacute stent thrombosis, successfully managed with antiplatelet therapy immediately after stent implantation ; and in - stent restenosis, prevention of which has been achieved with the development of drug - eluting stents. drug - eluting stents have become preferred therapy for patients undergoing coronary artery intervention, though reports of late stent thrombosis have led to uncertainty about the duration of antiplatelet therapy after drug - eluting stents placement. much controversy remains regarding perioperative management of patients with these devices, presenting for surgery or other invasive procedures. the purpose of this review is to provide an overview of the changing culture of coronary artery stenting, in addition to discussing perioperative management strategies and controversies surrounding coronary stents and antiplatelet therapy. a comprehensive literature search of medline was conducted using as keywords : antiplatelet therapy, non - coronary surgery, drug - eluting stents, and stent thrombosis. there is no definite standard of care for the perioperative management of drug - eluting stents in patients with drug - eluting stents. however, there is a growing understanding of the importance of continuation of drug - eluting stents in the perioperative period in order to prevent stent thrombosis along with a concern about the possibility of increased bleeding. appropriate timing of surgery after coronary artery stenting, team approach to the perioperative management of such patients with involvement of cardiologist, anesthesiologist, and surgeon, and development of an individual plan for each patient, weighing that patient s risk of thrombosis vs the risk of bleeding, could improve patient safety and optimize outcome.
atopic dermatitis affects 15 to 20% of preschool children in western countries [14 ]. the lifetime prevalence of eczema in children aged 14 years in vrmland county, sweden, was assessed in 2000 in the cohort study dampness in building and health to be 22%. the burden of disease has been assessed to be at least comparable to other chronic illnesses, such as diabetes or neurological disorders [68 ]. for population - based, epidemiological investigations, parental written questionnaires detecting eczema during childhood are advantageous. however, current questionnaires have been developed for older children and adults. given that in small children atopic dermatitis is the most common inflammatory disease, a questionnaire should be suitable to the general population and therefore evaluated in such a setting. furthermore, it should be applicable to eczema with different severity categories. a new questionnaire detecting atopic dermatitis in young children could probably be based on previous tools for older children. for the current study, a questionnaire was based on the existing isaac (international study of asthma and allergies in childhood) questions for school children. the aim of the current study was to estimate the diagnostic precision of the children 's eczema questionnaire compared to physicians ' clinical diagnoses as the gold standard in preschool children from the age of two months. further, we evaluated whether the parents considered the questions to be understandable and suitable for diagnosing eczema in this age group. a case control design similar to that used by williams. in his validation study on atopic dermatitis was chosen. as seen in figure 1, 35 children with eczema and 24 without eczema according to medical records and aged two months to six years were recruited consecutively from a preventive care register in arvika, vrmland county, sweden. these 60 children 's parents answered a questionnaire on eczema (the questionnaire to be evaluated). physician diagnosis, based on the presence of at least three major and three minor criteria of hanifin and rajka 's diagnostic criteria [12, 13 ], was used as the gold standard to validate the questionnaire. in clinical eczema cases, severity was measured with the scoring atopic dermatitis (scorad) index. the qualitative part of the investigation was carried out by means of a semistructured, one - to - one interview with the parents. the interview started with a general discussion regarding the understanding of the eczema questions and relevance of named issues on infants ' and preschool children 's eczema. each interview was conducted by the same physician, the first author of this paper, to ensure consistency. the regional ethical committee in uppsala approved the protocol for the study, dnr - c2007/41. inclusion criteria for index cases were eczema, treated either with emollients or topical glucocorticoids. inclusion criteria for controls were an age of two month to six years and no eczema. exclusion criteria were eczema treated with systemic treatment, other chronic diseases such as diabetes, acute infections, or if parents did not understand or speak swedish fluently. a power analysis showed we would need 60 children to assess sensitivity and specificity with a power of 0.8 and alpha 0.05. a total of 62 children were invited to participate, but there were two denials. atopic dermatitis was defined by hanifin and rajka as pruritic, chronically relapsing dermatitis, with typical features and distribution. our questionnaire was constructed to discriminate whether these essential signs of eczema existed in each child or not. the questions we used for this were derived from the isaac protocol, which was developed for school children. we used the main outcome measure of the isaac protocol, has your child ever had an itchy rash which was coming and going for at least 6 months ?, and the question, which is an additional question in the isaac protocol has this itchy rash at any time affected any of the following places : the folds of the elbows, behind the knees, in front of the ankles, under the buttocks, or around the neck, ears or eyes ?. the main adoption was that we divided the main outcome measure of the isaac protocol into two parts, thus we asked for chronic relapsing dermatitis and itch in two separate questions. further, we extended the list of involved sites, since more sites can be involved in younger children. involved sites were in agreement with halkjaer 's description of eczema distribution in young children, and face validity was confirmed by comparing our criteria with the uk diagnostic criteria for atopic eczema. does your child have or has your child had a red rash / eczema which can come and go ? has this red rash / eczema affected any of the following areas (during the last week) : around the eyes, ears, scalp, cheeks, forehead, neck, trunk, folds of the elbows / behind the knees, wrist or ankle, outer arms / legs ? a questionnaire - diagnosed eczema was compared to a physician 's diagnosis which was the gold standard. sensitivity and specificity as well as the positive predictive value (ppv) and negative predictive value (npv) of the sample population and stratified age groups were assessed. ppv was calculated as (prevalence sensitivity)/(prevalence sensitivity + (1 prevalence) (1 specificity)). npv was calculated as (1 prevalence)/((1 prevalence) + prevalence (1 sensitivity)/specificity). the most commonly mentioned and coded topics formed the basis of further defining themes and patterns. findings were summarized regarding the understanding of the eczema concept and relevance of the diagnostic questions. eczema occurred in 35 children, who reported dry skin twice as much as children without eczema (table 1). both children with and without atopic dermatitis manifested other skin disorders at the same rate as miliaria, haemangioma, keratosis pilaris, and melanocytic naevi. in the eczema group, eleven (33%) children awoke several times per week, and 2 (6%) awoke occasionally because of skin symptoms according to parental reports. a combination of affirmative answers to all three key questions, rash, itch, and location, predicted clinical eczema with good sensitivity 0.91 (95% ci 0.77 to 0.98) and specificity 1 (95% ci 0.86 to 1). most parents judged the questions to be understandable and suitableeven if i do not deal much with asthma and allergy. according to the parents, the questionnaire covered all the important facts regarding the eczema diagnosis. however, disease terms were regarded to be difficult ; eczema is too strong an expression to answer yes to whereas questions concerning symptoms were thought to be easily answeredeasy, one can already see itching at the age of 6 months. of 35 parents to children with eczema, 21 expressed feelings of stress and worry about their child 's health state and felt responsible for it. four parents reported not wishing their children to be diagnosed with eczema at all because of fear of cortisone treatment. itching and awakening due to itching with three key questions, atopic dermatitis in preschool children can be identified via a parental questionnaire. the high sensitivity and specificity our questionnaire provides a feasible diagnostic tool with high diagnostic precision, which can add to the field of epidemiological research in early childhood eczema. the semistructured interview confirmed high face and content validity, which might be one reason for the high proportion of children with detectable eczema. the ppv of the questionnaire is the proportion of children with a positive result who actually had physician - diagnosed eczema. predictive values are, however, related to the prevalence of eczema in the population, which could not be assessed with this study design. the high prevalence of eczema, 22%, in the population was derived from data from the same county. the results suggested that the risk of diagnosing eczema in healthy infants and small children was low. even the measured sensitivity was high, indicating that not many eczema diagnoses were missed. the questionnaire did not suggest healthy children to be diseased, as shown by the high specificity. it is important to estimate the severity of eczema, both for the description of the study population and for an assessment of generalizability. a score combining an assessment of disease extent with clinical features, duration plus intensity (scorad) was used. based on this assessment, participating children showed mild - to - severe eczema that allows applying the questionnaire in all severity groups. the strength of the study is the population - based setting, as the questionnaire is intended to be used in a similar setting. because all children were registered in a preventive care register and almost all invited children participated, it is unlikely that selection bias occurred, even though the sample was not randomly selected. assessing the diagnostic accuracy of the diagnostic test on a sample of the general preschool child population allows generalizing results. the narrow confidence intervals suggest that the sample size was adequate and that estimates of the population value are within a reasonable range. compared to a standardized skin examination protocol, the isaac questionnaire performed well in predicting eczema prevalence at the population level. however, on an individual level, a high proportion of flexural eczema was not confirmed by skin examination. even here a high proportion of the isaac cases were false and many physician - diagnosed cases were missed. this does not mean that our questionnaire is to be preferred but that the questions which we adopted to our cultural environment might be useful in sweden as shown by the validation against a gold standard. in different linguistic and cultural contexts, an adoption of the questions and new validation should be considered. it is for example possible that the questionnaire performs differently in populations with less access to health care. this study makes a contribution in solving diagnostic problems where there has been a lack of diagnostic questionnaires for small children. the questionnaire can be used in a population - based setting and across different severity groups of eczema.	aim. to develop and validate a questionnaire for detecting atopic dermatitis in infants and small children from the age of 2 months. methods. parents to 60 children answered a written questionnaire prior to a physical examination and individual semistructured interview. qualitative and quantitative analyses of validity, sensitivity, specificity, and predictive values of the questionnaire were performed. results. a total of 27 girls and 33 boys, aged 2 to 71 months, 35 with and 25 without physician - diagnosed eczema, participated. validation of the questionnaire by comparisons with physicians ' diagnoses showed a sensitivity of 0.91 (95% ci 0.770.98) and a specificity of 1 (95% ci 0.861). conclusions. three questions in a parental questionnaire were sufficient for diagnosing eczema in infants and small children.
acute myeloid leukemia (aml) develops from malignant transformation of immature hematopoietic cells through a complex multistep process that requires cooperation of different types of genetic alterations. alterations associated with aml include recurrent fusion genes such as t(8 ; 21), inv(16), and t(15 ; 17) that affect transcription factors or components of the transcriptional coactivation complex, resulting in impaired differentiation and/or aberrant self - renewal capacity by hematopoietic progenitors. in addition to these, a group that comprises mutation in genes involved in the proliferation and survival potential of hematopoietic progenitors includes receptor tyrosine kinases (flt3, kit, and ras), oncogenic transcription factor (cebp / alpha), and nucleophosmin (npm1) mutation. many of these gene mutations figure importantly as a provisional clinicopathologic entity in the revised world health organization (who) 2008 classification of myeloid neoplasms. fms - like tyrosine kinase-3 (flt3) is a class iii receptor tyrosine kinase located on chromosome 13q12. wild - type flt3 mrna is expressed on normal hematopoietic stem cells and in the majority of aml blast cells. the most common mutation in the flt3 gene is internal tandem duplication (flt3/itd) of the region coding for the juxtamembrane (jm) domain of the flt3 receptor. the mutated flt3 gene is always transcribed in frame and coded mutant flt3 with a long jm domain. another mutation reported is missense mutation in the tyrosine kinase domain (tkd) of the flt3 gene. the most frequent mutations are point mutations and deletions of codons 835/836 in second tkd of the gene. in addition, a frameshift mutation in exon 12 of the npm1 that interferes with cell cycle regulation has also been reported to occur frequently in aml. this mutation is an alternative leukemogenetic mechanism rather than chromosomal translocations that was discovered in 2005. due to its unique molecular, genotypic, immunophenotypic, and prognostic features, aml with mutated npm1 and another category of aml with cebpa were included as two separate provisional entities in the 2008 who classification of myeloid neoplasm. in leukemia and lymphoma, npm1 is also a partner in chromosomal translocations, where the dislocated cytoplasmic nucleophosmin appears to contribute to oncogenesis. npm1 is a nucleus - cytoplasm shuttling protein that is ubiquitously expressed and is highly conserved. the npm1 protein has been shown to contribute to many basic cellular processes such as the regulation of centrosome function, biosynthesis of ribosomes, and preventing aggregation of proteins in the nucleolus, and it participates in (arf - p53) tumor suppressor pathway. to date, there are only few studies specifically investigating the prevalence and clinical characteristic of npm1 and flt3 mutations in aml in indian population. ahmad., 2009, reported lower frequency of npm1 mutation in indian population compared to western countries but did not study the clinical relevance of npm1 mutation in aml patients. another study reported no difference in the cr rate of patients with and without npm1 mutation though it found poor prognosis for flt3/itd mutation in aml patients. moreover, in our previous study on flt3 mutation no association with the clinical outcome in aml patients with normal cytogenetic was reported. the study presented here investigates the prevalence and clinical characteristics of the exon 12 npm1 and flt3 (itd / d835) mutations in slightly different cohort of 161 de novo aml patients including adults and children. we also evaluated the association of these two gene mutations with immunophenotypic markers and other genetic alterations prevalent in aml. one hundred sixty - one patients (116 adults and 45 children) with confirmed diagnosis of aml admitted to the division of hematology, safdarjung hospital, new delhi, india, were consecutively included in the study from the year 2006 to the year 2010. our earlier study was based on 133 patients (90 men and 43 women) who were screened for the detection of only flt3 mutation. this study included the overlapping cases of around 25 subjects (18 adults and 7 children) from the earlier study for whom we have both dna and rna available for the detection of mutation and translocation. the diagnosis of aml was made on routinely stained bone marrow aspiration / biopsies and peripheral blood smears and evaluated according to the french - american - british (fab) criteria. cytochemical stains were used to differentiate aml from all and to subclassify them into fab subtypes. written informed consent was obtained from all patients and all in vitro procedures were performed according to the protocol approved by the ethical committee of the institute and safdarjang hospital. clinical data, including age, gender, whole blood cell count (wbc), hemoglobin (hb), platelet count, and presence of organomegaly, were collected at the time of diagnosis. peripheral blood samples (4 - 5 ml) were obtained from all patients in heparinised vials (1050 u / ml of blood) and transported to the laboratory on ice. in addition, bone marrow aspirates were collected in heparin containing phosphate buffered saline (pbs) when possible. one ml of whole blood / bone marrow sample was used for immunophenotyping studies by flow cytometry and the remaining 3 - 4 ml of blood / bone marrow was used for separation of pbmc using histopaque-1077 solution (sigma, usa) for genetic studies. the following monoclonal antibodies, all obtained from becton - dickinson biosciences, usa, were included : cd3-fitc, cd5-fitc, cd7-fitc (t cell specific), cd10-pe (calla), cd19-fitc (b cell specific), cd13-pe, cd33-fitc, hla - dr - fitc, mpo - fitc (myeloid cell specific), and tdt - fitc. cd45-pe and cd34-fitc antibodies were included in the panel to gate the blast cell population. for myeloperoxidase (mpo) antigen and tdt (intracellular), flow cytometric analysis was performed on a facscalibur flow cytometer (becton - dickinson, usa), and cellquest software was used to acquire and analyze the data. results were obtained by gating the blast cells using side scatter (ssc) versus forward light scatter (fsc) parameters and examining the associated fitc and pe fluorescence on fl1 and fl2 detectors, respectively. in addition, cd45 intensity versus side scatter (ssc) was used to detect blast cells and separate them from the normal populations. a case was considered as positive for specific antigen if the antigen was expressed in at least 20% of the leukemic cells of individual sample (20% cut - off level) and when the fluorescence intensity was clearly separated from negative controls (isotypic controls or negative cell population for the examined antigen). 340486) bead using facscomp software version 2.0. to ensure data extraction from clinical samples, instrument settings were also optimized using a normal donor 's peripheral blood samples (weekly) for fsc and ssc gains, fsc threshold, and fluorescence compensation levels. briefly, all patients included in the study were treated with the standard 3 + 7 induction chemotherapy or all - trans - retinoic acid (atra) therapy. therapy - related aml cases and patients treated with other than the standard 3 + 7 induction chemotherapy were excluded from the study. all patients included in this study received the standard 3 + 7 induction chemotherapy protocol as described earlier : daunorubicin (45 mg / m per day) for 3 days and cytarabine (100 mg / m per day as a continuous 24 h intravenous infusion) for 7 days followed by g - csf (5 g / kg) daily from day 8 till counts are recovered (neutrophils > 1.5 10/l and platelet count > 100 10/l). consolidation is comprised of three courses of high - dose cytosine arabinoside (3 g / m every 12 h on days 1, 3, and 5 ; total 18 g / m) followed by g - csf (5 g / kg) daily from day 8 till counts are recovered. a complete remission was defined as the absence of abnormal clinical symptoms and presence of less than 5% of myeloblasts in the bone marrow. in this study, clinical response of the patients complete response (cr) to induction chemotherapy was defined clinically by disappearance of abnormal physical findings attributable to the leukemia and by the of return of the patient to good physical health and hematologically by the return of the peripheral blood to normal values with respect to hemoglobin, total and differential white blood cell count and platelets, and the presence of less than 5% morphologically normal blast cells in a marrow preparation of normal cellularity. patients who did not meet the criteria of cr after induction therapy were considered as noncomplete response (nr). briefly, genomic dna from aml patients was extracted by using himedia kit (mumbai, india). npm1 exon 12 was amplified by genomic pcr using primers reported by gale., 2008. the reaction mixture contained 10 pmol of each primer, 100 ng of genomic dna, 200 m dntps, 2.0 mm mgcl2, and 1 unit of taq dna polymerase (mbi fermentas, uk) in the buffer provided by the manufacturer. amplification was performed in a thermal cycler (ptc 200 ; mj research, usa), at an initial denaturation at 95c for 5 minutes, followed by 30 cycles of 95c for 2 minutes, 62c for 45 seconds, 72c for 45 seconds and followed by a hold at 72c for 10 minutes and a cooldown. the 6-fam - labeled pcr products were diluted tenfold in water, and 0.5 l was mixed with 9.5 l of hidi formamide (applied biosystems, inc., usa) and 0.25 l of genescan liz 250 internal size standards (applied biosystems, inc., usa) and heated to 95c for 2 minutes snap chill down on ice. the samples were run on an abi 3130xl genetic analyzer using 16 cm capillaries. wt product size was 198 bp. for flt3 mutation analysis, polymerase chain reaction (pcr) and dna sequencing method were used to detect flt3/itd and flt3/d835 mutations in aml samples as described previously. to validate the result from capillary gel electrophoresis, pcr products larger than the wild - type product amplified fragments were cut and extracted from agarose gels using qiagen minelute kit (qiagen, hilden, germany) according to the manufacturer 's instructions. approximately 100 ng purified pcr product was directly sequenced with 3.3 pmol primers as described above with the big dye terminator cycle sequencing kit v3.1. sequence analysis was performed on an abi 3130xl sequence detection system (applied biosystems, inc. association of npm1 and flt3 mutations with commonly occurring translocation was also studied in 120 patients (91 adults and 29 children) in whom rna samples were available. the reverse transcription polymerase chain reaction (rt - pcr) was performed for the detection of fusion genes bcr / abl, pml / rar, and aml / eto. aml / eto and pml / rar fusion genes were detected by simple rt - pcr using primers and conditions according to the biomed-1 concerted action protocol. the presence of the fusion gene bcr / abl was detected by multiplex rt - pcr. for the analysis of categorical variables, either fischer 's exact or pearson 's x test was applied. mann - whitney rank sum test was used for the differences in the median variable in hemoglobin level, wbc / platelet count, and peripheral blast percentage. the main clinical and hematologic characteristics of the patients included in the study are summarized in table 1. of 161 de novo aml patients included in the study, 116 were adults (median age : 32 ; range 1685) and 45 were children (median age : 10 ; range 115). m2 (60/161, 37%) subtype was found to be the most common fab subtype followed by m5 (26/161, 16%) and m0/m1 (24/161, 15%) subtype. hepatosplenomegaly (hsm) was very common and seen in 92 patients (57.1%). eight samples were randomly selected from 34 positive samples for sequence analysis of npm1 mutation. six different sequence variants were observed, all leading to a frame shift in the region encoding the c - terminal of the npm1 protein. all mutations consisted of insertion of 4 bp insertions either between nucleotides 960 - 961 or between nucleotides 964 - 965 (see supplementary figure 1 available online at http://dx.doi.org/10.1155/2013/582569). the most frequent mutation (type a mutation) was the duplication of a tctg tetranucleotide found in 3 cases (table 2). these aberrations include flt3/itd in 35 (22%) cases and flt3/d835 mutation in 5 cases (3%). thirteen (38%) of the 34 samples with npm1 mutation had also flt3/itd mutation. baseline characteristics of patients with npm1 and flt3 (it d and d835) mutations are summarized in table 1. npm1 mutation was found more often in females (27%, 14/51) than males (18%, 20/110) (p = 0.24). the npm1 mutation was found more frequently in adults as compared to children (26% versus 9% ; p = 0.01). further stratification of age showed that frequency of npm1 mutation increases with age, 38% (12/32) being the highest frequency observed in aml patients above 45 yrs of age (p = 0.02) (figure 1). patients with npm1 mutation had significantly higher platelet count (p = 0.05) as compared to patients without any npm1 and flt3 mutations. further, platelet count was significantly higher in npm1 mutation group than in no mutation group in adult patients (56 10/l versus 20 10/l ; p = 0.02) and in children (25 10/l (2050 10/l) ; p = 0.62). moreover, npm1 mutation was found to be significantly associated with the absence of extramedullary diseases such as hsm (p = 0.01). no significant difference in age or gender was found in patients with flt3/itd (p = 1.0 and p = 0.28) and flt / d835 mutation as compared to those patients without any mutations. similar to npm1 mutation, flt3/itd mutation was found more often in adults (23%) as compared to children (18%) ; however, this difference was not statistically significant (p = 0.29). other clinical parameters except wbc count showed no association with either flt3/itd or flt3/d835 mutations. patients with flt3/itd mutation had significantly higher wbc count 60.2 10/l (range 1.4428 10/l) compared to patients without mutations 27.4 10/l (range furthermore, adult patients (> 15 years) had significantly higher median wbc count in flt3 mutation group compared to no mutation group (56.9 10/l versus 27.6 10/l ; p = 0.03). however, there was no significant difference in the wbc count in children between flt3/itd mutation and no mutation groups (113 10/l versus 25.6 10/l ; p = 0.21). though the median count was much higher in cases with flt3/itd mutation but not significant, this can perhaps be accounted for by the small numbers of patients with flt3/itd mutations in children. patients with both npm1 and flt3/itd mutations had higher wbc count as compared to patients without mutation (p = 0.04). this group similar to npm1 mutation was also associated with the absence of hsm (p = 0.03). however, other clinical parameters were not significantly different between patients with both npm1 and flt3/itd mutations and without mutations. npm1 and flt3/itd mutations status did not affect significantly the induction of cr rate in the study probably due to the smaller number of patients (29) in the clinical outcome category. immunophenotypically, npm1 mutation was found to be associated with the lower positivity of cd34 (29% versus 70% ; p < 0.001) and hla - dr expression (29% versus 81% ; p = 0.001) compared to the group without npm1 and flt3/itd mutations. in contrast to this, flt3/itd mutation was associated with the higher positivity of cd7 marker (33% versus 14% ; p = 0.04). further, there was no difference in the positivity of cd34 in the group with flt3/itd mutation compared to those without flt3/itd mutation (61% versus 70% ; p = 0.38). however, the group with both npm1 and flt3/itd mutations was associated with the lack of cd34 expression (p = 0.03). other markers such as cd3, cd5, cd10, cd19, cd14, cd13, cd33, tdt, hla - dr, and mpo were not significantly associated with npm1 and flt3/itd mutations status (table 3). aml / eto was the most frequent translocation detected (23%, 28/120) in aml patients (table 4). pml / rar translocation was detected in 4% (5/120) while 9% (11/120) of samples were positive for bcr / abl translocation. all the five positive cases of pml / rar were of typical m3 aml morphology with highly granulated blast cells, nuclear size irregular and highly variable. all the cases were strongly mpo positive and pas negative. correlation with gene mutation data in the patients showed that aml / eto translocation was inversely associated with the flt3/itd mutation (p = 0.04). frequency of aml / eto translocation was low in flt3/itd mutation group compared to flt3/itd wild group (12% versus 28% ; p = 0.04). no association of npm1 and flt3/itd mutations was observed with other translocation due to smaller number of cases positive for the translocation. bcr / abl translocation was detected in two patients positive for flt3/itd mutation while in one patient with npm1 mutation. pml / rar translocation was detected only in one case of both flt3 and npm1 mutations. genetic alterations in aml are known to be major determinants of the patient response to therapy and outcome besides their role in the pathogenesis of the disease. in addition to the aberrations that are revealed by conventional karyotyping, submicroscopic lesions such as flt3 and npm1 mutations which are undetectable at chromosome level have been described in aml [1, 6 ]. flt3 and npm1 mutations have been shown to be the most prevalent somatic alterations in aml, particularly in cytogenetically normal aml. these mutations play significant role in the diagnosis, risk assessment, and guidance of therapy [2, 13 ]. in the current study, the frequency of npm1 and flt3/itd mutations was 26% (30/116) and 23% (27/116) in adults, respectively, similar to the frequency reported in japanese (24.9% npm1, 22.6% flt3/itd) and in german adult aml population (27.4% npm1, 21% flt3/itd). when compared to patients from southeast asian region, the frequency of npm1 mutation was equivalent to our study (26%) ; however, the frequency of flt3/itd mutations was higher in southeast asian region as compared to our study (33% versus 23%). the frequency of npm1 mutation was 9% in children (15 years) and 26% in adult patients (15 years) in the current study (p = 0.02). the frequency of npm1 mutation in adult is nearly comparable to reports from other countries including japan, germany, and england (2541%) [12, 14, 15 ]. similarly, the frequency of npm1 mutations was 7.5% overall in pediatric aml patients (range 012%) [1719 ]. observed variation in the frequency of npm1 mutations might be due to the differences in the sample size of the study, ethnicity, and geographical distribution of the population studied. moreover, we observed a striking relationship between incidence of npm1 mutation and age of the aml patients in adults. the incidence of npm1 mutation appears to increase with age such that patients between 30 and 45 years have frequency of 22% and patients older than 45 years of age have frequency of 38%. similar age - dependent increase in the incidence of npm1 mutation has also been reported earlier [6, 17 ]. ahmad., 2009, have also reported a higher frequency of npm1 mutation in adults as compared to children in indian population (23.6 versus 2.5%). certain associations between the two gene mutations and clinical characteristics have been found in the current study, for instance, a higher platelet count (p = 0.05) and absence of hepatosplenomegaly (p = 0.01) with npm1 mutation and high wbc count with flt3/itd mutation., 2006, also observed that blasts with npm1 mutation may retain a certain capacity for thrombocytic differentiation as demonstrated by in vitro experiments of hsu., 2003 further, platelet count was significantly higher in npm1 mutation group than in no mutation group in adults only not in children. in concurrence with our report, falini., 2005 also found correlation of mutated npm1 with high platelet counts in adult aml patients [6, 21 ]. similarly in line with our study, braoudaki., 2010, too did not find association of npm1 mutation with platelet count in pediatric aml patients. we found a significantly increased wbc count in patients with flt3/itd mutation (p = 0.01) but not with flt3/d835. flt3/itd mutation leads to constitutively activated flt3 protein which causes proliferation, inhibits apoptosis, and suppresses differentiation of leukemic cells. it is reported that ligand independent constitutive activation of flt3 induced by it d mutation could activate some downstream signal molecules including mitogen activated protein (map) kinase, signal transducer and activator of transcription 5 (stat5), and serine threonine kinases akt, which contribute to cell proliferation and survival advantages [4, 23, 24 ]. this supports the observation reported in earlier studies that flt3/itd but not the flt3/d835 mutation is associated with higher wbc count in adult aml patients [25, 26 ]. however, median wbc count in pediatric patients with flt3/itd mutation was higher than in the patient without mutation but was not statistically significant, although others disagree with this. immunophenotyping analysis showed that npm1 mutation was associated with the lower positivity of cd34 expression (29 versus 70% ; p < 0.001) and lack of hla - dr expression (29 versus 81% ; p < 0.05). in several european studies on aml, npm1 mutation is inversely associated with the expression of cd34 marker [15, 21 ]. similar report of lack of cd34 expression in npm1 mutation positive has also been reported from south asian countries [16, 17 ]. all these studies including ours are in agreement with the fact that the lack of cd34 expression in npm1 mutated cases could be an indicator for good response to induction chemotherapy. though flt3/itd patients did not display a unique immunophenotype, a significant association of flt3/itd mutation with aberrant expression of cd7 was found (p = 0.04)., 2008 has also shown the association of coexpression of cd7 antigen with flt3/itd mutation [10, 28 ]. in concurrence with other reports, there was no significant association of npm1 mutation with the aml / eto, pml / rar, and bcr / abl translocations in the current study [14, 17, 22 ]. however, aml / eto translocation was found to be negatively associated with the flt3/itd mutation (p = 0.04). this is quite contrast to the study of schessl., (2005) which reports that aml / eto collaborates with flt3-lm (length mutation) in inducing acute leukemia in a murine bm transplantation model. of note, both myeloblastic and lymphoblastic leukemias of b cell and t cell types developed in this model of aml / eto and flt3-lm cooperation suggest that additional mutations in aml / eto positive progenitors are necessary for the transformation into leukemia - initiating cells. in summary, the frequency of npm1 and flt3 mutations was 21% and 25% in de novo aml patients comparable to other reports despite geographic and ethnic differences. distinct clinical and immunophenotypic characteristics associated with the npm1 and flt3/itd mutation were found which seems to characterize a distinct entity of aml. flt3/itd was found to be inversely associated with the presence of aml / eto translocation, indicating requirement of other mutations to cause aml. further, comprehensive studies on the biological effects of npm1 and flt3/itd mutations and their interactions with other genetic and epigenetic alterations are needed to gain insight into the molecular mechanism of these mutations involved in the pathogenesis of aml.	background. mutations in npm1 and flt3 genes represent the most frequent genetic alterations and important diagnostic and prognostic indicators in patients with acute myeloid leukemia (aml). objective. we investigated the prevalence and clinical characteristics of npm1 and flt3 mutations in 161 patients of de novo aml including adults and children. results. npm1 mutation was found in 21% and flt3 mutation in 25% of the aml patients. thirteen (8%) samples were positive for both npm1 and flt3/itd mutations. adult patients had significantly higher frequency of npm1 mutation than children (25.8% versus 8.8% ; p = 0.02). further, npm1 mutation was found to be more frequent in patients above 45 years of age (p = 0.02). npm1 mutation was significantly associated with higher platelet count (p = 0.05) and absence of hepatosplenomegaly (p = 0.01), while flt3/itd mutation was associated with higher white blood count (p = 0.01). immunophenotypically, npm1 mutation was associated with the lack of cd34 (p < 0.001) and hld - dr expression (p < 0.001), while flt3/itd mutation was positively associated with the expression of cd7 (p = 0.04). no correlation was found between npm1 mutation and fusion gene. interestingly, flt3/itd mutation was found to be inversely associated with aml / eto fusion gene (p = 0.04). conclusions. the results suggest that distinct clinical and immunophenotypic characteristics of npm1 and flt3/itd mutations present further insight into the molecular mechanism of leukemogenesis.
positron emission tomography (pet) is a form of molecular imaging that relies on derangement of physiological and biochemical processes for the detection of many diseases. long recognised as being a useful technique in neurology and cardiology, recent growth has been largely driven by its role in oncology. this growth has been based almost exclusively on the imaging characteristics of a single tracer, fluorine-18 fluorodeoxyglucose (fdg), that simply visualises a basic biochemical function of most living cells ; their ability to utilise glucose as an energy substrate. disease processes can either up - regulate or down - regulate glucose metabolism. despite the excellent diagnostic performance of fdg broadly, these relate, first, to the non - specificity of glucose - metabolic changes and, second, to lack of contrast between physiological and pathological uptake, limiting sensitivity. lack of specificity of glucose metabolism as a target for disease detection is particularly relevant for the detection of cancer. since enhanced glycolytic metabolism is a characteristic of many cancers but also of active inflammatory processes, cancer staging can be compromised by apparently focal accumulations of fdg due to infectious diseases, granulomatous lesions and inflammatory healing responses related to previous interventions or therapies. although pattern recognition can play an important role in differentiating these processes, biopsy may be necessary to confirm the nature of fdg pet abnormalities definitively. even when cancer is known to be present, high uptake in adjacent tissues due to physiological processes can reduce lesion contrast and thereby impair the sensitivity of fdg pet for the detection of disease. the most obvious example of this is in the brain where high glucose use by the normal cortex can mask the presence of brain tumours. low fdg - avidity can also occur with certain tumours, reducing contrast and, therefore, sensitivity even in the absence of high background activity. although both its specificity and sensitivity are imperfect, the overall diagnostic accuracy of fdg in cancer is so good in most clinical situations there is little room for improvement with respect to new tracers, which have to match and then improve on this already high sensitivity and specificity. nevertheless, new tracers offer the potential to address both these limitations and also to establish new applications for pet, particularly in niche clinical applications. the ability of pet to go beyond the anatomical information provided by imaging modalities such as magnetic resonance imaging (mri) and computed tomography (ct), or even the physiological information obtained from standard nuclear medicine procedures, down to the biochemical level is one of its major advantages. in an era where molecular profiling is identifying specific and mechanistically important alterations in diseased cells, a logical progression of pet tracer development is to go beyond probing of basic biochemical processes to looking at more specific features of cancer biology. the process of developing new tracers for human use is complex, but can be broken into logical steps. on one hand, there are phases related to identifying and meeting a clinical need, and on the other, phases involved with the development of the tracer itself. successful tracer development requires that these elements go hand in hand. for clinical development of new tracer there are five major steps : (1) definition of clinical need (2) establishment of an appropriate pre - clinical model for testing (3) evaluation in an appropriate clinical setting (4) comparison with existing standards (5) documentation of safety and efficacy. (1) definition of clinical need (2) establishment of an appropriate pre - clinical model for testing (3) evaluation in an appropriate clinical setting (4) comparison with existing standards (5) documentation of safety and efficacy. for the design and production of new tracers another five steps are involved : (1) identification of an appropriate target (2) selection of precursor ligands (3) development of labelling strategies (4) optimisation of labelling and qc (5) documentation of drug master file (dosimetry, toxicity etc.). (1) identification of an appropriate target (2) selection of precursor ligands (3) development of labelling strategies (4) optimisation of labelling and qc (5) documentation of drug master file (dosimetry, toxicity etc.). these 10 steps are all required to develop a new tracer to the point of clinical application but can not occur as independent processes. the most important step in our opinion is the recognition of clinical need based on existing limitations of available tracers and shortfalls in other imaging modalities. from this need and an understanding of disease causation, identification of potential imaging targets target discovery is a complex and, sometimes, serendipitous process but, in line with the process of drug discovery, is becoming increasingly sophisticated and technology - driven. in particular, it is now often based on genomic and proteomic analysis of diseased tissues. once a target is selected, then appropriate models for testing potential ligands need to be established. ideally, these are cells over - expressing the particular target and that can be both grown in culture for in vitro studies and induced in animals, either through direct implantation, by physical means, or by genetic modification, allowing in vivo testing. the selection of precursor ligands is driven by the target identified but also by an understanding of available labelling techniques. many biological interactions relate to stereochemistry and therefore, initial precursor selection can occur from libraries tested in silico before subsequent in vitro and in vivo testing. the development of labelling strategies leads to production of a radioligand suitable for further testing in the chosen model system. if the radiotracer appears promising, further work can proceed to optimise labelling and produce the compound in sufficient quantity and sufficient purity to enable testing in human subjects. the ideal means of confirming the potential utility of a new tracer is to test it in an appropriate population with the disease for which the tracer was developed. these preliminary human studies are consistent with recently promulgated micro - dosing principles for tracer compounds. if biodistribution studies look encouraging and the disease process can be visualised, further work to improve the synthesis yields, and to obtain more complete toxicity data in animals, is worthwhile. on the clinical validation side, subsequent comparison with existing standards is pertinent to demonstrate the incremental benefits of the new agent compared to existing diagnostic tests. the final phase of development relates to the documentation of clinical efficacy and safety, and of a regulatory approval file relating to the production and quality assurance of the tracer. as mentioned above, increasing understanding of molecular biology enables identification of novel potential targets for the diagnosis and treatment of diseases. these targets can be as specific as a mutation in a key gene or reflect downstream consequences as generic as substrate utilisation by cells. small animal pet is being increasingly used by academic institutions and pharmaceutical companies as a platform technology for translational research and drug development. the ability to extrapolate from animals to human studies makes pet a logical technique for both pre - clinical testing of new therapeutic drugs and for the validation of new tracers that might be relevant to the evaluation of human diseases. development of appropriate animal models for testing targeted tracers is an important step in this process. manipulation of cells and tissues to produce animal models that mimic human diseases provides a useful system to test new tracers. however, interspecies differences in the handling of chemicals may significantly influence the biodistribution and kinetics of novel tracers, requiring caution in predicting behaviour of these tracers in man. at a pragmatic level, a short feedback loop between studies in mice and then in man is important. the marked variability in the biodistribution of fluorine-18 3 -deoxy-3 -fluorothymidine (flt) in various animal models is but one salutary example of the limitations in evaluating new tracers in animal models. while unrestrained proliferation has been described as a hallmark of cancer and is a target of several targeted anti - cancer therapeutics, due to differences in blood thymidine levels and in the metabolism of flt, both uptake and excretion of this tracer show significant differences in biodistribution in different species. thus they may not necessarily provide the same information, nor necessarily reflect the process that they purport to evaluate. for example, dogs have low uptake of flt in the liver, whereas intense uptake is seen in man. nevertheless, the cellular uptake of flt provides evaluation of cellular proliferation, primarily through reflecting thymidine kinase 1 (tk1) activity. in animal and human clinical studies, our experience of various other tracers in mice and man have indicated that such interspecies differences also exist for amino - acid analogues like flourine-18 fluoro - ethyl - tyrosine (fet) and tracers of cell membrane formation like fluorine-18 fluorocholine (fch). the uptake of fet in most human xenografts that we have evaluated demonstrate excellent tumour - to - background contrast whereas in solid tumours in man, relatively low contrast compromises the sensitivity of this agent for tumour detection relative to fdg (fig. 1). on the other hand, uptake of the choline analogue, fch, in almost all xenograft models that we have evaluated is very poor but this tracer shows excellent uptake in some tumours that can have relatively low fdg - avidity, like prostate cancer (fig. the effects of micro - environmental factors and species - specific antigenic characteristics are likely to further increase the complexity of assessing tracers that rely on metabolic or immunogenic targeting. sap is an exciting tool but needs to be used in full recognition of both its strengths and weaknesses. based on established principles of pet metabolic imaging using tracers of substrate metabolism, development of radiolabelled amino acids has been seen as a means to address the limitations of fdg with respect to specificity for cancer and also for sensitivity of lesion detection in the brain where high background uptake can mask tumoral radiotracer accumulation. up - regulation of amino acid transport and enhanced protein synthesis is a hallmark of cancer. carbon-11 methionine has been shown to have similar sensitivity to fdg for detection of various cancers, including head and neck and lung cancer, but a somewhat higher specificity due to a reduced tendency to be accumulated in inflammatory lesions. as such, it is one of the few radiopharmaceuticals to compete favourably with fdg in terms of diagnostic accuracy in cancer. unfortunately the short physical half - life of c-11 makes this an impractical tracer for routine clinical applications. fet was developed by german researchers as an alternative cancer - imaging agent and in comparative studies with c-11 methionine was shown to have similar characteristics but has the practical advantages of using f-18 as the imaging radionuclide. this agent demonstrates very low uptake in the normal brain and relatively higher uptake in brain tumours ; it provides more sensitive detection of disease and better characterisation of the extent of tumoral involvement than does fdg (fig. extensive efforts have been made to develop pet tracers to enable the non - invasive imaging of hypoxia. fluorine-18 fluoromisonidazole (fmiso) is the most extensively studied agent both in animal models and humans. in particular, demonstration of the ability of fmiso to stratify prognosis in patients receiving conventional radiotherapy and targeted therapy of hypoxia has been demonstrated. however, fmiso is a relatively lipophilic compound and demonstrates relatively low uptake in hypoxic tissue relative to normal tissue, and slow clearance from normal tissues requiring delayed scanning with consequences on contrast and image quality. this has led to the development of other hypoxic tracers with more favourable imaging properties. preliminary results have been obtained at our institution with the use of faza to image hypoxia within head and neck squamous cell carcinomas (fig. 4). these suggest that faza is likely to be a superior agent for hypoxia imaging. fluorine-18 fluoromethyl - dimethyl-2-hydroxyethyl - ammonium or fluorocholine (fch) is an interesting new tracer. tumour tissues have a requirement for increased synthesis of phosphatidylcholine, an important constituent of cell membranes. increased rates of transmembrane transport and subsequent phosphorylation of choline by the enzyme choline kinase in tumours has been demonstrated. carbon-11 labelled choline has been used to successfully image a variety of tumours including prostate cancer, brain tumours, and oesophageal cancer and lung cancer but because of the short half - life of carbon-11 (20 min), fluorinated choline analogues, like fch, are potentially more suitable for clinical studies. preliminary studies suggest that fch may be more sensitive than fdg for detection of nodal and bone metastases in prostate cancer. high uptake in the liver and kidneys is a limitation of this tracer in detecting primary or secondary tumours in these organs. due to the favourable imaging characteristics and ready availability of fluorine-18, there has been an increasing focus on fluorinated tracers. however, a wide range of both cyclotron and generator produced isotopes are becoming available. these tracers lend themselves to complexing with biological macromolecules, such as peptides and antibodies. the availability of long - lived positron emitting radionuclides offers particular opportunities for translational research in the labelling of larger molecular species, including monoclonal antibodies, which can have slow accumulation in tissues. the tale of a tracer s journey from concept to clinical utility is long and littered with failures, not least because of the great success of the existing yardstick, fdg. nevertheless, there remain many opportunities for new tracers to complement existing diagnostic methods and potentially to supplant them in some situations. tracer development is not solely the domain of radiochemists delivering products for testing by nuclear medicine specialists. it is a multidisciplinary effort combining the skills and expertise of clinicians and scientists from many specialties. the commonality of goal needs to be delivering better diagnosis and, thereby, better health. a comparison of fdg and fet uptake in a primary lung cancer demonstrates substantially higher contrast with fdg and therefore, despite theoretically higher specificity for malignancy, the lower sensitivity decreases confidence in the veracity of a negative result. a comparison of fdg and fch uptake in nodal metastases demonstrates higher contrast and therefore, greater sensitivity of fch in this case. a comparison of fdg and fet in malignant brain tumour demonstrates substantially higher contrast with fet than fdg, despite lower absolute uptake as reflected by the standardised uptake value (suv). a comparison of faza and fmiso shows comparable lesions but higher contrast on faza, aiding clinician confidence in the images and region - of - interest assignment.	pet scanning is an emerging technology for the clinical evaluation of many disease processes in man. the vast majority of clinical positron emission tomography (pet) studies are performed using a single tracer, fluorodeoxyglucose. despite the excellent diagnostic performance of this tracer, it has recognised limitations. new tracers offer the potential to both address these limitations, and to establish new applications for pet. small animal pet is a logical technique for validating new tracers relevant to human diseases. however, interspecies differences in the handling of chemicals may significantly influence the handling of novel tracers. this requires caution in extrapolating findings in animals to expectations of performance in man. already there are several examples where biodistribution studies in mice would not have predicted the clinical utility of existing pet tracers. nevertheless, application of a systematic approach to tracer development is likely to speed transition of new tracers from animals into man.
hilar cholangiocarcinoma (hcca), or klatskin tumor, is a rare cancer arising at the confluence of the right and left hepatic ducts. radical surgery is still the only curative treatment, although this can only be performed in a minority of patients. nonetheless, only 5060% of patients who are surgically explored are ultimately amenable to a potentially curative resection, due to peritoneal, nodal, or liver metastases, as well as locally advanced disease. already in 1975 the bismuth - corlette (bc) classification was proposed, which has been modified in 1992. this classification system is based on proximal, ductal extent of the tumor. since the bc classification was not able to predict resectability [4, 5 ], the memorial sloan - kettering cancer center (mskcc) developed a new presurgical t - staging system in 1998 that also took into account portal vein involvement, lobar atrophy, and ductal extent of the tumor. lastly, in 2009 the tnm staging system for malignant tumors was revised in the seventh edition of the staging manual by the american joint committee on cancer (ajcc) and the international union against cancer (uicc) and also included some changes for extrahepatic cholangiocarcinomas. this system, however, is based on pathological criteria and, thus, is not applicable in the preoperative setting. over the years, surgical approach, resectability criteria, and options to achieve r0 resection have changed significantly. more specifically, hilar resection in combination with extended liver resection including the caudate lobe is considered essential for long - term survival [9, 10 ], whereas bilateral ductal involvement (bc type iv) is no longer considered per se a criterion of unresectability. resectability of hcca patients is defined by factors that are important when considering patients for any major liver resection, such as physical condition, age, and size and function of the future remnant liver. in addition, resectability is defined by factors specific for hcca, including invasion of the portal vein and hepatic artery, lymph node status, and proximal ingrowth into the segmental bile ducts. the aim of this study was, firstly, to evaluate the prognostic impact of these specific preoperative parameters on resectability, incidence of metastases, and yield of laparoscopy, and, secondly, to evaluate the currently used staging systems in hcca. all patients suspected of hcca and managed in the amc from january 2003 through august 2010 were evaluated. only patients with a tumor arising at the biliary confluence or the right or left main hepatic ducts were included. patients with tumors originating in the proximal common hepatic duct were included if the tumor extended into the biliary confluence. as previously described, cross - sectional imaging studies such as ct and mri were used in addition to ultrasound with duplex to assess liver parenchymal invasion, vascular invasion in portal vein and/or hepatic arteries and hepatic metastases, lymph node metastases, or extrahepatic metastases. imaging studies were preferably performed in our own center, and imaging studies from referring centers were only accepted, when comparable with our own high - quality imaging studies. patients considered to have potentially resectable tumors underwent further evaluation using staging laparoscopy and, in the last three years of the study, an additional pet - ct. staging laparoscopy was routinely performed in resectable patients, when feasible, although staging laparoscopy was omitted in some patients with limited bc type i or ii tumors, as we described recently in more detail. since we found no advantage of laparoscopic ultrasound for the staging of lymph nodes and hepatic artery involvement, laparoscopic ultrasound was not used in the current study. furthermore, tc - mebrofenin scintigraphy [15, 16 ] was performed in conjunction with ct volumetry to determine function of the future remnant liver. portal vein embolisation was performed in 6 patients, because the future remnant liver function or volume was deemed insufficient. when no metastases were found during further evaluation, patients were planned for resection, and preoperative biliary drainage was performed, of at least the future remnant liver, either percutaneously or endoscopically. during laparotomy, the abdomen was inspected for peritoneal seeding, distant metastases, or lymph node involvement outside the liver hilum, in the hepatoduodenal ligament and along the common hepatic artery until the celiac axis. if more causes of unresectability were found, the one discovered first was reported. more specifically, extrahepatic metastases, liver metastases, positive lymph nodes, and locally advanced disease were reported in decreasing order. in case of distant metastasis, only a cholecystectomy was performed. in patients with unresectable disease, biliary drainage was accomplished by definitive internal stenting by either ptc or ercp using metal expandable stents, usually within the same hospital admission. chemotherapy was not routinely administered until the results of the abc-02 trial came out in 2010. chemotherapy, palliative radiotherapy, or photodynamic therapy was administered in 38, 12, and 9 patients, respectively. in addition, most patients without a pathological diagnosis died during followup as described earlier in more detail. because resectability, as defined above, is determined by vascular involvement, ductal involvement, and lymph node status, we primarily evaluated these items for their prognostic impact on resectability. patients who were unresectable at initial imaging were not included in this analysis, since prediction of resectability in unresectable patients obviously is useless. for every potentially resectable patient we scored the absence or presence of tumor involvement of the portal vein, or hepatic artery, suspicious lymph nodes as well as lobar atrophy, or bilateral ductal involvement. these parameters were retrieved from reports of ct, duplex ultrasound, and mrcp, in which these parameters were accurately described. vascular status was based on narrowing of the vessels, encasement, proximity of tumour, and circumference of tumour. lymph nodes were assessed based on nodal shape, signs of necrosis, and short - axis diameter (< 1 cm). in some cases direct cholangiography, by either ptc or ercp, was also used to assess the extent of proximal ductal involvement. we then evaluated the prognostic impact of the different variables on the main outcome parameters, namely, resectability rate, yield of laparoscopy, and incidence of metastasis. during the study period however, we do not consider bilateral segmental ductal involvement any longer a criterion of unresectability, nor tumor invasion in the bifurcation of the portal vein. hence, we applied the following criteria for resectability : absence of hepatic or extrahepatic metastases, with the exception of lymph nodes metastasis confined to the hepatoduodenal ligament. in addition, a future remnant liver featuring sufficient volume (3040%) is required, in combination with tumor - free portal venous, arterial supply, and options for biliary reconstruction. reasons for unresectability of the patients were described earlier in more detail [13, 18 ]. patients were ultimately scored as resectable, when r0 or r1 resection had been performed. survival data was obtained from our own database or that of local hospitals and updated if necessary, by contacting the primary care physicians. survival (in months) was measured from the date of initial presentation at our center to the date of death, or the date of last contact when alive. we used univariate logistic regression to calculate odds ratios of portal vein involvement, hepatic artery involvement, lobar atrophy, suspicious lymph nodes, and bilateral ductal involvement for resectability, yield of laparoscopy, and presence of metastasis. survival probabilities were estimated using the kaplan - meier method and compared by the logrank test. as shown in figure 1, 289 hcca patients were seen in our center from january 2003 through august 2010. preoperatively, patients were classified by the bismuth - corlette classification as type i (n = 19), type ii (n = 20), type iiia (n = 100), type iiib (n = 55), or type iv (n = 95). 135 (47%) patients were considered unresectable (according to the resectability criteria described earlier) based on imaging findings that were usually confirmed by cytological puncture. also 23 patients were unfit for major surgery, predominantly because of age and significant comorbidities. another 23 patients were considered unresectable at staging laparoscopy, or by subsequent imaging after staging laparoscopy. 131 (45%) patients underwent explorative laparotomy, of whom a resection could be performed in 83 (63%) patients. the 48 patients who were deemed unresectable during laparotomy had metastases (n = 13), positive lymph nodes outside the hepatoduodenal ligament (n = 15), or locally advanced tumor (n = 20). the sites of positive lymph nodes precluding resection were celiac trunk in 5 patients, peripancreatic in 3 patients, periduodenal in 1 patient, and common hepatic artery in 6 patients (not resectable by regional lymphadenectomy). resection encompassed hilar resection in combination with (extended) hemihepatectomy including segment 1 in 71 (86%) patients or hilar resection only in 12 (14%) patients. 32 patients underwent (extended) left hemihepatectomy, 36 underwent (extended) right hemihepatectomy, and 3 patients underwent a central liver resection. bilateral ductal involvement (bc type iv) seemed important for the presence of metastasis, and consequently for the yield of laparoscopy. the involvement of the portal vein often coincided with involvement of the hepatic artery, yet the involvement of the hepatic artery seemed more predictive of unresectability as well as for incidence of metastasis. all potentially resectable patients based on imaging were staged according to the bc classification, and the mskcc staging system. the bc classification also correlated with the incidence of metastasis, with an incidence of 40% in the type iv patients as compared to 12% in the type i and ii patients. the yield of laparoscopy and overall survival was not statistically different between the staging groups. in both groups the high stage patients (t3, and bc type iv) represented only a small proportion of the resectable patients (16%). complete survival data could be retrieved from all but 2 patients. at the time of analysis, patients with benign disease at final pathology (n = 6) were not included in this assessment. as expected, patients who had undergone resection had a significantly longer median survival than those who did not undergo resection (37 versus 14 months ; p < 0.001), as shown in figure 2. as a result, survival differences found within the different groups of the staging systems will very likely be a result of differences in resectability. nonetheless, neither the mskcc staging system nor the bc type did correlate with overall survival, as shown in table 2. this study represents the common denominator of 289 patients referred to our center with hcca during a 7-year period. the large number of patients enabled us to evaluate different preoperative factors important for resectability, metastasis, and yield of laparoscopy and to evaluate the current staging systems. since the recognition of the disease in 1965 by gerald klatskin, much progress in management has been made, including developments in imaging, surgical techniques, and pre- and postoperative care. these developments have markedly increased the 5-year survival rate of patients who underwent resection to over 40% in recent series, including our own. also, as mentioned above, two different classification systems have been introduced in the past, that is, the bc classification system and the mskcc preoperative staging system [2, 3, 7 ], offering the possibility to compare data between centers, stratify patients before surgery in subgroups with different resectability rates, and select patients at risk who could benefit from additional imaging or staging laparoscopy. considering the latter, the additional value of staging laparoscopy has decreased dramatically in the last decade with improvements in imaging. the bc classification system is based on proximal ductal infiltration of the tumor and is still very useful in communications within a hospital as well as between centers. yet, its correlation with resectability or survival has been questioned, because it is more a classification system than a staging system. in 1997 the mskcc introduced a preoperative staging system, which also incorporated involvement of the portal vein and the presence of lobar atrophy. this group also pioneered the definition of resectability criteria, which differed largely between centers. furthermore, they showed that their proposed staging system could predict resectability, need for hepatic resection, and survival. yet, this system also has its limitations : the system is complicated and does not include in its assessment the presence of nodal or distant metastases or the involvement of the right or left hepatic artery. however most importantly, patients are divided into three categories that also include patients that are unresectable based on imaging. since the system was designed with the aim of predicting resectability, the inclusion of unresectable patients in the staging groups is less appropriate. we have also staged our patients according to the mskcc classification, as shown in table 2. the system predicted resectability, yet the predictive power for the other parameters (metastasis, yield of laparoscopy, and survival) was less impressive and not significant. the mskcc system incorporated lobar atrophy and portal vein involvement in addition to proximal ductal infiltration as classified in the bc system, yet according to our results, involvement of the right or left hepatic artery was more predictive than portal vein involvement. therefore, the incorporation of invasion of the right or left hepatic artery should probably be added to the system. furthermore, the presence of suspicious lymph nodes obviously also correlated with resectability and should probably also be used to predict resectability. lobar atrophy, however, was not predictive of one of the categories and therefore seems a less essential component of the system. in addition to the mskcc and bc classification systems, a new staging system was recently proposed by deoliveira.. this system is designed to consider the extent of cancer in the bile duct, portal vein, and hepatic artery, as well as tumor size, nodal involvement, future remnant liver size, and distant metastases, as well as the volume of the putative remnant liver after resection. the authors herewith aim to standardize the reporting of hcca and, consequently, allow comparisons of studies among centers or over time. in addition, the authors opened a registry, in which centers can prospectively and in a standardized fashion record features of this rare disease. since the system is merely designed to standardize reporting, rather than predicting resectability and guiding therapy, the system is more a classification system than a staging system. we analyzed 289 consecutive patients with hcca presenting in our centre during a 7-year period until 2010. this allowed us to secure to some extent a homogeneity in patient evaluation, surgical treatment, and imaging protocols. we aimed to include all preoperative data, of which we expected any influence on resectability, on yield of staging laparoscopy or on presence of metastasis, and to evaluate their individual impact. imaging has much improved in the last years, and all patients are usually evaluated by doppler ultrasound as well as state - of - the - art cross - sectional imaging. yet, accuracy is still not 100% for these imaging tools, and consequently, the accuracy of the analysis will suffer from the inaccuracy of the imaging modalities used., this limitation applies to all preoperative parameters and, unfortunately, is hardly avoidable. in the future, well - performed imaging studies should assess sensitivity, specificity, and accuracy of the different imaging modalities. until now, evidence on the accuracy of imaging studies for staging of hcca is scarce and inconclusive. secondly, resection criteria are not universally adopted worldwide, and consequently, the primary outcome parameter in our study, that is, resectability, depends on the criteria used in our center. in conclusion, current staging systems do predict resectability, yet there is clearly room for improvement in patient selection. future improved staging systems will hopefully enable us to select patients for additional treatment (adjuvant or neoadjuvant) or additional imaging or staging laparoscopy. according to our results, hepatic artery involvement and nodal status are also important for prediction of resectability, metastasis, and yield of laparoscopy and should therefore be incorporated in future staging systems after appropriate validation.	objectives. to evaluate, in hilar cholangiocarcinoma (hcca), the prognostic impact of specific preoperative radiologic parameters on resectability, metastases, and yield of laparoscopy, and to evaluate the currently used staging systems. methods. consecutive patients with hcca presenting in our center from january 2003 through august 2010 were evaluated. suspicion on lymph node metastasis, portal vein and hepatic artery involvement, lobar atrophy, and proximal extent of ductal invasion was scored. the prognostic value of these parameters for predicting resectability, yield of diagnostic laparoscopy, likelihood of metastatic disease, r0 resection, and survival was assessed. the bismuth - corlette classification and mskcc staging system were evaluated. results. of all 289 evaluated patients, 158 patients (55%) had unresectable disease based on cross - sectional imaging studies or diagnostic laparoscopy ; 131 patients (45%) underwent exploration. 83 patients (64%) underwent resection, of whom 67 (87%) had a radical (r0) resection. suspicious lymph nodes and involvement of the hepatic artery were important prognostic factors for resectability. predictive power of the evaluated staging systems was limited. conclusions. current staging systems predict resectability, but there is room for improvement. hepatic artery involvement and nodal status might be important factors for prediction of resectability and should be considered in future staging systems.
since strong and jako1 2 proposed laser endoscopic surgery for the treatment of larynx cancer in the seventies, transoral laser microsurgery (tlm) has gained territory in larynx oncology and established itself as an effective option in the treatment of glottic, supraglottic, and hypopharynx tumors. advantages such as the magnification that the microscope generates limits the number of resections by allowing the differentiation of healthy tissue from affected, thus, preserving disease free areas, reducing the number of tracheostomies and the use of nasogastric tubes.3 4 5 there are oncological benefits as well, similar to those in open surgery in patients with early stages and correctly selected, in addition to the good results in terms of quality voice after tlm comparable with radiotherapy itself in the treatment of early glottis tumors (t1). in different studies published on the treatment of laryngeal tumors, the 5-year local control ranges from 7894% for t16 and a 4791% for t2, using tlm, open partial laryngectomy, or radiotherapy.7 8 for this reason, the aim of this study was to review oncologic outcomes obtained by the treatment of a series of glottic tumors treated by tlm for t1a, t1b, and t2 glottic squamous cell carcinomas (scc) in a tertiary university hospital. endpoints for analysis in this study were locoregional control, overall and disease - specific survival, and larynx preservation rate. we performed a retrospective analysis of patients previously untreated, diagnosed with squamous cell carcinoma of the glottis (t1a, t1b, t2), n - / +, m0 according to criteria of the union internationale contre le cancer (uicc) and the american joint committee on cancer (ajcc) in a tertiary university hospital. we included patients treated with curative intent by tlm between january 2009 and january 2012. we identified through informative research of the records of our service, using the international classification of diseases (icd-9). we obtained demographic data (age, sex), comorbidities, tumor stage, additional test information, histological results, complications, survival, type of surgery, among others, by reviewing medical history. all the patients received information on treatment options (tlm versus rt versus open surgery). however, we actually try to avoid the use of open surgery in early tumor stages because we prefer a less invasive treatment option at first ; we only use this approach in patients for which it is not possible to achieve a correct exposure of the larynx during the suspension laryngoscopy. in addition, we usually perform a ct scan in all patients to confirm the disease local extension and to assess the neck. next, all cases were discussed in an interdisciplinary committee of head and neck tumors. patients with lesions suspicious for malignancy were scheduled for laryngeal microsurgery with biopsy followed by laryngeal laser resection in cases that were positive. after surgery, we presented tnm in the committee, which assessed the need for reoperation or additional radiotherapy (rt). to perform the surgical procedure, we used general anesthesia with orotracheal intubation in all patients, and performed resection using a lumenis co2 laser device (yokneam, israel) with a power setting of 48 w, in super pulsed mode and continuous setting, varying size and shape of the spot according to the moment of the surgery by using the micro - manipulator acuspot - acublade (lumenis, yokneam, israel). we classified type of cordectomy according to the european laryngological society (elsoc)9 proposal. in the case of small tumors (t1a, t1b), whenever possible, we attempted en bloc resection and, after resection, we pinned and oriented the piece on a corkboard. sometimes we added a picture or a diagram on the orientation of the surgical specimen in the larynx, as it is very difficult for the pathologist to have a three - dimensional idea of the orientation of the piece. for bulky tumors (t2), most cases required piecemeal resections. laser vestibulectomy was performed when the lateral or anterior portion of the tumor was hidden by a ventricular fold. in all cases the surgeons tried to achieve a margin of healthy tissue of 23 mm, trying to preserve the functions without affecting the oncological radicality of the procedure. during surgery, we did not perform intraoperative biopsies if resection was satisfactory ; on the contrary case, we conducted examination of frozen section when the depth of cancer infiltration was difficult to estimate. resection margins were classified as free, uncertain, or affected. in case of having affected margins as for patients with uncertain margins, only if there was evidence a suspicious lesion suggestive of persistence or recurrence during follow - up did we perform a new surgery. in patients with positive neck ganglion (n1, n2), we performed a functional neck dissection in the same surgical act, while in n0 patients, we followed the necks. however, if during follow - up metastatic disease was evident, we performed a functional neck dissection, which was done in a second look surgery accompanied by microlaryngoscopy exploration. in our service, patients treated for head and neck malignant tumors are followed during at least 5 years at the department of head and neck oncology. however, for this study, we considered a group of patients that have been followed for a minimum of 36 months. statistical analysis was performed with the spss program for windows, version 20.0 (spss, inc. we calculated overall survival, specific survival, local control with laser, final local control with laser and organ preservation using the kaplan - meier survival analysis. for overall survival, we used as reference survival time from the surgery until death from all causes or last revision, while specific survival was considered from the time of surgery until death by tumor cause or until the performance of a total laryngectomy. we calculated the influence of various factors on survival using the log - rank method and we correlated the influence of the margins on the need for re - intervention as well as the influence of t stadium on the need to place a nasogastric tube or dysphagia by the pearson chi - square test. fifty - eight patients met the inclusion criteria : 57 (98.3%) were men and 1 (1.7%) were women. the mean age was 65.5 10.7 years (min : 46/max : 88). of these, 15 (25.9%) were diabetic, 31 (53.4%) were hypertensive, 53 (91.4%) were smokers, and 12 (20.7%) consumed alcoholic beverages. the tumor stage of the patients included 30 t1a, 11(19%) t1b, and 17 (29.3%) t2 (table 1). we classified 55 (94.8%) patients as n0, 2 (3.4%) as n1 (t1a, t2), and 1 (1.7%) as n2a (pt2). the mean follow - up was 43.1 11 months (min : 19 / max : 72). regarding the type of cordectomy, the most common was type 4 (27 = 46.55%) and type 5a (21 = 36.2%) (table 1). surgical margins were free in 41 (70.7%) patients, uncertain in 12 (20.7%), and affected in 5 (8.6%) cases (table 2). the mean hospital stay was 2.1 days (min : 1/max : 14) ; however, in our service, most patients are discharged the day after surgery. regarding immediate post - surgical complications, 1 (1.7%) patient had postoperative bleeding, which required the placement of a clip in the superior laryngeal pedicle. other patients presented an episode of atrial fibrillation and needed to be in anesthetic resuscitation for 24 hours. nine (15.5%) patients required nasogastric (ng) in the immediate postoperative period with an average of 2.06 days (min : 1 / max : 6), being more frequent in patients with the most advanced tumors (p = 0.009). in none of the patients was it necessary to place a percutaneous gastrostomy tube (peg). during follow - up, 6 (10.3%) patients had some degree of postoperative dysphagia, which improved with swallowing rehabilitation. in the late postoperative period, no patients presented complications associated with surgical technique, such as aspiration pneumonia, chondritis of the thyroid cartilage, among others. regarding associated metastasis to the glottis scc, 3 (5.2%) patients had palpable metastasis at the moment of diagnosis. in two cases (n1), we performed bilateral functional neck dissection and, in one case (n2a), we performed a modified radical neck dissection. we achieved local control in the first surgery in 46 (79.3%) patients, in that 80.48% were t1 (32/41) and 76.47% were t2 (13/17). in 9 (15.5%) patients, we performed a second look operation (4 t1a, 4 t2, 1 t1b), and in another three patients, 2 re - interventions (3 t1a) were necessary to achieve local control. in one case (t2), a total laryngectomy was necessary, achieving 98.3% laser local control as well as functional laryngeal preservation. in terms of margins, 41 (70.6%) specimens were free of tumor involvement, 12 (20.6%) were uncertain, and 5 (8.6%) were affected. in the group of uncertain margins were where most re - interventions were needed : up to 8 of the 12 patients (table 2) (p < 0.001). the mean time elapsed until reoperation was 6.7 months (min : 2 / max : 11). regional control was possible in 56 (96.6%) patients, while we observed cervical nodal disease in two (3.4%) patients during follow - up. median time to onset of positive lymph was 11 months (min : 10/max : 12) and a functional neck dissection was performed in both. the use of complementary therapies in our study was limited because most patients were in early stages. three patients received additional rt, having positive metastases after making the neck dissection, two (3.4%) of these patients belong to the free margins group, and another patient to the affected margins group. in another two patients in the uncertain margins groups, which also had regional recurrence during follow - up, we performed a cervical neck dissection and subsequent treatment with rt. for the n+ patient, rt courses were scheduled within 46 weeks postoperatively and delivered weekly over a period of 6 to 12 weeks to reach a total dose of 45 to 60 gy. regarding the occurrence of second primary, 4 (6.8%) patients were affected, 2 of them for lung cancer, one bladder cancer, and another for malignant melanoma. the mean time to onset of the second primary was 20.25 months (min : 15 / max : 31). overall survival according to t stage was 90% (t1a), 90.9% (t1b), and 82.4% (t2), with no statistically significant difference between them (p = 0.590). as for n, the three - year survival of the 3 patients with metastases at diagnosis and 2 developed metastases during follow - up was 60%. we were unable to obtain statistical significance probably due to the limited sample n+ (p = 0.625). kaplan - meier three - year overall survival according to tumor stage and for all causes of death (87.9%). kaplan - meier three - year specific survival according to tumor stage (98.3%). during follow - up, 6 (10.3%) patients died, one patient (1/58 = 1.7%) by regional disease progression, 4 patients by second synchronous or metachronous neoplasms, and another one because of a stroke. in this retrospective study, we analyzed the results of a group of 58 patients with early laryngeal carcinomas (t1a, t1b and t2), treated by first intention with tlm. however, we must remember that when we are treating these kind of tumors, in addition to endoscopic techniques, we have open surgery and radiotherapy (rt) - three tools with similar effectiveness that do not allow us to favor one over the others in the treatment of these tumors based on oncologic outcomes.10 in different published studies of the treatment of laryngeal tumors, the 5-year local control ranged from 7894% for t16 and a 4791% for t2, using the 3 modes previously described.7 8 regarding our study, 79.3% local control was achieved with one surgery alone (t1 : 80.48% ; t2 : 76.47%), reaching a 98.3% local control after a second look intervention, similar results to those described in the literature by lee, who obtained exclusive local control with laser in 94.2% of patients.11 in the whole group, a total laryngectomy was required only in one case, achieving a rate of organ preservation of 98.3%, similar to those obtained by lester, who got 98.1%12 and lee, who got 96.2%11 of organ preservation. several authors suggest that in those patients in whom the anterior commissure, arytenoid, subglottic, or vocal muscle are affected will have a higher risk of local recurrence.11 13 14 while other factors may directly influence the outcome of surgery and the likelihood of tumor recurrence will be an adequate laryngeal exposure during surgery. to accomplish this, we had to mobilize the laryngoscopy blade during surgery looking for the best exposure and resect the ventricular fold when the exposure of the anterior commissure or laryngeal ventricle was not possible.11 at the glottal level, resection margins over 12 mm will be considered adequate,11 although some authors have suggested the effectiveness of margins less than 0.5 mm for in situ or t1 carcinomas.15 a different concept from the average of 5 mm was suggested for open surgery in head and neck cancer.16 ansarin concluded there was a need for additional treatment for patients with positive margins (reoperation or rt), since the presence of positive margins was associated with a greater tendency to recurrence. these results correlate with those obtained by crespo, who showed 37.5% local recurrence rates in patients who had positive margins against 0% recurrence in the group of patients with negative margins.18 however, both lee and hartl showed no statistically direct relationship between the presence of positive margins and the tendency to recurrence, suggesting the possibility of reoperation for these patients based on the findings during follow - up.11 19 this is precisely where follow - up plays a key role in the success of surgery, because, despite being able to get uncertain or positive margins, the phenomenon of vaporization after tumor resection will allow a safety margin of 23mm20 and, for this reason, in our center as well as in others, the presence of uncertain or positive margins in all cases is not associated to reoperation. to classify the degree of involvement of surgical margins, we decided to use the classification proposed by blanch, who aside from setting margins as positive or negative, also included the presence of uncertain margins.20 in our sample, 70.6% of patients had clear margins, 20.6% presented uncertain margins, and 8.6% presented affected margins. it was the uncertain margins group that required the most second - look interventions, being necessary in up to 66.6% (8/12) of patients. in our study, the three - year overall survival rate was 89.7% for all causes of death, the ultimate local control only with laser was 96.5%. the three - year specific survival rate including salvage surgery was 98.3%, while the laryngeal preservation rate was 98.3%, comparable with other results reported in the literature. lester achieved a five - year overall survival rate of 88.8%, a five - year disease - specific survival rate of 96.6%, and 98.1% of laryngeal preservation.12 lee achieved a five - year overall survival rate of 87.9%, an ultimate local control only with laser of 94.2%, a five - year specific survival rate of 99%, and 96.2% of laryngeal preservation.11 lucioni, in turn, reported an overall survival rate of 90.8%, an ultimate local control only with laser of 94.3%, a disease specific survival rate of 98.8%, and 97.7% of laryngeal preservation with a minimum follow - up of 24 months. all these results were based on studies in t1-t2 stages.13 in our study, only three cases (5.2%) presented complications in the immediate postoperative period, none of them mortal. we must remember, however, that the percentage of tumor complications in all stages operated by experimental surgeons oscillates between 56%, a hypothesis which we were able to confirm when comparing tumor size, tumor location, and degree of surgeon experience with the appearance of complications. in any case, of all of the complications described in the literature, the postoperative hemorrhage is most fearful due to the vital risk it brings to a patient without tracheostomy.13 21 22 other important factors to consider when contextualizing this surgery (mtl) is quality of life. vilaseca, in a prospective study that included patients treated for carcinoma in early and moderately advanced stages, obtained favorable results regarding the improvement in quality of life after surgery for most of their subjects, speech preservation being again the factor assessed.23 voice is undoubtedly one of the main factors to take into consideration when treating laryngeal tumors in early stages. van gogh, in a study that prospectively evaluated the voice in patients treated with rt or laser surgery in t1a carcinomas, showed that vocal recovery was faster in the group treated with laser. three months after surgery they did not find differences from normal voice, except for the fundamental frequency and at the highest pitch. they concluded that the mtl should be the treatment of choice for t1a tumors.24 however, remmelts found evidence against the use of laser in patients with deeply infiltrative t1a lesions and t1b lesions, since voice quality might become worse when a laser resection is performed in such patients. more recently, a meta - analysis conducted by greulich about voice outcomes following radiation versus laser microsurgery for t1 glottic carcinoma concluded that vhi scores were comparable following xrt and tlm for t1 glottic carcinoma, according to reports from current literature, suggesting no clinically significant difference in functional voice outcomes between treatment types.26 unfortunately, we have not included the results regarding vocal quality of patients in our study, because, despite having a voice unit, patients were not assessed systematically in the postoperative period. finally, we must note the limitations of our study. remembering that it is a retrospective study, in which we did not have a control group to compare our results (open surgery and/or rt) and we did not evaluate the voice or the quality of life in our patients after surgery. several studies support the use of tlm in the treatment of laryngeal tumors t1a, t1b, and t2, as well as for well - selected advanced tumor stages. our results coincide with those given in the literature, showing that tlm is a safe and effective option in the treatment of glottic carcinomas associated with less morbidity and a high percentage of local control, overall survival, specific survival, and organ preservation.	introduction transoral laser microsurgery (tlm) has won territory in larynx oncology, establishing itself as an effective option in treatment of glottic, supraglottic, and hypopharynx tumors. its advantages include limited resections, a reduction in number of tracheostomies, and the use of nasogastric tubes. moreover, its oncological benefits are similar to those from open surgery in patients with early or advanced stages, when correctly selected.objective the objective of this study is to review oncologic outcomes obtained with the treatment of a series of glottic tumors, treated by tlm.methods retrospective analysis of patients previously untreated, diagnosed with squamous cell carcinoma of the glottis (t1a, t1b, t2) in a tertiary university hospital. endpoints for analysis were local control, overall and disease - specific survival, and larynx preservation rate.results the study group included 58 patients that met the inclusion criteria : 57 (98.3%) men and 1 (1.7%) woman. mean age was 65.5 10.7 years (min : 46/max : 88). the tumor stages of the patients included were 30 t1a, 11 (19%) t1b, and 17 (29.3%) t2. three - year overall survival rate was 89.7% (fig. 1), and three - year disease - specific survival rate was 96.5%, three - year local control rate was 98.3%, and three - year organ preservation rate was 98.3%.conclusion tlm is a safe and effective option in the treatment of glottis carcinomas, associated with less morbidity and a high percentage of local control, overall survival, specific survival, and organ preservation.
co - culture of macrophages with calcifying vascular cells or human vascular smooth muscle cells enhanced alkaline phosphatase activity and mineralization potential. tumor necrosis factor and oncostatin m have been suggested as molecular mediators of macrophage - derived vascular calcification. in addition, depletion of macrophages reduced osteophyte formation in osteoarthritic models [8 - 10 ], and macrophages have been implicated in the sites of pathologic bone loss in inflammatory bone disorders. depletion of macrophages in primary calvarial osteoblast cultures in vitro has been shown to delay osteogenic differentiation and mineralization. in the macrophage fas - induced apoptosis (mafia) transgenic mouse model, short - term depletion of macrophages with treatment of a synthetic ligand in vivo showed a quantitative reduction of bone formation sites in endocortical bones. in a recent study, in vivo long - term depletion of macrophages in young (321 days) and adult (1622 months) mafia mice demonstrated an osteopenic phenotypes with suppressed serum bone turnover markers. this study reinforced the hypothesis that osteal macrophages play a pivotal role in bone anabolism. another independent study with lysozyme m - deficient mice also showed that pre - natal macrophage depletion led to early skeletal growth retardation and progressive osteoporosis. the latter two studies clearly showed that functional osteoclasts were not significantly affected in these macrophage - deficient models. taken together, these results suggest that osteal macrophages play an essential role in normal bone development and remodeling, especially through anabolic actions. one of the critical concerns with osteal macrophages is how to distinguish bone marrow resident macrophages from osteoclasts, since they share the same precursors. studies of mafia mice or lysozyme m - deficient mice showed functionally active osteoclast activities, while macrophages were remarkably depleted, but still there were possible impacts of subtle changes in osteoclasts on overall bone metabolism. a recent study showed more clear evidence supporting the independent presence of functioning osteal macrophages apart from osteoclasts. ccl5- deficient mice showed decreased f4/80-positive macrophages at the endocortical bone surface, following reduced bone formation compared to the wild - type mice. previous studies have demonstrated that macrophages are present during multiple stages of fracture healing, and produce mesenchymal growth factors. macrophages are also associated with more stable callus formation and healthy union. keeping pace with the studies of osteal macrophages in bone metabolism, several groups have extensively studied how osteal macrophages participate in fracture healing of bone. both systemic and local depletions of macrophages delayed fracture healing and impaired woven bone formation, while treatment of colony stimulating factor 1 increased macrophage recruitment into the injury sites and supported woven bone formation. this study showed that macrophages were essential for collagen type i - positive matrix formation and bone mineralization. similarly, an independent study also showed that depletion of macrophages during fracture repair, even after several days later to fracture, led to impaired bone union with incomplete callus formation accompanied with more fibrotic changes. they observed that macrophages were also involved in promoting the osteogenic differentiation of marrow mesenchymal progenitor cells. moreover, a recent study clarified that inflammatory m1-macrophages (f4/80 mac-2) played a crucial role in the initiation of early inflammation, and both inflammatory (f4/80 mac-2) and resident (f4/80 mac-2) macrophages derived anabolic signals for endochondral callus formations in murine fracture models. taken together, accumulating evidence suggests that macrophages and their specific molecular mediators contribute to fracture healing in a phase - specific polarization - dependent manner. to date, osteal macrophages have been considered a third cellular component, in addition to osteoblasts and osteoclasts. macrophages construct a cellular canopy structure over bone remodeling sites, coordinate osteoclast - to - osteoblast coupling, and drive anabolic cytokines for bone formation. in addition, macrophages might play a role in bone and marrow interactions especially at the osteoblastic stem cell niche. targeting osteal macrophages or their molecular mediators could be potent therapeutic challenges for developing anabolic therapies for bone disease. further studies are needed to develop specific targets that could be distinguished from osteoclast or inflammatory macrophages.	macrophages have been shown to have pleiotropic functions in various pathophysiologies, especially in terms of anti - inflammatory and regenerative activity. recently, the novel functions of bone marrow resident macrophages (called osteal macrophages) were intensively studied in bone development, remodeling and tissue repair processes. this review discusses the current evidence for a role of osteal macrophages in bone modeling, remodeling, and fracture healing processes.
a 34-year - old man presented with abrupt onset of intermittent exercise - induced pain in the left calf that improved with rest. past medical history was significant for 14 years of tobacco abuse, and 1-year use of electronic cigarette. he denied history or symptomatology concerning for vasculitis, autoimmune, connective tissue, thrombotic or rheumatologic disorders. physical examination revealed normal vital signs with palpable left femoral but absent popliteal, posterior tibial, and dorsalis pedis pulses. in spite of the absence of pulses on the left lower limb, magnetic resonance angiogram of abdomen and pelvis with three - vessel runoff revealed widely patent common femoral, profunda femoral, and proximal - mid superficial femoral arteries. however, distal segment of the left superficial femoral and above - knee popliteal arteries showed abrupt occlusion, with the occluded segment spanning 9 cm (figure 1). mild medical deviation of bilateral above - knee popliteal artery was also present suggesting extrinsic compression laterally, although with patent right arterial system (figure 2). this deviation was concluded to be related to the proximal and lateral aberrant insertion of the medial head of the gastrocnemius muscle in the left leg and due to a fibrous band or muscle slip in the right leg (figures 13). catheter - based angiogram was performed 3 days later and confirmed the diagnosis of distal superficial femoral and popliteal artery occlusion with possible popliteal entrapment syndrome as the etiology (figure 4). it also revealed significant infrapopliteal artery disease suggestive of thromboembolic event versus buerger s disease (figure 5). management was started with enoxaparin (80 mg) subcutaneous injection twice daily, aspirin (81 mg) once daily and lipitor (40 mg) at night. due to the severity of his symptoms, the patient underwent catheter - based lytic therapy using tissue plasminogen activator at 1 mg / h delivered through a multi - sidehole infusion catheter with heparin at 500 units / h through the sidearm of the sheath over 48 hours (figure 6), followed by left lower extremity superficial femoral artery - to - below - knee popliteal artery bypass using ipsilateral reverse saphenous vein graft tunneled beneath the sartorius and below - knee popliteal artery endarterectomy, with good results. following surgery, a repeat duplex arterial ultrasound was performed 2 weeks post - surgery and showed a widely patent distal superficial femoral artery to below - knee popliteal vein bypass graft without evidence of stenosis. popliteal artery entrapment syndrome (paes) is an important but infrequent condition that occurs as a result of compression of the popliteal artery by adjacent muscles and tendons,1 often resulting in ischemia and vascular claudication.2 most commonly seen in young and middle - aged men,3 paes can be a source of significant disability and impairment in affected patients. type i is characterized by an atypical course of the popliteal artery, type ii occurs as a result of an abnormal muscular insertion, and type iii exhibits both of these anatomic irregularities.4 this abnormal anatomy creates extrinsic compression which can over time cause vascular compression that can lead to damage.5 symptoms often occur as a result of arterial wall degeneration and compression, depending on the degree of compression, magnitude of forces acting on the popliteal artery, and duration of compression.6 if untreated, the compression mechanism frequently results in deterioration of the popliteal artery which may progress to an occlusion.7 recurrent popliteal artery compression can cause intimal damage, thrombosis,8 distal embolization, post - stenotic dilation, and formation of true aneurysm,6 which makes accurate diagnosis and timely management imperative.7 unfortunately, delayed diagnosis due to young patient age and lack of atherosclerotic risk factors is common, making progressive deterioration more frequent. the diagnosis is further complicated because paes can be difficult to separate from other causes of lower limb pain.9 clinical diagnosis is made with careful history, physical exam, imaging testing including ultrasonography, magnetic resonance angiography (mra), computed tomography angiography (cta), or catheter - based angiography.7 the most common presentation is intermittent claudication in young or middle - aged athletic patients without any context of atherosclerosis.4 doppler ultrasound is a non - invasive modality that provides a good overview of the popliteal fossa, popliteal artery, and arterial flow at a reasonable cost.10 magnetic resonance angiography and cta allow physicians to visualize the popliteal fossa and arterial anomalies, providing better arterial depiction than ultrasonography with excellent visualization of stenosis, dilation, and arterial wall abnormalities.9 furthermore, magnetic resonance angiography with high - contrast resolution offers precise analysis of muscle courses and their potential conflict with popliteal vessels as well as allowing classification, making it ideal for diagnosis.11 however, cta is less expensive and more available, making it a close second. once the diagnosis of paes has been made, several treatment options are available, with the treatment objective being to release the popliteal artery from compression and preserve popliteal arterial flow.5 conventional surgery, endovascular surgery, thrombolysis, or a combination of these modalities are all reasonable treatment options depending on the patient s clinical symptomology and anatomy.9 this decision making can be very challenging. if the artery is occluded, stenotic, or aneurysmal, vascular reconstruction is mandatory in addition to the division of any entrapping structure. in patients with an acute occlusion, thrombolysis and angioplasty prior to surgery are appropriate, but have a high risk of reocclusion and will not be effective without removing the underlying reason for the popliteal artery entrapment.12 our patient represents a typical case of paes in that he is a middle - aged man presenting with acute progressive calf claudication. he was progressively symptomatic for 2 weeks before seeking treatment, resulting in a total occlusion. patient has irregularity of the infrapopliteal arteries mostly consistent with thromboembolic disease and less likely to represent buerger s disease. this is based on the repeat catheter - based angiogram images showing improvement of the infrapopliteal arteries following lytic therapy. a diagnosis of buerger s disease made via angiography is based on the presence of segmental occlusions and corkscrew collaterals around the area of the occlusion.13 as these observations were not made in the repeat angiography, it was concluded that the diagnosis of buerger s disease was less likely. the final decision was made for bypass alone since the patient s artery had been damaged over time, and since muscular release surgery is a significant surgery posing more risk to the patient. although other cases of paes have been published,14,15 this case remains relevant due to the severity of presentation. the patient presented with complete occlusion of the left popliteal artery, which required multidimensional treatment. a combination of catheter - based lytic therapy, anticoagulation therapy, and surgical intervention was successfully implemented. we present a challenging case of paes with complicated decision making regarding an appropriate revascularization option with lowest risk. if untreated, the compression caused by this abnormal anatomy frequently results in damage to the popliteal artery which may give rise to an occlusion,8 making timely diagnosis and management essential.4	popliteal artery entrapment syndrome (paes) is an uncommon condition resulting from an abnormal anatomic relationship between the popliteal artery and the surrounding musculature. the compression created by this variance in anatomy can lead to ischemia and vascular claudication. the diagnosis of paes requires a thorough patient history and physical exam, a high index of suspicion, and dedicated imaging techniques. several treatment options are available, including surgical intervention, thrombolysis, or a combination of these depending on the clinical indication. we present a case of paes in a 34-year - old man who presented with typical symptoms illustrative of the complicated decision making related to this disorder.
growing evidence supports that post - acs depression is an independent risk factor for future cardiac events and mortality [24 ], and females are disproportionately affected. an estimated 7.5 million females live with a history of acs and up to 47% of females report symptoms of depression when screened for depression during hospitalization for acs. depression is the most common mental illness in females and females have twice the risk of major depressive disorder (mdd) as compared with males [7, 8 ]. data from national comorbidity survey (ncs) supports the gender difference life time risk for mdd (21% in females and 13% in males). population studies from canada, germany, and switzerland all report that females are at least twice as likely as males to suffer from mdd. in addition to the gender difference in prevalence of depression episodes, the depression symptoms that differ between males and females are heterogeneous with complex clusters of depressive symptoms [13, 14 ]. data from large epidemiological studies reveals that males and females often report distinct differences in the self - reported somatic depressive symptoms of depression [1517 ]. somatic depressive symptoms include sleep disturbance, appetite disturbance, and fatigue for at least two weeks [1517 ]. silverstein interviewed college students and found that female students had higher somatic depressive symptoms than male students. self - reported data from ncs also indicated that females reported twice the frequency of somatic depression symptoms as compared to males. although higher rates of depression have been reported in patients with acs, no known studies have specifically evaluated gender differences in specific depressive symptoms reported during the acs event. the purpose of this study was to examine the gender differences and prevalence of self - reported depressive symptoms and self - reported somatic depressive symptoms, as measured by the beck depression inventory ii (bdi - ii), in patients hospitalized for acs. this was a cross - sectional observational study with a convenience sample of 789 adults (248 females and 541 males) hospitalized for acs. the study included all participants meeting the inclusion criteria of a larger study looking at the interactions of genetics and depression on acs outcomes. the overall rate for males verses females was consistent with the gender ratio for acs admitted to the facility. participants were recruited during hospital admission for acs and were screened for self - reported depressive symptoms, by trained research nurses, using the bdi - ii. depression was defined as a bdi - ii score of 14 indicating at least moderate symptoms of depression. the study was approved by the university 's committee for the protection of human subjects internal review board. after informed consent was obtained, by trained research nurses, the bdi - ii was administered when the patients were stable enough during their hospital stay to answer the questions (13 days after hospital admission). inclusion criteria consisted of a confirmed diagnosis of acs and 18 years of age or older. acs was defined as chest pain or symptoms suggestive of acs lasting for more than 15 minutes with new transient or persistent st - wave ischemic electrocardiogram (ecg) changes and ultimate disposition as (1) acute myocardial infarction (ami), including st - wave elevation infarction and non - st wave elevation infarction or (2) unstable angina. the bdi - ii is a reliable and well - validated 21-item depression screening scale that uses a forced - choice 4-alternative response format that has been used in the acs population. the bdi - ii has been used for 35 years to identify and assess depressive symptoms and reports high reliability (.80) regardless of the population [21, 22 ]. the construct validity of bdi - ii has been established and has shown to be able to differentiate between depressed and non - depressed individuals. higher coefficient alphas (.92 for outpatients and.93 for college students) have been reported for the bdi - ii when compared to the previous version of the beck depression inventory (bdi). test - retest reliability was studied with baseline data obtained on 26 outpatients and therapy sessions one week apart (correlation.93, p < 0.001). the internal consistency of the bdi - ii in patients with acs has an alpha value of 0.87. bdi - ii scores of 14 indicate a moderate level of depressive symptoms [20, 24 ]. this cut - off score has successfully been used to screen patients hospitalized for acs (odds ratio, 7.82 ; 95% ci, 2.42 to 25.26 ; p = 0.0002) and the internal consistency of the bdi - ii in patients with acs has an alpha value of 0.87. somatic depressive symptoms were measured using bdi - ii question 16 (sleep disturbance), question 18 (appetite disturbance), and question 20 (fatigue). a patient was identified as positive for somatic depressive symptoms when the patient rated all three items as 1 or above. if a patient rated 0 on any of the three items, he or she was categorized as negative for somatic depressive symptoms. pearson chi - square tests for independence (categorical variables) or t - tests for independent samples (continuous variables) were used to examine differences in demographic characteristics between males and females. comparisons of depression ordinal scores and symptoms by gender were conducted with wilcoxon rank sum and pearson chi - square tests for independence, respectively. degrees of freedom for the pearson chi - square test were defined as follows : (r 1) (c 1), where r = the number of rows and c = the number of columns. significance level was set at p < 0.05. the sample consisted of 789 adult (248 females and 541 males) patients admitted with acs (table 1). a total of 88 (35%) females and 120 (22%) males had bdi - ii scores of 14 or higher. the majority of the patients were caucasian males (64%), with a mean age of 61.35 years (s.d. 27% for males, p < 0.0001), lived alone (25.5% for females and 17.6% for males, p = 0.0098), and were less physically active (35% for females and 24% for males, p = 0.0002) than males. more than half of females in the study had less than high school education (52%, p < 0.0001), while the majority of males had at least some college or college degree (60%). females were more likely to currently be taking antidepressants (21.3%) than males (10.57%) (p < 0.0001) and significantly more females (31.7%) than males (16.3%) (p < 0.0001) reported a history of depression diagnosis or treatment. results showed that depression during acs episodes are different between males and females. significantly more females (43.5%) reported feeling depressed over the past year than compared to males (27.2%). during admission for acs, the average bdi - ii scores were significantly higher for females (mean = 11.89, s.d. = 7.93) (p < 0.0001) indicating that the females reported greater depressive symptoms. females reported significantly more sadness, self dislike, crying, indecisiveness, sleep disturbance, appetite disturbance, and loss of interest in sex than males (table 2). significantly more females (7.66%) were identified as positive for somatic depression symptoms (reported 1 or higher scores on all three questions) than males (2.22%) (p = 0.0003). the prevalence of depression in this study was significantly higher in the females (35%) than males (22%) (bdi - ii scores 14). of the 21 depressive symptoms evaluated by the bdi - ii, 19 depressive symptoms were reported more frequently by females ; 7 out of the 19 symptoms reaching statistical significance (p < 0.05). although our results support the increase in somatic symptoms in females (sleep and appetite disturbances), as reported by wenzel, self - reported symptoms of fatigue were not statistically different between males and females. the sample was a convenient sample largely made up of caucasians with a high number of males, and participants were recruited from two large tertiary medical centers with high levels of acuity. although similar patterns of somatic depressive symptoms have been reported from several epidemiological studies [1517 ], other comorbid psychiatric, medical conditions, and treatments that may affect somatic symptoms should be considered. some of the self - reported somatic symptoms may have been caused by the current acs event. despite these limitations, results from this study raise the possibility that females may have heterogeneous symptomology with various depression subtypes that may affect their morbidity and mortality after acs events. in conclusion, results from the present study support the hypothesis that there are gender differences in depression prevalence and self - reported somatic depressive symptoms, in patients hospitalized for acs. somatic symptoms may be important indicators of depression among acs patients, especially female patients. the ability to diagnose and treat depression in these patients is an important intervention that would decrease subsequent acs events. future studies are needed to identify the biological and psychological mechanisms to explain this gender difference.	this study examined the prevalence of self - reported depressive symptoms and the self reported somatic depressive symptoms as measured by the beck depression inventory - ii (bdi - ii) among patients hospitalized for acute coronary syndrome (acs), and explored the impact of gender on both. a convenience sample of 789 adults (248 women and 541 men) was recruited for the study during hospital admission for acs and participants were screened for self - reported depressive symptoms. bdi - ii scores of 14 indicate a moderate level of depressive symptoms and this cut - off score was used to categorize patients into depressed and non - depressed groups. pearson chi - square tests for independence (categorical variables) and t tests for independent samples (continuous variables) were used for gender comparisons. results showed that depressive symptoms during acs episodes were different between women and men. women reported greater overall depressive symptoms (bdi - ii mean = 11.89, s.d. = 9.68) than men (bdi - ii mean = 9.00, s.d. = 7.93) (p < 0.000). significantly more women (7.66%) were identified positive for somatic depressive symptoms (sleep and appetite disturbances and fatigue) than men (2.22%) (p = 0.0003). findings support that there are gender differences in depressive symptoms experienced by patients hospitalized for acs. somatic symptoms of depression may be important indicators of depression especially among female acs patients.
since its introduction in 1968, endoscopic retrograde cholangiopancreatography (ercp) has become a very common procedure for the evaluation and treatment of biliary and pancreatic diseases. sphincterotomy is used mainly to remove biliary stones, drain the biliary tree, and the placement of stents in the common bile and pancreatic duct (1). however, the rate of complications, such as pancreatitis, bleeding, cholangitis, and perforation has been reported to range from 4% to 30% (2). duodenal injury was reported to have an incidence of 0.3% to 1.3% (35) with a relatively high mortality rate of 16% to 18% (6). in other studies, the rate of mortality after ercp was reported between 1.0% to 1.5% (7, 8). the incidence of post - ercp pancreatitis (pep) varies from 1%up to 7% (913), it may exceed 25% in very high risk populations (14). the majority of cases are due to papillotomy, whereas intraperitoneal perforations are less common and caused by the endoscope itself (5)., some authors classified ercp - related perforations according to the site and mechanisms of injury and suggested management guidelines accordingly (3, 6, 15). however, despite these guidelines, the classification of perforations remains poorly defined and the management of ercp - related perforation still remains controversial. the aim of this study was to investigate our findings and treatment outcomes in patients with ercp - related complications. between april 2006 till july 2010, 2447 ercp procedures with or without sphincterotomy were performed at the taleghani hospital, in tehran, iran. a retrospective review of ercp - related duodenal perforations was conducted to identify their incidence, management, and clinical outcome. medical records were reviewed for the following data : patient demographics, ercp indications, clinical presentation after the suspected perforation, diagnostic laboratory and radiologic studies, intraoperative findings, length of hospital stay, clinical course and final outcome. 2447 ercp procedures were performed in our hospital, of which 65% were therapeutic and 35% were diagnostic. complications developed in 168 (7%) cases : pancreatitis in 154 (6%), hemorrhage in 4 (0.16%) and duodenal perforation in 10 (0.4%). the patients mean age was 66 6 years with females / males : 1432(58%)/ 1015(42%). abdominal pain, nausea, leukocytosis and hyperamylasemia were most common findings in these patients (table 1). ercp indications are showed in table 2. clinical and paraclinical findings in patients after ercp according to complications pr : pulse rate, rr : respiratory rate, ot : oral tempreture, abd. tend : abdominal tenderness, cxr : chest x - ray, axr : abdominal x - ray ercp indications and patient 's data in each group meanstandard deviation sod : sphincter of oddi dysfunction 154 cases (6.29%) suffered from mild to severe pancreatitis. on examination, most patients with pep had abdominal tenderness, usually localized to the epigastric and periumblical regions, without rebound tenderness or abdominal distention. patients were treated with intravenous fluids and broad - spectrum intravenous antibiotics such as imipenem, ceftriaxone, and metronidazole (even if patients were afebrile). one case was undergone computerised tomography (ct) guided percutaneous external drainage because of pancreatic necrosis. the mean length of hospital admission was 114 days with 3.5 1 day on an intensive care unit (table 3). descriptive statistics of patient 's data (n=2447) pr : primary repair, d : drainage, gi : gastrointestinal, ab : antibiotic, ed : external drainage, ob : observation 10 cases (0.4%) had duodenal perforation. on examination all had abdominal tenderness at epigasteric and/or periumbilical with rebound tenderness and abdominal distention. patients were kept nil by mouth for bowel rest and treated with intravenous fluids, antibiotics and analgesia. four cases with duodenal perforation had no surgical procedures and were treated by conservative management. they had no any rebound tenderness or free air in chest x - ray. in this group, all of them were discharged in good condition without any complication. in the surgically treated group there was bile collection at laparatomy in two cases and 1 - 2 cm perforations were found in lateral wall of duodenum after kocher 's maneuver. surgical procedures included duodenal primary repair and drainage, common bile duct exploration and choledocho - duodenostomy (n=1). patients were admitted to intensive care unit for 3 1days and then discharged on the 72 postoperative days. generally, ercp is regarded as a safe procedure in the hands of experienced gastroenterologists. however, the rate of major complications approaches 10%, with bleeding, pancreatitis, cholangitis, and perforation among the most commonly reported serious complications. the overall mortality rate of the procedure is approximately 1% to 1.5% (7, 8), but in our study there was no any mortality. careful patient selection combined with skilled cannulation minimizes complications and higher - risk procedures should be performed in specialist centers. our reported rates of pep are within the range of other cohort studies and randomized, clinicaltrials (1621) perforation of the duodenum is a major complication that may lead to great mortality if left untreated. as a serious complication after ercp, perforations have been reported in some series to occur in 0.35% to 2.1% of patients (3, 4, 8) and it was 0.28% in our study. ercp - related perforations are diagnosed more frequently by experienced endoscopists, because of either contrast extravasations or appearance of retro-/intraperitoneal air during the procedure. there are some reports that scope - related perforations presented in gastric, esophageal, and lateral wall of duodenum and jejunum tend to be large and remote from the ampulla and require immediate surgery (3, 6). in our series, early surgery, with primary repair the surgical procedure selected is based upon the mechanism and degree of injury and patient 's condition. some authors performed pyloric exclusion and gastrojejunostomy in patients with duodenal perforation and failed conservative management (6). options include choledochotomy with stone extraction and t - tube drainage, repair of the perforation, drainage of abscess or phlegmon, choledochojejunostomy, or pancreatoduodenectomy (22, 23). whereas, duodenal diversion has been reported frequently in patients with peri - vaterian perforations and those operated on late (6). in our study none of the patients were treated with pyloric exclusion, gastrojejunostomy and duodenal diversion. the decision to treat patients with primary repair appears justified given the favorable outcomes that we reported. if the physical examination after ercp is indicative of perforation, then a decision regarding operative or nonoperative treatment must be made. clinical decisions can be supported, but not guided by invetsigations such as an erect chest x - ray, supine and upright abdominal radiographs and an abdominal ct scan in patients with severe pancreatitis if their symptoms and clinical parameters worsen. the extent of the operation was proportional to the degree of injury and the intra - abdominal contamination. the two basic principles of our surgical procedures were : a) primary repair of the perforation site and b) extensive drainage and/or debridement. our patients hospital stay were significantly less than another studies (less than11 days versus 31days in ji hun kim study (24) and 21 days in dimitrios v. avgerinoss study (25) and mean icu station was 3.66 days vs. 9 days of their studies (24, 25). in conclusion, patients should be kept fasting while receiving hydration, nasogastric suction and/or intravenous antibiotics. percutaneous drainage may be an alternative to surgical drainage in patients who develop retroperitoneal collections. surgery should be recommended for patients with toxic condition, peritonitis signs, abdominal distention and free air in chest x - ray.	aimthe aim of this study was to analyze clinical findings and treatment outcomes of patients with endoscopic retrograde cholangiopancreatography complications.backgroundendoscopic retrograde cholangiopancreatography has become a very common procedure for the evaluation and treatment of biliary and pancreatic diseases.patients and methodsa retrospective review of 2447 endoscopic retrograde cholangiopancreatography procedures and their complications since apr 2006 till dec 2010 was conducted to identify their incidence, optimal management, and clinical outcomes.results2447 endoscopic retrograde cholangiopancreatography procedures were performed. overall, complications developed in 168 (6.9%) cases : perforation in 10 (0.4%), hemorrhage in 4 (0.16%) and mild to severe pancreatitis in 154 (6.3%). the patients mean age was 66 6 yrs with females/ males of 1432(58.5%)/ 1015(41.5%). abdominal pain, nausea, leukocytosis and hyperamylasemia were most common findings in these patients. surgery was performed for 6 patients (0.24%). the most hospital station was 20 days : surgical group 72 days, pancreatitis 11 4 days and average 6 days for others.conclusionendoscopic retrograde cholangiopancreatography remains the endoscopic procedure that carries a high risk for morbidity and or mortality. the majority of events are of mild - to - moderate severity and when surgery should be done, it depends upon the clinicopathological condition and we do n't advise pyloric exclusion, gastrojejunostomy and duodenal diversion for these patients.
brain metastases are the most common type of intracranial neoplasm, and lung cancer is the most frequent neoplasm of brain metastases. non - small cell lung carcinoma (nsclc) has a 2040% chance of developing brain metastases in the disease course. whole - brain radiotherapy or platinum - based chemotherapy have been the standard therapy choices for patients with brain metastases, but the prognosis of patients with brain metastases is still disappointing. penetration of chemotherapeutic drugs into the central nervous system (cns) is limited primarily by the blood - brain barrier (bbb). gefitinib is an oral agent and epidermal growth factor receptor (egfr) tyrosine kinase inhibitor (tki), which has been reported to be effective in the treatment of brain metastases from nsclc by overcoming the bbb. an egfr mutation is a predictive biomarker for a good response to egfr - tki, even in brain metastases. here, we report the case of a woman with diffuse brain metastases from lung cancer who experienced total regression of the metastases under gefitinib treatment. the patient, a 58-year - old married taiwanese woman, was referred to our hospital with a complaint of severe headache and unsteady gait for 2 months. the patient had had a 10-year history of hypertension and a 4-year history of hyperthyroidism with regular follow - up. physical examinations revealed tenderness over her posterior neck and drowsy consciousness, with a glasgow coma scale score of e2, v4, m4, and an ecog performance status of grade 4. on admission, the patient underwent a brain mri which revealed diffuse metastatic lesions of variable sizes in the cerebra and cerebellum (fig. a chest x - ray revealed a focal mass in the left infrahilar region, and a chest ct scan showed a lobulated mass, measuring 3.5 2.1 cm, in the superior segment of the left lower lobe (lll) of the lung with pleural effusion (fig. therefore, adenocarcinoma of the lll portion of the lung, with brain and bone metastases, ct2n3m1, stage iv was diagnosed. an analysis of the egfr mutation elucidated a glu746_ala750 deletion in exon 19 (fig. 3). the patient received egfr - tki therapy with gefitinib 250 mg per day. her subjective symptoms improved gradually within 1 month of gefitinib therapy. after 6 months of treatment, a chest ct scan revealed partial remission of the primary pulmonary tumor, measuring 3.0 1.5 cm (fig. a brain mri showed the dramatic total regression of the diffuse brain metastatic lesions (fig. currently, the patient continues to receive egfr - tki therapy, and her survival time has exceeded 18 months since the initial diagnosis. so far, the patient has had a good ecog performance status of grade 1 and no obvious recurrence elsewhere. nsclc with brain metastases is usually associated with a poor outcome, and treatment is palliative in most cases. multiple brain metastases are associated with an even worse prognosis, and median overall survival is only a few months. standard treatment options include symptomatic therapy with whole - brain radiotherapy and corticosteroids, which lead to a median survival of 36 months. the use of conventional chemotherapy for brain metastases has been limited because of a presumed lack of effectiveness due to poor penetration beyond the bbb. the bbb is a highly specialized structure of astrocytes, pericytes, and vascular endothelium that maintains cns homeostasis. in a mouse model, the bbb has already been found to be permeable to brain metastases of tumors < 0.25 mm in diameter. ct scans and mri have detected edema, and contrast enhancement in brain metastases has confirmed the disruption of the bbb, which is no longer intact when brain metastases become clinically detectable. further studies show that egfr - tki has a low molecular weight and could penetrate beyond the bbb. therefore, it might be efficacious in the treatment of brain metastasis [5, 6 ]. many studies have proposed several factors which could make it possible to predict the response to gefitinib in patients with advanced nsclc, for example, asian female, non - smoker, with adenocarcinoma, and the presence of an egfr mutation. to our knowledge, the most common types of egfr mutation are an exon 19 deletion and an exon 21 l858r mutation. in our case, an asian female had adenocarcinoma of the lung with diffuse brain metastases and was treated with gefitinib therapy because of an egfr exon 19 deletion. after 6 months of follow - up, the dramatic response of total regression of the brain metastases was proved by brain mri. as of now, our patient has survived more than 18 months since the initial diagnosis. in conclusion, therefore, due to its ability to reverse poor performance status and achieve the unbelievable response of total regression of brain metastases, egfr - tki therapy could be considered as a first - line treatment for advanced adenocarcinoma of the lung, positive for an egfr mutation, with brain metastases.	we report the case of a woman with diffuse brain metastases from lung cancer who experienced total regression of the metastases under gefitinib treatment. the 58-year - old woman was referred to our hospital with a complaint of severe headache. a brain mri revealed diffuse metastatic lesions in the cerebra and cerebellum. adenocarcinoma of the lung with multiple brain metastases was diagnosed. the tumor was positive for an epidermal growth factor receptor (egfr) exon 19 deletion mutation. she was treated with gefitinib 250 mg per day. one year later, the diffuse brain metastases had totally resolved. egfr - tyrosine kinase inhibitor therapy could be a first - line treatment for patients with advanced adenocarcinoma of the lung with egfr mutation, especially in those with brain metastases.
anaemia is a common morbidity in the neonatal intensive care unit (nicu) and this arises predominantly from frequent iatrogenic blood draws necessitated by the need for laboratory investigations and monitoring. the high prevalence of haemolytic conditions in nigeria has also contributed to increased neonatal transfusion rates in the country [1, 2 ]. there are different formulas for estimating volume of required packed cells with varying degrees of accuracy [35 ]. this may result in variability in the correlation between the volume of transfused blood and posttransfusion packed cell volume (pcv). following red blood cell transfusion in stable patients, haematocrit equilibration occurs over time before a stable pcv level is reached. knowledge of the duration of this equilibration process is pertinent in the accurate timing of posttransfusion pcv check which should be done at the earliest possible time to avoid needless prolongation of hospital stay as well as delay in completing packed cell replacement in those whose posttransfusion pcv may fall below the expected values. although there is no consensus on the most appropriate timing of posttransfusion pcv, some studies have suggested early (2% (0.6 g / dl) were assumed to be unstable (ongoing blood loss or haemolysis). babies who required whole blood transfusion as well as those who died before completing the posttransfusion pcv measurements were also excluded. in our unit, furosemide is not routinely administered during red cell transfusion except for babies with clinical evidence of heart failure or fluid retention. the ethics committee of the hospital granted ethical approval for the study while parental consent for participation in the study was obtained from the mothers before commencement of study. for every eligible patient, relevant sociodemographic maternal and neonatal data were documented. the volume of packed cells to be transfused was calculated as v (ml) = 3 wt (kg) (expected change in haemoglobin.). this volume was given as a continuous infusion using an infusion pump or given in aliquots over a period of 3 - 4 hours. capillary blood samples were then obtained at 1 hour, 6 hours, 12 hours, 24 hours, and 48 hours after completion of transfusion by our residents for determination of pcv using biofuge.haemo centrifuge (heraeus instruments) at 10,000 revolutions per minute. mean (sd) values were computed for pre- and posttransfusion pcv at the specified periods and, using the student t - test, levels of statistical significance (p values) were generated for the differences in the mean values of pcv. levels of significance were also computed for the difference between the target posttransfusion pcv and the mean pcv observed at the specified times. forty - seven infants who met the inclusion criteria were prospectively recruited into the study. seven of them were excluded from analysis because of incomplete data (missing posttransfusion pcv results). these consisted of neonates and young infants aged 1 to 91 days (22.7 20.6), with a gestational age range of 26 weeks to 40 weeks. twenty - six babies (65%) had blood transfusion within the neonatal period while most of the babies with anaemia of prematurity were transfused beyond the 28-day postnatal age. eighteen babies (45%) had anaemia of prematurity while 7 had haemolytic anaemia associated with jaundice (abo isoimmunization = 6, sepsis = 1). the pretransfusion pcv was 32.1% 6.2% and this was significantly lower than the 1-hour posttransfusion pcv (48.5% 5.5%, p = 0.0000). as shown in table 2, 1-hour posttransfusion pcv was similar to the 6-hour posttransfusion pcv (47.8% 5.6%) p = 0.516, but both were significantly different from the 12-hour, 24-hour, and 48-hour posttransfusion pcvs. the 12-hour posttransfusion pcv was similar to the 24-hour and 48-hour posttransfusion pcvs (p = 0.237 and 0.063, resp.). the difference between the 1-hour posttransfusion pcv and the 1248-hour pcv varied from 1.3% to 3.5% (0.4 the mean 1-hour and 6-hour posttransfusion pcvs were significantly different from the target posttransfusion pcv (p = 0.048 and 0.046, resp.). however the 12-hour, 24-hour, and 48-hour posttransfusion pcvs were similar to the posttransfusion target pcv (p = 0.157, 0.495, and 0.683, resp.). figure 1 is an illustration of the changes in pcv following packed red cell transfusion. the immediate rapid increase (16 hours) was followed by a gradual drop in 612 hours and maintenance of a steady state between 12 and 48 hours. our study shows the expected immediate rise in pcv posttransfusion agreeing with results of previous studies. although this significant difference between the pre- and posttransfusion pcv was sustained throughout the period of observation (48 hours), the change in pcv posttransfusion followed a unique pattern which suggests that the equilibration process that probably began while red blood cell transfusion was ongoing terminated at about 12-hour posttransfusion beyond which no significant difference in pcv was noted. transfused packed red blood cells contain extracellular electrolytes (sodium, potassium, chloride, and bicarbonates) which contribute some solute load 34 mmol / l to the extracellular space, resulting in the movement of water from the intracellular compartment into the extracellular compartment. this may account for the significant drop in pcv values observed between 6 hours and 12 hours after transfusion in our study. we chose stable babies for this study to ensure elimination of the confounding effect of ongoing blood depletion (haemorrhage / haemolysis) on the equilibration process. the results from our study would suggest that pcv check could be done 12 hours after completing red blood cell transfusion in neonates. we considered the effect of repeated posttransfusion blood draws on the declining pcv from 1 hour to 48 hours. the average blood volume per capillary draw is 0.07 ml and 0.35 mls for 5 samples. the mean weight of babies in the study group was 3.3 kg. given that 6 mls / kg of whole blood transfused to a baby increases the haematocrit by about 3%, 0.35 mls/3.3 kg of blood drawn from baby for pcvs will expectedly produce a deficit of 0.05% in pcv from 1-hour posttransfusion to 48-hour posttransfusion. in our study the difference in pcv from 1 to 48 hours after transfusion was 2.7%, which exceeded the deficit estimated from the blood draws. while a coefficient of variation of about 7% has been reported for capillary pcv, some of the precautions we took while taking the samples (avoiding squeezing out blood from prick site and ensuring babies were in supine position) would necessarily limit the changes in body fluid distribution that may partly account for this variability. although elzik. demonstrated that pcv at 15 minutes and 6 hours after red cell transfusion in stable neonates were similar and both were significantly different from pretransfusion pcv, it is difficult from their study design to conclude that haematocrit equilibration process had been completed at 6 hours since they did not check pcv levels beyond 6 hours. similarly, adedoyin. reported that 1-hour and 7-hour posttransfusion haemoglobin differed only by 0.2 g / dl but there were no estimated pcv levels after 7 hours. our study showed a posttransfusion haemoglobin difference of 0.4 g / dl between 1 hour and 12 hours, increasing to 0.9 g / dl by 24 hours. saugel. reported a significant immediate and 2-hour increase in haematocrit among patients in icu who received red blood cell transfusion. pcv check was, however, not repeated beyond 2 hours and the study, which was conducted among adult patients, was not designed to measure the duration of posttransfusion haemoglobin equilibration. this was however not the case with the findings of elizalde. who did posttransfusion pcv on 32 adults with normovolemic anaemia following bleeding episodes. posttransfusion samples were taken at 15 min, 30 min, 60 min, 120 min, and 24 hours for pcv and no difference was found in the values, suggesting rapid haematocrit equilibration. our study was carried out on neonates whose haemodynamic responses to transfusion may differ from those of adults. while the differences in the duration of posttransfusion haematocrit equilibration process between our study and previous reports may not be easily explained, it can be speculated that the difference in the age group of patients in the various studies may be partly responsible. furthermore, the duration of blood transfusion and the use of diuretics before or after transfusion may affect the haematocrit equilibration process. a large multicenter age - stratified study, specifically designed to analyze post - rbc transfusion haematocrit equilibration, would be required to resolve these differences. we did not check the pcv in the interval between 6 and 12 hours after transfusion. doing that for this study would have considerably increased the number of times each baby was pricked for capillary blood sampling. we believe that this can be addressed in further studies specifically designed to check posttransfusion pcv values between 6 hours and 12 hours after red blood cell transfusion. a pilot study to determine the natural coefficient of variation for capillary pcv would have been quite helpful. our study has shown that, in stable nonhaemorrhaging and nonhaemolysing neonates, haemoglobin equilibration that probably begins during the period of transfusion ends by 12 hours after transfusion. although the limited sample size and the seemingly marginal differences in the values of pcv between 1 hour and 12 hours after transfusion may limit the clinical application of our findings, we suggest that posttransfusion pcv be checked by 12 hours and recommend further studies involving larger sample sizes for further validation.	anaemia is a common morbidity in the nicu and often requires transfusion of packed red blood cells. haematocrit equilibration following red cell transfusion occurs over time ultimately resulting in a stable packed cell volume (pcv). knowledge of this equilibration process is pertinent in the accurate timing of posttransfusion (pt) pcv. we conducted a prospective study to determine an appropriate timing for pt pcv estimation on 47 stable anaemic babies at the neonatal unit of national hospital, abuja. values of pcv were determined before transfusion and at 1, 6, 12, 24, and 48 hours posttransfusion. forty of the recruited neonates and young infants were analyzed. their gestational age range was 26 to 40 weeks. 1-hour pt pcv (48.5% 5.5%) was similar to the 6-hour pt pcv (47.8% 5.6%) p = 0.516, but both were significantly different from the 12-hour (46.8% 5.9%), 24-hour (45.9 5.8%), and 48-hour (45.4% 6.2%) pt pcvs. the 12-hour pt pcv was similar to the 24-hour and 48-hour pt pcvs (p = 0.237 and 0.063, resp.). we concluded that, in stable nonhaemorrhaging and nonhaemolysing young infants, the estimated timing of haematocrit equilibration and, consequently, posttransfusion pcv is 12 hours after red blood cell transfusion.
methotrexate (mtx) is the most common second - line therapeutic agent used to treat juvenile idiopathic arthritis (jia) worldwide. regardless of age or disease subtype, considerable interindividual variability in clinical response and adverse reactions exists with mtx, and thus far, there have been no predictive variables for outcomes in patients taking this medication. since the onset of clinical response may take months to manifest, the risk to benefit ratio early in treatment is altered, as there is risk for toxicity for several weeks to months before knowing if the medication has resulted in a clinical benefit. side effects may compromise efficacy due to patient noncompliance, clinician dose adjustment, or discontinuation even if the drug eventually is medically effective. medication dose alteration or discontinuation in the face of active disease is unacceptable when the alternatives for therapy in childhood arthritis are few and poorly studied in this population. by utilizing pharmacogenomic principles and a personalized therapeutic strategy, we hope to improve efficacy and prevent adverse drug reactions in children taking mtx to treat jia. when exploring the variability in response and toxicity to any medication used in children, a concept often overlooked is ontogeny. the effects of development can be applied at every level of drug disposition and response. these effects range from differences in gastric ph [2, 3 ] and gastric emptying which may affect absorption, to changes in circulating plasma proteins with age that may affect drug distribution. developmental changes in phase i drug biotransformation and phase ii conjugating enzyme expression have the potential to alter drug metabolism, and developmental differences in glomerular filtration rates will affect drug excretion in children compared to adults. common drug biotransformation pathways are also shared with endogenous compounds involved in growth and development, such as testosterone, cortisol, and vitamin d3, so it is not surprising that some of these pathways may be affected by the rapid growth and maturation of the pediatric patient, for example, during infancy and puberty. the developmental expression of these pathways at different rates or trajectories may also lead to variability in drug disposition and response. although pharmacogenetics appropriately strives to identify the correct dose of the correct drug for the correct person, the impact of development on an individual 's response to a drug must be taken into account. genotyping an individual for variations that affect function is an important step to understanding variability in outcomes ; however, knowing if and when that gene is expressed is a concept important to fully understanding genotype - phenotype relationships in children [9, 10 ]. an approach to investigating hypotheses related to drug outcomes in children can be guided by the following questions. what gene products are quantitatively important in the disposition (absorption, distribution, metabolism, and excretion) of the drug in question ? for each gene product, what is the developmental trajectory for the acquisition of functional activity ? is allelic variation in the gene(s) of interest associated with any functional consequences in vivo ? is there any evidence that allelic variation affects the developmental trajectory of the drug disposition phenotype ? what is the developmental context in which the gene(s) of interest is / are operating ? this process is also relevant for genes / gene products involved in drug response. partnered with the understanding of genetic variation in an individual, appreciating the changes in gene expression throughout growth and development will allow us to manage the complexity of therapeutics in children. we strive to individualize therapy for children rather than extrapolate from adult experience, which traditionally has been the norm. juvenile idiopathic arthritis (jia), formerly termed juvenile chronic arthritis (jca) or juvenile rheumatoid arthritis (jra) is one of the most common chronic diseases of childhood, and is an important cause of morbidity and disability in children. this disease is characterized by idiopathic peripheral arthritis with an immunoinflammatory pathogenesis, thought to be triggered by an external antigen. there is also speculation of a genetic predisposition for the disease [1215 ]. jia has a heterogeneous phenotypic expression, and includes several disease subtypes, whose classification continues to be revised and validated by clinicians worldwide. despite differences in disease expression between adults and children, like in many pediatric diseases, children are treated with generally the same armamentarium of drugs used to treat ra in adults. with the advent of disease modifying antirheumatic drugs (dmards), the philosophy of treatment changed from simple pain control to prevention of erosions and long - term damage of the joints. methotrexate (mtx), a folic acid antagonist, was approved by the food and drug administration for the treatment of ra in 1988, and several uncontrolled descriptive studies suggested, and a randomized placebo - controlled double blind clinical trial demonstrated the effectiveness of mtx in children with jia [1621 ]. mtx has subsequently become the most common second - line therapeutic agent used to treat juvenile idiopathic arthritis (jia) worldwide. although the collective clinical experience with mtx has been vast, there are still unanswered questions about its mechanism of action, and considerable interindividual variability in clinical response and adverse reactions exists [2224 ]. thus far, there have been few predictors for efficacy or toxicity in pediatric patients taking this medication, and clinicians essentially dose by trial and error. factors that could contribute to this variability are extensive, and some are unique to the pediatric population. we would like to explore the potential sources of variability that may contribute to outcomes on mtx in jia. methotrexate, a folic acid analog and potent inhibitor of several enzymes within the folate pathway, has been used in low doses for the treatment of rheumatic disease over the last several decades. for rheumatic conditions, the dose range in pediatrics spans 10-fold, ranging from 0.1 mg / kg / dose to 1 mg / kg / dose, administered on a weekly basis. options include oral and subcutaneous administration, but intramuscular and intravenous administration are possible, although less practical in the outpatient setting. before being taken into the body, contributors to variability that can not be overlooked include patient compliance, differences in administered dose, and route of administration. children, who are a fraction of the weight of their adult counterparts, are dosed with the same absolute mtx dose, despite their smaller size. although attention has been brought to this phenomenon, there continues to be little understanding of why children appear to require, relative to body weight, higher doses of mtx or how these doses are tolerated. serum mtx concentrations have not been found useful to predict response or toxicity with little correlation with dose or outcome [2527 ]. it is known that mtx acts as a folate antagonist, entering the cells primarily through the reduced folate carrier (rfc / slc19a1). once intracellular, mtx is bioactivated to a polyglutamated form by folylpolyglutamyl synthase (fpgs), which enhances the pharmacological activity and intracellular retention of mtx. in the ra and pediatric oncology literature, current evidence indicates that the enzymatic addition of glutamate residues to the mtx molecule in vivo (polyglutamation / mtxglun) is critical for pharmacologic activity by increasing the intracellular concentration of the drug and increasing its affinity for its therapeutic targets, thereby allowing more opportunity for its inhibitory effects to be exerted upon its target enzymes [2931 ]. the initial target of mtx to be identified was dihydrofolate reductase (dhfr), which forms tetrahydrofolate, a precursor required for one carbon donation for synthesis of thymidylate, purines, methionine and serine, remethylation of homocysteine to form methionine, and provision of methyl donors for multiple methyltransferase enzymes. inhibition of thymidylate synthetase (tyms), both directly and indirectly via depletion of tetrahydrofolate, leads to inhibition of pyrimidine biosynthesis with a resultant antiproliferative effect. the interruption of dna synthesis was thought to be the basis for rapidly dividing cell death in cancer cells. subsequently, the list of target genes has been extended to include amino - imidazole carboxamide ribonucleotide (aicar) transformylase (gene name, atic), which inhibits de novo purine synthesis and promotes the accumulation of aicar ribotide, inhibiting adenosine deaminase and leading to a build up of adenosine, a potent anti - inflammatory agent [32, 33 ]. adenosine 's effect is also mediated by adenosine a2 receptors (adora2), which are present on neutrophils, monocytes, lymphocytes and basophils, generally suppressing the immune function of these cells. gamma glutamyl hydrolase (ggh), the enzyme responsible for glutamate removal from mtx, transforms mtx into a form that can be effluxed from the cell by the atp - binding cassette (abc) family of transporters. due to the rapid decline in serum drug concentrations, serum mtx concentrations are of limited utility in determining appropriate dosing or management of this medication [2527 ]. on the other hand, rbc folate concentrations are established during erythropoiesis and represent the average folate status over the preceding 120 days. by extension, mtx concentrations in rbcs represent a reasonable surrogate biomarker of average drug exposure over a similar period of time. in vitro studies have revealed that the cellular response to folate deprivation is associated with increased expression of fpgs and decreased expression of abcg2, suggesting that the adaptive cellular response to low folate involves increased polyglutamation to promote the retention of folate. homozygosity for the variant allele of slc19a1 (80a / a genotype) has been associated with increased concentrations of intracellular mtxglun compared to heterozygous or wt genotypes in ra patients. although the data are limited, these examples reveal that allelic variation in these genes resulting in increased or decreased activity may be associated with inter - individual variability in intracellular mtxglun. recent associations between mtxglun and clinical outcomes have also been reported in the adult rheumatology literature [33, 36, 37 ]. (defined in adults as mtxglu3 or greater, parent mtx is mtxglu1) were associated with a number of improved response measures in ra. the relationship between mtxglun and side effects of the medication has not been established. in children, experience is limited to a single study reporting a total of 38 jia patients, and no relationship between intracellular total mtxglun concentrations and likelihood of response was apparent. as individual mtx polyglutamates differ with respect to their inhibitory effects on target enzymes and inhibition is modulated by folate polyglutamates, it is likely that multiple as yet unidentified factors contribute to variability in the relationship between mtxglun concentrations and efficacy and/or toxicity. in order to better identify factors that may contribute to the inconsistencies in response and toxicity to mtx, we sought to characterize the extent of variability of intracellular mtxglun concentrations in our jia patient population, and to investigate variables that may contribute to mtxglun variability. we have measured intracellular mtxglun concentrations in a cohort of 104 jia patients. in this cohort, total intracellular mtxglun (mtxglutot, the sum of all individual mtxglun) concentrations varied 40-fold with a mean of 85.4 48.8 concentrations of mtxglu1 - 7 were measured individually and as a percentage of each patient 's mtxglutot. mtxglu3 was the most prominent subtype identified, comprising 42% of mtxglutot, and was most highly correlated with mtxglutot (r = 0.96). higher concentrations of mtxglu1 + 2 were observed in patients receiving oral doses of mtx, whereas higher concentrations of mtxglu3 - 5 were observed in patients receiving subcutaneous doses of mtx (p t allele resulted in much higher mtx auc in female patients. additionally, in ra patients, increased mtx toxicity was noted in subjects carrying snps in the abcb1 and abcc2 genes [64, 65 ]. variation in the function of intestinal transporters, such as pcft, may result in differences in mtx bioavailability, and may play a part in explaining why children, whose body surface area and weight are much less than adults, still require a similar mtx dose as adults to maintain an appropriate level of disease control. the combination and relation of both influx and efflux transporter function will likely need to be better elucidated before final associations with genotype and phenotype can be made. after being absorbed from the gut, mtx is further metabolized in the liver to its primary metabolite 7-oh - mtx, and influx and efflux transporters within the liver can also be contributors to interpatient variability in systemic availability and response to the drug, especially when considering the inherent risk for hepatotoxicity. the role of hepatic specific transporters such as organic anion transporter polypeptides (oatp1b1/slco1b1, oatp1b3/slco1b3) have received more attention with the reported influence of the slco1b1 5 haplotype (c.51 t > oatp1b1 is located at the sinusoidal membrane of hepatocytes, and its transcript has been detected in hepatocytes. it has been shown to transport mtx in vitro, and in pediatric patients with all, variations in oatp1b1 were associated with clearance of mtx as well as gi toxicity. variations in the genotype of hepatic efflux transporters such as abcb1/mdr-1 and abcc2/mrp-2 may also play a role in drug response and toxicity. in abcc2-deficient rats, deficient mice, a dramatic increase in mtx and 7oh - mtx concentrations are found in the liver, as well as prolonged systemic exposure to both compounds after mtx administration compared to single gene knockout mice. there is currently a knowledge deficit regarding the ontogeny of these transporters. if dysfunction in efflux transporters such as abcc2, abcc3, and abcg2 leads to prolonged systemic exposure and elevated liver concentrations of mtx, perhaps more effective or efficient function of these transporters in children allow high doses to be tolerated and hepatic toxicity to be minimal. the complexity involved in predicting the cellular effects of mtx is only amplified by the multiple steps required to complete the journey to the cellular folate cycle, and recognizing that cell- and tissue - specific differences in cellular uptake and retention processes exist. the role of ontogeny adds a layer of complexity to an already intricate network of genes. very little is known about differences in gene expression with age within the network during normal growth and development, after perturbation following administration of mtx. extensive variability in outcomes and intracellular mtxglun concentrations in our pediatric jia population, as well as the observation that children need and tolerate the same absolute doses of the drug routinely prescribed to adults, begs the question : what role does ontogeny play ? although mtx is widely used in clinical practice in a number of disease entities, at different doses and by different routes of administration there remains significant lack of understanding of its mechanisms of action and the factors that contribute to the variability in toxicity and response seen clinically. given the time lag between initiation of treatment and the first indication of patient response, this knowledge is essential to determine a priori the probability of beneficial therapeutic response and also take into consideration the probability of toxicity so that the best informed clinical decisions can be made. in addition to differences in drug administration, (i.e., dose, route, compliance) factors that affect pharmacokinetics and pharamcodynamics such as genetic variation may explain individual differences in drug biotransformation. however, the pediatric population has an additional factor to consider, namely, the ontogeny of gene expression, which may invariably affect the relative expression of genes within the pathway as one carbon resources which are allocated to the different functions of the folate cycle (purine and pyrimidine biosynthesis, homocysteine remethylation to methionine, one carbon donor for methyltransferases) during periods of dynamic change in folate supply and demand. by taking into account not only the genes in question that may affect drug disposition, but also the developmental trajectory of genes involved in drug response, we may begin to better understand what makes children different, and identify and prevent unique adverse drug reactions in this population. future areas of study in this area may include investigating the developmental expression of tissue specific transporters, which may explaining the higher rate of subcutaneous administration and the higher doses of mtx used in children compared to adults. additionally, having a better understanding of how cellular folate concentrations and patterns change with age may also help explain the variation seen in mtx response. with newer techniques and a developmentally aware approach	although methotrexate is widely used in clinical practice there remains significant lack of understanding of its mechanisms of action and the factors that contribute to the variability in toxicity and response seen clinically. in addition to differences in drug administration, factors that affect pharmacokinetics and pharmacodynamics such as genetic variation may explain individual differences in drug biotransformation. however, the pediatric population has an additional factor to consider, namely the ontogeny of gene expression which may result in variation throughout growth and development. we review the current understanding of methotrexate biotransformation and the concept of ontogeny, with further discussion of how to implement a developmental pharmacogenomics approach in future studies.
all procedures were approved by the university of washington institutional animal care and use committee. male sprague - dawley rats (charles river, ca) were pair - housed upon arrival and given ad libitum access to water and lab chow and maintained on a 12-hour light / dark cycle. recovery surgeries were performed under isoflurane anesthesia on rats weighing 300350 g (~6070 days old), after which rats were single - housed. carbon - fiber electrodes targeting the nacc (relative to bregma : 1.3 mm anterior ; 1.3 mm lateral ; 7.0 mm ventral) and a ag / agcl reference electrode were implanted for voltammetry experiments. implantation of guide cannulas for microinjection experiments targeted the vta (relative to bregma : 5.6 mm posterior ; 0.5 mm lateral, 7.0 mm ventral) and/or the ppt (relative to bregma : 8.0 mm posterior ; 1.5 mm lateral, 5.8 mm ventral). for non - recovery voltammetry surgeries, rats were anesthetized with urethane (1.5 g / kg), additional holes were drilled above the ppt (relative to bregma : 8.0 mm posterior ; 2.0 mm lateral) or the bnst (relative to bregma : 0.3 mm posterior ; 1.5 mm lateral), and only the guide cannula above the vta was cemented into place. after at least 2 weeks of recovery from surgery, rats were placed and maintained on mild food restriction (~15 g / day of standard lab chow) to target 90% free - feeding weight, allowing for an increase in weight of 1.5% per week. behavioral sessions were performed in operant chambers (med associates, vt) that had sloped floors, a house light, and contained a food tray and two cue lights above two retractable levers on a single wall. the cue lights and their corresponding levers were located on either side of the food tray. rats were exposed to pr or fr experimental sessions (one session per day) according to the schedule presented in the supplementary table, which was previously shown to elicit stable behavior and was designed to minimize inflexible behaviors by alternating the side of the active lever across sessions. this behavioral schedule also accommodated at least two days of recovery time between intra - vta pharmacological manipulations. behavioral sessions began with both levers extending, and illumination of the house light and the cue light over the active lever. completion of the correct number of lever presses led to the delivery of food rewards (45-mg food pellets, bioserv, nj), retraction of the levers, and the cue and house lights turning off for a 30-s inter - trial interval (iti). food rewards were earned on a fr4 reinforcement schedule during fr sessions that consisted of 60 trials. pr sessions were identical to fr4 sessions except that the operant requirement on each trial (t) is the integer (rounded - down) of 1.4 lever presses, starting at 1 lever press (i.e. 1, 1, 1, 2, 3, 5, 7, 10, 14, 20, 28, 40, 56, 79, 111, 155, 217, 304, 426, etc). pr sessions ended after 15 min elapsed without completion of the response requirement in a trial. rats completed at least two baseline pr sessions before the reward magnitude was changed or drugs were administered intra - cerebrally. acute stress was administered by placing the rat in a tail vein restrainer for 20 minutes. intra - cerebral injectors extended 1 mm past the end of the guide cannula, targeting a final depth below the skull surface of 8.0 mm for intra - vta injections and 6.8 mm for intra - ppt injections. infusions were performed 15 min prior to the start of the behavioral session and were visually monitored to ensure successful infusion. injectors remained in place for 1-minute post - injection to minimize backflow of the drug. injections of helical crf (500 ng in 1% acetic acid in saline vehicle) and crf (100 ng, 200 ng, 1 g, or 2 g in artificial cerebral spinal fluid) were administered into the vta in a volume of 0.5 l. intra - ppt injections of baclofen / muscimol (0.3 nmol / 0.03 nmol) were delivered in saline vehicle at a volume of 0.3 l. drugs were purchased from bachem (crf), tocris (baclofen and muscimol) and sigma (-helical crf). during experimental recording sessions in behaving rodents, the chronically - implanted carbon - fiber microelectrodes were connected to a head - mounted voltammetric amplifier for dopamine detection by fast - scan cyclic voltammetry as described elsewhere. the potential applied to the carbon fiber was ramped from 0.4 v (vs ag / agcl) to + 1.3 v and back at a rate of 400 v / s during a voltammetric scan and held at 0.4 v between scans at a frequency of 10 hz. to confirm that electrodes were capable of detecting dopamine, unexpected food pellets were delivered before and after a recording session to elicit dopamine release. chemical verification of dopamine was achieved by obtaining high correlation of the cyclic voltammogram (electrochemical signature) to that of a dopamine standard (correlation coefficient r 0.75 by linear regression). the voltammetry data for a session were not analyzed if food pellet delivery did not elicit dopamine release that satisfied the chemical verification criteria. for anesthetized experiments, dopamine release was evoked by stimulating the ppt (relative to bregma : 8.0 mm posterior ; 2.0 mm lateral, 6.57.5 mm ventral) or the bnst (relative to bregma : 0.3 mm posterior ; 1.5 mm lateral, 6.57.5 mm ventral) with a bipolar stimulating electrode (60 pulses delivered at 60 hz, 200 a). stimulations were performed every 5 min until a stable baseline for 20 min was achieved (50% of planned experiments due to technical complications. rats were excluded for analysis if cannulas did not target the region of interest, save for those illustrating the lack of an effect of crf when administered outside of the vta (supplementary fig. general motor activity was assessed using the rate of head entries into the food tray. following completion of the experimental sessions, animals were anesthetized with ketamine / xylazine (100 mg / kg) and the recording site was marked by making a small electrolytic lesion at the electrode tip by passing a current (~70 a) through the carbon fiber microelectrode for 20 seconds. animals were subsequently perfused transcardially with 4% paraformaldehyde in phosphate - buffered saline at ph = 7.4 before the brains were removed and post - fixed in the paraformaldehyde solution. the brains were then placed in 30% sucrose solution in phosphate - buffered saline for 48 hours, flash frozen, and sectioned coronally (60 m). all procedures were approved by the university of washington institutional animal care and use committee. male sprague - dawley rats (charles river, ca) were pair - housed upon arrival and given ad libitum access to water and lab chow and maintained on a 12-hour light / dark cycle. recovery surgeries were performed under isoflurane anesthesia on rats weighing 300350 g (~6070 days old), after which rats were single - housed. carbon - fiber electrodes targeting the nacc (relative to bregma : 1.3 mm anterior ; 1.3 mm lateral ; 7.0 mm ventral) and a ag / agcl reference electrode were implanted for voltammetry experiments. implantation of guide cannulas for microinjection experiments targeted the vta (relative to bregma : 5.6 mm posterior ; 0.5 mm lateral, 7.0 mm ventral) and/or the ppt (relative to bregma : 8.0 mm posterior ; 1.5 mm lateral, 5.8 mm ventral). for non - recovery voltammetry surgeries, rats were anesthetized with urethane (1.5 g / kg), additional holes were drilled above the ppt (relative to bregma : 8.0 mm posterior ; 2.0 mm lateral) or the bnst (relative to bregma : 0.3 mm posterior ; 1.5 mm lateral), and only the guide cannula above the vta was cemented into place. after at least 2 weeks of recovery from surgery, rats were placed and maintained on mild food restriction (~15 g / day of standard lab chow) to target 90% free - feeding weight, allowing for an increase in weight of 1.5% per week. behavioral sessions were performed in operant chambers (med associates, vt) that had sloped floors, a house light, and contained a food tray and two cue lights above two retractable levers on a single wall. the cue lights and their corresponding levers were located on either side of the food tray. rats were exposed to pr or fr experimental sessions (one session per day) according to the schedule presented in the supplementary table, which was previously shown to elicit stable behavior and was designed to minimize inflexible behaviors by alternating the side of the active lever across sessions. this behavioral schedule also accommodated at least two days of recovery time between intra - vta pharmacological manipulations. behavioral sessions began with both levers extending, and illumination of the house light and the cue light over the active lever. completion of the correct number of lever presses led to the delivery of food rewards (45-mg food pellets, bioserv, nj), retraction of the levers, and the cue and house lights turning off for a 30-s inter - trial interval (iti). food rewards were earned on a fr4 reinforcement schedule during fr sessions that consisted of 60 trials. pr sessions were identical to fr4 sessions except that the operant requirement on each trial (t) is the integer (rounded - down) of 1.4 lever presses, starting at 1 lever press (i.e. 1, 1, 1, 2, 3, 5, 7, 10, 14, 20, 28, 40, 56, 79, 111, 155, 217, 304, 426, etc). pr sessions ended after 15 min elapsed without completion of the response requirement in a trial. rats completed at least two baseline pr sessions before the reward magnitude was changed or drugs were administered intra - cerebrally. acute stress was administered by placing the rat in a tail vein restrainer for 20 minutes. intra - cerebral injectors extended 1 mm past the end of the guide cannula, targeting a final depth below the skull surface of 8.0 mm for intra - vta injections and 6.8 mm for intra - ppt injections. infusions were performed 15 min prior to the start of the behavioral session and were visually monitored to ensure successful infusion. injectors remained in place for 1-minute post - injection to minimize backflow of the drug. injections of helical crf (500 ng in 1% acetic acid in saline vehicle) and crf (100 ng, 200 ng, 1 g, or 2 g in artificial cerebral spinal fluid) were administered into the vta in a volume of 0.5 l. intra - ppt injections of baclofen / muscimol (0.3 nmol / 0.03 nmol) were delivered in saline vehicle at a volume of 0.3 l. all drug treatments were administered in a counterbalanced manner across pr sessions. drugs were purchased from bachem (crf), tocris (baclofen and muscimol) and sigma (-helical crf). during experimental recording sessions in behaving rodents, the chronically - implanted carbon - fiber microelectrodes were connected to a head - mounted voltammetric amplifier for dopamine detection by fast - scan cyclic voltammetry as described elsewhere. the potential applied to the carbon fiber was ramped from 0.4 v (vs ag / agcl) to + 1.3 v and back at a rate of 400 v / s during a voltammetric scan and held at 0.4 v between scans at a frequency of 10 hz. to confirm that electrodes were capable of detecting dopamine, unexpected food pellets were delivered before and after a recording session to elicit dopamine release. chemical verification of dopamine was achieved by obtaining high correlation of the cyclic voltammogram (electrochemical signature) to that of a dopamine standard (correlation coefficient r 0.75 by linear regression). the voltammetry data for a session were not analyzed if food pellet delivery did not elicit dopamine release that satisfied the chemical verification criteria. for anesthetized experiments, dopamine release was evoked by stimulating the ppt (relative to bregma : 8.0 mm posterior ; 2.0 mm lateral, 6.57.5 mm ventral) or the bnst (relative to bregma : 0.3 mm posterior ; 1.5 mm lateral, 6.57.5 mm ventral) with a bipolar stimulating electrode (60 pulses delivered at 60 hz, 200 a). stimulations were performed every 5 min until a stable baseline for 20 min was achieved (50% of planned experiments due to technical complications. rats were excluded for analysis if cannulas did not target the region of interest, save for those illustrating the lack of an effect of crf when administered outside of the vta (supplementary fig. general motor activity was assessed using the rate of head entries into the food tray. following completion of the experimental sessions, animals were anesthetized with ketamine / xylazine (100 mg / kg) and the recording site was marked by making a small electrolytic lesion at the electrode tip by passing a current (~70 a) through the carbon fiber microelectrode for 20 seconds. animals were subsequently perfused transcardially with 4% paraformaldehyde in phosphate - buffered saline at ph = 7.4 before the brains were removed and post - fixed in the paraformaldehyde solution. the brains were then placed in 30% sucrose solution in phosphate - buffered saline for 48 hours, flash frozen, and sectioned coronally (60 m). supplementary figure 1. acute restraint stress and intra - vta injections of crf did not elicit gross impairments of motor function. behavioral effects of unilateral or bilateral injections of crf on motivated behavior during pr sessions. the effect of crf on motivated behavior was absent when guide cannula did not target the vta. nacc dopamine release in response to rewards and their predictions is sensitive to rewards size in pr sessions. behavioral data from the rats included in voltammetry experiments assessing the effect of crf in the vta on motivation. crf in the vta reduced dopamine release to unexpected reward delivery after completion of the pr session. dopamine release in the nacc elicited by stimulating vta afferent pathways and its modulation by crf. intra - vta injections of crf further attenuated motivation in rats in a reduced motivational state.	stressors impact dopamine - dependent behaviors such as motivation, though the underlying neurobiological mechanism is not well defined. we report that corticotropin - releasing factor (crf) acts within the ventral tegmental area (vta) to reduce the motivation to work for food rewards. crf in the vta regulates dopamine output in a stimulus- and pathway - specific manner, collectively offering a mechanism by which acute stress selectively regulates information transmission via the vta to reprioritize motivated behavior.
many of the difficulties of higher - order calculations in qft can be traced back to the treatment, in the framework of dimensional regularization (dr), of the infinities arising in the intermediate steps of the computation. ultraviolet (uv), infrared (ir), and collinear (cl) divergences are first dimensionally regulated, and then renormalized away in the uv case or canceled by combining virtual and real contributions, or reabsorbed in the collinear behavior of the initial state parton densities. in order to attack the problem numerically, it is often necessary to subtract and add back approximations of the ir / cl singular structures. at one loop, several well - tested subtraction procedures have been introduced in the last two decades [28 ]. at two loops and beyond, the situation is more involved, but progress is under way [914 ]. the first obvious ingredient, which may lead to a significant simplification in the above picture, is a computational procedure in which all parts of the calculation can be directly treated in four dimensions. as for the virtual contribution, the fdr approach has been recently introduced in reference, which allows a subtraction of the uv divergencesat the level of the integrand, leaving a four - dimensional integration over the loop momenta. in the same work, the use of fdr as an ir regulator in qed with massive fermions is also suggested. in this paper, i present the first application of the fdr ideas in the context of fully massless qcd, where the issues related to gauge invariance are much more subtle than in the qed case. i concentrate, in particular, on the calculation of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathcal{o}(\alpha _ s)$$\end{document}o(s) gluonic corrections to the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h \rightarrow gg$$\end{document}hgg decay in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_\mathrm{top } \rightarrow \infty $ $ \end{document}mtop limit, and re - derive the well - known fully inclusive result [16, 17]1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \vargamma (h \rightarrow gg) = \vargamma ^{(0)}(\alpha _ s(m_h^2)) \left [1+\frac{95}{4}\,\frac{\alpha _ s}{\pi } \right ], \end{aligned}$$\end{document}(hgg)=(0)(s(mh2))1 + 954s,where2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \vargamma ^{(0)}(\alpha _ s(m_h^2))= \frac{g_f \alpha _ s^2(m_h^2)}{36 \sqrt{2 } \pi ^3 } m^3_h \end{aligned}$$\end{document}(0)(s(mh2))=gfs2(mh2)3623mh3is the lowest order contribution, with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n_f= 0$$\end{document}nf=0 in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha _ s(m_h^2)$$\end{document}s(mh2), since only gluons are considered. despite its simplicity, all key ingredients of massless qcd are present in this process, such as the simultaneous occurrence of ir / cl divergences and uv renormalization. the fact that the correct expression is reproduced shows that fdr is a valid and consistent approach in massless qfts, and it gives confidence in its potential to simplify multi - leg / loop computations. section 2 provides the set - up of the calculation. in sect. 3, i review the fdr treatment of the uv divergences and discuss its interplay with the ir and cl infinities. section 4 presents the fdr computation of the virtual part, while sect. 5 deals with the real contribution and its merging with the one - loop piece. the effective interaction of one higgs field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h$$\end{document}h with two, three and four gluons mediated by an infinitely heavy top loop is described by the lagrangian [18, 19]3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \mathcal{l}_\mathrm{eff } = -\frac{1}{4 } a h g^{a}_{\mu \nu } g^{a,\mu \nu }, \end{aligned}$$\end{document}leff=-14ahgaga,,where4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } a = \frac{\alpha _ s}{3\pi v}\left (1+\frac{11}{4}\frac{\alpha _ s}{\pi } \right) \end{aligned}$$\end{document}a=s3v1 + 114sand \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v$$\end{document}v is the vacuum expectation value, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v^2= (g_f \sqrt{2})^{-1}$$\end{document}v2=(gf2)-1. the corresponding feynman rules are given in, and the diagrams for the decay rate \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vargamma (h \rightarrow gg)$$\end{document}(hgg) are drawn in fig. 1virtual and real diagrams contributing to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h \rightarrow g g (g)$$\end{document}hgg(g) at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathcal{o}(\alpha _ s^3)$$\end{document}o(s3). the gray blobs in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v_6$$\end{document}v6 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v_7$$\end{document}v7 represent gluon wave - function corrections and the dashed line stands for the higgs field. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_1(p_i, p_j, p_k)$$\end{document}r1(pi, pj, pk) corresponds to three diagrams with permuted gluons virtual and real diagrams contributing to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h \rightarrow g g (g)$$\end{document}hgg(g) at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathcal{o}(\alpha _ s^3)$$\end{document}o(s3). the gray blobs in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v_6$$\end{document}v6 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v_7$$\end{document}v7 represent gluon wave - function corrections and the dashed line stands for the higgs field. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_1(p_i, p_j, p_k)$$\end{document}r1(pi, pj, pk) corresponds to three diagrams with permuted gluons there are five graphs contributing to the virtual part \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vargamma _ { v}$$\end{document}v without counting gluon wave - function corrections and four diagrams for the real radiation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vargamma _ { r}$$\end{document}r. in the following, i separately compute, in fdr, the two pieces, showing how ir / cl divergences drop in the sum5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \vargamma _ { v}(h \rightarrow gg) + \vargamma _ { r}(h \rightarrow ggg). consider the one - loop quadratically divergent rank - two tensor6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \int d^4q \frac{q_\alpha q_\beta } { d_0 d_1 }, \end{aligned}$$\end{document}d4qqqd0d1,with7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } d_i ~=~ q^2-d_i,~~~d_i ~=~ m^2_i - p^2_i- 2 (q\cdot p_i),~~~p_0=0.\nonumber \\ \end{aligned}$$\end{document}di = q2-di, di = mi2-pi2 - 2(qpi),p0=0.its uv convergence can be improved by first deforming the propagators by a vanishing amount \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu ^2$$\end{document}218\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } d_i \rightarrow \bar{d}_i~=~d_i-\mu ^2, \end{aligned}$$\end{document}didi = di-2,and then by repeatedly using the identity9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \frac{1}{\bar{d}_i } = \frac{1}{\bar{q}^2 } \bigg (1+\frac{d_i}{\bar{d}_i } \bigg), \end{aligned}$$\end{document}1di=1q2(1+didi),where10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \bar{q}^2~=~q^2-\mu ^2. \end{aligned}$$\end{document}q2=q2-2.note that the prescription in eq. (6) can then be rewritten as11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \frac{q_\alpha q_\beta } { \bar{d}_0 \bar{d}_1}&= q_\alpha q_\beta \left (\left [\frac{1}{\bar{q}^4 } \right ] + \left [\frac{d_0+d_1}{\bar{q}^6 } \right ] + \left [\frac{d_1 ^ 2}{\bar{q}^8 } \right ] + \frac{d_1 ^ 3}{\bar{q}^8 \bar{d}_1 } \right. + \frac{d_0 d_1}{\bar{q}^6 \bar{d}_1 } + \frac{d_0 ^ 2}{\bar{q}^4 \bar{d}_0\bar{d}_1 } \right), \end{aligned}$$\end{document}qqd0d1=qq1q4+d0+d1q6+d12q8+d13q8d1+d0d1q6d1+d02q4d0d1,where the terms in square brackets are uv divergent but depend only on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu ^2$$\end{document}2. the fdr definition of the integral in eq. (6) is obtained by integrating the expansion in eq. (11), after dropping the divergent pieces, and taking the physical limit \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu \rightarrow 0$$\end{document}0:12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&b_{\alpha \beta } (p_1 ^ 2,m_0 ^ 2,m_1 ^ 2) = \int [d^4q ] \frac{q_\alpha q_\beta } { \bar{d}_0 \bar{d}_1 } \nonumber \\&\quad \equiv \lim _ { \mu \rightarrow 0 } \int d^4q \, q_\alpha q_\beta \left (\frac{d_1 ^ 3}{\bar{q}^8 \bar{d}_1 } + \frac{d_0 d_1}{\bar{q}^6 \bar{d}_1 } + \frac{d_0 ^ 2}{\bar{q}^4 \bar{d}_0\bar{d}_1 } \right) \!.\nonumber \\ \end{aligned}$$\end{document}b(p12,m02,m12)=[d4q]qqd0d1lim0d4qqqd13q8d1+d0d1q6d1+d02q4d0d1.the r.h.s. of eq. (12) corresponds to a well - defined four - dimensional integral, in which all uv divergences are explicitly subtracted. the gauge - invariance properties of this definition are discussed in detail in [15, 21 ]. in the rest of this section, i mostly concentrate on cl and ir infinities, and, in particular, on the matching between virtual and real contributions. a convenient starting point to study the cl singularities (12):13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&b_{\alpha \beta } (0,0,0) = \lim _ { \mu \rightarrow 0 } \int d^4q \, \frac{q_\alpha q_\beta d_1 ^ 3}{\bar{q}^8 \bar{d}_1 } \nonumber \\&\quad = -8 p_1^\rho p_1^\sigma p_1^\tau \lim _ { \mu \rightarrow 0 } \int d^4q \frac{q_\alpha q_\beta q_\rho q_\sigma q_\tau } { \bar{q}^8 \bar{d}_1 } = 0, \end{aligned}$$\end{document}b(0,0,0)=lim0d4qqqd13q8d1=-8p1p1p1lim0d4qqqqqqq8d1=0,which vanishes, after tensor decomposition, since \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_1 ^ 2= 0$$\end{document}p12=0. analogously, one proves that14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&b_{\alpha } (0,0,0) = \int [d^4q ] \frac{q_\alpha } { \bar{d}_0 \bar{d}_1 } = 0, \nonumber \\&b(0,0,0) = \int [d^4q ] \frac{1}{\bar{d}_0 \bar{d}_1 } = 0. \end{aligned}$$\end{document}b(0,0,0)=[d4q]qd0d1=0,b(0,0,0)=[d4q]1d0d1=0.those results coincide with dr in which scale - less integrals are zero and are due to a cancelation between two \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\ln (\mu ^2)$$\end{document}ln(2) of cl and uv origin, respectively. for example,15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } b(p^2,0,0)&= -i \pi ^2 \lim _ { \mu \rightarrow 0}\int _ 0 ^ 1 \mathrm{d}x\, \nonumber \\&\times \,\,\left [\ln (\mu ^2 -p^2 x (1-x)) -\ln (\mu ^2) \right ] \ !, \end{aligned}$$\end{document}b(p2,0,0)=-i2lim001dxln(2-p2x(1-x))-ln(2),where the first logarithm develops a cl singularity in the limit \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p^2 \rightarrow 0$$\end{document}p20. thus, the virtual cl infinities, generated by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$1 \rightarrow 2$$\end{document}12 splittings of massless particles, are naturally regulated by the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu ^2$$\end{document}2-deformed propagators inside the loop, while the external momenta remain massless, as illustrated in fig. 2 a and c. the real counterpart of this procedure 2b and d, and corresponds to a phase space in which all would be massless external particles are given a common mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu $ $ \end{document} and the internal ones stay massless. in other words, one has to replace216\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \frac{1}{2(p_i \cdot p_j) } \rightarrow \frac{1}{(p_i+p_j)^2 } \end{aligned}$$\end{document}12(pipj)1(pi+pj)2 in any possible singular denominator of the real matrix element squared, integrate over the aforementioned massive phase space, and take the limit \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu \rightarrow 0$$\end{document}0.fig. the one - gluon cut in a contributes to the virtual part, the two - gluon cut in b to the real radiation. f represent typical cut - diagrams contributing to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h \rightarrow gg(g)$$\end{document}hgg(g) gluon splitting ir / cl singularities regulated by massive (thick) gluons. the one - gluon cut in a contributes to the virtual part, the two - gluon cut in b to the real radiation. f represent typical cut - diagrams contributing to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h \rightarrow gg(g)$$\end{document}hgg(g) as for the ir divergences, the reasoning follows the same lines. for example, the only ir / cl divergent scalar one - loop three - point function generated by the cut in fig. 2e is317\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } c(s)= \int [d^4q ] \frac{1}{\bar{d}_0 \bar{d}_1 \bar{d}_2 } = \lim _ { \mu \rightarrow 0 } \int d^4q \frac{1}{\bar{d}_0 \bar{d}_1 \bar{d}_2 }, \end{aligned}$$\end{document}c(s)=[d4q]1d0d1d2=lim0d4q1d0d1d2,with18\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } m_0 ^ 2 = m_1 ^ 2 = m_2 ^ 2 = p_1 ^ 2= p_2 ^ 2= 0,~~~s= -2(p_1 \cdot p_2).\nonumber \\ \end{aligned}$$\end{document}m02=m12=m22=p12=p22=0,s=-2(p1p2).by denoting19\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \mu _ 0=\frac{\mu ^2}{s }, \end{aligned}$$\end{document}0=2s, one computes20\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } c(s)&= \lim _ { \mu \rightarrow 0 } \frac{i \pi ^2}{2 s } \ln ^2\bigg (\frac{\sqrt{1 - 4\mu _ 0}+1}{\sqrt{1 - 4\mu _ 0}-1}\bigg) \nonumber \\&= \frac{i \pi ^2}{s } \left [\frac{\ln ^2(\mu _ 0)-\pi ^2}{2 } + i\,\pi \ln (\mu _ 0) \right ], \end{aligned}$$\end{document}c(s)=lim0i22sln2(1 - 40 + 11 - 40 - 1)=i2sln2(0)-22+iln(0),which is indeed fully matched by the inclusive real contribution in fig. i use the described approach to uv / cl / ir infinities to compute \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vargamma _ { v}(h \rightarrow gg)$$\end{document}v(hgg) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vargamma _ { r}(h \rightarrow ggg)$$\end{document}r(hggg). the calculation is greatly simplified by eqs. (13) and (14). in fact, only the diagrams \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v_1$$\end{document}v1 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v_2$$\end{document}v2 in fig. 1 one computes21\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \vargamma _ { v}(h \rightarrow gg)=-3 \frac{\alpha _ s}{\pi } \,\vargamma ^{(0)}(\alpha _ s)\,m^2_h\, \mathcal{r}e \left [\frac{c(m^2_h)}{i \pi ^2}\right ].\nonumber \\ veltman decomposition, the only subtlety being the fdr treatment of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu ^2$$\end{document}2 [15, 21 ] : for consistency with eq. (8), a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q^2$$\end{document}q2 appearing in the numerator of a diagram should also be deformed,22\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } q^2 \rightarrow \bar{q}^2, \end{aligned}$$\end{document}q2q2,and integrals involving \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu ^2$$\end{document}2, such as23\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } { \tilde{b}}(p_1 ^ 2,m_0 ^ 2,m_1 ^ 2) = \int [d^4q ] \frac{\mu ^2}{\bar{d}_0 \bar{d}_1 }, \end{aligned}$$\end{document}b~(p12,m02,m12)=[d4q]2d0d1,require the same integrand expansion as if\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu ^2= q^2$$\end{document}2=q2. (12),24\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&{\tilde{b}}(p_1 ^ 2,m_0 ^ 2,m_1 ^ 2)\nonumber \\&\quad = \lim _ { \mu \rightarrow 0 } \int d^4q \, \mu ^2 \left (\frac{d_1 ^ 3}{\bar{q}^8 \bar{d}_1 } + \frac{d_0 d_1}{\bar{q}^6 \bar{d}_1 } + \frac{d_0 ^ 2}{\bar{q}^4 \bar{d}_0\bar{d}_1 } \right) \nonumber \\&\quad = \frac{i \pi ^2}{2}\left (m_0 ^ 2+m_1 ^ 2-\frac{p_1 ^ 2}{3}\right). (20) into (21),25\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \vargamma _ { v}(h \rightarrow gg)=\frac{3}{2 } \frac{\alpha _ s}{\pi } \,\vargamma ^{(0)}(\alpha _ s)\, \left (\pi ^2-\ln ^2\frac{m^2_h}{\mu ^2 } \right). the unpolarized matrix element squared, derived from the real emission diagrams in fig. 1, reads26\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&\|m\|^2 = 192\,\pi \alpha _ sa^2 \bigg [\frac{s^3_{23}}{s_{12}s_{13 } } + \frac{s^3_{13}}{s_{12}s_{23 } } + \frac{s^3_{12}}{s_{13}s_{23 } } \nonumber \\&\quad + \,\, \frac{2(s^2_{13}+s^2_{23})+3 s_{13}s_{23}}{s_{12 } } + \frac{2(s^2_{12}+s^2_{23})+3 s_{12}s_{23}}{s_{13 } } \nonumber \\&\quad + \,\, \frac{2(s^2_{12}+s^2_{13})+3 s_{12}s_{13}}{s_{23}}+ 6 (s_{12 } + s_{13 } + s_{23})\bigg ], \end{aligned}$$\end{document}\|m\|2=192sa2[s233s12s13+s133s12s23+s123s13s23 + 2(s132+s232)+3s13s23s12 + 2(s122+s232)+3s12s23s13 + 2(s122+s132)+3s12s13s23 + 6(s12+s13+s23)],where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{ij}= (p_i+ p_j)^2$$\end{document}sij=(pi+pj)2. this expression is obtained from the massless result with the replacement \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2(p_i \cdot p_j) \rightarrow s_{ij}$$\end{document}2(pipj)sij, in accordance with eq. (16). as described in sect. 3, in order to match the virtual ir / cl singularities, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\|m\|^2$$\end{document}\|m\|2 should be integrated over a massive three - gluon phase space with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_i^2= \mu ^2$$\end{document}pi2=2, which can be parametrized as27\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \int d \phi _ 3 = \frac{\pi ^2}{4s } \int ds_{12 } ds_{13 } ds_{23}\, \delta (s - s_{12}-s_{13}-s_{23}+3\mu ^2), \end{aligned}$$\end{document}d3=24sds12ds13ds23(s - s12-s13-s23 + 32),where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sqrt{s}$$\end{document}s is the higgs mass. it is convenient to introduce the dimensionless variables28\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } x= \frac{s_{12}}{s}-\mu _ 0,~~y= \frac{s_{13}}{s}-\mu _ 0,~~z= \frac{s_{23}}{s}-\mu _ 0, \end{aligned}$$\end{document}x = s12s-0,y = s13s-0,z = s23s-0,with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu _ 0$$\end{document}0 given in eq. (19), in terms of which, using the condition,29\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } x+y+z = 1, \end{aligned}$$\end{document}x+y+z=1,all ir / cl divergent bremsstrahlung integrals can be reduced to the following ones:30\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&i(s) = \int _ { r } \mathrm{d}x \mathrm{d}y\, \frac{1}{(x+\mu _ 0) (y+\mu _ 0) }, \nonumber \\&j_{p}(s) = \int _ { r } \mathrm{d}x \mathrm{d}y\, \frac{x^p}{(y+\mu _ 0)}\,\,~~(p \ge 0), \end{aligned}$$\end{document}i(s)=rdxdy1(x+0)(y+0),jp(s)=rdxdyxp(y+0)(p0),where the integration region reads, in the fully inclusive case,31\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \int _ { r } \mathrm{d}x \mathrm{d}y \equiv \int _ { 3 \mu _ 0}^{1 - 2\sqrt{\mu _ 0 } } \mathrm{d}x \int _ { y_-}^{y_+ } \mathrm{d}y, \end{aligned}$$\end{document}rdxdy301 - 20dxy - y+dy, with32\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } y_{\pm } & = \frac{1}{4(x + \mu _ 0) } \left [(1-\mu _ 0)^2-(r_0 \mp r_1)^2\right ] -\mu \mu _ 0}.\nonumber \\ \end{aligned}$$\end{document}y=14(x+0)(1-0)2-(r0r1)2-0,r0=(x-0)2 - 402,r1=(1-x)2 - 40.thus33\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&\vargamma _ { r}(h \rightarrow ggg)= 3 \frac{\alpha _ s}{\pi } \,\vargamma ^{(0)}(\alpha _ s)\, \nonumber \\&\quad \times \left (\frac{1}{4}+i(m^2_h)-\frac{3}{2}j_0(m^2_h)-j_2(m^2_h) \right). \end{aligned}$$\end{document}r(hggg)=3s(0)(s)14+i(mh2)-32j0(mh2)-j2(mh2).finally, one computes, up to terms which vanish in the limit \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu _ 0 \rightarrow 0$$\end{document}00,34\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } i(s) = \frac{\ln ^2(\mu _ 0)-\pi ^2}{2 } \end{aligned}$$\end{document}i(s)=ln2(0)-22and35\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&j_{p}(s) = -\frac{1}{p+1 } \ln (\mu _ 0) + \int _ 0 ^ 1 \mathrm{d}x \,x^p\left [\ln (x)+ 2 \ln (1-x) \right ] \nonumber \\&\quad = -\frac{1}{p+1 } \ln (\mu _ 0) \!-\!\frac{1}{p+1}\left [\frac{1}{p+1}\!+\!2 \sum _ { n=1}^{p+1}\frac{1}{n } \right ] (p \ge 0),\nonumber \\ \end{aligned}$$\end{document}jp(s)=-1p+1ln(0)+01dxxpln(x)+2ln(1-x)=-1p+1ln(0)-1p+11p+1 + 2n=1p+11n(p0),so that36\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&\vargamma _ { r}(h \rightarrow ggg)= \frac{3}{2 } \frac{\alpha _ s}{\pi } \,\vargamma ^{(0)}(\alpha _ s)\, \nonumber \\&\quad \times \left (\ln ^2\frac{m^2_h}{\mu ^2}-\pi ^2+\frac{73}{6 } -\frac{11}{3}\ln \frac{m^2_h}{\mu ^2 } \right). (4), one obtains37\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \vargamma (h \rightarrow gg) = \vargamma ^{(0)}(\alpha _ s) \left [1+\frac{\alpha _ s}{\pi } \left (\frac{95}{4}-\frac{11}{2 } \ln \frac{m^2_h}{\mu ^2 } \right) \right ].\nonumber \\ \end{aligned}$$\end{document}(hgg)=(0)(s)1+s954 - 112lnmh22.all cl / ir \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\ln (\mu ^2)$$\end{document}ln(2) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\ln ^2(\mu ^2)$$\end{document}ln2(2) cancel in eq. (37), so that the remaining \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu $ $ \end{document} is directly interpreted as the renormalization scale. this is a typical procedure in fdr : since the uv infinities are subtracted from the very beginning, the unphysical left - over \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu $ $ \end{document} dependence is eliminated, on the perturbative level one is working at, by a finite renormalization, which fixes the bare parameters in terms of the observables. this is obtained, in the case at hand, by simply replacing \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vargamma ^{(0)}(\alpha _ s) \rightarrow \vargamma ^{(0)}(\alpha _ then the logarithm is reabsorbed in the gluonic running of the strong coupling constant,38\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \alpha _ s(\mu ^2)}{1+\frac{\alpha _ s}{2 \pi } \frac{11}{2 } \ln \frac{m_h^2}{\mu ^2 } }, \end{aligned}$$\end{document}s(mh2)=s(2)1+s2112lnmh22,and eq. this is particularly important in qcd, where nlo calculations have to be matched with the running of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha _ s$$\end{document}s and parton densities, conventionally derived in dr. i consider uv, cl, and ir divergences in turn, showing the equivalence of fdr with the dimensional reduction version of dr, widely used in supersymmetric theories. i start by establishing the connection between the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$1/\epsilon $ $ \end{document}1/ dr regulator and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\ln (\mu ^2)$$\end{document}ln(2) appearing in fdr. as for the uv infinities, (15) (with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p^2 \ne 0$$\end{document}p20) reads39\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \int d^nq \frac{1}{q^2(q+p)^2}= i \pi ^2 \int _ { 0}^{1 } \mathrm{d}x\, [\delta -\ln (-p^2 x (1-x))],\nonumber \\ \end{aligned}$$\end{document}dnq1q2(q+p)2=i201dx[-ln(-p2x(1-x))],where40\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } n = 4 + \epsilon \,~~~\mathrm{and}~~~\delta = -\frac{2}{\epsilon } - \gamma _ e -\ln \pi. \end{aligned}$$\end{document}n=4+and=-2-e - ln.thus, dr and fdr uv regulators are linked through the simple \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{ms}}$$\end{document}ms replacement41\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \delta \rightarrow \ln (\mu ^2). virtual singularities follow the same pattern, as can be inferred from the exact uv / cl cancelation in eqs. (13) and (14). as a consistency check, the dr version of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_{p}$$\end{document}jp reads42\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&j^\mathrm{dr}_{p}(s) \!=\ ! \frac{(\pi s)^{\frac{\epsilon } { 2}}}{\vargamma \left (1+\frac{\epsilon } { 2}\right) } \int \mathrm{d}x\,\mathrm{d}y\,\mathrm{d}z\,\frac{x^p}{y}\delta (1\!-\!x\!-\!y\!-\!z) (xyz)^{\frac{\epsilon } { 2 } } \nonumber \\&\quad = -\frac{1}{p+1 } (\delta -\ln (s)) -\frac{1}{p+1}\left [\frac{1}{p+1}+2 \sum _ { n=1}^{p+1}\frac{1}{n } \right ], \nonumber \\ \end{aligned}$$\end{document}jpdr(s)=(s)21+2dxdydzxpy(1-x - y - z)(xyz)2=-1p+1(-ln(s))-1p+11p+1 + 2n=1p+11n, which indeed coincides with eq. (35) if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta = \ln (\mu ^2)$$\end{document}=ln(2). finally, the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\ln ^2(\mu ^2)$$\end{document}ln2(2) terms generated by overlapping ir / cl singularities drop, together with the full constant part, when adding virtual and fully inclusive real contributions, which can be traced back to the following relation:43\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \mathcal{r}e \left [\frac{c(s)}{i \pi ^2}\right ] = \frac{1}{s } i(s)\, \end{aligned}$$\end{document}rec(s)i2=1si(s)between eqs. an easy calculation shows that the same happens in dr. in fact44\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&\mathcal{r}e \left [\frac{1}{i \pi ^2 } \int d^nq \frac{1}{q^2(q+p_1)^2(q+p_2)^2 } \right ] \nonumber \\&\quad = \frac{1}{s } (\pi s)^{\frac{\epsilon } { 2}}\, \vargamma \left (1-\frac{\epsilon } { 2}\right) \left [\frac{4}{\epsilon ^2}-\frac{2}{3 } \pi ^2 \right ], \end{aligned}$$\end{document}re1i2dnq1q2(q+p1)2(q+p2)2=1s(s)21-242 - 232,where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_1 ^ 2= p_2 ^ 2= 0$$\end{document}p12=p22=0 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s= -2(p_1\cdot p_2)$$\end{document}s=-2(p1p2), and45\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } i^\mathrm{dr}(s)&= \frac{(\pi s)^{\frac{\epsilon } { 2}}}{\vargamma \left (1+\frac{\epsilon } { 2}\right) } \int \mathrm{d}x\,\mathrm{d}y\,\mathrm{d}z\,\frac{1}{xy}\delta (1\!-\!x\!-\!y\!-\!z) (xyz)^{\frac{\epsilon } { 2 } } \nonumber \\&= (\pi s)^{\frac{\epsilon } { 2}}\,\vargamma \left (1-\frac{\epsilon } { 2}\right) \left [\frac{4}{\epsilon ^2}-\frac{2}{3 } \pi ^2 \right ]. however, an important difference between dr and fdr follows from self - contractions of metric tensors coming from the feynman rules. in dr \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\alpha \beta } g^{\alpha \beta } = n$$\end{document}gg=n, while \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\alpha \beta } g^{\alpha \beta } = 4$$\end{document}gg=4 in fdr. (41), and the fdr treatment of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu ^2$$\end{document}2 discussed in sect. 4, makes explicit the equivalence between fdr and dimensional reduction in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{\mathrm ms}}$$\end{document}ms scheme. having established this, all the well - known transition rules between dimensional reduction and dr [26, 27 ] can be directly applied to fdr. in the case of eq. (37), it turns out that the expression is the same in both dimensional reduction (or fdr) and dr. therefore, the correct strong coupling constant to be used is the customary \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha _ s(\mu ^2)$$\end{document}s(2) in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\mathrm{ms}}$$\end{document}ms scheme, proving that the fdr result coincides with eq. i have presented an fdr calculation of the gluonic qcd corrections to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h \rightarrow gg$$\end{document}hgg in the large top effective theory, demonstrating that ultraviolet, collinear, and infrared divergences can be simultaneously and successfully regulated in four dimensions. i have proved the equivalence, at the one - loop level, of dimensional reduction and fdr, making the latter approach attractive also in supersymmetric calculations, where the fermionic and bosonic sectors must share the same number of degrees of freedom. the advantage of directly working in the four - dimensional minkowsky space is expected to lead to considerable simplifications in higher - order qft computations, especially in connection with numerical techniques. this issue, together with the extension of fdr to more loops, is currently under study.	i apply fdr a recently introduced four - dimensional approach to quantum field theories (qfts)to the computation of the nlo qcd corrections to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h \rightarrow gg$$\end{document}hgg in the large top mass limit. the calculation involves all key ingredients of qcd namely ultraviolet, infrared, and collinear divergences, besides \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha _ s$$\end{document}s renormalization and paves the way for successful use of fdr in massless one - loop qft computations. i show in detail how the correct result emerges in fdr, and discuss the translation rules to dimensional regularization.
granuloma annulare manifests various skin lesions, such as erythematous, plaque, papular, nodular and ulcerative forms, in addition to the typical annular lesion. we report a case of generalized erythematous granuloma annulare with a remission after lipid - lowering diet. a 62-year - old japanese woman presented with itchy erythematous skin lesions on the left lower abdomen, the right side of the chest and both axillae. the skin lesions were first noticed one year earlier, and subsequently spread to the chest and back in spite of treatment with topical steroid and oral epinastine hydrochloride. physical examination showed a well - demarcated erythema of approximately 10 cm in diameter with limited fine scales on the left lower abdomen (figure 1a). some of the macules on the chest and axillae measured up to 5 cm and were palpable pinkish erythemata surrounded by small red papules at the periphery (figure 1b, c). laboratory tests showed hba1c 6.2% (normal : 4.35.8), aspartate aminotransferase (ast) 76 iu / l (normal : 540), alanine aminotransferase (alt) 258 iu / l (normal : 535), total cholesterol (t - chol) 235 mg / dl (normal : 110210), and triglyceride 962 mg / dl (normal : 55129) (table 1). histopathological examination of biopsy material obtained from the left and right abdomen and stained with hematoxylin and eosin (h&e) showed similar changes, including homogenized but poorly stained collagen fibers and granulomatous infiltration of histiocytes, lymphocytes and giant cells between the fibers in the upper and middle dermis. elastica - van - gieson staining showed diminished fractured elastic fibers, but no phagocytosis of giant cells. alcian - blue staining confirmed the presence of mucin deposition between collage fibers (figure 2b). a diagnosis of a generalized erythematous papular type of granuloma annulare was established based on the clinical and histopathological findings together with the distribution pattern of the lesion (on more than one anatomic site). consultation with the department of internal medicine regarding glucose intolerance, liver dysfunction and hyperlipidemia was followed by a lipid - lowering diet for a period of three months. one month later, the skin lesions showed clear remission, together with improvement in laboratory data (table 1). granuloma annulare often shows generalized distribution of the skin manifestations that cover more than one anatomic site. the generalized form of granuloma annulare also tends to exhibit various types of skin lesions. the frequency of the erythematous type is reported to be approximately 16%, and 14 japanese cases, including the present case, have been reported since 1966 [415 ]. the age distribution of the reported japanese cases ranged from 50 to 70, with no gender difference, with a distribution mainly on the extremities (table 2). the association of granuloma annulare with diabetes mellitus is well documented, together with other less frequent complications of autoimmune disorders and internal malignancy. the association of granuloma annulare with dyslipidemia was recently reported. in the study by wu, dyslipidemia was more common in generalized than localized / disseminated disease, and the annular lesion morphology was associated with hypercholesterolemia. our review of the japanese literature on generalized erythematous granuloma annulare showed that 6 out of 14 had glucose intolerance, 1 had rheumatoid arthritis, and 1 had internal malignancy. the distinct features of the present case were the following : 1) the skin lesions were mainly noted on the trunk, 2) high serum triglyceride level (962 mg / dl), 3) mild glucose intolerance, and 4) liver dysfunction. in this regard, moreover, granuloma annulare often spontaneously resolves after a biopsy especially in the erythematous type. the clinical improvement noted in the present case could have been triggered by the biopsy, topical application of the steroid ointment and/or beneficial changes in both glucose intolerance and hyperlipidemia following lipid - lowering diet. however, one can not dismiss the possible involvement of hyperlipidemia in the activity of granuloma annulare. therefore, the presented case suggests the possible relationship between granuloma annulare and hyperlipidemia, with possible improvement of granuloma annulare by lipid - lowering diet. with the increased concern on adult lifestyle - related diseases in recent years, physicians need to pay attention to granuloma annulare and we should also be aware of generalized lesions, which can be easily overlooked as non - specific skin lesions, and conduct skin biopsy to establish a definitive diagnosis. this case was presented at the 74th tokyo division meeting of the japanese dermatological association.	granuloma annulare has been associated with systemic disease including diabetes mellitus. we report a case of a 62-year - old japanese woman with generalized erythematous granuloma annulare who showed remission after substantial improvement in hyperlipidemia following a strict lipid - lowering diet. the lesion appeared in the lower abdomen one year before current presentation and subsequently spread to other areas of the trunk despite treatment with topical steroid and oral epinastine hydrochloride. physical examination showed a well - demarcated erythematous plaque measuring 10 cm in diameter with fine scales on the left abdomen, and slightly indurated pinkish plaques of up to 5 cm in diameter on the right side of the abdomen and axillae. clinical laboratory tests showed mild glucose intolerance (hba1c 6.2%), mild liver dysfunction (ast : 86 iu / l, alt : 76 iu / l), slight hypercholesterolemia (total cholesterol : 235 mg / dl), and severe hyperlipidemia (triglyceride : 962 mg / ml). histopathological examination of the lesions showed homogenization of collagen fibers and granulomatous infiltrates between fibers in the upper and middle dermis. a diagnosis of generalized erythematous granuloma annulare was established based on the clinical and histopathological findings, especially with the distribution on more than one anatomic site. a lipid - lowering diet for three months resulted in major improvement of hyperlipidemia and remission of the skin lesions.a review of generalized erythematous granuloma annulare in the japanese literature indicated a well - known association of granuloma annulare with diabetes mellitus, however, the relation with hyperlipidemia was described only recently. this case suggests a possible relationship between granuloma annulare and hyperlipidemia, with possible improvement of granuloma annulare with a lipid - lowering diet.
infections caused by carbapenem - resistant klebsiella pneumoniae (crkp) represent a significant public health concern worldwide. infections caused by crkp isolates are associated with high morbidity and mortality rates, particularly in patients with haematological malignancies. infection and colonization by these multiresistant bacteria may represent a challenge in sct recipients for the management of post - transplantation complications, as well as for the eligibility to receive a transplant in patients who acquire the pathogen prior to the procedure (1,2). we herein report the case of a blast - phase chronic myeloid leukemia (bp - cml) patient with a highly resistant, crkp - associated tricuspid valve endocarditis, who was treated with a combination of systemic antimicrobial therapy and surgical valve repair, and subsequently underwent successful allogeneic stem cell transplantation (allo - sct). to the best of our knowledge, a 50-year - old female patient with bp - cml was referred to the istanbul medipol university in january 2015. the disease was in chronic phase, so treatment with imatinib was started by her physicians. after treatment with imatinib for 3 months, the patient developed progressive disease with myeloid blastic transformation and 7 + 3 induction chemotherapy was initiated. during this treatment, the patient was intubated for severe pneumonia with sepsis and had to stay in the intensive care unit (icu) for ~2 months in a libyan hospital. the bone marrow biopsy that was performed after the infection had resolved was consistent with complete remission of the leukaemia ; thus, the patient was referred to our hospital for an allo - sct. on admittance to the hospital, the patient was febrile. peripheral and central venous port blood cultures were collected, which grew kp that was susceptible only to tigecycline. as venous port infection was suspected, the patient 's venous port was removed and she was treated with tigecycline and meropenem (table i, 1st bacteraemia). the patient was in good condition and preparations for allo - sct were initiated. during her work - up, the blood cultures again revealed kp, with the same resistance pattern (table i, 2nd bacteraemia). the fever resolved and the acute - phase parameters returned to normal levels within 5 days. treatment was again discontinued after 2 weeks, but the patient developed fever shortly after. repeated blood cultures again grew pan - resistant kp (table i, 3rd bacteraemia). rectal swabs that were collected during the hospitalisation period were all negative for the carrier state of crkp. transthoracic echocardiogram was performed for repeated bacteraemia, and revealed enlarged right cardiac chambers, moderate tricuspid valve insufficiency and a mass sized 1.51.4 cm on the tricuspid valve, compatible with a septic vegetation. with the diagnosis of tricuspid valve endocarditis due to a multiresistant pathogen, the patient was operated for excision of the vegetation and tricuspid valve repair (fig. the patient was treated with imipenem - cilastatin 1 g intravenously every 6 h, plus gentamicin 80 mg intravenously every 8 h, plus sulbactam 1 g intravenously every 6 h for 4 weeks. during the treatment period of endocarditis, the patient was also treated for cml with nilotinib. however, polymerase chain reaction (pcr) for bcr / abl remained positive, indicating residual disease. after the infection had resolved, unmanipulated peripheral blood allo - sct from a full - matched sibling donor was performed in november, 2015. the reduced intensity conditioning (ric) regimen included fludarabine at 30 mg / m / day for 5 days and busulfan at 3.2 mg / kg / day for 2 days. cyclosporin a, short - course methotrexate and antithymocyte globulin were used for acute graft - vs.- host disease (gvhd) prophylaxis. allo - sct was performed without complications ; neutrophil engraftment was achieved on day + 15 and thrombocyte engraftment was achieved on day + 14 post - transplantation. the patient is currently in complete remission with complete chimerism, without evidence of any gvhd, for > 3 months after allo - sct. the pcr of bcr / abl is also negative, showing no signs of residual disease. written informed consent was obtained from the patient for the publication of the case details. kp is one of the most important causes of health care- and icu - acquired infections (3) and has been reported as the second overall cause of gram - negative bloodstream infections (bsi) after escherichia coli. however, klebsiella species are a particularly rare cause (1.5%) of gram - negative bacterial endocarditis. klebsiella species cause 1.2% of the cases of native valve endocarditis and 4.1% of the cases of prosthetic valve endocarditis. it has been reported that the most commonly affected valve is the aortic valve, followed by the mitral and tricuspid valves. the most common source of bacteraemia is defined as urinary tract infection and infection of a pacemaker (4,5). in our patient, to the best of our knowledge, this is the first reported case of tricuspid valve endocarditis due to crkp. the main predictors of crkp infection are poor functional status, high severity of underlying conditions, icu stay and prior exposure to antimicrobial agents. patients with haematological malignancies and haematopoietic stem cell transplant recipients are particularly vulnerable to crkp infections due to chemotherapy - induced gastrointestinal mucositis, prolonged hospitalizations and neutropenia, as well as the frequent use of broad - spectrum antibacterial agents (7). our patient combined several risk factors, namely poor functional status, serious haematological malignancy, history of active chemotherapy, prolonged hospitalization with icu stay and the use of broad - spectrum antibiotics, making her treatment more challenging. treatment selection is generally based on the results of retrospective and/or single - center studies of crkp bsis. it is currently widely accepted that the combination of a first - line antibiotic, mainly carbapenems, at higher doses to overcome resistance, and a second - line antibiotic with gram - negative activity for which resistance has hopefully not yet developed (for example, colistin, tigecycline, gentamicin, fosfomycin) is the most suitable treatment option for crkp bsis (4,8). our patient was successfully treated with a combination of high - dose imipenem - cilastatin, gentamicin and sulbactam. in tricuspid valve endocarditis, surgery is indicated for persistent right - sided heart failure despite medical therapy, recurrent pulmonary septic emboli, septic shock, abscess formation or failure of antimicrobial therapy to control the infection (7). in our patient, however, the main limitation of tricuspid valve replacement was the need for long - term anticoagulation, which would be a major problem for performing heart surgery, as well as for performing allo - sct in our patient. in case of the latter condition, data are limited, as there is only one reported case of allo - sct being performed in a patient with a prosthetic valve (9). another problem is the increased risk of bleeding, infectious complications and reoperation rates of patients with haematological malignancies undergoing cardiac surgery (10). in our case, no cardiac complications occurred, without the need for additional measures during allo - sct. in cml optimal outcomes are achieved with allo - sct after obtaining complete remission with induction chemotherapy (11). in our patient, however, recent crkp endocarditis was a challenge when deciding to perform allo - hsct. satlin published a retrospective analysis of 18 malignant haematological patients with carbapenem - resistant enterobacteriaceae bsi. in that study, initial empirical antimicrobial therapy was active in only 11% of the patients and the mortality rate was 56% (12). in another multicenter retrospective study, girmenia assessed the epidemiology and prognostic factors of crkp infections in an sct setting. crkp infections were reported in 53.4% of the centers and were documented in 0.4% of autologous and 2% of allogeneic scts. the infection - related mortality rate was 16% in autologous and 64.4% in allogeneic sct. a pre - transplant crkp infection and an inadequate first - line treatment were associated with an increased mortality in allo - sct patients who developed a crkp infection. indeed, despite the administration of first - line crkp - targeted antibiotic therapy (ctat), 55% of the patients who received ctat succumbed to the disease (13). zuckerman evaluated the eradication of crkp carrier state in 15 patients undergoing intensive chemotherapy or sct. in that study, the eradication rate was 66% and all the patients in whom eradication failed succumbed to the disease, usually due to active crkp infection (14). according to the results of kuruvilla, recurrence of endocarditis during allo - sct was the worst outcome, exhibiting a very high mortality rate (15). for these reasons, we decided to reduce the transplantation - associated risks with complete eradication of the endocarditis and the crkp infection prior to the transplantation, and opted for performing a less invasive surgery rather than a mechanical tricuspid valve, which would require a higher level of anticoagulation. we also planned to use an ric regimen instead of myeloablative conditioning during the transplantation. in conclusion, we herein report a case of a bp - cml patient with a highly resistant, crkp - associated tricuspid valve endocarditis, who was treated with a combination of systemic antimicrobial therapy and surgical valve repair, and subsequently underwent successful allo - sct. to the best of our knowledge, this is the first reported crkp tricuspid valve endocarditis and the first reported case of crkp endocarditis that proceeded to undergo a successful allo - sct in the medical literature. this report demonstrated that extensively resistant crkp endocarditis may be treated with combination therapy without mechanical heart valve replacement in patients with haematological malignancies.	in chronic myeloid leukemia (cml), the occurrence of blastic transformation is rare. treatment outcome is generally poor. allogeneic stem cell transplantation (allo - sct) is the only potentially curative treatment option for advanced - phase cml. infections caused by carbapenem - resistant klebsiella pneumoniae (crkp) isolates are associated with high morbidity and mortality rates, particularly in patients with haematological malignancies. infection and colonization by these multiresistant bacteria may represent a challenge in sct recipients for the management of post - transplantation complications, as well as for the eligibility to receive a transplant in patients who acquire the pathogen prior to the procedure. we herein report the case of a blast - phase cml patient with a highly resistant, crkp - associated tricuspid valve endocarditis, who was treated with a combination of systemic antimicrobial therapy and surgical valve repair, and subsequently underwent a successful allo - sct.
focused assessment with sonography in trauma (fast) is a modality to rapidly detect free fluid (usually blood) in the peritoneal, pericardial, or pleural spaces in trauma patients (1). it can be performed in the emergency department (ed) to provide noteworthy information in a short span of time. therefore, fast exam is applied in blunt trauma algorithms as an initial evaluation procedure. it is performed immediately after the primary survey of the advanced trauma life support (atls) protocol and is the basis for immediate decisions for further evaluation and management of the patient (2). interest and experience with fast grew among surgeons and emergency physicians during the early 1990s, when it was no longer exclusively performed by radiologists (3 - 5). in 2001, american college of emergency physicians (acep) published the first formal comprehensive guidelines ultrasound use in emergency medicine that contained the application of fast as a core application (6). in 2008, the acep offered recommendations for the training of fast by emergency physicians (3). as an ultrasound imaging method, fast is an operator - dependent technique, i.e. the skill of the operator is critically important for correct diagnosis. many studies have shown that trained nonradiologist physicians are capable of performing an expedient fast as accurately as formally trained radiologists (7 - 9). nonetheless, some radiologists believe that a high level of knowledge and experience is needed to perform an accurate and reliable fast in trauma patients (10). the present study was conducted to compare the diagnostic accuracy of fast performed by emergency medicine and radiology residents for the detection of peritoneal free fluid in trauma patients. this prospective observational study was conducted between november 2012 and november 2013 in al - zahra educational hospital (isfahan, iran) of isfahan university of medical sciences. patients of any age or sex admitted to the emergency department (ed) of hospital for blunt abdominal trauma, high energy trauma (box 1) (11), and multiple trauma were deemed eligible for participation in the study. after arrival in ed and a primary trauma survey, fast was carried out by previously trained emrs. patients on whom performing fast would potentially delay emergency procedures, those with penetrating abdominal trauma and preexisting peritoneal fluid, and pregnant women were excluded from the study. the patients were evaluated in supine position with arms abducted slightly or above the head. all scans were done by the same ultrasound machine (dc-7, mindray medical ltd., china) in ed, while a low frequency (5 - 2 mhz) curved array transducer was selected with a focused depth based on the patient 's body. in pediatric patients, a higher - frequency linear array transducer was selected to produce sound waves with adequate depth penetration to obtain better resolution (12). a typical fast examination was performed to obtain the 4 standard views (subxiphoid (pericardial) 4-chamber view, right coronal and intercostal oblique view, left coronal and intercostal oblique view, and suprapubic (pelvic) view) to examine 4 specific areas of the thoracoabdominal region (13). history and physical examination findings were noted and fast scan findings (negative or positive for abdominal free fluid) were collected and documented on a data sheet for each case. any amount of free fluid in any of the 3 windows (hepatorenal, splenorenal, suprapubic) was considered positive for abdominal free fluid (haemoperitoneum). after fast was completed by emrs, the patients were transferred to radiology department and underwent fast by radiology residents (second year radiology residents) using the same technique and machine as emrs. all fast scans were completed within 4 minutes. for the patients whose transfer to rd was dangerous or impossible and those who were hemodynamically unstable, the bedside fast scan was down by emrs and rrs. ultrasound findings for each patient were compared with the results of computed tomography (reported by the attending radiologist), operative exploration, or observation. for definitive imaging of the abdomen in positive or suspected reports of each resident group a ct scan of the abdomen and pelvis with intravenous contrast was performed. in addition, patients with negative fast results were observed for 6 - 12 hours in the emergency department. patients with negative fast scan whose abdominal physical examination was suspicious, underwent ct scan of the abdomen and pelvis at the request of the senior em resident or an attending emergency physician. patients with positive fast scan who were hemodynamically unstable and did not respond to 2 l iv fluid administration while no other source was found for the instability, underwent exploratory laparotomy without further imaging (14). all statistical analyses were performed using the statistical package of social sciences (spss) version 19.0 (spss, chicago, il, usa). to evaluate the diagnostic agreement between the results of the two groups of residents and also between fast results and final diagnostic method, kappa score analysis was run. sensitivity, specificity, positive, and negative predictive values of fast performed by emrs and rrs were calculated and compared using chi - square analysis. one hundred and forty participants with a mean age of 28.5 13.8 years (range 4 - 65 years) were included in this study. about 71% of patients were male. of remaining 138 patients, 17 patients had positive fast scan by one or both resident groups. five patients (with positive fast scan from both groups) had moderate or greater amount of intraperitoneal free fluid in fast and underwent operation without having other diagnostic modalities and laparotomy proved the presence of haemoperitoneum in all cases. in 6 patients with negative fast, finally, fast scan findings were compared to the results of surgical exploration in 5 patients (3.62%), to ct scan in 18 patients (13.04%), and to clinical observation in the remaining 115 patients (83.33%). good diagnostic agreement was noted between the results of fast scans performed by emrs and rrs (= 0.701, p < 0.001), also between the results of emrs - performed fast and the final outcome (= 0.830, p < 0.0010), and finally between the results of rrs - performed fast and the final outcome (= 0.795, p < 0.001), (complete agreement would be equal to a of 1, and chance agreement would be equal to 0) (table 1). sensitivity, specificity, positive, and negative predictive values of fast scans done by emrs and rrs are presented in table 2. there were no differences in these parameters between fast scans performed by emrs and rrs (figure 1). emrs - performed fast and final outcome, kappa = 0.830 and p < 0.001. between rrs - performed fast and final outcome, kappa = 0.830 and p < 0.001. in this study, we compared the diagnostic value of fast scan between rrs and emrs. this investigation showed that er - performed sonography had an acceptable diagnostic value in comparison to rr - performed fast. rapid detection of intraperitoneal fluid in patients with blunt abdominal trauma to select an appropriate approach is highly critical in reducing mortality and improving the outcome. diagnostic peritoneal lavage (dpl), ct imaging, and fast are the main paraclinical modalities to evaluate patients with blunt abdominal trauma. ct scan is the gold standard and provides the evidence of bleeding plus detailed information of injured organ. dpl is sensitive in the detection of abdominal bleeding (16) but is invasive and time consuming and may bring about some complications to patients (17). although it can not replace ct scan, it is highly valuable in the initial evaluation of patients with abdominal trauma and could save the time and provide highly valuable information to make decisions about the further management of patients in ed (13). therefore, it is very important for physicians working in ed to be able to do an accurate and reliable fast. the results of these studies demonstrated a sensitivity of 52% - 100% and specificity of 96% - 99% for fast performed by nonradiologists (18 - 20). some studies have shown that the ability to detect haemoperitoneum using the fast method is equally accurate if it is performed by nonradiologists (nr) and radiologists alike (9, 14). they reported a sensitivity of 73% and specificity of 97% for surgeon - performed fast and sensitivity of 79% and specificity of 99% for radiologist - performed fast. evaluated the accuracy of fast done by nonradiologists and compared it to radiologists - performed fast in the emergency department of a trauma center in india. sensitivity values of fast done by nr and rr were 100% and 95.6% and specificity was 97.5% in both groups (8). the results of our study suggest that the accuracy of fast performed by trained emrs is comparable to rrs performed fast. reported a sensitivity of 78% and specificity of 99% for fast performed by uk emergency physicians (7). ingeman. reported that fast performed by emergency physicians had a sensitivity of 75%, a specificity of 96% and an accuracy of 91% (19). in our study, sensitivity and specificity for rrs - performed fast were 84% and 97%, respectively. currently, fast is being done by residents and specialists of radiology, emergency medicine, and surgery in iran. shojaee. evaluated the accuracy of fasts performed by emrs with respect to the detection of abdominal free fluid following blunt trauma and compared it to rrs performed fasts. both rrs- and ers - performed fasts had about 60% sensitivity and 99% specificity in comparison to the final outcome based on the findings of abdominal ct scans and clinical follow - up (21). kakaei. evaluated the role of fast in assessing injured people in iran 2012 earthquake and reported that sensitivity of fast did not change when it was performed by rrs, but its specificity increased (compared to em and surgery residents) (22). therefore, the results of our study are comparable with previous studies conducted in iran and other countries. all emrs in hospitals affiliated to isfahan university of medical sciences have substantial training in fast as a part of residency training programs, including didactic sessions and practical examinations, each with at least 200 fast examinations at the end of the first year of residency (according to the log book). it has been suggested by most investigators that sensitivity and specificity begin to plateau after 25 to 50 fast exams (23 - 25), while some others have recommended 200 examinations (26). in addition, during the second year of residency training, they are required to complete 1 month training in an em radiology rotation. hence, it is no surprise that emrs of this center provide accurate fast exams. first, emrs were more involved with the patients and had more information about them which could affect their performance in fast. second, we could not perform ct scan as gold standard for all patients. and finally, the timing of fast performed by each group of residents is different in this study. emrs performed fast just after patients arrival to ed (after primary survey) and the patients were transferred to rd after the primary evaluations in the ed. regarding the role of time in the fluid accumulation it could affect the result of fast scan. although the patients were transferred to radiology department as soon as possible to eliminate this effect, this limitation is inevitable when we decide to perform the fast scan on the same subjects by both groups of residents. further studies with separate subjects for each group of residents or doing serial fasts on patients may practically eliminate the effect of time on the performance of each group. we conclude that, trained emrs have the ability to perform fast scan with high diagnostic value (specificity, sensitivity, ppv, and npv), similar to rrs for the detection of haemoperitoneum in patients with blunt abdominal trauma.	background : focused assessment with sonography in trauma (fast) is a method for prompt detection of the abdominal free fluid in patients with abdominal trauma.objectives:this study was conducted to compare the diagnostic accuracy of fast performed by emergency medicine residents (emr) and radiology residents (rrs) in detecting peritoneal free fluids.patients and methods : patients triaged in the emergency department with blunt abdominal trauma, high energy trauma, and multiple traumas underwent a fast examination by emrs and rrs with the same techniques to obtain the standard views. ultrasound findings for free fluid in peritoneal cavity for each patient (positive / negative) were compared with the results of computed tomography, operative exploration, or observation as the final outcome.results:a total of 138 patients were included in the final analysis. good diagnostic agreement was noted between the results of fast scans performed by emrs and rrs (= 0.701, p < 0.001), also between the results of emrs - performed fast and the final outcome (= 0.830, p < 0.0010), and finally between the results of rrs - performed fast and final outcome (= 0.795, p < 0.001). no significant differences were noted between emrs- and rrs - performed fasts regarding sensitivity (84.6% vs 84.6%), specificity (98.4% vs 97.6%), positive predictive value (84.6% vs 84.6%), and negative predictive value (98.4% vs 98.4%).conclusions : trained emrs like their fellow rrs have the ability to perform fast scan with high diagnostic value in patients with blunt abdominal trauma.
about 20%30% of the patients with tle develop medication resistance and may benefit from a surgical treatment. for patients with medically refractory tle, surgical excision of the affected temporal lobe while anterior temporal lobectomy (atl) has proven to be effective in the treatment of tle patients resistant to medical therapy, successful surgical outcome depends not only on an accurate localization of the epileptogenic focus, but also on the ability to map and preserve the eloquent cortex. in particular, the lateralization and localization of language cortex are of paramount importance for atl in the dominant hemisphere. as demonstrated by haglund., the distance from the language cortex to the resection boundary is the most important predictor of developing postoperative language deficits. currently, invasive tests such as the intracarotid amobarbital procedure (iap, also called the wada test) and electrocortical stimulation mapping (esm), both of which present the patient with additional risks, are the common means for lateralization and localization of the functional regions in brain. noninvasive imaging techniques may reduce the need for such procedures, and, in particular, functional magnetic resonance imaging (fmri) is an alternative to replace these invasive procedures for surgical planning of the patients with tle. in the following section, we review the basics of fmri, while sections 3 and 4 review previous studies comparing fmri to the wada test for language lateralization and fmri to esm for language localization. in section 5, we discuss the role of fmri in studying brain plasticity for language function after temporal lobectomy (tly). fmri employs the blood - oxygenation - level - dependent (bold) contrast mechanism as an indirect measure of underlying neuronal activity. when certain functional areas in brain are activated, there is an increase in local metabolism and oxygen consumption. because of the neurovascular coupling between regional changes in brain metabolism and cerebral blood flow (cbf), the activated local areas in the brain experience a decrease in oxyhemoglobin and an increase in deoxyhemoglobin in the postcapillary vascular bed. since hemoglobin has different magnetic properties depending on its state of oxygenation, being diamagnetic when oxygenated and paramagnetic when deoxygenated, these oxygen - related changes to the blood lead to local magnetic field inhomogeneities, which in turn result in detectable changes in the magnetic resonance signal measurable by mri. to elicit the activated functional areas in brain using the bold signal effectively and robustly, most fmri studies employ a block design, in which tasks are alternated to generate brain response in different states (in the simplest form, two states : rest versus activation). event - related designs have also been employed in the last few years to examine language function and lateralization. in contrast to a block design, in which the conditions are alternated within a block (resting, task1, resting, task2) with each block having a fixed duration, in an event - related design, events of different types are randomly intermixed. some studies have shown a more robust activation of language areas in event - related than in block design paradigms. because the magnetic signal changes caused by the hemodynamic behaviour are usually very small, the selection and design of the experimental paradigm is of utmost importance. various auditory and visual stimulation paradigms have been developed to examine the language functions, and since language tasks are most likely to activate not only language areas but also other functional regions, the experimental paradigm requires controlled tasks that activate nonlanguage cortex equally. a common example used for language study that can be performed easily in patients for lateralization purposes is the verb generation and verbal fluency task. in this type of design, participants are instructed to covertly generate action verbs for each noun (e.g., for the word knife one can think to cut, to slice, to throw). the resting phase involves participants looking at a cross in the center of the screen while not actively engaging in any language function. alternatively, subjects may be presented with nonsense collections of letters to washout the nonlinguistic aspects of the task. this task provides relatively consistent activation of the anterior language areas (figure 1). another commonly used language task employed at our institution is a sentence completion paradigm participants are informed they will see a sentence on the screen, which they must covertly complete, such as i called the, women wear, we cleaned, task demonstrates more widely distributed networks, including the anterior and posterior language areas. in this type of design, for example, the subjects are instructed to compare the samples in terms of their meaning and to select two out of three words that are most alike. the most commonly used imaging sequence in fmri studies is echo planar imaging (epi), due to its fast acquisition time. however, the temporal resolution of such a sequence is in the order of several seconds, as it requires some time to produce detectable hemodynamic changes after stimulus onset and the spatial resolution is usually significantly lower than that of anatomical mri. another drawback is that epi is sensitive to field inhomogeneities, leading to geometric distortion of the images in certain brain regions. since the bold signal is extremely sensitive to motion, one of the common problems during any fmri experiment is subject motion, which can compromise the entire experiment. motion can range from gross head movement to the minimal brain motion associated with cardiac or respiratory cycles, a.k.a. the cardioballistic effect. before any subsequent analysis can be performed, the individual images are commonly realigned and coregistered to minimize the motion effect. next, the signal time course in each voxel of the images and the time course of different tasks are correlated using statistical modeling. various statistical tests (e.g., student 's t - test) can then be applied on a voxel - by - voxel basis to examine the probability that a particular voxel with an increased signal is associated with a particular functional state of the brain. the result of this process is a map of the voxels that show statistically significant changes associated with the brain function under investigation. this step is critical since the fmri signal changes are usually very small (in the order of 0.5%5%), leading to a high probability of false negative results. to better appreciate the anatomical location of the origin of the signal, these statistical maps are usually registered and fused to a high - resolution anatomical image (figure 2). as described above, fmri analysis requires considerable mathematical, statistical, and image processing that is provided by a variety of free or commercial software packages, such as statistical parametric mapping (spm) (http://www.fil.ion.ucl.ac.uk/), fsl (http://www.fmrib.ox.ac.uk/fsl/), afni (http://afni.nimh.nih.gov/afni), and brain voyager (http://www.brainvoyager.com/). the wada test consists of unilateral injection of sodium amobarbital into the internal carotid artery (ica), which temporarily anaesthetizes the hemisphere ipsilateral to the injection site. while one hemisphere is anaesthetized, language and memory functions of the hemisphere contralateral to the injection site can be tested. to test cerebral dominance for language, the patient is asked to perform a number of tasks involving expressive and receptive language. for example, tasks involving counting numbers, naming the months of the year, or naming objects are often used to examine frontal language areas, while repetition, responding to verbal commands, and reading are employed to explore language functions served by the posterior brain regions. these protocols can be adapted or simplified for children or mentally challenged individuals, as long as they possess expressive language and are able to understand the instructions. the test is administered before the injection to provide a baseline measure and is repeated after the anaesthesia has taken effect. if the hemisphere dominant for speech is anaesthetized, the patient is temporarily rendered mute, which is not the case when the nondominant hemisphere is deactivated. when each hemisphere retains some language functions following unilateral injection, bilateral representation is confirmed. although the wada test has long been considered as the gold standard for preoperative language and memory testing, the method has several drawbacks, since it is highly invasive and quite uncomfortable for most patients, and there is a small risk of morbidity. figure 3 shows axial t2 (a) and axial diffusion - weighted (b) images of a patient with medically intractable tle of left temporal lobe origin who had experienced a right middle cerebral artery stroke secondary to a traumatic dissection of the right ica during the wada test. another major drawback for the wada test is that the procedure requires the patient to respond verbally, making it difficult to obtain reliable results from young children and mentally challenged patients. finally, the wada test only provides information about lateralization, but not localization of cognitive functions. these important limitations have led many epilepsy centers to seek alternative means to probe language and memory functions in patients prior to epilepsy surgery. a number of studies have examined the utility of fmri as compared to the wada test for language lateralization in patients with various neurological conditions, with reports being related specifically to epilepsy and tle. one of the first published studies to compare fmri with the wada test in patients with tle was described by desmond., where the authors employed a semantic task during which the patients pressed a button when they recognised a word as being abstract as distinct from being concrete. the hemispheric language laterality index was calculated using the formula (1)li=(vlvr)(vl+vr)100, where vl and vr are activation volumes for the left and right hemispheres, respectively. found that the fmri laterality indices were in agreement with the wada test in all of their seven patients. used a semantic decision task, in which the patients pressed a button if they heard a word that was both the laterality indices were in concordance with the wada test for all of their 22 patients with epilepsy. following these two studies, bahn. studied four epilepsy patients with two relatively simple covert word generation tasks and found 100% concordance between fmri and the wada test in all their four patients with epilepsy. in a study conducted by yetkin., a group of 13 patients with medically intractable epilepsy were tested with a verbal fluency task. good concordance between fmri and the wada test with the laterality correlation coefficient of 0.96 between the two techniques was reported except for one patient. studied ten patients with tle using a panel of language paradigms including verbal fluency, story - listening, and sentence repetition tasks. they found that fmri laterality indices were highly concordant with the wada test in the frontal lobe, although this was not the case in the temporal lobe. moreover, they found that a verbal fluency task was more effective than other tasks in achieving better correlation with the wada test. while the previous studies have shown promising concordance between fmri and the wada test, the patients in these studies had mainly unilateral dominant epilepsy. enrolled seven patients with unilateral and six with bilateral tle to investigate the predictive power of fmri for language lateralization. they concluded that using a combination of language tasks that included verb generation, verb fluency, picture naming, and sentence comprehension, they were able to better predict the lateralization of these patients. another study by sabbah. also tried to investigate the effectiveness of fmri in lateralization of atypical language in patients with epilepsy. by using two different semantic fluency paradigms, they reported that 19 out of 20 patients had concordant lateralization result from both fmri and the wada test. comparing fmri - based and wada - based laterality quotients for speech in tle patients, benke. found agreement in 89.3% of right tle patients and in 72.5% of left tle patients. however, while fmri correctly detected atypical right hemisphere speech in all cases, it missed left hemispheric dominance of speech in 17.2% of patients with tle. furthermore, the method was less sensitive to bilateral speech representation. while higher magnetic field strength is desirable for fmri studies, this may not be necessary for language lateralization. for instance, deblaere. used a simple word generation paradigm in an 1.0 t mr scanner and showed that fmri reliably lateralized the language function in a clinical setting. although most of the previous studies focused on expressive speech, gaillard. conducted an fmri study in which a combination of expressive and receptive language paradigms was employed. they reported that there was a complete agreement between the wada and fmri in 21 of 25 patients and that the use of multiple tasks increased the accuracy in determining hemispheric dominance for language function. to date, the largest study on comparison between fmri and the wada test was conducted by woermann., who enrolled 100 patients with epilepsy, of whom 69 had tle. they employed a single covert word generation language paradigm and derived the laterality based on visual inspection of fmri statistical maps. concordance of 91% was observed, and the discordance may have been the result of visually assessing often bilateral, although mainly asymmetric fmri activations. generally, fmri and the wada test agree very well with each other. because of the relative advantages of fmri, more and more epilepsy centers are replacing the wada test with fmri for presurgical language mapping. however, we also note some divergent results from the previous studies, which can be attributed to several factors. first of all, the fmri paradigms used to evaluate language lateralization vary considerably between studies. most of the paradigms are capable of activating the left frontal lobe in healthy control subjects and patients with typical representations for language functions. in patients with atypical representations for language, the use of multiple language tasks increases the likelihood of accurate lateralization of the cerebral hemispheres for language functions. other limitations of fmri for language mapping include the variability in statistical thresholds employed to calculate the activated volumes, difficulties in determining the extent to which the right hemisphere participates in language processing in patients with bilateral representations for language networks, and because not all areas involved in a task may be activated by a particular fmri paradigm. another disadvantage is that the patients must lie motionless in the scanner during image acquisition, making this technique less suitable for children and other special populations. one of the limitations of the previous fmri studies in language mapping of patients with epilepsy has been their small sample sizes, resulting in low statistical power. while the largest study enrolled over 100 patients with epilepsy, even for a study with this size, it is difficult to distribute the subjects uniformly over the different subgroups of epilepsy. for example, some studies may have more patients with typical dominance for language than patients with atypical dominance for language. systematic review with meta - analysis is one statistical technique that may overcome the small sample size problem faced by many clinical studies. meta - analysis combines the results of several studies that address a set of related research hypotheses, with the general aim being to more powerfully estimate the true effect size as opposed to a smaller effect size derived in a single study under a given single set of assumptions and conditions. one example is a meta - analysis of english speaking healthy adult subjects performed by vigneau. they separated the contrasts into three groups phonology, semantics, and sentence processing, and were able to find the relative clusters associated with each group. a similar study was performed by medina. who combined several related studies to investigate the role of fmri to replace the wada and esm tests for epilepsy surgery using the bayesian analysis approach. they concluded that the use of fmri increased the final posttest probabilities of hemispheric dominance for language in patients with epilepsy. this procedure involves direct application of electrical currents to the cerebral cortex of the awake patient, which either produces a response (e.g., movement) or disrupts function (e.g., speech arrest). esm is believed to directly identify cortex essential for a specific task and can be performed extraoperatively in patients with implanted electrodes or intraoperatively with the cerebral cortex exposed at surgery. tool for localization of language function during tly. figure 4 demonstrates the esm stimulation sites from 13 tle patients who underwent surgery, normalized to mni space, and fused with the mni the red dots concentrated around the inferior frontal gyrus in the frontal lobe represent broca 's areas, while the cluster of red dots situated near the superior temporal gyrus represent wernike 's area. although it is widely accepted as the most reliable method of localizing language functions in the brain, esm has several major drawbacks, including invasiveness, low spatial resolution due to limited sampling of the exposed cortex intraoperatively, the potential to induce epileptic after - discharges, as well as prolonging the operating time. moreover, this technique is not always feasible in clinical practice, since it requires full collaboration of the patient as well as the clinical expertise of the surgical team. in contrast to esm, fmri offers several appealing features for language localization, including noninvasiveness, preoperative data that can be employed to guide surgery and lead to reduced operating time, and superior spatial and temporal resolution for whole brain analysis. accordingly, some studies have investigated the utility of fmri to replace esm for preoperative function localization in tle surgical planning [2429 ]. one of the first studies that compared fmri and esm included 28 patients, of whom 22 had epilepsy. two language tasks, number counting and word generation, which consistently activated mostly the inferior frontal lobe, were employed in this study, with results indicating a correspondence within 2 cm between fmri and esm using the word generation task. another early investigation to compare fmri and esm was performed by fitzgerald., who employed an array of five fmri language tasks using both auditory and visual input to probe the language areas of 13 patients (one with epilepsy and eight with brain tumors). the sensitivity and specificity of fmri for identifying language regions were calculated based on the esm results. concluded that the combination of language tasks was more effective than any single task and that fmri was sufficiently sensitive to localize the language areas in these patients. schlosser. studied 33 patients with tle, brain tumour, and avm, using a single auditory comprehension task during the course of the study. they showed in 23 patients a consistent fmri activation that was similar to that observed in healthy control subjects. because in some patients the lesions were remote from the language areas, only 16 received intraoperative esm for language localization. while four of their five tle patients had a successful matching between fmri and esm, their correlation studies were rather qualitative and not discussed in detail. although some fmri language paradigms have been developed and shown to activate language networks consistently in the normal population, it is expected that neurological disorders could affect the organization of the network and make these paradigms less effective in this population. carpentier. conducted a study in which a control group was compared with the diseased cohort to determine how the neurologically impaired group performed relative to the normals. while they found that their language tasks (visual and auditory comprehension) were able to successfully activate both broca 's and wernike 's areas in the healthy control group, the same array of language tasks resulted in lower activation and greater bilateral representations in the diseased group nevertheless, the concordance between fmri and esm was 100% (15 positive language sites identified by esm matched with fmri activation within a 1 cm range). while a single language task activated some aspects of language network to achieve a good correlation with esm in some of the previous studies, it is unlikely that this limited approach could fully explore the complete language networks. therefore, several studies have been conducted to investigate the utility of multiple tasks to increase the likelihood of concordance between esm and fmri. pouratian. employed a battery of linguistic tasks, including visual object naming, word generation, auditory responsive naming, visual responsive naming, and sentence comprehension to identify language areas of 10 patients with vascular malformation. they found fmri to be very sensitive, but rather unspecific, in identifying which cortical areas are essential for language. employed a battery of language tasks (i.e., verb generation, picture naming, verb fluency, and sentence comprehension) in their study and found that a single task could not elicit all the critical language areas that are elicited by esm. they demonstrated that despite high sensitivity, on average only 51% of fmri sites were confirmed by esm. in contrast, in 10 out of 11 patients, the absence of fmri activity was 100% concordant with the absence of critical language areas in esm. roux. performed a study in which naming and verb generation tasks were administered to a group of 14 brain tumour patients. they also found that by combining the two language tasks they were able to increase sensitivity to 59% and specificity to 97%. eight of their patients underwent postoperative fmri, but only three of them showed an agreement between preoperative fmri, postoperative fmri, and esm. in general, good but not complete agreement between fmri and esm can be achieved when using a combination of carefully designed language paradigms. first of all, while the choice of language paradigm is of outmost importance to effectively activate the language network, there is still no consensus as to what combination of language paradigms is the most suitable for language mapping in an fmri study, and this topic remains an active area of research. furthermore, the classical human language system model consists of two primary regions : the expressive broca 's area in the posterior inferior frontal lobe, and the receptive wernicke 's area in the posterior superior temporal lobe. it has been shown that certain language tasks can activate broca 's area more effectively than wernike 's region. therefore, it is expected that the sensitivity of fmri to map language areas, and the concordance between fmri and esm may be task and lobe dependent. however, most studies performed so far have not examined the concordance at the lobe level. the significance threshold is used to define the extent of language activation from the fmri studies. a very strict threshold would result in very poor correlation (high type ii error) while a relaxed threshold would produce false positive correlations (high type i error). the selection of threshold has usually been subjective and based on the previous experiences in these studies, and this variation may be partially responsible for the different results obtained. in addition, using a single fixed threshold across multiple subjects does not take into account the variability of activated language areas among individuals [25, 30 ]. an alternative approach to this problem is to apply an adaptive threshold that, based on the physiological observation of the extent of the language area, confines the area to be around 1 to 2 cm on the cortical surface. while this approach makes the comparison more consistent for all patients, in practice this can be very laborious. finally, different registration approaches are used to align esm and fmri maps for comparison purpose. in several studies [23, 27 ], 2d esm maps captured intraoperatively using a camera were manually correlated to the 2d fmri maps and visually inspected, an approach that is considered to be subjective and qualitative. in contrast, other studies employed a relatively simple 2d-2d automatic rigid registration [24, 25, 28 ]. however, since 2d-2d registration does not account for the perspective effect in the 2d camera images, errors of several mm can remain in the registration. however, such devices are not always available in many procedures, while simply imaging exposed cortical surface with a digital camera is a very common practice and requires less time to perform. an additional complicating effect is that the brain can deform up to 10 mm after opening the dura matter. without correction for this deformation, the accuracy of the reconstruction of the 3d coordinates of the stimulation sites is compromised by using the current navigation systems. figure 5 demonstrates our approach, which employs automatic 2d to 3d projective registration to fuse an interoperative cortical photographic image onto an mr brain image that incorporates a preoperative fmri activation map. in, figure 5(a) the label f is a speech arrest site elicited by esm that corresponds well to the peak of fmri activation in the frontal lobe as shown in figure 5(b). another area in which fmri can play a role in the treatment of tle is to study the language plasticity and reorganization before and after surgery. previous fmri studies have shown that atl had a differential impact on the language functions of the left and right tle patients. wong. studied a group of 24 tle patients who underwent atl procedures to examine the impact of atl on the cortical organization of language processing, using a verb generation task on both left and right tle patients to compare their preoperative and postoperative fmri response. after the surgery, the right tle patients activated the same cortical network as before the surgery, while the left tle patients elicited less activation. a subtraction analysis between the preoperative and postoperative bold response showed that the right inferior frontal gyri (ifg) and the left middle frontal gyri (mfg) were less activated after the surgery in the left tle patients. the attenuated correlation between the language scores and the postoperative bold response within the ifg and mfg in both patient groups indicated cortical reorganization after the atl. these findings suggest that the cortical organization of language processing is affected differently by the left and right tle and is subsequently reorganized after atl. another finding of this study is that the right tle patients shifted the correlation between their language scores and bold signals from the typical language areas (i.e., ifg and mfg) to the anterior cingulate cortex (acc) after atl. fmri is a noninvasive technique that has replaced the invasive tests for presurgical assessment of the language network in some epilepsy centers. however, there are still some requirements that fmri must meet to enable its wider utility in the clinical practice for language mapping. first, a highly effective language paradigm or a battery of paradigms needs to be developed that can be administered to the patients in a clinically feasible time. second, a robust statistical analysis methodology that removes individual biases (in selecting threshold levels, e.g.) must be developed. finally, because the population being studied is, in general, atypical with respect to language localization, a high predictive power is required for critical language areas in the brain. these issues are currently being addressed one by one. with the availability of higher field strength mris, faster imaging sequences, better study paradigms, and improved postprocessing tools	functional magnetic resonance imaging (fmri) is a noninvasive technique that is increasingly used to understand the cerebral cortical networks and organizations. in this paper, we describe the role of fmri for mapping language networks in the presurgical workup of patients with medically intractable temporal lobe epilepsy (tle). studies comparing fmri with the intracarotid sodium amobarbital (wada) test and fmri with intraoperative cortical stimulation mapping for language lateralization and/or localization in medically intractable tle are discussed.
a postgraduate medical entrance examination (pgee) is the qualifying examination that is offered to the medical graduates willing to get enrolled in postgraduate programs. any pgee that aims at selecting candidates with highest academic and professional excellence must incorporate different measures to evaluate the knowledge (cognitive domain), skills (psychomotor domain) and attitudes (affective domain) of an aspirant. various methods of assessment have been employed from time to time by institutions and universities across the globe to fulfill these expectations. in india, however, thinking abilities of the candidates in the cognitive domain (knowledge) alone are mostly evaluated in all kinds of pgees. multiple choice questions (mcqs) are the usual kinds of items that are presented in these examinations to accomplish this objective. it is generally presumed that mcqs, if framed carefully, can assess one 's thinking skills in all the six levels of knowledge domain, namely, 1. these levels actually represent the degrees of difficulty : i.e., the lower levels being simpler and higher levels being more complex and difficult. this categorization of levels from lower order to higher order in the knowledge domain is one of the vital components of bloom 's taxonomy. bloom 's taxonomy of educational objectives happens to be one of the most often used models while designing the different training, learning and examination methods. benjamin bloom in order to promote higher forms of thinking in education, such as analyzing and evaluating concepts, processes, procedures, and principles, rather than just remembering facts,,. though there are many arguments highlighting the limitations of mcqs, they are the most commonly used testing items for undergraduate and postgraduate medical entrance examinations. it is generally accepted that mcq testing is an efficient, objective and reliable way of assessing the abilities in the cognitive domain. however, in the current scenario of ayurveda education, the picture appears to be quite different. during the postgraduate education (after completing which, the students become eligible for teaching assignments), the evaluation and assessment skills do not form a part of training in general. further, there exists no formal mechanism to train ayurveda teachers in framing mcqs. a few of the previous studies have shown that graduate level of ayurveda education is more memory - oriented than being competence based,,,. therefore, the kind of postgraduate scholars who get enrolled in different institutions through competitive process often may not meet the expected levels of academic and professional excellence. keeping this possibility in view, we conceived the present study with an objective of evaluating the levels of thinking skills that are actually assessed through mcqs in different pgees of ayurveda, conducted in different universities across india. this is a retrospective observational study aimed at evaluating the ayurveda pgee question papers of last five years (20102014). we divided india into four conventional zones : north, west, east and south. we consulted the online repositories of pgee question papers such as www.ayurvedpg.com and www.liveayurved.com, and also the specific websites of different universities spread across india, to access the authentic papers. we ensured the authenticity of the question papers by choosing to include the original scanned copies only. pg entrance guides available in the market deliberately because the authenticity of the question papers given in these books is often unreliable. the total number of questions presented in pgees in different universities was not constant ; it varied from 80 (in jodhpur ayurveda university jau) to 250 (in maharashtra university of health sciences - muhs) per year. however, except for muhs, the average number of questions being presented per year was between 100 and 200 in most of the universities. the guiding principle followed in this study was either to have full sets of question papers for five years from each zone (north, south, west and east), or, to have at least 700 questions for five - year period from each zone. (the number 700 was arrived at after taking the average of 100 and 200 questions, i.e., 150 questions per year for 5 years, and then allowing a deletion rate of 10 questions per year for possible ambiguity). whenever we could not access the full sets of question papers from a single university for 5 years, we clubbed those question papers with other universities in the same zone so that we could achieve our target number. finally, seven different universities spread across north, west, east and south zones of india were shortlisted : banaras hindu university (bhu), dr. sarvapalli radhakrishnan rajasthan ayurveda university (dsrrau), gujarat ayurveda university (gau), maharashtra university of health sciences (muhs), rajiv gandhi university of health sciences (rguhs), university of jammu (uj), and uttarakhand ayurveda university (uau). though the prescribed action verbs were taken into consideration during this exercise to assign the bloom 's taxonomy level, they did not serve the purpose completely since we were assessing only mcqs. therefore, a group of two investigators, previously trained in the concept of bloom 's taxonomy, read out aloud each of these mcqs and came to a consensus unanimously regarding the level of thinking skills the particular mcq assessed. whenever there was a lack of consensus, a higher level was agreed upon among the two levels proposed. ambiguous and incorrect table 1 shows the details of zonal distribution, number of questions, number of question papers, and the specific universities that were included in the study. it also shows the number of questions included in each year as per different universities in each zone. the table 2 shows the assigned bloom 's taxonomy levels of the studied sample of questions. we observed that out of 3299 mcqs, 3079 (93.3%) assessed only the application (level-3) was only 0.3% (11), whereas the percentage of mcqs that assessed the analysis component (level-4) was a mere 0.2% (5). it may be noted that there was not even a single question to assess the synthesis and evaluation (level-5 and level-6) components for the entire five - year period in our sample. we observed that 78.9% of the questions were from ayurveda subjects and 21.1% were from the current biomedical sciences. among ayurveda subjects, a significant number of questions were from chikitsa (14.5%), siddhanta (11.3%), dravyaguna (8%), shaarira (7.5%) and vikriti vijnyana (7.1%). the subjects that contributed most to the biomedical sciences category were medicine (5.6%), physiology (3.8%), anatomy (2.7%) and surgery (2.5%). however, it was interesting to note that only 2.5% of questions were related to the integrative knowledge of both current biomedical sciences and classical ayurveda. it was further interesting to note that, out of all questions evaluated, there were 28.8% of questions that required the candidate to know a specific reference / chapter of a specific classical ayurveda textbook, and 13.4% of questions required the candidate to know the numerical values such as the correct number of shastra karma (surgical procedures), the correct number of chapters in a given ayurveda textbook, etc. the table 2 shows the assigned bloom 's taxonomy levels of the studied sample of questions. we observed that out of 3299 mcqs, 3079 (93.3%) assessed only the application (level-3) was only 0.3% (11), whereas the percentage of mcqs that assessed the analysis component (level-4) was a mere 0.2% (5). it may be noted that there was not even a single question to assess the synthesis and evaluation (level-5 and level-6) components for the entire five - year period in our sample. we observed that 78.9% of the questions were from ayurveda subjects and 21.1% were from the current biomedical sciences. among ayurveda subjects, a significant number of questions were from chikitsa (14.5%), siddhanta (11.3%), dravyaguna (8%), shaarira (7.5%) and vikriti vijnyana (7.1%). the subjects that contributed most to the biomedical sciences category were medicine (5.6%), physiology (3.8%), anatomy (2.7%) and surgery (2.5%). however, it was interesting to note that only 2.5% of questions were related to the integrative knowledge of both current biomedical sciences and classical ayurveda. it was further interesting to note that, out of all questions evaluated, there were 28.8% of questions that required the candidate to know a specific reference / chapter of a specific classical ayurveda textbook, and 13.4% of questions required the candidate to know the numerical values such as the correct number of shastra karma (surgical procedures), the correct number of chapters in a given ayurveda textbook, etc. our study confirms the notion that ayurveda education, in general, is more memory - oriented than being competence based. this is because, the recall - based questions dominate the present ayurveda pgees, irrespective of the region and university. many of the categories of mcqs, such as those focusing on the numeric values, may not have much clinical applicability. even remembering the specific references and specific chapter numbers of a textbook, may not help in gaining practically useful knowledge because the searchable digital versions of samhitas are already available. therefore, the results of our study suggest that a higher order of cognition is not being assessed in these examinations and hence, the system seems to have been designed to specially select the students with only a good ability to memorise over those who have critical thinking skills to pursue postgraduate programs in ayurveda. the standards of ayurveda education are being challenged over the past few years repeatedly by the experts, researchers and policy makers,,,,,,,. the seriousness of this challenge is reflected by the fact that the governing body, central council of indian medicine (ccim), has repeatedly attempted to introduce numerous changes to the curricula of graduate and postgraduate programs during last ten years or so the situation is of real concern because, the education system, when not healthy, is likely to produce mediocre physicians and teachers. mediocrity not only dilutes the present day standards, but also gets perpetuated among the coming generations of students. in this context, selecting academically and professionally excellent students for pursuing postgraduate programs becomes essential to keep up the standards of education and practice. it must be noted that, till recently, there existed no mechanism to train ayurveda teachers in examination and evaluating skills, including the preparation of mcqs. the recent initiative, training of teachers, taken up by the ccim is an admirable step in this regard. we propose that such training programs must include the following topics and that all the teachers must be trained in these domains on a compulsory basis : a. fundamental principles of assessment in medical education, b. good practices of question paper setting, c. good practices of preparing multiple choice questions, d. good practices of conducting viva voce examination, and e. good practices of conducting practical examination. it has been shown in a study that effective faculty development programs are useful in training the teachers in framing good mcqs. there is in fact, a scarcity of standard peer - reviewed scholarly curriculum - oriented textbooks in ayurveda. there are ambiguous contents and inter - author differences in the currently available textbooks. because of the compulsion to provide evidence in the form of references to substantiate the proposed answer in the answer key, the question paper setters often prefer playing safe by asking recall - based questions to stay away from being dragged into any such controversies. further, there is also a dearth of literature related to effective teaching, learning, and evaluation methods relevant for ayurveda. therefore, there is a need for generating literature related to methods in ayurveda education while also sensitizing the teachers regarding these issues. in the following section we provide examples for mcqs that assess various levels of thinking skills in the cognitive domain of bloom 's taxonomy, taking the applied kriya sharir (ayurveda physiology) as the area of study. we have also taken into consideration the need for integrative approach while framing these items. we have also explained the mental processes involved in solving these questions and the reasons for assigning the specific levels of bloom 's taxonomy. the two types of pliha vriddhi (splenic enlargement) described by chakrapani are : a.mridu vriddhi (soft enlargement) and kathina vriddhi (hard enlargement)b.sashoola vriddhi (painful enlargement) and ashoola vriddhi (painless enlargement)c.chyuta vriddhi (splenic displacement) and achyuta vriddhi (splenic enlargement in situ)d.ghana vriddhi (solidified enlargement) and aghana vriddhi (liquefied enlargement) mridu vriddhi (soft enlargement) and kathina vriddhi (hard enlargement) sashoola vriddhi (painful enlargement) and ashoola vriddhi (painless enlargement) chyuta vriddhi (splenic displacement) and achyuta vriddhi (splenic enlargement in situ) ghana vriddhi (solidified enlargement) and aghana vriddhi (liquefied enlargement) the correct option in this case is c. the common words that are used to evaluate the recall component are : define, label, state, list, match, write, recall, name, underline, repeat, choose, record, mark, identify and recognize. to answer this question correctly, a student needs to recall the classification of pliha vriddhi (splenic enlargement) as described by chakrapani in charaka samhita, chikitsa sthana, 13th chapter. thus, this question requires the knowledge of classification and nomenclature, which is of first level in bloom 's taxonomy. b. understanding (level-2) based on which of the following information you can draw the relationship between vrikka according to ayurveda physiology?a.vrikka is related to meda, and sveda is also related to meda. udaka means water.d.all the above options are wrong and there is no relationship between vrikka and water homeostasis. vrikka is related to meda, and sveda is also related to meda. the functions of sveda and mutra are related to kleda. all the above options are wrong and there is no relationship between vrikka and water homeostasis. the correct option in this case is a. to answer this question correctly, the student needs to identify the following relationships : 1. further, the student needs to recall that the sweat and urine represent the two important routes of water excretion. the student further needs to identify that the following distracting phrases, i.e., vrikka is said to drain the water into basti and vrikka is a synonym of kloma are incorrect. the common words that are used to evaluate the comprehending ability are : describe, rephrase, restate, discuss, explain, express, locate, tell, indicate, condense, outline and report. this item actually requires the student to mentally outline and restate the concept of water homeostasis in ayurveda. c. application (level-3) praseka (excessive salivation) may manifest as a feature of kapha vriddhi, and dhattura (datura metel) has been described to possess now, choose the most appropriate statement among the following : a.dhattura can not block excessive salivation because salivation is not a muscarinic actionb.dhattura may be effective in conditions such as parkinson 's disease and motion sickness because its ' chemical constituents are muscarinic antagonistsc.dhattura can block salivation because salivation is mediated through nicotinic receptorsd.dhattura can cause contraction of gut smooth muscles and can stimulate the secretion of hcl because they are muscarinic actions dhattura can not block excessive salivation because salivation is not a muscarinic action dhattura may be effective in conditions such as parkinson 's disease and motion sickness because its ' chemical constituents are muscarinic antagonists dhattura can block salivation because salivation is mediated through nicotinic receptors dhattura can cause contraction of gut smooth muscles and can stimulate the secretion of hcl because they are muscarinic actions the common words that are used to evaluate the application ability are : apply, use, demonstrate, illustrate, chart, solve, operate, implement, practice, employ, dramatize and show. to answer this question, a student needs to know that the plant described (d. metel) contains alkaloids such as atropine and hyoscine. the student must have an understanding that salivation is a muscarinic action and occurs in parkinson 's disease and in motion sickness. this is how the student 's ability to remember, understand and apply the knowledge in a new situation is tested. all other options are factually incorrect, and hence, the correct option in this case is b. this item expects the student to actually use and apply the understanding related to the physiology of autonomic nervous system and the pharmacology of d. metel. d. analysis (level-4) ayurveda employs kedari - kulya and khale - kapota theories to describe the processes of the tissue nourishment. considering the nature of energy expenditure involved, these processes can be categorized into : a.sympathetic and parasympathetic effects respectivelyb.passive and active transportation respectivelyc.inspiration and expiration respectivelyd.systole and diastole respectively sympathetic and parasympathetic effects respectively passive and active transportation respectively inspiration and expiration respectively systole and diastole respectively the correct option in this case is b. the common words that are used to evaluate the analyzing ability are : distinguish, discriminate, analyze, compare, contrast, diagram, differentiate, relate, classify, examine, categorize. to arrive at the correct answer, the student must have an understanding of the analogies that have been used to describe these theories. kedari - kulya theory makes use of a situation where the water from a reservoir flows passively through small canals into different pieces of land where grains are grown. khale - kapota theory uses an instance where pigeons from different places approach a heap of collected grains and return to their nests after picking up the grains of their choice. the student must be able to break down this information into two distinct aspects of energy expenditure : the flow of water occurs student must also know that the technical terms used to describe the two methods of transport in biology are passive and active respectively and must be able to relate these terms with the above analogies. the student must also be able to eliminate other confusing distractors, which are again related to energy expenditure but in an improper order. this item therefore, requires a student to break down the elements and to find the relationships among two different concepts to arrive at a final answer. thus this question assesses understanding, comparison, categorization, and analytical skills in a student which is of fourth level in bloom 's taxonomy. e. synthesis (level-5) read the following paragraph carefully and select the most appropriate hypothesis that can be generated on the basis of the information provided : pakvashaya (large intestine) is the major site of vayu and vayu has been ascribed with many functions of brain. the large intestine has a unique composition of microorganisms. recent research suggests that diet can affect certain autoimmune diseases by inducing changes in gut microbe communities. takra, (buttermilk) a known probiotic substance, is indicated in the management of gastrointestinal disorders in ayurveda. necrotizing entero - colitis in infants is being implicated in subsequent neurodevelopmental defects such as cerebral palsy and microcephaly. rajayapana basti has been reported to be effective in improving motor disabilities among children suffering from cerebral palsy. now, read the following hypotheses 1.considering the fact that the diet can influence the gut microbe communities, takra may be effective in non - infective diarrhea associated with lactose intolerance2.because microbial flora is linked with the development of brain, asava and arishta may be effective in treating ischemic infarction in brain3.because rajayapana basti has been reported to be effective in cerebral palsy, its action may be through correcting the dysbiosis in the gut4.because overuse of antibiotics impairs the composition of gut microbiota, it may lead to dysautonomia in future considering the fact that the diet can influence the gut microbe communities, takra may be effective in non - infective diarrhea associated with lactose intolerance because microbial flora is linked with the development of brain, asava and arishta may be effective in treating ischemic infarction in brain because rajayapana basti has been reported to be effective in cerebral palsy, its action may be through correcting the dysbiosis in the gut because overuse of antibiotics impairs the composition of gut microbiota, it may lead to dysautonomia in future a.all may be plausible but 1 is most appropriateb.all may be plausible but 2 and 3 are most appropriatec.all may be plausible but 3 is most appropriated.all may be plausible but 1 and 3 are most appropriate all may be plausible but 1 is most appropriate all may be plausible but 2 and 3 are most appropriate all may be plausible but 3 is most appropriate all may be plausible but 1 and 3 are most appropriate many experts believe that framing mcqs to assess however, carefully framed questions can assess this ability to certain extent. to answer this question, the student must evaluate the intra- and inter - relationships within the given points. for example, he / she must be able to recognize that takra is contraindicated in lactose intolerance and ischemic infarction is not a neurodevelopmental disorder. further, antibiotic overuse leading to dysautonomia is not a plausible assumption considering the given information, because dysautonomia has not been shown to have a neurodevelopmental link. therefore, the correct option in this case is c since basti is directly administered into the large bowel. this involves combination, comparison, and analysis of diverse elements described in the paragraph and synthesis of new hypothesis. thus, this question assesses the knowledge, understanding, application and analysis with skills of generation, or creation (synthesis) which is of level - five in bloom 's taxonomy. the common words that are used to evaluate the synthesizing ability are : create, design, plan, organize, generate, develop, propose, assemble, compose, make, construct, formulate, prepare, invent, adapt and elaborate. f. evaluation (level-6) a study was carried out among healthy male adult volunteers. jatharagni in these volunteers was assessed thrice in a year using a validated questionnaire in spring (vasanta), rainy (varsha) and winter (hemanta) seasons. these volunteers were also tested for their serum t3 and t4 levels during these seasons to see if there is a relationship between the agni, season and serum thyroid hormone levels. the major observations of this study have been given below : the mean serum t3 and t4 levels showed a strong tendency towards an increase in spring season in comparison to the other two seasons. the mean jatharagni scores showed a strong tendency towards a decrease in spring season in comparison to the other two seasons. now, read the following interpretations proposed by three students based on the above observations, evaluate the appropriateness of these interpretations, and choose the correct option from the given list that follows.1.considering t3 and t4 levels to be corresponding to dhatvagni, it can be stated that the relationship between dhatvagni and jatharagni may be inversely proportional and there could be a negative feedback type of mechanism operating between dhatvagni and jatharagni2.considering t3 and t4 levels to be corresponding to dhatvagni, it can be stated that the relationship between dhatvagni and jatharagni may be directly proportional and there could be a positive feedback type of mechanism operating between dhatvagni and jatharagni.3.the mean jatharagni scores do not increase along with serum t3 and t4 levels. the questionnaire to assess agni was inappropriate, or the methods employed to record serum t3 and t4 levels were wrong. considering t3 and t4 levels to be corresponding to dhatvagni, it can be stated that the relationship between dhatvagni and jatharagni may be inversely proportional and there could be a negative feedback type of mechanism operating between dhatvagni and jatharagni considering t3 and t4 levels to be corresponding to dhatvagni, it can be stated that the relationship between dhatvagni and jatharagni may be directly proportional and there could be a agni must intensify as serum t3 and t4 levels increase. either the questionnaire to assess agni was inappropriate, or the methods employed to record serum t3 and t4 levels were wrong. a.3 is the most appropriate interpretationb.both 1 and 3 are possible interpretations, but 1 is most appropriatec.2 is the most appropriate interpretation, while 1 and 3 are wrongd.both 2 and 3 are correct interpretations, while 1 is wrong 3 is the most appropriate interpretation both 1 and 3 are possible interpretations, but 1 is most appropriate 2 is the most appropriate interpretation, while 1 and 3 are wrong both 2 and 3 are correct interpretations, while 1 is wrong this question requires student 's knowledge about basic concepts of metabolism according to ayurveda and contemporary science. he / she should also have skills to find out and generate possible links between two sciences. the student must also analyze the research results given and the way in which the research problem was investigated. the option a is inappropriate because there is no substantial information to justify the 3rd interpretation in the given paragraph. finally, after making judgments of the given possible interpretations the student must be able to evaluate the alternatives and must arrive at the correct option, which is b. the commonly used words to assess the evaluating ability are : appraise, critique, judge, weigh, evaluate, select, rate, defend, prioritize, value, score, measure and assess. in this question, the solving process involves the ability to evaluate and judge, hence is of level-6 in bloom 's taxonomy. the study supports the notion that the current formal ayurveda education is more memory - oriented than being competence based. we therefore propose that, there is a need for including an appropriate proportion of the mcqs to assess higher order thinking in the pgees. further, ayurveda teachers are required to be trained well in the skills related to writing good mcqs since mcqs are able to assess more or less all the six levels of bloom 's taxonomy in cognitive domain. we further suggest that our study may be taken as a lead to introduce the required reforms in pgees.	backgroundthe standards of ayurveda education in india are being questioned in the recent years and many suggestions related to educational reforms are being put forth by educators and health policy experts. however, the post graduate entrance examinations (pgees) that are carried out to select the candidates to pursue postgraduate programs have received little attention in this context.objectivesthe objective of this study was to classify the multiple choice questions (mcqs) from ayurveda pgees conducted in different universities of india during the five year period (ranging from 2010 to 2014) into six levels of bloom 's taxonomy in cognitive domain.methodsthis is a retrospective observational study. the sampling method followed was purposive sampling. totally, 3299 mcqs obtained out of 25 question papers from seven universities spread across four zones of india (north, south, west and east) were included in the study and were classified based on the bloom 's taxonomy.resultsabout 93.3% of mcqs assessed only the recall component whereas 6.2% of the mcqs assessed comprehension. percentage of mcqs that assessed application level was a mere 0.3% whereas the percentage of mcqs that assessed the analysis component was found to be only 0.2%. there was not even a single question to assess the synthesis and evaluation components.conclusionswe conclude that an appropriate proportion of mcqs assessing higher order thinking are required to be included in ayurveda pgees. while it is possible to frame mcqs to assess all six levels of bloom 's taxonomy in cognitive domain, the teachers are required to be trained well in the skills of mcq writing. we propose that our study may be taken as a lead to introduce the required reforms in pgees.clinical trial registration no. : not applicable.
nanotechnology is the creation of functional materials, devices and systems through control of matter on the nanometer length scale and exploitation of novel phenomena and properties (physical, chemical, biological, mechanical, electrical) at that length scale. in the area of cosmetics and anti - aging, in particular, as well as in the pharmaceutical arena, nanotechnology has played an important role in delivering active ingredients to the skin, in both patch delivery and timed release application. nanoparticles / nanospheres sound like futuristic technology the application of nanotechnology concepts to medicine joins two large - scale cross - disciplinary fields with an unprecedented societal and economical potential arising from the natural combination of specific achievements in the respective fields. as the need for the development of new medicines is pressing and given the inherent nanoscale functions of the biological components of living cells, nanotechnology has been applied to diverse medical fields such as oncology and cardiovascular medicine. indeed, nanotechnology is being used to refine discovery of biomarkers, molecular diagnostics, drug discovery and drug delivery which could be applicable to management of these patients. to achieve these aims, nanotechnology strives to develop and combine new materials by precisely engineering atoms and molecules to yield new molecular assemblies on the scale of individual cells, organelles or even smaller components, providing a personalized medicine. because of increased use of nanotechnology, the risk associated with exposure to nanoparticles, the routes of entry and the molecular mechanisms of any cytotoxicity need to be well understood. in fact, these tiny particles are able to enter the body through the skin, lungs or intestinal tract, depositing in several organs and may cause adverse biological reactions by modifying the physiochemical properties of living matter at the nanolevel.[57 ] nanotechnology is being applied to biomarker - based proteomics and genomics technologies. nanoparticles can be used for qualitative or quantitative in vivo or ex vivo diagnosis by concentrating, amplifying and protecting a biomarker from degradation, in order to provide more sensitive analysis. nanoparticles are currently being tested for molecular imaging to achieve a more precise diagnosis with high - quality images. in fact, contrast agents have been loaded onto nanoparticles for tumor and atherosclerosis diagnosis. different nanoparticles have been used for molecular imaging with magnetic resonance images (mri), ultrasound, fluorescence, nuclear, and computed tomography imaging. nanotechnology is being applied extensively to provide targeted drug therapy, diagnostics, tissue regeneration, cell culture, biosensors and other tools in the field of molecular biology. various nanotechnology platforms like fullerenes, nanotubes, quantum dots, nanopores, dendrimers, liposomes, magnetic nanoprobes and radio - controlled nanoparticles are being developed. nanomedicine, with its broad range of ideas, hypotheses, concepts, and undeveloped clinical devices, is still in its early stage. this article outlines the present developments and future prospects for the use of nanotechnology techniques in experimental in vivo and in vitro studies and in engineering nanodevices and biosensors for clinical and investigative use in diagnosis and therapy in the fields of genetics, oncology, cardiology, dermatology and also in the field of medicine, in general, and molecular biology, in particular. the early genesis of the concept of nanomedicine sprang from the visionary idea that tiny nanorobots and related machines could be designed, manufactured, and introduced into the human body to perform cellular repairs at the molecular level. nanomedicine today has branched out in hundreds of different directions, each of them embodying the key insight that the ability to structure materials and devices at the molecular scale can bring enormous immediate benefits in the research and practice of medicine. nanomedicine is the application of nanotechnologies in medicine for maintenance and improvement of human life using the knowledge on human organism at a molecular level [figure 1 ]. application of nanoparticles and nanomaterials for the diagnostic and therapeutic purposes is now significantly extended in nanomedicine. nanomedicine involves utilization of nanotechnology for the benefit of human health and well - being. the use of nanotechnology in various sectors of therapeutics has revolutionized the field of medicine where nanoparticles of dimensions ranging between 1 and 100 nm are designed and used for diagnostics, therapeutics and as biomedical tools for research. many modern cosmetic or sunscreen products contain nano - sized components [figure 2 ]. nanoemulsions are transparent and have unique tactile and texture properties ; nanocapsule, nanosome, noisome, or liposome formulations contain small vesicles (range : 505000 nm) consisting of traditional cosmetic materials that protect light- or oxygen - sensitive cosmetic ingredients. transdermal delivery and cosmetic research suggests that vesicle materials may penetrate the stratum corneum (sc) of the human skin, but not into living skin. modern sunscreens contain insoluble titanium dioxide (tio2) or zinc oxide (zno) nanoparticles (np), which are colorless and reflect / scatter ultraviolet (uv) more efficiently than larger particles. also, nanomaterials such as nano - sized vesicles or tio2 and zno nanoparticles which are currently used in cosmetic preparations or sunscreens pose no risk to human skin or human health, although other np may have properties that warrant safety evaluation on a case - by - case basis before human use. the first generation of lipid nanoparticles was introduced as solid lipid nanoparticles (sln) and the second, improved generation as nanostructured lipid carriers (nlc). identical to the liposomes, the lipid nanoparticles appeared as products first on the cosmetic market. the cosmetic benefits of lipid nanoparticles include enhancement of chemical stability of actives, film formation, controlled occlusion, skin hydration, enhanced skin bioavailability and physical stability of the lipid nanoparticles as topical formulations. nlc are on the market as concentrates to be used as cosmetic excipients, special formulation challenges for these products and they also appeared in a number of finished cosmetic products worldwide. nanotechnology in dermatology due to the lower risk of systemic side effects, topical treatment of skin disease appears favorable, yet the sc counteracts the penetration of xenobiotics into viable skin. since epidermal lipids are found in high amounts within the penetration barrier, lipid carriers attaching themselves to the skin surface and allowing lipid exchange between the outermost layers of the sc and the carrier appear promising. besides liposomes, sln and nlc have been studied intensively. today, sln and nlc exhibit many features for dermal application of cosmetics and pharmaceutics, i.e. controlled release of actives, drug targeting, occlusion and penetration enhancement and also increase of skin hydration associated with it [figure 3 ]. the potential of topically applied np to penetrate human skin and produce a systemic health risk has been suggested in reviews by hoet. mechanism of nanoparticle drug delivery via two main mechanisms passive and active targeting with advances in nanotechnology, pure silver has been recently engineered into nanometer - sized particles (diameter < 100 nm) for use in the treatment of wounds. in conjunction with other studies, it was demonstrated that the topical application of silver nanoparticles (agnps) can promote wound healing through the modulation of cytokines. nonetheless, the question as to whether agnps can affect various skin cell types keratinocytes and fibroblasts during the wound - healing process still remains. a study was conducted for evaluating in vitro skin penetration of agnps and the results showed that agnps absorption through intact and damaged skin was very low but detectable, and that in case of damaged skin it was possible to increase permeation of silver when applied as nanoparticles. moreover, agnps could be detected in the sc and the outermost surface of the epidermis by electron microscopy, and silver applied as nanoparticles coated with polyvinylpirrolidone is able to permeate the damaged skin in an in vitro diffusion cell system. the topical administration of the lyotropic liquid crystal nanocube to sc rapidly broke down the lipid lamella structure which would be recognized as a wound without organ change and the observations suggested that a structural change in lipid can stimulate and trigger recognition of a slight injury in the wound defense and a repair response as homeostasis and this actually succeeded in improving photo - induced hyperpigmentation on human face. skin can be exposed to solid nanoscale particles through either intentional or non - intentional means. intentional dermal exposure to nanoscale materials may include the application of lotions or creams containing nanoscale tio2 or zno as a sunscreen component or fibrous materials coated with nanoscale substances for water- or stain - repellent properties. non - intentional exposure could involve dermal contact with anthropomorphic substances generated during nanomaterial manufacture or combustion. provided compelling evidence that nanoparticles could penetrate the skin in demonstrating epidermal and dermal penetration by fluorescent microspheres (0.51.0 m) in human skin in an in vitro flexed skin model. the interaction of nanoparticles with skin has received significant attention recently because of the increasing use of nanoscale particles in stain - resistant clothing, cosmetics, and sunscreens. the dermal route of exposure is also important because of the tendency of agglomerated airborne nanoparticles to settle on surfaces and the difficulties in preventing dermal contact with these settled particles. several studies have been conducted examining the ability of nanoscale tio2, used as an ultraviolet (uv)-absorbing component in sunscreens, to penetrate the epidermis in human volunteers and animal and in vitro models.[2428 ] the primary tio2 particles used in these studies ranged in size from 10 to 60 nm, with larger - sized aggregates present. in all of these studies, nanoparticles did not appear to penetrate past the sc of the epidermis, though in some cases, accumulation in the hair follicles was observed. studies have found 20-nm, negatively charged polystyrene spheres to behave similarly to nanoscale tio2 with regard to their follicular localization and lack of sc penetration. in contrast to the studies of nanoscale tio2 and polystyrene, a study examining the dermal penetration of quantum dots has shown limited dermal penetration. this study utilized scanning fluorescent confocal microscopy to examine the influence of size, shape and surface charge on quantum dot penetration in an in vitro, flow - through diffusion porcine skin model. a small fraction of the applied dose for some quantum dot species was shown to penetrate the sc, with an even smaller fraction escaping the epidermis and accumulating within the dermis. this penetration was dependent upon size, shape and surface charge of the quantum dots, with smaller, spherical particles demonstrating greater penetration than larger, ellipsoidal particles. none of the quantum dot species were shown to pass through the entire skin thickness to the opposing perfusate side of the diffusion cell. this limited dermal penetration has also been shown recently for < 10-nm maghemite and iron nanoparticles using an in vitro human skin model, suggesting that smaller nanoparticles may have this ability. though studies have yet to examine the effect of skin condition on nanoparticle absorption, damaged skin would be expected to provide less of a barrier, and this issue may be important in certain situations such as the application of nanoparticle - containing sunscreens to sun - damaged skin. in support of this idea, the flexing of skin in vitro has been shown to increase dermal penetration of micron - sized dextran particles and derivatized fullerenes. together, the available data suggest that healthy, intact skin is a significant barrier to certain nanomaterials. this area of research is anticipated to lead to the development of novel, sophisticated, multifunctional applications which can recognize cancer cells, deliver drugs to target tissue, aid in reporting outcome of therapy, provide real - time assessment of therapeutic and surgical efficacy, and most importantly, monitor intracellular changes to help prevent precancerous cells from becoming malignant. although the expectations from nanotechnology in medicine are high and the potential benefits are endlessly enlisted, the safety of nanomedicine is not yet fully defined. use of nanotechnology in medical therapeutics needs adequate evaluation of its risk and safety factors. however, it is possible that nanomedicine in future would play a crucial role in treatment of human diseases and also in enhancement of normal human physiology. with concurrent application of nanotechnology in other fields, its utility is likely to extend further into diagnostics, molecular research techniques and tools. nanotechnology has a bright future with multiple applications in many areas including engineering, optics, energy, and consumer products., there are as many challenges to get it right, and recognize and avoid potential risks associated with these new developments. essential for the successful present and future development is a multidisciplinary team approach involving material scientists, physicians and toxicologists who work closely together. we will also need to push the existing knowledge as far as it will go in the service of protecting people this must be administered strategically and targeted to addressing specific issues in a timely manner. failing to take these steps will ultimately lead to people 's health being endangered and emerging nanotechnologies floundering. but with foresight, sound science and strategic research, we have the opportunity to ensure that emerging nanotechnologies are as safe as possible, while reaching their full potential. ongoing efforts by scientists, researchers, and medical personnel can sincerely ensure to do big things using the very small. but as such, the applications of nanotechnology in dermatology are still a developing and challenging field to dermatologists. nanotechnology is beginning to change the scale and methods of vascular imaging and drug delivery. indeed, the national institutes of health (nih) roadmap 's nanomedicine initiatives envisage that nanoscale technologies will begin yielding more medical benefits within the next 10 years. this includes the development of nanoscale laboratory - based diagnostic and drug discovery platform devices such as nanoscale cantilevers for chemical force microscopes, microchip devices, nanopore sequencing, etc. the national cancer institute has related programs too, with the goal of producing nanometer scale multifunctional entities that can diagnose and deliver therapeutic agents and monitor cancer treatment progress. these include design and engineering of targeted contrast agents that improve the resolution of cancer cells to the single cell level and nanodevices capable of addressing the biological and evolutionary diversity of the multiple cancer cells that make up a tumor within an individual. thus, for the full in vivo potential of nanotechnology in targeted imaging and drug delivery to be realized, nanocarriers have to get smarter. pertinent to realizing this promise is a clear understanding of both physicochemical and physiological processes. these form the basis of complex interactions inherent to the fingerprint of a nanovehicle and its microenvironment, the examples of which include carrier stability, extracellular and intracellular drug release rates in different pathologies, interaction with biological milieu, such as opsonization and other barriers en route to the target site, be it anatomical, physiological, immunological or biochemical, and exploitation of opportunities offered by disease states (e.g. tissue - specific receptor expression and escape routes from the vasculature). inherently, carrier design and targeting strategies may vary in relation to the type, developmental stage, and location of the disease. therefore, it is essential that fundamental research be carried out to address these issues if successful efficient application of these technologies has to be achieved. the future of nanomedicine will depend on rational design of nanotechnology materials and tools based on a detailed and thorough understanding of biological processes rather than forcing applications for some materials currently in vogue. nanotechnology is an emerging branch of science for designing tools and devices of size : a)1100 mmb)1100 nmc)1100 cmd)1100 m nanotechnology is the creation of : a)functional materialsb)control on the nanometer length scalec)exploitation of propertiesd)all of the above tiny particles enter the body through : a)skinb)lungs or intestinal tractc)a and bd)eyes nanotechnology is being applied extensively to provide targeted : a)drug therapyb)diagnosticsc)tissue degenerationd)all of the above early genesis of the concept of nanomedicine sprang from the visionary idea : a)endoscopeb)nanorobots and related machinesc)magnetic resonance imagesd)genetic engineering nanoparticles are : a)transparentb)opaquec)turbidd)greasy stratum corneum counteracts the penetration of : a)antibioticsb)xenobioticsc)deodorantsd)conditioners silver nanoparticles can promote wound healing through the modulation of : a)lymphocytesb)leukocytesc)plateletsd)cytokines increasing use of nanoscale particles in : a)stain resistant clothingb)cosmeticsc)sunscreensd)all of the above scientists, researchers and medical personnel can sincerely ensure to do : a)small things using the very bigb)big things using the very smallc)both of the aboved)none of the above nanotechnology is an emerging branch of science for designing tools and devices of size : a)1100 mmb)1100 nmc)1100 cmd)1100 m nanotechnology is the creation of : a)functional materialsb)control on the nanometer length scalec)exploitation of propertiesd)all of the above control on the nanometer length scale exploitation of properties tiny particles enter the body through : a)skinb)lungs or intestinal tractc)a and bd)eyes lungs or intestinal tract nanotechnology is being applied extensively to provide targeted : a)drug therapyb)diagnosticsc)tissue degenerationd)all of the above early genesis of the concept of nanomedicine sprang from the visionary idea : a)endoscopeb)nanorobots and related machinesc)magnetic resonance imagesd)genetic engineering nanorobots and related machines magnetic resonance images a)transparentb)opaquec)turbidd)greasy stratum corneum counteracts the penetration of : a)antibioticsb)xenobioticsc)deodorantsd)conditioners silver nanoparticles can promote wound healing through the modulation of : a)lymphocytesb)leukocytesc)plateletsd)cytokines increasing use of nanoscale particles in : a)stain resistant clothingb)cosmeticsc)sunscreensd)all of the above stain resistant clothing scientists, researchers and medical personnel can sincerely ensure to do : a)small things using the very bigb)big things using the very smallc)both of the aboved)none of the above small things using the very big big things using the very small	nanotechnology and nanomedicine are complementary disciplines aimed at the betterment of human life. nanotechnology is an emerging branch of science for designing tools and devices of size 1100 nm, with unique functions at the cellular, atomic and molecular levels. the concept of using nanotechnology in medical research and clinical practice is known as nanomedicine. today, nanotechnology and nanoscience approaches to particle design and formulations are beginning to expand the market for many drugs and forming the basis for a highly profitable niche within the industry, but some predicted benefits are hyped. under many conditions, dermal penetration of nanoparticles may be limited for consumer products such as sunscreens, although additional studies are needed on potential photooxidation products, experimental methods and the effect of skin condition on penetration. today, zinc oxide and titanium dioxide nanoparticles (2030 nm) are widely used in several topical skin care products such as sunscreens. thus, in the present scenario, nanotechnology is spreading its wings to address the key problems in the field of medicine. the benefits of nanoparticles have been shown in several scientific fields, but very little is known about their potential to penetrate the skin. hence, this review discusses in detail the applications of nanotechnology in medicine with more emphasis on the dermatologic aspects.
xylan is a noncrystalline complex polysaccharide consisting of a backbone of -d-1, 4-linked xylopyranoside units substituted with acetyl, glucuronosyl, and arabinosyl side chains. xylans are the main carbohydrate in the hemicellulosic fraction of vegetable tissues and form an interface between lignin and the other polysaccharides. the polysaccharides are mainly found in secondary plant cell walls, and their characteristic of adhesion helps to maintain the integrity of the cellular wall. cellulose is a linear polymer of d - glucose units linked by 1, 4--d - glucosidic bond and is crystalline in nature. cellulose is the main constituent of plants and thus the most abundant biopolymer on earth comprising approximately 3550% of plant dry weight. hydrolysis of xylan and cellulose are essential steps towards the efficient utilization of lignocellulosic materials in nature. lignocellulosic waste forms a large proportion of solid waste in our cities, thus constituting an environmental problem. the use of microbial enzymes in lignocellulosic waste treatment has been shown to be an alternative that is efficient and cost - effective. therefore, considering the industrial potentials of xylanases and cellulases, and their potential use in lignocellulolytic waste treatment, it becomes imperative to obtain new enzymes and enzyme - producing microbial strains that produce highly active xylanases and cellulases at low cost. chemical hydrolysis of lignocellulose is accompanied with the formation of toxic components that are toxic to the environment, hence the need to explore the use of microorganisms and their enzymes, which have high specificity, mild reaction conditions, negligible substrate loss, and side product generation and are environmentally friendly, in lignocellulose hydrolysis. xylanases and cellulases are widely abundant in nature ; they are produced by bacteria, fungi, protozoa, algae, gastropods, arthropods, nematodes, and so forth. filamentous fungi have been reported to be good producers of lignocellulolytic enzymes from industrial point of view due to extracellular release of the enzymes, higher yield compared to yeast and bacteria, and also the production of several auxiliary enzymes that are necessary for debranching of substituted polysaccharides. the application of xylanases and cellulases has been mainly considered for the bioconversion of lignocellulosic materials, especially residues and wastes produced by agriculture and forestry to produce higher value products such as ethanol fuel and other chemicals. other potential applications of the enzymes include bread making, fruit juice extraction, beverage preparation, increasing digestibility of animal feed, converting lignocellulosic substances to feedstock, and fibre separation and in paper and pulp industries. therefore, considering the potentials of using xylanases and cellulases in industries and lignocellulolytic waste treatment, it becomes imperative to obtain enzyme - producing microbial strains that produce highly active xylanases and cellulases. genetic manipulations by classical mutation techniques and by use of recombinant dna technology have been used to increase the expression levels of a large number of microbial enzymes. the use of modern techniques to improve the production of metabolites does not invalidate the search for wild organisms producing useful metabolites. in fact the screening of naturally occurring microorganisms may be the best way to obtain new strains and/or enzymes for commercial applications. due to the need to obtain xylanases and cellulases with specific processing characteristics, especially in developing countries with low technological capabilities, this study was undertaken to screen fungal cultures for cellulose and xylan degrading enzymes. fungi were isolated from soil samples, decaying logs of wood, and sawdust using a tenfold serial dilution - plating technique on potato dextrose agar (pda) plates into which 30 g of chloramphenicol was added. this was incubated at room temperature, that is, 28 2c. the culture was observed daily and fungal growth was subcultured onto fresh plates of pda until pure isolates were obtained. the pure cultures were then transferred to pda slants and maintained by subculturing every four weeks. the isolated fungi were identified after growth on pda medium by observing their macroscopic (colour, texture, appearance, and diameter of colonies) and microscopic (microstructures) characteristics according to barnett and hunter, domsch. smears of the isolated fungi were prepared in lactophenol cotton blue and examined with the x40 objectives of a compound binocular microscope for microscopic appearance. the relative occurrence of the fungi was determined by the following formula : (1)relative occurrence(%)=toti100, where to is the total number of occurrence of a particular fungus and ti is the total number of isolates of all fungi. the basal medium composition for production of enzyme by submerged fermentation was based on a modified previous medium containing (gl) : 1.4 (nh4)2so4, 2 kh2po4, 0.3 cacl2, 0.3 mgs047h20, 2 cocl2, and 1 ml trace elements. the composition of the trace element solution was (gl) mnso4h2o, 1.56 ; feso47h2o, 5 ; znso47h2o, 1.4. the carbon source, carboxymethyl cellulose (cmc) (sigma chemical co.) for cellulase production, was added to the basal medium at 1% concentration and then sterilized at 121c for 15 min. 1 ml of sterilized trace elements was then added to the medium after cooling. oat - spelt xylan (sigma chemical co.) was used as carbon source instead of cmc in the medium for production of xylanase. twenty millilitres (20 ml) of the sterile basal medium prepared as earlier described (with the appropriate carbon source) in a 50 ml conical flask was inoculated with 1 ml of standardized fungal spore suspension (1 10 spores ml). after statically incubating the conical flasks for 6 days at 30c the content of each flask was filtered through whatman filter paper no.1. xylanase activity was determined by incubating 0.1 ml of culture filtrate with 0.9 ml of 1% (w / v) oat - spelt xylan (sigma chemical co., st. louis, mo.) in 0.05 m citrate buffer, ph 5.0 at 50c for 30 min. the reaction was terminated by adding 1 ml of dinitrosalicylic acid (dnsa) reagent. the reaction mixture was then placed in a boiling water bath at 100c for 5 min and thereafter cooled to room temperature. absorbance was read at 540 nm using a pye unicam sp6 - 250 visible spectrophotometer. xylanase activity was expressed as 1 mol of reducing sugar (xylose equivalent) released per minute per milliliter of enzyme solution. carboxymethyl cellulase (cmcase) activity was measured by determining the amount of reducing sugar released from low viscosity carboxymethyl cellulose (cmc) (sigma chemical co., st. the reaction mixture consisted of 0.9 ml 1% (w / v) cmc in 0.1 m citrate buffer, ph 5.0, and 0.1 ml culture filtrate. after incubation at 50c for 30 min the reaction was stopped by addition of 1 ml dnsa acid followed by boiling in a water bath at 100c for 5 min. after cooling the reaction mixture to room temperature, the absorbance values were read at 540 nm using a pye unicam sp6 - 250 visible spectrophotometer. one unit (u) of cmcase activity was expressed as 1 mol of reducing sugar (glucose equivalent) released per minute per milliliter of enzyme solution. a total of 110 fungi were isolated from saw dust, soil, and decaying wood (table 1). (20.0%), aspergillus niger (16.3%), trichoderma viride (13.6%), and aspergillus sp. and the relative occurrence of members of the genera trichoderma and aspergillus was 42.6% and 40.8%, respectively, thereby making them the fungi most isolated. the fungi of lowest incidence was a. fumigatus (0.9%), followed by a. flavus, t. pseudokoningii, and penicillium sp. the highest occurrence of fungi was recorded from soil samples (52%), followed by saw dust (29%), and least was in decaying wood (19%)., rhizopus sp., and t. longibrachiatum were isolated from sawdust and soil samples only. aspergillus sp., a. niger, a. ustus, trichoderma sp., and t. viride were isolated from all the three different types of samples used for the isolation of the fungi. this finding is in line with previously reported studies [2226 ] that members of the genera aspergillus and trichoderma were the dominant fungi in forest and agricultural soils. fungi have many different functions in soils, which include either active roles, such as the degradation of dead plant material, or inactive roles where propagules are present in the soil as a resting stage. cellulolytic and xylanolytic activities of the isolated fungi were determined by estimating the amount of reducing sugar released by the fungi when grown in carboxymethyl cellulose (cmc) and oat - spelt xylan, respectively. isolates ff2, ff12, and ff6 from decaying logs of wood produced significantly high amount of reducing sugar (1.20 mg ml, 1.02 mg ml, and 1.00 mg ml, resp.). the lowest value of 0.35 mg ml reducing sugar from oat - spelt xylan was observed in isolate ff13. a significantly high amount of reducing sugar (0.60 mg ml) from cmc was given by isolate ff9 and the least (0.10 mg ml) was given by isolates ff3, ff13, and ff20 (table 2). table 3 shows the amounts of reducing sugar produced during screening of isolated fungi from saw dust for cellulase and xylanase production. isolates fd26 (1.35 mg ml) and fd18 (1.30 mg ml) produced the highest amount of reducing sugar when grown on oat - spelt xylan. other high reducing sugar producing isolates from oat - spelt xylan were fd7 (1.25 mg ml), fd12 (1.20 mg ml), fd16, fd23, and fd25 (1.00 mg ml). the lowest amount of reducing sugar was produced by isolate fd28 (0.15 mg ml) acting on oat - spelt xylan. isolates fd4, fd7, and fd18 produced the highest amount of reducing sugar from cmc. isolates fd14 (0.75 mg ml) and fd26 (0.70 mg ml) were also high reducing sugar producers from cmc. the lowest amount of reducing sugar (0.01 mg ml) from cmc was produced by isolate fd5. the amount of reducing sugar produced when screening fungal isolates from soil for hydrolytic enzyme activity is shown in table 4. the highest amount of reducing sugar was produced by isolate fs3 (1.30 mg ml) when grown in oat - spelt xylan. three other high reducing sugar producing isolates from oat - spelt xylan were fs34 and fs48 (1.20 mg ml), fs7 (1.15 mg ml), fs10, fs35, and fs50 (1.00 mg ml). isolate fs4 produced the least amount of reducing sugar (0.10 mg ml) from oat - spelt xylan. when the isolated fungi were grown in cmc, isolate fs48 (0.75 mg ml) was the highest reducing sugar producer, followed by isolates fs47 (0.60 mg ml) and fs39 (0.55 mg ml). isolate fs17 (0.05 mg ml) was the lowest reducing sugar producer on cmc. seventeen fungal isolates that produced high amount of reducing sugar (1.00 mg ml) when grown on oat - spelt xylan were selected for further screening. the protein content of the culture filtrates obtained after growing the selected fungal isolates in basal medium containing oat - spelt xylan was determined and the specific activity of the enzyme produced by the organisms calculated. the result is presented in table 5. the selected fungal isolate with highest specific activity of 1.30 u mg) were significantly better (p < 0.05) than that of the other isolates. this was followed by isolates fd12 and fs48 with specific activities of 1.08 and 1.05 u mg proteins, respectively. the isolates with low specific activities were ff6 (0.54 u mg proteins), fd25 (0.63 u mg proteins), fs10 (0.65 u mg protein), and ff2 (0.68 u mg proteins). the result of the specific activities of seven selected fungal strains that produced high reducing sugar when grown in cmc is shown in table 6. the isolate fd4 produced a significantly higher (p 0.05) specific activity of 1.23 u mg proteins. the lowest specific activity (0.61 u mg proteins) was obtained by isolate fd26. the fungal strains that gave specific activities of 1.0 in tables 5 and 6 were chosen for further studies of their enzymatic potentials. many species of the genus have been identified to possess all component of the cellulase complex. trichoderma has been listed as a common and effective cellulase producer [25, 2932 ]. there are many reports on isolation of cellulose and xylanase producing fungi from soil, lignocellulosic waste from the vinegar industry, waste paper, cotton waste, bagasse, and leaf litters. fungi are well known agents of decomposition of particularly xylan and cellulose containing organic matter. xylan and cellulose degrading enzymes have been used in food processing, detergent formulation, textile production, feed preparation, production of wine, beer, and fruit juice, and in bioconversion of lignocelluloses to fuel ethanol [31, 33 ]. in this study, trichoderma and aspergillus had a higher relative rate of occurrence in saw dust, log of wood, and soil. the organisms also produced xylanase and cellulase with high specific activities compared to the other isolates. the fungal cultures will be further studied for their enzymatic potentials in the bioconversion of lignocellulosic waste to useful products.	the aim of this work is to select filamentous fungal strains isolated from saw dust, soil, and decaying wood with the potential to produce xylanase and cellulase enzymes. a total of 110 fungi were isolated. fifty - seven (57) of these fungi were isolated from soil samples, 32 from sawdust, and 19 from decaying wood. trichoderma and aspergillus had the highest relative occurrence of 42.6% and 40.8%, respectively. trichoderma viride fd18 showed the highest specific activity of 1.30 u mg1 protein for xylanase, while the highest cellulase activity of 1.23 u mg1 was shown by trichoderma sp. f4. the isolated fungi demonstrated potential for synthesizing the hydrolytic enzymes.
cerebral vasospasm (cv) is the narrowing of the major cerebral arteries following aneurysmal subarachnoid hemorrhage (asah) and is a leading contributor to the morbidity and mortality associated with asah. the annual incidence of asah in the united states is between 21,000 and 33,000 people. of these, approximately 67% will develop vasospasm [2, 3 ]. in the setting of asah the acute phase typically begins 3 to 4 hours after hemorrhage, with rapid, spontaneous resolution. in contrast, the chronic phase begins 3 to 5 days later, with maximum narrowing between days 6 and 8, resolving after about 14 days. angiographic vasospasm refers to the observed narrowing of contrast medium in the major cerebral arteries. radiologic modalities used to diagnose cv include computed tomography angiography (cta), magnetic resonance angiography (mra), and catheter angiography. clinical vasospasm is the sequelae of neurocognitive deficits presumably as a result of a prolonged ischemic state. angiographic and clinical vasospasms appear to be distinct phenomena, with asah patients presenting with angiographic cv only (43% of patients), both angiographic and clinical cv (33% of patients), or none of them (24% of patients). although there are many hypotheses on the pathogenesis of cv, it still remains a poorly understood phenomenon. in 1944, zucker observed that lysed erythrocytes incited smooth muscle contraction of cerebral arteries in mammals. a later clinical angiographic study by ecker and riemenschneider in 1951 found that the degree of vasospasm is directly related to the volume of subarachnoid blood observed on head cts, leading to the use of the fisher grade in predicting vasospasm onset. the inciting event of cv is likely an inflammatory response to extravasated blood products in the subarachnoid space, leading to prolonged and deregulated contraction of vascular smooth muscle cells (vsmcs). lysed rbcs in subarachnoid space surrounding the cerebral vasculature can generate inflammatory downstream effects that result in endothelial damage and smooth muscle contraction. in particular, extracorpuscular oxyhemoglobin is the potent inflammatory compound and has been shown to increase the formation of reactive oxygen species (ros), decrease nitric oxide (no) concentration, increase prostaglandin synthesis, and increase lipid peroxidation [9, 1113 ]. such changes in the vascular equilibrium can lead to the activation of pro - vasospasm signaling pathways and the synthesis of inflammatory gene products. this process occurs in parallel to the delayed cerebral ischemia (dci) resulting from microthromboses and cortical spreading ischemia [14, 15 ]. while much work has been done to characterize the signaling pathways implicated in cv, the field still lacks a definitive explanatory model with robust predictability and therapeutic targets. for example, clazosentan, an endothelin receptor antagonist, has shown great attenuation of angiographic vasospasm in preclinical and clinical studies but no improvement in neurological outcomes. in contrast, nimodipine, a calcium channel blocker, improves functional outcomes without a parallel reduction in angiographic vasospasm. an active area of research is the exploration of the genetic basis for cv, which has previously been supported by population studies. new advances in molecular genetics have revealed several genes whose products are presumptive mediators of cv and genetic polymorphisms that portend increased cv risk and/or poorer outcomes. understanding the genetic mechanism of disease generation may provide insight into novel therapeutic avenues. in this review, we will summarize the most recent research in the following areas regarding genetics and cv : (1) prognostic role of genetics, (2) key signaling mediators involved, and (3) gene therapy and gene delivery. stratifying risk based on next generation sequencing is gradually becoming integrated into medical practice. in cardiovascular medicine, indications on the use of drugs such as clopidogrel, warfarin, and statins genetic screening of family members of patients diagnosed with familial hypercholesterolemia is currently recommended in the uk. close family members of patients with aneurysms may be screened for their own susceptibility for aneurysm formation and rupture. genomic biomarkers may also be used to stratify sah patients for more intensive monitoring according to vasospasm risk, as well as informing medication administration. genomewide and other gene association studies have identified several genes that may play a larger role in developing quick and inexpensive screening protocols in sah. this represents an update on a genetics review by ducruet and colleagues, with attention to recent discoveries reported in the literature. the following is a summary of genetic markers associated with sah and cv, with evidence for disease mechanisms as well as implicated polymorphisms (table 1). catecholamines have been implicated in the development of acute cv following sah [37, 38 ]. comt, a key enzyme in the degradation of catecholamines, has been shown to play a role in acute cv. a study of 167 chinese han sah patients showed that patients with the comt - a allele, a / a genotype were more likely to develop acute cv. this polymorphism may be a biomarker for predicting poor outcomes in patients with aneurysms that may later rupture. endothelial nitric oxide synthase (enos) gene, which is found on chromosome 7q35, plays an important role in cv and other cardiovascular diseases. enos is present in the endothelium of the major cerebral arteries and produces nitric oxide (no), a potent vasodilator. constitutive levels of no inhibit platelet aggregation, vascular smooth muscle proliferation, and inflammation [9, 23, 4244 ]. perivascular oxyhemoglobin released after sah scavenges no generated by enos, decreasing no - mediated vasorelaxation and contributing to the onset of vasospasm [4547 ]. clinically, decreased levels of no in the csf have been reported in asah patients. overexpression of the enos gene has been shown to be vasoprotective in humans and canines in the setting of sah [48, 49 ]. polymorphisms of the enos genes are linked to intracranial aneurysm formation [50, 51 ] and coronary vasospasm. genetic association studies have shown that enos 7 - 786 gene snps predispose asah patients for vasospasm [1923 ]. however, the various findings are contradictory, showing either an effect with the t allele, c allele, or no clear association. this apparent discrepancy is likely attributable to the heterogeneity of vasospasm definition as well as the complex regulation of the enos gene. the activity of enos is tightly regulated at many stages, including transcription, substrate availability, cofactors, protein - protein interactions, posttranslational modifications, and dimerization [59, 60 ]. the literature is mixed on whether enos activity is increased, decreased, or unchanged [6366 ] following sah. further, it is still unclear what role sah plays in the phosphorylation of enos. enos is physiologically a homodimer but under pathological conditions decouples and forms a ferrous - dioxygen complex which has a tendency to form superoxide radicals instead of no. recent studies by sabri and colleagues have shown that sah leads to increased phosphorylation of enos on ser1177 and subsequent uncoupling, which decreases no availability while simultaneously increasing the production of superoxide. superoxide may further react with remaining no to form peroxynitrite, which continues to deplete residual no. while it is known that simvastatin can mitigate vasospasm [63, 68 ], recent work suggests that simvastatin may work by recoupling enos to increase no production and decrease the production of superoxide. this may further explain its mechanism of attenuating vasospasm clinically and in other animal models [63, 64 ]. another recent study showed that preconditioning (pc) of mice in hypoxic chambers prior to induction of sah selectively increases the expression and activity of enos, increasing no levels and reducing vasospasm. in addition, decreased no availability with no change in enos activity was observed in sah, which may also support the pathologic uncoupling hypothesis. haptoglobin (hp) is a serum protein produced primarily by hepatocytes that binds to free hemoglobin released by lysed erythrocytes. the bound complex is taken up by macrophages through the cd163 receptor, presumably reducing extracorpuscular hemoglobin toxicity. it is composed of an alpha subunit and a beta subunit, which are encoded by the haptoglobin alpha gene and haptoglobin beta gene, respectively. the hp gene exists in 2 alleles in humans, and 3 genotypes are possible : hp 1 - 1, hp 2 - 2, or hp 2 - 2. the hp 1 product is a linear dimer, the hp 2 - 1 product is a linear polymer, and the hp 2 - 2 product is a cyclical polymer [73, 74 ]. these structural differences confer different binding affinities, with greater binding and clearing of hemoglobin in hp 1 - 1 individuals and weaker binding and clearance in hp 2 - 2 individuals [45, 75 ]. as a result, the hp 2 - 2 allele is thought to be more proinflammatory than the hp 1 - 1 allele, leading to increased inflammation, immune response, oxidation, and vasoconstriction [9, 47 ]. as such, the hp gene has been shown to be upregulated in a canine model of cv. a study of 32 fisher grade 3 sah patients conducted in 2006 showed that individuals with the hp 2 allele had greater likelihood than hp 1 individuals of developing vasospasm. clinically, the allelic distribution of hp in western patients roughly corresponds to the prevalence of cv in western sah patients [5, 77 ]. in recent years the hp 2 - 2 mouse has been investigated as a novel animal model for cv and its treatment [78, 79 ]. mice with the hp 2 - 2 genotype display greater arterial narrowing and neurological deficits compared to hp 1 - 1 mice following experimental sah. pai-1 is a protein encoded by the serpinei gene, found on the 7q21.3-q22 chromosome. it is antifibrinolytic, functioning as the principal inhibitor of tissue plasminogen activator and urokinase, which themselves activate plasmin. the 4 g allele has been previously associated with higher plasma concentrations of pai-1 in the acute setting and poorer survival after trauma as well as increased pai-1 activity in myocardial infarction. pai-1 has also been investigated in the setting of sah for a possible link to thrombosis - related dci, thought to contribute to the neurological deficits resulting from cv. a study of 126 asah patients in 2004 found that the presence of the 4 g allele in the 4g/5 g promoter snp is associated with increased risk for cerebral ischemia and poorer 3-month gos outcomes relative to the 5 g allele. however, another study of 183 asah patients in 2009 found no association between the pai-1 snp and poor outcomes on the 1-year gos - e scale. given the multifactorial nature of vasospasm, more work must be done to characterize this apparent discrepancy. apoe, a polymorphic protein encoded by the apoe gene, is associated with plasma lipoproteins and is involved in lipid transport and metabolism in the central nervous system. three common alleles have been reported in humans (2, 3, and 4) on the 19p13 chromosome, encoding 3 different isoforms (apoe2, apoe3, and apoe4). the 4 allele predisposes patients to poor neurological outcomes in cv, traumatic brain injury, and ischemic stroke [27, 8688 ]. however, the purported role of this allele in vasospasm following asah is still unclear. while this polymorphism does not appear to play a role in generating vasospasm, it seems to inhibit normal neuronal plasma membrane repair following cerebral ischemia, ostensibly worsening outcomes for the patients that do end up developing vasospasm. within the past decade the promoter region of apoe has come under more scrutiny for its role in cv after sah. polymorphisms in the promoter region have previously been associated with poorer outcomes in traumatic brain injury. a study in 2003 found that carriers of the g-219 t allele had more unfavorable gos functional scores. another study reported that in e4 carriers the 491aa snp contributed to poor outcomes in traumatic brain injury. a study of 101 sah patients in china in 2010 showed that patients carrying the 219 t allele had an increased risk of cv. this effect is possibly mediated through decreased transcriptional activity of apoe, which decreases its protective effect in the setting of inflammation. a recent study of apoe knockout mice observed enhanced vasoconstriction in response to endothelin-1, a vasoconstrictive compound associated with cv. administration of cilostazol was reported to decrease endothelial dysfunction in knockout mice, which likely increases enos phosphorylation. this suggests that apoe may play a vasoprotective role in cv and that underexpression of the gene may be overcome with medication for patients with normal enos expression in the setting of sah. ryanodine receptors (ryrs) are a family of ca intracellular calcium channels that mediate the calcium - induced calcium release (cicr). there are three subtypes (ryr1, ryr2, and ryr3), which are all present in vascular smooth muscle [94, 95 ]. activation of these receptors facilitates ca sparks, which promote vasorelaxation through hyperpolarizing vsmcs [9698 ]. ryrs are involved in the regulation of cerebral artery luminal diameter [99, 100 ], and recent evidence indicates that ryrs may play a role in the pathogenesis of vasospasm after sah. koide and colleagues found a 50% reduction in ca spark activity coupled with a 65% reduction in ryr2 expression following induced sah in rabbits. these changes appear to come from a combination of reduced ryr2 expression as well as increased fkbp12.6 expression, a stabilizer of ryr2 channels. a 2011 study of 46 patients in germany revealed that sah patients who were heterozygous for the c.6178g > t polymorphism of the ryr1 gene were more likely to develop symptomatic vasospasm. cystathionine -synthase (cbs) is an enzyme that converts homocysteine and serine to form cystathionine, releasing h2s in the process. h2s is a vasodilator and neuromodulator and is known to function in the cerebral circulation, although the nature of its interaction warrants further study [30, 102, 103 ]. cbs is the predominant source of h2s in the brain and therefore may play a role in cerebrovascular disease. a study of 87 asah patients found that those with the 699ct and tt (gain of function) genotypes had increased angiographic vasospasm, but no increase in delayed cerebral ischemia. this study suggests that h2s - mediated signaling is neuroprotective in asah, and this protection may not be dependent on vasoprotection. more work must be done to characterize these prognostic genes and generate more consistent findings on their role in vasospasm. differences in study designs, sample sizes, and vasospasm - monitoring modalities must be reconciled for more definitive explanations. however, these remain the most studied genes and may therefore play a role in future stratification of sah patients. inflammatory molecules generated from the breakdown of blood products following sah incite several known cascades of cellular signaling enzymes. in particular, compounds such as endothelin-1, oxyhemoglobin, bilirubin oxidation products (boxes), and ros activate cytokine and cellular signaling pathways [9, 104, 105 ]. these can lead to the alteration of expression of cv - related genes, the mechanisms of which are still currently being investigated. it is hypothesized that spasmogens, when released from lysed blood cells surrounding vascular tissue, lead to the sequential activation of phospholipase c (plc), inositol-1,4,5-trisphosphate (ip3), and diacylglycerol (dag). this pathway mobilizes ca and activates protein kinase c (pkc), which together activate protein tyrosine kinase (tk). tk phosphorylates ras to form the gtp - bound activated form of ras, which is associated with cell cycle regulation, cell adhesion, and migration. ros, as those generated following sah, have also been associated with ras activation. ras has been shown to be activated in a rabbit model of sah, peaking at day 3. gtp - bound (activated) ras interacts with raf-1 to phosphorylate downstream effectors such as extracellular signal - regulated kinase (erk). as such, inhibition of the ras - erk pathway is associated with a reduction in vasospasm in rabbit and canine models of sah [107, 109, 110 ]. mapk is associated with vasoconstriction, impaired vasorelaxation, tissue proliferation, apoptosis, and inflammation [106, 111 ]. an in vitro model using human vascular smooth muscle cells found that inhibition of p38 mapk resulted in decreased vasospasm and cytokine production. it is hypothesized that caldesmon and calponin, which are substrates for mapk, are associated with vasoconstriction, but the exact interaction has not yet been determined [106, 113 ]. these proteins are inhibitors of ca - dependent smooth muscle contraction, and inhibition of them by mapk may lead to sustained vascular smooth muscle contraction. the jak / stat signaling pathway is an important mediator of downstream cytokine and growth factor activity and may be involved in the pathogenesis of cv. cytokines such as il-6 are known to be elevated in sah patients and are also known activators of jaks. jak1 has been shown to be activated in a rat model of sah following production of il-6. stat proteins have been shown to be activated in the basilar artery in a rat model of sah, with stat3 expressed in the intima and media and stat1 expressed in the adventitia. while stat3 is activated in response to cytokines, stat1 is activated by the free radicals generated by oxyhemoglobin metabolism following sah. jak2/stat3 signaling is associated with the expression of the apoptotic genes bcl-2 and bcl - xl in the intima of the basilar arteries in rabbits. these early gene products may mediate the generation of vasospasm, as fibrosis of the cerebral arteries is associated with vasospasm following sah. cox-2 products, including prostaglandins and thromboxanes, are known to lead to endothelial dysfunction through endothelial - dependent contractions. inflammatory changes in the adventitia may result in decreased vessel compliance and may contribute to the vessel stiffness observed in cv. taken together, these findings suggest that the jak / stat pathway may be an important mediator of vasospasm. rho - kinase, the effector of rho, plays an important role in the cardiovascular system through its interaction with the myosin light chain (mlc) in vsmc contractions. rho - kinase also phosphorylates myosin light chain directly, generating sustained contraction in a similar manner as the ca / calmodulin - dependent mlc kinase pathway. rho - kinase has been shown to be involved in the pathogenesis of both coronary and cerebral vasospasm [124, 126131 ]. in addition, the rho / rho - kinase pathway decreases no production through the production of cyclophilin a (cypa), which decreases enos expression. cypa itself stimulates erk1/2, akt, and jak in vsmcs, which contributes to increased ros production [133, 134 ]. rho - kinase is also known to play a role in vascular smooth muscle through increasing vascular smooth muscle proliferation, ros production, inflammation, and endothelial damage [123, 134 ]. fasudil, an inhibitor of rho - kinase, has shown some benefit in treating vasospasm in sah patients. we summarize the current status of gene therapy in cv (table 2). within the past decade viral vector - mediated gene therapy has been explored in the context of vasospasm and other vascular diseases [136, 137 ]. in a proof - of - concept experiment reported in 1997, muhonen and colleagues demonstrated that -galactosidase could be transferred to cerebral blood vessels and surrounding tissue during vasospasm using a virus vector. in 2002 ono and colleagues showed that the hmox1 gene can be transferred to the rat basilar artery adventitia through adenovirus using transcisternal injection. this was associated with increased heme oxygenase-1 mrna and activity, with increased basilar artery diameter and cbf [55, 138 ]. in the last decade this method has shown efficacy in preclinical sah models using genes such as calcitonin gene - related peptide (cgrp), enos, and superoxide dismutase. there are no published clinical models of such therapy to date in cv. however, overexpression of the serca2a gene by viral transfer has shown success in improving outcomes in heart failure patients [139141 ]. intramuscular injection of vegf - carrying adenovirus has improved peripheral artery occlusive disease and coronary artery disease in clinical trials [137, 142 ]. despite the promise gene therapy holds, there are several challenges to the translation of preclinical protocols to humans in the setting of cv. the route of administration is typically through the cisterna magna, which requires more invasive procedures in critically ill patients ; however, external ventricular drains may provide appropriate csf access. it is difficult to ensure adequate and accurate tissue distribution, especially to the endothelium. in addition, humans have increased body weight relative to small laboratory animals. therefore, greater loads of gene - carrying viruses will be required. as cv is a polygenic disorder, single gene therapy alone may not be sufficient. it may be more difficult to express genes within deeper layers of blood vessels with perivascular administration. perhaps the development of endovascular administration with vectors with enhanced transfer efficiency may be a solution. there have also been problems with expressed genes remaining functional for only short periods of time, perhaps from weeks to months. however, given the relatively short time course of vasospasm, this may not matter as much. safety concerns such as inflammation, viral cytotoxicity, and random viral dna integration into host cells still persist. these events may be especially problematic in cv, as an increase in inflammation may exacerbate existing vasospasm, and such responses have been shown in adenovirus gene therapy. fortunately in human trials of angiogenesis gene transfer for vascular disease, no increases in tumors, retinopathy, kidney failure, or cardiovascular endpoints have been reported. there are other potential approaches to targeting cv - related genes which involve delivering gene products to vascular tissue. presumptive gene products can be conjugated with 1020 amino acid polypeptides and delivered into somatic cells. this form of protein therapy can be used for therapeutic overexpression of genes in cv. in 2011 ogawa and colleagues demonstrated that hmox1 can be conjugated with an 11 arginine polypeptide and introduced by transcisternal injection in a rat model. they found that this method transduced the hmox1 gene into all layers of the basilar artery and was vasoprotective in an experimental sah model. such a method may be applicable to transduction of other vasoprotective genes previously discussed in order to mitigate cv. however, concerns such as short time course of action, the need for continuous administration, imprecise delivery, and the need for large doses may make this less practical. crossing the blood - brain barrier (bbb) has been a historical challenge in neurotherapeutics. in addition, decreased cerebral blood flow (cbf) in the perivasospasm period decreases the delivery of intra - arterial administered drugs. in 2011 ogawa and colleagues demonstrated that intranasal delivery of calcitonin gene - related protein (cgrp) can be an effective and minimally invasive way of bypassing the bbb. this study, performed in a rat model of sah, attenuated vasospasm of the basilar artery as well as neurological deficits. more work will need to be done to see if this method is amenable to other known medications and gene products. within the past decade there has been increased knowledge in the genetic basis of cv along with refinements in gene therapy. advances in genetic technology could add genetic screening and gene delivery to the armamentarium of future providers caring for sah patients. while further study will be required to translate the available knowledge into clinical practice, the field of genetics holds great promise for the management of cerebral vasospasm.	cerebral vasospasm (cv) is a major source of morbidity and mortality in aneurysmal subarachnoid hemorrhage (asah). it is thought that an inflammatory cascade initiated by extravasated blood products precipitates cv, disrupting vascular smooth muscle cell function of major cerebral arteries, leading to vasoconstriction. mechanisms of cv and modes of therapy are an active area of research. understanding the genetic basis of cv holds promise for the recognition and treatment for this devastating neurovascular event. in our review, we summarize the most recent research involving key areas within the genetics and vasospasm discussion : (1) prognostic role of genetics risk stratification based on gene sequencing, biomarkers, and polymorphisms ; (2) signaling pathways pinpointing key inflammatory molecules responsible for downstream cellular signaling and altering these mediators to provide therapeutic benefit ; and (3) gene therapy and gene delivery using viral vectors or novel protein delivery methods to overexpress protective genes in the vasospasm cascade.
the role of ventriculo - peritoneal (vp) shunt in the treatment of hydrocephalus is well established for several decades. it however results in several complications, the commonest among which is shunt blockage and revisions. multiple shunt revisions can be a difficult situation to handle particularly if the shunt has been placed in all quadrants of the abdomen. we are presenting a case where a patient with congenital hydrocephalus had fourteen shunt revisions and sixteen surgeries in his life span for hydrocephalus and its sequels. the patient was offered endoscopic third ventriculostomy (etv) an alternative to repeated shunt revisions. a 16-year - old male child presented with history of headache, vomiting, and impaired vision for last 5 months. the patient also suffered an episode of generalised tonic clonic (gtc) seizure one month ago. he also had complaints of fever and swelling over right mastoid and peritubal cerebrospinal fluid (csf) leak along the tract of va shunt for the last 15 days. patient was in altered sensorium with glasgow coma scale (gcs) = e4 v4 m5. he had no vision in right eye whereas in left eye only hand movements could be perceived. abdomen of the child with multiple scars of shunt revisions patient was operated elsewhere at the age of 7 months in the year 1987 for congenital hydrocephalus. he had presented with increasing size of the head (51.5 cm) with bulging anterior fontanelle. right vp shunt (chhabra ; surgiwear india) was performed, which worked well till the age of 3 years. from year 1990 to 1999, vp shunt was revised 6 times for blockage at the abdominal end. in 2000 he had 2 shunt revisions 6 months apart. in 2001 within a small duration of 5 months patient had 6 shunt revisions for shunt malfunction. in all, the shunt tube was placed in almost all quadrants of abdomen. in 2001 patient presented with headache, later shunt tube was reinserted in suprahepatic space but it was again blocked very soon. this va shunt worked for nearly 9 years before patient presented to us. in all, he had sixteen operations in his life for hydrocephalus and sequel of shunt. the computed tomography (ct) scan showed marked dilatation of lateral and third ventricles with aqueductal stenosis. note the absence of csf in subarachnoid spaces as the patient had shunt placement at almost every part of the abdomen and va shunt was also malfunctioning, the option of etv was considered in him with informed consent. there were some adhesions distal to floor of third ventricle, which were broken with fogarty catheter. after etv, there was a remarkable improvement in level of consciousness and there was some improvement in vision (6/18) in both eyes as well. patient was able to do finger counting at a distance of 3 feet one month after the surgery. at a follow - up of six months, there were no signs of any clinical deterioration. the post op ct scan showed presence of csf in subarachnoid spaces without a significant reduction in size of ventricles. patient was in altered sensorium with glasgow coma scale (gcs) = e4 v4 m5. he had no vision in right eye whereas in left eye only hand movements could be perceived. patient was operated elsewhere at the age of 7 months in the year 1987 for congenital hydrocephalus. he had presented with increasing size of the head (51.5 cm) with bulging anterior fontanelle. right vp shunt (chhabra ; surgiwear india) was performed, which worked well till the age of 3 years. from year 1990 to 1999, vp shunt was revised 6 times for blockage at the abdominal end. in 2000 he had 2 shunt revisions 6 months apart. in 2001 within a small duration of 5 months patient had 6 shunt revisions for shunt malfunction. in all, the shunt tube was placed in almost all quadrants of abdomen. in 2001 patient presented with headache, vomiting and intestine obstruction due to large infected intra abdominal pseudocyst. later shunt tube was reinserted in suprahepatic space but it was again blocked very soon. after 15 days right ventriculo - atrial shunt was done. this va shunt worked for nearly 9 years before patient presented to us. in all, he had sixteen operations in his life for hydrocephalus and sequel of shunt. the computed tomography (ct) scan showed marked dilatation of lateral and third ventricles with aqueductal stenosis. note the absence of csf in subarachnoid spaces as the patient had shunt placement at almost every part of the abdomen and va shunt was also malfunctioning, the option of etv was considered in him with informed consent. there were some adhesions distal to floor of third ventricle, which were broken with fogarty catheter. after etv, there was a remarkable improvement in level of consciousness and there was some improvement in vision (6/18) in both eyes as well. patient was able to do finger counting at a distance of 3 feet one month after the surgery. at a follow - up of six months, there were no signs of any clinical deterioration. the post op ct scan showed presence of csf in subarachnoid spaces without a significant reduction in size of ventricles. however, repeated revisions can cause very difficult challenge to surgeon as well as to the patient. there can be situations where putting a shunt in previously operated cases in the abdomen is difficult, more so if the patient had undergone repeated shunt revisions. etv has emerged as an important alternative to vp shunt in the treatment of all types of hydrocephalus.[24 ] its role in patients with previous vp shunt surgery is being studied worldwide. there are reports of successful etv in patients who were previously shunted with encouraging results.[58 ] there would still be some reluctance to do etv in previously shunted patients due to risk of its failure. however, the acceptance of etv is likely to be more when repeated shunt failures have already occurred. etv can provide shunt independent life to such group of patients, particularly the patients who had tragic experience of repeated shunt revisions as in this case. the long term follow - up of such cases would be subjected to experience of similar cases in various institutions. however, the patient and the family were very happy as another shunt revision was avoided.	the role of endoscopic third ventriculostomy (etv) is getting more popular for all types of hydrocephalus. it has several advantages and is also being considered for malfunctioning of ventriculo - peritoneal shunt. a 16-year - old child had fourteen shunt revisions in his life. he was eventually treated with etv with successful result. repeated shunt failure can be an additional indication of etv.
in previous decades, some cataract surgeons prescribed a low myopia - inducing pseudophakic implant (usually 1.0 d) for one or two eyes of patients who wanted to comfortably see close objects. boerner and thrasher first described monovision design in pseudophakic patients in 1984, but there have been few studies published since that time, even though pseudophakic monovision is widely practiced. a survey in 2003 of the american society of cataract and refractive surgery (ascrs) members showed that in the united states, 86% of surgeons preferred monovision or modied monovision, and only 13% preferred an array or other multifocal intraocular lenses (iols). however, in a survey in 2007, monovision or modied monovision was recommended by 61% of ascrs members, while the restor multifocal iol (alcon) was preferred by 17.5%. a survey in new zealand in 2004 was similar to that in 2007 in that monovision or modied monovision was preferred by 81%, but in 2007, it decreased to 50%, while multifocal iol increased to 31%. although the use of monovision has decreased, it is still a preferred and common surgical approach to spectacle independence. as mentioned above, studies on pseudophakic monovision are few but its actual clinical practice is common. in 2002, greenbaum showed that most patients with bilateral eye cataracts or bilateral high ametropia have good results. handa., then described ocular dominance and patient satisfaction after monovision induced by iol implantation. chen., compared monofocal acrysof iol using a blended monovision formula with a multifocal array iol for independence from spectacle after cataract surgery. ito and shimuzu compared the reading ability of pseudophakic monovision with refractive multifocal intraocular lenses., also evaluated patient satisfaction and visual function after pseudophakic monovision. marques and marques and then hayashi., studied the optimal target anisometropia for pseudophakic monovision. stanojcic., compared visual fields of patients with implantation of multifocal iols with monofocal iols of monovision design. therefore, there are only a few studies on pseudophakic monovision, although monovision plays an important role in presbyopia correction. because some visual functions, such as stereopsis, contrast sensitivity and visual fields, can be reduced after monovision correction, patient selection is a very important issue in presbyopia surgeries, as well as for pseudophakic monovision. surgeons often select patients who not only have a strong desire to be free of glasses after surgery, but also fully understand monovision design and its drawbacks.[1314182022 ] ito., and shimizu[18202930 ] emphasized patient selection, and strict criteria were used. their inclusion criteria were patients older than 18 years, a previous cataract surgery in one eye with good uncorrected distance vision in that eye (snellen visual acuity 20/30 or better), and good visual potential in the phakic eye. exclusion criteria were : (1) corneal astigmatism greater than 1.50 d ; (2) ocular deviation (strabismus, exophoria > 10.0 prism diopters) ; (3) strong ocular dominance ; (4) patients over 60 years of age ; and (5) small pupil diameter to enhance near vision performance. the hole - in - card test (patients were asked to look at a landolt c target at 50 cm and at 5 m through a 1-cm hole in the center of a piece of cardboard) was used to determine sighting dominance for distant vision. hand., studied the relationship between ocular dominance and patient satisfaction with monovision induced by iol implantation. they stressed the importance of thorough examination of the quantity of ocular dominance for monovision design. pattern visual evoked cortical potentials are sometimes used to assess the interaction of binocular signals in the visual cortex., had less restrictions for selection of patients for pseudophakic monovision ; corneal astigmatism greater than 1.0 d was excluded in the study by finkelman., and 1.5 d greater than was excluded in the study by marques. greenbaum had almost no restrictions for patient selection, except for corneal astigmatism greater than 2.0 d, even including patients who had high ametropia and could not be corrected by lasik surgery. handa., stressed the importance of ocular dominance in patient selection. currently, little advice has been proposed for selection of patients for monovision by ppps in refractive surgery, and there are no clearly defined limitations for pseudophakic monovision. in monovision design, one eye is corrected for distance vision and the other eye for near vision. in clinical practice, traditional (or conventional) monovision is where the dominant eye is corrected for distance and the nondominant eye is corrected for near vision. the reason for this could be that it is easier to suppress blurred vision in the nondominant eye than in the dominant eye. there is another design called cross monovision, in which the dominant eye is corrected for near vision and the nondominant eye is corrected for distance. the dominant eye was corrected to 0 to 0.25d, and the nondominant eye was corrected to 2.00 0.50 d. shimizu preferred customized monovision (taking into account that the pseudophakic eye exhibits appreciable accommodation based on pupil diameter) with multifocal iols and mild monovision (1.00 to 1.50 d, slight myopia in the nondominant eye with pseudoaccommodation in patients with pupil diameters of < 2.5 mm). they also used hybrid monovision (implantation of a monofocal iol in the dominant eye and a diffractive multifocal iol in the nondominant eye) for patients under 60 years from 2009. greenbaum also used a traditional design, with the dominant eye corrected to emmetropia, and the nondominant eye corrected to 2.75 d. however, finkelman., and marques., did not account for eye dominance. in the study by finkelman., just one eye was corrected to stigmatism and the other eye was corrected to 1.0 to 1.5 d. in the study by marques., the first eye was corrected to + 0.5 d and the second eye was corrected to 2.0 d. marques and marques also used toric iol in patients with relevant corneal astigmatism. in the study by hayashi., approximately 1.50 d of anisometropia was thought to be optimal for successful monovision. leaming 's survey of practice styles and preferences of ascrs members showed that approximately 46% preferred a modified monovision approach (nondominant eye set to the 0.5 to 1.0 range regardless of age) in pseudophakic monovision practice. in a 2007 survey, a new zealand survey in 2004 showed that modified monovision was preferred in 62%, and in 2007 it was 50%. there are no strict criteria set for pseudophakic monovision, and therefore, various designs have been used by different physicians. both retrospective and prospective studies have shown that most patients achieved the desired results after pseudophakic monovision, similar to monovision practiced in lasik, conductive keratoplasty (ck) and traditional contact lenses to correct presbyopia. boerner and thrasher performed a retrospective study of the postoperative use of glasses in 100 patients with pseudophakic monovision. a total of 11% of patients wore glasses out of habit, even though their visual acuities subjectively needed no correction after surgery. greenbaum 's study included 120 patients for removal of visually significant cataracts, and 20 patients had correction of high ametropia (+ 8.00 to 14.00 d). one hundred and twenty - nine patients (92%) achieved 20/30 or better uncorrected distance acuity, j1 or better uncorrected near acuity, or both. one hundred and ten patients (91%) in the cataract group and 19 (95%) in the clear lens group also achieved this level. patient acceptance was 90% in the cataract group and 100% in the clear lens group. ito., evaluated 82 patients (age 4987 years) with pseudophakic monovision, and found that most patients had a binocular uncorrected visual acuity of 0.10 logmar. shimizu performed further studies and analyzed the outcomes of conventional monovision (69 patients) or customized monovision (20 patients). he found that the mean refraction in the nondominant eye was 2.20 0.39 d for patients having conventional monovision and it was 1.34 0.24 d in the customized monovision group, but there were no significant differences of uncorrected visual acuity between the two groups. uncorrected distance visual acuity was at least 20/30 in 96% of patients, with 92% achieving n8 (j4) or better uncorrected near acuity. one fourth of the patients were completely independent of spectacles and one patient was totally dependent on spectacles. no patient required iol exchange or other refractive corrective procedures or better vision at all distances. all patients achieved uncorrected distant visual acuity of 20/40 or better and uncorrected near visual acuity of j3 or better, and intermediate visual acuity of j3 in 90%. marques and marques combined the pseudophakic monovision technique with toric iol in patients with relevant corneal astigmatism, and all patients achieved uncorrected distance visual acuity 20/30 and uncorrected near visual acuity j2, and none of them required spectacle correction at the sixth postoperative month. chen., compared acrysof monofocal iol in the mini monovision formula (n = 20) with the array multifocal iol (n = 20) for patients wanting glasses independence after cataract surgery. the acrysof monofocal iol group showed similar and even slightly better results than the array multifocal iol group. however, a further study by chen., showed that the restor iol had a significantly higher proportion of postoperative independence from glasses than acrysof monofocal iol in the mini monovision formula., compared reading ability after bilateral cataract surgery in patients who had pseudophakic monovision achieved by monofocal iol implantation and patients who had refractive multifocal iol implantation. stanojcic., assessed the difference in binocular visual fields in patients who underwent bilateral cataract surgery with either multifocal (mf ; tecnis zm900, amo) iols or monofocal iols with powers adjusted to give monovision (akreos ao, bausch and lomb). there was no statistically significant difference in the incidence of suboptimal visual field results in these two groups (p=0.662), but visual fields of monovision had various defects. some visual functions such as stereopsis, contrast sensitivity, and visual fields can decrease after monovision correction. pseudophakic monovision has the same problems as monovision induced by lasik, ck and contact lenses, especially when there is high postoperative anisometropia. ito and shimuzu showed that near stereopsis is slightly decreased compared with almost complete refractive correction, although it remains within the normal range (within 100 seconds of arc). the most frequently cited reasons for dissatisfaction were asthenopia and spectacle dependence. in the study by marques., the titmus test showed a mean 197 of arc with reduction of stereo acuity in 31 patients (total, 38 patients)., showed the same problems ; stereopsis and contrast sensitivity were slightly decreased but they were still within the normal range. greenbaum 's report had little discussion regarding complications of pseudophakic monovision, but the incidence of halos or glare was 20% overall. handa., found that the reason for unsuccessful pseudophakic monovision was that the reversal thresholds (i.e., exclusive visibility of the nondominant eye crosses over that of the dominant eye) were at high decreasing contrast (20%), or not at all. however, in the successful monovision group, the reversal thresholds were displayed only at low decreasing contrast (80% and 60%). although some patients have had some complaints, the rate of satisfaction of surgery results was high in all patients. first, many studies have been carried out on monovision and a series of theories have been proposed. second, new iols[3234 ] such as accommodating iol and multifocal iol have attracted more physicians and patients than older methods such as pseudophakic monovision. the first reason is that binocular vision of pseudophakic monovision is established after surgery of two cataract eyes, which can not see clearly preoperatively, and this is not the case in nearly normal eyes with lasik, ck or contact lenses. in addition, the theories of binocular vision development are very complicated, and therefore these theories may need modification. second, new iols still have some drawbacks ; for example, accommodating iols do not have sufficient accommodating capability, and multifocal iols cause glare and halos. pseudophakic monovision can use common iols, and it can be reversed temporarily with contact lenses or glasses if patients are taking long driving trips or if they have difficulty adapting, but when using multifocal iols, waxy vision or glare is hard to correct. the term monovision does not exactly describe two eyes being able to complement each other, but it has a very long history and there is no other term that can replace it. nevertheless, the term pseudophakic monovision should not be restricted to monofocal iol implantation in two eyes, but instead should include various iols such as aspherical iol, toric iol, multifocal iol, accommodating iol and light - adjustable iol to complement each other.[123942 ] in addition, visual function after cataract surgery is not merely restored, but can be reconstructed, including binocular visual function, which is very important to pseudophakic monovision. there are few studies comparing iol - induced monovision with other methods of presbyopia correction. it is difficult to determine which method is better or which is the best when financial interest is excluded., have investigated the effect of pupil size on visual acuity in pseudophakic monovision. finally, a definitive term should have a formal abbreviation, but pseudophakic monovision does not have such an abbreviation.	there are few studies on pseudophakic monovision even though it is widely applied. we reviewed the published literature on pseudophakic monovision. surgeons select patients who not only have a strong desire to be free of glasses after surgery, but also fully understand monovision design and its drawbacks. however, other criteria adopted for pseudophakic monovision are very different. both traditional monovision and cross monovision are used in pseudophakic monovision, and the target binocular anisometropia ranges from 1.0 d to 2.75 d. postoperative results were acceptable in every study and most patients were satisfied, with vision being improved and presbyopia corrected. complications were decreased stereopsis, contrast sensitivity, and visual fields, similar to other types of monovision. the term pseudophakic monovision should include more than just monocular intraocular lens implantation in two eyes, and further studies are required.
childbirth is a challenging and critical experience in women s life with its important psychological, sensational, and physical impact (1). the studies showed that fear of childbirth pain is a common feeling among pregnant women. the findings of a research conducted in sweden, showed that at least 10% of women suffer from fear of labor. negative impact of fear not only leads to adverse birth outcomes but also may increase the likelihood of cesarean section (2). indeed, pain is a unique, complex, and personal experience with multiple responses to stimulation of the senses (3). the studies revealed that the main reason for cesarean operation is fear from childbirth pain. in iran, episiotomy and fatigue are due to lack of tolerance for labor pain (4 - 6). in choosing strategies for coping with pain, fear, and fatigue, using techniques to increase comfort and reduce pain and stress of delivery is not a new concept. currently, the complementary therapy is used to release the tension and relax the muscles of pregnant women with intense pain. interventions to relieve sever pain often includes immersion in water (7), acupuncture and acupressure (8), transcutaneous electrical nerve stimulation (tens) (9), breathing techniques, yoga (10), hypnosis (11), physical and emotional support for coping with pain (12, 13), music therapy (14), listening to quran, and massage therapy (15). a researcher reported various non - pharmacological methods for pain management with their proven effectiveness. meanwhile, the efficacy of these interventions requires more investigation due to various limitations, which make the mother more dependent on her caregivers (16). however, multi - sensory room offers a sense of safety and enhances the self - control behavior of the subjects. ability to satisfy basic needs like eating, drinking, and so on will lead to more favorable results. for example, acupuncture is unpleasant for some due to fear of needle penetration. also, acupuncture, acupressure, immersion in water, hypnosis, and tens should be performed by a technically proficient practitioner (skilled in recognizing and determining trigger points) (17), while the participants of the present study could walk, eat, drink, and had opportunity to divert their attention to other things such as aquarium or optical motion pictures (18). controlled multi - sensory environment or snoezelen room is a dutch term that means a room designed to stimulate the senses of people. it provided muscles relaxation for the people who suffered from severe pain (19). this has become a part of complementary medicine, which promotes efficient comfort and relaxation causing endorphins release and relieving person s pain coping capacities. a snoezelen environment can be used as a distraction from pain by focusing on various senses, including visual and auditory. it provides participants with the feelings of comfort, relief, and self - control (20). snuffelen (to seek out or explore) and doezelen to relax (20). multi - sensory environment does not have a persian translation ; therefore, based on available literature we refer to it as cave relaxation room. the room design was a combination of light music, fish tank, optical illusions, and aromatic oils (19). release of endorphins help relieve the pain, relax the muscles (16), and divert the attention from the pain (17). the mechanism of effect of music on pain is as follows : the central part of the brain is specialized for attention, cognition, and emotional functions and nerve impulses go down to the posterior horn of the spinal cord, while music therapy impedes the transmission of nerve impulses to the spinal cord (15). the first snoezelen room was built in 1997 for treating children with serious and life - threatening conditions that suffered from pain (18). controlled multi - sensory environment was used for treatment of people with disabilities too (19). it was aimed to provide an environment for relaxation through gentle stimulation and opportunities for interaction and reducing fatigue. a study evaluated the effect of snoezelen therapy on chronic pain management through the comparison of traditional treatment method with multi - sensory stimulation to promote relaxation. it showed significant reduction in pain intensity and fatigue during the first month of utilizing this method (p < 0.03) (20). it seems necessary to provide this type of environment during childbirth process, which may reduce emotional stress of the mothers. the present study aimed to determine the effect of snoezelen room on the first and the second stages of delivery, labor pain intensity, perinea status, and number of cesarean operations. the study population included all eligible primiparous women admitted to the labor units in first stage of labor and had 4 cm of cervix dilation. one of the rooms in hospital was designed as a snoezelen room (we called it seeking relaxation room). an aquarium with live fishes was placed there aimed to distract the patient s attention from the labor. oil - burning candles were put in the room adding a drop of lavender essential oil (under supervision of the ministry of health) depending on the subject s request., the entire room was floored by soil - colored carpet (figure 1). the study was conducted at 2 birth rooms in one of the central state and educational hospitals affiliated to iran university in tehran, with approximately 600 monthly births. the inclusion criteria were being primiparous with : 1) 4-cm dilated cervix ; 2) intact membrane ; 3) no indication for cesarean section ; 4) no history of asthma, and allergy ; 5) no pulmonary or heart diseases, high blood pressure, diabetes, or infertility ; and 6) no cervical cerclage in the current pregnancy (21). after randomizing, the participants who underwent an emergency caesarean section were excluded. in the present study, the required sample size was estimated by a pilot study on 20 subjects to test the mean and standard deviation (sd) of the pain scores (vas scale) at 6 and 8 cm of cervical dilation. in the intervention and control groups, these pain scores were 6.04 (1.10) and 7.59 (1.49), respectively, which showed a difference of 1.55 in pain score between two groups. with statistical power of 0.8 and rejection of a null effect at 0.05 significant levels, we calculated that 38 subjects are needed in each group. assuming a possible attrition rate of 35%, the final target sample size was set at 50 per each group. participants were randomly assigned to intervention (snoezelen room) and control groups. in order to evenly distribute the participants, a block randomization list (with block - sizes of 4 at 6 variation) was prepared by a statistician, and was given to two persons in reception unit in the form of sequentially numbered, sealed opaque envelopes, to provide concealment allocation to the group. the experimental group entered into snoezelen room where they could walk, sit, or lay down based on their wishes. aquarium with fishes was placed in front of them at a distance of 1.5 m from the mothers and the projector with green light alternately changed pictures. the lavender essence was dropped into water dish and a candle burned and evaporated into atmosphere. it is not possible to blind both midwife and subjects to the intervention. as a research assistant, a qualified midwife working in that hospital serviced all participants of t2 groups. visual analogue scale (vas) is a 10-cm horizontal line with a no pain at all at the left end and worst possible pain at the right end. women indicated their level of pain intensity by placing a cross on the line (23). this scale is the standard scale that many researchers used (24 - 26). pain was measured at entering the room, the first 3 hours (every hour), and in the second stage (from full dilation to onset of fetal head crowning). the first stage was measured from the mothers entering to the room until full cervix dilation. the second stage was measured from full cervix dilation until the onset of fetal head crowning. mann - whitney test, chi - square test and independent t test were conducted in statistical analyses. the statistical analysis showed no significant difference with regard to age (t = 1.43, p = 0.83, df = 85), educational degree, job, and perinea status between the two groups. the chi - square analysis found significant differences in the perinea status (table 1). the independent t test indicated significant difference between the mean score of two groups during the first stage, but did not show significant difference in the second stage. using independent t test, no significant difference was seen between the mean score of pain intensity in mothers when entering to labor, and during the first hour and the second hour, while significant difference was observed between the mean score of pain intensity in the third hour and in the second stage between two groups. the statistical analysis showed no significant difference in labor augmentation or newborns apgar score in the first minute (table 2). friedman test was used to match the samples for repeated data during that time and wilcoxon pairs signed - rank test was used for pain intensity within groups. the results of friedman test revealed a significant overall difference (p < 0.000). for paired comparison with bonferroni correction, p value was 0.008. all paired comparisons were shown significant differences in sequential hours in the control group (p < 0.000), which were expected. because the process of cervix dilation was normal and accordingly pain was increasing. in intervention group, no significant difference was seen between pain intensity at the third hour with the first and the second hours. the result revealed that the intensity of pain in the intervention group increased less than that of the control group. the mean score of pain intensity at the third hour in the intervention and control groups were 5.26 (0.86) and 9.56 (1.48), respectively. another study reported that in the first three hours of active phase, the subjects reported pain intensity reduction in the intervention group compared to control group, but there was no difference in the subsequent hours between two groups (27). it supports the pain - relieving effects of multi - sensory room, which used as a midwifery intervention for pain relieve in childbearing. aromatherapy is one of the common elements in snoezelen room that increases the production of endorphins, decreases pain intensity, releases muscle tensions, and diverts attention from pain. the effect of music therapy is also one of the elements in snoezelen room. the central part of the brain is specialized for attention, cognition, and emotional functions and nerve impulses go down to the posterior horn of the spinal cord ; in music therapy, transmission of nerve impulses to the spinal cord is impeded (16). findings indicated that the first stage duration of labor in the intervention group was less than the control group and observed significant difference. sosa showed that the length of time from mothers admission to delivery was shorter in the experimental group compare to the control group (8.8 vs. 19.3 hours, p < 0.001) without a supportive companion (27). there are unpleasant consequences like insomnia, delayed in initial attachment between mother and newborn, and improper breast feeding position of mothers (28, 29). evidence shows that the prevalence of perinea trauma is lower among women who gave birth at home, because they are mentally and physically are prepared for the birth and the environment is attractive and comfortable for the mother ; so they with the physiological process, without hurrying, which creates a sense of security as home environment can help healthy perinea (30). the delivery process is controlled by providing calm and relaxed environment in hospital, undertaking maximum self - control ability, and increasing pressure in lower abdominal pressure to help easier delivery of fetus s head, so that childbirth would be implemented with minimum injury to perineum. the present study had some advantages and limitations. according to the results of this study, there was less pain and shorter duration in the first labor phase in mothers staying in snoezelen room than the mothers without this facility. therefore, snoezelen room may be an effective option for mother s satisfaction in childbearing and reduction in cesarean rate. since pain relief occurred a couple of hours after entering to labor in our study, it seemed that those mothers did not have any experience for childbirth in such places. it is better that the mothers got familiar with this type of delivery room in antenatal period. the limitation in our study was related to the different level of anxiety and fear in mothers entering the hospital. childbirth is a stressful event, and anxiety and lower satisfaction are associated with a traumatic experience. acute pain is often accompanied by anxiety and distress and almost all women experience it in childbirth (31, 32). taking information about the procedure of hospital admission, genital anatomy, physical examinations, and laboratory tests can reduce anxiety and increase the sense of self - control over labor. they were also told that they were free to answer any questions or withdraw from the study at any time. all participants were encouraged to ask any questions or raise any concerns about their care or participation in the study.	background : one of the strategies for a good outcome and pain free childbearing is to design the delivery room.objectives:the aim of this study was to evaluate the effects of snoezelen room on childbearing outcome such as pain intensity, duration of labor, and perinea status in nulliparous women.patients and methods : this study was a randomized controlled clinical trial consists of 100 childbearing women. they were randomly divided into 2 groups. the experimental group went to snoezelen room when their cervix dilation was 4 cm, while the control group went to physiologic delivery room with the same cervix dilation.results:the mean sd of vas (visual analogue scale) pain intensity of the experimental and control groups before the intervention were 5.1 1.95 and 5.58 1.62, respectively (p = 0.13). the mean sd of vas pain intensity scores of the experimental and control groups after 3 hours spending in their assigned rooms were 5.26 0.86 and 9.56 1.48, respectively (p = 0.01). the mean sd of the first stage scores of the experimental and control groups were 6.95 0.97 and 8.41 0.67, respectively (p = 0.042). about 92% of participants intervention vs. 66% of control participants had perinea laceration (p = 0.041).conclusions : according to the findings of the present study, distracting senses in snoezelen room decreases mother s pain intensity, the length of labor, and incidence of episiotomy.
chyluria is the presence of a milky white lymphatic fluid that is rich in protein and dietary lipids in the form of chylomicrons in urine. the most important cause of chyluria is impaired circulation of lymph flow by thoracic duct obstruction which arises from a variety of causes, including filariasis, trauma, surgical procedure, tumor and aneurysm. it is commonly caused by the parasitic filarial nematode wuchereria bancrofti (w. bancrofti). a strict low - fat diet is recommended in the conservative management of chyluria for the purpose of reducing intralymphatic pressure via decreasing chylous lymph absorption from the intestine. however, it is uncertain whether inhibition of cholesterol absorption with ezetimibe reduces intralymphatic pressure in filarial patients. we report a case of filariasis presenting with severe chyluria and nephrotic - range proteinuria in which inhibition of cholesterol absorption with ezetimibe has been remarkably effective. a 75-year - old woman was admitted to our department with proteinuria and bilateral lower - extremity edema. she lived in the prefecture of nagasaki, a filarial endemic region, from birth to 74 years of age. the patient first presented with chyluria in her 40s and had been diagnosed by lymphangiography with chyluria due to filariasis in 1980. since then she developed lower - extremity edema three months before the current admission, and her primary - care doctor referred her to our department for treatment on september 2010. on examination, she looked well and her vital signs were stable ; blood pressure was normal (125/73 mmhg) and pulse rate was 76 beats / min. laboratory examinations revealed a white blood cell count of 4.36 10/l (4.36 10/l). g / l) and total cholesterol level was 266 mg / dl (6.88 mmol / l), with triglycerides at 229 mg / dl (2.59 mmol / l). urinalysis showed the presence of protein (3 +) and occult blood (3 +) using a dipstick and massive proteinuria (8.9 g/24 h). there was no evidence of urinary stone or intra - abdominal space - occupying lesion or ascites fluid on the computed tomography scan of abdomen. technetium-99m - human serum albumin (tc-99m - hsa) lymphoscintigraphy revealed the presence of communications between the lymphatic system and the urinary tract (figure 1a). 1.(a) technetium-99m - human serum albumin (tc-99m - hsa) lymphoscintigraphy (pre - administration of ezetimibe) shows the presence of communications between the lymphatic system and the urinary tract (arrow). (b) tc-99m - hsa lymphoscintigraphy (three months post - administration of ezetimibe) shows a reduction of lymph flow to the urinary tract (arrow). (c) cystoscopy reveals the presence of chyluria flowing out from the right ureteral orifice (arrow). (a) technetium-99m - human serum albumin (tc-99m - hsa) lymphoscintigraphy (pre - administration of ezetimibe) shows the presence of communications between the lymphatic system and the urinary tract (arrow). (b) tc-99m - hsa lymphoscintigraphy (three months post - administration of ezetimibe) shows a reduction of lymph flow to the urinary tract (arrow). (c) cystoscopy reveals the presence of chyluria flowing out from the right ureteral orifice (arrow). the diagnosis of chyluria was confirmed by the ether test, which showed complete clearing of urinary opacity by the addition of ether to her milky urine. there were no obvious findings of glomerulonephritis in the renal biopsy (figure 2). on the basis of the clinical course, the laboratory findings and imaging studies, filariasis was considered to be involved in the pathogenesis of chyluria and nephrotic - range proteinuria as well in this case. fig. 2.(a, b) light microscopy of the patient 's renal biopsy showing a slight degree of tubulointerstitial atrophy, fibrosis and intimal thickening of arteries. (a, b) light microscopy of the patient 's renal biopsy showing a slight degree of tubulointerstitial atrophy, fibrosis and intimal thickening of arteries. effacement of the foot processes is not found. because this patient showed moderate dyslipidemia, ezetimibe (10 mg / day) moreover, the urinary protein excretion decreased markedly to 0.2 g/24 h and hematuria also disappeared. as the urinary protein decreased, the edema in the lower extremities disappeared and the hypoproteinemia improved (albumin level, 4.2 g / dl or 42 total cholesterol level had decreased to 210 mg / dl (5.43 mmol / l), with triglycerides at 69 mg / dl (0.779 mmol / l) following remission. tc-99m - hsa lymphoscintigraphy showed a reduction of lymph flow to the urinary tract three months later (figure 1b). our patient with filariasis showed remarkable improvement in both nephrotic - range proteinuria and severe chyluria after treatment with ezetimibe. the renal biopsy in our patient showed benign nephrosclerosis, which did not explain such a massive amount of proteinuria. many reports have previously documented the incidence of glomerulonephritis associated with acute filarial infection, which is called using light microscopic examination, these reports have described various types of renal injuries such as membranous, mesangiocapillary and chronic sclerosing glomerulonephritis [47 ]. using immunofluorescence microscopy, granular deposits of igg and c3 have also been reported [5, 7 ]. in our patient, thus, mechanisms other than renal lesions, such as retrograde lymphatic hypertension and the rupture of lymph vessels into the urinary tract, may have played an important role in the development of proteinuria and chyluria. the histological changes of lymphatics in filariasis result from inflammatory damage to the lymphatics caused by adult worms. adult worms migrate to lymphatics and stimulate inflammatory processes which cause lymphatic obstruction and prevent intestinal lymph outflow. as a result, the elevated intralymphatic pressure triggers the rupture of vulnerable sites in the mucous membranes of the renal pelvis and calyx, which causes a leakage of chyle into the urinary tract. in our case, tc-99m - hsa lymphoscintigraphy also showed communications between the lymphatic system and the urinary tract. inhibition of cholesterol absorption with ezetimibe may be remarkably effective in the treatment of massive proteinuria and chyluria in filariasis, as occurred in our patient. ezetimibe is the first of a new class of drugs that inhibits dietary and biliary cholesterol absorption by blocking at the brush border of the intestine. the molecular target for ezetimibe was recently identified to be the niemann - pick c1-like 1 (npc1l1), the intestinal cholesterol transport protein at the small intestine, especially expressed in the brush border. in our patient, therapeutic intervention with ezetimibe may have resulted in a reduction of chylous lymph absorption from the intestine and the prevention of mucosal rupture in the renal pelvis and calyx via reduced intralymphatic pressure. this fact may suggest an important role of intralymphatic pressure in the pathogenesis of chyluria and nephrotic - range proteinuria in filariasis. there is no definitive treatment for filarial chyluria. in mild cases, rest and a strict low - fat diet with high protein and fluid intake surgical treatment is indicated in the patient who presents with refractory severe chyluria with large amounts of proteinuria, hypoproteinemia, progressive weight loss and an immunodeficiency state from hypogammaglobulinemia. from the viewpoint of invasiveness and the difficulties of a second surgery, the risks of surgical treatment should not be considered less serious. there are some reports in the literature that spontaneous remission can be seen in up to 50% of patients with chyluria [11, 12 ] ; in our patient, any other factors contributing to the continuation or remission of chylous urine were not apparent, so we can not completely exclude the possibility that her improvement was a spontaneous remission unrelated to ezetimibe. however, the average duration of chyluria in a series of cases with spontaneous remission was 44.3 months. the recurrence of chyluria after discontinuation of ezetimibe and a remission following re - administration would have more convincingly clarified the contribution of this agent to the remission of the chyluria. however, the patient expressed a strong wish to continue on ezetimibe due to concerns about the relapse of chyluria and has therefore continued to receive ezetimibe and a fat - restricted diet through our department. in the six months since discharge, we describe a case of filariasis with remarkable improvement in both severe chyluria and nephrotic - range proteinuria on treatment with ezetimibe. treatment with ezetimibe may have the potential to become an effective and safe treatment for the management of chyluria, and should be considered when filarial patients present with chyluria and massive proteinuria before resorting to invasive surgical procedures.	we describe a case of filariasis presenting with severe chyluria and nephrotic - range proteinuria. there were no obvious findings of glomerulonephritis in the renal biopsy. technetium-99m - human serum albumin (tc-99m - hsa) lymphoscintigraphy revealed the presence of communications between lymphatic channels and the urinary tract. ezetimibe (10 mg / day) was administered during hospitalization. chyluria was decreased within a few days following the administration of ezetimibe. moreover, a remission was obtained from nephrotic - range proteinuria. tc-99m - hsa lymphoscintigraphy showed a reduction of lymph flow to the urinary tract three months later. in our patient, therapeutic intervention by ezetimibe may have resulted in a reduction of chylous lymph absorption from the intestine and the prevention of mucosal rupture into the renal pelvis and calyx via reduced intralymphatic pressure. ezetimibe may be an effective and safe treatment for this indication, and should be considered when filarial patients present with chyluria and massive proteinuria before employing invasive surgical procedures.
despite the fact that physical inactivity has been known as a risk factor for non - communicable, chronic diseases, reduced participation in physical activities is one of the changes that has occurred recently in lifestyle. this problem is not specific to a particular population, and adolescents are at risk of physical inactivity as well. based on the centres for disease control and prevention report, in 2011, only 29% of high school students in the usa had satisfactory physical activity. however, the us department of health and human services recommends that performing at least 60 min of physical activity per day is necessary to maintain and improve adolescents health. these physical activities must be repeated 3 days or above per week, and include strength exercises to increase the capability of main muscles of the body and limbs and aerobic activities to increase cardiorespiratory capacity. contrary to public perceptions, adolescents physical inactivity is also highly prevalent in developing communities, as in developed communities. a study published in 2010 demonstrated that over 60% of adolescents in iran showed lack of adequate physical activity. available evidence shows that there is an obvious decline in physical activity after childhood and inactivity is likely to become a personal habit continuing in older ages. therefore, the researchers highlight the need to conduct studies aimed to increase the participation of adolescents in regular physical activities and promote community 's health. physical activity, as with many other behaviors, is influenced by several individual, inter - personal, and environmental determinants. the spread of access to digital technologies and the reduced need for physical activities to do daily tasks and for transportation are some of the most important changes in environmental factors related to physical activity. the historical balance between energy intake and expenditure in humans in such a situation, the importance of intra - personal factors, alongside self - care, reassuring efforts, and protective behaviors, increases further. self - regulation is one of these determinants which exerts indirect effects on physical activity at inter - personal and environmental levels, in addition to individual level. despite different definitions have been offered, self - regulation refers to a variety of strategies and behaviors used to adopt and/or maintain the behaviors associated with health. according to bendura, self - regulation is exerted through a variety of cognitive and behavioral mechanisms such as self - monitoring, goal setting, feedback, self - reward, self - instruction, enlistment of social support, and enables the individual to control individual and environmental health - related factors. through maintaining provision of practical guidelines and offering effective feedbacks on behavior and its outcomes, self - regulation enables the individual to have a high control of his / her behavior. many times, exercise and physical activity require disregard of short - term joys to achieve a more valuable benefit in the future. bearing short - term negative consequences in order to achieve long - term positive outcomes, as one of the known human capacities, is accomplished by self - regulation mechanisms. some studies have demonstrated self - regulation as a mediator between self - efficacy construct and physical activity, alongside its direct role in physical activity. given the role of self - regulation construct in analysis and moderation of physical activity - related behaviors, availability of an appropriate instrument to measure this psychological construct is particularly important. so, different types of questionnaires have been presented up to now for the assessment of self - regulation about leisure time physical activity. one of the commonly used instruments to measure physical activity - related self - regulation is the 43-item questionnaire (pasr-43) introduced by petosa in 1994. this questionnaire, which has been developed to measure self - regulation in middle - aged population, consists of six main sub - constructs. today, the questionnaires such as pasr-43 have been translated into different languages, including persian, and are commonly used to measure self - regulation construct. although one of the benefits of administering standard questionnaires is to save research resources, the efficiency of each questionnaire is mostly dependent on the social and cultural specifications of the populations which are going to be measured by the questionnaire. one of the specifications of a good questionnaire is the ability to uniformly assess the individuals of various age groups, gender, and ethnic populations with specific educational level and other demographic characteristics. although fairness of a questionnaire makes the findings of a study in which it has been administered highly generalizable, lack of a quantitative approach for assessing this characteristic and validity 's dependency on specifications of the target population have caused failure to address fairness satisfactorily. as a result, several studies have demonstrated the weakness of common questionnaires in view of fairness. 's study conducted in 2009 on the elderly adults, construct validity of petosa questionnaire was demonstrated as unacceptable. similar studies conducted recently have shown the significance of use of specific measurement instruments in populations with different cultural, language, age, and gender characteristics. in addition, the passage of time could influence an instrument 's capability of measuring psychological constructs in a particular population. despite the significance of using specific instruments to measure the factors related to physical activity, no evidence has yet been obtained regarding a specific instrument to measure self - regulation construct in adolescents in iran. therefore, the present study seeks to show the steps of development and psychometric evaluation of a specific 16-item questionnaire to measure self - regulation construct in iranian male adolescents. this questionnaire is expected to be administered with fewer items than in similar questionnaires to conduct observational and interventional studies. this study is a cross - sectional, descriptive study conducted in april and may, 2013, in isfahan, central iran. isfahan was divided into three regions, privileged, semi - privileged, and sub - privileged, based on relevant studies of socioeconomic status and the viewpoints of municipal experts and healthcare professionals. then, four high schools were randomly enrolled from each region. after explaining the research purposes, we gave necessary explanations to the students regarding the confidentiality of data and volunteer participation. the inclusion criteria for the study were : age 15 - 19 years, lack of a major health problem preventing regular physical activity, and no membership in professional sports teams. thus, 650 male adolescents of age 15 - 19 years, living in isfahan, participated in the study. the rate of physical activity during leisure time was determined by the long form of international physical activity questionnaire (ipaq). this questionnaire was developed by a group of italian researchers in 1998 and administered in observational and interventional studies after being translated into various languages, including persian. reliability and validity of the persian version of ipaq were assessed in several studies. in a study conducted in tabriz, iran, content validity index (cvi) and content validity ratio (cvr) of ipaq were derived and found to be 0.85 and 0.77, respectively. in addition, cronbach 's alpha of this questionnaire in a persian - speaking population was estimated as 0.7 and spearman 's correlation coefficient was estimated as 0.9 by test - retest. using this questionnaire, the individuals could be assigned to three groups : lowly active (less than 600 met - min / week), moderately active (between 600 and 3000 met - min / week), and severely active (higher than 3000 met - min / week). the instrument recommended for self - regulation measurement consists of 16 items. this instrument measures the rate of use of self - regulation mechanisms in the past 4 weeks by propositions of always, often, sometimes, rarely, and never. after review of the literature, 25 items were developed to measure self - regulation construct about leisure time physical activity. some of the items were already used identically in previous studies and translation of these items into persian was done by translation - back translation method by two health experts who had mastered the language. the items were examined for face validity and cultural adaptation by five experts of health education to evaluate qualitatively as per some criteria including compliance with the principles and rules of persian language, simplicity, comprehensibility, relevance, appropriateness to the construct of interest, and lack of ambiguity. cvr of each item was also assessed according to lawshe method by 10 other experts of health education who formed the expert panel. this panel commented on each item as essential, useful but non - essential, and non - essential. to determine cvi, simplicity, specificity, and clarity were considered. for simplicity, relatively simple, and not simple. for specificity, the options were highly relevant, relevant, moderately relevant, and irrelevant. for clarity, they were quite clear, clear, comprehensibility of the questionnaire was assessed in a pilot study of 35 members of the target population who were not enrolled in the final study, using the options fully comprehensible, comprehensible, relatively comprehensible, and not comprehensible. the number of fully comprehensible and comprehensible ticked items was divided by 35 to derive comprehensibility coefficient. to derive reliability coefficient, we administered the questionnaire to 75 members of the target population who were not enrolled in the final study and the internal consistency criterion was used. after analysis of the items, exploratory factor analysis (efa) was run to estimate the construct validity of the questionnaire. since the recommended instrument contained a number of sub - divisions, the extraction step was done with the presupposition of principle components. because of the potential correlation among these sub - divisions, the type of rotation of the items was determined promax. the study was started after 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 sciencesthe purpose and procedures of the study were explained to the participants, and researcher 's emphasis on confidentiality of data and voluntary nature of participation.parental informed consent and student dissent were considered as an inclusion criteria.the investigators guaranteed that there were no any conflicts of interest. the study was started after 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 the purpose and procedures of the study were explained to the participants, and researcher 's emphasis on confidentiality of data and voluntary nature of participation. this study is a cross - sectional, descriptive study conducted in april and may, 2013, in isfahan, central iran. isfahan was divided into three regions, privileged, semi - privileged, and sub - privileged, based on relevant studies of socioeconomic status and the viewpoints of municipal experts and healthcare professionals. then, four high schools were randomly enrolled from each region. after explaining the research purposes, we gave necessary explanations to the students regarding the confidentiality of data and volunteer participation. the inclusion criteria for the study were : age 15 - 19 years, lack of a major health problem preventing regular physical activity, and no membership in professional sports teams. thus, 650 male adolescents of age 15 - 19 years, living in isfahan, participated in the study. the rate of physical activity during leisure time was determined by the long form of international physical activity questionnaire (ipaq). this questionnaire was developed by a group of italian researchers in 1998 and administered in observational and interventional studies after being translated into various languages, including persian. reliability and validity of the persian version of ipaq were assessed in several studies. in a study conducted in tabriz, iran, content validity index (cvi) and content validity ratio (cvr) of ipaq were derived and found to be 0.85 and 0.77, respectively. in addition, cronbach 's alpha of this questionnaire in a persian - speaking population was estimated as 0.7 and spearman 's correlation coefficient was estimated as 0.9 by test - retest. using this questionnaire, the individuals could be assigned to three groups : lowly active (less than 600 met - min / week), moderately active (between 600 and 3000 met - min / week), and severely active (higher than 3000 met - min / week). this instrument measures the rate of use of self - regulation mechanisms in the past 4 weeks by propositions of always, often, sometimes, rarely, and never. after review of the literature, 25 items were developed to measure self - regulation construct about leisure time physical activity. some of the items were already used identically in previous studies and translation of these items into persian was done by translation - back translation method by two health experts who had mastered the language. the items were examined for face validity and cultural adaptation by five experts of health education to evaluate qualitatively as per some criteria including compliance with the principles and rules of persian language, simplicity, comprehensibility, relevance, appropriateness to the construct of interest, and lack of ambiguity. cvr of each item was also assessed according to lawshe method by 10 other experts of health education who formed the expert panel. this panel commented on each item as essential, useful but non - essential, and non - essential. to determine cvi, simplicity, specificity, and clarity were considered. for simplicity, the options used were quite simple, simple, relatively simple, and not simple. for specificity, the options were highly relevant, relevant, moderately relevant, and irrelevant. for clarity, they were quite clear, clear, comprehensibility of the questionnaire was assessed in a pilot study of 35 members of the target population who were not enrolled in the final study, using the options fully comprehensible, the number of fully comprehensible and comprehensible ticked items was divided by 35 to derive comprehensibility coefficient. to derive reliability coefficient, we administered the questionnaire to 75 members of the target population who were not enrolled in the final study and the internal consistency criterion was used. after analysis of the items, exploratory factor analysis (efa) was run to estimate the construct validity of the questionnaire. since the recommended instrument contained a number of sub - divisions, the extraction step was done with the presupposition of principle components. because of the potential correlation among these sub - divisions, the type of rotation of the items was determined promax. the study was started after 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 sciencesthe purpose and procedures of the study were explained to the participants, and researcher 's emphasis on confidentiality of data and voluntary nature of participation.parental informed consent and student dissent were considered as an inclusion criteria.the investigators guaranteed that there were no any conflicts of interest. the study was started after 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 the purpose and procedures of the study were explained to the participants, and researcher 's emphasis on confidentiality of data and voluntary nature of participation. mean age of the participants in the study was 16.3 1.0 (range : 15 - 19) years. as per the comments of the five - member expert panel, 12 out of total 25 items were revised and changed for phrasing and 6 items were deleted. the expert panel believed that addition of these two items enhances the questionnaire 's coverage of various dimensions of self - regulation construct and promotes its validity. 8 addresses an individual 's effort to find a solution when facing the barriers to physical activity. item no. 16 reflects an individual 's requesting others around to be able to run one 's physical activity. then, cvr of the remaining items was calculated and on using the relevant formula, the cvr of five items did not register the threshold level, and hence, the items were excluded. given that 0.79 was considered as the items cvi acceptability to remain in the questionnaire, all the remaining items (n = 16) were satisfactory with regard to cvi. after administering the questionnaire in the pilot study, we determined 0.79 as the acceptable comprehensibility and all items met the comprehensibility requirements. also, the questionnaire 's cronbach 's alpha was investigated in a pilot study of 75 members of the target population and was found to be 0.84. after exclusion of outliers, the mean physical activity was found to be 2324 1437 met - min / week and the mean physical activity in leisure time was 842 831 met - min / week. also, 293 (49%) participants had low activity, 278 (46%) had moderate activity, and 32 (5%) had high activity. the questionnaire 's cronbach 's alpha was found to be 0.90 after 603 participants filled it up. by correlation matrix, all the recommended items had a correlation coefficient of higher than 0.4 with the other items (p 0.005). all the items were determined as acceptable and none of them was excluded [table 1 ]. item total statistic of self - regulation questionnaire abut leisure time physical activity in iranian male adolescents kaiser meyer olkin index was found to be 0.92 and bartlett 's test of sphericity was significant at 95% ci (= 4139, df = 120, p = 0.00). given the adequacy of sample size and correlation matrix 's appropriateness to factor analysis, the data were included in the efa. by this test, four main factors were generated, which explained 63.6% of the variance in self - regulation. the first factor included the items 9, 11, 12, 14, 15, and 16. this factor alone explained 42.3% of the variation in self - regulation total score and the internal consistency coefficient of the items relevant to this factor was estimated as 0.85 by cronbach 's alpha. this item addressed requesting others around to remind the individual of the time of physical activity. 11 yielded the lowest (0.607) correlation ; it reflected the individual 's conversations with others about the ways to develop and implement physical activity and also correlated with item no. 2 (0.408). the second factor included the items 5, 6, 7, 8, and 11. this factor explained 9.6% of the variation in self - regulation and the cronbach 's alpha of the items relevant to this factor 's total items was found to be 0.81. 7 representing schedule development to do physical activity yielded the highest (0.703) correlation with item no. 11 yielded the lowest (0.408) correlation [table 2 ]. rotated component and structure matrix with pca and promax rotation for items of self - regulation questionnaire related to leisure time physical activity in iranian male adolescents the third factor included the items 1, 2, 3, and 4 this factor explained 6.3% of the variation in self - regulation and the cronbach 's alpha of the items relevant to this factor 's total items was found to be 0.82. 2 addressing the required facilities for physical activity yielded the highest (0.785) correlation with item no. 4 yielded the lowest (0.650) correlation and it addressed the benefits of physical activity. 10 (0.834) was related to self - reward after each accomplishment in performing physical activity or its constituents. the internal consistency of the items related to this factor was derived as 0.68 by cronbach 's alpha. as per the comments of the five - member expert panel, 12 out of total 25 items were revised and changed for phrasing and 6 items were deleted. the expert panel believed that addition of these two items enhances the questionnaire 's coverage of various dimensions of self - regulation construct and promotes its validity. 8 addresses an individual 's effort to find a solution when facing the barriers to physical activity. item no. 16 reflects an individual 's requesting others around to be able to run one 's physical activity. then, cvr of the remaining items was calculated and on using the relevant formula, the cvr of five items did not register the threshold level, and hence, the items were excluded. given that 0.79 was considered as the items cvi acceptability to remain in the questionnaire, all the remaining items (n = 16) were satisfactory with regard to cvi. after administering the questionnaire in the pilot study, we determined 0.79 as the acceptable comprehensibility and all items met the comprehensibility requirements. also, the questionnaire 's cronbach 's alpha was investigated in a pilot study of 75 members of the target population and was found to be 0.84. after exclusion of outliers, the mean physical activity was found to be 2324 1437 met - min / week and the mean physical activity in leisure time was 842 831 met - min / week. also, 293 (49%) participants had low activity, 278 (46%) had moderate activity, and 32 (5%) had high activity. the questionnaire 's cronbach 's alpha was found to be 0.90 after 603 participants filled it up. by correlation matrix, all the recommended items had a correlation coefficient of higher than 0.4 with the other items (p 0.005). all the items were determined as acceptable and none of them was excluded [table 1 ]. item total statistic of self - regulation questionnaire abut leisure time physical activity in iranian male adolescents kaiser meyer olkin index was found to be 0.92 and bartlett 's test of sphericity was significant at 95% ci (= 4139, df = 120, p = 0.00). given the adequacy of sample size and correlation matrix 's appropriateness to factor analysis, the data were included in the efa. by this test, four main factors were generated, which explained 63.6% of the variance in self - regulation. the first factor included the items 9, 11, 12, 14, 15, and 16. this factor alone explained 42.3% of the variation in self - regulation total score and the internal consistency coefficient of the items relevant to this factor was estimated as 0.85 by cronbach 's alpha. this item addressed requesting others around to remind the individual of the time of physical activity. 11 yielded the lowest (0.607) correlation ; it reflected the individual 's conversations with others about the ways to develop and implement physical activity and also correlated with item no. 2 (0.408). the second factor included the items 5, 6, 7, 8, and 11. this factor explained 9.6% of the variation in self - regulation and the cronbach 's alpha of the items relevant to this factor 's total items was found to be 0.81. 7 representing schedule development to do physical activity yielded the highest (0.703) correlation with item no. 11 yielded the lowest (0.408) correlation [table 2 ]. rotated component and structure matrix with pca and promax rotation for items of self - regulation questionnaire related to leisure time physical activity in iranian male adolescents the third factor included the items 1, 2, 3, and 4 this factor explained 6.3% of the variation in self - regulation and the cronbach 's alpha of the items relevant to this factor 's total items was found to be 0.82. 2 addressing the required facilities for physical activity yielded the highest (0.785) correlation with item no. 4 yielded the lowest (0.650) correlation and it addressed the benefits of physical activity. 10 (0.834) was related to self - reward after each accomplishment in performing physical activity or its constituents. the internal consistency of the items related to this factor was derived as 0.68 by cronbach 's alpha. this study was conducted to evaluate the psychometrics of a 16-item questionnaire specifically developed to measure self - regulation related to leisure time physical activity in iranian male adolescents. for this, reliability, content validity, and the questionnaire 's reliability was assessed by internal consistency and cronbach 's alpha in two steps. in the pilot study, cronbach 's alpha coefficient was derived as 0.84 and total internal consistency was found to be satisfactory. the findings of the present study are consistent with the reliability coefficient of the 53-item questionnaire introduced by petosa and calculated by test - retest. in addition, cronbach 's alpha was found to be 0.85 for enlistment of social support, 0.81 for goal setting and schedule development, 0.82 for self - instruction, and 0.68 for self - monitoring, representing satisfactory consistency of the items associated with any sub - construct of the questionnaire except self - monitoring. in petosa 's study, although the cronbach 's alpha was reported as 0.79 for self - monitoring, this index was found to be 0.62 for one of the measured sub - constructs, i.e. reinforcement. in petosa 's opinion, the emphasis of the items related to a sub - construct on the behavioral mechanisms of self - regulation and failure to address cognitive behaviors could explain the relatively low internal consistency of the items. hence, future studies are expected to enhance the internal consistency of this sub - construct by introducing one or two items related to cognitive processes of self - regulation. to achieve satisfactory face and content validity, we elicited the comments of health experts to determine the cvi and cvr of the 25 recommended items and then unsatisfactory items were excluded. finally, 16 items remained in accordance with the experts comments elicited in different steps. brons and gorow have offered literature review and eliciting the comments of experts and the representatives of the target population as the most important measures to achieve content validity for measurement instruments, which should be considered at the step of instrument development. therefore, the comments of five health experts outside the research team were elicited after literature review in the present study. these comments helped the researchers consider different dimensions of physical activity - related self - regulation and the contribution of each factor in the final items of the questionnaire at the step of instrument development. the first factor was mostly correlated with the items 9, 11, 12, 13, 14, 15, and 16, all of which are related to a sub - division of self - regulation construct addressing an individual 's effort to garner social and environmental support. therefore, this factor was named enlistment of social support. the associated items with this factor address thinking about the influence of others around on physical activity schedule, seeking assistance from others around to remind one of the time of physical activity, requesting them to eliminate the existing barriers to performing physical activity, and talking about the purposes and ways of performing physical activities with others. the second factor including the items 5, 6, 7, 8, and 11 exhibited the highest correlation this factor, namely goal setting, included the issues concerning setting goal and developing physical activity schedule. the items included in the second factor sub - division address the issues including development of a regular schedule and setting of specific output and behavioral goals for physical activity, planning for performing physical activity schedule along with others, planning for solving current problems, as well as talking about the purposes and methods of performing physical activity with others. 11 was concurrently correlated with the first and second factors, concerning talking with others about the purposes and ways of performing physical activity schedule. as this item is related to both planning and enlistment of social support, its correlation with both first and second factors could be justified. the third factor, namely self - instruction, included the items 1, 2, 3, and 4. these items assessed the rate of thinking about physical activity benefits, the ways to increase physical activity, and the facilities of and barriers to performing physical activity. these two items address the registry of performed physical activities and rewarding self for accomplishment of physical activity performance. however, the questionnaire developed by petosa has identified six sub - constructs consisting of goal setting, self - monitoring, time management, enlistment of social support, reinforcements, and relapse prevention. fleury introduced the index of self - regulation in 1998 to measure the self - regulation related to physical activity. the initial target population of this 9-item questionnaire includes middle - aged adults and only three dimensions, including stimulus control, behavior maintenance, and behavior monitoring, have been considered. in recent years, some items of this questionnaire were used to develop specific questionnaires to particular target populations and the validity and reliability of the new questionnaires were reported. the difference in the number of generated sub - constructs between the present study and two other studies could be attributed to the development and selection of the items as per the experts comments. these items were introduced based on the demographic and cultural specifications of the target community. have introduced a single sub - construct, namely self - reward, the associated behaviors with self - reward were assigned to the self - monitoring sub - construct. as the associated behaviors with self - reward are a subdivision of self - communication skills, it is also associated with social and cultural specifications of different communities, which could contribute to the number of the items selected by the expert panel to measure self - reward - related behaviors in iranian male adolescents. in addition, the selection of the present questionnaire 's items with the assistance of expert panel was made in such a way that led to elimination of relapse prevention and time management sub - constructs. failure to implement the qualitative extraction of the items using the target population and estimate concurrent and predictive validity could be the most important limitation of the present study. however, use of a considerable number of the target population members (n = 603) was a strength of this study. in addition, division of isfahan into three regions, privileged, semi - privileged, and sub - privileged, and enrollment of participants from all the regions could increase the generalizability of the findings. although the findings indicated that the 16-item questionnaire used in this study enjoyed satisfactory validity and reliability to measure physical activity - related self - regulation construct in leisure time of male adolescents in iran, the reassessment of validity and reliability of the new instrument in this population and others, and assessment of concurrent and predictive validity with regard to other main constructs of socio - cognitive theory could be helpful.	background : self - regulation is one of the current psychological concepts that have been known as a determinant of leisure time physical activity. due to cultural and social diversity in different societies and age groups, application of specific questionnaires is essential to perform investigations about physical activities. the aim of this study is development and evaluation of psychometric properties of a self - regulation questionnaire about leisure time physical activity in iranian male adolescents.materials and methods : this cross - sectional study was conducted in 2013, and data of 603 male students from 12 high schools in isfahan were collected. a comprehensive literature review and similar questionnaire review were conducted and 25 items were selected or developed to measure self - regulation. comprehensibility of items was evaluated in a pilot study and an expert panel evaluated face and content validity. exploratory factors analysis (efa) was used for evaluation of construct validity and extraction of sub - constructs of self - regulation. leisure time physical activity was assessed using international physical activity questionnaire (ipaq).results : the mean age of the participants was 16.3 years (sd = 1.0) and the range was 15 - 19 years. cronbach 's coefficient of the questionnaire in the pilot and main study was 0.84 and 0.90, respectively. efa resulted in four sub - constructs including enlistment of social support, goal setting, self - construction, and self - monitoring, which explained 63.6% of the variance of self-regulation.conclusions:results of this investigation provide some support to the validity and reliability of the 16-item questionnaire of self - regulation abut leisure time physical activity in the target group.
nonketotic hyperglycemia (nkh) is known to cause focal motor or secondarily generalized seizures. occipital seizures in nkh are seldom reported, especially with visual hallucinations and persistent homonymous hemianopia (hh) with characteristic radiological and electroencephalographic (eeg) findings. our patient was a middle - aged man who presented with a new onset, single episode of generalized tonic examination revealed persistent complete right hh and he was observed to have complex partial seizures. magnetic resonance imaging (mri) showed subcortical t2 hypointensity within the left occipital lobe in t2w and flair images. random blood glucose at presentation was 581 mg / dl with hba1c of 11.4%. the seizure and visual field deficits were successfully terminated by the introduction of antiseizure medication and glycemic control. the hh is reversible with apt treatment primarily including glycemic control with or without antiseizure medication. nonketotic hyperglycemia (nkh) signifies presence of high blood sugars in the absence of ketosis or hyperosmolar state. only a few case reports describe occipital seizures in nkh associated with a particular finding on mri brain and eeg. our patient was a 53-year - old indian male, known to have hypertension, type ii diabetes mellitus, and chronic kidney disease. on the day of presentation, he had complaints of visual impairment in both eyes, followed a few minutes later by a single brief episode of generalized tonic however, he had a history of skin abscesses secondary to poor glycemic control. in the er, he was seen in the postictal period and was agitated and drowsy suggestive of global brain dysfunction. laboratory results were significant for hyperglycemia with random plasma glucose level of 581 mg / dl and hba1c of 11.4% ; ketones were negative, findings suggestive of nkh. serum sodium and creatinine were 136 mg / dl and 2.9 mg / dl respectively. the cerebrospinal fluid analysis was done to rule out the possibility of concomitant inflammatory or infective process, which turned out to be normal. later, after regaining consciousness, his neurological examination showed a complete right hh. during examination, multiple episodes of complex partial seizures were observed characterized by sudden head and eye turning to the right, right - sided nystagmus, and cessation of speech and awareness. because of the abnormal renal function tests, mri brain with contrast was not performed. during conventional scalp eeg recording, the patient had three seizures, each one lasting about 1 min, associated with high amplitude fast activity over the left occipital region with ipsi- and contralateral spread followed by gradual reducing in frequency and amplitudes in the postictal phase (fig. therefore, oxcarbazepine was introduced following which the hh gradually disappeared after 3 days of treatment. our patient was a 53-year - old indian male, known to have hypertension, type ii diabetes mellitus, and chronic kidney disease. on the day of presentation, he had complaints of visual impairment in both eyes, followed a few minutes later by a single brief episode of generalized tonic in the er, he was seen in the postictal period and was agitated and drowsy suggestive of global brain dysfunction. laboratory results were significant for hyperglycemia with random plasma glucose level of 581 mg / dl and hba1c of 11.4% ; ketones were negative, findings suggestive of nkh. serum sodium and creatinine were 136 mg / dl and 2.9 mg / dl respectively. the cerebrospinal fluid analysis was done to rule out the possibility of concomitant inflammatory or infective process, which turned out to be normal. later, after regaining consciousness, his neurological examination showed a complete right hh. during examination, multiple episodes of complex partial seizures were observed characterized by sudden head and eye turning to the right, right - sided nystagmus, and cessation of speech and awareness. the mri brain showed hypointensity on t2wi and flair sequences as shown in fig. 1. because of the abnormal renal function tests, mri brain with contrast was not performed. during conventional scalp eeg recording, the patient had three seizures, each one lasting about 1 min, associated with high amplitude fast activity over the left occipital region with ipsi- and contralateral spread followed by gradual reducing in frequency and amplitudes in the postictal phase (fig. therefore, oxcarbazepine was introduced following which the hh gradually disappeared after 3 days of treatment. hyperglycemia is known to present with diverse neurological phenomena including focal seizures including epilepsia partialis continua. mostly reported cases are of motor origin, with scanty reports of occipital seizures especially in the setting of nkh. the occipital seizures may have a variety of visual symptoms like visual hallucinations, blurred vision, flickering lights and objects, as well as visual field defects as seen in our patient., with nkh, visual hallucinations, and versive seizures associated with similar mri brain and eeg findings as in our patient. however, in our patient, it was interesting to note that there was persistent hh lasting for almost 3 days, recovering only after initiation of a second antiseizure medication along with better glycemic control. this is contrary to the abovementioned report where seizures were controlled promptly with phenytoin and lorazepam within an hour.this prolonged visual deficit has previously been reported in hyperglycemia, possibly a postictal todd 's phenomena or epilepsia partialis continua. in the absence of motor seizure possible differential diagnosis includes migraine, stroke / transient ischemic attack, or posterior reversible encephalopathy. occipital seizures in hyperglycemic states have been described with blood glucose levels ranging from 200 mg / dl to over 500 mg / dl. only a few cases have mentioned the hba1c, usually high, similar to our case, indicating poor long - term control (6). hence, it is suggested that occipital seizures may be a result of long - term poor glycemic control rather than an acute hyperglycemic state. recently, katp channels have been implicated in the pathogenesis of hyperglycemia - related seizures. variable eeg and visual evoked potential findings have been described including ictal, interictal, and postictal epileptiform discharges. our patient had 3 seizure episodes during the eeg with progressive electrographic changes of high - amplitude, high - frequency sharp / spike - and - waves activity in the left occipital region with secondary spread to the contralateral side (fig. 2). magnetic resonance imaging brain finding is typically similar to our case, i.e., focal subcortical hypointensity on t2w and flair images. the postulated mechanisms include a free radical injury with iron deposition due to hypoxic ischemic injury (not supported by an animal model), transient seizure effect (not supported by the typical mri findings in postictal states i.e., hyper intensity in the affected cortical and subcortical regions on t2wi), and intracellular dehydration due to the seizure. the mainstay of treatment is good glycemic control, hydration, and use of antiseizure medication although the need for long - term use is not established yet. notably, phenytoin has been known to worsen hyperglycemia, and in some reported cases, it failed to control the seizures as well, like in our patient. our patient continued to have recurrent seizures after initiation of phenytoin that later remitted promptly with initiation of oxcarbazepine. a persistent visual field defect could represent a postictal todd 's phenomenon or epilepsia partialis continua. the optimal duration of antiseizure medications remains unknown considering the symptomatic nature of these seizures, due to a potentially reversible cause. prompt recognition is thus crucial in order to begin timely management, thus, prevent consequences like complex partial status epilepticus and neuronal damage.	introductionnonketotic hyperglycemia (nkh) is known to cause focal motor or secondarily generalized seizures. occipital seizures in nkh are seldom reported, especially with visual hallucinations and persistent homonymous hemianopia (hh) with characteristic radiological and electroencephalographic (eeg) findings.summaryour patient was a middle - aged man who presented with a new onset, single episode of generalized tonic clonic seizure and nkh. he complained of seeing intermittent colorful stripes in his right visual field. examination revealed persistent complete right hh and he was observed to have complex partial seizures.magnetic resonance imaging (mri) showed subcortical t2 hypointensity within the left occipital lobe in t2w and flair images. the eeg showed electrographic seizures originating from the left occipital region. random blood glucose at presentation was 581 mg / dl with hba1c of 11.4%.the seizure and visual field deficits were successfully terminated by the introduction of antiseizure medication and glycemic control.conclusionoccipital seizures with visual field deficits can occur in hyperglycemic states. these can be associated with specific mri brain and eeg changes. the hh is reversible with apt treatment primarily including glycemic control with or without antiseizure medication.
the data presented here are part of a larger study on the efficacy of a new psychoeducational intervention for individuals with fear of blushing (dijk. in press). participants were 52 individuals who applied for treatment because of their fear of blushing and 27 non - fearful controls. fearful participants applied for treatment after reading articles about a fear of blushing treatment study that appeared in both local and national media. control participants were all indirect acquaintances of the staff members of the department of clinical psychology of the university of groningen who were asked via these staff members to serve as control participants in a study on fear of blushing. they were screened with the blushing scale of the blushing trembling sweating questionnaire (btsq, bgels and reith 1999). control participants scoring over 50 on the blushing scale of the btsq were considered fearful of blushing and therefore excluded (n = 1). mean btsq blushing scores for the control group were lower than those of the fearful group (table 1). the two groups were similar with respect to gender, age, and educational level. table 1means and standard deviations of variables as a function of groupvariablesgroupt - statisticsnfcfbn = 27n = 49gender, % female77.873.50.41age34.30 (12.68)39.43 (11.35)1.81educational level 2.67 (0.92)2.61 (0.93)0.25stiat blushing, d - measure0.13 (0.47)0.37 (0.42)2.29stiat blushing, % error trials overall6.62 (6.24)8.28 (6.17)1.12 pairing blush - positive5.65 (6.48)11.77 (10.92)3.07 pairing blush - negative5.59 (6.95)4.80 (4.23)1.90others evaluations 6.00 (1.62)3.23 (1.56)7.32blushing questionnaire 9.16 (6.97)73.77 (12.59)24.62stiat single target implicit association test, nfc non - fearful controls, fb individuals with fear of blushingeducational level in categories of 1 tot 4 for higher education9-item subscale of the conditional cognition scale5-item subscale of the blushing, trembling, and sweating questionnairep 0.1). a two group (fb, nfc) anova on explicit blushing cognitions showed a significant main effect for group (f(1,74) = 53.64, p 0.1). a two group (fb, nfc) anova on explicit blushing cognitions showed a significant main effect for group (f(1,74) = 53.64, p < 0.01, partial = 0.42) indicating that individuals with fear of blushing had more negative explicit expectations about the social costs of their blushing than the control group. the stiat scores and explicit cognitions were shown to be largely unrelated (r = 0.17, n.s.). this was also evident when correlations were computed for each group separately (fearfuls : r = 0.03, n.s. ; the present study was designed as a first step in getting more insight into the role of automatic associations between blushing and social costs in fear of blushing in a treatment - seeking sample of individuals with fear of blushing. in line with our predictions, the results showed that the fearful group was characterized by stronger automatic associations between blushing and negative social outcomes (and/or weaker automatic associations between blushing and positive outcomes) than the control group. in addition, individuals with fear of blushing had more negative explicit expectations about the social costs of their blushing than the non - fearful control group. interestingly, explicit cognitions and automatic associations were shown to be largely unrelated, attesting to the relevance of measuring both automatic and explicit blushing associations for fear of blushing. in an attempt to explain the highly negative evaluation of blushing in individuals with fear of blushing, it has been proposed that fearful individuals overestimate the social costs of their blushing (dijk. furthermore, it has been argued that in addition to explicit beliefs, more automatic dysfunctional associations may be critically involved in fears, such as fear of blushing (e.g., ouimet. 2009). in line with the latter, the present study provided evidence that treatment - seeking individuals with fear of blushing show stronger automatic associations between blushing and social costs than non - fearful controls. in addition, the present findings regarding the explicit index of the anticipated social costs of displaying a blush replicated earlier findings from analogue samples (dijk. 2010). thus, the present results indicate that individuals with fear of blushing are characterized both by explicit negative expectations about the social costs of their blushing and by more automatic associations between blushing and social costs. while the pattern of results was in the same direction for automatic as well as explicit blushing associations, we did not find a significant correlation between both types of associations. this fits well within recent information - processing models concerning fear that consider explicit and automatic associations as distinct cognitive processes that influence different kinds of behaviors (e.g., gawronski and bodenhausen 2006 ; ouimet. 2009). in individuals with fear of blushing, automatic associations between blushing and social costs might trigger automatically initiated fear responses such as fearful thoughts and behavioral responses (cf. strack and deutch 2004). even when individuals with a fear of blushing have sufficient time and cognitive resources, they will probably not correct these initial dysfunctional associations on a more explicit level, because their explicit beliefs about blushing are similarly negative and dysfunctional. this way automatic and explicit cognitions about blushing may both act to sustain or even enhance the preoccupation with blushing in individuals with fear of blushing (cf. this signifies the importance of measuring both types of cognitions in order to obtain a more comprehensive understanding of the processes that may underlie fear of blushing. to further examine the causal influence of automatic blushing associations, it will be important to test whether the experimental manipulation of these automatic associations leads to changes in fear of blushing (cf. furthermore, the differential predictive validity of automatic versus explicit blushing associations should be tested for actual fearful behaviour in social contexts that might elicit a blushing response (cf. if dysfunctional automatic associations do indeed play a causal role in the generation and/or maintenance of anxiety symptoms, then both explicit and automatic levels of information processing should be addressed in therapy. following this, it seems important to investigate whether dysfunctional automatic associations decrease or even disappear under the influence of conventional cbt strategies. on the one hand, it seems possible that cbt might change automatic processes, for example via explicit attitude change (cf. gawronski and bodenhausen 2006) or via behavioral experiments or repeated exposure - in - vivo. on the other hand, it could be that residual dysfunctional automatic associations after cbt are involved in the recurrence of symptoms (cf. existing treatments should be adjusted or extended in a way that dysfunctional cognitions are targeted on both explicit and more automatic levels of information processing. while the present findings suggest that automatic and explicit associations seem to be distinct cognitive processes, the observed group differences for explicit associations however, it can not be ruled out that the difference in effect sizes is largely attributable to the methodological approach used. since fear of blushing and explicit blushing associations were both measured via self - report measures, considerable method variance could be shared between explicit blushing associations and fear of blushing that is not shared with the automatic associations, making it a much more stringent test for the automatic associations. secondly, it should be acknowledged that the categories used in the stiat were not explicitly labeled in terms of social costs but more generally as negative vs. positive outcomes. future research will have to determine whether more specific automatic cost associations are indeed important in fear of blushing, or whether individuals with fear of blushing are characterized by negative automatic associations with blushing in general. thirdly, the control group in the present design was not assessed for clinical status. possibly the degree of psychopathological symptoms was low in the present control group, and, therefore, it can not be ruled out that other clinical groups besides individuals with fear of blushing might also show negative associations with blushing. lastly, it would be interesting to compare social anxiety patients with and without a fear of blushing to see whether negative blushing associations are specific for individuals with a fear of blushing or a more general characteristic of social phobia. this comparison could also rule out the possibility that socially anxious individuals have a general tendency to judge situations more negatively, irrespective of blushing per se. to conclude, the present study not only showed that individuals with fear of blushing explicitly indicate that blushing is associated with negative outcomes, it also revealed that at a more automatic level blushing elicits negative automatic associations in this group. both types of processes appear to be separate constructs that may help to increase our understanding of fear of blushing. these results imply that it might be valuable to include assessments of automatic associations in addition to the more traditional self - report questionnaires, for example as pre and post measures in the evaluation of treatment (e.g., huijding and de jong 2009 ; teachman. when conventional treatments do not sufficiently alter dysfunctional automatic associations, it might also be relevant to design new ingredients of cbt that more directly target these automatic associations. as a case in point, it has recently been shown that a computerized association task was effective in reducing social anxiety via modifying participants dysfunctional automatic associations (clerkin and teachman 2010). together, these findings may help to further increase our understanding of the cognitive processes that underlie fear of blushing.	to explain fear of blushing, it has been proposed that individuals with fear of blushing overestimate the social costs of their blushing. current information - processing models emphasize the relevance of differentiating between more automatic and more explicit cognitions, as both types of cognitions may independently influence behavior. the present study tested whether individuals with fear of blushing expect blushing to have more negative social consequences than controls, both on an explicit level and on a more automatic level. automatic associations between blushing and social costs were assessed in a treatment - seeking sample of individuals with fear of blushing who met dsm - iv criteria for social anxiety disorder (n = 49) and a non - anxious control group (n = 27) using a single - target implicit association test (stiat). in addition, participants explicit expectations about the social costs of their blushing were assessed. individuals with fear of blushing showed stronger associations between blushing and negative outcomes, as indicated by both stiat and self - report. the findings support the view that automatic and explicit associations between blushing and social costs may both help to enhance our understanding of the cognitive processes that underlie fear of blushing.
the bba mandated that hcfa implement risk - adjustment capitated payments for medicare+choice organizations (m+cos) in the year 2000. prior to this, the method for determining payments to capitated health plans was based on a beneficiary 's location and was adjusted by demographic factors only. the demographic factors associate a predicted relative costliness with demographic characteristics, e.g., age, sex, and medicaid eligibilty starting in 2000, measures of health status were to be added to adjust payments. the use of inpatient data was deemed a feasible beginning in applying risk - adjustment methods to capitated payments. hospital admissions are relatively low in frequency compared with physician visits, making them a good first step in data collection and transmission by health plans. admissions are also a marker for a group of people that tend to be costly to the medicare program in the future. inpatient data could also be used for 2000 because an inpatient - based risk adjustment model was available., the model uses beneficiary demographic characteristics and categories of hospitalizations in one year to predict medicare costs in the next year. the model has been applied to the medicare beneficiaries eligible because of age or disability, but not, at this time, to the people having end - stage renal disease (esrd). the demographic characteristics used are : age, sex, status of medicaid eligibility, and whether a person over age 65 was originally medicare eligible due to disability. there are 15 mutually exclusive cost categories into which a hospitalization may be categorized if the stay is in one of the disease groups predictive of future year costs. the principal diagnosis (international classification of diseases, 9th revision, clinical modification) determines classification in most cases. for each individual, the stay with the greatest predicted cost implications determines the incremental payment. as described by pope. (2000), some hospitalizations are subsumed into the base payment categories that use only demographics to predict payment. these stays may have principal diagnoses that do not predict increased costs, diagnoses that are vague, or the stays may have been for fewer than 2 days. each applicable demographic and pip category is associated with an increment to a person 's risk - adjustment factor ; the sum of these increments is the final factor for an individual. (2000) describes the model in a table that contains the medicare incremental factors. one last factor enters near the end of the payment computation process. because medicare 's financial liability is reduced when a beneficiary has a primary insurer other than medicare, a working aged the risk - adjusted payment is multiplied by the working aged factor if an enrollee is reported to have coverage by another primary payer in a given month. risk - adjustment models are usually calibrated with total expenditures as the dependent variable of a regression, and the coefficients of the demographic and disease variables estimated as incremental expenditures. the risk - adjustment scores are applied, however, as adjustments to standardized payment rates, rather than as direct predictors of expenditures. the coefficients are converted to relative adjustment factors by dividing them by the national mean predicted per capita expenditure for the ffs medicare population. to implement the system it is necessary to compute the appropriate standardized payment rates and the national mean. the following discussion addresses these and other aspects of hcfa 's implementation of the pipdcg system : the integration of a demographic module for newly eligible persons, computation of the national mean, derivation of the standardized rates (the risk - adjustment ratebook), the data flow for hmo encounter data, and the transition schedule for implementation. those beneficiaries designated as having esrd were excluded from the modeling and will continue to be capitated using current esrd rates and demographic factors. the largest group not covered by the model are the beneficiaries new to medicare, for whom hcfa does not have sufficient data for risk adjustment. these beneficiaries may be of any age ranging from new young disabled eligible persons, to some who start their part b medicare benefits when considerably older than age 65. because the pipdcg model is being implemented only for people with a full year of diagnosis data available, some of the new enrollees can not be risk adjusted for 2 years. factors were developed based on age, sex and medicaid status (table 1). because most of the enrollees with insufficient eligibility time are age 65 through 67, the 65 - 69 age group, which is usually averaged, most older persons in this 5-year age group will be paid under the risk model, not the new enrollee module. including their higher expenditures in computing the mean for new enrollees in the age group using claims for the standard 5 percent sample of medicare beneficiaries, average expenditures were computed for all age / sex groups, with and without medicaid status. to derive factors for the 65 through 69 single - year age / sex groups, the first step was to compute average expenditures for beneficiaries age 66 through 79. using a regression model, a best - fitting line was estimated and projected for beneficiaries age 65 through 69. this was done separately for the medicaid and non - medicaid beneficiaries so that a medicaid increment could be computed. the resulting demographic model has been incorporated as a module in the pipdcg software distributed by hcfa. with all the aged and disabled beneficiaries covered by the system, risk adjustment was ready to be integrated into hcfa 's payment system. the pipdcg model could have been implemented as an adjuster to a national capitation rate, however, hcfa has traditionally used the county as the area for which base rates are computed. the bba did not change this, but provided some flexibility prior to the bba, using the adjusted average per capita cost (aapcc) method, a standardized payment rate was assigned to each county. this rate was equal to 95 percent of the projected per capita ffs costs in the county, divided by the average demographic factor for the county ffs population. as previously discussed, the demographic factor is a measure of a person 's expected expenditures, predicted from demographic characteristics, relative to the national average. each resulting county rate is the medicare per capita payment as it would be for the national average medicare beneficiary, given the county 's price and utilization patterns. hmos were paid each enrollee 's county rate adjusted for the individual 's factors, based on age, sex, medicaid status, etc. the rates computed using risk - adjustment factors are not necessarily the same as those computed using the demographic factors. the average county risk factors can be quite different numerically from the purely demographic factors. such a risk ratebook could have been computed for each year in parallel with the aapcc demographic ratebook, but the bba mandated changes in the way both the demographic and risk ratebooks would be computed for 1998 and beyond. the bba ratebook changes are intended to reduce the variation in rates over time and across counties, and to encourage m+cos to expand into counties without capitated plans. the new demographic ratebooks are based on the aapcc rates in effect in 1997 and follow a specified algorithm projecting forward. starting in 1998, the new county rate would be the greatest of : a 2-percent increase over the prior year rate ; a minimum or floor amount ; or a blend of a national rate with an area - specific or local county rate. which of these applies depends finally upon comparing total projected payments under this greatest of rule to projected payments under the local rates alone. if projected payments exceed those using the local rate method, the excess is reduced (eliminated if possible) by diminishing or eliminating the blend rates till one of the other this comparison of two simulated total payment amounts is the budget neutrality referred to in bba. payments can and do exceed the budget neutral amounts because the floor and 2 percent minimum increase provisions limit the ability to match the local rate payments. for purposes of the rate calculation, the local rate is the 1997 aapcc rate trended forward by the national m+c growth rate projected to the payment year. first a standardized mean payment is computed : the sum of payments resulting if all medicare beneficiaries were enrolled in capitated entities, divided by the sum of the beneficiaries adjustment factors. the payments for each person are the product of the local standardized rates and the appropriate adjustment factor. this standardized rate is then adjusted for input price levels for each local area before being blended with the local rate. applying the bba method other bba rate changes are : subtraction of the portion of per capita costs related to paying teaching hospitals for medical education ; shifting of a portion of home health expenditures to the part b per capita amount from part a ; and using a national average split, rather than county - specific proportions of expenditures that are allocated to part a and part b. the portion of the rates associated with graduate medical education payments is phased out in 20 percentage point increments starting in 1998 ; 100 percent in 2002. health status risk adjustment based on hospital inpatient data was mandated to start in 2000 and had to incorporate the bba ratebook requirements. the law was interpreted to require that the demographic bba ratebook should continue to be published each year, even if risk adjustment was in effect. because the bba demographic ratebook would not be the appropriate base to be multiplied by pipdcg factors, a process of rescaling it to the risk ratebook was required. as the ratebooks follow a complicated path over time, the rescaling factors are not constant. the risk ratebook process started with the computation of a risk ratebook for the bba base year, 1997. the average risk factor for the ffs beneficiaries in each county was computed using the pipdcg model using the new - enrollee module where appropriate. each county average expenditure prediction was divided by the national mean predicted expenditure toproduce an average risk factor for the county. three years of base year data, 1994, 1995, and 1996, were used to compute three sets of factors. variation across years was small in almost all cases and the mean factor was used by hcfa 's actuaries for most counties. for the numerators of the rates, the actuaries computed the per capita costs for each county, aggregating the part a and part b expenditures for aged persons and disabled persons. they divided 95 percent of the per capita costs by the county average risk factors to standardized them, and produce the 1997 base risk ratebook. the base risk ratebook was then projected forward following the same bba rules governing the demographic ratebook. with both ratebooks prepared for each year, rescaling factors, which convert the demographic rates to the risk rates, they are the ratios of the county risk rates to the county demographic rates. in march 1999, hcfa published for 2000 the bba demographic ratebook and the rescaling factors for risk adjustment. the risk - adjustment system incorporates both aged and disabled beneficiaries ' total expenditures in a unified system, with factors relative to the mean for the whole group. the demographic method maintains four sets of factors and rates, for part a and part b expenditures for aged persons and disabled persons. in order to rescale the aged rates, the county aged rescaling factor is set to the ratio of the risk rate for the county to the sum of the aged part a and part b demographic rates. a second, if the bba process were linear, without the greatest of provision, a simpler rescaling could have been done. however, because a given county can be, for example, at the floor or blend in one ratebook and at the rate reflecting a 2-percent increase in the other, it is necessary to compute the rescaling factor after the rates have been computed for both ratebooks. the process thus far described has emphasized the use of ffs data in computing factors for beneficiaries to set the rate - book. however, data from hmos do play a part in the computation of the national rate and the budget neutrality calculation in the bba ratebook process. for the national rate, they are needed because both ffs and managed care populations are used to calculate the mean payment for all beneficiaries. for budget neutrality this is in addition to the primary role of the data in determining factors and payments for individual enrollees. with all beneficiaries being potential managed care enrollees, complete data are needed on each person. by combining data from both ffs and m+cos, this was done for the implementation year starting january 2000. to facilitate data flow, three modes of data submission hmos could elect to have their participating hospitals submit standard billing form ub-92 bills to hcfa fiscal intermediaries ; plans could themselves submit standard ub-92s ; or plans could submit an abbreviated ub-92 with fewer elements and fewer fiscal intermediary edits. during the second data year, only the latter two methods were allowed. the abbreviated record requires elements sufficient to determine eligibility and compute an amount that would have been paid under standard medicare pricing. priced encounter records can be used along with ffs information in future recalibrations of risk - adjustment models. the implementation data collection year is not a calendar year. in consultation with the industry, it was decided to collect data from july through june to be used to compute factors for use in the following calendar year. for payments in 2000, bills and encounter records with discharge dates from july 1998 through june 1999 were used. the 6 months following allow data flow after the june service date, and time for computation of factors for all beneficiaries. this method was chosen over the alternative : collection of data january - december, paying based on interim factors, and reconciliation when a final set of factors becomes available midyear. much of the industry preferred knowing the final factor at the start of the year. since that determination, however, as a startup modification, it has been decided to let lagging july through june data continue to flow into hcfa and to reconcile final factors after the payment year. the process of collecting the data required setting up communications between plans and fiscal intermediaries ; training sessions were organized and much intensive consultation was needed by some plans. on the hcfa side, modifications had to be made to claims processing systems to allow processing the new claim types without confusion with ffs claims. the first year of data collection, service period july 1997-june 1998, was used to estimate the impact of the fully implemented pipdcg system on m+cos. the best estimate at that time indicated an aggregate payment reduction compared with a purely demographic method of 7 percent. (this is not necessarily a decrease in payment from one year to the next. refinement of the estimate was done after collection of the second year 's data. although the profile of inpatient stays was similar to year 1, the estimated impact was smaller because final values of some parameters in the system became available after the earliest estimate for the first year. the current impact estimate of a fully phased - in pipdcg model is a aggregate payment reduction between 5 and 6 percent relative to the demographic system. most estimated plan level impacts were negative, but there were a few plans that would receive increases based on their adverse selection detected by the pipdcg model. to soften the impact and mitigate concerns about data problems at some hmos, a decision was made to phase - in the risk adjustment system. a weighted average (or transition blend) of the demographic and risk adjustment system payments is to be paid. congress mandated the following weights on the risk - adjusted part of the average : in 2000, 10 percent ; in 2001, 10 percent ; in 2002, not more than 20 percent. as it is hcfa 's goal to move to a more comprehensive system, using additional diagnoses from ambulatory settings, in 2004, it has not been determined what, if any, transition will occur at that time. to compute and report to m+cos the payments using a blend of two systems, hcfa had to develop a new payment and m+co reporting system. the new payment system computes payments under the demographic and risk systems separately and computes the weighted average of the final numbers. monthly enrollment reports are sent to m+cos, as they have been, but they have gotten more complex because the characteristics needed to determine adjustment factors for each system differ. the report now includes the characteristics for both systems, including the pipdcg group to which the person is assigned. bill and encounter data from all sources were compiled and merged with demographic data to produce relative risk factors for all medicare beneficiaries. the ratebooks for 2000 had been published the previous march, as directed by bba. in december, january enrollments were determined and the payments for both parts of the transition blend were computed and averaged. enrollment reports went out to the m+cos and payments were made on schedule for january 2000. those beneficiaries designated as having esrd were excluded from the modeling and will continue to be capitated using current esrd rates and demographic factors. the largest group not covered by the model are the beneficiaries new to medicare, for whom hcfa does not have sufficient data for risk adjustment. these beneficiaries may be of any age ranging from new young disabled eligible persons, to some who start their part b medicare benefits when considerably older than age 65. because the pipdcg model is being implemented only for people with a full year of diagnosis data available, some of the new enrollees can not be risk adjusted for 2 years. factors were developed based on age, sex and medicaid status (table 1). because most of the enrollees with insufficient eligibility time are age 65 through 67, the 65 - 69 age group, which is usually averaged, most older persons in this 5-year age group will be paid under the risk model, not the new enrollee module. including their higher expenditures in computing the mean for new enrollees in the age group using claims for the standard 5 percent sample of medicare beneficiaries, average expenditures were computed for all age / sex groups, with and without medicaid status. to derive factors for the 65 through 69 single - year age / sex groups, the first step was to compute average expenditures for beneficiaries age 66 through 79. using a regression model, a best - fitting line was estimated and projected for beneficiaries age 65 through 69. this was done separately for the medicaid and non - medicaid beneficiaries so that a medicaid increment could be computed. the resulting demographic model has been incorporated as a module in the pipdcg software distributed by hcfa. with all the aged and disabled beneficiaries covered by the system, risk adjustment was ready to be integrated into hcfa 's payment system. the pipdcg model could have been implemented as an adjuster to a national capitation rate, however, hcfa has traditionally used the county as the area for which base rates are computed. the bba did not change this, but provided some flexibility prior to the bba, using the adjusted average per capita cost (aapcc) method, a standardized payment rate was assigned to each county. this rate was equal to 95 percent of the projected per capita ffs costs in the county, divided by the average demographic factor for the county ffs population. as previously discussed, the demographic factor is a measure of a person 's expected expenditures, predicted from demographic characteristics, relative to the national average. each resulting county rate is the medicare per capita payment as it would be for the national average medicare beneficiary, given the county 's price and utilization patterns. hmos were paid each enrollee 's county rate adjusted for the individual 's factors, based on age, sex, medicaid status, etc. the rates computed using risk - adjustment factors are not necessarily the same as those computed using the demographic factors. the average county risk factors can be quite different numerically from the purely demographic factors. such a risk ratebook could have been computed for each year in parallel with the aapcc demographic ratebook, but the bba mandated changes in the way both the demographic and risk ratebooks would be computed for 1998 and beyond. the bba ratebook changes are intended to reduce the variation in rates over time and across counties, and to encourage m+cos to expand into counties without capitated plans. the new demographic ratebooks are based on the aapcc rates in effect in 1997 and follow a specified algorithm projecting forward. starting in 1998, the new county rate would be the greatest of : a 2-percent increase over the prior year rate ; a minimum or floor amount ; or a blend of a national rate with an area - specific or local county rate. which of these applies depends finally upon comparing total projected payments under this greatest of rule to projected payments under the local rates alone. if projected payments exceed those using the local rate method, the excess is reduced (eliminated if possible) by diminishing or eliminating the blend rates till one of the other this comparison of two simulated total payment amounts is the budget neutrality referred to in bba. payments can and do exceed the budget neutral amounts because the floor and 2 percent minimum increase provisions limit the ability to match the local rate payments. for purposes of the rate calculation, the local rate is the 1997 aapcc rate trended forward by the national m+c growth rate projected to the payment year. per capita cost adjusted downward (till 2003) by a fraction of a percentage point. first a standardized mean payment is computed : the sum of payments resulting if all medicare beneficiaries were enrolled in capitated entities, divided by the sum of the beneficiaries adjustment factors. the payments for each person are the product of the local standardized rates and the appropriate adjustment factor. this standardized rate is then adjusted for input price levels for each local area before being blended with the local rate. applying the bba method other bba rate changes are : subtraction of the portion of per capita costs related to paying teaching hospitals for medical education ; shifting of a portion of home health expenditures to the part b per capita amount from part a ; and using a national average split, rather than county - specific proportions of expenditures that are allocated to part a and part b. the portion of the rates associated with graduate medical education payments is phased out in 20 percentage point increments starting in 1998 ; 100 percent in 2002. health status risk adjustment based on hospital inpatient data was mandated to start in 2000 and had to incorporate the bba ratebook requirements. the law was interpreted to require that the demographic bba ratebook should continue to be published each year, even if risk adjustment was in effect. because the bba demographic ratebook would not be the appropriate base to be multiplied by pipdcg factors, a process of rescaling it to the risk ratebook was required. as the ratebooks follow a complicated path over time, the rescaling factors are not constant. the risk ratebook process started with the computation of a risk ratebook for the bba base year, 1997. the average risk factor for the ffs beneficiaries in each county was computed using the pipdcg model using the new - enrollee module where appropriate. each county average expenditure prediction was divided by the national mean predicted expenditure toproduce an average risk factor for the county. three years of base year data, 1994, 1995, and 1996, were used to compute three sets of factors. variation across years was small in almost all cases and the mean factor was used by hcfa 's actuaries for most counties. for the numerators of the rates, the actuaries computed the per capita costs for each county, aggregating the part a and part b expenditures for aged persons and disabled persons. they divided 95 percent of the per capita costs by the county average risk factors to standardized them, and produce the 1997 base risk ratebook. the base risk ratebook was then projected forward following the same bba rules governing the demographic ratebook. with both ratebooks prepared for each year, rescaling factors, which convert the demographic rates to the risk rates, they are the ratios of the county risk rates to the county demographic rates. in march 1999, hcfa published for 2000 the bba demographic ratebook and the rescaling factors for risk adjustment. the risk - adjustment system incorporates both aged and disabled beneficiaries ' total expenditures in a unified system, with factors relative to the mean for the whole group. the demographic method maintains four sets of factors and rates, for part a and part b expenditures for aged persons and disabled persons. in order to rescale the aged rates, the county aged rescaling factor is set to the ratio of the risk rate for the county to the sum of the aged part a and part b demographic rates. if the bba process were linear, without the greatest of provision, a simpler rescaling could have been done. however, because a given county can be, for example, at the floor or blend in one ratebook and at the rate reflecting a 2-percent increase in the other, it is necessary to compute the rescaling factor after the rates have been computed for both ratebooks. the process thus far described has emphasized the use of ffs data in computing factors for beneficiaries to set the rate - book. however, data from hmos do play a part in the computation of the national rate and the budget neutrality calculation in the bba ratebook process. for the national rate, they are needed because both ffs and managed care populations are used to calculate the mean payment for all beneficiaries. for budget neutrality this is in addition to the primary role of the data in determining factors and payments for individual enrollees. with all beneficiaries being potential managed care enrollees, complete data are needed on each person. by combining data from both ffs and m+cos, this was done for the implementation year starting january 2000. to facilitate data flow, three modes of data submission hmos could elect to have their participating hospitals submit standard billing form ub-92 bills to hcfa fiscal intermediaries ; plans could themselves submit standard ub-92s ; or plans could submit an abbreviated ub-92 with fewer elements and fewer fiscal intermediary edits. during the second data year, only the latter two methods were allowed. the abbreviated record requires elements sufficient to determine eligibility and compute an amount that would have been paid under standard medicare pricing. priced encounter records can be used along with ffs information in future recalibrations of risk - adjustment models. the implementation data collection year is not a calendar year. in consultation with the industry, it was decided to collect data from july through june to be used to compute factors for use in the following calendar year. for payments in 2000, bills and encounter records with discharge dates from july 1998 through june 1999 were used. the 6 months following allow data flow after the june service date, and time for computation of factors for all beneficiaries. this method was chosen over the alternative : collection of data january - december, paying based on interim factors, and reconciliation when a final set of factors becomes available midyear. much of the industry preferred knowing the final factor at the start of the year. since that determination, however, as a startup modification, it has been decided to let lagging july through june data continue to flow into hcfa and to reconcile final factors after the payment year. the process of collecting the data required setting up communications between plans and fiscal intermediaries ; training sessions were organized and much intensive consultation was needed by some plans. on the hcfa side, modifications had to be made to claims processing systems to allow processing the new claim types without confusion with ffs claims. the first year of data collection, service period july 1997-june 1998, was used to estimate the impact of the fully implemented pipdcg system on m+cos. the best estimate at that time indicated an aggregate payment reduction compared with a purely demographic method of 7 percent. (this is not necessarily a decrease in payment from one year to the next. refinement of the estimate was done after collection of the second year 's data. although the profile of inpatient stays was similar to year 1, the estimated impact was smaller because final values of some parameters in the system became available after the earliest estimate for the first year. the current impact estimate of a fully phased - in pipdcg model is a aggregate payment reduction between 5 and 6 percent relative to the demographic system. most estimated plan level impacts were negative, but there were a few plans that would receive increases based on their adverse selection detected by the pipdcg model. to soften the impact and mitigate concerns about data problems at some hmos, a decision was made to phase - in the risk adjustment system. a weighted average (or transition blend) of the demographic and risk adjustment system payments is to be paid. congress mandated the following weights on the risk - adjusted part of the average : in 2000, 10 percent ; in 2001, 10 percent ; in 2002, not more than 20 percent. as it is hcfa 's goal to move to a more comprehensive system, using additional diagnoses from ambulatory settings, in 2004, it has not been determined what, if any, transition will occur at that time. to compute and report to m+cos the payments using a blend of two systems the new payment system computes payments under the demographic and risk systems separately and computes the weighted average of the final numbers. monthly enrollment reports are sent to m+cos, as they have been, but they have gotten more complex because the characteristics needed to determine adjustment factors for each system differ. the report now includes the characteristics for both systems, including the pipdcg group to which the person is assigned. bill and encounter data from all sources were compiled and merged with demographic data to produce relative risk factors for all medicare beneficiaries. the ratebooks for 2000 had been published the previous march, as directed by bba. in december, january enrollments were determined and the payments for both parts of the transition blend were computed and averaged. enrollment reports went out to the m+cos and payments were made on schedule for january 2000. hcfa and the hmo industry have shown that a prospective risk - adjusted capitation system can be implemented on a large scale, even in the complex environment created by the bba. risk - adjustment factors have been computed, not only for the more than 6 million medicare hmo enrollees, but for nearly all the approximately 40 million medicare beneficiaries. the accuracy of payments for m+cos is improved over demographic methods even when only 10 - 12 percent of enrollees are distinguished by disease - based measures, as in the pipdcg model. for the future, hcfa has sponsored much research into more comprehensive models that use ambulatory data, and has found them even more accurate at the individual and group level. with the cooperation of the industry the primary goal of risk adjustment has been to make payments that match the projected needs of enrollees. there are many health insurance systems that face the problem of making proper payments for enrollee groups with atypical health status. in some states the decreasing cost of electronic data processing and communications has made encounter - based risk - adjustment systems feasible. the data flows used for risk adjustment, particularly from ambulatory care, have other important uses, however. capitated entities will have the data needed for better management of patients and for better management of costs. in addition, the data allow the development of many of the quality measures being used by industry today. special data collection efforts to determine the rates of screenings, immunizations, tests and procedures can be substantially reduced. the implementation of medicare 's first health - status based system for capitated payment indicates the feasibility of further improved payment systems and collection of data for outcomes and quality studies.	the health care financing administration (hcfa) implemented risk adjustment for medicare capitated organizations january 2000. the risk adjustment system used, the principal inpatient diagnostic cost group (pipdcg) method, had to be incorporated into the payment structure mandated by the balanced budget act of 1997 (bba). this article describes how risk adjustment was integrated into the payment system within the rules of the bba, and how fee - for - service (ffs) and health maintenance organization (hmo) data are collected and used in the determination of payment.
when the elderly fall and injure themselves, they may be bedridden and require full - time care. various factors contribute to the incidence of falls, including a decreased ability to control posture. postural control involves the appropriate use of the muscles attached to the hips and ankles and an adequate stepping strategy. stepping strategies involve movements to form a new base of support in the event of balance disturbances in order to prevent falling2,3,4,5,6. a time delay during stepping has been reported to be associated with a tendency to fall by the elderly7. many studies have performed motion analyses to contribute toward prevention of falls in this population. more effective exercises can be prescribed for the elderly by identifying the muscles involved in successful stepping strategies. the purpose of this study was to investigate and evaluate the timing and amount of muscle activity in the forward - stepping motion of young persons. seven healthy subjects (3 males, 4 females ; age, 21.3 9 years ; height, 164.9 6.5 cm ; weight, 56.9 7.2 kg) participated in this study. all subjects were informed of the aims of the study and gave their written informed consent before participation. all test protocols were approved by heisei college of medical technology ethics committee [h23 - 9 ]. the stepping distances were 50 cm (condition 1) and 80 cm (condition 2) from the center point. emg data were recorded using a telemyosystem g2 em-601 and myo research xp (noraxon), and data were sampled at 1,000 hz. muscle activities of the tibialis anterior, gastronemius lateral head, rectus femoris, biceps femoris, vastus medialis, and soleus of the stance and swing legs were recorded simultaneously. the electrodes were 55 mm in diameter and were positioned on each muscle in line with the fiber direction. emg data were rectified and the root mean square (rms) value over 50 ms was determined. all measured waveforms were calculated as % mvc normalized rms value at the time of mvc for 3 s. the analysis focused on two parameters of the motion task. the first was the reaction time (rt), the time elapsing from the light signal to the plantar surface leaving the ground (foot - off). the second was the movement time (mt), the time elapsing from foot - off to the plantar surface making contact with the ground again. to determine the foot - off time of the plantar surface and the time when it contacted the ground again, a foot switch was used. this switch was pasted to the side of the heel and to the ball of the stepping foot and was synchronized with the emg. the paired t - test was used to test the significance of the differences in rt and mt between the two experimental conditions. the rate of change of condition 2 to that of condition 1 (condition 2/condition 1 = rate of change) in rt and mt, and the amount of muscle activity were calculated. pearson s correlation coefficient was used to examine the relationship between the rate of change in the amount of muscle activity and rt and mt. values are meanssd. : p<0.05, : p<0.01 the results are presented in tables 1, 2, 3. the average value of rt in condition 1 was 0.49 s, while that in condition 2 was 0.54 s, a significant increase. the average value of mt in condition 1 was 0.33 s, and in condition 2 it was 0.40 s, a significant increase. a high negative correlation was found between the rate of change in the amount of tibialis anterior muscle activity of the stance leg (r = 0.885) and rt (table 4). high positive correlations were found between the rates of change in the gastronemius (r = 0.725) and soleus (r = 0.799) muscles and rt of the swing leg. the results of this study suggest a relationship between the tibialis anterior muscle of the stance leg, the soleus muscle, the gastrocnemius muscle of the swing leg, and rt. the tibialis anterior muscle of the stance leg moves the leg forward by quickly changing the center of gravity. rt is adequate when the activity of the tibialis anterior muscle is high, as evidenced by the negative correlation between values for this muscle activity and rt. in contrast, high positive correlations between rt and the gastrocnemius and soleus muscle activities were found for the swing leg. these muscles change the center of gravity by swinging the leg forward and placing the foot forward. thus, more rt is required when the activity of these muscles is high. one is to swing the leg out taking a step forward using the gastrocnemius and soleus muscles of the swing leg. the other is to take a step forward using the tibialis anterior muscle of the stance leg. increasing the activity of the tibialis anterior muscle of the stance leg may lead to taking a step forward rapidly. no correlations between mt and muscle activities were found in this study. the initiation of movement and changes in the center of gravity were observed during rt measurements. we would like to study a large number of subjects who are much older than the participants of the present study in the future, and we would like to conduct exercises for the stance leg during therapy for the elderly.	[purpose ] the purpose of this study was to investigate and evaluate the timing and amount of muscle activity during forward - stepping motion. [subjects and methods ] seven healthy subjects participated in this study. the task was to step forward from a static standing position. timing and amount of muscle activity were measured during the task. muscle activities of the stance leg and the swing leg were measured using surface electromyography (emg). [results ] a high negative correlation was found between the rate of change in the amount of tibialis anterior muscle activity of the stance leg and the reaction time. high positive correlations were found between the rates of change in the gastrocnemius and soleus muscles and the reaction time of the swing leg. [discussion ] forward - stepping motion can be accomplished using two strategies. one is to swing the leg out taking a step forward using the gastrocnemius and soleus muscles of the swing leg. the other is to take a step forward using the tibialis anterior muscle of the stance leg. increasing the activity of the tibialis anterior muscle of the stance leg may lead to taking a step forward rapidly.
longipalpis sandflies were allowed to feed on 2 hounds naturally infected with l. infantum, strain mcan / us/2001/foxymo1 or a closely related strain. during 20072011, the hounds had been tested for infection with leishmania spp. by elisa, pcr, and dual path platform test (chembio diagnostic systems, inc. medford, ny, usa (table 1). l. infantum development in these sandflies was assessed by dissecting flies starting at 72 hours after feeding and every other day thereafter. migration and attachment of parasites to the stomodeal valve of the sandfly and formation of a gel - like plug were evident at 10 days after feeding (figure 1), indicating successful parasite development. serologic results determined by immunofluorescence antibody testing : < 64 indicates negative (). pcr results : indicates no amplification ; borderline indicates amplification on 1 of 3 tests ; positive (+) indicates amplification on 2 of 3 or 3 of 3 tests. development of l. infantum (mcan / us/2001/ foxymo1) in laboratory - reared lutzomyia longipalpis sandflies led to stomodeal valve blockage 1013 days after infection. a) dissected gut of infected sandfly, showing stomodeal valve (cardia) obstructed by leishmania parasites (dashed box). b) parasites obstructing stomodeal valve and parasite - secreted plug (dashed box). original magnification 40. c) parasite plug dissected from the stomodeal valve, showing metacyclic promastigote parasites attached to plug (arrow), as well as free - swimming parasites (arrowheads). next, to determine sandfly capacity to transmit the us strain of l. infantum to a susceptible vertebrate host, we allowed l. infantum naive and l. infantum infected sandflies to feed on 7 l. infantum naive hamsters for 13 days. for confirmation of l. infantum infection, we dissected the alimentary tract of sandflies that fed on the hamsters (table 2). a total of 30 sandflies were used for feeding on hamsters ; 11 flies fed and were subjected to leishmania detection by pcr, which confirmed leishmania positivity for 5 sandflies. hamster l. infantum dna was detected in hamster blood by quantitative pcr (qpcr) and was present in hamster nos. 1 (at 2 mo), 2 (at 3 mo), 5 (at 4 mo), and 6 (at 4 mo) with cycle thresholds of 43.88, 28.27, 34.38, and 45 respectively. 5, a cutaneous lesion consistent with leishmania infection persisted for 1 month. tissue from this lesion was harvested to assay for leishmania infection. bacteria found in the tissue probably represented secondary infection, a common sequela of canine vl. no l. infantum parasites were observed on slides stained with hematoxylin and eosin, periodic acid schiff, or giemsa, and lesion tissue was negative for l. infantum by qpcr (data not shown). qpcr was performed to quantify parasite load within common leishmania - infected organs from all hamsters. the mean quantities of leishmania dna amplified from spleen, bone marrow, and lymph node from hamsters on which leishmania - infected sandflies had fed were 12-, 22-, and 11-fold greater than that from hamsters on which leishmania - naive sandflies had fed (figure 2). according to extrapolation from a pcr standard curve similar to one previously used (5), the highest parasite load was in bone marrow, which contained an average of 1,238 (282) parasites / mg tissue. leishmania spp.specific quantitative pcr was performed, and parasite load was calculated from a standard curve. statistical significance was determined by 1-way analysis of variance with bonferroni posttest between 6 naive and 15 infected groups, by tissue type. in the united states, parasites from vl - symptomatic, naturally infected hunting hounds remain highly infectious to leishmania - competent lu. parasites that had fed on l. infantum infected hounds were able to develop fully within sandflies and to be subsequently transmitted to and disseminated within hamsters. longipalpis sandflies to acquire l. infantum from naturally infected dogs has been compared in leishmaniasis - endemic brazil (13). although lower infection rates were observed in lu. longipalpis (36%) sandflies, the intensity of infection (200500 promastigotes / fly) was higher in lu. shannoni sandflies are commonly found within the united states and also in areas where l. infantum infected hounds were reported (4). these data demonstrate the risk for vectorborne transmission of zoonotic vl from these dogs in the united states. despite our use of an l. infantum strain that is primarily, if not solely, transmitted via vertical transmission between dogs in the united states, we were able to measure substantial parasite loads in sandflies that fed on these dogs and in the bone marrow, spleen, and peripheral lymph nodes of hamsters on which infected sandflies had fed (figures 1, 2). parasite dna was not amplified in the liver, possibly because of lower parasite loads in the liver during later infection, as demonstrated in experimental vl infections of mice (14). therefore, the us strain of l. infantum that is circulating in north american hunting hounds has not lost virulence factors that facilitate adherence to sandfly gut and facilitate transmission, and subsequent dissemination, in a secondary host. this study focused on the possibility that domestic hounds serve as reservoir hosts for l. infantum within the united states ; however, other potential l. infantum reservoirs include coyotes, foxes, and opossums. when leishmaniasis was found to be reemerging among hounds in the united states in 2000, a total of 291 wild canids were trapped and tested (15). no serologic evidence of infection was found, but these studies were limited to the southeastern united states ; further study is needed to rule out the possibility that enzootic cycles of transmission do not exist within wild canids. the range covered by lu. shannoni sandflies overlaps that of reservoir species including coyotes, foxes, and hunting hounds. occurrence of leishmania vectors in areas of naturally infected hounds indicates a coalescence of components for establishment of a sylvatic and/or domestic cycle of l. infantum. diagnostic testing and preventive measures should be considered for dog breeds known to harbor l. infantum. in the united states, l. infantum is circulating among dogs. despite the fact that vertical transmission maintains vl within the hound population (5), l. infantum was able to fully develop in sandflies and be further transmitted to a susceptible vertebrate host. l. infantum strain mcan / us/2001/foxymo1, similar to the common european zymodeme mon-1, circulating dog - to - dog in north america maintained all necessary requirements for complete development within sandflies. shannoni, and l. infantumi) in hounds may put companion dogs and humans at risk and could pose an emerging risk for l. infantum triggered clinical disease in at - risk populations in north america. technical appendix. photograph and histologic images of lesion on hamster on which leishmania infantum infected sandflies fed.	leishmaniasis is a zoonotic disease caused by predominantly vectorborne leishmania spp. in the united states, canine visceral leishmaniasis is common among hounds, and l. infantum vertical transmission among hounds has been confirmed. we found that l. infantum from hounds remains infective in sandflies, underscoring the risk for human exposure by vectorborne transmission.
inability to reach the papilla due to duodenal obstruction related to neoplastic invasion, by extrinsic compression or as a consequence of benign pathologies, may preclude ercp performance. percutaneous transhepatic cholangiography or eus - guided biliary access are mini - invasive alternative treatments associated with a morbidity up to 31 % and 17 %, respectively 1. provisional insertion of removable covered self - expanding metal stents (csems) has been introduced in the last decade with the goal of dilating benign strictures in the gastrointestinal tract caused by pyloric 2 3 or esophageal peptic stenosis, gastrointestinal anastomosis 4 5, and radiation - induced stenosis. csems have been used as well in the treatment of gastrointestinal leaks 6. moreover, the use of temporary duodenal stents has been described in literature for management of perforated duodenal ulcer or perivaterian perforation after ercp 7 8. provisional deployment of sems to overcome duodenal strictures may be a therapeutic option to allow an ercp procedure after a few days. sems removal, usually after 7 days, does not represent a problem and, in most cases, allows an ercp to be performed, as shown in anecdotal reports 9. the aim of this retrospective study is to report overall technical success rates, clinical outcomes, and rates of adverse events associated with deployment of temporary fully or partially covered duodenal stents (fcsems / pcsems) as a bridge to ercp in cases of inaccessible papilla due to duodenal strictures in a single tertiary endoscopic center. between january 2010 and november 2015, a total of 9000 ercps were performed by our team at our private institution. sixty - six patients (26 male, 40 female) with an average age of 73 years (sd 13.7) in whom ercp could not be performed due to inability to reach the papilla underwent duodenal csems deployment as a bridge to ercp. included were all consecutive patients with a duodenal stricture impeding performance of ercp and no prior history of biliary endoscopic interventions. ten patients in whom the same approach was successful were excluded due to previously performed biliary procedures. this exclusion criterion was adopted to evaluate the efficacy of our technique only in cases of nave papillae. technical and clinical outcome plus complication rates for temporary duodenal stenting and subsequent stent removal and ercp performance were recorded. comparison of the results according to stricture etiology and location and type of duodenal stent used was performed to search for potential predictors of success with our approach. informed consent, including need for multiple endoscopic sessions, was obtained from all patients. duodenal stricture was present in all patients. in 54 patients (82 %) there was no notion of duodenal stricture before index endoscopy. meanwhile 12 patients (18 %) presented with nausea and vomiting on hospital admission. considering mutignani s bilioduodenal stricture classification 10, stricture location was at the level of the duodenal bulb or upper duodenal genu in 49 patients and affecting the second part of the duodenum with papilla involvement in the remaining 17 patients. all procedures were performed under general anesthesia ; prophylactic antibiotics were administered in all cases and continued until the first endoscopic control in the 11 patients with cholangitis. duodenal stricture was defined as inability to reach the papilla of vater due to duodenal neoplastic invasion or extrinsic compression (fig.1) even after balloon dilation up to 20 mm. before considering provisional duodenal stenting,. 11 endoscopic view of pyloric and duodenal stricture due to exstrinsic compression. in 16 patients the examination was started with an array echo - endoscope (gfue140, gfue180, olympus linear, japan) and stopped at the pylorus or bulb because of stenosis. however, in 10 of 16 patients fna - eus was successfully performed from the bulb or stomach for 6 pancreatic masses, 2 lymph nodes and 2 bile duct lesions. in all other patients a standard duodenoscope (tjf160 vr, tjf180 v, olympus, japan) was used. after reaching the stenosis, through - the - scope contrast injection was always performed to evaluate the length and degree of the stenosis. several attempts to overcome the stricture were done with change of the patient s position (supine and left lateral side) and, if no neoplasia was present, performance of hydrostatic dilation up to 20 mm. balloon dilation was not performed in case of tumoral invasion of the duodenal wall due to the high risk of perforation related to such a procedure. in case of failure even after balloon dilation, the subject was enrolled in the study and a tandem catheter (boston scientific, massachusetts, usa) was used to insert a 450-cm guide wire in the distal duodenum. afterward a partially or fully covered self expandable metal stent (pcsems or fcsems) (hanarostent, m. i. tech, seoul, south korea taewoong medical, co., ltd, gimpo - si, south korea) was inserted (fig. 2). all sems were 20 mm in diameter whereas stent length (9, 10, 11 or 13 cm) and the use of pcsems or fcsems were at the discretion of the endoscopist. as a general rule the proximal end of the stent was delivered across the pylorus while the location of the distal end was related to stricture length irrespective of papilla location. we were not concerned about the stent covering the papillary region because the mesh in the stent prevented watertight closure of the papilla. pcsems was generally preferred in cases of short or low - grade stricture as an attempt to reduce the risk of migration. repeat endoscopy was performed between at a minimum of 2 and a maximum of 7 days. the time span for each patient was determined based on the individual s clinical condition and degree of tightness so as to maximize stent expansion across the narrowing and to reduce the risk of migration and ingrowth or overgrowth of inflammatory tissue (fig. b radiological view before stent removal showing full expansion and a slight migration of the stent. during the second endoscopic session a plain abdominal x - ray afterward duodenoscopy across the stent was attempted and if it was not possible, hydrostatic intra - stent dilation up to 20 mm was performed using a cre balloon (boston scientific, massachusetts, usa). thereafter stent removal was easily carried out using a foreign body forceps. only after successful stent removal was permanent duodenal stenting was performed, if clinically necessary, at the end of the ercp, delivering an uncovered duodenal sems. technical success was defined as the ability to deploy the pc / fcsems across the duodenal stenosis. complete expansion of the pc / fcsems was defined as complete resolution of the waist at the level of stenosis. bleeding, perforation, pancreatitis, dysphagia, and stent migration were evaluated and considered as procedural / technical complications related to sems deployment / retrieval. clinical success was defined as the capability to perform a successful therapeutic ercp following dilation of the duodenum with pc / fc sems with decrease of jaundice or resolution of cholangitis. complications related to subsequent ercp and duodenal permanent stenting were evaluated and considered as follow : bleeding, pancreatitis, perforation, cholangitis, cholecystitis, dysphagia, and vomiting due to stent malfunctioning. mean follow up was 45 (7 90) days. because most patients had end - stage disease, longer follow up was not feasible. frequency, percentages, means (sd), and medians were used for descriptive analysis. categorical variables were compared using the chi - squared test (or fischer s exact test when necessary). statistical analysis were performed using ibm spss statistics 20 (ibm company, arnock, new york, usa). all procedures were performed under general anesthesia ; prophylactic antibiotics were administered in all cases and continued until the first endoscopic control in the 11 patients with cholangitis. duodenal stricture was defined as inability to reach the papilla of vater due to duodenal neoplastic invasion or extrinsic compression (fig.1) even after balloon dilation up to 20 mm. before considering provisional duodenal stenting,. 11 endoscopic view of pyloric and duodenal stricture due to exstrinsic compression. in 16 patients the examination was started with an array echo - endoscope (gfue140, gfue180, olympus linear, japan) and stopped at the pylorus or bulb because of stenosis. however, in 10 of 16 patients fna - eus was successfully performed from the bulb or stomach for 6 pancreatic masses, 2 lymph nodes and 2 bile duct lesions. in all other patients a standard duodenoscope (tjf160 vr, tjf180 through - the - scope contrast injection was always performed to evaluate the length and degree of the stenosis. several attempts to overcome the stricture were done with change of the patient s position (supine and left lateral side) and, if no neoplasia was present, performance of hydrostatic dilation up to 20 mm. balloon dilation was not performed in case of tumoral invasion of the duodenal wall due to the high risk of perforation related to such a procedure. in case of failure even after balloon dilation, the subject was enrolled in the study and a tandem catheter (boston scientific, massachusetts, usa) was used to insert a 450-cm guide wire in the distal duodenum. afterward a partially or fully covered self expandable metal stent (pcsems or fcsems) (hanarostent, m. i. tech, seoul, south korea taewoong medical, co., ltd, gimpo - si, south korea) was inserted (fig. 2). all sems were 20 mm in diameter whereas stent length (9, 10, 11 or 13 cm) and the use of pcsems or fcsems were at the discretion of the endoscopist. as a general rule the proximal end of the stent was delivered across the pylorus while the location of the distal end was related to stricture length irrespective of papilla location. we were not concerned about the stent covering the papillary region because the mesh in the stent prevented watertight closure of the papilla. pcsems was generally preferred in cases of short or low - grade stricture as an attempt to reduce the risk of migration. repeat endoscopy was performed between at a minimum of 2 and a maximum of 7 days. the time span for each patient was determined based on the individual s clinical condition and degree of tightness so as to maximize stent expansion across the narrowing and to reduce the risk of migration and ingrowth or overgrowth of inflammatory tissue (fig. b radiological view before stent removal showing full expansion and a slight migration of the stent. during the second endoscopic session a plain abdominal x - ray was always performed to evaluate the degree of expansion of the stent. afterward duodenoscopy across the stent was attempted and if it was not possible, hydrostatic intra - stent dilation up to 20 mm was performed using a cre balloon (boston scientific, massachusetts, usa). thereafter stent removal was easily carried out using a foreign body forceps. only after successful stent removal was permanent duodenal stenting was performed, if clinically necessary, at the end of the ercp, delivering an uncovered duodenal sems. technical success was defined as the ability to deploy the pc / fcsems across the duodenal stenosis. complete expansion of the pc / fcsems was defined as complete resolution of the waist at the level of stenosis. bleeding, perforation, pancreatitis, dysphagia, and stent migration were evaluated and considered as procedural / technical complications related to sems deployment / retrieval. clinical success was defined as the capability to perform a successful therapeutic ercp following dilation of the duodenum with pc / fc sems with decrease of jaundice or resolution of cholangitis. complications related to subsequent ercp and duodenal permanent stenting were evaluated and considered as follow : bleeding, pancreatitis, perforation, cholangitis, cholecystitis, dysphagia, and vomiting due to stent malfunctioning. mean follow up was 45 (7 90) days. because most patients had end - stage disease, longer follow up was not feasible. frequency, percentages, means (sd), and medians were used for descriptive analysis. categorical variables were compared using the chi - squared test (or fischer s exact test when necessary). statistical analysis were performed using ibm spss statistics 20 (ibm company, arnock, new york, usa). indications for ercp were as follow : jaundice in 51 patients, cholangitis in 11 cases, chronic pancreatitis - related pain in 3 patients, and biliary leak following cholecystectomy in 1 subject. underlying pathologies responsible for biliary symptoms were : 40 pancreatic cancer, 6 bile duct cancer, 1 duodenal cancer, 5 chronic pancreatitis, 7 common bile duct (cbd) stones, and 7 diffuse metastatic disease from different cancers (2 breast, 1 gastric, 1 ovarian, 1 kidney and 1 colon). sixty - six stents were delivered for the following pathologies : neoplastic duodenal invasion in 15 patients, peptic stenosis in 5, and extrinsic compression in 46. the extrinsic compression group comprised 7 patients with no evidence of malignant disease (5 chronic pancreatitis and 2 undetermined non - malignant compression) and 39 patients with eus - evident tumoral compression on the duodenal wall. seventeen of 66 (25.8 %) patients had a pcsems and 49 (74.2 %) patients had a fcsems. the length of pcsems was 9 cm in 13 cases and 11 cm in 4, with the length of deployed fcsems as follow : 10 cm in 32 cases, 11 cm in 9, and 13 cm in the remaining 8. there was no bleeding, pancreatitis, dysphagia or perforation following stent deployment. at an average of 3.15 days (1 7) follow up, endoscopy showed 53 stents fully expanded, 2 migrations, and 11 stents that were incompletely expanded. in the latter 11 patients, a hydrostatic dilation up to 20 mm was carried out. stent length was 13 and 10 cm, respectively, while original pathology was pancreatic cancer in the first patient and chronic pancreatitis in the second, which in both cases caused extrinsic compression. migration was diagnosed 3 days after sems deployment even if no stricture resolution was noted. one patient expelled the stent spontaneously per ano while the other patient underwent colonoscopy for retrieval. statistical analysis showed a numeric but not statistically significant difference between pc and fcsems groups (p = 0.0634) concerning the migration rate. no statistically significant differences in terms of the migration rate were highlighted in the study when considering stricture characteristics (neoplastic invasion vs peptic stricture vs extrinsic compression) with a p value = 0.638. nor were days of stenting significant (3 days vs > 3 days) with a p value = 0.509. all stents were removed without complication after 1 day in 9 patients, 2 days in 10, 3 days in 18 subjects, 4 days in 22, 5 days in 2 patients, and after 7 days in the remaining 3 subjects. subsequent duodenoscopy and successful therapeutic ercp could be performed in 56 out 66 patients (85 %) with delivery of biliary plastic or metal stent, stone removal in 1 patient, and pancreatic prosthesis in 2 patients. reasons for failure included : inability to recognize the papilla due to neoplastic invasion in 3 patients, persistence of tight duodenal narrowing in 5 cases, and early stent migration and stricture persistence in the remaining 2 cases. stricture location failures occurred in 2 patients with a duodenal bulb stricture and in 8 patients with a stricture involving the papillary region. m, male ; f, female ; ptbd, percutaneous transhepatic biliary drainage eight out of 10 patients underwent same - day percutaneous biliary drainage by means of transhepatic cholangiography (ptbd) and 2 underwent transduodenal eus biliary drainage (eus - bd) with a choledocoduodenal anastomosis during the same session (fig. one of the 2 eus biliary drainage patients presented 24 hours later with a biliary leak and consequent peritonitis and underwent emergency surgery with a double gastric and biliary bypass. d eus cholangiography successfully performed after stent removal showing a neoplastic stricture of the intrapancreatic choledochus. following biliary drainage, permanent duodenal stenting with insertion of an uncovered duodenal stent (wallflex, boston scientific, massachusetts, usa) was performed in 40 out 66 patients due to tight duodenal stricture and clinically evident goo. after 5 days 1 patient with chronic pancreatitis underwent biliary and gastric by - pass. two patients developed moderate post - ercp pancreatitis, according to the cotton. there was no procedure related - mortality. at a mean follow - up of 45 days (7 90), liver enzymes and bilirubin levels improved in all 66 patients ; moreover no patient required revision of the duodenal stent or biliary re - stenting. differences in failure rate among the different groups were compared to search for statistically significant risk factors that influenced clinical outcome. however, no statistically significant differences were found comparing pcsems vs fcsems (p value = 0.717) and considering the different types of stenosis : neoplastic invasion vs peptic stricture vs extrinsic compression (p = 0.737) (table 3 and table 4). fc - sems, fully covered self - expanding metal stent ; pc - sems, partially covered self - expanding metal stent ; sems, self - expanding metal stent fc - sems, fully covered self - expanding metal stent ; pc - sems, partially covered self - expanding metal stent ; sems, self - expanding metal stent biliary endoscopic sphincterotomy (bes) has been accepted as the gold standard treatment for choledocholithiasis and malignant biliary - pancreatic pathologies 13. however the condition sine qua non is to pass through the duodenum and recognize the papilla. duodenal obstruction is a condition that can be present both in malignant pathologies such as biliopancreatic or duodenal cancer or diffuse metastatic pathologies and in benign ones such as peptic ulcer, benign pylorus stenosis or chronic pancreatitis. in case of nonresectable pathology and concomitant gastric outlet obstruction, a recent systematic review 14 showed the superiority of permanent duodenal stenting compared to palliative surgical approaches. covered duodenal stents are already used for the management of fistula and leak, gastrointestinal anastomotic strictures, radiation - induced stenosis, and benign pyloric stenosis. however, covered stents are burdened by a high migration rate due to the presence of the cover that minimizes hyperplastic tissue ingrowth through the mesh 15. in our study we propose expanding the indication for temporary duodenal stenting as a bridge to ercp for refractory duodenal strictures. however, our ercp success rate after temporary duodenal stenting is still lower than the 90 % success rate suggested as a reasonable minimal standard 16. first, even if our technique allowed us to dilate the stricture and reach the papilla, the duodenum, due to underlying pathology, was sub - stenotic and rigid in most cases. approach to the papillary region was cumbersome and ability to manipulate the duodenoscope and accessories was often limited. second, in two - thirds of the failed ercps (3 out of 10), it was possible to overcome the stenosis but impossible to visualize the papilla. in such circumstances, an eus and endoscopic rendezvous approach with anterograde introduction of a guidewire across the papilla could be attempted 17. in the 10 cases in which second - attempt ercp was unsuccessful, 8 patients underwent ptbd instead of eus - bd. ptbd was preferred in 3 subjects due to neoplastic invasion of the duodenum preventing creation of a choledochoduodenostomy and in 1 patient with chronic pancreatitis because in our institution, we currently prefer to perform ptbd for benign pathologies. meanwhile, in the remaining 4 patients, the decision to undergo to ptbd vs eus - bd was defined by the referring surgeon. according to our results, long and severe stricture, papillary region involvement, and tumoral invasion of the duodenal wall moreover all aforementioned features are typical of neoplastic etiology and are often present simultaneously. indeed, in our series, 9 of 10 patients in which the procedure failed had disease with a malignant etiology. despite a tertiary referral center for interventional biliopancreatic endoscopy, we adopt a step - up approach before more invasive treatments such as eus - bd ptbd as both are associated with high rates of morbidity and mortality 18 19. percutaneous transhepatic biliary drainage is the gold standard second - line treatment after failure of ercp or in case of altered anatomy that precludes ercp performance. since the first ptbd, major advances have led to biliary drainage becoming a well - established procedure worldwide 20. nowadays ptbd and subsequent stenting achieve an extremely high clinical success rate hindered, however, by a high morbidity rate 21. in our experience, in the near future, eus - bd is likely to become a fundamental tool for the biliopancreatic endoscopist ; however, before that happens, eus - bd techniques must be standardized and new dedicated devices developed. regarding standardization of the technique in the literature, there are few studies reporting eus - bd in patients with concomitant obstructive jaundice and duodenal obstruction 22 23 and even fewer comparing the 2 different eus - bd approaches available : hepaticogastrostomy (hgs) versus choledocoduodenostomy (cds). moreover, reported outcomes are discordant. in their retrospective study of 39 patients, ogura. 24, reported a longer stent patency and a lower ae rate for hgs. meanwhile dhir. in turn reported a statistically significant higher complication rate for the transhepatic route compared to that in a multicenter retrospective study on the transduodenal one 25. in a large comparative study, poincloux 26, reported no differences between cds and hgs in terms of technical success, clinical outcome, and complication rate. as reported by many experts, further prospective randomized controlled clinical trials are needed to validate the efficacy and safety of eus - bd. several studies in the literature 10 27 describe different approaches to manage malignant bilioduodenal stricture. however, in our opinion, such an approach is appropriate only in a tertiary referral hospital. performance of an ercp through the mesh of a duodenal stent is often cumbersome, demanding a high level of expertise and very often requiring technically demanding procedures such as balloon dilation of the mesh or stent trimming by means of foreign body forceps or apc. our technique, in constrast, requires only temporary duodenal stenting which is feasible even in less experienced centers. as aforementioned, kikuyama s 11 large balloon traction technique is a very useful tool for overcoming several but not all duodenal strictures. such an interesting technique can be attempted, whenever feasible, before considering provisional stenting. in the current study we excluded 10 patients in whom a biliary stent (plastic or metallic) nonetheless we adapted our technique even for those 10 patients, successfully removing and exchanging both plastic and metallic stents and avoiding a cumbersome eus or radiological procedure for stent replacement. we reported a lower stent migration rate compared to that reported in the current literature 28. our low migration rate may be explained by the short indwelling time of the stents (average 3.15 days), the degree and nature of the stricture treated, and some technical tricks. stent were left in place for such a short time because our aim was just to dilate the stricture and allow the passage of the duodenoscope. moreover, it typically takes up to 48 hours for a stent to achieve complete expansion so it is necessary to maintain it for that period to achieve maximal radial force. we always deploy at least half of the stent above the stenosis in order to allow a certain degree of physiologic migration and, as a general rule, we always try to release the proximal end of the stent across the pylorus. moreover it is important to avoid stent impaction at the horizontal portion of the duodenum in order to avoid occlusion. in our experience, the factor key to minimizing the rate of migration is keeping the stent as short as possible, just enough to achieve its full expansion. that may be due to the fact that pcsems were preferred in cases of slack or short strictures. similar to other authors 29 30, we did not experience complications related to mechanical occlusion of the papilla due to duodenal sems. that may be explained by the fact that covering the papillary region does not induce watertight closure. temporary covered duodenal stenting as a bridge to ercp for duodenal obstruction is safe and in 85 % of cases, allowed us to successfully perform therapeutic ercp. temporary duodenal stenting may be a sound option as a step - up approach before referring such patients for more complex techniques such as eus - bd or ptbd. such a technique could be very useful as a first - line approach, even in smaller endoscopic centers that do not have the expertise or the availability of an endosonographer or interventional radiologist. long and complete obstruction due to tumor infiltration of the second part of the duodenum, however, remains a factor predictive of failure requiring alternative biliary drainage techniques.	background and study aims : duodenal obstruction may prevent performance of endoscopic retrograde cholangiopancreatography (ercp). percutaneous transhepatic biliary drainage (ptbd) or endoscopic ultrasonograhy - guided biliary access (eus - bd) are alternative treatments but are associated with a higher morbidity and mortality rate. the aim of the study is to report overall technical success rate and clinical outcome with deployment of temporary fully or partially covered self - expanding duodenal stent (pc / fcsems) as a bridge to ercp in case of inaccessible papilla due to duodenal strictures. patients and methods : this retrospective study included 66 consecutive patients presenting with a duodenal stricture impeding the ability to perform an ercp. provisional duodenal stenting was performed as a bridge to ercp. a second endoscopic session was performed to remove the provisional stent and to perform an ercp. afterward, a permanent duodenal stent was delivered if necessary. results : sixty - six duodenal stents (17 pcsems and 49 fcsems) were delivered with a median indwelling time of 3.15 (1 7) days. two migrations occurred in the pcsems group, 1 of which required lower endoscopy for retrieval. no other procedure - related complications were observed. at second endoscopy a successful ercp was performed in 56 patients (85 %) ; 10 patients (15 %) with endoscopic failure underwent ptbd or eus - bd. forty patients needed permanent duodenal stenting. conclusions : provisional removable covered duodenal stenting as a bridge to ercp for duodenal obstruction is safe procedure and in most cases allows successful performance of therapeutic ercp. this technique could be a sound option as a step up approach before referring such cases for more complex techniques such as eus - bd or ptbd.
plenty of epidemiologic evidence demonstrated that estrogen might influence the incidence of colon cancer in women [13 ]. colon cancer risk increased after menopause and decreased after hormone replacement treatment (hrt). estrogen receptors were found in colon epithelium and the estrogen receptor beta was the dominant subtype. on cell models, many studies had found that estrogen could affect the growth of cells originated from colon mucosa [6, 7 ]. on an animal model of rats induced by dmh since the angiogenesis was vital for tumorigenesis and the estrogen was a well - known vasoactive hormone, it was worth investigating whether estrogen could influence angiogenesis in the course of colon carcinogenesis. there were several types of vasculation during carcinogenesis, including angiogenesis, vasculogenesi, and vasculogenic mimicry. in early stage of cancer, the main type of vessel formation was angiogenesis, triggered by proangiogenic factors. among the pro - angiogenic factors, vegf was the essential factor in angiogenesis [11, 12 ]. in the present study, we studied the effects of estrogen on the microvessel density (mvd) and the expression of vegf and its main upstream regulator hif-1. female sprague - dawley rats were purchased from the animal center of tongji medical college. protocols for animal experimentation and maintenance were approved by the animal ethics committee at our university and carried out in accordance with the institutional guidelines. thirty - six female rats (10 weeks of age) were housed in plastic cages (4 rats per cage) under standard laboratory conditions (21 1c temperature, 50 10% humidity, and 12 h of light time from 6 am to 6 pm) with normal food and tap water provided ad libitum. all rats were ovariectomized (ovx) at the age of 11 weeks. at the age of 12 weeks, these rats were randomly divided into 3 groups according to the following treatment : control group (n = 12), dmh group (n = 12), and dmh + e2 group (n = 12). in the control group, rats were subcutaneously and intraperitoneally administrated with vehicles once a week. in the dmh group, rats received intraperitoneal injections of dmh (20 mg / kg body weight) [13, 14 ] once a week. in the dmh + e2 group, rats received subcutaneous injections of 17-estradiol (40 g / kg body weight and dissolved in camellia oil) once a week, together with the weekly intraperitoneal injections of dmh (20 mg / kg body weight). six weeks after the last injection, all the animals were sacrificed by an overdose injection of chloral hydrate (600 mg / kg intraperitoneal injection). after sacrifice, the entire colorectums were collected and opened longitudinally and washed with pbs. the length (l), width (w), and height (h) of each polyp were measured, and the volume of each polyp was calculated using the formula v = l w h /6. the polyps with the volume ranging from 50 mm to 70 mm were cut into halves. one portion of the polyp was stored at 80c for rt - pcr and western blot, and the other half was fixed in 4% paraformaldehyde and embedded in paraffin block. the polyps with a volume of 70 mm were all fixed in 4% paraformaldehyde and embedded in paraffin block. polyps were fixed in 4% paraformaldehyde and embedded in paraffin. then the polyp samples were cut into 4 m sections using a microtome. sections were stained with hematoxylin and eosin (h & e) and examined histologically in a blinded manner. sections (4 m) were cut from paraffin - embedded polyp samples and mounted on poly - l - lysine - coated slides. immunohistochemical staining was performed using anti - cd34 antibody (boster, china) and anti - pcna antibody (cell signaling technology, usa) with the avidin - biotin - peroxidase complex (abc) method. the proliferation rate was assessed by the pcna index, defined as the percentage of pcna - positive cells. pcna index was determined by counting pcna - positive cells in a total of at least 1000 cells in different randomly selected areas at 400 magnification. the cd34-stained sections were initially scanned at low power (40 and 100) and the areas of specimens with the highest neovascularization stained by cd34 were selected as hot spots. subsequently, microvessel counting was carried out in four fields of the hot spots at 400 magnification. any brown - stained endothelial cells or cell cluster clearly separated from adjacent microvessels, tumour cells, and other connective tissue elements were considered as a single countable vessel. tissue samples from the polyps ranging in volume from 50 mm to 70 mm and the normal colonic mucosa samples in the control group were used. total tissue rna was extracted with trizol reagent (invitrogen) following the manufacture 's instruction. the cdnas from total rna were synthesized using primescript rt reagent kit (takara, japan). the mrna expression was evaluated by real - time pcr with an abi stepone plus (applied biosystems, singapore). the concentrations of the reagents were adjusted to reach a final volume of 20 l, containing 2 l cdna product, 10 l sybr premix ex taq ii (takara, japan), and 0.8 l of forward and reverse primers. the reaction was carried out by 45 amplification cycles of 95c for 5 s and 60c for 30 s. pcr primers were designed by primer 5.0 and blast search to check specificity. tissue samples from the polyps ranging in volume from 50 mm to 70 mm and the normal colonic mucosa samples in the control group were used. protein was extracted with protein extraction kit (beyotime, china), separated on 10% sd - spage, and transferred to polyvinylidene difluoride (pvdf) membranes (millipore, usa). membranes were blocked in 5% nonfat milk diluted in tbst for 1 hour at room temperature and then were incubated overnight at 4c with the following primary antibodies : anti - hif-1 (abcam, cambridge, uk) and anti - vegf - a (abcam, cambridge, uk). after rinsing with tbst for three times, the membrane was incubated with horseradish peroxidase - conjugated secondary antibodies for 1 hour at room temperature. the outcome was visualized by the ecl plus western blotting detection system according to the manufacturer 's instructions. data were evaluated by anova in which multiple comparisons were performed using the least - significant difference method, while those data in heterogeneity of variance were analyzed by kruskal - wallis test. the volumes of polyps in the two experimental groups were evaluated by student 's t test. colon polyp incidence was expressed as percentages, and results were statistically analyzed using the chi - square test. eleven out of 12 (91.7%) rats in dmh group and 8 out of 12 (66.7%) rats in dmh + e2 group developed colon polyps (figure 1), while none of the rats in control group developed colon polyps. the incidence of colon polyps in dmh group was higher than that in dmh + e2 group (91.7% versus 66.7%), though this difference was not statistically significant (p > 0.05) (table 2). the polyp multiplicity (mean number of polyps per rat) in dmh group was significantly higher than that in dmh + e2 group (6.8 3.1 versus 3.0 1.1, p < 0.05) (table 2). at the same time, the average volume of polyps in dmh group was significantly bigger than that in dmh + e2 group (102.97 77.67 versus 45.85 43.40, p < 0.05) (table 2). the pcna index of the polyps from dmh group ranged from 19.6% to 31.2%, with an average of 27.1% 5.2%. when dmh was administrated together with estradiol in dmh + e2 group, the pcna index decreased significantly to an average of 18.5% 2.9% (27.1% 5.2% versus 18.5% 2.9%, p < 0.05). the pcna index in control group was significantly lower than that in the other two groups (figure 2). the mvd in control group was significantly lower than that in the other two groups (figure 3). the mvd elevated to an average of 32.13 3.98 per field in dmh group. when dmh was administrated together with estradiol, the mvd decreased to 19.0 4.24 per field. the mvd in dmh + e2 group was significantly lower than that in dmh group (p < 0.05) (figure 3). the mrna expression of hif-1 in the dmh group or dmh + e2 group was significantly upregulated compared to that of the control group (figure 4(a)). and the mrna transcripts in dmh group were almost 2-fold higher than that in dmh + e2 group (figure 4(a) ; p < 0.05). we observed the similar tendency of hif-1 expression at protein level (figure 4(c)). vegf expression in the dmh group or dmh + e2 group was higher than that in control group at both mrna (figure 4(b)) and protein (figure 4(c)) levels. when we compared dmh + e2 group with dmh group, we found that estrogen treatment caused a significant decrease (1.43-folds) in vegf mrna expression (figure 4(b) ; p < 0.05). epidemical studies showed that postmenopausal women were at increased risk of colorectal cancer (crc) compared with premenopausal women, and data from prospective randomized trials showed that hrt reduced the risk of crc in postmenopausal women by 30 to 40%. many studies indicated that estrogen could inhibit proliferation and induce apoptosis in colon cancer cells [19, 20 ]. the study of estrogen on tumorigenesis of colon cancer in animal models was scarce. in the present study, we investigated the effect of estrogen on colon polyp formation in an ovariectomized rat model. as a result, we found that the multiplicity and volumes of polyps in the estrogen - dmh simultaneous treated group were lower than those in the dmh treated group. this result confirmed that estrogen could inhibit colon carcinogenesis in dmh - induced rat colon cancer model. and this result was accordant with the epidemiological results and the results found in colon cell models. in our study, we found that the pcna index of polyps in dmh + e2 group was significantly lower than that in dmh group. this result suggested estrogen could inhibit tumor formation by inhibiting the proliferation ability of colon stimulated by the carcinogen. this result was also in agreement with the findings in the cell model which indicated estrogen could inhibit the proliferation of colon cancer cell. many studies had demonstrated that estrogen could negatively regulate cellular proliferation by estrogen receptor beta in several types of cancers, such as ovarian cancer and prostate cancer [21, 22 ]. as estrogen receptor beta was the dominant estrogen receptor subtype in the colon tissue of rats, it was possible that estrogen might exert its antiproliferative effect by estrogen receptor beta in rats. for the growth of tumor, increased proliferation must be accompanied by increased blood supply and this is achieved by angiogenesis and increased blood microvessel density. tumor angiogenesis is regulated by various activating and suppressive factors. among the activating factors, vegf plays a central role in the induction of angiogenesis. as an important transcription factor, estrogen stimulates angiogenesis in the uterine endometrium. while in breast cancer, estrogen was found to induce the expression of vegf however, the effects of estrogen on angiogenesis or proangiogenic factors were distinct in different conditions. and the difference might be related to the different predominant subtype of estrogen receptors expressed in the tissues. for example, estrogen could induce the expression of vegf and activation of hif-1 in uterus mainly expressing estrogen receptor, but estrogen inhibited angiogenesis and reduced the expression of vegf in breast cancer which mainly expressed estrogen receptor beta. in another study in prostate cancer, er beta was reported to repress the transcription of vegf and destabilize hif-1. these studies suggested that estrogen could inhibit angiogenesis and depress proangiogenic factors by er beta. in our study, we found estrogen significantly depressed the microvessel densities in dmh - induced colon polyps. accordingly, our study indicated that estrogen reduced the expression of vegf and hif-1 in dmh - induced colon polyps. as er beta was also the predominant er subtype in the colon of rats, we hypothesized that estrogen might inhibit angiogenesis of colon polyps by downregulating hif-1 and vegf via er beta. in summary, the present study demonstrated that estrogen could inhibit colon polyp formation in a rat model of colon cancer induced by dmh. meanwhile, estrogen depressed the microvessel densities and reduced the expression of vegf and hif-1. we supposed that estrogen might inhibit colon carcinogenesis by downregulating hif-1 and vegf, eventually reducing angiogenesis.	objective. to study the effects of estrogen on colon polyp formation, proliferation, and angiogenesis on a rat model of colon cancer induced by dimethylhydrazine (dmh). methods. thirty - six female ovariectomized (ovx) rats were randomly divided into 3 groups : (i) control group (administrated with vehicles weekly), (ii) dmh group (administrated with dmh weekly), and (iii) dmh + e2 group (administrated with dmh and 17-estradiol weekly). the incidence, volumes, and multiplicity of colon polyps in each group were evaluated. the microvessel density (mvd), the expressions of proliferating cell nuclear antigen (pcna), and the expressions of hif-1 and vegf in polyps were detected in each group. results. estrogen reduced the multiplicity, volumes, and the pcna expressions of dmh - induced colon polyps. the mvd in dmh + e2 group was significantly lower than that in dmh group. estrogen treatment decreased the hif-1 and vegf expressions at both mrna and protein level. conclusion. estrogen replacement was protective for ovariectomized rats from dmh - induced carcinogenesis, and one of the mechanisms for this was due to estrogen 's inhibitive effects on blood vessel formation by downregulating vegf and hif-1 expressions.
	arthropod - borne viruses (arboviruses) are a growing threat to global health. complex vector virus host interactions lead to unpredictable epidemiological patterns. difficulties in accurate surveillance including imperfect diagnostic tools impair effective response to outbreaks. with arboviral infections causing a wide spectrum of disease severity, from asymptomatic infection to fatal neuroinvasive and haemorrhagic fevers, the potential impact on blood safety is significant. asymptomatic or presymptomatic individuals may introduce virus into the blood supply by donation, while recipients can potentially suffer severe consequences. dengue, west nile and chikungunya outbreaks have led to responses by blood transfusion services which can inform future planning. reports of transfusion - associated transmission demonstrate the potentially fatal consequences of lack of haemovigilance. south - east asia remains vulnerable to arboviruses with permissive climate and high levels of endemic transmission as well as the potential for emerging and re - emerging arboviral diseases. resource limitations constrain the use of expensive technologies for donor screening. continued surveillance and research will be required to manage the arboviral threat to the blood supply.
telomerase is a ribonucleoprotein complex that prevents the erosion of chromosomal extremities (telomeres) in eukaryotes. the telomerase enzyme is formed by an rna component and a reverse transcriptase, the catalytic subunit which is considered to be the primary determinant for its activity. the human - telomerase reverse transcriptase (h - tert) maintenance of telomere length is essential for tumorigenesis, as most human tumors stabilize their chromosome ends using telomerase. many types of tumors, including brain cancers, have been studied by utilizing these procedures. meningiomas, which may arise anywhere along the meninges, are the most common types of primary brain tumors in domestic animals ; they are usually discrete, slow growing masses with smooth surfaces and broad dural attachments. recently, a subset of 27 canine meningiomas was tested for telomerase immunoreactivity, and tert expression was found to be significantly correlated with the mib1 antibody labelling index. the aim of this study was to expand the present knowledge regarding telomerase expression in both canine and feline meningiomas. twenty - five cases of meningiomas (14 canine and 11 feline) were selected and classified according to the world health organization (who) scheme. replicate sections were tested for the presence of the enzymatic catalytic subunit of telomerase, h - tert. endogenous peroxidase activity was blocked by immersion in 0.3% hydrogen peroxide in methanol for 30 min ; slides were immersed in citrate buffer (ph 6.0), heated for two 5 min periods in a microwave oven at 750w. the primary antibody, an anti - human tert (clone 44f12, diluted 1 : 50 in pbs ; novocastra, uk), was applied to the slides overnight at 4, followed by a streptavidin - biotinperoxidase complex (lsab kit ; dako, netherlands) and diaminobenzidine (0.04% for 7 min) as chromogen. sections were then counterstained with papanicolaou hematoxylin, rinsed in tap water, dehydrated, and mounted. the h - tert index was assessed by an image analyzer (lucia 32g / mutech ; nikon, japan) in ten fields (20 lens) and expressed as the percentage of positive cells. furthermore, h - tert labelled cells were subjectively estimated using semiquantitative grading, in which 0 indicated the absence of positive cells, 1 was 1 - 10% of positive cells, 2 was 11 - 30% of positive cells, 3 was 31 - 60% of positive cells, and 4 was more than 61% of positive cells. mitoses were counted in five fields (40 lens) on replicate sections stained with a toluidine blue technique, which was modified to enhance the detectibility of mitotic figures. mitotic activity was then graded as low (l) : 0 - 5 mitoses, or high (h) : > 6 mitoses. parametric one - way analysis of variance (anova) was performed to compare h - tert values with histologic type, gender and species. simple logistic regression analysis was used to evaluate the association between the two tested methods (subjective and automated). a value of p 6 mitoses. parametric one - way analysis of variance (anova) was performed to compare h - tert values with histologic type, gender and species. simple logistic regression analysis was used to evaluate the association between the two tested methods (subjective and automated). a value of p < 0.05 was considered to be significant. in canine meningiomas, the histotypes most often observed were meningothelial (7/14) and anaplastic (4/14), followed by fibroblastic (1/14), transitional (1/14), and psammomatous (1/14), with a strong predominance of females (9/14). among cats, the transitional variety was mostly represented (6/11), followed by psammomatous (2/11) and anaplastic (2/11) ; a predominance of males was noted (7/11) (table 1). concerning ihc, h - tert protein was localized in the nucleus, notably the nucleolus, and was also occasionally found in the cytoplasm of neoplastic meningothelial cells (2 cases) (fig. 1a - c) (table 1). in three specimens out of 25, no signal was detected ; in the remaining samples, the staining intensity ranged from mild to strong, being very variable from case to case. the distribution of immunoreactive cells was speckled throughout the slide ; even the percentage was very variable, with the proportion of positive cells ranging from 0 to 90%. according to the adopted semiquantitative grading system of h - tert immunopositivity, five cases were classified in group 0, seven in group 1, two in group 2, seven in group 3, and four in group 4 (table 1). the comparison of the two tested counting systems indicated a direct positive correlation between the two methods (r = 0.881 ; p = 0.0000). h - tert expression was higher in meningioma samples from cats than in those from dogs (automated count in dogs : mean 23.3%, range : 0.71 - 73.91% ; automated count in cats : mean 42.3%, range 15.93 - 68.87% ; p = 0.06). no other association was evident between h - tert immunolabelling and sex or histotype for dogs or cats. 2) was useful to appreciate a very low mitotic activity in the vast majority of meningiomas (l group : 21 out of 25) ; only four cases evidenced a high number of mitotic figures (h group : 4 out of 25), including three dogs (2 anaplastic, 1 meningothelial) and one cat (anaplastic meningioma) (table 1). no correlation between the number of h - tert immunoreactive cells (telomerase expression) and their proliferative activity, assessed by mitotic count on toluidine blue - stained sections, was evident. in our series, canine meningiomas were more frequently found to be of the pure meningothelial type than of other types, although the who classification schemes report the transitional variety to be the most commonly observed in dogs ; nevertheless, the transitional variant shows features of both meningothelial and fibroblastic meningiomas. furthermore, meningiomas occurred more frequently in female dogs and male cats ; this finding mirrors the results of other reports, and has been putatively related to a dissimilar expression of estrogen, progesterone, and androgen receptors in canine and feline meningiomas. telomerase expression, which is assessed by h - tert ihc, was displayed in expected locations, mainly nucleolar and nuclear locations, as previously reported, and also was rarely found in cytoplasmic locations. strong nucleolar staining may be explained as the presence of the telomerase holoenzyme, which is assembled within the nucleolus. the nuclear labelling may represent the active telomerase complex at the ends of the chromosome. conversely, the cytoplasmic staining may be interpreted as the shuttling of telomerase holoenzyme from the nucleus, out to the cytoplasm, and then back into the nucleus during the assembly process. this machinery is well - known because the relevance of telomerase in the process of tumoral transformation has been investigated thoroughly. in fact, its activation is considered to be a fundamental step in the " immortalization " of neoplastic cells, and has been suggested to play a key role in the progression of several tumors including human intracranial meningiomas. the data obtained in the present study also seem to support this trend in canine and feline meningiomas. the correlation observed between the subjective semiquantitative grading and the automated count was highly significant, considering the two systems to be interchangeable in the evaluation of h - tert ihc. with both measurement systems, there was no appreciable relationship between h - tert expression and the meningioma histotype, or with other variables such as gender and age. the h - tert expression tended to be higher in samples from feline meningiomas than in canine meningiomas, and this may suggest a different role of the telomerase pathway between the two species, and may indicate a different prognostic significance. in most human and animal tumors, the proliferative activity of neoplastic cells is directly related to the biological behavior. for this reason, the proliferative activity is considered to be a relevant prognostic factor that can be assessed by the counting of mitoses, or by the immunohistochemical evaluation of proteins expressed throughout the cell cycle (i.e. ki67 antigen recognized by mib1 antibody). in this respect, arachnoid " cap " cells, from which meningiomas arise, have a slow rate of cell division, and therefore a high mitotic activity can not be expected in their tumoral transformations. this behavior can be explained with prolonged storage of samples (in formalin or in paraffin blocks), which is responsible for subcellular modifications (i.e. cross - linking between proteins and/or nucleic acids that provides a strong steric hindrance creating an intricate physical barrier to antibodies employed in ihc). therefore, the proliferative activity of tumor cells has been evaluated by counting mitoses in sections stained with a modified toluidine blue technique, which enhances the detectibility of mitotic figures. most cases prompted a very low proliferative activity (l group : 21 out of 25) ; only four cases displayed relatively high mitoses counts, which suggested a more aggressive phenotype, and three of these were grouped in the anaplastic histotype, that, among other meningioma variants, behaves in an aggressive fashion and carries a much poorer prognosis than do benign meningiomas (malignant meningioma). several studies have attempted to assess the proliferative potential and the existence of an association between meningioma recurrence and mib1 labelling index in patient groups with low and high risk of recurrence, attributing a prognostic significance to the mitotic index and to the ki-67 labelling index ; however, the actual prognostic relevance of these parameters remains controversial, heterogeneous, and of uncertain value. in this respect, it has been postulated that h - tert expression might be a relevant marker of progression towards recurrence and malignancy of meningiomas. in the present study, no significant association between h - tert expression and the histological type of meningioma or the mitoses count was evident, although a recent paper reports an association between h - tert and mib1 staining in canine brain tumors (meningioma and oligodendroglioma). a possible explanation for this is that telomerase activation, a fundamental parameter of potential malignant transformation, may occur independently of the signal to proliferate, thereby supplying the cells with unlimited growth capabilities. these preliminary findings suggest that telomerase expression could play a role as a prognostic indicator that acts independently of kinetic parameters, although this should be evaluated using a larger dataset with available clinical information.	telomere length maintenance is regarded as a fundamental step in tumorigenesis, as most human brain tumors, including meningiomas, stabilize the ends of their chromosomes using telomerase. this investigation represents an introduction to telomerase expression in canine and feline meningiomas. twenty - five archived cases (14 dogs and 11 cats) were immunohistochemically tested for human - telomerase reverse transcriptase (h - tert), scored, and quantified ; furthermore, mitoses were counted on sections stained with a modified toluidine blue. the h - tert antibody immunolabelled the nucleus and nucleolus of meningeal neoplastic cells, with an intensity ranging from mild to strong and a speckled distribution ; a significantly higher expression in cats was noted, while no significant association between h - tert immunolabelling and sex or histotype was evident in dogs or cats. the telomerase enzyme represents a fundamental parameter of potential malignant transformation, which may occur independently of the signal to proliferate, thereby supplying the cells with unlimited growth capabilities. telomerase expression could be a prognostic indicator independent of the kinetic parameters, although this should be evaluated using a larger dataset with available clinical information.
as the field of biomedical informatics continues to evolve and grow into subspecialties, it is important to track the literature generated in the field and to understand which topics attract the most attention by readers and by authors who use and cite particular articles in their own work. universities are increasingly making use of bibliometrics to evaluate academic progress, and some already require that candidates include the total number of citations to their articles, the impact factor for each publication, the author 's h - index (or equivalent), in addition to an account of the author 's contribution to each article.1 2 some institutions provide guidance as to which journals should be considered first tiered, second tiered, etc. following the pattern of academic institutions that attempt to measure productivity with bibliometrics, some funding agencies have also established guidelines for progress reports in high visibility programs. the measures include quantity and quality of publications, with a proxy for the latter represented in the form of journal impact factors. to respond to this increasing demand for quantification of academic production, web services to track citations and h - indices, such as the institute for scientific information 's (isi) web of knowledge,3 scopus,4 and google scholar5 have been created. some journals also provide the number of article views and/or downloads per month (dpm). even though, as pointed out by several authors, these measures are imperfect,69 they may provide useful clues to define trends in biomedical informatics and to start taking into account how citation in subspecialized fields varies. although the number of downloads and citations is not the only way to measure information dissemination and scientific impact, they are relatively easy to track. it is well known that the citation rates of journals from different disciplines vary significantly due to many factors such as the average number of authors, average number of citations in an article, size of the community, and so on.1012 however, little is known about how citation rates vary within biomedical informatics topics. jamia is a generalist journal in biomedical informatics. to understand the publications that readers consider interesting and cite - worthy, we expanded our initial analysis of citation rates by topic and free access status done in late 2011 (which covered only articles published from 2009 to 2010),13 to articles published in 2011 and 2012. the distribution of citations for jamia peaks between 3 and 4 years, so our analyses for 200911 are reasonably accurate, whereas those for 2012 are still very preliminary. we present here an analysis that shows the yearly rates of publication and average citations for jamia articles according to selected mesh topics in the past 4 years (200912) to understand whether different subfields of biomedical informatics have different citation distributions and how they are changing over time. in this analysis, we take into account the fact that the number of articles with free immediate access when published online or in print has increased rapidly in this period due to the option for authors to select an open access model. we report the associations among downloads, number of citations, and free access status. in july 2013 we collected the number of yearly citations to jamia articles published between 2009 and 2012 from the isi web of science database. we removed non - peer reviewed materials such as highlights, editorials, letters, and american medical informatics association (amia) messages, which left us with a total of 564 articles to analyze. these articles described research and applications, brief communications, perspectives, reviews, and case reports. note that jamia had slightly different article category names and several article subcategories before 2011 (for example, model formulation, synthesis of research). we recorded dates for the online and in - print publication in a regular issue of the journal, or in a special online issue when applicable. we recorded whether the article was open (that is, freely available immediately at online release, before inclusion in a specific journal issue), free (that is, freely available when it was included in a print or online journal issue), or embargoed (that is, freely available to non - subscribers only after 1 year of embargo from the date of publication in an issue of the journal). we also recorded the number of downloads for abstracts, full - text in html, and full - text in pdf from the jamia web site (http://www.jamia.org). we utilized the same methodology first described by kim to calculate the number of citations per month (cpm) from the date at which the article became available online, and calculated dpm using the number of full - text online views plus the number of pdf downloads. for articles in which no date of online publication was available, we calculated p values for comparisons that were sufficiently powered. with an effect size () of 0.8 in cpm, the required sample size was 25 (for each group) for a desired statistical power (1-) of 0.8 at a significance level () of 0.05 using a two - sample t - test. the nine most frequent topic categories, which were also employed in our 2011 article,13 were used to classify the articles. two authors independently selected the most relevant topic for an article, by retrieving mesh terms from pubmed, when available, and reading the titles and abstracts. another author served as an arbitrator when there was a conflict, selecting a topic from the two proposed topics (there were 75 disagreements). the visual trends for the nine most frequent mesh topics are plotted in figure 1 to show the changes in the past 4 years. the figure shows the total number of articles in each of the categories (right vertical axis), and the breakdown of the average number of cpm (left vertical axis). a topic for which the block of bars is displayed in the group 201x in the x - axis shows the average number of cpm since publication for articles published each year between 2009 and 201x, with one bar representing each year, as indicated in the legend. breakdown of number of accepted articles (line plot, right axis), average number of citations for articles published each year from 2009 to 2012 (bar plot, left axis), grouped by year in which the citation appeared (x - axis). (a) and (b)(j) show the breakdown of citations for all articles and for articles in nine different categories, respectively. note that the scale for the right axis, which represents the number of published articles, varies depending on the topic, while the left axis is fixed. medical record systems was the most frequently assigned topic (124/573), while prevention and control represented the smallest group (24/573). medical informatics included articles related to biomedical and health informatics education, as well as some policy and other articles that could not be well classified into the other categories. as shown in figure 1, medical record systems, algorithms, and methods are the fast growing categories in terms of accepted articles, and they also show an increasing trend in citations. natural language processing, medical informatics, information storage and retrieval, and methods have a consistent pattern of increased citations. topics related to computer science, such as algorithms and user computer interaction tend to have relatively fewer citations than those related to healthcare. the correlations between dpm and cpm are significant in all years that is, 2009 (p<0.01), 2010 (p<0.01), 2011 (p<0.01) and 2012 (p=0.06)at a 0.1 threshold. the cpm versus dpm plots are illustrated in figure 2, in which we also show the linear regression model for each year. downloads per month (dpm) versus citations per month for all articles in the past 4 years. figure 3 shows the boxplots displaying cpm for open access (freely available on publication online), free (free access on publication in a journal issue), and embargoed articles (for 2009 there were only embargoed articles, and for 2010 there was a small number of free access articles but no open access option). (a) boxplots of open access plus free articles and embargoed articles, and p values for comparisons that are powered to detect significant differences (=0.2). (b) comparisons among open access (that is, free on online publication), free (that is, free on publication in a journal issue), and embargoed articles for 2011 and 2012. note that 2009 did not have open access or free articles, and 2010 did not have an open access option. analyses for 2012 are preliminary given the relatively short time from publication to the time all data were collected in mid-2013. figure 3 also shows a number of comparisons based on the free availability of papers from 2010 to 2012. in figure 3a, we combined open and free access articles into one category (o+f), then compared to embargoed articles. o+f articles received significantly higher cpm in 2011 when tested with a two - tailed t - test (p=0.0036). this trend continued in the preliminary analysis for 2012, and the difference was statistically significant (p=0.0410). the free (typically either editor 's choice or amia - endorsed) articles received higher cpm than the embargoed articles in the year 2011 (analysis not sufficiently powered given only 21 free articles) (see figure 3b). the open access articles in 2012 also had a higher cpm than the embargoed articles in the same year, but the difference was not statistically significant (p=0.3600). as the embargoed articles are made freely available after 1 year of publication, there could be a stronger effect in citations accrued in the short term (that is, in the same year of publication and the subsequent year). we thus compared the mean number of citations (for two consecutive years including the year of publication) for freely available and embargoed articles published in 2010 and 2011 (see figure 4). for 2012, there was incomplete information as the total number of citations for 2013 was not yet available. results show that this difference was significant for 2011 (p=0.0034), the only year for which the test had sufficient power. citations (in two consecutive years including year of publication) for articles published in 2010 and 2011. the difference between freely available (free in 2010 or open access plus free in 2011) and embargoed articles was evaluated using a two - sample t - test. table 1 lists the most cited 200912 papers for two consecutive years starting with the one in which they were published in a journal issue and gives an idea of topics and types of articles that attracted most short - term citations. most cited articles when considering two consecutive years including the year of publication in a journal issue ties are broken by date of publication, with most recent articles ranking higher. most downloads from 2009 to 2012 note that the total number of downloads is calculated as the sum of the full text access and pdf downloads, and does not include abstract access. jamia has published several special focus issues in the past 2 years, and the scope of the journal was expanded to include translational bioinformatics,38 6174 economic evaluations of health information technology,49 7580 and brief communications of large national initiatives for sharing of tools and data.45 47 51 8186 it is too early to know whether these topics will result in higher, the same, or lower numbers of citations than other topics. some topics appear to have an increasing number of citations in the past few years, and the distribution of articles across the topics also appears to be changing. we could have chosen two or more per article. fixing the number of topics per article was considered to be important so an article that was assigned several topics would not have a greater influence in the citation count than an article that was assigned fewer topics. by assigning just the main topic to an article, the patterns of citations for different topics vary considerably. for a small but highly varied field such as biomedical informatics, in which some researchers are doing research more closely aligned with computer science than medicine, some form of weighting should be developed to account for the fact that the average number of citations to a computer science topic is smaller than the average number of citations to a health science topic.87 the analyses displayed for 2012 are very preliminary, as the number of cpm is still expected to increase. citations were collected mid - year in 2013, and are significantly lower than the total number of citations expected in 2013. the number of downloads may serve as a proxy for upcoming citations in these years. however, this proxy has limitations. although correlations between dpm and cpm are all statistically significant, it is easy to see in figure 2 that some highly downloaded articles have relatively few citations and vice versa. it is possible that some articles appeal to many readers, but only some of these appeal both to readers and authors. that some articles are highly cited but have relatively few downloads is harder to explain, but it is possible that authors are citing articles after only reading their abstracts online, or that they are using a paper copy of the journal. more research is needed to determine patterns of articles for which dpm and cpm are not correlated. an important trend towards an increased number of online full - text views and downloads was observed. free downloads and online views were not always associated with increased citations, but may be important for the dissemination of biomedical informatics to journal non - subscribers. some influential articles may not result in a large number of citations, but may have a significant impact on the dissemination of knowledge into clinical practice and public policy.88 it is possible that this is happening with some jamia articles. however, it is too early to assess the impact of these articles in clinical practice and health policy. in addition, the assessment of download frequencies can be biased (for example, it is possible to generate a large number of downloads to any particular article by developing automated programs to access those articles) and hence it is difficult to use download frequency in isolation as a measure of academic productivity and impact. in addition, we do not report here on pubmed central download frequencies, which are likely to constitute a large portion of downloads.89 it is interesting to note that freely available 2012 articles dominate the list of most downloaded articles (that is, five were from 2012, and eight were freely available on publication in a journal issue). we will follow the trend of citations for those articles in the upcoming years to see whether they result in an overall higher number of citations than articles that were not freely available on publication. as, according to davis,90 social stratification may result in most citations originating from authors who are based in institutions that have access to subscription journals, it is possible that citation rates will not be associated with free access status. the analyses presented here show trends in the field that emphasize association but not causation. these are typically amia communications endorsed by its board of directors, and usually reporting on the results of workshops, or consensus statements, and jamia editor 's choice articles, which represent embargoed articles that the editor considered important to be freely disseminated in the short term. therefore, it could be the case that this designation was the main driver for higher exposure and higher citation rates. in addition, many of these articles had corresponding freely available webinars that attracted thousands of viewers,91 and some had news media or social media coverage.92 similarly, some authors may have elected the open access option depending on the importance they placed on short - term dissemination. this could result in self - selection of the authors best work, which might have been more highly cited anyway. in a retrospective study like this, it is impossible to eliminate these potential biases, but they do not invalidate the observation that freely accessible articles generate a larger number of online full - text views and downloads, and that they have a tendency to be more cited. our analyses showed that, for the jamia articles studied, the average monthly number of online full - text views plus article downloads (dpm) was highly correlated with the average number of cpm, and hence it may potentially serve as a short - term surrogate for the latter. we also showed an overall tendency for a higher number of citations to jamia articles that are freely available at the time of publication. citation differences between free and embargoed articles were significantly higher for the former in 2011 (p=0.0096).	in a growing interdisciplinary field like biomedical informatics, information dissemination and citation trends are changing rapidly due to many factors. to understand these factors better, we analyzed the evolution of the number of articles per major biomedical informatics topic, download / online view frequencies, and citation patterns (using web of science) for articles published from 2009 to 2012 in jamia. the number of articles published in jamia increased significantly from 2009 to 2012, and there were some topic differences in the last 4 years. medical record systems, algorithms, and methods are topic categories that are growing fast in several publications. we observed a significant correlation between download frequencies and the number of citations per month since publication for a given article. earlier free availability of articles to non - subscribers was associated with a higher number of downloads and showed a trend towards a higher number of citations. this trend will need to be verified as more data accumulate in coming years.
increasing clinical epidemiologic studies revealed that some common chronic diseases (such as coronary heart disease [chd ], diabetes mellitus [dm ], and cerebral infarction [ci ]) are closely associated with depression, and they were reciprocally causative and had an interaction [14 ]. to the best of our knowledge, the mechanisms underlying the concomitant depression in patients with chronic diseases are usually ascribed to the disruption of the hypothalamic - pituitary - adrenal (hpa) axis and the involvement of varieties of proinflammatory cytokines. in the past decade depression may not only be a result of vascular diseases, but also a risk factor of vascular diseases. the relationship between depression and vascular endothelial dysfunction (a feature in the early stage of vascular diseases) is currently an important research area. according to embryology further studies revealed that endothelial cells also have endocrine characteristics and are able to regulate inflammatory reactions and coagulation. endothelial dysfunction is mainly characterized by endothelium - dependent diastolic dysfunction, which is ascribed to the reduction in nitric oxide (no) production. currently, 2 methods are employed to detect endothelial function : intravascular injection of acetylcholine and pressurization with a cuff. in terms of intravascular injection of acetylcholine, when the endothelial function is normal, acetylcholine may induce the vascular dilation via no pathway ; however, when the endothelial dysfunction is present, vasoconstriction occurs after injection of acetylcholine. although this method is regarded as the criterion standard for evaluating endothelial function, it is not widely used in clinics because it is invasive, time - consuming, and expensive. when pressurization with a cuff is used to block the blood flow of the brachial artery, rapid blood flow after deflation may cause a high shear stress to the endothelial cells and induce them to release no, resulting in vascular dilation. this method may be influenced by many factors and highly dependent on the level of detection ability, which results in low repeatability and reliability. endopat is a peripheral arterial tonometry in which the change in the finger arterial pulse volume is measured to evaluate the endothelial function by reactive hyperemia peripheral arterial tonometry index (rhi). it has no disadvantages related to the above mentioned methods and can be widely applied in clinical practices. a recent meta analysis showed that both severe and transient depression were related to endothelial dysfunction. however, whether the endothelial function becomes normal or is still abnormal during the following remission of depression or even complete recovery from depression is still unclear (that is endothelial function and depression status are independent characteristics or mutually dependent) [1316 ]. this may be ascribed to the fact that the vascular endothelial dysfunction is influenced by a variety of factors such as lifestyle, concomitant diseases, and pharmacotherapy. studies have been conducted to investigate the endothelial function in patients who have recovered from depression by undergoing anti - depression therapies. however, these studies focused on the influence of antidepressant treatment on endothelial function. few studies have been conducted to investigate the relationship between endothelial function and depression recovery after excluding the influence of antidepressants. we hypothesized the following : depression is closely related to endothelial dysfunction ; the remission of depression symptoms is accompanied by the improvement of endothelial function ; and that the endothelial function is worst in depression patients, best in non - depression patients, and moderate in remission patients. patients were consecutively recruited from the cardiology, neurology, endocrinology, and clinical psychology clinics of beijing chaoyang hospital from march 2013 to june 2014. these patients were aged 3070 years, and had the ability to provide consent and cooperate with the questionnaire survey. subjects for the 3 groups were matched for age (3y) and body mass index (1 kg / m) at a ratio of 1:1:1 and there were 62 subjects in each group. this cross - sectional study was approved by the ethics committee of beijing chaoyang hospital. depression group : depression was diagnosed according to the chinese version of the modified structured clinical interview for dsm - iv diagnostic criteria for patient version by 2 trained clinical psychiatrists. all patients were drug - nave and in their first episode of illness (they experienced a depressive episode for the first time), and had 17-item hamilton rating scale for depression total scores (hamd17) 17. remission group : subjects had a self - reported history of physician - diagnosed depression, hamd17 7, and antidepressants had been discontinued for at least 3 months. control group : all healthy controls were interviewed with the structured clinical interview for dsm - iv, non - patient version. they were free of depression, other psychiatric or neurological illnesses, history of head injury, and had hamd17 7. exclusion criteria were : severe cardiovascular disease (massive myocardial infarction and coronary artery bypass), acute diseases in internal medicine (such as acute nerve deafness), severe mental disorders (schizophrenia and bipolar disorder), and cognition impairment. demographics characteristics, including gender, age (in years), and smoking history, were obtained by self - report. height was measured using a wall - mounted stadiometer and weight was assessed by using calibrated electronic scales. body mass index (bmi) was calculated as kg / m. systolic blood pressure (sbp) and diastolic blood pressure (dbp) were measured on the left arm. fasting glucose (fg), triglycerides (tg), total cholesterol (tc), high - density lipoprotein cholesterol (hdl) and low - density lipoprotein cholesterol (ldl), and self - reported history of physician - diagnosed diseases (such as chd, dm and ci) were also recorded. current depressive symptoms were determined with hamd17, which is the most common scale used for clinical evaluation of depression status. endopat2000 (itamar medical, caesarea, israel) has been widely used to measure the endothelial function, which is a beat - to - beat plethysmographic recording of the finger arterial pulse - wave amplitude (pwa) with pneumatic probes. a variety of clinical studies have confirmed that this technique is convenient and easy to handle and had good reliability and intraclass correlation coefficients (0.78) [2027 ]. the sensitivity and specificity of this technique were as high as 80% and 85%, respectively. a cuff was inflated to obstruct the blood flow of the brachial artery for 5 min, followed by deflation and subsequent vascular dilation. endopat software (version 2.3.2) was employed to calculate the ratio of signal amplitude before flow obstruction to that after flow obstruction. the higher the rhi was, the better the endothelial function. the demographics and clinical characteristics were compared with analysis of variance (anova) or kruskal - wallis test for continuous variables and chi - square test for categorical variables. the linear regression model was used to analyze the effect of candidate covariates on rhi, and the candidate covariates included gender, age, bmi, sbp, dbp, fg, tc, hdl, ldl, tg, cardiovascular disease (yes / no), cerebrovascular disease (yes / no), and dm (yes / no). the general linear model was employed to compare rhi among the 3 groups after adjusting the covariates, and bonferroni method was used for the correction of multiple comparisons among groups. patients were consecutively recruited from the cardiology, neurology, endocrinology, and clinical psychology clinics of beijing chaoyang hospital from march 2013 to june 2014. these patients were aged 3070 years, and had the ability to provide consent and cooperate with the questionnaire survey. subjects for the 3 groups were matched for age (3y) and body mass index (1 kg / m) at a ratio of 1:1:1 and there were 62 subjects in each group. this cross - sectional study was approved by the ethics committee of beijing chaoyang hospital. depression group : depression was diagnosed according to the chinese version of the modified structured clinical interview for dsm - iv diagnostic criteria for patient version by 2 trained clinical psychiatrists. all patients were drug - nave and in their first episode of illness (they experienced a depressive episode for the first time), and had 17-item hamilton rating scale for depression total scores (hamd17) 17. remission group : subjects had a self - reported history of physician - diagnosed depression, hamd17 7, and antidepressants had been discontinued for at least 3 months. control group : all healthy controls were interviewed with the structured clinical interview for dsm - iv, non - patient version. they were free of depression, other psychiatric or neurological illnesses, history of head injury, and had hamd17 7. exclusion criteria were : severe cardiovascular disease (massive myocardial infarction and coronary artery bypass), acute diseases in internal medicine (such as acute nerve deafness), severe mental disorders (schizophrenia and bipolar disorder), and cognition impairment. demographics characteristics, including gender, age (in years), and smoking history, were obtained by self - report. height was measured using a wall - mounted stadiometer and weight was assessed by using calibrated electronic scales. body mass index (bmi) was calculated as kg / m. systolic blood pressure (sbp) and diastolic blood pressure (dbp) were measured on the left arm. fasting glucose (fg), triglycerides (tg), total cholesterol (tc), high - density lipoprotein cholesterol (hdl) and low - density lipoprotein cholesterol (ldl), and self - reported history of physician - diagnosed diseases (such as chd, dm and ci) were also recorded. current depressive symptoms were determined with hamd17, which is the most common scale used for clinical evaluation of depression status. endopat2000 (itamar medical, caesarea, israel) has been widely used to measure the endothelial function, which is a beat - to - beat plethysmographic recording of the finger arterial pulse - wave amplitude (pwa) with pneumatic probes. a variety of clinical studies have confirmed that this technique is convenient and easy to handle and had good reliability and intraclass correlation coefficients (0.78) [2027 ]. the sensitivity and specificity of this technique were as high as 80% and 85%, respectively. a cuff was inflated to obstruct the blood flow of the brachial artery for 5 min, followed by deflation and subsequent vascular dilation. endopat software (version 2.3.2) was employed to calculate the ratio of signal amplitude before flow obstruction to that after flow obstruction. then, rhi was obtained. the demographics and clinical characteristics were compared with analysis of variance (anova) or kruskal - wallis test for continuous variables and chi - square test for categorical variables. the linear regression model was used to analyze the effect of candidate covariates on rhi, and the candidate covariates included gender, age, bmi, sbp, dbp, fg, tc, hdl, ldl, tg, cardiovascular disease (yes / no), cerebrovascular disease (yes / no), and dm (yes / no). the general linear model was employed to compare rhi among the 3 groups after adjusting the covariates, and bonferroni method was used for the correction of multiple comparisons among groups. there were no significant differences in gender, age, bp, bmi, fg, ldl, hdl, tc, tg, or history of chronic diseases among the 3 groups (table 1). results showed that the demographics and clinical characteristics were comparable among three 3 groups and were matched for age and bmi. the influence of 15 variables (e.g., gender, age, and clinical indicators of rhi) was analyzed by linear regression analysis (table 2). the results showed that dbp, tg, chd, and hamd17 had significant effects on rhi (p<0.05). in terms of distribution balance of all covariates, a general linear model was used to compare rhi among the 3 groups without adjustments. the mean rhi was 1.93, 2.34, and 2.19 in the depression, control, and remission groups, respectively (figure 1). the difference of rhi among the 3 groups was statistically significant (p=0.0004). compared with the controls (2.3370.697), rhi in the remission group (2.1940.426) was markedly higher than in the depression group (p=0.027). however, there was no significant difference between remission and control groups (p=0.3363). after matching for age and bmi, patients in the 3 groups had comparable demographics and clinical characteristics. the results showed that depression was closely related to endothelial dysfunction, and endothelial function should be improved as symptoms of depression were relieved. the endothelial function in patients with remission from depression was comparable to that in non - depression patients. endothelial dysfunction is a critical step in the development of cardiovascular disease, such as hypertension, atherosclerosis, and thrombosis. several studies have demonstrated that endothelial function has relevance to blood pressure and lipids, which are cardiovascular risk factors. the relationship between depression symptoms and endothelial function is complicated. some cross - sectional studies and retrospective studies [14,3032 ] showed that depression in adolescence, climactereium and senility, unipolar depression, bipolar depression, major depressive episodes, and minor depressive symptoms were closely associated with endothelial dysfunction. regarding biological mechanisms, depression may reduce synthesis of nitric oxide synthase and create imbalance between dilation (by no) and contraction (by et-1) of blood vessels, which finally causes endothelial dysfunction., depression may cause obesity, reduced exercises, and disturbed metabolism of glucose and lipid, which indirectly result in endothelial dysfunction. however, several studies found a relationship between depression and chd and dm. in the present study, patients were recruited from a general hospital in china, and results showed that dbp, tg, chd, and hamd total scores had significant effects on rhi, consistent with previous research. another important result in the present study was that the improvement of depression was accompanied by improvement of endothelial function. this may be ascribed to the following reasons : 1) endothelial dysfunction is dependent on depressive status. the improvement of depression may cause the cortisol secretion to become normal in the hpa axis. in the presence of stress, metyrapone, a drug able to inhibit the secretion of cortisol, is able to prevent endothelial dysfunction. in our study, the endothelial function was measured on the basis of no and was mainly characterized by vascular dilation. after anti - depressant therapy, the receptor might become normal, leading to vascular dilation. pizzi. found the improvement of depression in chd patients with depression following anti - depressant therapy was accompanied by the improvement of endothelial function. there is evidence showing that the endothelial dysfunction is still present in depression patients after anti - depressant therapy. among clinical studies, only the sadhart study indicated that ssris might reduce the morbidity and mortality related to cardiovascular and cerebrovascular diseases. the evidence is still not enough to establish that successful anti - depressants therapy can reduce the incidence of cardiovascular events and/or increase the survival rate of these patients. although the effect of ssris on nitric oxide synthase and no production is ambiguous, anti - depressants as a confounding factor might obscure the relationship with endothelial function. in the present study, the influence of anti - depressants therapy on this relationship was excluded and results confirmed that the endothelial function recovered 3 months after discontinuation of anti - depressants therapy when clinical remission of depression was confirmed, and the endothelial function in remission patients was comparable to that of non - depression patients. our findings are consistent with previously reported research and demonstrate that the endothelial function after anti - depressants treatment could recover to the level of healthy controls. endopat2000 is able to identify atherosclerosis at an early stage and predict the adverse cardiovascular outcomes within 7 years. this study for the first time employed endopat for the non - invasive quantitative evaluation of vascular endothelial function in outpatients recruited from a general hospital. our findings are consistent with results obtained with other methods for the detection of endothelial function. thus, this technique is applicable in clinical studies on general screening and our results may be generalized. this was a cross - sectional study that failed to evaluate the causative relationship between depression and endothelial function. this causative relationship needs to be confirmed in future prospective studies with larger sample sizes. although paired comparisons were done among the 3 groups after balancing the clinical characteristics of subjects in these groups, which is a novelty of this study, the course of depression and details of anti - depressants therapy were not analyzed and evaluated. in future studies, investigators may focus on the relationship of changes in depression status with changes in endothelial function of a specific individual. taken together, depression in outpatients from a general hospital in china is closely related to endothelial dysfunction. the improvement of depression after anti - depression therapy is accompanied by improvement of endothelial function, which returns to a level comparable to that in healthy controls.	backgroundthis study aimed to investigate the endothelial function by reactive hyperemia index (rhi) in patients with depression, subjects recovering from depression, and subjects without a history of depression.material/methodsoutpatients were recruited from a general hospital in china ; 62 patients diagnosed with depression and the 17-item hamilton rating scale for depression (hamd17) total scores 17 were enrolled as the depression group, 62 patients with a history of depression, discontinuation of antidepressants therapy at least 3 months ago, and hamd17 7 were recruited as remission group, and 62 subjects without a history of depression served as the control group (hamd17 7).resultsthe mean rhi was 1.93, 2.34, and 2.19 in depression, control, and remission groups, respectively, showing a significant difference among the 3 groups (p=0.0004). in addition, a marked difference in rhi was found between depression and control groups (p=0.0003) and between depression and remission groups (p=0.0270). however, there was no significant difference between remission and control groups (p=0.3363).conclusionsthere is a relationship between depression and endothelial dysfunction in outpatients from a general hospital in china. the improvement of depression is synchronous with the improvement of endothelial function.
dietary consumption of olive oil is considered a key component to explain the association of mediterranean diet with a lowered incidence of metabolic disorders and cardiovascular diseases [13 ]. the beneficial action of extra virgin olive oil is mainly attributed to its seco - phenolic compounds and their secondary metabolites, which have highly been investigated as both whole extracts and individual components [4, 5 ]. in particular, the proactive ingredient oleuropein (ole) and its derivative hydroxytyrosol (ht) have demonstrated several beneficial effects in in vitro and in vivo experimental models [69 ]. ole is a natural polyphenolic compound belonging to the secoiridoids and is present in high amount in the leaves and unprocessed olive drupes. after crushing, ole is hydrolyzed by endogenous esterase giving rise to its aglycone derivate that is the active secoiridoid present in virgin olive oil. after oral administration, it is rapidly absorbed with maximum plasma concentration occurring 2 h after administration and excreted in urine mainly as glucuronides or as free form in very low concentrations [10, 11 ]. the ability of ole and ole derivatives to scavenge reactive oxygen species and their antioxidant activity [6, 8, 12, 13 ] have been associated with the protection against cardiovascular diseases and metabolic disorders [1417 ]. two other mechanisms have been proposed to explain the hypoglycemic effect of ole and ht : the improvement of glucose - induced insulin release and the increased peripheral uptake of glucose [14, 18 ]. moreover, an effect of ole on insulin action and secretion has been described in overweight middle - aged men, who received a diet supplemented with olive leaf polyphenols. such an effect was independent by fat distribution, dietary intakes, and physical activity and was similar to that obtained with antidiabetic drug treatment. in addition, ole has been successfully used to prevent hepatic steatosis in mice fed with high fat diet [9, 2022 ]. in our laboratory, we have synthesized a peracetylated derivative of ole (ac - ole) in good yields and very mild conditions by applying nonconventional synthetic methodologies on the natural substrate ole extracted from renewable raw material [23, 24 ]. in previous works, ac - ole has shown in vitro higher antioxidant and antiproliferative effects on thyroid and breast cancer cells than the natural molecule [25, 26 ]. a relevant model to study metabolic disorders in mice is the highly palatable cafeteria (caf) diet : this dietary intervention is closer to western food habits than the traditional high fat diet, creating therefore a phenotype of exaggerated obesity with glucose intolerance and inflammation. in the present study, we analyzed the effects of oral subchronic administration of ole and ac - ole in c57bl/6jolahsd mice fed with a caf diet. the nutritional compositions of the standard (std) diet (teklad global 18% protein rodent diet, harlan laboratories s.r.l., udine, italy) and the caf diet are reported in table 1. caf diet is a mixture of std diet and commercial food as snack, chocolate, biscuits, and wafers. ole (mw 540, dissolved in water at the dose of 0.037 mmol / kg, equivalent to 20 mg / kg) and its semisynthetic derivative ac - ole (mw 792, dissolved in corn oil at the dose of 0.025 mmol / kg, equivalent to 20 mg / kg) were obtained as described. male c57bl/6jolahsd mice (n = 20, aged 4 weeks) were purchased from harlan laboratories s.r.l., housed in a temperature (2022c) and humidity (64%) controlled animal room and maintained on a 12 : 12 h light / dark cycle. after 2 weeks of quarantine the mice were divided into four separate groups (n = 5 in each group) of equal average body weight (19.4 1.2 g) : mice fed with a std diet (named std), mice fed with a caf diet (named caf), mice fed with a caf diet supplemented with daily oral administration of ole (0.037 mmol / kg ; named ole), and mice fed with a caf diet supplemented with daily oral administration of ac - ole (0.025 mmol / kg ; named ac - ole). a std diet (harlan laboratories s.r.l.), suggested for this strain of mice, was used as control to (a) analyze the effects of caf diet and (b) investigate the effects of treatment with ole and ac - ole. animals of all groups received the same vehicle used for the peracetylated compound and had free access to food and water. every day, ole and ac - ole were prepared freshly before administration and added to caf diet pellet : it was checked that each mouse had ingested the food containing the daily dose of ole and ac - ole. food intake was calculated subtracting every week the aliquot of food not ingested by the total food added to each cage. body weight, nasoanal (n - a) length, and girth waist of all animals were recorded at weekly interval. at the end of treatment, after overnight fasting, mice were deeply anesthetized by intraperitoneal injection of tiletamine hydrochloride and zolazepam hydrochloride (zoletil, virbac, france) at dosage of 80 mg / kg, and medetomidine hydrochloride (domitor, orion corporation, finland) at dosage of 6.6 mg / kg. blood samples were obtained by cardiac venipuncture and collected into blood collection tubes (vacuette, greiner bio - one gmbh bad haller str. the whole liver, abdominal fat, thoracic aorta, kidney, and lung were removed and weighed and then were washed in normal saline solution and immediately fixed in 10% neutral buffered formalin (sigma - aldrich s.r.l., all efforts were made to minimize the number of animals used in this study and their suffering. blood samples, collected at the end of the treatments, were centrifuged at 1700 g for 10 min at room temperature and serum was stored at 20c until use. by using commercial reagents (siemens healthcare diagnostics s.r.l., milan, italy) and an automated biochemistry analyzer (dimension exl, siemens healthcare diagnostics s.r.l.) the levels of basal glucose, triglycerides, total cholesterol, low density lipoproteins cholesterol (ldl), high density lipoproteins cholesterol (hdl), alanine aminotransferase (alt), and aspartate aminotransferase (ast) were determined. the concentration of insulin and leptin was measured using elisa kits (rat / mouse insulin elisa kit ; mouse leptin elisa kit, emd millipore corporation, darmstadt, germany) according to the manufacturers ' instructions. approximate insulin resistance (ir) was calculated using the homeostasis model assessment (homa)-ir using the following formula : [glucose (mmol / l) insulin (u / ml)]/22.5. tissue samples were embedded in paraffin after dehydration through a graded ethanol series followed by xylene. several sections (thickness 4 - 5 m) from each paraffin block were cut, mounted on slides and, after deparaffinization and rehydration, were stained with haematoxylin and eosin according to standard methods for histological assessment under the light microscopy. in liver of each mouse, two different sections were evaluated, each of them cut along the major axis of the gland. results are expressed as mean standard deviation (sd). the data relative to body weight, naso - anal length, girth waist, food consumption, energy intake, liver weight, and hematological and clinical chemistry parameters were analyzed using sigma plot version 12 (systat software, inc.). one - way anova followed by tukey multiple comparison test was performed for different treatment groups. mice were fed for 15 weeks with std, caf, or caf diet supplemented with ole or ac - ole. during the experimental period, the mice showed no signs of suffering and no significant differences appeared in hematological parameters at the end of the treatments (table 2). the body weight increased in all groups, in accordance with the physiological growth of the mice (figure 1(a)). in caf mice, this increase was significantly higher than std (figure 1(a), p < 0.01), in parallel with an enhanced abdominal girth (figure 1(b), p < 0.05) and an increase in abdominal fat (figure 1(c), p < 0.01). supplementation of caf diet with ole or ac - ole reduced the body weight increase (figure 1(a), p < 0.01), as well as both abdominal girth and final abdominal fat (figures 1(b) and 1(c), p < 0.05 and p < 0.01, resp.). the food intake did not differ among the four groups during the experimental period, whereas a significant increase of energy intake was produced by caf diet (table 3, p < 0.001), and the addition of the two compounds did not cause significant changes in ole and ac - ole - treated groups when compared to caf group (table 3). liver weight was significantly increased in caf (1.6 0.4 g) versus std (1.1 0.1 g). treatment with ole and ac - ole prevented hepatic enlargement, as evident by the weight values, both absolute and normalized for the body weight, similar to those of the std mice (figures 2(a) and 2(b)). histologic analysis of liver tissue sections representative of the four groups of animals is shown in figure 2(c) and the quantitation of steatosis is reported in figure 2(d) : caf diet induced a significant steatosis, which was greatly reduced in ole and ac - ole groups. in terms of inflammatory cell infiltration, no significant difference was detectable among the four groups of animals ; indeed, in all mice only a mild and very focal lymphocytic infiltration in the portal triad was detectable. gomori staining indicates the absence of fibrosis in all four groups of animals (data not shown). caf diet induced no detectable differences in the other examined tissues (figure 3). caf fed mice showed significantly higher levels of plasma total, hdl, and ldl cholesterol than the std fed mice (table 4). ole and ac - ole supplemented caf diet determined a lower increase of such levels and, interestingly, ldl - cholesterol levels were maintained in the normal range described for these animals. no significant differences were observed in the levels of plasmatic triglycerides (table 4). in addition, caf diet induced an elevation in alt and ast levels when compared with std group. following treatment with ole, only the alt levels returned to the values of the std, while ac - ole determined a reduction of both enzymes (table 4). however, transaminase serum levels remained in the normal range in all four groups of mice. fasting glucose levels were slightly but significantly higher in caf animals compared to mice fed with std diet, although still in the normal range (figure 4). instead, ole and ac - ole groups showed values similar to std (figure 4). interestingly, insulin levels were significantly higher in caf mice as compared to std group (figure 4, p < 0.01), while, in both ole and ac - ole animals, they were significantly lower compared to the caf group (figure 4, p < 0.01). homa - ir, used as measure of insulin sensitivity, indicated that caf group showed statistically significant increase in insulin resistance compared to std group (figure 4, p < 0.001) while the supplementation of ole and ac - ole on caf diet reversed this effect (figure 4, p < 0.001 versus caf). also leptin content was significantly changed in caf as compared to std mice (figure 4, p < 0.01) ; again, lower levels were observed in the groups treated with ole or its peracetylated derivative (figure 4, p < 0.01 versus caf). polyphenolic compounds of extra virgin olive oil are known to exert a series of beneficial effects, mainly due to their antioxidant activity. a large number of studies that performed in vitro and in vivo experimental models have focused on the activity of ole revealing a large array of biological effects either when used as single compound or in a mixture of phenolic extracts from olive oil drupes or leaves [4, 6, 7, 17 ]. in view of a possible use in pharmaceutical field as single drug or as additive in the food, we have synthesized and tested in our laboratory a peracetylated derivative of ole [23, 25, 26 ]. peracetylation and deacetylation occur naturally in cells thanks to the activity of mitochondrial acetyl - coa dependent enzymes [3032 ]. it has been demonstrated that unprotected drugs, such as n - substituted aromatic compounds, are n - acetylated inside the cells in order to limit their adverse biological effects. similarly, peracetylated natural compounds such as peracetylated resveratrol or peracetylated epigallocatechin-3-gallate can act as prodrugs because they are deacetylated inside the cells. since peracetylation improves the chemical stability and permeability without affecting the transportation pathway through the membrane, the intracellular deacetylation results in an augmented dose of unprotected natural active principle in the intracellular medium [25, 26, 32 ]. we have previously reported the beneficial effects of ole and ac - ole in vitro. in this study, they have been investigated in vivo to analyze the effects versus the metabolic alterations caused by a caf diet. this diet is composed of a mixture of fat and carbohydrates and resembles in a better way the western type food habits compared to traditional lard - based high fat diet. indeed, mice fed with a caf diet develop metabolic syndrome more severely than those fed with high fat diet. in our study, mice fed with this diet for 15 weeks presented an increased body weight associated with histological signs of hepatic steatosis, high serum levels of total and ldl - cholesterol and hyperinsulinemia. only slight changes and inside the normal range levels were observed for transaminases, probably due to the limited period of exposure to this diet, as already reported in the other study. interestingly, the presence in caf mice of an increase in insulin levels seems to reproduce the well - known phase of hyperinsulinemic normoglycemia observed in obese individuals before the development of diabetes mellitus 2 (dm2). in our caf fed mice, administration of 0.037 mmol / kg / day of ole and 0.025 mmol / kg / day of ac - ole, determined the following : (a) reduction in the body weight increase due to minor accumulation of fat in the abdominal adipose tissue, with unchanged food intake gain ; (b) significant decrease of macroscopic and microscopic steatosis ; (c) lower levels of serum lipids, with normalization of the levels of ldl - cholesterol ; (d) correction of elevated glucose plasmatic levels without increase in insulin levels, associated with normal homa - ir. in a recent study, van der stelt. demonstrated that high dose of ole supplementation (758 mg / kg body weight) in mice fed with a high fat diet resulted in decrease of body weight, serum lipids, hepatic lipid accumulation, and liver weight after 12 weeks of treatment. in our study, the same results were observed using a much lower dose of ole or ac - ole (20 mg / kg body weight). prevention of body weight increase in animals (mice or rats) fed with high fat diet without acting on food intake was also reported using a similar dosage of ole in similar experimental conditions [36, 37 ]. as in our study, no action on food intake was observed when ole was added as single compound, while a decrease was detected when olive leaf phenolic extracts were tested, probably owing to the presence of apigenin [39, 40 ]. also, the caloric intake of ole and ac - ole groups was significantly higher with respect to std but they did not gain weight ; we may hypothesize that ole and ac - ole act by increasing the energy expenditure and/or reducing energy retention, by regulating the expression of genes involved in adipogenesis and thermogenesis. our present data showed that the same prevention of body weight increase was obtained with lower doses of ac - ole (0.025 versus 0.037 further studies on the gene expression profile modified by both compounds will help to identify the molecular pathways involved in such an effect. also prevention of hepatic steatosis exerted by ole has already been described in animal models [21, 22 ]. as for the protective action on body weight increase, an identical effect was obtained in our experimental model using ac - ole. again, as for the anti - flogistic and in vitro antineoplastic effects [25, 26, 41, 42 ], this semisynthetic compound demonstrated high efficacy. the most interesting findings of the present study, in our opinion, appear to be those regarding the metabolic effects of the compounds. in our experimental model, exposure to a caf diet determined a significant increase in both total and ldl - cholesterol levels. ole and ac - ole treatment was able to partially prevent such effects and, most importantly, normalize the ldl - cholesterol levels. [43, 44 ] demonstrated that ole (20 mg / kg) decreased cholesterol and triglycerides levels in hypercholesterolemic rabbits. instead, we found no significant changes in triglycerides, probably because in this strain of mice treatments longer than 15 weeks are required to get such alteration. however, insulin levels resulting are higher than mice fed with std diet, so that we may suppose that it was the parallel increase of insulin secretion which counteracted the effects of caf diet. treatment with ole and ac - ole obtained the same result with levels of insulin maintained in the normal range, with normalization of the values of homa - ir. in the absence of euglycemic hyperinsulinemic clamp, it is not possible to fully demonstrate the occurrence of an increased insulin sensitivity, as postulated in other studies using olive leaf polyphenols. another possibility is that normal insulin levels depend on the lack of body weight increase. in any case, however, we believe that prevention of damage associated with prolonged exposure to hyperinsulinemia that characterizes the pathologies associated with metabolic syndrome, dm2, and obesity deserves to be underlined. an interference between ole (or ac - ole) and substances present in elements of caf diet (as well as any kind of commercial diet used in this kind of studies) can not be excluded. in conclusion, these findings demonstrate, in this experimental model of metabolic damage caused by caf diet, the beneficial effects of ac - ole, exerted at even lower dose in moles than ole. further studies, including the administration of different dosages of ac - ole and even for shorter periods will allow characterizing the molecular mechanism involved in such an action, in view of a potential use of this compound for prevention of human metabolic disorders.	the high consumption of olive tree products in the mediterranean diet has been associated with a lower incidence of metabolic disorders and cardiovascular diseases. in particular, the protective effects of olive oil have been attributed to the presence of polyphenols such as oleuropein (ole) and its derivatives. we have synthesized a peracetylated derivative of ole (ac - ole) which has shown in vitro antioxidant and growth - inhibitory activity higher than the natural molecule. in this study, male c57bl/6jolahsd mice were fed with a standard (std), cafeteria (caf) diet, and caf diet supplemented with ole (0.037 mmol / kg / day) and ac - ole (0.025 mmol / kg / day) for 15 weeks. we observed a significant reduction in the caf diet - induced body weight gain and increase of abdominal adipose tissue. also, ole and ac - ole prevented the development of hepatic steatosis. finally, ole and ac - ole determined a lower increase of hdl and ldl - cholesterol levels and corrected caf diet - induced elevation of plasma glucose concentrations by improving insulin sensitivity. the observed beneficial properties of ole and ac - ole make these compounds and in particular ac - ole promising candidates for a potential pharmaceutic use in metabolic disorders.
aflatoxins are highly toxic, mutagenic, carcinogenic, and teratogenic compounds contaminating a wide range of food commodities [1, 2 ]. the major naturally occurring aflatoxins, namely, afb1, afb2, afg1, and afg2, constitute a class of structurally related toxic fungal metabolites. they are extremely potent carcinogens and can have significant economic impacts, making them important targets for detection and quantification. commodities frequently contaminated by aflatoxins include cereals, nuts, dried fruits, spices, and pulses [4, 5 ]. when animals consume afb1 contaminated foodstuffs, the toxin is metabolized in the liver and excreted as afm1 via milk and urination. humans are exposed to the deleterious effects of aflatoxins either directly by eating contaminated grains or indirectly via animal products. evidence of hazardous human exposure to aflatoxins through various foods including dairy products has been shown by several investigators [13, 7 ]. many analytical methods have been developed for estimation of aflatoxins in agricultural commodities. among them the optical detection method is regarded as one of the sensitive techniques for aflatoxin analysis. the optical signal is not influenced by electrical, magnetic, or ionic fields. table 1 gives an account of some of the reported biosensors for analysis of afm1 and afb1. it is evident that the need for a highly selective multianalyte detection system is acute. simultaneous analysis of aflatoxins is essential to minimize the consumption of contaminated food and feed and import surveillance programs and controlling quality of products [5, 8, 9 ]. there is a need for development of single platform for simultaneous analysis of these toxins. few attempts have been made towards development of immunosensors capable of both discriminative detection and class - selective multianalyte analysis. in one such approach, multianalysis of aflatoxins was investigated by development of an immunoaffinity column. this was done by covalently coupling monoclonal antibody 1c11 against aflatoxins to amino - silica gel microparticles and then packing these into a cartridge. this immunoaffinity column was subsequently used for selective extraction of aflatoxins in agri - products. in a recent article, novel peptide mimics of aflatoxin (mimotopes) 5 such mimotopes were obtained and used in an indirect competitive elisa for analyzing total aflatoxin concentration in peanut and feedstuffs. in another interesting article, multianalysis of different triazine herbicides using cr of antibodies coupled to pattern recognition has been reported. a similar approach could be implemented for multianalysis of different aflatoxins which are structurally similar (figure 1). in this work, we performed fluorimetric analysis of afm1 using fitc conjugated secondary (2) antibody in 384 microwell plate as shown in figure 2. the microwell plate was coated with anti - afm1 primary (1) monoclonal antibody (mab) which reacted with afm1 and partly recognized structurally similar aflatoxins such as afm2, afb1, and afg1. subsequently we analyzed 2 real milk samples using standard addition method, and recovery rate was studied. afm1, bovine serum albumin (bsa), tween 20, and certified reference material (crm) erm - bd282 (afm1 in whole milk powder, 100% recovery was obtained, the concentration (400 pg / ml) was much below the recommended level (2 g / l). for afg1 analysis, the recovery was 100.4%. in this case, the recovery for s1 and s2 was obtained as 92.6% and 93.6%, respectively. the mixture analysis was done by adding 50 and 25 pg / ml of afm2 and afb1 (individually) in varying amount of afm1 in the range 1100 pg / ml as shown in figures 7(a) and 7(b). it was observed that, at 12.5 pg / ml of afm1, the afm1 + afm2 (25 pg / ml) mixture showed 4% and the afm1 + afm2 (50 pg / ml) mixture showed 10% lesser signal intensity than that of only afm1. similarly at 25 pg / ml of [afm1 ], the mixture of both afm1 + afm2 (25 and 50 pg / ml) showed 2% and 4% decreases in signal intensity, respectively, as compared to only [afm1 ]. at the eu cutoff limit or 50 pg / ml only 1.5% of decrease in signal intensity was obtained for afm1 + afm2 (25 pg / ml) mixture. but when analyzed for afm1 + afm2 (50 pg / ml) mixture, it showed 4% lesser signal when compared to only afm1. at higher concentration of afm1 (100 pg / ml), the afm1 + afm2 (25 pg / ml) showed 4% and the afm1 + afm2 (50 pg / ml) showed 5% lesser signal intensity when compared to that of only afm1. afm2 showed almost 50% cr with afm1, and its presence as cocontaminant in the mixture resulted in small variation of 1.5 to 4% decrease at lower concentration (25 pg / ml), whereas at 50 pg / ml, the signal suppression was observed in the range of 4 to 10% variation. the mixture analysis was also carried out for afb1 and afm1 using anti - afm1 antibody as shown in figure 7(b). the standard calibration curve for afm1 was plotted against mixture of afm1 and afb1 (25, 50 pg / ml). in this case, further decline in signal intensities was observed. at 12.5 pg / ml of [afm1 ], [afb1 ] for 50 and 25 pg / ml showed 7.5% and 11.5% decrease in signal, respectively. for 25 and 50 pg / ml of [afm1 ], the afm1 + afb1 (25 pg / ml) mixture showed the same decrease in signal of about 8%, but for afm1 + afb1 (50 pg / ml) mixture it was 11% and 9.5%, respectively. at higher concentration of afm1 (100 pg / ml), the afm1 + afb1 (25 pg / ml) and afm1 + afb1 (50 pg / ml) mixtures showed 9 and 9.5% lesser intensities, respectively. the curve representing the mixture of [afm1 ] with 100 pg / ml [afb1 ] showed 12 and 10% less signal intensity of 25 and 50 pg / ml, respectively, when compared to only [afm1 ]. in all the mixtures of afb1, the signal suppression was observed in a higher order of 7.5 to 11.5% variation when compared to the mixture analysis of afm2. this difference has occurred for the reason of difference in cr of afb1 with afm1 (23%) which differs from 50% cr of afm2 with afm1. this mixture analysis can play significant role in detecting and differentiating the presence of different aflatoxins which exist as cocontaminants found in the food commodities. based on their cr properties, afm1, afm2, and afb1 this work illustrates a simple and sensitive fluorimetric multianalyte microwell plate based immunoassay for aflatoxins. ultrasensitive analysis of afm1 was successfully carried out by the developed technique with lod as low as 1 pg / ml. elisa for multianalysis of different aflatoxins was investigated in pbs, certified reference material for afm1 and in real samples by fluorimetric technique in the microwell plate in presence of various types of competing analytes. structurally analogous aflatoxins were analyzed using the cr concept. from the assay, it was observed that afm2 showed the highest cr as compared with afb1. the multianalysis of different aflatoxins was also verified by the mixture analysis of afm1 with afb1 and afm2. assay on microwell plate allowed testing of highly toxic aflatoxins done with low sample volume and with easy handling. besides the sensitivity and minimal reagent consumption, such multianalysis would lead to simultaneous screening of multiple analytes of aflatoxins.	a sensitive fluorimetric elisa was developed for the analysis of aflatoxins. the assay was performed in a 384 microwell plate, wherein high specificity monoclonal antibody against afm1 (mab - afm1) was used as capture antibody and fitc conjugated secondary antibody was used for detection and quantification of the analyte. the linear range of the immunoassay was found to be 6.2550 pg / ml. afm1 as low as 1 pg / ml was detected by this method with assay volume 40 l. the multi - analysis of different aflatoxins was also investigated in the microwell plate, based on the cross - reactivity (cr) approach. real milk samples were tested along with certified reference material by standard addition method and recovery analysis was done. the mab - afm1 showed 23.2% cr with afb1, 50% cr with respect to afm2, and least cr towards afg1 (< 1%). furthermore, mixture analysis of afm2 and afb1 was carried out at specific concentrations of afm1. the advantages of this developed immunoassay are high sensitivity, high throughput, multianalyte detection, versatility, and ease of handling.
diabetes mellitus type 1 (dm1) is associated with defects in tnf- signaling which result in an altered balance between tnf 's prosurvival and proapoptotic effects. one of the manifestations of this is prolonged survival of immune b cells and both cd4 and cd8 t lymphocyte subsets [1, 2 ]. on the other hand, in patients with dm1 enhanced apoptosis is prevalent, occurring in pancreatic cells, endothelial retinal cells, and various renal tissues [35 ]. the apoptosis defect has also been found in nonobese diabetic (nod) mice, a spontaneous model of human dm1. lymphocytes from these animals are more susceptible to tnf--induced apoptosis than those from control animals. moreover, apoptosis develops in the liver of streptozotocin - induced diabetic rats and is mediated by the tnf - r1 signaling pathway. neutrophils from diabetic patients display an enhanced apoptotic activity whereas those from diabetic mice show a reduced apoptotic rate [7, 8 ]. molecular studies in nod mice have revealed several genetic defects that alter the immune function within the mhc genomic region. a point mutation has been localized in the shared promoter - enhancer region of genes encoding lmp2 and tap-1 proteins, which negatively regulates the transcription of these genes. this mutation impairs proteolytic cleavage and activation of the nf-b transcription factor, which is physiologically bound to the inhibiting i-b subunit. nod mice, therefore, do not show degradation of the i-b subunit, which permanently suppresses the nf-b - dependent transcription of cell survival - promoting proteins. thus, stimulation of lymphocytes by tnf- preferentially triggers the proapoptotic pathway [5, 9, 10 ]. in humans, the genetic defects that are important for altered apoptosis are distinct from those in nod mice. an important role has been ascribed to a mutation identified in a gene - coding small ubiquitin - like modifier 4 (sumo4). a single amino acid substitution defect was found to prevent the nf-b - dependent activation. such negative regulation of the nf-b transcription factor hampers transcription of the prosurvival and antiapoptotic proteins [11, 12 ]. analysis of several clinical studies on different human populations has enabled an association to be found between multiple polymorphism in and near the gene sumo4 and susceptibility to diabetes mellitus type 1. many more molecular defects are expected in dm1, but these have not yet been detailed. different malfunctioning proteins have already been found in the tnf - r2 signaling pathway, resulting in inefficacies in the tnf - r2 pathway which is important for cell survival and cell proliferation. thus, in the case of nonactive tnf - r2 receptors, the only way of signaling by tnf- may be through the proapoptotic tnf - r1-dependent path. all these data imply that in diabetic patients, monocytes, cells that are important for innate and specific immune responses, may be vulnerable to apoptosis. it has been recognized that enhanced apoptosis underlies retinopathy and nephropathy, the late diabetic microvascular complications [4, 5 ]. tnf-, the chief inflammatory mediator of microangiopathy, triggers a sequence of biological events that culminate in apoptosis of vascular endothelial cells. the exact mechanism of apoptosis of endothelial cells has been understood in retinopathy. in the stages preceding overt retinopathy, the retinal capillary dysfunction is initiated by permanent infiltration by leukocytes. these cells adhere tightly to the endothelial cells as a result of the stimulating effect of tnf- on the expression of adhesive molecules on both endothelial cells and leukocytes. adhering inflammatory cells produce an array of angiogenic, inflammatory, and fibrogenic factors that promote endothelial cell - junction breakdown, blood - retinal barrier loss, and injury and apoptotic death of retinal endothelium and pericytes [1416 ]. long before overt complications occur in the animal model of diabetes, monocytes form the main constituent of infiltration within the lumen of the retinal microvessels. a large accumulation of monocytes and granulocytes is responsible for capillary leukostasis, which mechanically blocks blood flow and increases injury. a subset of monocytes, the so called nonclassical cd14cd16 monocytes comprise about 10% of the whole cd14 monocyte population. they are enriched in genes associated with the differentiation processes for an antiproliferative and proapoptotic state. the cd16 subsets are expanded in different kinds of inflammatory disease, such as rheumatoid arthritis, crohn 's disease, hiv, hepatitis, severe asthma, coronary artery disease, end - stage renal disease, sarcoidosis, tuberculosis, and stroke [1922 ]. in vitro experiments on whole blood cell cultures have revealed that the cd16 monocytes may be generated by tnf- treatment to approximately 30% of the total monocytes. in the blood of septic patients, the number of these cells correlated with the blood levels of tnf-. the dendritic cells originating from them were better equipped with adhesion molecules, showed properties of migratory cells, and stimulated more strongly the proliferative activity of tcd4 cells as compared to those originating from classical monocytes. thus, they infiltrate capillaries, small veins, and arteries and strongly attach to the endothelial cells. in addition, these cells are chief producers of tnf-, releasing this cytokine preferentially on the layer of endothelial cells and not spilling it over to the blood stream. cd14cd16 cells ; it constitutes the main monocyte pool, comprising about 85% of the whole monocyte population. they are mobile cells and may be rapidly recruited from the blood to the infected tissues, where they undergo activation and transform into the cd14cd16 forms. when stimulated, these cells produce a set of inflammatory mediators including il-8, ccl-2, ccl-3, il-6, tnf-, no, and ros. the characteristic clustering of genes found in monocyte subsets indicates that the classical monocytes are highly versatile and capable of responding to a variety of external stimuli and mediating tissue repair and immune functions. although the distinction between monocyte subsets has been demonstrated in different diseases and in healthy volunteers, there are no data to support the notion that such differentiation also exists among healthy juveniles and dm1 patients. moreover, bearing in mind the general apoptotic defect in dm1, it would be interesting to characterize the spontaneous apoptotic potential of these monocyte subsets, if it exists. to date the aim of our study therefore was to provide quantitative data on cd14cd16 and cd14cd16 monocyte subsets in juvenile - onset complication - free diabetes mellitus type 1 and to compare them with the corresponding values in the healthy age- and sex - matched control group. next, we planned to enumerate the spontaneously apoptotic monocytes within both subsets in the dm1 patients and to compare the results with the values for the healthy subjects. moreover, since the data imply that the proapoptotic potential of a dm1 patient 's cells may be dependent on an overactive tnf - r1 pathway, it would be interesting to determine the expression of tnf - r1 receptors on monocytes and then to manipulate this with tnf - r1 blocking or enhancing agents so that its effect on monocyte apoptosis may be observed. finally, we wanted to assess the effect of infliximab, which is a chimeric monoclonal antibody against tnf- with a murine variable region and a human immunoglobulin constant region, on the apoptosis of monocytes of dm1 patients and healthy controls. the group examined consisted of 60 randomly selected children and adolescents aged 14.5 3.2 years (28 boys and 32 girls) with long - standing dm1 (a disease duration of 5.8 3.6 years, hba1c = 8.38 2.21%, and an albumin excretion rate of 15.35 7.9) from the diabetology outpatient clinic at the medical university of gdask, poland. patients with microvascular complications and those with coexisting autoimmune, chronic, and acute inflammatory diseases were excluded from the study. in all the patients, examined c - peptide levels were below 0.5 ng / ml. the control group consisted of 30 healthy age- (13.5 2.8 years) and sex - matched (14 males and 16 females) volunteers. informed consent was obtained from the parents of all the children enrolled in the study, while juveniles over 16 years old consented in person. the study was approved by the ethics committee of the medical university of gdask and was carried out in accordance with the code of ethics of the world medical association (declaration of helsinki 1975 and its amendments 1983, 1989, 1996 jama 1997 ; 277:925 - 926) for experiments involving humans. hba1c was measured by an immunoturbidometric method using the unimate 3 set (hoffmann - la roche ag, d) with a normal range of 3.06.0%. fasting glucose was measured by an enzymatic test (roche diagnostics gmbh, d). urinary albumin excretion was expressed as the average of three 24-hour collections obtained during the six months prior to enrolment in the study. microalbuminuria was defined as albumin excretion within the range of 30299 mg/24 hours in at least two out of three urine samples. urinary albumin excretion was measured by the immunoturbidometric assay using tina - quant (boehringer mannheim gmbh, d). peripheral blood mononuclear cells were obtained by density gradient centrifugation on the ficoll / isopaque (pharmacia, s). isolated peripheral blood mononuclear cells were placed on 24-well plates in rpmi 1640 containing 5% fcs (sigma aldrich, usa) at a density of 1 10/well. fc receptors were blocked with 1 g of human igg/10 cells for 15 minutes at room temperature. the first was left without additives and the second was kept with 50 l of infliximab (remicade, janssen biologics b.v. ; the third set was incubated with one of the following substances : anti - tnf - r1 mab (clone h398) 10 g / ml (serotec l.t.d., usa), anti - tnf - r2 mab (clone m1) 10 g / ml (serotec l.t.d., usa), 16 m, sodium salicylate 10 mm, and 4-bromophenacyl bromide (bpb) (sigma aldrich, usa). plates were incubated for 24 h at 37c with 5% co2. after incubation plates were kept for 5 minutes on ice. the tubes were rinsed three times with cold pbs and then centrifuged and the supernatants removed. the sedimented cells were suspended in 200 l of 5% rpmi and the following mabs were added : 4 l anti - cd14 percp (clone mp9 ; igg2b) (bd biosciences, usa), 3 l anti - cd16 apc - cy7 or fitc (clone 3g8 ; igg1) (bd biosciences, usa), and 5 l anti - tnf - r1 pe (clone16803 ; igg1) (r&d systems, usa). cells of the healthy volunteer group were divided into only two parts, those without a modifier and those with infliximab. plates were incubated for 30 minutes in the dark on ice and were then centrifuged and the supernatant was discarded. after being washed with pbs, the cellular pellet was suspended in 70 l of annexin v binding buffer (bd biosciences, usa), supplemented with 3 l of annexin - fitc (bd biosciences, usa). after incubation the tubes were filled with 400 l of annexin v binding buffer and read on a cytometer. the measurements were performed using a lsrii flow cytometer (bd biosciences, usa) and equipped with a solid state coherent sapphire blue laser (20 mw, 488 nm), a solid state uv laser - light wave x - cyte laser (20 mw, 355 nm), and a helium - neon gas laser (17 mw, 633 nm). the expression of cell surface markers was assessed after gating on live cells determined by scatter characteristics. data were analyzed using the facs diva 6.0 software (becton dickinson, usa). the region containing monocytes was generated on the basis of their forward versus right - angle light scatters. the monocyte subsets were analyzed from dot plots representing cd14 versus cd16 staining. the threshold level for the fluorescence of positive cells was set for each sample from a difference between curves obtained from the specific mab and isotype control mab staining. for comparison of the skew - distributed variables the nonparametric kruskal - wallis anova test was applied. the significance level was set at p < 0.05 and two - sided tests were performed as the standard. by means of flow cytometry we identified two monocyte subsets in healthy volunteers and dm1 patients : the cd14cd16 and cd14cd16 cells (figure 1). the monocytes counts in the dm1 patients and the healthy group are compared in table 1. the absolute number of cd14cd16 monocytes was significantly lower in dm1 patients as compared with the healthy volunteers. the values of cd14cd16 monocytes showed an inverse trend being higher in the patients than in healthy controls. since defects of apoptosis in different tissues have been recognized as pathogenic in dm1, the next analysis was a comparison of the apoptotic potential of the dm1 patients ' monocytes and those of the healthy subjects. for this purpose the binding of anexinn v to phosphatidylserine on cell surface was used. the cells were stained for visualization of the cd14 and cd16 molecules and then with fluorochrome - conjugated annexin v. the number of apoptotic, annexin v - positive cells was calculated and is presented in table 2. the absolute values and percentages of annexin v binding cells in dm1 patients were found to be about twice that found in the healthy volunteers (table 2). this applied to both of the subsets examined. generally, about half of the cells of the patients ' monocytes bound to annexin v. there were no differences between subsets in this respect. in addition, we observed a significant gradual increase in the pool of cd16 and cd16 apoptotic cells with disease duration (figure 2). the tnf - r1 receptor is recognized as a main mediator of the tnf--induced apoptotic signal. in dm1 patients, however, defective apoptosis may be associated with an altered signal transduction through the tnf - r2 receptor. the next step was therefore for the factors blocking or augmenting tnf - r1 receptor expression to be added to the incubation medium, and apoptosis was assessed on cd16 and cd16 monocytes. it appeared that anti - tnf - r1 antagonistic mab significantly decreased the percentage of apoptotic cells only within the cd16 monocyte subset. sodium salicylate (ss), an irreversible inhibitor of cyclooxygenase (cox) activity, significantly reduced the percentage of cd16 apoptotic monocytes. anti - tnf - r2 antagonistic mab had no significant effect on the parameters examined (data are not presented here) (figure 3). bromophenacyl bromide (bpb), an agent potentiating apoptosis, increased the percentage of the apoptotic cells in both cd16 and cd16 monocyte subsets by more than 90%. our results indicate therefore that the tnf - r1 receptor transduces the apoptotic signal in the monocytes of dm1 patients. it is easy to manipulate the apoptotic ability of diabetic monocytes through tnf - r1 modifiers (figure 3). the next stage was to pose the question of whether infliximab, an approved anti - tnf drug, may also control the apoptotic potential of monocytes. the effect of infliximab was analyzed on the tnf - r1 apoptotic monocytes in the dm1 patients and the healthy controls. figure 4 shows gating strategy used for analysis of apoptotic tnf - r1 annexin v - positive monocytes. infliximab binds and neutralizes the soluble and transmembrane forms of tnf- and hinders its access to the tnf - r1 and tnf - r2 receptors. its effect on dm1 patients ' monocytes has not been known. in order to learn whether infliximab may influence the apoptotic cd16 and cd16 monocytes, peripheral blood mononuclear cells were incubated in a culture medium for 24 hours either without additives or with 50 l of infliximab. after incubation the mab against cd14, cd16, and tnf - r1 were applied together with annexin v for visualization of the tnf - r1 apoptotic monocytes. the effect of infliximab on the dm1 patients ' monocytes is presented in figure 5. infliximab reduced the percentage of cd16tnf - r1 apoptotic monocytes while having no effect on the corresponding values of the cd16tnf - r1 apoptotic cells (figure 5). in contrast to its effect on monocytes in the dm1 patients, infliximab did not reduce the percentage of the cd16tnf - r1 and cd16tnf - r1 apoptotic monocytes in the healthy control group (figure 6). we found that two monocyte subsets in dm1 patients exhibit an increased spontaneous apoptotic activity in vitro in comparison with those in the healthy group. both subsets showed this defect to the same degree. our results complement those already published on enhanced apoptosis affecting various cells and tissues in diabetic patients and diabetes type 1 model animals [38 ]. it seems therefore that enhanced apoptosis is a general phenomenon found in diabetes type 1. while it is obvious that apoptosis of different cells and tissues poses a serious risk of injury to the organs targeted and of functional failure in these organs, not much is known about the role of apoptotic monocytes in dm1. it can, however, be deduced that apoptotic monocytes cause serious deterioration of the target organs of late diabetic complications. apoptotic monocytes may directly stimulate endothelial cell activation and enhance adhesion of blood cells to endothelium. this study revealed that the tumor necrosis factor - related apoptosis - inducing ligand (trail)/apo2 protein, which is responsible for transduction of the tnf - dependent apoptotic signal, simultaneously generates enhanced expression of adhesion molecule icam-1 on endothelium in conjunction with enhanced expression of its ligand, the cd18 molecule, on blood cells. the (trail)/apo2 protein is able to accelerate apoptosis of blood cells, making them adhere firmly to the endothelial wall of small vessels. in this cascade, monocytes that are undergoing apoptosis may exacerbate the process of leukostasis in small renal and eye vessels, directly hindering blood flow and inducing endothelial ischemia. the phagocytic function of the monocytes, which are physiologically responsible for the removal of other apoptotic cells, is also impaired because they die of apoptosis themselves. at the end, trail induces migration of endothelial cell progenitors and, as a consequence, the formation of new vessel tubes, a prerequisite for retinopathy. greater apoptosis of monocytes thus favors damage to the small blood vessels and promotes angiogenesis in the target organs of microangiopathic diabetic complications. in order to determine which of the tnf- receptors initiate the apoptotic process, the technique of annexin v binding was used for enumeration of the apoptotic cells. this technique detects early apoptotic events and enables the tnf - r receptors to be identified on cells in initial phase of apoptosis. our results therefore indicate that the proapoptotic potential of a dm1 patient 's monocytes is dependent on the tnf - r1 pathway. in this paper, we presented that manipulation with drugs that inhibit tnf - r1 expression [3335 ] may diminish the pool of cd16 monocytes undergoing apoptosis, while cd16 monocytes appear nonreactive towards tnf - r1 inhibitors. on the other hand, augmentation of apoptosis by pbp reached similar values in both monocyte subsets. this indicates that tnf- modifiers may be useful for controlling excessive monocyte apoptosis in diabetic patients. a reduction in cd16 apoptotic monocytes may be achieved by means of such widely used tnf - r1 blockers as sodium salicylate. we have recently documented an expansion of the cd16 monocytes in juvenile - onset complication - free dm1 patients during a five - year observation period, during which the percentage and absolute number of the cd14cd16 monocytes doubled. the present study reveals that the expanded monocyte population is not fully functional, since about half entered the early phase of apoptosis. our previous study indicated that the size of the nonclassical monocyte subset correlated with the blood level of tnf-, suggesting that this cytokine plays an important role in the expansion of the cd16 monocyte pool. from experiments conducted in vitro with monocytes, when apoptosis starts the tnf - r1 receptors are still on the monocyte surface, but later the tnf--tnf - r1 complexes become internalized and the number of tnf - r1-positive monocytes diminishes. since an enlarged pool of apoptotic monocytes in dm1 patients increases the danger of leukostasis in the small vessels of the target organs of microangiopathy, it would be desirable to reduce the number of these cells in these patients. our results suggest that tnf - r1 inhibitors such as sodium salicylate and its derivatives may be useful for eliminating the cd16 apoptotic monocytes, while infliximab may be appropriate for the reduction of apoptotic cd16 cells. our experiment with sodium salicylate as tnf - r1-suppressing factor indicates that this widely used anti - inflammatory agent may confer benefits on diabetic patients. hitherto this substance has not been recommended in dm1 therapy. however, previously published results for nod mice indicate that sodium salicylate derivatives are effective in protection from retinopathy, which is in keeping with our data. infliximab has, to our knowledge, not been used in experimental therapy for dm1 patients. however, a recent clinical study indicates the beneficial effect of etanercept, the other general tnf - inhibitor, on the clinical manifestation of dm1. moreover, in clinical studies infliximab has been used successfully with patients with dm1 when this disease coexisted with other autoimmune diseases [40, 41 ]. in these studies, infliximab appeared to be beneficial for clinical remission of polymyalgia rheumatica as a complication of dm1 and for regression of diabetic sight - threatening macular edema. in animal studies, infliximab inhibited urinary albumin excretion in experimental diabetic rats and ameliorated diabetic neuropathy in mice. in our study it has been known to neutralize soluble and membrane tnf- and by this means moderates the degree of inflammation. infliximab causes the tnf - r2 receptors to be shed from monocytes and stimulates interleukin-10 secretion in monocyte cultures in vitro. this indicates that infliximab is directing the immune response towards the th2 profile, a condition required for the effective removal of different apoptotic cells. moreover, infliximab is able to inhibit inflammation - induced angiogenesis and selectively deplete immature blood vessels. its putative role in the prevention of microvascular diabetic complications should therefore be further clarified. it is important to realize that modification of tnf- activity in type 1 diabetes mellitus should be undertaken in complication - free phase of the disease. the study done by group of researches analyzing monocytes in patients with advanced diabetes type 1 and type 2 showed that in patients with late diabetic complications prevails the anti - inflammatory profile with an enhanced il-10 blood level and cd16 monocytes are at lower frequency as compared with patients without complications. this paper suggests also that the disappearance of cd16 monocytes from blood is associated with their transformation into macrophages. macrophages probably settle down in tissues and especially on endothelium and become insensitive to action of tnf- modifiers. in conclusion, diabetes mellitus type 1 is associated with greater apoptosis of monocyte subsets which may contribute to the development of microvascular complications. tnf - r1 modifiers, together with infliximab, appear to ameliorate monocyte apoptosis and should be further examined for their assistance in the treatment of diabetes mellitus as protective agents against late diabetic microvascular complications. they should join other experimental forms of treatment currently under consideration for preventing capillary leukostasis, such as treatment with statins, a ppar agonist, or anti - vegf agents.	diabetes mellitus type 1 is associated with an enhanced apoptosis of different cells and tissues, accelerating occurrence of diabetic microvascular complications. the aim of our study was to determine spontaneous apoptotic potential of the monocyte subsets in juvenile - onset complication - free diabetes mellitus type 1 and to compare them with the corresponding values of the healthy. moreover, we wanted to assess effects of tnf - r1 blocking agents and those of general tnf- blocker (infliximab) on spontaneous apoptosis of monocytes. sixty randomly selected dm1 patients (14.5 3.2 years) and 30 healthy (13.5 2.8 years) volunteers were enrolled in the study. our results indicate that three monocyte subsets are distinguishable in the groups of young diabetic patients and the healthy, similarly to in the blood of adults. dm1 patients were characterized by higher values of apoptotic monocytes than the healthy. the manipulation with drugs inhibiting tnf - r1 expression diminished the pool of cd16 + apoptotic monocytes. infliximab reduced the apoptotic cd16 cells. in conclusion, diabetes mellitus type 1 is associated with greater apoptosis of three monocyte subsets which may contribute to the development of microvascular complications. tnf- modifiers appear to ameliorate monocyte apoptosis. they may be useful for controlling excessive monocyte apoptosis in diabetic patients.
in order to understand how cells become disorganised in tumours it is vital to understand the key players and regulators that control and maintain cell polarity, which lead to epithelial tissue organisation. as expected, the molecular complexes involved in the establishment and maintenance of cell polarity are largely conserved throughout the metazoa, despite the wide range of epithelial tissue types and biological processes that require them. three major complexes involved in the regulation of apical basal cell polarity of epithelial cells have been described : the crumbs - pals1 (stardust)-patj - lin-7 (crumbs complex) and par3 (bazooka)-par6-apkc (par complex) complexes, which are found apically, and the lethal giant larvae (lgl)-scribble (scrib)-disc large (dlg) proteins (scribble complex) that localise at the basolateral membrane. both the par and crumbs complexes promote apical membrane identity, whereas the scribble complex promotes basolateral membrane identity by antagonising the other two (figure 1). among the three polarity complexes, the par complex is the best studied in the context of apical basal polarity in epithelial cells. classically, the proteins considered to be the core of the par complex include the two par pdz domain - containing proteins par3 and par6, with the addition of atypical protein kinase c (apkc), and the par complex has been shown to be required for the establishment and the maintenance of apical basal polarity and apical domain development in epithelial cells. phosphorylation has a key role in controlling polarity, and this is reflected by the fact that par1 and par4, homologous in mammals to microtubule affinity regulating kinases (marks) and lkb1, respectively, are serine / threonine kinases and par5 is a member of the 14 - 3 - 3 family of proteins that generally binds ser / thr phosphorylated proteins. par3 is phosphorylated on ser827 of its apkc - binding region by apkc itself, resulting in decreased affinity for apkc. rho kinase also prevents the interaction between par3 and apkc by phosphorylating thr883 of par3, thereby suppressing the activity of the par complex. the activity of the par complex is further regulated by the dynamic nature of par3 's association with the stable par6apkc complex. a compelling study in drosophila demonstrated that par3 is in fact excluded from the apical domain by the par6apkc complex. instead, the drosophila par3 homologue baz localised independently of apkc and par6 in the follicular epithelium, and below them at the level of the apical / lateral junction. this correlates with the observation that in many epithelial tissues, including in mammals, par3 and the par6apkc complex do not colocalise. however, in mammals, the apical / lateral domain is formed by tight junctions, which are more apical and distinct from the adherens junctions, thus par3 is essentially localised at the level of tight junctions where it colocalises with zonula occludens-1 (zo-1). this model for par3 exclusion from the apical domain involves both the par6apkc complex and the crumbs complex, in order to prevent the interaction between par3 and the par6apkc complex. on one hand, apkc phosphorylates par3 on ser827 in mammalian par3 to decrease their affinity for each other while, on the other hand, crumbs and stardust compete with par3 to interact with the same domain of par6 (figure 1). this exclusion mechanism is crucial to restrict the extent of the apical / lateral junction and define the border between the apical and lateral domains in drosophila epithelial cells. further investigation is required, but the existing evidence suggests that the observations outlined above may be generalised to epithelial tissues in mammals. the three members of the scribble complex have been shown to interact genetically, with dlg and scribble physically interacting through a protein called guk - holder in drosophila neuronal synapses. however, there is little evidence for their physical interaction in mammalian epithelial cells and, as a result, the term module ' is sometimes used when referring to the scribble complex. more recently, scribble and lgl2 have been reported to interact directly in polarised mammalian epithelial cells, although this interaction has not yet been reported in other experimental systems. lgl, by competing with par3 for par6apkc, restricts the par complex to the apical domain. furthermore, phosphorylation and inactivation of lgl at the apical domain by apkc restricts the par and scribble complex apically and basolaterally in epithelial cells, respectively. interestingly, in drosophila, apkc is also able to phosphorylate crumbs to promote the apical localisation of the crumbs complex. key phosphorylation events and protein / protein interactions therefore result in the exclusion of scribble from the apical domain, and of the crumbs and par complexes from the basolateral domain. these membrane domains consequently acquire unique identities that set the basis for the establishment of apical basal polarity in epithelial cells. asymmetric cell division is pivotal for the maintenance of epithelial tissue homeostasis. when a stem cell divides asymmetrically, it generates two daughter cells : one with an identical cell fate and the other with a different one. asymmetric cell division relies on the asymmetric distribution of cell fate determinants (numb, pros, brat, pon and mira) and, as a result, core polarity proteins and the correct orientation of mitotic spindles. emerging evidence, in particular from studies of neuroblasts in drosophila, indicates that asymmetric division functions as a mechanism of tumour suppression. when polarity genes or cell fate determinants are deleted or mutated, neuroblasts divide symmetrically, leading to tissue overgrowth and transplantable tumours that are similar to mammalian cancers (figure 2). for example, dlg / scrib / lgl mutant neuroblasts have defects in basal protein targeting, a reduced apical cortical domain and reduced apical spindle size, which is thought to lead to symmetric divisions and, as a result, to the accumulation of cells and the development of tumours. in drosophila, a number of proteins such as pins, mud and the mitotic kinases aurora - a and polo have been shown to have a major role in regulating mitotic spindle orientation and asymmetric cell division. intriguingly, adenomatous polyposis coli 2 (apc2) was discovered to be part of the centrosome complex of drosophila germline cells, where it functions in establishing the correct orientation of the mitotic spindles. moreover, deletion of both drosophila apc genes results in asymmetric stem cell division defects as a result of mitotic spindle misorientation. have developed an induced cortical polarity ' assay in drosophila s2 cells that should facilitate the identification of further proteins, domains and amino acids that regulate spindle orientation. in mammals, probably as a consequence of their redundancy, the function of core polarity proteins in the regulation of asymmetric cell division has been harder to elucidate. however, lgl1 knockout mice present some defects in the asymmetric division of neural progenitors, that may result in overproliferation and a lack of differentiation. recently, in a compelling study using in vivo electroporation of mouse embryos and cortical slice cultures, par3 has been shown to regulate the asymmetric division of neural progenitor cells via the control of the notch signalling pathway in the developing neocortex. this study has also shown that par3 is distributed and inherited asymmetrically as cells divide, suggesting that its subcellular distribution regulates the mode of progenitor cell division and daughter cell fate specification. the transition from an epithelial to mesenchymal phenotype that occurs during emt has been associated with metastatic progression ; apical basal polarity is lost during this process and cell cell junctions are weakened and disrupted. several lines of evidence suggest that core polarity proteins are important for the formation and maintenance of the ajc, suggesting that their loss could induce or at least contribute to emt (figure 3). for example, it has been shown that par3 depletion in mammalian epithelial cells disrupts the formation of tight junctions. moreover, it has been suggested that the par6apkc complex, together with cdc42, can control adherens junction remodelling through the regulation of arp2/3-dependent endocytosis. another example comes from knock - down experiments showing that patj is important for the proper polarisation of mammalian epithelial cells and the formation of tight junctions. sirna - mediated knockdown of scrib in sk - co15 cells inhibits tight junction reassembly. tgf, in cooperation with the ras pathway, is a key inducer of emt and promotes invasion and metastasis. several studies suggest that disrupting cell polarity may work in concert with tgf signalling or facilitate tgf-mediated emt. for instance, it has recently been shown that disruption of the crumbs complex predisposes to emt in eph4 cells, a cell line normally insensitive to tgf-mediated emt. another study shows that tgf signalling is able to downregulate the expression of par-3 through the induction of mir-491 - 5p in rat proximal tubular epithelial cells, suggesting that tgf may induce the disruption of cell polarity. probably, one of the most direct links between the disruption of cell polarity and emt comes from the finding that tgf receptors directly interact with par6 at the level of adherens junctions. tgf is able to induce phosphorylation of par6 at ser345, enabling the recruitment of the e3 ubiquitin ligase smurf1, which is required for localised tgf-induced degradation of the actin regulator rhoa. importantly, this phosphorylation event is required for tgf-dependent emt in mammary gland epithelial cells, as mutation of this residue blocks emt and tight junction disruption. furthermore, in a mammary fat pad assay, over - expression of par6/s345a in emt-6 cells reduced their ability to induce metastasis to the lung, further demonstrating the importance of tgf/par6 signalling in emt and the development of metastasis. taken together, these studies show that polarity proteins are important emt regulators in epithelial cells. underlying their potential role as tumour suppressors, core polarity proteins are often targeted and disrupted by oncogenic signalling. for example, scrib - deficient mutants cooperate with oncogenes to mediate transformation in drosophila. normally, scrib - deficient mutant clones in the eye imaginal discs are eliminated by jnk - dependent apoptosis. however, in the presence of activated oncogenic pathways such as ras or notch, apoptosis is inhibited and neoplastic tumours occur. in addition, in support of these data, loss of hscrib has been shown to cooperate with h - ras to promote cell invasion through deregulation of mapk signalling in an organotypic culture system. a number of viral oncogenes have been found to directly interact with polarity proteins, suggesting that disruption of polarity is important. for example, the human t - cell leukemia virus type 1 tax protein binds to hscrib and alters its subcellular localisation in infected t - cells. in addition to scrib, tax and the oncoprotein 9orf1 have been shown to interact with dlg. the interaction of tax and hdlg was shown to affect the function of hdlg in controlling cell growth, and tax disrupted the interaction between hdlg and apc. however, it remains to be shown whether disruption of the hdlg / apc complex is the critical event that leads to deregulated cell growth. e6 oncogenes in human papilloma virus (hpv) have also been found to interact with pdz - containing polarity proteins. for example, e6 proteins from hpv-16 and hpv-18 have the ability to interact with, and induce the proteasomal degradation of, dlg and hscrib. intriguingly, e6 has also been shown to target phosphorylated forms of dlg, and patj of the crumbs polarity complex, for degradation. these findings suggest that key pdz - containing polarity proteins are common cellular targets of viral oncoproteins. moreover, it seems that viral proteins have evolved additional ways to target cell polarity, strongly suggesting that its disruption is necessary for malignant transformation. in addition to viral oncogenes, core cell polarity mechanisms are targeted by abnormally activated growth factor signalling pathways. both the erbb2 and tgf signalling pathways have been shown to be directly involved in the regulation of polarity independently of their transcriptional response. in conclusion, many activated oncogenic pathways target and disrupt epithelial polarity in order to achieve malignant transformation, supporting the importance of cell polarity in suppressing tumour formation. scrib, dlg and lgl were identified as tumour suppressors in drosophila, in screens for mutations causing cancerous overgrowth of the larval imaginal discs and brain. interestingly, the phenotypes of these mutants can be rescued by the mammalian homologues of these genes, showing that they are functionally conserved and suggesting that they may have a tumour suppressive function in human cells. a growing amount of data showing mislocalisation, decreased expression or complete loss of the products of these genes in primary tumours from human patients further indicate their involvement in mammalian tumourigenesis. over the past few years, there has been an increasing body of evidence that the deregulation of these core polarity complexes, both in terms of expression level and subcellular localisation, may have a causal link to disease and cancer in particular (for an exhaustive list of changes in expression and localisation of polarity proteins in carcinoma cell lines and primary tissues see huang and muthuswamy). perhaps the best - studied polarity complex in human tumours is the scrib / lgl / dlg complex. in particular, the deregulation of scrib has been reported to promote the transformation of mammary epithelial cells in vitro and in vivo, by disrupting morphogenesis and cell polarity and by inhibiting myc - induced apoptosis, thus providing novel insight into how core polarity proteins regulate cell transformation. in this study, loss of scrib was shown to cooperate with myc to induce mammary tumours, correlating with a drosophila study that showed that lgl requires myc to promote clonal malignancy. in human keratinocytes, scrib loss has been shown to result in erk activation which might contribute to cancer progression. scrib was found to be mislocalised or downregulated in several cancer types including cervical, colon adenocarcinoma and endometrial. scrib, as well as dlg1 and lgl1, was also mislocalised and downregulated in a transgenic mouse model of cancer. in a screen of 60 tumour samples, hugl-1 (the human homologue of lgl) transcripts were often reduced or lost in tumour tissues of the breast (76%), prostate (53%), lung (63%), ovarian (50%) and colon carcinoma (75%). however, the numbers in this study are relatively small and it will be important to verify these data on a larger scale. hugl-1 was lost in 75% of tumour samples in a cohort of 94 patients undergoing surgery for colorectal cancer, and was associated with advanced stage and lymph node metastases. loss or downregulation of hugl-1 expression was also observed in malignant melanoma and associated with an advanced stage of the disease. a study of 80 hepatocellular carcinomas showed that hugl-1 mrna is frequently mutated by aberrant splicing, and that two of the variants were able to promote hepatocellular carcinoma in nude mice. dlg expression levels and localisation have also been shown to be affected in high - grade premalignant cervical neoplasia, invasive squamous cell carcinoma and colon adenocarcinoma. the deregulation of core polarity proteins in cancer is not limited to scrib, dlg and lgl, however, as almost every protein involved in the core apical basal polarity machinery of epithelial cells has been shown to be affected in some way. for example, the par3 gene is deleted in 15% of primary oesophageal squamous cell carcinomas and downregulated in a number of tumour tissues. a recent study identified homozygous intragenic microdeletions, involving genes encoding components of the core polarity complexes in a genome - wide screen of 684 cell lines. interestingly, among these genes, par3 was found to be the most commonly targeted and was disrupted in both cell lines and some primary tumours. this study will undoubtedly be complemented by data from ongoing cancer genome sequencing efforts such as the cancer genome atlas. taken together, these data strongly underline the causal link between the deregulation of core polarity proteins and human cancer. over the past decade, a number of tumour suppressor pathways have been directly linked to epithelial cell polarity, suggesting that the integrity of apical basal polarity is crucial for the prevention of tumour development. although lkb1 deficiency does not cause a gross defect in cell polarity in the intestine, for example, it may regulate cell polarity through its ability to phosphorylate members of the ampk - related kinase (ark) family in other tissues. hence, par4/lkb1 is able to activate par1/mark in the drosophila oocyte and, in epithelial cells, may have its role through par1/mark activation. alternatively, par4/lkb1 may regulate polarity through the activation of ampk (amp - dependent activated protein kinase), which in turn activates myosin ii. among all of the par genes identified in model organisms, par4/lkb1 is the most well - established tumour suppressor. all of these factors suggest that regulators of cell polarity may have an important role in suppressing tumours. in agreement with this, for example, the tumour suppressor von hippel - lindau exerts its regulation on polarity at several levels : it is able to directly interact with apkc and mediate its ubiquitination and subsequent degradation, and its interaction with the par complex is involved in the regulation of polarised microtubule growth and formation of primary cilia. another tumour suppressor that regulates cell polarity is phosphatase and tensin homolog (pten), which is likely to be involved in different aspects of epithelial cell polarity. however, as apical accumulation of phosphatidylinositol 4,5 biphosphate is dependent on apical targeting of pten, and as membrane targeting of par3 is mediated by direct binding to phosphoinositide lipids, pten may be instrumental in the apical localisation of par3. for example, hdlg can interact with the apc tumour suppressor, and their interaction negatively regulates cell cycle progression. apc was also found to interact with hscrib and it has been suggested that scrib controls its localisation at the level of the adherens junctions in epithelial cells. recently, apical basal polarity in epithelial cells has been linked to another tumour suppressor, with the discovery that aspp2 is a new binding partner of par3. aspp2 is critical for the localisation of par3 at the level of tight junctions in epithelial cells and, therefore, has a crucial role in the establishment and maintenance of apical basal polarity in epithelial cells in culture. in vivo, aspp2 deficiency results in defects arising during the development of the central nervous system, characterised by a loss of apical basal polarity and an expansion of neural progenitor cells. aspp2 colocalises with par3 at the level of tight junctions in a variety of epithelial cells and tissues, suggesting that its role in controlling apical aspp2 is a transcriptional target of e2f1 and was first identified as a p53 regulator that specifically promotes its apoptotic function. tumour studies in mice have identified aspp2 as a haploinsufficient tumour suppressor gene, and aspp2 levels have been shown to be deregulated in human tumours and tumour cell lines, suggesting that its tumour suppressive role is conserved in humans. studies in drosophila have shown that daspp, the unique aspp protein in drosophila, localises at adherens junctions. these studies suggest that daspp regulates retinal morphogenesis by acting in concert with drassf8 to promote dcsk activity, thus, the function of aspp2 in the establishment and maintenance of apical basal polarity in epithelial cells appears to have been conserved from drosophila to humans. basal polarity is linked to its ability to regulate p53 and its family members, p63 and p73. one possibility is that aspp2, upon external stimuli, may be able to shuttle from tight junctions to the nucleus to have its transcriptional role, in a similar way to other junctional proteins such as -catenin or zo-1. therefore, it will be of great interest to investigate the extent to which aspp2 's regulation of the localisation of par3 and the apical basal polarity of epithelial cells contributes to its tumour suppressive function, and how this interplays with its role in regulating p53, the most mutated tumour suppressor in human cancers, and p63, a key regulator of epithelial stratification. there are a number of studies that also suggest that mitotic spindle orientation is crucial for asymmetric cell division in mammals. for instance, p63 has been shown to be important for mitotic spindle orientation during asymmetric cell division of epidermal stem cells. finally, emerging evidence suggests that there is also a direct relationship between loss of cell polarity of stem cells and asymmetric division and tumour initiation in mammals. for example, the tumour suppressor p53 has recently been linked with the regulation of asymmetric divisions of mammary stem cells. in the future, due to their redundancy in mammals, the real challenge will be to investigate whether core polarity proteins regulate asymmetric stem cell division in mammalian epithelia, and whether their deregulation consequently drives tumour initiation as a result of asymmetric stem cell division defects. taken together, these studies emphasise the role and importance of known tumour suppressors in the control of epithelial cell polarity. many of those tumour suppressors regulate the functions of core polarity proteins through direct interactions suggesting that, in addition to their better - known roles in the control of cellular proliferation, these roles are crucial for the prevention of tumour development. underlying their potential role as tumour suppressors, core polarity proteins are often targeted and disrupted by oncogenic signalling. for example, scrib - deficient mutants cooperate with oncogenes to mediate transformation in drosophila. normally, scrib - deficient mutant clones in the eye imaginal discs are eliminated by jnk - dependent apoptosis. however, in the presence of activated oncogenic pathways such as ras or notch, apoptosis is inhibited and neoplastic tumours occur. in addition, in support of these data, loss of hscrib has been shown to cooperate with h - ras to promote cell invasion through deregulation of mapk signalling in an organotypic culture system. a number of viral oncogenes have been found to directly interact with polarity proteins, suggesting that disruption of polarity is important. for example, the human t - cell leukemia virus type 1 tax protein binds to hscrib and alters its subcellular localisation in infected t - cells. in addition to scrib, tax and the oncoprotein 9orf1 have been shown to interact with dlg. the interaction of tax and hdlg was shown to affect the function of hdlg in controlling cell growth, and tax disrupted the interaction between hdlg and apc. however, it remains to be shown whether disruption of the hdlg / apc complex is the critical event that leads to deregulated cell growth. e6 oncogenes in human papilloma virus (hpv) have also been found to interact with pdz - containing polarity proteins. for example, e6 proteins from hpv-16 and hpv-18 have the ability to interact with, and induce the proteasomal degradation of, dlg and hscrib. intriguingly, e6 has also been shown to target phosphorylated forms of dlg, and patj of the crumbs polarity complex, for degradation. these findings suggest that key pdz - containing polarity proteins are common cellular targets of viral oncoproteins. moreover, it seems that viral proteins have evolved additional ways to target cell polarity, strongly suggesting that its disruption is necessary for malignant transformation. in addition to viral oncogenes, core cell polarity mechanisms are targeted by abnormally activated growth factor signalling pathways. both the erbb2 and tgf signalling pathways have been shown to be directly involved in the regulation of polarity independently of their transcriptional response. in conclusion, many activated oncogenic pathways target and disrupt epithelial polarity in order to achieve malignant transformation, supporting the importance of cell polarity in suppressing tumour formation. scrib, dlg and lgl were identified as tumour suppressors in drosophila, in screens for mutations causing cancerous overgrowth of the larval imaginal discs and brain. interestingly, the phenotypes of these mutants can be rescued by the mammalian homologues of these genes, showing that they are functionally conserved and suggesting that they may have a tumour suppressive function in human cells. a growing amount of data showing mislocalisation, decreased expression or complete loss of the products of these genes in primary tumours from human patients further indicate their involvement in mammalian tumourigenesis. over the past few years, there has been an increasing body of evidence that the deregulation of these core polarity complexes, both in terms of expression level and subcellular localisation, may have a causal link to disease and cancer in particular (for an exhaustive list of changes in expression and localisation of polarity proteins in carcinoma cell lines and primary tissues see huang and muthuswamy). perhaps the best - studied polarity complex in human tumours is the scrib / lgl / dlg complex. in particular, the deregulation of scrib has been reported to promote the transformation of mammary epithelial cells in vitro and in vivo, by disrupting morphogenesis and cell polarity and by inhibiting myc - induced apoptosis, thus providing novel insight into how core polarity proteins regulate cell transformation. in this study, loss of scrib was shown to cooperate with myc to induce mammary tumours, correlating with a drosophila study that showed that lgl requires myc to promote clonal malignancy. in human keratinocytes, scrib loss has been shown to result in erk activation which might contribute to cancer progression. scrib was found to be mislocalised or downregulated in several cancer types including cervical, colon adenocarcinoma and endometrial. scrib, as well as dlg1 and lgl1, was also mislocalised and downregulated in a transgenic mouse model of cancer. in a screen of 60 tumour samples, hugl-1 (the human homologue of lgl) transcripts were often reduced or lost in tumour tissues of the breast (76%), prostate (53%), lung (63%), ovarian (50%) and colon carcinoma (75%). however, the numbers in this study are relatively small and it will be important to verify these data on a larger scale. hugl-1 was lost in 75% of tumour samples in a cohort of 94 patients undergoing surgery for colorectal cancer, and was associated with advanced stage and lymph node metastases. loss or downregulation of hugl-1 expression was also observed in malignant melanoma and associated with an advanced stage of the disease. a study of 80 hepatocellular carcinomas showed that hugl-1 mrna is frequently mutated by aberrant splicing, and that two of the variants were able to promote hepatocellular carcinoma in nude mice. dlg expression levels and localisation have also been shown to be affected in high - grade premalignant cervical neoplasia, invasive squamous cell carcinoma and colon adenocarcinoma. the deregulation of core polarity proteins in cancer is not limited to scrib, dlg and lgl, however, as almost every protein involved in the core apical basal polarity machinery of epithelial cells has been shown to be affected in some way. for example, the par3 gene is deleted in 15% of primary oesophageal squamous cell carcinomas and downregulated in a number of tumour tissues. a recent study identified homozygous intragenic microdeletions, involving genes encoding components of the core polarity complexes in a genome - wide screen of 684 cell lines. interestingly, among these genes, par3 was found to be the most commonly targeted and was disrupted in both cell lines and some primary tumours. this study will undoubtedly be complemented by data from ongoing cancer genome sequencing efforts such as the cancer genome atlas. for example, deregulation of par6 was observed in er - positive breast tumours. taken together, these data strongly underline the causal link between the deregulation of core polarity proteins and human cancer. over the past decade, a number of tumour suppressor pathways have been directly linked to epithelial cell polarity, suggesting that the integrity of apical basal polarity is crucial for the prevention of tumour development. although lkb1 deficiency does not cause a gross defect in cell polarity in the intestine, for example, it may regulate cell polarity through its ability to phosphorylate members of the ampk - related kinase (ark) family in other tissues. hence, par4/lkb1 is able to activate par1/mark in the drosophila oocyte and, in epithelial cells, may have its role through par1/mark activation. alternatively, par4/lkb1 may regulate polarity through the activation of ampk (amp - dependent activated protein kinase), which in turn activates myosin ii. among all of the par genes identified in model organisms, all of these factors suggest that regulators of cell polarity may have an important role in suppressing tumours. in agreement with this, for example, the tumour suppressor von hippel - lindau exerts its regulation on polarity at several levels : it is able to directly interact with apkc and mediate its ubiquitination and subsequent degradation, and its interaction with the par complex is involved in the regulation of polarised microtubule growth and formation of primary cilia. another tumour suppressor that regulates cell polarity is phosphatase and tensin homolog (pten), which is likely to be involved in different aspects of epithelial cell polarity. however, as apical accumulation of phosphatidylinositol 4,5 biphosphate is dependent on apical targeting of pten, and as membrane targeting of par3 is mediated by direct binding to phosphoinositide lipids, pten may be instrumental in the apical localisation of par3. for example, hdlg can interact with the apc tumour suppressor, and their interaction negatively regulates cell cycle progression. apc was also found to interact with hscrib and it has been suggested that scrib controls its localisation at the level of the adherens junctions in epithelial cells. recently, apical basal polarity in epithelial cells has been linked to another tumour suppressor, with the discovery that aspp2 is a new binding partner of par3. aspp2 is critical for the localisation of par3 at the level of tight junctions in epithelial cells and, therefore, has a crucial role in the establishment and maintenance of apical basal polarity in epithelial cells in culture. in vivo, aspp2 deficiency results in defects arising during the development of the central nervous system, characterised by a loss of apical basal polarity and an expansion of neural progenitor cells. aspp2 colocalises with par3 at the level of tight junctions in a variety of epithelial cells and tissues, suggesting that its role in controlling apical aspp2 is a transcriptional target of e2f1 and was first identified as a p53 regulator that specifically promotes its apoptotic function. tumour studies in mice have identified aspp2 as a haploinsufficient tumour suppressor gene, and aspp2 levels have been shown to be deregulated in human tumours and tumour cell lines, suggesting that its tumour suppressive role is conserved in humans. studies in drosophila have shown that daspp, the unique aspp protein in drosophila, localises at adherens junctions. these studies suggest that daspp regulates retinal morphogenesis by acting in concert with drassf8 to promote dcsk activity, thus, the function of aspp2 in the establishment and maintenance of apical basal polarity in epithelial cells appears to have been conserved from drosophila to humans. however, it remains unclear whether the function of aspp2 in regulating apical basal polarity is linked to its ability to regulate p53 and its family members, p63 and p73. one possibility is that aspp2, upon external stimuli, may be able to shuttle from tight junctions to the nucleus to have its transcriptional role, in a similar way to other junctional proteins such as -catenin or zo-1. therefore, it will be of great interest to investigate the extent to which aspp2 's regulation of the localisation of par3 and the apical basal polarity of epithelial cells contributes to its tumour suppressive function, and how this interplays with its role in regulating p53, the most mutated tumour suppressor in human cancers, and p63, a key regulator of epithelial stratification. there are a number of studies that also suggest that mitotic spindle orientation is crucial for asymmetric cell division in mammals. for instance, p63 has been shown to be important for mitotic spindle orientation during asymmetric cell division of epidermal stem cells. finally, emerging evidence suggests that there is also a direct relationship between loss of cell polarity of stem cells and asymmetric division and tumour initiation in mammals. for example, the tumour suppressor p53 has recently been linked with the regulation of asymmetric divisions of mammary stem cells. in the future, due to their redundancy in mammals, the real challenge will be to investigate whether core polarity proteins regulate asymmetric stem cell division in mammalian epithelia, and whether their deregulation consequently drives tumour initiation as a result of asymmetric stem cell division defects. taken together, these studies emphasise the role and importance of known tumour suppressors in the control of epithelial cell polarity. many of those tumour suppressors regulate the functions of core polarity proteins through direct interactions suggesting that, in addition to their better - known roles in the control of cellular proliferation, these roles are crucial for the prevention of tumour development. in this review, we have highlighted how epithelial cell polarity may contribute to tumour suppression (figure 4), through its role in controlling asymmetric cell division and the integrity of the ajc. loss of cell polarity is a hallmark of cancer, however, studies in transgenic mouse models have so far been unable to clearly answer the question of whether core polarity proteins are tumour suppressors or not. future studies using multiple and/or conditional knockout mouse models will be essential to finally demonstrate their direct role in tumour suppression. due to the vital importance of core polarity proteins in maintaining tissue homeostasis, redundancy mechanisms have evolved in mammals that may make this issue too complex to address. nonetheless, it is emerging that an increasing number of well - known tumour suppressors have a pivotal role in regulating cell polarity.	the correct establishment and maintenance of cell polarity are crucial for normal cell physiology and tissue homeostasis. conversely, loss of cell polarity, tissue disorganisation and excessive cell growth are hallmarks of cancer. in this review, we focus on identifying the stages of tumoural development that are affected by the loss or deregulation of epithelial cell polarity. asymmetric division has recently emerged as a major regulatory mechanism that controls stem cell numbers and differentiation. links between cell polarity and asymmetric cell division in the context of cancer will be examined. apical basal polarity and cell cell adhesion are tightly interconnected. hence, how loss of cell polarity in epithelial cells may promote epithelial mesenchymal transition and metastasis will also be discussed. altogether, we present the argument that loss of epithelial cell polarity may have an important role in both the initiation of tumourigenesis and in later stages of tumour development, favouring the progression of tumours from benign to malignancy.
the life cycle of toxocara canis, a common round worm residing in the small intestine of dogs, is complex. infection of dogs occurs by ingestion of eggs that are passed in the feces and contained in soil. the ingested embryonated eggs hatch in the stomach and small intestine, and the larvae liberate. the larvae invade the intestinal mucosa, enter into the mesenteric and portal veins, reach the liver, and then pass to the lungs. from the lungs, some larvae pass through the bronchioles, trachea, and pharynx, and are swallowed and develop into adults in the small intestine. other larvae, from the lungs, are carried to the heart and are distributed throughout the body by systemic circulation, mainly to the lungs, liver, kidneys, and muscles. the larvae can be transferred to pups through transplacental circulation or transmammary passage through milk during lactation [1 - 4 ]. infection to other animals takes place by ingestion of egg - contaminated soil in dooryards and parks. the infected larvae, which are 0.5 mm long, reach the liver, may become encapsulated, and dormant in the liver parenchyma, and move slowly from place to place (i.e., visceral larva migrans), or may migrate to the lungs and may continue to be distributed in other tissues. these arrested larvae in animal tissues do not grow into an adult ascarid, but are capable of transmitting to other animals that eat the infected tissue harboring the encapsulated larvae. this mode of transmission among carnivous vertebrates has been experimentally established [4 - 8 ]. if these animals form a part of the food chain of the definitive host, i.e., dogs and cats, the life cycle of the parasite is completed. a large variety of non - canid animals may be infected. for t. canis, the known paratenic hosts include mice, rats, chickens, pigeons, lambs, pigs, and cows. thus, animals are infected by ingestion of embryonated eggs in contaminated soil or by ingestion of encapsulated larvae in the tissues of paratenic hosts through cannibalism. as in animals, human infection occurs in 2 ways ; by ingestion of embryonated eggs or, alternatively, by transfer of the encapsulated larvae of t. canis in the tissues of a paratenic host to humans. in certain ethnic groups, some adults tend to eat uncooked animal tissues that contain encapsulated infective larvae. after swallowing, the encapsulated larvae hatch in the small intestine, liberate and penetrate the intestinal wall, get into the portal vein and reach the liver and lungs, and again encapsulate and remain alive for a certain period. uncooked livers of cows [8 - 11 ], pigs, lambs, and chickens have been reported as the sources of human infections. a recent retrospective study with korean patients with peripheral blood eosinophilia showed that a recent history of eating raw cow liver was related to an increased risk of toxocariasis. however, epidemiological data on the role of ingestion of raw cow liver in the transmission of t. canis from animals to humans is limited, and there has been no explicitly designed study. in order to assess the role of eating raw cow liver for human infection of t. canis, we performed a cross - sectional study. since the goal of this study was to assess the role of ingestion of raw cow liver in toxocariasis in the general population, we enrolled a population of healthy adults who visited a health - screening center from january to december 2009. the interviewer asked for voluntary individual participation in this study and the subjects were provided with a questionnaire and a document for informed consent. the subjects were told of the possibility of t. canis infection through ingestion of uncooked animal tissues or breeding dogs in the house or garden. there were 122 men and 28 women (age range, 25 - 71 years ; median age, 50 years ; the institutional review board approved this study and the informed consent was obtained from each patient. at a face - to - face interview, a trained nurse interviewer used a structured questionnaire to collect data concerning social characteristics of the subjects. the data included the history of ingestion of raw cow liver or raw liver of other animals, the history of ingestion of raw cow meat or of raw meat of other animals, the history of ingestion of raw blood of animals, and the history of ingestion of raw freshwater fish. the data also included the time and frequency of ingestion, the number of occasions and the amount consumed, the species of animals, and any history of keeping a dog in the house or garden. the study coordinator and medical doctors made every efforts to keep the interviewer blind to the clinical diagnosis of the participants. after a thorough review of the questionnaire filled - in by the interviewer, an experienced physician without knowledge of any other information concerning the participants determined whether the subject had a significant history of eating raw tissues of animals or freshwater fish. the subject was considered to have a significant history of recent ingestion of raw tissues if the subject of the following criteria : 1) definite experience of ingestion of raw tissues of animals or fish, 2) the amount consumed was more than a single mouthful, and 3) the time of ingestion within 1 year. an elisa kit (bordier affinity products sa, crissier, switzerland) was used for the diagnosis of human toxocariasis through detection of human igg antibodies to toxocara excretory / secretory (e / s) antigens. although some cross reactions may occur by other human helminthiases, such as trichinosis, fascioliasis, and strongyloidiasis, the titers in these helminthiases are lower than the titers of positive control serum samples of individuals infected with t. canis. all individuals underwent serological (elisa) tests for common parasites (clonorchis sinensis, paragonimus westermani, sparganosis, and cysticercosis) in our country. we evaluated the relationship of adults with positive results of toxocara elisa and the history of recent ingestion of raw cow liver or raw liver of other animals, raw meat of animals, or raw blood of animals, a recent history of eating raw freshwater fish, a history of keeping dogs, and the serologic test results of other parasites with the use of multivariate statistical analysis. odds ratios (or) of results of toxocara elisa, together with the corresponding 95% confidence intervals (ci), in relation to 7 variables, were derived using multiple logistic regression analysis. a p - value of < 0.05 was considered significant. data analyses were performed with a commercially available software program (pasw statistics, release 17.0.2 ; spss, chicago, illinois, usa). since the goal of this study was to assess the role of ingestion of raw cow liver in toxocariasis in the general population, we enrolled a population of healthy adults who visited a health - screening center from january to december 2009. the interviewer asked for voluntary individual participation in this study and the subjects were provided with a questionnaire and a document for informed consent. the subjects were told of the possibility of t. canis infection through ingestion of uncooked animal tissues or breeding dogs in the house or garden. there were 122 men and 28 women (age range, 25 - 71 years ; median age, 50 years ; the institutional review board approved this study and the informed consent was obtained from each patient. at a face - to - face interview, a trained nurse interviewer used a structured questionnaire to collect data concerning social characteristics of the subjects. the data included the history of ingestion of raw cow liver or raw liver of other animals, the history of ingestion of raw cow meat or of raw meat of other animals, the history of ingestion of raw blood of animals, and the history of ingestion of raw freshwater fish. the data also included the time and frequency of ingestion, the number of occasions and the amount consumed, the species of animals, and any history of keeping a dog in the house or garden. the study coordinator and medical doctors made every efforts to keep the interviewer blind to the clinical diagnosis of the participants. after a thorough review of the questionnaire filled - in by the interviewer, an experienced physician without knowledge of any other information concerning the participants determined whether the subject had a significant history of eating raw tissues of animals or freshwater fish. the subject was considered to have a significant history of recent ingestion of raw tissues if the subject of the following criteria : 1) definite experience of ingestion of raw tissues of animals or fish, 2) the amount consumed was more than a single mouthful, and 3) the time of ingestion within 1 year. an elisa kit (bordier affinity products sa, crissier, switzerland) was used for the diagnosis of human toxocariasis through detection of human igg antibodies to toxocara excretory / secretory (e / s) antigens. although some cross reactions may occur by other human helminthiases, such as trichinosis, fascioliasis, and strongyloidiasis, the titers in these helminthiases are lower than the titers of positive control serum samples of individuals infected with t. canis. all individuals underwent serological (elisa) tests for common parasites (clonorchis sinensis, paragonimus westermani, sparganosis, and cysticercosis) in our country. we evaluated the relationship of adults with positive results of toxocara elisa and the history of recent ingestion of raw cow liver or raw liver of other animals, raw meat of animals, or raw blood of animals, a recent history of eating raw freshwater fish, a history of keeping dogs, and the serologic test results of other parasites with the use of multivariate statistical analysis. odds ratios (or) of results of toxocara elisa, together with the corresponding 95% confidence intervals (ci), in relation to 7 variables, were derived using multiple logistic regression analysis. a p - value of < 0.05 was considered significant. data analyses were performed with a commercially available software program (pasw statistics, release 17.0.2 ; spss, chicago, illinois, usa). sixty - eight (79.1%) of 86 seropositive adults for t. canis had a recent history of ingestion of raw cow liver within 1 year, while 29 (45.3%) of 64 seronegative adults had a history of raw cow liver ingestion. ten adults ate raw livers of animals other than cows, such as pigs (n=7), dogs (n=3), goats (n=2), chickens (n=2), ducks (n=1), or geese (n=1). table 1 presents the distribution of the 86 seropositive adults and 64 negative adults for the toxocara elisa. according to multivariate analyses, a recent history of ingestion of raw cow liver was related to an increased risk of toxocariasis (or, 4.4 ; 95% ci, 1.9 - 10.2 ; p=0.001). a history of keeping dogs also showed an increased risk of toxocariasis (or, 3.7 ; 95% ci, 1.2 - 11.6 ; p=0.022). a recent history of ingestion of raw liver of other animals, raw meat of animals, raw blood of animals, raw freshwater fish, and the serologic results for other parasites were not related with the increased risk of toxocariasis. chops of raw cow liver is one of the popular dishes in korea, served in meat restaurants or buffet restaurants, sometimes along with chops of raw cow meat. chops of raw liver and meat are served together with the usual cooked or barbecued meats. some people believe that raw liver or raw meat is good for health, particularly the raw liver for eyesight. in our study population, about 2/3 (64.7%, 97 of 150) of adults had an experience of ingestion of raw cow liver. in the korean society, some people eat chops of raw cow liver frequently when they dine at meat restaurants. not infrequently, raw livers of chickens, goats, pigs, ducks, geese, or rabbits are also consumed. usually, these people are checked by clinicians because of eosinophilia or sometimes because of incidentally found small nodular lesions in the liver and/or lungs as seen on radiological imaging, such as sonography or ct. the results of our study suggest that ingestion of raw cow liver substantially increases the risk for infection with t. canis. the seropositive rate for t. canis in adults that ate raw cow liver (70.1%, 68 of 97) was more than 2 times higher than in adults that did not consume raw cow liver (34.0%, 18 of 53). in a retrospective study with korean patients with peripheral blood eosinophilia, 87.5% of seropositive patients had a recent history of eating raw cow liver, and 25.0% of seronegative patients had this history. in 20 individuals that ate raw cow liver frequently (4 times or more per year), the seropositive rate was 95.0% (19 of 20). in 35 individuals that ate raw cow liver within 6 months prior to the interview, the seropositive rate was 94.3% (33 of 35). it appeared that the seropositive rate was higher in people with a history of ingestion of raw cow liver frequently and recently. in spite of being seropositive for t. canis, 18 (20.9%) of 86 individuals denied a history of ingestion of cow livers. ingestion of raw livers of pigs, lambs, and chickens have been reported as routes of human infections. we attempted to establish a role of eating habits of raw animal livers other than cows, such as goats, chickens, pigs, ducks, or geese for t. canis infection. there were 10 seropositive individuals with a history of consuming raw livers of other animals, but they had a history of ingestion of cow livers as well. therefore, we could not verify the relation between ingestion of raw livers of other animals and toxocariasis. five of 18 subjects that denied a history of ingestion of cow livers were keeping dogs ; 2 dogs lived in the house and the other 3 dogs lived in gardens. infection might take place by ingestion of t. canis eggs that were spread by the dogs. in our study, the history of keeping dogs was statistically significant with t. canis infection. however, a retrospective study with korean patients with peripheral blood eosinophilia could not show that the history of keeping dogs was related to an increased risk of toxocariasis. in another 7 subjects who were seropositive without a history of raw cow liver ingestion, infection might have occurred by ingestion of raw cow meat. there have been several case reports describing toxocara infection that probably took place by ingestion of raw meat of animals. in animal experiments, toxocara larvae were recovered in the lungs, liver, kidneys, brain, and muscles. suggested the possibility of zoonotic risk of t. canis infection through ingestion of animal meat. in our study, many adults (84.5%, 82 of 97) that ate raw cow liver also ate raw cow meat. therefore, we could not make a differentiation of subjects infected with toxocara through ingestion of raw cow liver from subjects infected through ingestion of raw cow meat., such as t. cati. also, there may be cross reactions with other helminthiasis, such as trichinosis, fascioliasis, and strongyloidiasis. the rate has been reported as 18% in a rural area in china, 20% in malaysia, 26% in iran, 6 - 36% in the czech republic, 2 - 5% and 14 - 37% in urban and rural areas of the midi - pyrenees area in france, 5% in switzerland, and 5% in a rural area in korea. as dogs and cats are popular pets, there is widespread contamination of the environment with infective stage eggs and, therefore, toxocariasis is a worldwide disease, irrespective of developed or developing countries. in a total of 150 subjects enrolled in this study, 86 (57.3%, 86 of 150) considering the seroprevalence rate in a rural population in korea (5%), the seropositive rate in this study was very high. higher seroprevalences have been reported in indonesia (68%) and nepal (81%). fan. assumed that the high seroprevalence among healthy adults of taiwanese aboriginal populations (46%) was due to the habit of eating raw liver of wild boar. the population enrolled in this study were healthy subjects who voluntarily visited the clinic for health screening. therefore, these people were relatively wealthy and they dine at meat restaurants more frequently than other people, and they might have had more chances to consume raw cow livers. the study was of a cross - sectional nature and thus temporal associations could not be inferred. therefore, we could not ascertain the inconsistency between seronegative subjects and remote infection by ingestion of raw cow livers. a bias arising from recall data based on the ability of an individual to recall the history of ingestion of raw tissue of animals could not be avoided. we did not count the number of chops of animal tissues ingested in determining the seropositivity of the enrolled participants. the study population might not be a representative of the general population as a nurse interviewer chose the participants randomly by selecting those subjects who responded voluntarily. its sensitivity and specificity are around 90%. with the limitations of the study in mind, we again suggest that ingestion of raw cow liver and keeping dogs increases the risk of t. canis infection in korea. in view of many adults with subclinical toxocariasis, ingestion of uncooked livers of paratenic hosts is an important source for human infections that can not be ignored.	the aim of this study is to ascertain the relationship between ingestion of raw cow liver and toxocara canis infection. a total of 150 apparently healthy adults were divided into 2 groups ; 1 group consisted of 86 adults with positive results of toxocara elisa, and the other group of 64 adults with negative results. one researcher collected the history of ingestion of raw cow liver within 1 year and recent history of keeping dogs. among 86 seropositive adults for t. canis, 68 (79.1%) had a recent history of ingestion of raw cow liver. multivariate statistical analysis showed that a recent ingestion of raw cow liver and keeping dogs were related to an increased risk of toxocariasis (odds ratios, 4.4 and 3.7 ; and 95% confidence intervals, 1.9 - 10.2 and 1.2 - 11.6, respectively). a recent history of ingestion of raw cow liver and keeping dogs was significantly associated with toxocariasis.
the anatomy and function of the superior laryngeal nerve (sln) are well described ; however, the consequences of sln injury remain variable and ill defined. the prevalence of sln injury as a consequence of cervical spine surgery is difficult to discern, as its clinical manifestations are often inconstant and frequently of a subclinical degree. sln injury is best characterized in the head and neck surgery literature, where its prevalence has been reported to occur in up to 60% of conventional procedures such as total thyroidectomy. as it pertains to cervical spine procedures, specifically anterior cervical discectomy and fusion (acdf), sln injury remains poorly characterized. vocal cord paralysis following acdf is traditionally attributed to recurrent laryngeal nerve (rln) traction. however, sln injury may also produce vocal cord paralysis in addition to reduced laryngeal sensation, dysphagia, and decreased laryngeal cough reflex predisposing to aspiration and impaired vocal quality. thus unrecognized sln injury may be an underappreciated cause of persistent dysphonia following anterior cervical spine surgery. the sln is vulnerable to injury when operating at the rostral levels of the cervical spine, thus understanding its detailed anatomy and vascular supply remains critical to avoiding its stretch or ligation during cervical spine procedures. we have conducted a retrospective multicenter study involving 21 high - volume surgical centers from the aospine north america clinical research network, selected for their excellence in spine care and clinical research infrastructure and experience. medical records for 17 625 patients who received cervical spine surgery (levels from c2 to c7) from january 1, 2005 to december 31, 2011, inclusive, were reviewed to identify occurrence of 21 predefined treatment complications. the complications included reintubation requiring evacuation, esophageal perforation, epidural hematoma, c5 palsy, recurrent laryngeal nerve palsy, superior laryngeal nerve palsy, hypoglossal or glossopharyngeal nerve palsy, dural tear, brachial plexopathy, blindness, graft extrusion, misplaced screws requiring reoperation, anterior cervical infection, carotid artery injury or cerebrovascular accident, vertebral artery injuries, horner s syndrome, thoracic duct injury, tetraplegia, intraoperative death, revision of arthroplasty and, pseudomeningocele. trained research staff at each site abstracted the data from medical records, surgical charts, radiology imaging, narratives, and other source documents for the patients who experienced one or more of the complications from the list. copies of crf forms were transferred to the aospine north america clinical research network methodological core for processing, cleaning, and data entry. in the current study involving 21 centers and 17 265 cases of cervical spine surgery, 8887 patients underwent an anterior cervical operation. in this subset of patients, this population consisted of 141 males and 117 females with a mean age of 57.1 years (sd = 13.2) and a mean body mass index (bmi) of 28.8 kg / m (sd = 6.5). the mean operative time was 212.7 minutes (sd = 110.8) and mean blood loss was 461.1 ml (sd = 725.5). a total of 139 patients (53.9%) underwent anterior surgery, 101 patients (39.1%) underwent posterior surgery, and 18 patients (7.98%) underwent combined anterior / posterior surgery. the prevalence ranged from 0% to 1.25% across the various centers involved in the study. the sln palsy occurred in a 51 year - old man with a bmi of 30.4 kg / m who presented with severe degenerative cervical spondylosis and diffuse disc pathology at c3/4 and c4/5. clinically, the patient endorsed neck pain, radiculopathy, and early signs of myelopathy. he underwent a c4 corpectomy and c3 - 5 fusion via a left sided approach. the operation lasted 357 minutes, and total blood loss was 300 ml. although the patient was noted to have a short neck, cervical traction was not used, and his neck was not manipulated intra - operatively. formal otolaryngology evaluation with laryngoscopy, strobovideolaryngoscopy, and a modified barium swallow test confirmed the diagnosis of sln palsy. the patient underwent placement of a temporary percutaneous gastrostomy tube and was discharged from the hospital 8 days later with the tube still in place. a repeat barium swallow test 6 weeks later demonstrated normal swallowing function suggesting resolution of his sln palsy, and the gastrostomy tube was removed. retrospective studies by bulger and ballotta reported a 1% prevalence of permanent and transient sln palsy following acdf and carotid endarterectomy respectively. another study from china by zhao found 3 patients in 282 (1.1%) who developed sln palsy after acdf ; all 3 cases resolved within 1 week after surgery. in this multicenter study, the prevalence ranged from 0% to 1.25%. the current study supports previous findings that sln injury as a consequence of anterior cervical spine surgery is an extremely rare phenomenon. the diagnosis of sln palsy is challenging and can not be made on clinical suspicion alone. initial evaluation of impaired phonation and dysphagia may include laryngoscopy ; however, it provides variable results and may be insufficient as a stand - alone test for the diagnosis of sln palsy. in cases where laryngoscopy is inconclusive, the most common therapy recommended for sln palsy is voice therapy targeted at strengthening the cricothyroid muscle. several surgical procedures have been described in the literature ranging from laryngoplasty and cricothyroid muscle approximation to nerve - muscle pedicle techniques for reinnervation. however, most of these studies are small series that lack long - term data and require further research to delineate the role of these procedures in the treatment of sln palsy. in cases of vocal cord paralysis, injection therapy, although temporary, is often effective in restoring the cough reflex, preventing aspiration and improving voice quality. early diagnosis and treatment often results in good clinical outcomes, as sln palsy following anterior cervical surgery is transient in most cases. it is important to note that the prognosis of sln injury is difficult to determine because it is extremely uncommon following anterior cervical spine surgery and the symptoms are often subclinical in nature and extent. confirmed cases requiring treatment are likely more severe and may predispose to observational bias. the sln arises from the nodose ganglion as a branch of the vagal nerve within the carotid sheath midway between the jugular foramen and the carotid bifurcation (at the level of the c2 vertebra). it then runs medially and caudally approximately 1.5 cm toward the thyrohyoid membrane before dividing into the internal and external branches. the internal branch of the sln is the larger of the 2 branches and continues medially with the superior laryngeal artery, which most commonly arises from the superior thyroid artery. it follows a transverse trajectory as it courses inferior to the hyoid bone, pierces the thyrohyoid membrane and divides into 3 terminal branches. the internal branch of the sln provides sensory and parasympathetic innervation from the larynx to the level of the vocal folds in addition to a small motor branch to the interarytenoid muscle. these are critical pathways mediating the afferent limb and subsequent recruitment of the laryngeal cough reflex. parenthetically, the prevalence of aspiration pneumonitis as a consequence of anterior cervical spine surgery remains low due to the laryngeal mucosa s bilateral sln innervation. the smaller external branch maintains a course medial to the carotid arteries as it runs distally toward the superior thyroid. the external branch is consistently located posterior to the superior thyroid artery, thus serving as a potential landmark. it is a purely motor branch providing innervation to the inferior pharyngeal constrictor muscles and most important, the cricothyroid. the cricothyroid muscle plays an important role in phonation by regulating tension of the vocal folds. injury to the external branch of the sln results in impairment of high - pitched tone and early fatiguability, an important consideration when performing anterior cervical surgery in singers. the course and divisions of the sln are essentially symmetric between the right and left sides of the neck, thus neither side imposes an increased risk of injury when compared with the other. the trunk of the sln and proximal divisions are at greater risk of injury during high - cervical exposures. the sln consistently originates at the c1 or c2 level and the internal branch pierces the thyrohyoid membrane, entering the surgical field at the level of the c4 vertebral body in the majority of cases. after exiting the carotid sheath, the sln courses medially and lies within the pretracheal fascia overlying the longus colli en route to the thyrohyoid membrane. when exposing the higher levels of the cervical spine above the carotid bifurcation, division of the longus colli and fascia deep to the carotid arteries must be cautiously performed to avoid inadvertent injury to the trunk of the sln. prior to placement of self - retaining retractor blades in the upper cervical spine, it is important to adequately dissect the longus colli muscles laterally on either side of the vertebral bodies in order to ensure that the blades remain beneath the muscle layer. if the retractor blades are positioned superficial to the longus colli, they may create significant traction on one or both branches of the sln as they course from the carotid sheath to the larynx within the pretracheal fascia. as the external branch takes an aggressive medial course, it is less vulnerable to injury during this portion of the exposure compared with the internal branch, which takes off at a more acute angle from the trunk of the sln. the superior thyroid artery serves as a useful landmark for the sln, as both internal and external branches arise near it. therefore, aggressive dissection near the artery or bipolar cautery may cause injury to the proximal portions of one or both divisions of the sln. the external branch is more vulnerable to injury than the internal branch during ligation of the superior thyroid artery, as the distal portions lie immediately deep to the artery. identification of the cricothyroid muscle and the upper lobe of the thyroid gland are useful landmarks for the external branch. once the self - retaining retractor blades are appropriately placed, care should be taken to limit dissection and electrocautery to the confines of the blades to avoid injury to the distal divisions of the sln, especially the internal branch. the cricrothyroid artery and superior laryngeal arteries run closely with the external and internal branches, respectively. excessive distraction of the retractor blades medially may cause bleeding from either of these arteries prompting excessive use of bipolar electrocautery along the pretrachial fascia, that could lead to injury to either division of the sln. exposing the anterior cervical spine is more challenging in patients who have undergone prior neck surgery due scar tissue formation and altered anatomy. patients who undergo multilevel spine surgery or a corpectomy require additional tissue dissection that may increase the risk of sln injury. intraoperative neuromonitoring with electromyography may be useful in avoiding sln injury during difficult cases or those requiring a more difficult exposure. jonas and bahr described a technique for monitoring the external branch of the sln during thyroid surgery by placing a bipolar electrode in the cricothyroid muscle and stimulating the nerve or suspected nerve with a nerve stimulator. they found the technique 97% effective in both positive and negative identification of the external sln. however, the true prevalence is likely higher than reported in the spine literature as the signs and symptoms of sln palsy are often subtle and transient. meticulous dissection, thoughtful retraction, and a comprehensive understanding of anatomic constraints are suggested to minimize prevalence of sln injury.	study design : a retrospective multicenter case - series study ; case report and review of the literature.objective:the anatomy and function of the superior laryngeal nerve (sln) are well described ; however, the consequences of sln injury remain variable and poorly defined. the prevalence of sln injury as a consequence of cervical spine surgery is difficult to discern as its clinical manifestations are often inconstant and frequently of a subclinical degree. a multicenter study was performed to better delineate the risk factors, prevalence, and outcomes of sln injury.methods:a retrospective multicenter case - series study involving 21 high - volume surgical centers from the ao spine north america clinical research network. medical records for 17 625 patients who received subaxial cervical spine surgery from 2005 to 2011 were reviewed to identify occurrence of 21 predefined treatment complications. descriptive statistics were provided for baseline patient characteristics. a retrospective review of the neurosurgical literature on sln injury was also performed.results:a total of 8887 patients who underwent anterior cervical spine surgery at the participating institutions were screened, and 1 case of sln palsy was identified. the prevalence ranged from 0% to 1.25% across all centers. the patient identified underwent a c4 corpectomy. the sln injury was identified after the patient demonstrated difficulty swallowing postoperatively. he underwent placement of a percutaneous gastrostomy tube and his sln palsy resolved by 6 weeks.conclusions:this multicenter study demonstrates that identification of sln injury occurs very infrequently. symptomatic sln injury is an exceedingly rare complication of anterior cervical spine surgery. the sln is particularly vulnerable when exposing the more rostral levels of the cervical spine. careful dissection and retraction of the longus coli may decrease the risk of sln injury during anterior cervical surgery.
ldiflares were measured in age-, height-, and bmi - matched groups of type 1 diabetic (n = 16), igt (n = 14), and healthy control (n = 16) subjects. those with type 1 diabetes presented with a typical history or ketoacidosis and were treated with insulin from diagnosis. subjects with ankle brachial pressure indexes of 300 pu) and measured. additionally, the maximum hyperemia (ldimax) in the skin immediately beneath the heater was measured using the imager. unlike the flare, ldimax is mediated by nonneurogenic means and reflects maximum microvascular hyperemia (1). in all subjects clinical neuropathy was excluded using the neuropathy disability scale (9) ; vibration perception thresholds (neurothesiometer) ; and quantitative sensory testing of warming, cooling, and vibration perception (computer aided sensory evaluator iv). variables were correlated using pearson coefficient. with coefficients of variation (cvs) for ldiflare and ldimax conservatively estimated at 20% (actual cvs : ldiflare, 13% ; ldimax, 6%), a prestudy power calculation suggested 16 participants per group would detect a 20% difference with 80% power. after 20 min acclimatizing in a temperature - controlled room (25 1c), the neurogenic flare was induced by heating a 1-cm diameter area on the dorsum of the foot to 44c for 20 min. the area was scanned using the imager, and the flare area was identified (with a hyperemic response > 300 pu) and measured. additionally, the maximum hyperemia (ldimax) in the skin immediately beneath the heater was measured using the imager. unlike the flare, ldimax is mediated by nonneurogenic means and reflects maximum microvascular hyperemia (1). in all subjects clinical neuropathy was excluded using the neuropathy disability scale (9) ; vibration perception thresholds (neurothesiometer) ; and quantitative sensory testing of warming, cooling, and vibration perception (computer aided sensory evaluator iv). variables were correlated using pearson coefficient. with coefficients of variation (cvs) for ldiflare and ldimax conservatively estimated at 20% (actual cvs : ldiflare, 13% ; ldimax, 6%), a prestudy power calculation suggested 16 participants per group would detect a 20% difference with 80% power. the ldiflare was significantly lower in the igt compared with the control (2.78 1.1 vs. 5.23 1.7 cm, p = 0.0001) and type 1 diabetic (5.16 2.3 cm, p = 0.002) groups. in contrast, the ldiflares of the type 1 diabetic and control groups were not different. the relatively favorable lipid profile in the diabetic group may be related to greater use of lipid - lowering therapy. in the combined igt and healthy control groups, fasting triglycerides (r = 0.39 ; p = 0.044) and 2-h glucose (r = 0.48 ; p = 0.0066) were inversely correlated with ldiflare. summary of results for igt, diabetic, and control groups data are means sd or n (%) unless otherwise indicated. all groups not significantly different unless marked : p < 0.05 vs. igt ; p = 0.0001 vs. igt. first, the ldiflare technique detects early small - fiber dysfunction when conventional tests, including computer aided sensory evaluator iv, are normal. the potential for earlier detection and the noninvasive nature of the method are advantages over existing techniques such as skin biopsy, particularly when repeated measurements are required. second, the detection of small - fiber dysfunction in the feet in the igt group is consistent with other studies but is novel in that functional rather than structural integrity was examined (1012). given that functional defects may precede structural changes and are more likely to be reversible, the method may be particularly valuable in assessing interventions to prevent or delay progress of neuropathy. further studies comparing ldiflare technique with other functional tests such as cardiac autonomic function and the newer structural techniques, intraepidermal and corneal nerve fiber density, would help determine its relative value. third, small - fiber dysfunction in the igt group contrasts with the apparent normality in the type 1 diabetic subjects. the latter group was unusual, being relatively free from complications, but this was necessary to exclude endoneural microangiopathy, which itself is associated with neuropathy (13). it is possible that other tests such as cardiac autonomic function may have been abnormal in this group. the difference in small - fiber function between these two groups suggests factors other than hyperglycemia are implicated in the development of small - fiber dysfunction. the significant association with certain features of the metabolic syndrome, namely 2-h glucose and triglyceride levels, suggests a common metabolic link. the flare, although dependent on intact small fibers, requires sufficient microvascular vasodilatation for it to be detected by the imager. this was so in all subjects, demonstrated by the ldimax measurements. in this context, the absence of any correlation between ldimax and ldiflare measurements confirms that they reflect different parameters. additionally, ldiflare was undiminished in the type 1 diabetic group despite, as expected from our previous study, a reduction in their ldimax (14). ldimax is believed to reflect endothelial function (15), and impaired endoneurial function is implicated in the development of neuropathy (13). however, that ldimax did not correlate with small - fiber function might indicate that endothelial dysfunction only affects neural function at more advanced stages when there is significant microangiopathy. in summary, the ldiflare technique has adequate sensitivity to detect early small - fiber dysfunction. the presence of small - fiber dysfunction in those with igt and not in those with long - duration type 1 diabetes in the absence of significant microvascular disease suggests that factors other than hyperglycemia contribute to small - fiber dysfunction in those with igt.	objectivethis study explored the importance of glycemic burden compared with features of the metabolic syndrome in the pathogenesis of diabetic neuropathy by comparing c - fiber function in people with type 1 diabetes to that in people with impaired glucose tolerance (igt).research design and methodsthe axon reflex elicited flare areas (ldiflares) were measured with a laser doppler imager (ldi) in age-, height-, and bmi - matched groups with igt (n = 14) and type 1 diabetes (n = 16) and in healthy control subjects (n = 16).resultsthe flare area was reduced in the igt group compared with the control (2.78 1.1 vs. 5.23 1.7 cm2, p = 0.0001) and type 1 diabetic (5.16 2.3 cm2, p = 0.002) groups, whereas the flare area was similar in the type 1 diabetic and control groups.conclusionsthis technique suggests that small - fiber neuropathy is a feature of igt. the absence of similar small - fiber neuropathy in those with longstanding type 1 diabetes suggests that glycemia may not be the major determinant of small - fiber neuropathy in igt.
parkinson 's disease (pd) is a progressive neurological disease that affects millions of people world wide, with 1.8% of the population at age > 65 years (1). the death of dopaminergic neurons in the substantia nigra is a pathological feature of the disease. pd is a complicate disease that both environmental and genetic factors play various roles in its causation and development. the genetic evidence is that first degree relatives of the familial pd patients are more vulnerable to pd than the general population, especially significant for early - onset pd (2,3). however, reliable biomarkers or tests to facilitate early and accurate diagnosis are currently not available (4). genetic testing, if available, could complement clinical diagnostic criteria. several genetic mutations and variants (point substitution, deletion, insertion or even polymorphisms) park2, lrrk2, pink1, snca, uchl1 and park7 are the most frequently studied genes (7). some of the genetic variants are considered as causal factors while others may cause neuronal dysfunction indirectly. for example, mutations in the 5-utr of nr4a2 have significantly decreased the expression of nr4a2 gene and its downstream gene tyrosine hydroxylase (8). at present, over 100 genes have been reported to associate with pd in various forms. some of these genetic variants are widespread in patients while others are ethnic - related risk factor. lrrk2 g2019s is a common pathogenic mutation found in 57% of familial pd and 12% of sporadic pd worldwide (9,10). at the same time, this mutation shows specific ethnic prevalence with exceptionally high frequency in north african arabs (3742% in familial and 41% in sporadic pd) (11) but is rare in chinese (12,13). both r496h and c.84insggfs are found in patients from ashkenazi jews (14,15) and have not been reported in other ethnic groups. genetic screening and treatment strategies could be improved if genetic features have been well characterized. further elucidation of such information may also lead to new developments in diagnostic methods and early treatment. due to advances in genetic technology and the polygenic nature of pd, genetic information of the disease the amount of data is a daunting challenge for individual researchers in searching and examining desirable information. for instance, there are over 1300 reports in the pubmed database if parkinson 's disease and the combination of gene names, official symbols and aliases in literature reports further amplifies the difficulty of retrieving relevant information. in addition, although information of gene function, gene sequences, protein structure and mutation reports are searchable, their availability are scattered across various databases such as entrez gene (16), genbank (17), swiss - prot (18), omim (19) and pubmed (http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed). the human gene mutation database [hgmd ; (20) ], which covers mutation information for over 2800 human genes, is the most comprehensive but its public free - access version is limited to less - updated information. if one searches the hgmd for pd, one may only get the mutation information for less than 20 genes. thus, to obtain desired information, researchers have to perform at least two time - consuming tasks : (i) examine large volume of data ; and (ii) query a number of different databases. there are a few databases that specialize in pd mutation information, but they are limited in either coverage or functionality. for example, the lovd parkinson 's disease mutation database developed by the parkinson 's institute in leiden university (http://www.grenada.lumc.nl/lovd2/tpi/home.php) briefly covers six genes with total of 71 variants. the pdgene (http://www.pdgene.org/) is more comprehensive and contains the most updated list of pd candidate genes with emphasis in genetic association studies. however, it lacks the functionality for ethnic comparison and it does not provide summary and statistic reports. to address some of the current limitations of pd databases and to facilitate effective and comprehensive information acquisition, we have developed the mutation database for pd (mdpd). through a single online location with user - friendly interfaces, researchers are able to retrieve the latest information on pd, covering genetic variation (mutation and polymorphism), population studies, literature evidence and gene sequences. various cross - references to public databases are incorporated to assist further exploration and evaluation. mdpd is a specialist database that presents the human mutation information relevant to pd as an online resource. mutation evidences are extracted from pubmed database (from 1995 to 10 june 2008) based on keyword search [parkinson 's disease / genetics (mesh) ]. sequences, variants and general gene information are obtained from other databanks as mentioned above (the latest update of mdpd was 4 september 2008). important information, such as size of study sample, control group, population, age of onset, type of pd (sporadic versus familial), mutation outcome, its reference sequence and possible impact of the variant, have been manually extracted from the data source. in addition to mutation, single nucleotide polymorphism (snp) shows the linkage between genotype and the susceptibility of disease (21). snps can fall within the coding region and non - coding region in most cases. in non - coding region, a snp does not change protein sequence but may still have consequences on the risk of disease by affecting the splicing site or transcription factor binding site. some snps have protective effects while others may increase the risk of pd, or show no significant outcomes and need further research. as we believe snp information plays an important role in the design of genetic tests and also in understanding the mechanisms of the disease, they are included in our database. on the other hand, variations that lack precise genetic locations, such as variants in approximate chromosome regions or markers in inter - gene regions, we believe genetic location is very critical in mutation study and such approximate results need to be resolved before inclusion in the database. control studies and genome - wide association studies (gwas) have shown that pd is a polygenic disease that multiple gene mutations are responsible for the malfunction (7,22). a single nucleic acid substitution may lead to a multitude of possible outcomes, including amino acid exchange (missense mutation), no amino acid change (silent mutation), peptide truncation (nonsense mutation), absence of the protein (deletion) and even the production of a different protein (frame shift or insertion). other types of mutation, such as duplication and compound mutation (more than one type of mutation) are also found in pd. as such, we categorize the genetic variations into the following : missense mutation, silent mutation, nonsense mutation, compound mutation, deletion, insertion, duplication, triplication, frame shift, short repeat and snp. all of these classifications are based on the primary reference as the trusted data resource. we believe that such categorization can help the user to examine and compare the variations more efficiently. another classification in mdpd is the variations impact to reflect the outcomes of the variations. divergent impacts are expected due to differences in the method used, sample size and the studied population. one variation may have more than one impact. for example, the v380l substitution in park2 gene is marked associated since it has been found in the sample of early onset pd patient (23). negative result in other occasion due to the lack of significant association with patients (24). such inevitable discrepancy of impact reflects the complicated nature of pd in which multiple genetic factors play various roles and that interactions between genetic and environmental factors may influence the end result dynamically. according to the found effects of each variation, we classified its impact as protective factor, risk effect has been mentioned in the primary reference of a variance, we assign its impact as protective factor or risk factor. if a variance has been reported in both patients and controls without statistic difference, we label it as questionable. the classification aims to help user recapitulate information according to comparison outcome. mdpd is designed to be a publicly accessible online resource with user - friendly interface. a web - based user interface to the database is provided via an apache 2.0 http server with php scripting engine. mdpd contains the following functional pages : browser, search, compare, statistics and variation report. three searching options are available in the search page : a variation search can be based on the gene name, gene i d or swiss - prot accession number. in accordance to the recommendations from the hugo nomenclature committee, official gene symbols are used in each record, but mdpd also provides all known aliases in the result pages for easy reference and retrieval. searching mutation information based on geographic region or the author 's name in reference collection are two other helpful options. searching for mutation information based on a geographic region enables a user to quickly know what genes have been studied in the region (or population) and the number of related publications may indicate regional research efforts. on the other hand, searching for mutation information based on the reference author 's name can help researchers in the community easily identify various leading authors ' research interests and their collaborators. various useful features have been built into mdpd and span several web pages for ease of use and navigation. the complete list of web pages and their corresponding features are listed in table 1. we highlight the variation report page here as we believe that it is a useful feature of mdpd. in addition to providing the generic gene information, hyperlinks to entrez, swissport and omim, the variation sequence (in both amino acid and nucleic acid levels) and pubmed collection. for variation impact and variation type, we do not attempt to modify the findings from the primary reference, as we have mentioned previously. users are advised to judge the classification based on his / her knowledge and the up - to - date research. to understand the impact of a genetic substitution, the user should be aware of any conflicting results from divergent ethnic groups or from specific subsets of patients. researchers may also use it to identify key genetic factors, namely those for which impactful variations have been reported in many geographic regions (or ethnic groups). for example, mdpd includes 74 literature reports that described 258 different variants of park2 from 33 geographic regions. deletion, duplication, triplication, insertion, missense mutation, nonsense mutation, silent mutation and compound mutations were all found in this gene. among them, missense mutation and deletion are the most frequently conveyed with 140 and 86 records, respectively. based on such information, a user can make reasonable inference about the importance of park2 instantly. to further confirm his / her speculation hot sport through variation sequence. table 1.functional summary of mdpdweb pagecontentsbrowsealphabetic list of gene symbolchromosomal location of the genelinks to entrez gene and swiss - prot databasesentry to summary pagesearchsearch gene name, symbol, aliases (allow partial name), gene i d, swiss - prot idsearch studied geographic regions (ethnic group)search author 's name for primary referencesummaryabout the genenumber of records for the genenumber of variants reported in swiss - prot databaselink to omim databasenumber of pubmed reference for the gene in mdpdvariation reportlist of variation impactlist of variation typelist of studied geographic regions (ethnic groups)variation sequence (in both amino acid and nucleic acid levels)list of pubmed reference for the geneentry to individual variation report (sample size, control group, age, gender, testing variation, impact, geographic and comments)comparisoncomparing genetic data from any two geographic regionsstatisticskey statistics in mdpdtop 10 genes with most literature reportstop 10 genes with most reported negative variantstop 10 countries / regions with most studies done functional summary of mdpd allowing users to compare mutations between ethnic groups is another helpful element of mdpd. users can readily obtain a list of mutation genes of an interested ethnic group from search. comparing the mutations between two ethnic groups of interest can also be done easily in comparison. currently, more than 2300 entries covering 202 human genes are stored in mdpd. through systematic data mining on the integrated information, mdpd offers researchers new means for inspecting and making sense of the mutation evidences in published findings. gene mutations and variations have become the focus of pd research in the last decade. linkage mapping, case control study, pedigree analysis and gwas are powerful approaches to identify and correlate genetic contribution to pd. however, how various mutations and variants affect the disease and shape its development remains unclear. various research biases and errors contribute to fewer reproducible association findings and diminish the assessing power between genetic variants and the risk of common disease (25,26). to partially overcome the limitation of accumulated imperfect data, we intend to include all published literatures with precise number of sample size (both case and control), variation position, variation impact and geographic location., we remind users to be caution in interpretation of deductions from published data. many genes and multiple variants in a gene possessing the available information is an essential first step to further understand and eventually to elaborate effective strategies for diagnosis and treatment. it contains records for over 100 pd - associated genes verified from various genetic tests. among them, the top 10 most reported mutation genes are lrrk2, park2, snca, cyp2d6, mapt, pink1, uchl1, park7, maob and apoe (table 2). the data in mdpd also reveal that current research has been focused in certain key genetic targets the top 10 genes accounted for 1053 entries from 326 publications, which makes up to 44% of the total records and more than half of the literature reports (57%) in mdpd. at the same time, we realize that 202 genes are somewhat under the current research radar. table 2.top 10 genes with the most published reference in mdpdgene nameno. of recordno. of countries / regionsno. of referencelrrk22653876park23292866snca1172451cyp2d6611631mapt391420pink1781819uchl1481218park7581216maob37715apoe21814 top 10 genes with the most published reference in mdpd another interesting outcome from mdpd is the high frequency of negative result in the variation reports. negative in 9 out of the top 10 most reported genes (the exception is park2 with 15.2% negative reports). there are at least three implications : (i) many variants have low incident rates in pd patients and may not be a good screening target for survey ; (ii) these variants may have insignificant impact to pd ; and (iii) discrepancy may be caused by ethnic - related genetic variance, sample size, methods used or research errors. the variants with the most positive reports could be valuable genetic targets and further studies on them may warrant potential breakthrough in diagnosis and treatment. pd is a multi - factorial disease for which the environmental factors and genetic elements are likely to be equally important. studies have showed that lifestyles (such as smoking and coffee consumption), pesticides and metal exposure, and even well water drinking are factors that influence the risk of disease in both sporadic and early - onset pd (27,28,29). the involvement of multiple genes, the high incident rate in aging population and high percentage of sporadic cases suggest the possibility of multiple interactions and connections in etiology of pd. in many cases, it is difficult to isolate the environmental factors and to specify the short- and long - term exposure. as such, we did not include environmental study information in mdpd, but the user should be mindful about the potential interactions with environmental factors. discovering the relationships between the various genetic factors is an essential step toward understanding the mechanism of complex diseases such as pd. from mdpd, we know at least 202 genes have been examined for their possible involvement in pd. our future work would be to develop an information system that can assess the impact of disease - causing mutations in terms of the functional changes of their encoded proteins and the interactions. protein interactions, and the role of the genetic variants in various neurodegenerative pathways will hopefully provide insights that will lead to novel treatments for pd. institute for infocomm research (ir) ; agency for science, technology and research (astar), singapore.	parkinson 's disease (pd) is the second most common neurodegenerative disorder affecting millions of people. both environmental and genetic factors play important roles in its causation and development. genetic analysis has shown that over 100 genes are correlated with the etiology and pathology of pd. however, accessing genetic information in a consistent and fruitful way is not an easy task. the mutation database for parkinson 's disease (mdpd) is designed to fulfill the need for information integration so that users can easily retrieve, inspect and enhance their knowledge on pd. the database contains 2391 entries on 202 genes extracted from 576 publications and manually examined by biomedical researchers. each genetic substitution and the resulting impact are clearly labelled and linked to its primary reference. every reported gene has a summary page that provides information on the variation impact, mutation type, the studied population, mutation position and reference collection. in addition, mdpd provides a unique functionality for users to compare the differences on the type of mutations among ethnic groups. as such, we hope that mdpd will serve as a valuable tool to bridge the gap between genetic analysis and clinical practice. mdpd is publicly accessible at http://datam.i2r.a - star.edu.sg / mdpd/.
in patients with a congenital portosystemic shunt, circulating immune complexes (cics) enter the systemic circulation from the portal blood without hepatic clearance and may be deposited in the glomeruli, leading to nephropathy. this condition is known as membranoproliferative glomerulonephritis (mpgn) with a noncirrhotic portosystemic shunt (ncpss), and it is a rare disorder for which the optimum treatment remains controversial [14 ]. because a high shunt ratio may be important in the onset of mpgn, embolization of the shunt has been proposed, but its efficacy remains unclear. we present a patient with nephrotic syndrome, renal insufficiency and a congenital portosystemic shunt. a 75-year - old man with peripheral oedema was admitted on 4 november 2005. he had no history of hereditary or genetic disorders, and he did not drink alcohol. unspecified congenital liver disease and renal dysfunction were noted at a health check in 2004. on admission, physical examination revealed no evidence of inflammation, but he had marked lower limb oedema. g / l ; leukocytes, 7,000/l ; platelets, 12.5 10/l ; total protein, 43 g / l ; albumin, 22 g / l ; urea nitrogen, 8.93 mmol / l ; creatinine, 124 mol / l ; serum cholesterol, 10.09 mmol / l ; aspartate aminotransferase, 19 iu / l ; alanine aminotransferase, 15 iu / l ; gama - glutamyltranspeptidase, 21 iu / l ; alkaline phosphatase, 236 iu / l and total bilirubin 20.5 mol / l. / l, immunoglobulin a was 3.28 g / l, immunoglobulin m was 1.74 g / l, ch50 was 24.2 u, c3 was 43 u and c4 was 8 u. tests for hepatitis b virus, hepatitis c virus and cryoglobulins were negative. urinalysis showed > 100 red cells per high - power field and 14 leukocytes per high - power field, while the 24-h urinary protein excretion was 8.08 g. ultrasonography showed an increase of renal cortical echogenicity and a normal liver. there were global diffuse endocapillary proliferation, increased mesangial cellularity and matrix and a lobular appearance. capillary walls were thickened, and the glomerular basement membrane showed a double contour due to subendothelial deposits and mesangial interposition. there were also wire loop glomeruli, suggesting secondary glomerular disease (figure 1). immunofluorescence detected iga (predominantly), c3, c1q, igm and igg with an irregular, chunky capillary and mesangial distribution (figure 2). electron microscopy revealed massive subendothelial electron - dense deposits (edd) and endocapillary proliferation (figure 3). (a-1) first renal biopsy : there is diffuse endocapillary proliferation with an increase of mesangial cellularity and matrix, as well as a lobular pattern. the capillary walls are thickened, and the glomerular basement membrane shows a double contour due to the presence of subendothelial deposits and mesangial interposition. (a-2) first renal biopsy : there were also wire loop glomerular lesions, suggesting secondary glomerular disease. the double contour glomerular basement membrane, lobular appearance and wire loop lesions are no longer detected. (c) third renal biopsy : there are mesangial matrix expansion and an increase of mesangial cellularity. (a) first renal biopsy : iga(a) (predominant), igg(b), igm(c), c3(d) and c1q(e) show an irregular, chunky capillary and mesangial distribution. (b) third renal biopsy : there is diffuse positivity for iga(a), igm(b) and c3(c). (a) first renal biopsy : massive subendothelial electron - dense deposits (arrow) and endocapillary proliferation suggest a diagnosis of mpgn type 1 like. (b) third renal biopsy : subendothelial deposits and endocapillary proliferation have resolved. in december 2005, he suddenly lost consciousness. hepatic function tests and blood glucose were not abnormal, and computed tomography (ct) of the brain was also normal. however, the serum ammonia level was 184 mol / l, so encephalopathy due to hyperammonaemia was diagnosed. contrast - enhanced ct revealed a large shunt between the left branch of the portal vein and the inferior vena cava. technetium-99 m galactosyl human serum albumin (99mtc - gsa) scintigraphy showed normal hepatic function. histological examination of a liver biopsy specimen only revealed mild changes indicating portal hypertension, so he was not affected by cirrhosis. percutaneous transhepatic portal vein embolization (ptpe) was performed, reducing the shunt ratio to 54.9% of the preoperative level on per rectal technetium-99 m pertechnetate portal scintigraphy. by day 15 after ptpe, the 24-h urinary protein excretion decreased to 0.25 g. in march 2006, a second renal biopsy was performed. in december 2006, laboratory tests revealed a total protein of 60 g / l, serum albumin of 34 g / l, serum creatinine of 124 mol / l, ch50 of 36.2 u, c3 of 76 u and c4 of 13 u. the urinary protein was negative. accordingly, his renal function had improved. in december 2006, a third renal biopsy was performed, and 99mtc - gsa scintigraphy showed minimal deterioration of hepatic function at that time. the specimens contained 20 (march 2000) and 17 (december 2006) glomeruli, respectively.. however, endocapillary proliferation, the double - contour glomerular basement membrane and lobular mesangial interposition had all resolved (figure 1). on electron microscopy, subendothelial deposits and endocapillary proliferation had resolved (figure 3). there were global diffuse endocapillary proliferation, increased mesangial cellularity and matrix and a lobular appearance. capillary walls were thickened, and the glomerular basement membrane showed a double contour due to subendothelial deposits and mesangial interposition. there were also wire loop glomeruli, suggesting secondary glomerular disease (figure 1). immunofluorescence detected iga (predominantly), c3, c1q, igm and igg with an irregular, chunky capillary and mesangial distribution (figure 2). electron microscopy revealed massive subendothelial electron - dense deposits (edd) and endocapillary proliferation (figure 3). (a-1) first renal biopsy : there is diffuse endocapillary proliferation with an increase of mesangial cellularity and matrix, as well as a lobular pattern. the capillary walls are thickened, and the glomerular basement membrane shows a double contour due to the presence of subendothelial deposits and mesangial interposition. (a-2) first renal biopsy : there were also wire loop glomerular lesions, suggesting secondary glomerular disease. the double contour glomerular basement membrane, lobular appearance and wire loop lesions are no longer detected. (c) third renal biopsy : there are mesangial matrix expansion and an increase of mesangial cellularity. the findings resemble those of iga nephropathy. (a) first renal biopsy : iga(a) (predominant), igg(b), igm(c), c3(d) and c1q(e) show an irregular, chunky capillary and mesangial distribution. (b) third renal biopsy : there is diffuse positivity for iga(a), igm(b) and c3(c). (a) first renal biopsy : massive subendothelial electron - dense deposits (arrow) and endocapillary proliferation suggest a diagnosis of mpgn type 1 like. (b) third renal biopsy : subendothelial deposits and endocapillary proliferation have resolved. in december 2005, he suddenly lost consciousness. hepatic function tests and blood glucose were not abnormal, and computed tomography (ct) of the brain was also normal. however, the serum ammonia level was 184 mol / l, so encephalopathy due to hyperammonaemia was diagnosed. contrast - enhanced ct revealed a large shunt between the left branch of the portal vein and the inferior vena cava. m galactosyl human serum albumin (99mtc - gsa) scintigraphy showed normal hepatic function. histological examination of a liver biopsy specimen only revealed mild changes indicating portal hypertension, so he was not affected by cirrhosis. percutaneous transhepatic portal vein embolization (ptpe) was performed, reducing the shunt ratio to 54.9% of the preoperative level on per rectal technetium-99 m pertechnetate portal scintigraphy. by day 15 after ptpe, the 24-h urinary protein excretion decreased to 0.25 g. in march 2006, a second renal biopsy was performed. in december 2006, laboratory tests revealed a total protein of 60 g / l, serum albumin of 34 g / l, serum creatinine of 124 mol / l, ch50 of 36.2 u, c3 of 76 u and c4 of 13 u. the urinary protein was negative. accordingly, his renal function had improved. in december 2006, a third renal biopsy was performed, and 99mtc - gsa scintigraphy showed minimal deterioration of hepatic function at that time. the specimens contained 20 (march 2000) and 17 (december 2006) glomeruli, respectively.. however, endocapillary proliferation, the double - contour glomerular basement membrane and lobular mesangial interposition had all resolved (figure 1). on electron microscopy, subendothelial deposits and endocapillary proliferation had resolved (figure 3). first, in our patient with nephrotic syndrome and secondary immune complex - mediated mpgn who had no predisposing factors (infection, chronic liver disease, hereditary disorders, etc.) except for an ncpss, shunt embolization achieved remission of nephrotic syndrome and improved renal function without immunosuppressive therapy. second, renal histopathology changed from an mpgn type 1 like to an iga nephropathy - like pattern after shunt ratio reduction, while immune complex deposits changed from full house to predominantly iga and subendothelial edd disappeared. moreover, it was demonstrated that cics cause this disease and that reducing glomerular exposure to immune complexes is beneficial. to our knowledge, this is the first report of such findings. the embryologic development of the venous system of the trunk involves stages where there are complex collaterals between precursors to the caval system and precursors to the portal veins, persistence of which can lead to anomalous connections. noncirrhotic portosystemic shunt (ncpss) is synonymous with the term congenital intrahepatic portosystemic venous shunt (ipsvs). oguz. found that in most reported cases of congenital ipsvs, the patients were over 50 years old [610 ], although a few paediatric cases have also been reported. uchino. reviewed 51 cases of congenital ipsvs in japan, and 12 of the patients had hepatic encephalopathy at the time of diagnosis. the natural history of ipsvs depends upon the shunt ratio and on the patient 's age, since the frequency of hepatic encephalopathy increases with age. a decrease in the tolerance of the ageing brain to toxic metabolites may explain the late onset of clinical manifestations. large intrahepatic shunts are more often associated with hepatic encephalopathy than small shunts. as the shunt ratio increases, the amount of nitrogen - containing substances from the portal blood that have bypassed hepatic extraction increases in the systemic circulation and this can lead to hepatic encephalopathy. when the shunt ratio exceeds 60%, the risk of hepatic encephalopathy is reported to increase. there is much evidence to suggest that mpgn type 1 is a chronic immune complex - mediated glomerulonephritis. deposition of cics in the glomeruli is thought to be central to the pathogenesis of mpgn type 1, as well as to the occurrence of secondary mpgn type 1 in patients with known immune complex diseases, such as systemic lupus erythematosus, shunt nephritis, chronic bacteraemia and chronic hepatitis b or c. in addition, acute post - infectious gn and mpgn are frequent in patients with cirrhosis. in this patient, all such conditions were negative, so an ncpss was regarded as a predisposing factor for secondary mpgn type 1. shunt nephritis means immune complex - mediated glomerulonephritis that develops as a complication of chronic infection of a ventriculoatrial or (rarely) ventriculoperitoneal shunt inserted for the treatment of hydrocephalus. more than 150 cases have been reported, and the histological findings resemble those of mpgn type 1. development of shunt nephritis is due to chronic bacteraemia after insertion of a ventriculoatrial shunt. in contrast, our patient did not have shunt nephritis because there was no infection. also, our patient 's renal findings resembled lupus nephritis class iv (world health organization). overexpression of cytokines, chemokines and growth factors induced by the glomerular deposition of cics is important in the pathogenesis of lupus nephritis, and cics were also involved in causing nephritis in our patient. in both shunt nephritis and lupus nephritis, cics are thought to play a crucial role in the onset of immune complex - mediated glomerulonephritis, so the aetiology of lupus nephritis and our patient 's nephritis would seem to be identical or very similar to that of shunt nephritis. the outcome of shunt nephritis is good if early diagnosis and treatment are provided, including intravenous antibiotics and removal of the infected shunt, with the influence of cics being diminished by treatment of the primary disease. although there was no infection and our patient had a congenital portosystemic shunt, treatment of the primary disease also decreased exposure of the glomeruli to cics, which is the same factor that leads to the improvement of shunt nephritis. both soma. and karashima. suggested a causal relationship between ncpss and the development of nephritis due to reduced hepatic clearance of cics. karashima. also speculated that a high shunt ratio (> 90%) may trigger such nephritis. reducing the shunt ratio (54.9%) improved clinical and histological findings in our patient, supporting the hypothesis of karashima. our patient 's early renal findings resembled lupus nephritis class iv (world health organization). after shunt ratio reduction, the renal histology resembled mesangial proliferative glomerulonephritis with the predominant iga deposition. accordingly, it seems that various immunoglobulins accumulate in the glomeruli when the shunt ratio is high, while iga from the intestinal tract dominates after the shunt ratio decreases. although the shunt mechanisms differ, both diseases seem to be caused by reduced hepatic clearance of cics. the differences of renal pathology probably arise because the type, volume and size of the immune complexes deposited in the glomeruli depend on the shunt ratio. since ptpe may decrease liver function, the optimum reduction of the shunt ratio to achieve remission of nephritis and maintain adequate liver function needs to be determined in the future. in conclusion, the present case revealed that this type of nephritis is reversible at an early stage and that it can be treated by reduction of the shunt ratio. when immune complex - mediated nephrotic syndrome is refractory, early screening for ncpss may be important.	we present a case of a 75-year - old man with nephrotic syndrome and renal insufficiency caused by immune complex - mediated secondary membranoproliferative glomerulonephritis. he developed hepatic encephalopathy. a congenital portosystemic shunt was identified, indicating a diagnosis of membranoproliferative glomerulonephritis with noncirrhotic portosystemic shunt. proteinuria resolved after shunt ratio reduction by percutaneous transhepatic portal vein embolization. renal function and histopathological findings improved without immunosuppressive therapy. this case emphasizes the role of a high shunt ratio and reduced hepatic clearance of circulating immune complexes in such nephropathy. membranoproliferative glomerulonephritis with a shunt may cause refractory nephrotic syndrome, but embolization is effective.
we conducted an investigation to verify and validate the utility of a triage cc - based edss in southeastern ontario as a tool for monitoring respiratory disease by comparing it retrospectively with data from nacrs and telehealth ontario. this study was part of a broader research project approved by the queen s research ethics board and adheres to the principles and policies for the protection of personal health information charter. daily counts of discharges of persons with respiratory disease based on icd-10-ca codes were obtained from the nacrs database, and counts of respiratory ccs were likewise collected from edss. patient location was determined by forward sortation address (i.e., first 3 digits of postal code) and health unit code in the nacrs database and by specific reporting hospital in the edss data set. we categorized telehealth ontario calls into episodes of upper or lower respiratory disease on the basis of a priori classification schemes verified by other research (6,1113). all data were compiled into weekly totals (sunday saturday) from each of the 3 nonidentifiable data sets. during july 2004march 2006, edss contained 29,668 reports of respiratory diseases in persons seeking care at 1 of the 7 area hospitals. during the same period, telehealth ontario received 4,247 calls about upper and lower respiratory disease, and nacrs recorded 19,315 cases of respiratory disease from southeastern ontario. analysis comparing the edss respiratory ccs with the telehealth ontario calls about respiratory disease (figure) resulted in a spearman correlation coefficient of 0.91, indicating good correlation. analysis comparing the edss respiratory cc to all nacrs respiratory disease diagnoses resulted in a spearman correlation coefficient of 0.98, indicating very good correlation. correlations were highest and most significant when no time lags were included in the models. weekly totals of emergency department surveillance system (edss) respiratory chief complaints, national ambulatory care reporting system (nacrs) respiratory visits, and calls to telehealth ontario (th) about respiratory illness, ontario, canada, july 2004june 2006. this study verified that ed cc data can be used as a timely source of surveillance for respiratory diseases. ed ccs in southeastern ontario strongly correlated in time with nacrs respiratory discharge diagnoses and calls to telehealth ontario about respiratory disease. nacrs data are unavailable to public health stakeholders in a timely enough fashion to be useful for day - to - day monitoring of respiratory disease trends in the community, whereas ed ccs are available electronically and in real time. as expected, edss ccs about respiratory disease peaked during the influenza seasons (early november 2004mid - april 2005 and early december 2005early may 2006) (figure) ; however, edss ccs continued to fluctuate during the influenza off season, probably because of other respiratory pathogens. the nature of the edss data did not allow us to separate respiratory complaints related to influenza from those related to other pathogens. one consideration for interpreting edss data is that the hospitals in this surveillance system include large referral hospitals that have high volumes of patients visiting from outside our health unit boundaries. although this large referral area may result in higher counts of visits than reflected in the nacrs database, we believe it does not affect our interpretation of the data. even though each hospital is required to submit data, it is still possible that not all records from each hospital are sent on time to nacrs. although the public health system has accepted syndromic surveillance as a useful tool, doubts remain about its anticipated early warning benefits (14). these potential benefits can not be tested because no large - scale outbreaks have occurred since the inception of our real - time syndromic surveillance system. when a pandemic occurs, syndromic surveillance may be able to help healthcare workers recognize a potential outbreak, which theoretically could help them mitigate effects on society earlier. our study demonstrates that in southeastern ontario, ed ccs accurately reflect respiratory conditions of patients in the area. the correlations found strongly suggest that edss accurately monitors respiratory disease in the community and contributes to early detection of respiratory disease outbreaks. we continue to monitor the system from day to day and future studies will use laboratory data to assess the value of advanced warning on a number of syndromes captured in our system.	to validate the utility of a chief complaint based emergency department surveillance system, we compared it with respiratory diagnostic data and calls to telehealth ontario about respiratory disease. this local syndromic surveillance system accurately monitored status of respiratory diseases in the community and contributed to early detection of respiratory disease outbreaks.
the increased risks of vascular changes, including abnormal placentation, after repeated cesarean sections are well studied. herein, we describe a patient with delayed hemorrhage from a uterine avm, following dilation and curettage for a cesarean scar pregnancy. a 32-year - old g3p2 presented with a cesarean scar ectopic pregnancy managed with dilation and curettage, which incurred a 1,500-ml blood loss. within 6 weeks, she returned with 2 episodes of vaginal bleeding. the avm 's location, starting at the left lateral apex of the cesarean scar and extending into the parametrium, necessitated a radical hysterectomy. the prevalence of uterine avms has increased with the rise in surgical obstetrics. in patients with a failed prior interventional procedure, the location of the avm within the abnormal uterine scar tissue requires familiarity with radical pelvic surgical techniques that are normally used in cancer surgery in order to definitively treat this delayed obstetrical complication. uterine arteriovenous malformations (avms) are uncommon and can present as life - threatening hemorrhage, accounting for 12% of all genital hemorrhage. acquired uterine avms are typically a result of trauma or instrumentation, although they have also been associated with gestational trophoblastic disease, endometrial cancer, diethylstilbestrol exposure and intrauterine devices. initial imaging is done by ultrasonography, although mri is frequently necessary to identify the extent of the lesion. herein, we present a patient who required radical surgery despite selective embolization for persistent hemorrhage secondary to an acquired uterine avm. a 32-year - old g3p2012 presented with sonographic findings indicating a cesarean scar ectopic pregnancy. the lower uterine segment was 6 mm thick, incorporating the bladder, uterine serosa, myometrium, decidua and trophoblast. laparoscopically, the uterine serosa appeared normal. during the curettage she had a 1.5-liter postpartum hemorrhage, which stopped after placement of an intrauterine compression balloon as a tamponade. ultrasound revealed a multicystic structure with low - resistance arterial flow on doppler, inseparable from the anterior lower uterine segment (fig. 1). mri demonstrated multiple, enlarged vessels extending from the myometrium to the left broad ligament, which raised suspicion of an avm (fig. the left uterine arteriography suggested the presence of a large, high - flow arteriovenous fistula (avf), with drainage through the left internal iliac vein and left gonadal vein. the lesion was successfully embolized with coils, and an angiogram confirmed cessation of the flow. other multiple, small avfs supplied by both uterine arteries were considered clinically insignificant given the occlusion of the larger avf. repeat angiography demonstrated a hypervascular midline mass arising from the anterior division of the internal iliac arteries bilaterally, without a dominant feeding artery. angiographically, these represented multiple, small avfs, which were larger and more numerous than those previously noted, consistent with acquired avm (fig. 3). given her recurrence of symptoms despite vascular embolization with coils, a step - wise procedure was planned : gelfoam (pfizer, new york, n.y., usa) bilateral embolization of the internal iliac arteries, followed by a hysterectomy. gelfoam embolization of the internal iliac arteries resulted in a 4060% reduction in vascularity of the uterine avm (fig. 4). grossly, the avm originated from the prior hysterotomy site and extended into the left parametrium. dissection of the potential spaces of the pelvis was undertaken, and the bladder and left ureter were dissected away from the avm. the right and left round ligaments were ligated and the anterior and posterior leaves of the broad ligaments were incised. on the right side, the utero - ovarian ligament was transected and the right fallopian tube and ovary were preserved. on the left side, the avm extended into the mesosalpingeal plexus and involved the left gonadal vessels. the posterior broad ligament incision was extended to the left paracolic gutter. above the pelvic brim, the left gonadal vessels (infundibulopelvic vessels) were ligated above the extension of the avm, and the left fallopian tube and ovary were resected en bloc with the hysterectomy specimen (see below). the peritoneum overlying the anterior cul - de - sac and bladder flap was densely adherent to the cesarean section hysterotomy scar. the right paravesical and pararectal spaces were then opened and the right ureter was identified and freed up to the right ureteral tunnel. the right uterine artery was ligated at the level of the internal cervical os followed by the ligation of the right cardinal ligament. this mobilized the right side of the uterus down to the level of the upper one third of the vagina. the left parametrium contained the avm that extended from the midportion of the cesarean section hysterotomy site to the left pelvic sidewall. the ureter, which had been identified at the level of the left pelvic brim, was dissected free from the medial leaf of the left broad ligament. the left uterine artery and vein were isolated at their origin off the internal iliac vessels (hypogastric vessels) and ligated. the left paravesical space was opened, the superior vesical artery was ligated, and the space of retzius was opened ; this allowed the mobilization of the left lateral edge of the bladder. the left ureteral tunnel was then fully opened, and the ureter was completely mobilized away from the avm (fig. 5). next, the left side of the bladder was sharply dissected away from the avm and the hysterotomy scar. the left parametrium was ligated at the left pelvic sidewall, and the inferior aspect of the avm was released. the uterus, cervix, left adnexa and upper one third of the vagina were then resected (fig. upon pathologic examination, the enlarged vessels and arteriovenous communications were considered part of a placenta percreta with dehiscence of hysterotomy and subinvolution of the placental implantation site, which extended into the parametrium (fig. this case illustrates the need for radical pelvic surgical techniques, which are used in cervical cancer surgery, to safely resect a hysterotomy scar avm that presented with life - threatening hemorrhage. congenital and acquired uterine avms have a similar pathophysiology : avms are high - flow systems, with blood coursing through to the venous system under arterial pressure and flow. acquired avms of the uterus are characterized by single or multiple avfs which represent the direct connection of an artery and vein without an intervening capillary bed, as distinct from congenital avms which are characterized by a nidus of poorly differentiated blood vessels connecting the arterial and venous systems. the venous system is unable to accommodate the high - pressure, high - flow state, resulting in a higher propensity for bleeding. arteriography can distinguish between an avm and an avf by demonstrating the presence or absence of a nidus, respectively. avms require occlusion of the nidus, whereas avfs require occlusion of the fistulous communication or its feeding vessels. when the arterial territory distal to the avf can withstand ischemia and the distal arteries pose no risk of retroperfusion of the avf, the arterial feeders may be occluded with embolic particles (i.e. gelfoam). as acquired uterine avms contain single or multiple avfs, gelfoam embolization was performed in this patient because the risk of retroperfusion was considered less important given the planned hysterectomy. the technical and clinical success rates of uterine artery embolization for traumatic avms are reportedly 100 and 93%, respectively. treatment of acquired avms requires occlusion or resection of all arteriovenous communications to prevent further life - threatening hemorrhage. occlusion of only one large avf was achieved during the first interventional procedure ; it became evident that the extent of the avm was prohibitive for occlusion with particle embolization alone. the surgeon must have familiarity with the pelvic spaces and vasculature to perform the dissection and preserve anatomic function. the role of the advanced gynecologic surgeon is known in the acute setting, such as during intraoperative hemorrhage or unintended visceral injury, but should also be recognized in cases with distorted pelvic anatomy. abnormal implantation in the cesarean scar bed accounts for 6% of ectopic pregnancies in multiparas. this may result from failed obliteration of placental bed vessels in the absence of retained placental tissue. conversely, retained villi may be found within a uterine pseudoaneurysm, with subsequent recruitment of collateral vessels. this has been reported in placenta accreta and may be similar to our experience, given the pathologic diagnosis. the roles of the interventional radiologist and advanced gynecologic surgeon will continue to evolve in the management of delayed obstetrical complications given the pathophysiology and anatomic challenges of the scarred pelvis.	backgroundacquired arteriovenous malformations (avms) can develop after uterine instrumentation. the increased risks of vascular changes, including abnormal placentation, after repeated cesarean sections are well studied. herein, we describe a patient with delayed hemorrhage from a uterine avm, following dilation and curettage for a cesarean scar pregnancy.casea 32-year - old g3p2 presented with a cesarean scar ectopic pregnancy managed with dilation and curettage, which incurred a 1,500-ml blood loss. within 6 weeks, she returned with 2 episodes of vaginal bleeding. initial angiography demonstrated a high - flow arteriovenous fistula, which was coiled. vaginal hemorrhage recurred ; repeat angiography demonstrated a large avm. gelfoam embolization of the bilateral internal iliac arteries reduced the vascularity of the avm. the avm 's location, starting at the left lateral apex of the cesarean scar and extending into the parametrium, necessitated a radical hysterectomy. pathologic examination revealed a placenta percreta extending into the parametrium.conclusionthe prevalence of uterine avms has increased with the rise in surgical obstetrics. in patients with a failed prior interventional procedure, surgical management is necessary to prevent life - threatening hemorrhage. the location of the avm within the abnormal uterine scar tissue requires familiarity with radical pelvic surgical techniques that are normally used in cancer surgery in order to definitively treat this delayed obstetrical complication.
in thailand, cholangiocarcinoma (cca), a malignant tumor derived from bile duct epithelium, occurs with a high incidence in tropical countries where it is associated with liver fluke (opisthorchis viverrini) infestation and nitrosamine ingestion. cca shows high mortality and presents challenges in diagnosis ; so prognosis for cca patients is rather poor. the most widely used circulating marker for cca is carbohydrate antigen (ca) 19 - 9. however, (ca) 19 - 9 is also elevated in pancreatic cancer, gastric cancer, and primary biliary cirrhosis and has been shown it gives false positive results. carcinoembryonic antigen (cea) is the other common tumor marker used for detecting cca. cea is not specific, being mainly used for colorectal cancers, and can be elevated in other types of cancer, such as gastrointestinal or gynecologic malignancies. differences in expression profiles between normal liver and cca tissues were studied, because cca is contained in liver tissue and is suggested to arise from the same stem cells as hcc. we have previously compared cca and hcc cell lines using proteomic techniques in order to investigate potential cca markers for early diagnosis. comparison of 2d - page patterns for a cholangiocarcinoma cell line (hucca-1) and two hepatocellular carcinoma cell lines (hepg2 and hcc - s102) showed that cytokeratin 7 (ck7), cytokeratin 19 (ck19), an unknown proteins (u2/2), and galectin-3 were found in cca but not in hcc. an extension of this study investigated membrane proteins and cytosolic proteins, which showed that ten membrane proteins were found in hucca-1 but not in hcc - s102, including mitogen - activated protein kinase kinase kinase 2, calgizzarin, integrin alpha-6 precursor, ezrin, and hippocalcin - like protein 1. the subproteomic approach used here may be useful for developing potential biomarkers for early detection of cca. however, proteomic studies using cell lines have limitations, since it is not known whether the differentially expressed proteins will be present in accessible biological fluids such as plasma, serum, or urine or not. cells and tissues secrete proteins into the extracellular environment, the secretome, which may reflect a large variety of pathological conditions and may be a useful source of biomarkers. these proteins are not only components of the extracellular matrix and biological fluids but are also involved in blood coagulation, immune defense, signal transduction, and carcinogenesis. certain proteins secreted from cancer cells that enter the circulatory system can be utilized as targets for monitoring or screening for the presence of cancer cells. the cancer secretome, such as that from hepatocellular carcinoma, lung cancer, breast cancer, and oral squamous cell carcinoma, has been studied by many research groups to determine the release of the total proteins by cancer cells. this technique provides useful tools for the discovery of novel biomarkers, by using a cell culture model system in which the cells were grown in serum - free media for proteomic analysis [13, 14 ]. secretome analysis of nasopharyngeal carcinoma (npc) cell lines has been studied by sds - page and maldi - tof ms and revealed several potential npc protein markers [15, 16 ]. since the secretomes of cholangiocarcinoma have not previously been reported, we have used conditioned media to compare the secretomes of cholangiocarcinoma cell line (hucca-1) with those of hepatocellular carcinoma cell lines (hcc - s102, hepg2, sk - hep-1, and alexander). expression of proteins was studied by sds - page and lc / ms / ms, and overexpression of proteins in hucca-1 was confirmed by using 1-de and 2-de immunodetection. tissue samples were collected from department of surgery, and liver fluke and cholangiocarcinoma research center, faculty of medicine, khon kaen university. ethical clearance for the tissues was approved by the ethics committee for human research of khon kaen university (he471214). after resection, specimens were immediately taken to a pathologist, who sampled both the tumor itself and adjacent normal - appearing bile duct. both samples were then snap - frozen in liquid nitrogen within 10 minutes of removal from the patients and stored at 80c until analysis. the details of types, ages, genders, grades, and histopathology are shown in table 1. cholangiocarcinoma cell line, hucca-1 derived from bile duct tumor mass of thai patient, was kindly provided by professor sirisinha and grown in ham 's f12 culture medium (hyclone laboratories, logan, ut, usa), containing 15 mm hepes and supplemented with 10% fetal bovine serum (fbs, hyclone laboratories), 100 u / ml penicillin, and 100 g / ml streptomycin and 125 ng / ml amphotericin b. hepatocellular carcinoma cells, hcc - s102, established from thai patient was kindly provided by dr. sumalee tungpradakul and grown in rpmi 1640 (gibco, grand island, ny, usa) containing 25 mm hepes, supplemented with penicillin (100 u / ml), streptomycin (100 g / ml) amphotericin b (125 ng / ml) (gibco), and 10% fbs. another human hepatocellular carcinoma cell line, the alexander cell line, a human hcc - derived cell line secreting hbsag, originally obtained from american type culture collection (rockville, md, usa) was kindly provided by dr. ananda nisalak of the department of virology, the armed forces research institute of medical sciences (afrims), bangkok, thailand. the hepatoblastoma cell line, hepg2 and hepatocellular cell line, sk - hep-1 were purchased from american type culture collection. the alexander hepg2, and sk - hep-1 cell lines were grown in dmem (gibco) with 10% fbs, 100 u / ml penicillin, 100 g / ml streptomycin, and 125 ng / ml amphotericin b. all cells were maintained at 37c in a humidified atmosphere, 95% air, 5% co2 at 37c. after incubating the cells in complete media or serum - free media for 24 hours, the numbers of viable cells and dead cells were counted by using the trypan blue dye exclusion method. the percentage of cell viability was expressed as the ratio of total viable cells to the sum of total viable and dead cells. to obtain culture supernatants, approximately 3 10 cancer cells were grown to 80% confluence and then the cells were washed with serum - free medium 2 times before incubation in serum - free medium for 24 hours. after incubation, the conditioned medium was harvested and centrifuged 800xg at 4c for 10 minutes to remove suspended cells. the supernatant was dialyzed against distilled water using a dialysis membrane with molecular weight cutoff 3500 da (cellu sep, texas, usa) for 48 hours and then concentrated by speedvac. the concentrations of total proteins were determined by the bradford protein assay reagent (bio - rad laboratories, ca). twenty micrograms of conditioned media from five cell lines were resolved on 8%14% gradient sds - page (100 105 0.75 mm)., san francisco, ca, usa) at 12 ma, room temperature for 2 hours. the protein was stained with coomassie brilliant blue r-250 (serva electrophoresis gmbh, heidelberg, germany). protein extracts, separated by 12% sds - page and transferred onto nitrocellulose membrane (hybond ecl, ge healthcare, buckinghamshire, uk), were probed with monoclonal antibodies against ngal (santa cruz biotechnology, ca, usa). proteins of interest were detected using the enhanced chemiluminescence (ecl) plus detection system, with high - performance film (hyperfilm ecl ; ge healthcare). three hundred micrograms of conditioned media from hucca-1 were applied by overnight in - gel rehydration of 70 mm, using nonlinear ph 310 ipg gel strips (ge healthcare). the first dimension (ief) was performed to give a total of 8000 vh on an ipgphor (ge healthcare). focused ipg strips were equilibrated for 10 minutes in a solution (6 m urea, 30% glycerol, 1% sds 50 mm tris - hcl buffer (ph 6.8), and 1% dtt), and then for an additional 10 minutes in the same solution except that dtt was replaced by 2.5% iodoacetamide. the ipg strips were then applied to the second dimension 12% t sds - page (100 105 1.5 mm). tissues were homogenized in phosphate buffered saline containing 0.2 mm phenylmethanesulfonylfluoride, 2 g / ml pepstatin a, 1 g / ml bestatin, and centrifuged for 20 minutes at 15 000 rpm. samples (10 g protein) were mixed with sample buffer, boiled, and applied to 15% t sds polyacrylamide gels (100 80 0.75 mm)., san francisco, ca, usa) at 10 ma, room temperature for 1.5 hours, followed by electroblotting of proteins from the gel onto pvdf membranes (immobilon - p ; millipore, billerica, ma, usa) at 100 v for 30 minutes at 4c. after blocking in 5% nonfat dry milk, membranes were probed with 1 : 200 diluted anti - ngal monoclonal antibody (santa cruz biotechnology, santa cruz, ca, usa), repeatedly washed in 20 mm tris buffered saline, ph 7.6, containing 0.1% tween 20, and then incubated in 1 : 5000 rabbit antimouse immunoglobulin g (igg ; dako cytomation, glostrup, denmark) for 1 hour. after washing, the reaction was developed using the ecl plus detection system, with high - performance film (hyper - film ecl ; ge healthcare). the solvent were discarded and gel particles were dried completely by speed vac. reduction and alkylation were performed by swelling the gel pieces in 50 l buffer solution (0.1 m nh4hco3, 10 mm dtt and 1 mm edta) and incubating at 60c for 45 minutes. after cooling, the excess liquid was removed and quickly replaced by the same volume of freshly prepared 100 mm iodoacetamide in 0.1 m nh4hco3 solution. the iodoacetamide solution was removed and the gel pieces were washed with 50% acetonitrile in water, 3 times for 10 minutes each time, and the gel pieces were completely dried. aliquots of trypsin (promega corporation, wi, usa) (1 g trypsin fifty l of digestion buffer (0.05 m tris hcl, 10% acetonitrile, 1 mm cacl2, ph 8.5) and 1 l of trypsin were added to the gel pieces. after incubating the reaction mixture at 37c overnight the gel pieces were then extracted by adding 60 l of 2% freshly prepared trifluoroacetic acid and incubating for 30 minutes at 60c. the extract and the saved digestion buffer lc / ms / ms analyses were carried out using a capillary lc system (waters) coupled to a q - tof mass spectrometer (micromass, manchester, uk) equipped with a zspray ion source working in the nanoelectrospray mode. the tryptic peptides were concentrated and desalted on a 75 m i d 150 mm c18 pepmap column (lc packings, amsterdam, the netherlands). eluents a and b were 0.1% formic acid in 97% water, 3% acetonitrile, and 0.1% formic acid in 97% acetonitrile, respectively. six l of sample was injected into the nanolc system, and separation was performed using the following gradient : 0 minute 7% b, 35 minutes 50% b, 45 minutes 80% b, 49 minutes 80% b, 50 minutes 7% b, and 60 minutes 7% b. the database search was performed with proteinlynx screening swiss - prot and ncbi. for some proteins that were difficult to find, the mascot search tool available on the matrix science site screening ncbinr was used. the search parameters were set as follows : peptide mass tolerance = 1 da ; ms / ms ion mass tolerance = 1 da ; enzyme set as trypsin and allowance was set up to two missed cleavages ; peptide charges were limited to 2 + and 3 +. the proteins were identified with p - values.05 and mascot scores > 35 were considered as promising hits. the secreted proteins from conditioned media of hucca-1, hcc - s102, hepg2, sk - hep-1, and alexander cells were resolved by sds - page and visualized by coomassie blue as shown in figure 1. the secreted protein patterns were enriched in the conditioned media and showed patterns that differed significantly in each cell line. the effect of 24-hour serum starvation on the viability of the five cancer cells cultured was tested and the results showed that this treatment had little effect on cell viability (figure 2). because of the high sensitivity of mass spectrometry in identifying mixtures of proteins, the stained gels were marked and cut into 0.5 mm slices from the gel top to dye front as shown in figure 1. forty - eight total gel bands were subjected to in - gel tryptic digestion and the proteins were identified by lc / ms / ms. there are 83 proteins secreted from the hucca-1 cell line as shown in table 2. the secreted proteins from 4 hepatocellular carcinoma cell lines (hcc - s102, hepg2, sk - hep-1, and alexander) were collected in the form of a database, together with those of other cell lines including lung, breast, cervical, and oral cell lines, used in our group (manuscript in preparation). the secreted proteins from 4 hepatocellular carcinoma cell lines are shown in table 1 in supplementary material available online at doi:10.1155/2010/437143. the molecular weights shown in table 2 and supplementary table 1 are theoretical values from databases. some experimental molecular weights differed from theoretical values because the bands were from sds - page, in which the proteins were denatured and found as isozymes. from table 2 and supplementary table 1, some proteins containing one matched peptide were considered because the peptide score was greater than 35, higher than the mascot score of 20, which kristiansen. we also manually selected only the peptide match that had a length of at least 8 amino acids with sequence tag of at least three amino acids to be a good y - ion series.. then proteins were classified by their functions into various categories, namely, chaperone / stress response, cell cycle, cytoskeleton / mobility, dna replication / gene regulation, extracellular matrix, immunological response, ion channels, metabolism, protection and detoxification, protein synthesis and degradation, signal transduction, transport / binding proteins, tumor - associated proteins, and unannotated / function inferred. when the hucca-1 cell line is compared to the 4 hepatocellular carcinoma cell lines, all cell lines secreted distinct protein profiles. we also found distinct secreted protein profiles when we compared the secreted proteins to different types of cell lines (liver, lung, breast, cervical and oral). thus, some 30%50% proteins were expressed at the same level in 5 cell lines, while 50%70% proteins showed differences in expression. forty - nine secreted proteins were found only in hucca-1 which was marked in table 2, including laminin 5 beta 3, heat shock 90 kda protein 1, heat shock 70 kds protein 8 isoform 1 variant, grp78 precursor, desmoglein-4 nephroblastoma overexpressed precursor, neutrophil gelatinase - associated lipocalin (lipocalin 2, ngal), desmoplakin, cathepsin d, dnaj homolog subfamily b member 11, annexin iv variant, annexin a5, ras - related protein rap-1a, rhov protein, and rotatin isoform cra_e. of the overexpressed proteins found only in hucca-1, 15 appeared to be proteins related to cancer. 1-de immmunoblotting was used to verify the presence of 4 proteins by using specific antibodies for nephroblastoma overexpressed precursor and neutrophil gelatinase - associated lipocalin (lipocalin 2, ngal). immunodetection only confirmed the presence of lipocalin 2 (figure 3(b)), but not the presence of the other two proteins. expression of lipocalin 2 was found only in the conditioned media of hucca-1, but not in the conditioned media of the other 4 cell lines (figure 3(b)). furthermore, there was no expression of lipocalin 2 in the cell lysate of any of the 5 cell lines, including hucca-1 (figure 3(a)). the 2-de proteomic pattern of conditioned media of hucca-1 with coomassie blue staining is shown in figure 4(a) and 2-de immunoblotting of the same sample was used to detect the lipocalin 2 forms (figure 4(b)), revealing 4 spots with mw / pi 23.5/6.0, 23.5/6.5, 23.0/7.3, and 22.5/8.7. these different forms are likely to result from posttranslational modifications. to confirm that lipocalin 2 is highly expressed in cholangiocarcinoma, the 12 homogenate samples from the pairs of normal and cancer of cholangiocarcinoma tissues described in table 1 were resolved by sds - page gels and immunoblotted with monoclonal antibody to lipocalin 2. the results clearly showed a positive band of 23 kda in all cancer tissues (figure 5), with 9 out of the 12 cases showing high expression level of lipocalin 2, while the corresponding normal tissues were all negative. the study of the cancer secretomes by using proteomic technology has greatly accelerated in recent years. with the rapid progress in mass spectrometry (ms), bioinformatics, and analytical techniques, our present work has investigated the cell secretomes of cholangiocarcinoma (cca) compared to 4 hepatocellular carcinoma (hcc) cell lines. with each cell line, three repeat experiments were performed by collection of the conditioned media from cca and hcc cell lines, followed by concentration and precipitation by tca / acetone. the proteins were then run onto sds - page, gel bands excised consecutively, and proteins analyzed by lc / ms / ms. some bands showed low levels of matching, some bands showed blue color, and some were almost clear. the pattern on sds - page of secreted proteins and the total identified proteins were differed with each cell line. when the protein expression of the hucca-1 cell line is compared to the hcc cell lines, only 14, 14, 19, and 6 proteins from alexander, hcc - s102, hepg2, and sk - hep-1, respectively, matched with hucca-1 (data not shown). proteins secreted into conditioned medium may be tumor specific and can represent potential biomarkers that may circulate in the blood stream. some interesting secreted proteins expressed only in cholangiocarcinoma include laminin 5 beta 3, heat shock 90 kda protein 1, heat shock 70 kds protein 8 isoform 1 variant, grp78 precursor, desmoglein-4 nephroblastoma overexpressed precursor, neutrophil gelatinase - associated lipocalin (lipocalin 2, ngal), desmoplakin, cathepsin d, dnaj homolog subfamily b member 11, annexin iv variant, annexin a5, ras - related protein rap-1a, rhov protein, and rotatin isoform cra_e. for example, laminin-5 (lama3, lamb3, and lamc2) is a heterotrimeric glycosylated protein that belongs to th ln family and is formed by 332 chains assembled with disulfide bonds. ln-5 is widely expressed in the human body but shows differential expression in metastatic and nonmetastatic hepatocellular carcinoma [21, 22 ]. ln-5 also plays an important role in cell migration during tumor invasion and tissue remodeling, and laminin 5 2 chain and 3 chain have both been suggested to be important in the invasiveness of cancer cells. unfortunately, no commercial antibody appears to be available for laminin 5 3 chain ; expression of the protein could not be validated in cholangiocarcinoma tissues. laminin 2 chain exhibits aberrant expression in a stepwise manner through different aggressive stages of tumor progression. nephroblastoma overexpressed (nov) belongs to the ccn family of genes that encode secreted proteins associated with the extracellular matrix (ecm) and exert regulatory effects at the cellular level. nov is likely to play a role in cell growth regulation, in the progression and in the metastatic potential of melanomas. the expression of this protein appears to be associated with a higher risk of developing metastases in ewing 's sarcoma. we found the high expression of nov secreted in the media of the cholangiocarcinoma cell line. neutrophil gelatinase - associated lipocalin (ngal or lipocalin 2) is a prominent member of the lipocalin family. it was first isolated as a 25 kda glycoprotein covalently bound with matrix metalloproteinase-9 (mmp-9) in human neutrophil. it is a secreted acute phase protein, which is also upregulated in multiple cancers, including breast, lung, and pancreas. desmoplakin, a specialized adhesion junction protein and the principal plaque protein of desmosomes, has been found in various tissues including heart, skin, and meninges. the biological significance of desmoplakin has been recently reported in both autosomal dominant and autosomal recessive disorders from naturally occurring human gene mutation. desmoplakin was found to be abnormal in many conditions including autoimmune blistering diseases, epithelial malignancies, and blood vessel morphogenesis. desmoplakin was also found to be secreted by the cholangiocarcinoma cell line in our study. cathepsin d, the aspartic protease, is an independent marker of poor prognosis in human breast cancer. cathepsin - d was reported to play an essential role in multiple tumor progression steps, affecting cell proliferation, angiogenesis, and apoptosis. a cathepsin d - like protein has also been reported from the gut and other tissues of the human liver fluke opisthorchis viverrini where long - standing infection is associated with cancer of the bile ducts or cholangiocarcinoma. this protein was also found in the excretory and secretory products of cultured adult flukes, indicating a role in host - parasite relationships. the annexins are family of calcium - regulated phospholipid - binding proteins with diverse roles in cell biology. there are 12 human annexin subfamilies (a1a11 and a13) that have been found to have various intra- and extracellular roles in a range of cellular processes such as cell signalling, ion transport, cell division, and apoptosis [31, 32 ]. annexin a1 also has a role in controlling the inflammatory response while annexin a2 is present on the external surface of endothelial cells. annexin a2 and annexin a4 appeared to be potential markers of interest for diagnosis of colorectal cancer. the present studies showed that annexin a1, annexin a2, annexin iv variant, and annexin a5 were expressed in the cholangiocarcinoma cell line. the presence of the proteins nephroblastoma overexpressed precursor and neutrophil gelatinase - associated lipocalin (lipocalin 2, ngal) was validated in the cell lysates and conditioned media from all 5 cells by using specific antibodies. then 2-de immunoblotting of the conditioned media showed the expression of lipocalin 2 as 4 spots at mw / pi 23.5/6.0, 23.5/6.5, 23.0/7.3, and 22.5/8.7 (figure 4(b)). the posttranslation modification of the lipocalin 2 has not been reported yet and further studies will be needed to provide more information. finally, we verified the expression of lipocalin 2 in 12 homogenate samples from the pairs of normal and cancer tissues from cholangiocarcinoma patients. the lipocalin 2 antibody clearly detected a band of 23 kda in all cancer tissues, while the pairs of normal tissues were all negative. of the cancer tissues, 9 out of 12 cases showed high expression levels of lipocalin 2. however, since the cases studied here were in the late stage (grade 3 - 4) of cancer, it is of interest to see if tissues at the early stages of cholangiocarcinoma show lipoclain 2 expression. further studies will investigate the expression of lipocalin 2 in different types and stages of cholangiocarcinoma in order to understand its relationship to grades and types of cancer. proteomic techniques have been used to compare the secretomes or proteins secreted into the external environment, cholangiocarcinoma, and hepatocellular carcinoma cell lines. distinct proteins were found, in particular lipocalin 2, which may have potential value as a biomarker for cholangiocarcinoma.	cholangiocarcinoma (cca) and hepatocellular carcinoma (hcc) occur with relatively high incidence in thailand. the secretome, proteins secreted from cancer cells, are potentially useful as biomarkers of the diseases. proteomic analysis was performed on the secreted proteins of cholangiocarcinoma (hucca-1) and hepatocellular carcinoma (hcc - s102, hepg2, sk - hep-1, and alexander) cell lines. the secretomes of the five cancer cell lines were analyzed by sds - page combined with lc / ms / ms. sixty - eight proteins were found to be expressed only in hucca-1. examples include neutrophil gelatinase - associated lipocalin (lipocalin 2), laminin 5 beta 3, cathepsin d precursor, desmoplakin, annexin iv variant, and annexin a5. immunoblotting was used to confirm the presence of lipocalin 2 in conditioned media and cell lysate of 5 cell lines. the results showed that lipocalin 2 was a secreted protein which is expressed only in the conditioned media of the cholangiocarcinoma cell line. study of lipocalin 2 expression in different types of cancer and normal tissues from cholangiocarcinoma patients showed that lipocalin 2 was expressed only in the cancer tissues. we suggest that lipocalin 2 may be a potential biomarker for cholangiocarcinoma.
ankylosing spondylitis (as) is a chronic, inflammatory and rheumatic disease from the group of spondyloarthropathies (spas), which primarily involves the sacroiliac joint and spine, and less frequently peripheral joints, and can also present with non - joint involvement. the human leukocyte antigen (hla)-b27, present in the majority of the patients, contributes to pathophysiological mechanisms of the disease. non - joint involvement may be observed as ophthalmological, pulmonary, neurological, and cardiac manifestations. cardiac involvement in as patients has been reported at the rate of 2 - 10% [1 - 3 ]. cardiac involvement in as may be in the form of aortic insufficiency, aortitis at the ascending aorta, atrioventricular (av) block, or branch block. mitral insufficiency in as is rare, but can cause cardiac failure [4 - 7 ]. cardiovascular system involvement is more frequent in patients with long - term as and with peripheral joint involvement. the frequency of aortic dissection has been reported as approximately 2.9 in 100,000. the most frequently observed symptom in these patients is chest pain ; however, it may present with atypical findings, such as having no pain or as the presence of cardiac, neurological, or ischemic extremities [11, 12 ]. aortic dissection is generally observed between the ages of 50 and 70 years, and is found in men at a rate that is double of that observed in women. in addition, causes such as acute joint rheumatism, syphilis, traumatic aortic insufficiency, bacterial endocarditis, marfan s syndrome, and as weaken the tunica media of the aorta in particular, and form the basis for the dissection to develop. sources of stroke can appear within lesions, especially those within the proximal aorta, and paraplegia can develop in cases with distal lesions that involve the spinal arteries. even though stroke and paraplegia develop rarely, their incidence in cases of aortic dissection is between 2% and 8%. inflammatory cellular infiltrates, cytokines, genetic, and environmental factors are suspected to contribute to the etiology of as. many studies have demonstrated that chronic, systemic, and inflammatory states within the spas may also cause cardiac and vascular pathologies. although cardiac involvement may be clinically asymptomatic in patients with as, it can cause significant problems in some patients. the incidence of cardiovascular involvement in as ranges between 10% and 30%. when it is taken into account that cardiac changes may start before the clinical symptoms of as present, early diagnosis and treatment are even more important [17 - 19 ]. more than one method is described in the literature, such as 24-h holter monitoring and echocardiography, to assist in assessing the cardiac signs of rheumatic disease. yildirir have demonstrated that the calculation of qt dispersion on a standard ecg revealed valuable information regarding arrhythmias in a more economic, easily applicable and rapid manner. patients at an increased risk for arrhythmias could therefore be directed for more advanced examinations. the increased frequency of aortitis in patients with as was first found in the 1930s. today, it is known that as is not only related to aortic pathology, but also to cardiac complications such as left ventricular diastolic dysfunction, pericarditis and conduction disorders (av block or branch block), and rarely mitral insufficiency, cardiac failure and cardiomegaly [1, 20 - 22 ]. the most frequently observed cardiac sign in as patients is a conduction abnormality, which generally precedes other cardiac findings such as valve insufficiency. cardiac involvement has been detected in 5 - 10% of patients with as in the form of a conduction disorder or aortic insufficiency [23, 24 ]. although rheumatological signs of as are absent in hla - b27-positive patients, an increase in conduction disorders in these patients has been detected. indeed, 20% of patients with permanent pacemakers have been found to be positive for hla - b27. in a study by dik, first - degree av block conduction disorder was shown to have a significantly higher prevalence in as patients compared to the normal population and conduction disorders were associated with disease activity and duration. there are two common theories about the etiology of conduction disorders in patients with as : inflammation that leads to damage to the interventricular septum wall and av node dysfunction that forms as a result of damage to the arterial supply to the av node. these processes combine to contribute to av extrasystole and the formation of av block [19, 23 ]. toussirot suggested that autonomic nervous system irregularities can lead to conduction defects and arrhythmias, and therefore affect prognosis unfavorably. in patients with as, they have defined esr and crp levels and disease severity as factors associated with the autonomic nervous system [9, 25, 26 ]. yildirir did not find a statistically significant difference between as patients and the control group in terms of autonomic dysfunction, but accepted that the study was limited by the fact that all patients were young and on significant anti - inflammatory treatment, which could have slowed or stopped the progression of autonomic dysfunction. in as, the presence of aortic root and valve disease is related to the duration of underlying disease. one of the primary pathophysiological explanations for valve disease in as proposed by bulkley and roberts has been that aortic root dilatation results from fibrous growth along the intima, after they examined the autopsy findings of eight patients with as. in more detailed examinations, it has been observed that the cellular inflammatory process that leads to endarteritis around the aortic root and valve and is sustained by platelet aggregation leads to tissue thickening, as well as to aortic valve insufficiency by stimulating fibroblast hyperactivity [1, 28 ]. in their study, roldan examined the aortic root and valves using transthoracic echocardiography (tee) in patients with as, and found aortic root and valve disease in 82% of patients with as compared to the control group. other findings included aortic root thickening, increased aorta root stiffness and vessel dilatation. in their study with follow - up for > 39 months, they detected 25 patients (24%) who developed new aortic root or valve abnormalities, while current valve insufficiencies significantly worsened in 12%, and abnormalities were resolved in 20%. aortic valve insufficiency is mostly observed in patients with as ; however, mitral insufficiency does occur but at a lesser frequency. the suggested mechanism is that the fibrosis of sub - aortic tissues may reach the mitral valve leaflets. another potential mechanism that causes mitral insufficiency is mitral valve stenosis, caused by left ventricular hypertrophy as a result of severe aortic insufficiency. it has not been fully clarified whether cardiac changes as a result of as develop secondary to the primary involvement of cardiomyocytes or secondary to aortic and valvular involvement. in some trials in which the left ventricular function (lvf) of patients with as was examined, defects in systolic and diastolic functions were frequently reported [24, 29, 30 ]. brewerton studied myocardial tissue biopsies in 28 patients with as who had no ischemic heart disease, valve disease or hypertension, and observed a slight increase in diffuse interstitial connective tissue ; however, no inflammatory change or amyloid was detected. furthermore, no hypertrophy was observed in the hearts examined. of the studies in which systolic functions were examined, riberio detected varying degrees of systolic dysfunction in five patients with apparent contraction disorder in a series of 28 patients, while brewerton have detected this in 16 out of 30 patients. gould described several abnormalities in the myocardial functions of patients with as. the most important of these is diastolic dysfunction, suggesting a decrease in left ventricular compliance. in their studies, crowley detected diastolic dysfunction at the rate of 20% (59 patients), and brewerton et the authors suggested from their histopathological data that there was an increase in myocardial inflammation and the connective tissue / myocyte rate. in their studies, yildirir specifically examined the diastolic filling functions of as patients, showing a statistically significant slow early diastolic filling rate (e - wave rate), and a late rapid diastolic filling rate (a - wave rate). in these patients, early diastolic filling / late diastolic filling rate ratios were assessed as being generally lower than controls. the diastolic dysfunction in as is usually not sufficiently severe to cause diastolic heart failure. coronary flow reserve (cfr) some experts have emphasized the prognostic significance of this measurement in patients with atherosclerosis. in their study published in 2008, caliskan used transthoracic doppler echocardiography (ttde) to assess cfr in as patients, and determined that cfr levels were decreased. furthermore, crp and tnf - alpha levels correlated with cfr values in these patients. in another study performed in 2010 with a very limited sample size (17 patients), the myocardial infarct rate was examined in as patients, and an increase of 2 - 3 fold was found in the infarct rate of the as population. it is important that there is no difference between the use and type of anti - rheumatic treatment among as patients with or without a history of myocardial infarct. divecha showed in 2005 that not only the level of inflammatory markers (e.g., crp and il-6) increased in patients with as, but these caused insulin resistance and dyslipidemia as a result of their effects on lipid metabolism and insulin activity, resulting in atherogenesis. it has been reported that a chronic elevation at the levels of these markers, even though sub - clinical, would accelerate the risk for coronary heart disease in patients with no autoimmune diseases [33, 35 ]. the effects of as on the cardiovascular system significantly affect mortality and morbidity. if chest pain develops in at - risk individuals, aortic dissection should be considered, and urgent interventions should be performed as appropriate. therefore, the cardiological examination of patients with as should be emphasized especially in individuals with long - term disease and who are hla - b27-positive. the fact that cardiac involvement may occur before the emergence of clinical findings of as, and that an underlying disease such as as may be present in patients who have had aortic dissection, should be considered in affected patients.	ankylosing spondylitis is one of the subgroup of diseases called seronegative spondyloarthropathy. frequently, it affects the vertebral colon and sacroiliac joint primarily and affects the peripheral joints less often. this chronic, inflammatory and rheumatic disease can also affect the extraarticular regions of the body. the extraarticular affections can be ophthalmologic, cardiac, pulmonary or neurologic. the cardiac affection can be 2 - 10% in all patients. cardiac complications such as left ventricular dysfunction, aortitis, aortic regurgitation, pericarditis and cardiomegaly are reviewed.
periodontal diseases, including gingivitis and periodontitis, are a group of infectious diseases caused predominantly by different types of gram - negative anaerobic and micro - aerophilic bacteria that colonize the subgingival area resulting in long - term local and systemic elevation of pro - inflammatory prostaglandins and cytokines. several bacterial species such as porphyromonas gingivalis, prevotella intermedia, prevotella nigrescens, tannerella forsythia, treponema denticola, fusobacterium nucleatum, aggregatibacter actinomycetemcomitans, and campylobacter rectus have been identified in the subgingival biofilm of periodontal disease patients. a recent report suggested that high levels of a. actinomycetemcomitans, f. nucleatum, and p. intermedia were observed in the subgingival biofilm during the second and third trimesters of pregnancy, with increased susceptibility for gingivitis. changes in the physiological process during pregnancy can alter the inflammatory response by intensifying the gingival inflammation. clinical parameters such as bleeding on probing and pocket depth may increase during pregnancy, without associated increase in plaque index score, which reduces after parturition. this increased severity of periodontal disease is related to increased vascular permeability, reduced immune responses, and shifts in the composition of supra and subgingival microbial flora during the gestational period. it has been reported that the levels of a. actinomycetemcomitans and red complex bacterial species started to increase from 22 week of pregnancy in mothers of premature babies, while these bacterial levels remained stable in mothers at term delivery. moreover, after delivery, a 2.42-fold increase in the a. actinomycetemcomitans species was observed among mothers of premature babies, as compared to the mothers with term delivery. meta - analyses of reported studies have revealed a significant risk of preterm delivery and low birth weight among pregnant women with periodontitis. the odds ratio for preterm birth in mothers with periodontitis varied from 1.7 to 2.73, for low birth weight from 1.5 to 2.11, and for preterm birth and low weight from 2.35 to 3.57. about 25% of preterm low birth weight cases occur without even a suspected risk factor, but estimates suggest that 18.2% of all cases may be attributable to periodontal disease. it has also been reported that subjects with generalized periodontitis have a fivefold increased risk of preterm birth before 35 weeks of gestation and a sevenfold increased risk of delivery before 32 weeks of gestation. in addition, a significant association between preeclampsia and periodontal disease has been reported in the literature. hence, all meta - analyses have demonstrated that the periodontal disease could promote adverse pregnancy outcomes. studies have reported high prevalence of periodontal disease among pregnant women and a prevalence of preterm low birth weight babies of around 11.3% in saudi arabia. in addition, the risk of preterm low birth weight remained high despite controlling the other risk factors, suggesting possible correlation between periodontal disease and preterm low birth weight. all the previously mentioned associations between periodontal disease and adverse pregnancy outcomes and universal presence of periodontal disease provide strong rationale for gynecologists to enquire their patients about oral and periodontal health. gynecologists are the first - line healthcare professionals to come in contact with pregnant women. their awareness regarding association between periodontal disease and adverse pregnancy outcomes is extremely important in recognizing modifiable periodontal disease risk factors associated with pregnancy. also, incorporation of the periodontal care into gynecologic management may improve pregnancy outcomes, if early intervention is sought to reduce microbiologic load on oral tissues during pregnancy. until now, awareness of periodontal health and disease among practicing gynecologists remains undisclosed with lack of published literature from riyadh. hence, the present study was undertaken with an objective to determine knowledge of periodontal disease and pregnancy outcomes among the gynecologists practicing in riyadh, saudi arabia. this was a cross - sectional survey of a sample of gynecologists from riyadh, capital city of saudi arabia. sample size was calculated by considering the effect size of (chi - square tests) 0.3, alpha error probability of 0.05, and power of the study 0.94, with four degrees of freedom. it resulted in a sample size of 198, which was adjusted to the nearest number of 200. a list of all the government and private hospitals and polyclinics providing gynecologic / obstetric care was obtained from the directory of hospitals in riyadh. a total of 200 gynecologists working in these hospitals were then selected by employing simple random sampling technique. this study was approved by the research center of riyadh colleges of dentistry and pharmacy (rcsdp), riyadh. a structured, self - administered, closed - ended questionnaire with a letter mentioning the purpose of the study was distributed to the gynecologists during their consultation hours. instrument utilized in the present study was developed based on literature reviews of relevant published articles. a pilot study was conducted among a sample of 50 gynecologists by using modified version of the questionnaire in english language to ensure comprehensibility and reliability. a cronbach 's alpha of 0.87 was found, which meets the purpose of this study. the 50 questionnaires utilized in the pilot study were excluded from the final study sample. the questionnaire consisted of four sections with 12 closed - ended questions. section i included questions on age, gender, number of years in clinical practice, and practice sector (government or private) ; section ii included five questions on knowledge of periodontal disease and possible ways of prevention ; section iii included four questions on knowledge of periodontal disease and pregnancy outcomes ; and section iv included three questions on oral self - care attitudes among gynecologists. questionnaire responses were coded and entered into a statistical package for social sciences (ibm - spss version 19) software for analysis. chi - square test was applied to assess the differences in responses between different age groups, duration of practice, gender, and sector. this was a cross - sectional survey of a sample of gynecologists from riyadh, capital city of saudi arabia. sample size was calculated by considering the effect size of (chi - square tests) 0.3, alpha error probability of 0.05, and power of the study 0.94, with four degrees of freedom. it resulted in a sample size of 198, which was adjusted to the nearest number of 200. a list of all the government and private hospitals and polyclinics providing gynecologic / obstetric care was obtained from the directory of hospitals in riyadh. a total of 200 gynecologists working in these hospitals were then selected by employing simple random sampling technique. this study was approved by the research center of riyadh colleges of dentistry and pharmacy (rcsdp), riyadh. a structured, self - administered, closed - ended questionnaire with a letter mentioning the purpose of the study was distributed to the gynecologists during their consultation hours. instrument utilized in the present study was developed based on literature reviews of relevant published articles. a pilot study was conducted among a sample of 50 gynecologists by using modified version of the questionnaire in english language to ensure comprehensibility and reliability. a cronbach 's alpha of 0.87 was found, which meets the purpose of this study. the 50 questionnaires utilized in the pilot study were excluded from the final study sample. section i included questions on age, gender, number of years in clinical practice, and practice sector (government or private) ; section ii included five questions on knowledge of periodontal disease and possible ways of prevention ; section iii included four questions on knowledge of periodontal disease and pregnancy outcomes ; and section iv included three questions on oral self - care attitudes among gynecologists. questionnaire responses were coded and entered into a statistical package for social sciences (ibm - spss version 19) software for analysis. chi - square test was applied to assess the differences in responses between different age groups, duration of practice, gender, and sector. a shapiro wilk 's test (p > 0.05) showed that the mean knowledge scores were normally distributed among the gynecologists working in the government sector [skewness 0.121 (se = 0.314) and kurtosis 0.312 (se = 0.314) ] ; belonging to the age groups of 2529 years [skewness 0.632 (se = 0.456) and kurtosis 0.147 (se = 0.887) ], 3034 years [skewness 0.423 (se = 0.369) and kurtosis 0.165 (se = 0.724) ], 4549 years [skewness 0.176 (se = 0.409) and kurtosis 0.523 (se = 0.798) ] ; and having an experience of 2125 years [skewness 0.124 (se = 0.501) and kurtosis 0.429 (se = 0.972) ]. wilk 's test (p 0.05) showed that the mean knowledge scores were normally distributed among the gynecologists working in the government sector [skewness 0.121 (se = 0.314) and kurtosis 0.312 (se = 0.314) ] ; belonging to the age groups of 2529 years [skewness 0.632 (se = 0.456) and kurtosis 0.147 (se = 0.887) ], 3034 years [skewness 0.423 (se = 0.369) and kurtosis 0.165 (se = 0.724) ], 4549 years [skewness 0.176 (se = 0.409) and kurtosis 0.523 (se = 0.798) ] ; and having an experience of 2125 years [skewness 0.124 (se = 0.501) and kurtosis 0.429 (se = 0.972) ]. wilk 's test (p < 0.05) showed that the mean knowledge scores were not normally distributed for both male and female gynecologists, with a skewness of 0.400 (se = 0.246) and kurtosis of 0.041 (se = 0.488) for males and a skewness of 0.685 (se = 0.237) and kurtosis of 0.127 (se 0.469) for female gynecologists. similarly, knowledge score was not normally distributed among gynecologists belonging to the age groups of 3539 years, 4044 years, and 5054 years and experience groups of 15 years, 610 years, 1115 years, and 1620 years, as shown by shapiro wilk 's test (p < 0.05) with different skewness and kurtosis values. a total of 200 gynecologists belonging to different age groups, gender, duration of practice, and sector participated in the study. seventy - one percent of the gynecologists were private practitioners, with majority (35.5%) of them having an experience of 15 years, as shown in table 1. demographic data of the study participants figure 1 displays the responses of the gynecologists to the questions aimed at the assessment of the knowledge of periodontal disease. it is clear that only one - quarter (26%) of the gynecologists were able to define dental plaque correctly, while less than half (44%) of them knew that dental plaque is the cause of gum disease. high percentages (69%) of gynecologists were aware that bleeding gum indicates the presence of gum inflammation. more than half (56.5%) of the gynecologists agreed that periodontal disease can be prevented by brushing and interdental cleaning. also, 39.5% gynecologists considered nighttime before going to bed is the most important time for toothbrushing. awareness of dental plaque and gum diseases among gynecologists when participants were enquired about the knowledge of periodontal disease and pregnancy outcomes, more than half of the respondents (54.5%) said that plaque and negligence of toothbrushing were the main cause of gum disease during pregnancy. very high percentage (80%) of the respondents knew the serious effect of smoking on pregnancy and child birth. however, less than half (44.5%) of the gynecologists believed that there is a relationship between gum disease and premature labor [table 2 ]. knowledge of periodontal disease and pregnancy outcomes among study participants table 3 demonstrates the oral self - care attitudes of gynecologists. about 70% of them said that they use soft bristled toothbrush to remove dental plaque. more than half (54%) of the gynecologists visited their dentist on a regular basis. when asked about the reason for not visiting dentist regularly, 37% expressed fear, 23% mentioned the expensive nature of dental care, and 39.5% mentioned that they were not in need of dental care. oral self - care attitudes among study participants a significantly high percentage of female gynecologists and those working in the government sector were more aware of bleeding gum and prevention of gum disease by brushing and flossing, as compared to their counter parts (p < 0.05). similarly, a significantly high percentage of gynecologists working in private sector were aware of the timing of toothbrushing (p < 0.05) [table 4 ]. pearson chi - square test for dental plaque related oral diseases among study participants table 5 shows the oral health related pregnancy outcomes among gynecologists. a significantly high percentage of male gynecologists and senior gynecologists aged 5054 years answered that gum disease would lead to the delivery of a preterm or low - birth - weight infant (p < 0.05). likewise, a significantly high percentage of gynecologists working in the government sector knew about the negligence of toothbrushing and negative effects of smoking on the pregnant woman and her child (p < 0.05). recent research in periodontal medicine has shown the link between periodontal disease and poor pregnancy outcomes. gynecologists, as women 's healthcare specialists, can play an important role in oral health care, especially during pregnancy and child birth. therefore, their awareness regarding periodontal health is important to identify the modifiable oral risk factors to prevent adverse pregnancy outcomes. in general, the present study assessed gynecologists understanding of various pregnancy - related oral health issues along with their oral self - care attitudes. although the overall periodontal health knowledge and awareness was acceptable in this study, there still exist some lacunae in the knowledge in recognizing deposits on the tooth surface. in this study, 53% gynecologists answered that plaque was a hard deposit and only 26% were able to recognize plaque as a soft deposit. poor oral hygiene resulting from the accumulation of plaque and presence of local irritants has been associated with gingival changes leading to gingivitis. in the present study, 69% of the participants agreed that bleeding gum indicates gingival inflammation. this result is lower than that reported in the study conducted in united arab emirates. in addition, 56.5% of the gynecologists agreed that gum disease can be prevented by regular brushing and flossing. hormonal and vascular changes that accompany pregnancy often exaggerate the inflammatory response to local deposits. in the present study, about 37.5%, 36%, and 17% of the study participants said that hormonal changes, poor oral hygiene, and plaque, respectively, cause inflamed gums among pregnant women. in contrast, 98.3% of indian gynecologists agreed that hormonal changes during pregnancy could cause inflamed gums and 89.5% of the medical doctors in tanzania believed poor oral hygiene was the main cause of periodontal disease. it has been suggested that optimal oral health during pregnancy may have a beneficial impact on the personal health of the pregnant woman as well as that of her offspring. daily oral hygiene practices such as toothbrushing and flossing, which aid in preventing the accumulation of bacterial plaque, should be encouraged during pregnancy and after child birth. in the present study prevalence studies have suggested that 2050% of women from developed countries report smoking at the beginning of pregnancy. immediate effects of smoking on pregnancy outcomes may be miscarriage, low birth weight, preterm birth, and perinatal death. among the study participants, 80% were aware of the negative effects of smoking on pregnant women and their infants health. in the present study, 44.5% of the gynecologists believed that gum disease would lead to the delivery of a preterm or low - birth - weight infant. this result is lower than that reported in other studies and higher than that reported by some other studies. female gynecologists and those working in the government sector had better periodontal health knowledge than those working in the private sector. what do bleeding gums indicate ? and how can you prevent gum disease ? however, the question was responded by a significantly high percentage of gynecologists working in the private sector (p < 0.05). this indicates that gynecologists working in the government sector are more likely to have access to correct and relevant oral health information, as compared to the private sector gynecologists. male gynecologists, those working in the government sector, and those belonging to older age groups had better knowledge of periodontal disease and pregnancy outcomes. this was statistically significant (p < 0.05) for three questions : do you think that gum disease would lead to the delivery of a preterm or low - birth - weight infant ? what causes gum disease in pregnant women ? and do you think smoking has a bad effect on the pregnant woman and her child ? this could be attributed to the cumulative knowledge of oral health and pregnancy outcomes gained over a period of time, with better access to information and resources provided in the government sector. all the above - mentioned lower responses by gynecologists could be due to the lack of knowledge regarding outcomes of poor oral health, limited access to oral healthcare professionals, lack of time for prenatal oral health counseling and referral, and inadequate oral health training received during the previous years of medical education. with regards to the oral self - care attitudes among gynecologists, almost 71% stated that they use toothbrushes with soft bristles, while 8.5% of the gynecologists did not know the type of bristles of their toothbrushes. about half of the gynecologists declared that they visit their dentist on a regular basis. most of the participants who mentioned that they did not visit their dentist regularly (39.5%) believed that there was no need to visit their dentist periodically, while 37.5% stated fear as the main reason behind not visiting the dentist. almost 23% mentioned that the expensive cost of dental treatment is the reason that prevented them from visiting their dentist. this suggests the gap in the oral self - care attitudes among gynecologists considered in the present study. although oral self - care attitudes were anonymous, the responses might be biased to what the participants believed was ideal. caution must be taken in interpreting the applicability of the current data until these findings can be confirmed by larger, prospective investigations. gynecologists considered in the present study showed an acceptable level of knowledge and awareness toward periodontal health and the association between periodontal disease and adverse pregnancy outcomes. inter - professional cooperation between gynecologists and dental healthcare professionals is required to bridge knowledge gap. also, educational programs on periodontal health targeted toward gynecologists are needed to improve the periodontal health awareness, in order to reduce the incidence of preterm low - birth - weight babies.	objective : to determine the knowledge of periodontal disease and pregnancy outcomes among the gynecologists practicing in riyadh, saudi arabia.materials and methods : a cross - sectional survey of 200 randomly chosen gynecologists from riyadh was carried out by using a structured, self - administered, closed - ended questionnaire. knowledge of periodontal disease and possible ways of prevention ; knowledge of periodontal disease and pregnancy outcomes ; and oral self - care attitudes among gynecologists were assessed.results:one-quarter of the gynecologists knew the meaning of dental plaque. almost 57.5% believed that the frequency of toothbrushing should be increased during pregnancy. eighty percent of gynecologists were aware of the serious effects of smoking on the pregnant woman and her child. less than half (44.5%) believed that there is a relationship between gum disease and premature labor. gynecologists working in the government sector were significantly more aware about the causes of inflamed gum in pregnancy, gum disease and its prevention, and negative effects of smoking on pregnancy. a significantly higher percentage of male and senior gynecologists aged between 50 and 54 years answered that gum disease would lead to the delivery of a preterm or low - birth - weight infant.conclusion:gynecologists considered in the present study showed an acceptable level of knowledge and awareness toward periodontal health and the association between periodontal disease and adverse pregnancy outcomes.
prostate is a fibromusculoglandular organ encircling the neck of the bladder and male urethra, weighing up to 20 g in normal adults. prostatic enlargement or growth most commonly occurs due to nodular hyperplasia or due to neoplasia like adenocarcinoma of tubuloalveolar glands and also its precursor lesions prostatic intraepithelial lesion (pin). all these conditions arise in males after 50 years of age and are due to the effect of androgen released from testis. all these conditions cause bladder outlet obstruction giving rise to different clinical features like dysuria, retention of urine and eneuresis. low - back pain due to vertebral metastasis is common in the late stage of prostatic carcinoma. this malignancy, though uncommon in asian countries, the incidence is increasing in our country 1% every year. the incidence of pin is even higher, approximately 70% above the 70 years of age. to detect early this commonly occurring malignancy in men, screening tests are considered after the age of 40 years, which include digital rectal examination (dre), transrectal ultrasonography (trus) and estimation of prostate - specific antigen (psa) in serum. serum psa level above 4 ng / ml is noted in most cases of adenocarcinoma of prostate and if it is increased more than 0.15 per unit volume of prostate (psa density), it is indicative of adenocarcinoma of prostate. androgen receptor (ar) also known as nr3c4 is a type of nuclear receptor which is activated by binding of either of the androgenic hormones. the ars are closely related to progesterone receptor (pr) and progestins in higher dose can block the ars. ar remains important in the development and progression of prostatic carcinoma and ar expression is maintained throughout prostate carcinoma progression and the majority of androgen - independent or hormone - refractory prostate carcinoma express ar. estrogen receptor (er) and estrogen are implicated in prostatic carcinogenesis and tumor progression. the prs are also nuclear receptors and progressive emergence of prs during tumor progression obviously reflects the ability of metastatic and androgen - insensitive tumors to use estrogens through an er--mediated pathway., our objective of this study was to correlate the histopathology of prostatic tissues resected due to different growth with psa level in serum and expression of ar, er and pr by immunohistochemistry (ihc). the study was done in the departments of pathology and urology of ipgmer, kolkata, during the period of july 2008 to august 2010. a total of 50 cases were collected which were diagnosed in urology dpt. as cases of prostatic growth. among them, 35 were transurethral resection of prostate (turp) specimens with clinicoradiological diagnosis of nhp, 05 were trasurethral ultrasonography guided biopsy (trus - bx) specimens and remaining 10 specimens were of radical prostatectomy specimens with clinicoradiologic and biochemical markers favoring prostatic adenocarcinoma. gross details of sent specimens along with clinical, radiological and serum psa level of all patients were noted. hematoxylin and eosin (hande)-stained sections were prepared for routine histopathological evaluation to see nature of growth including gleason scoring in cases of carcinoma. poly - l - lysine - coated slides were used for immunohistochemical staining for ar, er and pr. the different reagents for ihc were provided by biogenex including positive control. statistical analysis was done by using statistica data analysis software version 6.0 [tulsa, oklahoma ; statsoft, inc., we studied total fifty (50) cases including turp, trus - bx and specimens of radical prostatectomy of which 34 cases were diagnosed as nhp (68%), 4 cases as pin (8%) and12 cases as prostatic adenocarcinoma (24%) as per routine histopathology [figure 1 ]. (b) histopathology of prostatic adenocarcinoma (h / e 400) the mean age was 68.66 years. among all only 7 patients were under 60 years, 22 were between 61 and 70 years, 17 were between 71 and 80 years and 4 patients were above 80 years of age. among the 34 cases of nhp, 6 cases were under 60 years, whereas 3 cases were above 80 years. all cases of pin were noted above 70 years of age. among the carcinoma cases 7 cases were seen in between 71 and 80 years of age, 3 cases were between 61 and 70 years, and one case each below 60 years and above 80 years. psa values > 10 ng / ml were noted in 11 cases of carcinoma and among which 7 cases had > 100 ng / ml. twenty - nine cases of nhp subjects had psa values 100 ng / ml. serum psa values were much higher in carcinoma than that of nhp with p value 0.00001, which is also corroborated by the results of study by aboseif. in our study, so, in our study, higher serum psa was seen in cases with higher gleason score showing a positive correlation. er expression in prostate tissues was negative in all cases of nhp, pin and prostate carcinoma. these ihc results were identical to that of other studies such as wernert and kang.. found the er and pr were demonstrated by ihc in nuclei of periglandular fibrocytes and smooth muscle cells and hyperplatic basal cells, but glandular secretory epithelium were negative and thus in prostatic carcinoma cases in their study er and pr were negative. they concluded that estrogens might contribute to nhp by triggering stromal proliferation with a secondary inductive epithelial growth. obviously, they do not act directly on prostatic carcinoma but inhibit growth via the hypophyseal - testicular axis. the pr expressions of carcinoma cells and stromal cells in prostatic carcinoma were found in 93.3% and 76.7%, respectively. the pr were immunoreactive in stromal cells around carcinoma cells, as demonstrated in studies done by kang. 82.35% of nhp cases showed pr expression (including weak expression also), whereas 41.67% of carcinoma cases showed pr expression. in fisher 's exact test (two - tailed), a p value was 0.021 between nhp and carcinoma cases, which is statistically significant. all the prostatic growth lesions in our study were positive for ar expression with varying staining intensity. only 11.78% of nhp cases, 50% of pin cases and 100% of carcinoma cases showed strongly positive ar expression. our study showed identical result of ar expression in prostatic lesions to the studies done by qui yi - q. and brolin. ar immunoreactivity is almost exclusively nuclear and was observed in tumor cells, non - neoplastic glandular epithelial cells and a proportion of peritumoral stromal cells. mean percentages of ar - positive cells were significantly higher in cancer tissues compared to that in normal prostatic tissues (p < 0.001). brolin. analyzed ar, pr and er contents in cytosol and salt - extractable nuclear subcompartments from 6 normal, 39 nhp and 7 malignant prostatic tissue specimens using the radioligand - binding assay technique. the highest content was found in the cytosol and nucleic acid from malignant prostatic tissues. from this study, we observed that among our patients with a mean age of 68.66 years whose specimens of prostate were sent from urology department to our pathology department for the histopathological study, nhp cases were most common and pin were least common. serum psa was within normal limits in nhp cases, but higher in prostatic carcinoma cases and also more in higher gleason score and grey - zone in pin cases. sex - steroid receptor status as seen by ihc was divergent in different types of prostatic growth lesions. but, all cases were er negative. with these findings, antiandrogen and antiprogesterone therapy may be indicated in the treatment of prostatic adenocarcinoma.	prostate gland is a fibromusculoglandular structure situated at the neck of urinary bladder. so, enlargement or growth of prostate due to nodular hyperplasia (nhp) or prostatic intraepithelial neoplasia (pin) or adenocarcinoma may give rise to bladder outlet obstruction. malignant growth i.e., pin or adenocarcinoma cases are associated with increased blood level of prostate - specific antigen (psa) and increased expression of different sex - steroid receptors because the growth is dependent on the interactions of androgen, progesterone and estrogen. the aim of our study is to correlate the histopathology, psa levels and expression of different sex - steroid receptors by immunohistochemistry in different prostatic growth lesions. among the total 50 cases received, inclusive of transurethral resection of prostate (turp), transrectal ultrasound - guided biopsy and radical prostatectomy, 34 cases were diagnosed as nhp, 4 cases as pin and 12 cases as adenocarcinoma histopathologically. serum psa values above 10 ng / ml were seen in 2 cases of pin and 11 cases of adenocarcinoma and none of nhp. estrogen receptor (er) () expressions were negative in all cases. progesterone receptor (pr) expressions were strongly positive in 35% cases of both nhp and adenocarcinoma, whereas androgen receptor (ar) expressions were strong among all cases of adenocarcinoma and only in four cases of nhp. by observing these findings it can be suggested that antiandrogen and antiprogesterone therapy simultaneously will do better than antiandrogen alone in treating prostatic growth lesions.
electrocardiography is nowadays one of the most widely used diagnostic methods in screening tests for early detection of cardiac diseases. examinations are noninvasive and have a large impact on clinical diagnosis and on further medical treatment. the ecg signals reflect the electrical activity of the heart muscle as it is sensed by electrodes placed on the body surface. in clinical practice, the most commonly used electrodes layout is 12-lead standard ecg system. standard ecg has, however, limited sensitivity (3070 %) and specificity (7095 %) in detection of acute coronary syndromes. to improve effectiveness of the ecg diagnostic, high - resolution measurement technique and body surface potential mapping (bspm) were proposed [3, 29 ] and validated [5, 15, 31 ]. however, due to time - consuming procedure of large number ecg electrodes placement, the method is still not widely used in clinical practice. there are many independent factors affecting the ecg examination results related to ecg measurement procedure and physiological inter - individual variability. one of the main sources of mistakes is inaccurate ecg electrode placement in suggested anatomical landmarks, e.g., in proper intercostal spaces. on the other hand, since differences in inter - individual human anatomies, both factors, dependent and independent on medical staff, can cause the change of the distance between the electrodes and source of the signal in the heart as well as the solid angle at which outline of ventricular mass is seen from the body surface. the displacements of the precordial electrodes located nearby the signal source have a greater influence on the ecg signal than shifts of the distant limb electrodes [20, 34 ]. precordial electrodes need sometimes to be shifted to apply bandages, drains, and to undertake an echocardiographic study. however, displacement of the ecg electrodes from determined standard positions can arise also from mistakes of medical staff [9, 18, 20, 23 ] as well as by patients at home who participate in ecg monitoring programs. a common mistake is placing v1 and v2 electrodes too high, in second or third intercostal space, which could result in superior misplacement of remaining precordial electrodes. electrodes v5 and v6 are also placed frequently in the fifth intercostal space and not in the recommended parallel position to electrode v4, which is usually not precisely positioned according to visual estimation of midclavicular line. thus, one important issue which should be taken into account is poor reproducibility of precordial lead placement in serial ecg recordings. kerwin. reported that correct lead positions with an error less than 1 cm were achieved by trained technicians only in case of 50 % of studied men and 20 % of studied women. they found that electrode placement error often was in the range of 23 cm, but occasionally reached even 6 cm. a number of methods for controlling variation in chest electrodes position were suggested and validated. soliman recently proposed to add simple measurement of the distance from suprasternal notch to the v1v2 position assuring the same position of electrodes between clinical trials. herman. invented a sliding ruler that facilitates correct lead placement and for documenting its position on the chest. advise to use the grid printed on non - stretchable material to record and later on, if needed, to relocate wrong positioned electrodes. unlikely, all methods, even the simplest, are not accepted by clinicians, meaning that ecg electrodes are often placed not precise according to subjective visual inspection. the analysis of ecg signals recorded from misplaced electrodes can lead to misinterpretation or even to significant diagnostic errors like incorrect recognition of anterior infarction, anteroseptal infarction, ventricular hypertrophy [9, 14 ], false diagnosis of ischemia, or brugada syndrome [16, 24 ]. have shown that incorrect electrode placement could lead to wrong diagnosis in 1724 % of patients. precordial electrode displacement could cause wrong diagnosis made by human expert as well as by computer - based analysis. the aim of this study was to investigate in detail the effect of displacement of the precordial ecg electrodes on the morphology of the recorded multilead high - resolution ecg signals, in particular, to answer the questions what kind of changes in the recorded ecg signal could be expected while moving the electrode in any direction at a short distance (up to 5 cm) and which precordial ecg leads are most sensitive to electrodes displacement. examinations were carried out in general hospital of medical university of vienna (austria) using a high - resolution ecg measurement system (biosemi). the 64 active electrodes were placed on the body surface according to modified amsterdam lead system [4, 29 ]. the electrodes positions on the chest surface used in this study are shown in fig. 1a.table 1basic data of studied groupmean sdnumber of patients with specified pathologyage (years)height (cm)weight (kg)bmi (kg / m)dsu (cm)dc (cm)micadicdbbbdcm62.74 11.74174.75 7.0887.49 12.8628.62 3.59110.27 7.5340.27 3.0339463372 mi myocardial infarction, bmi body mass index, cad coronary artery disease, bbb bundle branch block, dcm dilated cardiomyopathy, icd implantable cardioverter defibrillator, dsu distance between sternal notch and umbilicus, dc circumference of the thorax at level of iv intercostial space, sd standard deviationfig. 1 a bspm measurement layout and location grid of interpolated ecg signal around lead v2. the location grid contains 11 11 nodes spaced apart by 1 cm. b ecg signals obtained by interpolation for nodes of grid marked by diamond in panel a. c time - aligned - averaged superimposed ecg signals measured from 64 locations on thorax surface basic data of studied group mi myocardial infarction, bmi body mass index, cad coronary artery disease, bbb bundle branch block, dcm dilated cardiomyopathy, icd implantable cardioverter defibrillator, dsu distance between sternal notch and umbilicus, dc circumference of the thorax at level of iv intercostial space, sd standard deviation a bspm measurement layout and location grid of interpolated ecg signal around lead v2. the location grid contains 11 11 nodes spaced apart by 1 cm. b ecg signals obtained by interpolation for nodes of grid marked by diamond in panel a. c time - aligned - averaged superimposed ecg signals measured from 64 locations on thorax surface the multi - lead high - resolution ecg was recorded for 5 min with 4,096 hz sampling frequency and digitized with 24-bit amplitude resolution while subject was at rest in supine position. the study protocol was approved by an institutional ethical committee in accordance with declaration of helsinki, and informed consent was obtained from each patient. then, linear baseline wander estimation and removal procedure were applied (u - p segment used as isoelectric line). the cross - correlation method for beats alignment was used, and then, signal averaging in time was performed., the result for each subject was visually examined and edited based on the view of time - aligned superimposed heartbeats (fig. the position of ecg signal estimation points in close distance (15 cm) from precordial electrodes (v1v6) was determined for each subject as shown in fig. 1 for electrode v2. then, ecg signals were interpolated in 11 11 coordinates of rectangular grid centered on selected precordial lead position (fig. shapes of estimated signals were compared with shape of reference signal recorded in precordial lead location. the analysis was performed for depolarization (qrs complex) and repolarization (st - t - u segment) phases of cardiac cycle (fig. in order to precisely evaluate changes in ecg signal morphology caused by a shift of ecg electrodes, the distribution function method (dfm) proposed by rix and maleng was used. it was shown that dfm is independent of amplitude and timescale variation of a signal, assessing the real shape changes and provides valuable information for cardiac diagnosis [5, 13 ]. let s0(t) be a reference signal and sj(t) a signal to compare, whose supports are included in time interval [0, t ]. if s0(t) and sj(t) are equal in shape, signal s0(t) can be derived from sj(t) through increasing affine functions:1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{0 } \left (t \right) = k_{j } s_{j } \left ({ a_{j } t + t_{j } } \right) + c_{j } ; \quad a_{j } > 0, \, k_{j } > 0, $ $ \end{document}where kj, aj, tj, cj are, respectively, magnitude coefficient, scale coefficient, delay, and offset. since ecg baseline drift is subtracted in preprocessing stage, cj value can be omitted and eq. (1) is rewritten as follows:2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{0 } \left (t \right) = k_{j } s_{j } \left ({ a_{j } t + t_{j } } \right) ; \quad a_{j } > 0,\, k_{j } > 0,$$\end{document}which corresponds to the following:3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{j } \left ({ t ' } \right) = k_{j}^ { ' } s_{0 } \left ({ \frac{{t^ { ' } - t_{j } } } { { a_{j } } } } \right), \quad { \text{where}}\,t^ { ' } = a_{j } t + t_{j } \quad { \text{and}}\quad k_{j}^ { ' } = \frac{1}{{k_{j } } }.$$\end{document } assuming s0(t) and sj(t) are positive signals, the shape difference between two ecg waves was characterized by the function (t) defined by the relation:4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{j } \left (t \right) = s_{0 } \left ({ \varphi \left (t \right) } \right) \quad { \text{i } }. { \text{e}}.\,\varphi = s_{0}^ { - 1 } \circ s_{j }, $ $ \end{document}where sj(t) and s0(t) are the normalized integral functions of sj(t) and s0(t), respectively, rising from 0 to 1 on the signal support:5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{j } \left (t \right) = \frac{{\mathop \int \nolimits_{0}^{t } s_{j } \left (\tau \right){\text{d}}\tau } } { { \mathop \int \nolimits_{0}^{t } s_{j } \left (\tau \right){\text{d}}\tau } }, \quad s_{0 } \left (t \right) = \frac{{\mathop \int \nolimits_{0}^{t } s_{0 } \left (\tau \right){\text{d}}\tau } } { { \mathop \int \nolimits_{0}^{t } s_{0 } \left (\tau \right){\text{d}}\tau } }, $ $ \end{document}the interval [0, 1 ] is divided by m equidistant values yi : 0 1) or shrunken (the pearson correlation coefficient (r) and rmse between reference signal s0(t) and signals sj(t) were also calculated to compare obtained results with those available in the literature. for an ecg signal measured in particular precordial electrode position and ecg signal observed at a given distance from that location, the rmse was defined as follows:8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\text{rmse } } = \sqrt { \frac{1}{n}\sum\nolimits_{i = 1}^{i = n } { \left ({ s_{0 } (ti) - s_{j } (ti) } \right)^{2 } } }, $ $ \end{document}where n number of samples in the averaged ecg signal, s0(ti)the amplitude of measured ecg signal in ith sample (reference signal), sj(ti)the amplitude of interpolated ecg signal sj in ith sample. rmse parameter was normalized to range of the observed data in order to minimize the effect of inter - individual differences in ecg signal magnitude on mean rmse values computed in studied group.9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\text{nrmse } } = \frac{\text{rmse}}{{s_{{{\text{obs}},\hbox{max } } } - s_{{{\text{obs}},\hbox{min } } } } }.$$\end{document } the relative variability index (rv) defined by hoekema. 1a), for a given precordial lead, was defined as the averaged variances over all subjects, for each grid node, and each time instant divided by the overall power of recorded signal:10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\text{rv } } = \sqrt { \frac{{\frac{1}{l \cdot t}\mathop \sum \nolimits_{l = 1}^{l = l } \mathop \sum \nolimits_{t = 1}^{t } \frac{1}{k}\mathop \sum \nolimits_{i = 1}^{k } \left ({ v_{i, l, t } - \overline{v}_{l, t } } \right)^{2 } } } { { \frac{1}{l \cdot t \cdot k}\mathop \sum \nolimits_{l = 1}^{l } \mathop \sum \nolimits_{t = 1}^{t } \mathop \sum \nolimits_{i = 1}^{k } v_{i, l, t}^{2 } } } }, $ $ \end{document}where vi, l, t is the amplitude of ecg signal in grid node l, in time t, for subject i ; \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{v}_{l, t}$$\end{document } is the mean signal over all subjects in grid node l, in time t ; l is the number of nodes (l = 121) in data grid ; t is the number of time instants (t = 800) ; k is the number of studied subjects (k = 60). the relation is equivalent to ratio of standard deviation to root - mean - square value of all the measured ecg signals. additional analysis was performed in order to distinguish ecg changes caused by electrode displacement from physiological variability of ecg shape in time. five sets of precordial ecg signals (v1v6) averaged in time were computed separately for each subject from five consecutive 1-min recordings. examinations were carried out in general hospital of medical university of vienna (austria) using a high - resolution ecg measurement system (biosemi). the 64 active electrodes were placed on the body surface according to modified amsterdam lead system [4, 29 ]. the electrodes positions on the chest surface used in this study are shown in fig. 1a.table 1basic data of studied groupmean sdnumber of patients with specified pathologyage (years)height (cm)weight (kg)bmi (kg / m)dsu (cm)dc (cm)micadicdbbbdcm62.74 11.74174.75 7.0887.49 12.8628.62 3.59110.27 7.5340.27 3.0339463372 mi myocardial infarction, bmi body mass index, cad coronary artery disease, bbb bundle branch block, dcm dilated cardiomyopathy, icd implantable cardioverter defibrillator, dsu distance between sternal notch and umbilicus, dc circumference of the thorax at level of iv intercostial space, sd standard deviationfig. 1 a bspm measurement layout and location grid of interpolated ecg signal around lead v2. the location grid contains 11 11 nodes spaced apart by 1 cm. b ecg signals obtained by interpolation for nodes of grid marked by diamond in panel a. c time - aligned - averaged superimposed ecg signals measured from 64 locations on thorax surface basic data of studied group mi myocardial infarction, bmi body mass index, cad coronary artery disease, bbb bundle branch block, dcm dilated cardiomyopathy, icd implantable cardioverter defibrillator, dsu distance between sternal notch and umbilicus, dc circumference of the thorax at level of iv intercostial space, sd standard deviation a bspm measurement layout and location grid of interpolated ecg signal around lead v2. the location grid contains 11 11 nodes spaced apart by 1 cm. b ecg signals obtained by interpolation for nodes of grid marked by diamond in panel a. c time - aligned - averaged superimposed ecg signals measured from 64 locations on thorax surface the multi - lead high - resolution ecg was recorded for 5 min with 4,096 hz sampling frequency and digitized with 24-bit amplitude resolution while subject was at rest in supine position. the study protocol was approved by an institutional ethical committee in accordance with declaration of helsinki, and informed consent was obtained from each patient. then, linear baseline wander estimation and removal procedure were applied (u - p segment used as isoelectric line). the cross - correlation method for beats alignment was used, and then, signal averaging in time was performed., the result for each subject was visually examined and edited based on the view of time - aligned superimposed heartbeats (fig. the position of ecg signal estimation points in close distance (15 cm) from precordial electrodes (v1v6) was determined for each subject as shown in fig. 1 for electrode v2. then, ecg signals were interpolated in 11 11 coordinates of rectangular grid centered on selected precordial lead position (fig. shapes of estimated signals were compared with shape of reference signal recorded in precordial lead location. the analysis was performed for depolarization (qrs complex) and repolarization (st - t - u segment) phases of cardiac cycle (fig. in order to precisely evaluate changes in ecg signal morphology caused by a shift of ecg electrodes, the distribution function method (dfm) proposed by rix and maleng was used. it was shown that dfm is independent of amplitude and timescale variation of a signal, assessing the real shape changes and provides valuable information for cardiac diagnosis [5, 13 ]. let s0(t) be a reference signal and sj(t) a signal to compare, whose supports are included in time interval [0, t ]. if s0(t) and sj(t) are equal in shape, signal s0(t) can be derived from sj(t) through increasing affine functions:1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{0 } \left (t \right) = k_{j } s_{j } \left ({ a_{j } t + t_{j } } \right) + c_{j } ; \quad a_{j } > 0, \, k_{j } > 0, $ $ \end{document}where kj, aj, tj, cj are, respectively, magnitude coefficient, scale coefficient, delay, and offset. since ecg baseline drift is subtracted in preprocessing stage, cj value can be omitted and eq. (1) is rewritten as follows:2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{0 } \left (t \right) = k_{j } s_{j } \left ({ a_{j } t + t_{j } } \right) ; \quad a_{j } > 0,\, k_{j } > 0,$$\end{document}which corresponds to the following:3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{j } \left ({ t ' } \right) = k_{j}^ { ' } s_{0 } \left ({ \frac{{t^ { ' } - t_{j } } } { { a_{j } } } } \right), \quad { \text{where}}\,t^ { ' } = a_{j } t + t_{j } \quad { \text{and}}\quad k_{j}^ { ' } = \frac{1}{{k_{j } } }.$$\end{document } assuming s0(t) and sj(t) are positive signals, the shape difference between two ecg waves was characterized by the function (t) defined by the relation:4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{j } \left (t \right) = s_{0 } \left ({ \varphi \left (t \right) } \right) \quad { \text{i } }. { \text{e}}.\,\varphi = s_{0}^ { - 1 } \circ s_{j }, $ $ \end{document}where sj(t) and s0(t) are the normalized integral functions of sj(t) and s0(t), respectively, rising from 0 to 1 on the signal support:5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{j } \left (t \right) = \frac{{\mathop \int \nolimits_{0}^{t } s_{j } \left (\tau \right){\text{d}}\tau } } { { \mathop \int \nolimits_{0}^{t } s_{j } \left (\tau \right){\text{d}}\tau } }, \quad s_{0 } \left (t \right) = \frac{{\mathop \int \nolimits_{0}^{t } s_{0 } \left (\tau \right){\text{d}}\tau } } { { \mathop \int \nolimits_{0}^{t } s_{0 } \left (\tau \right){\text{d}}\tau } }, $ $ \end{document}the interval [0, 1 ] is divided by m equidistant values yi : 0 1) or shrunken (< 1) in time. the pearson correlation coefficient (r) and rmse between reference signal s0(t) and signals sj(t) were also calculated to compare obtained results with those available in the literature. for an ecg signal measured in particular precordial electrode position and ecg signal observed at a given distance from that location, the rmse was defined as follows:8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\text{rmse } } = \sqrt { \frac{1}{n}\sum\nolimits_{i = 1}^{i = n } { \left ({ s_{0 } (ti) - s_{j } (ti) } \right)^{2 } } }, $ $ \end{document}where n number of samples in the averaged ecg signal, s0(ti)the amplitude of measured ecg signal in ith sample (reference signal), sj(ti)the amplitude of interpolated ecg signal sj in ith sample. rmse parameter was normalized to range of the observed data in order to minimize the effect of inter - individual differences in ecg signal magnitude on mean rmse values computed in studied group.9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\text{nrmse } } = \frac{\text{rmse}}{{s_{{{\text{obs}},\hbox{max } } } - s_{{{\text{obs}},\hbox{min } } } } }.$$\end{document } the relative variability index (rv) defined by hoekema. 1a), for a given precordial lead, was defined as the averaged variances over all subjects, for each grid node, and each time instant divided by the overall power of recorded signal:10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\text{rv } } = \sqrt { \frac{{\frac{1}{l \cdot t}\mathop \sum \nolimits_{l = 1}^{l = l } \mathop \sum \nolimits_{t = 1}^{t } \frac{1}{k}\mathop \sum \nolimits_{i = 1}^{k } \left ({ v_{i, l, t } - \overline{v}_{l, t } } \right)^{2 } } } { { \frac{1}{l \cdot t \cdot k}\mathop \sum \nolimits_{l = 1}^{l } \mathop \sum \nolimits_{t = 1}^{t } \mathop \sum \nolimits_{i = 1}^{k } v_{i, l, t}^{2 } } } }, $ $ \end{document}where vi, l, t is the amplitude of ecg signal in grid node l, in time t, for subject i ; \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{v}_{l, t}$$\end{document } is the mean signal over all subjects in grid node l, in time t ; l is the number of nodes (l = 121) in data grid ; t is the number of time instants (t = 800) ; k is the number of studied subjects (k = 60). the relation is equivalent to ratio of standard deviation to root - mean - square value of all the measured ecg signals. additional analysis was performed in order to distinguish ecg changes caused by electrode displacement from physiological variability of ecg shape in time. five sets of precordial ecg signals (v1v6) averaged in time were computed separately for each subject from five consecutive 1-min recordings. distributions of computed mean parameters around precordial electrodes positions, called shape difference maps (sdm), are presented in figs. 2, 3, and 4. mean values of shape difference descriptors of qrs complex and st - t - u segment were calculated in the studied group and are denoted by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\alpha } $ $ \end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{rmse}}$$\end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{nrmse}}$$\end{document }, and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{r}$$\end{document}. color scales of sdm were normalized. the red (blue) color corresponds to the maximum (minimum) value for a given shape parameter. the neighboring interpolation points marked by white dots are separated by 1 cm.fig. 2 a body surface distributions of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\alpha } $ $ \end{document } parameter quantifying the scale change of qrs complex in time domain for positions of interpolated ecg signals in relationship to precordial leads location (marked by red dot in the center of a map), b averaged qrs complex from lead v3 for one of studied patient compared to qrs complexes interpolated in the 5 cm distance (to the left / right) from v3 position (colour figure online)fig. 3body surface distributions of mean values of ecg shape descriptors, rmse, nrmse, and r computed for qrs complex in the studied patients group. distributions of parameter values on the body surface quantitating the shape difference between qrs complex recorded in the standard position of selected precordial electrode (marked by red dot in the center of each map) and qrs signal interpolated in a given distance from the correct electrode position. the distance between neighboring interpolation points corresponds to 1 cm shift of the standard precordial electrode (colour figure online)fig. 4body surface distributions of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{rmse}}$$\end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{nrmse}}$$\end{document }, and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{r}$$\end{document } parameters computed for st - t - u segment in the studied patients group. distributions of parameter values on the body surface quantifying the shape difference between st - t - u segment recorded in the standard position of selected precordial electrode (marked by red dot in the center of each map) and st - t - u segment interpolated in a given distance from correct electrode position. the distance between neighboring interpolation points corresponds to 1 cm shift of the standard precordial lead (colour figure online) a body surface distributions of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\alpha } $ $ \end{document } parameter quantifying the scale change of qrs complex in time domain for positions of interpolated ecg signals in relationship to precordial leads location (marked by red dot in the center of a map), b averaged qrs complex from lead v3 for one of studied patient compared to qrs complexes interpolated in the 5 cm distance (to the left / right) from v3 position (colour figure online) body surface distributions of mean values of ecg shape descriptors, rmse, nrmse, and r computed for qrs complex in the studied patients group. distributions of parameter values on the body surface quantitating the shape difference between qrs complex recorded in the standard position of selected precordial electrode (marked by red dot in the center of each map) and qrs signal interpolated in a given distance from the correct electrode position. the distance between neighboring interpolation points corresponds to 1 cm shift of the standard precordial electrode (colour figure online) body surface distributions of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{rmse}}$$\end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{nrmse}}$$\end{document }, and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{r}$$\end{document } parameters computed for st - t - u segment in the studied patients group. distributions of parameter values on the body surface quantifying the shape difference between st - t - u segment recorded in the standard position of selected precordial electrode (marked by red dot in the center of each map) and st - t - u segment interpolated in a given distance from correct electrode position. the distance between neighboring interpolation points corresponds to 1 cm shift of the standard precordial lead (colour figure online) the distribution of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\alpha } $ $ \end{document } (eq. 6) for selected distances to the standard v2, v3, and v4 electrodes calculated for qrs complex is presented in fig. remaining maps of parameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\alpha } $ $ \end{document } computed for other precordial electrodes are not shown due to negligible observed changes (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\alpha } $ $ \end{document } values were in the range from 0.994 0.129 to 1.010 0.136.) scaling effect of st - t - u segment in time for all precordial leads was not observed. (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\alpha } $ $ \end{document } values varied from 0.988 0.129 to 1.002 0.131.) averaged qrs complexes for one case are presented in fig. the 5 cm displacement of v3 electrode in direction to v2 position causes stretching of qrs complex in time by factor = 1.04, while the v3 shift by 5 cm in opposite direction causes shrinking of qrs complex in time by factor = 0.98. the representative maps of standard deviations from mean shape difference descriptors are shown in fig. sd values for qrs complex were in the range from 0 to 0.15 for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\alpha } $ $ \end{document }, from 0 to 3.5 for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document }, from 0 to 250 v for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{rmse}}$$\end{document }, from 0 to 10 % for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{nrmse}}$$\end{document }, and from 0 to 0.4 for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{r}$$\end{document}. the observed sds in case of st - t - u segment varied from 0.13 to 0.14 for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\alpha } $ $ \end{document }, from 0 to 1.5 for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document }, from 0 to 70 v for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{rmse}}$$\end{document }, from 0 to 10 % for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{nrmse}}$$\end{document }, and from 0 to 0.5 for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{r}$$\end{document}.fig. (sd) from a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document } parameter computed in qrs complex and st - t - u segment for a given distance from precordial electrodes v2 and v3 (positioned in the center of the map) distribution of standard deviations (sd) from a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document } parameter computed in qrs complex and st - t - u segment for a given distance from precordial electrodes v2 and v3 (positioned in the center of the map) table 2 presents the numerical values of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{rmse}}$$\end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{nrmse}}$$\end{document }, and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{r}$$\end{document } parameters quantifying the ecg shape changes due to electrode shifts at 1 and 5 cm. notice that the largest values are marked in bold font.table 2maximal observed changes of ecg morphology at 1 and 5 cm distance from precordial electrode positionsparametercardiac phaseprecordial electrodev1 v2 v3 v4 v5 v6 1 cm \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document } (ms)qrs complex0.7 0.4 0.9 1.9 0.5 0.80.2 0.10.1 0.10.1 0.1st - t - u segment 0.3 0.2 0.2 0.20.2 0.20.2 0.10.1 0.10.1 0.1 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{rmse}}$$\end{document } (v)qrs complex41 2248 28 67 35 41 2328 1321 10st - t - u segment13 911 8 13 9 8 55 44 3 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{nrmse}}$$\end{document } (%) qrs complex4 23 2 4 3 3 22 13 2st - t - u segment 6 5 3 35 44 33 33 2 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{r}$$\end{document } qrs complex 0.99 0.03 0.99 0.010.99 0.010.99 0.010.99 0.010.99 0.01st - t - u segment 0.98 0.07 0.99 0.010.99 0.030.99 0.020.99 0.030.99 0.015 cm \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document } (ms)qrs complex 2.7 2.7 2.5 3.32.6 2.41.2 0.90.8 0.40.7 0.4st - t - u segment 1.6 1.4 0.7 0.81.2 1.00.9 0.80.6 0.50.6 0.5 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{rmse}}$$\end{document } (v)qrs complex235 126292 158 344 192 235 140145 65125 63st - t - u segment 73 53 69 5263 4446 3327 1923 19 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{nrmse}}$$\end{document } (%) qrs complex15 816 8 18 9 14 1012 412 5st - t - u segment 19 11 16 916 1015 913 614 7 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{r}$$\end{document } qrs complex0.85 0.280.83 0.28 0.78 0.28 0.82 0.320.94 0.130.93 0.10st - t - u segment0.85 0.330.90 0.24 0.82 0.31 0.86 0.320.90 0.220.91 0.22bold font highlights the largest (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{rmse}}$$\end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{nrmse}}$$\end{document }) and smallest (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{r}$$\end{document }) values sd of ecg shape difference descriptors maximal observed changes of ecg morphology at 1 and 5 cm distance from precordial electrode positions bold font highlights the largest (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{rmse}}$$\end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{nrmse}}$$\end{document }) and smallest (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{r}$$\end{document }) values sd of ecg shape difference descriptors obtained results from analysis of time variability of ecg signal shape in precordial leads are summarized in table 3. the mean values sd of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{rmse}}$$\end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{nrmse}}$$\end{document }, and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{r}$$\end{document } were shown. the values highlighted in bold were used as threshold to decide about significance of shape changes due to electrode displacement, and they represented the largest observed ecg signal changes in time.table 3time variability of ecg morphologyparametercardiac phaseprecordial electrodev1 v2 v3 v4 v5 v6 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document } (ms)qrs complex0.34 0.26 1.15 0.71 0.30 0.180.08 0.060.07 0.050.05 0.03st - t - u segment0.11 0.10 0.17 0.13 0.16 0.120.12 0.080.09 0.070.05 0.04 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{rmse}}$$\end{document } (v)qrs complex18 13 25 17 23 1520 1418 1211 79st - t - u segment8 7 12 9 9 77 55 43 2 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{nrmse}}$$\end{document } (%) qrs complex1.1 0.81.2 0.91.2 0.81.2 0.8 1.6 1.1 1.6 1.1st - t - u segment3.1 2.52.4 1.84.1 3.04.2 2.8 4.4 3 4 3 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{r}$$\end{document } qrs complex0.999 0.0010.998.0010.999.0010.999.001 0.997.002 0.998.001st - t - u segment0.996 0.0050.997.003 0.978.017 0.988.0110.984.0160.990.001bold font highlights the largest (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{rmse}}$$\end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{nrmse}}$$\end{document }) and smallest (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{r}$$\end{document }) values sd of ecg shape difference descriptors for a given cardiac phase time variability of ecg morphology bold font highlights the largest (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{rmse}}$$\end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{nrmse}}$$\end{document }) and smallest (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{r}$$\end{document }) values sd of ecg shape difference descriptors for a given cardiac phase in obtained results, distinct changes of ecg morphology were observed when precordial electrodes were displaced. in fig. 1b, the example of ecg signal recorded from lead v2 at correct and displaced position is presented. the changes in ecg signal morphology are clearly visible while moving the electrodes from their correct positions. distributions of all evaluated parameters (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{rmse}}$$\end{document},\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{nrmse}}$$\end{document }, and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{r}$$\end{document }) in relation to electrode shift in a given direction were coincident. the dispersion of the signal morphology was growing with the increase in the distance from the reference point (tables 2 and 3). that shape disparity was observed both in the mean shape difference values and its standard deviation values (shape difference maps in figs. 3 and 4 versus corresponding sd maps presented in fig. 5). the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{r}$$\end{document } parameter was less sensitive to changes of precordial electrode positions compared with other calculated parameters. the shift up to 1 cm from precordial leads in any direction has the negligible impact on the ecg morphology (table 2). mean values of shape difference descriptors were in the range of physiological variability of ecg signal morphology in time (table 2). these results are in agreement with the outcome of study presented by szakolczai. where the mean morphology change of a whole cardiac cycle observed in v2 electrode shifted by 1 cm in vertical and horizontal directions was r = 0.98 0.04, rmse = 67 28 v. in our analysis, more prominent morphology changes of ecg waves were found for electrode displacements of 2 cm or higher. this supports the results of simulation study performed by turzova. where the mean relative error between body surface potential maps estimated in standard and vertically shifted electrodes positions remained less than 5 % up to 2 cm shift. beyond the threshold value set to 2 cm differences in bspm steeply increased. in our study, mean values of rmse sd calculated for st - t - u segment were in the range from 12 9 to 73 53 v for the vertical shift of electrodes by 5 cm. (2010) reported similar trend of rmse values changes in response to vertical shift (0.55 cm) of precordial electrodes. in their work, the median of rmses calculated for st - t segment in 5 cm distance from correct precordial electrodes positions ranged between 30 v and 130 v. lower values of rmse parameter obtained in our study may result from longer segment of ecg signal used for calculations as well as influence of inter - individual differences in measured signal amplitude. a degree of morphology changes depends on ecg lead, shift magnitude, direction of displacement, and on the ecg segment selected to analysis. we found that leads v2 and v3 are the most sensitive to displacement (figs. 3, 4). for example, a 5 cm left displacement of v3, in direction to v2 position, causes significant changes in qrs shape and st - t - u curves (figs. 3, 4). they report the v2 as the most affected, but they indicated electrode v3 together with v5 and v6 as the least sensitive to displacement. it could be due to the fact that they compared ecg morphology changes in electrodes placed in one specific nonstandard ecg leads arrangement. besides the shift magnitude, directions of precordial lead displacement have significant impact on ecg signal morphology (fig. lead v1 is more sensitive to horizontal than vertical displacement, and ecg morphology changes more prominently while shifting electrode toward v2 position. negligible differences in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document } parameter values caused by the shift of the v1 electrode in vertical directions, with observed differences in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{nrmse}}$$\end{document } values (figs. 3, 4), suggest that ecg signal is changing mainly due to change in the amplitude without significant changes in the main waveform pattern (amplitude scaling effect). the v2 displacement affects more the qrs complex morphology than st - t - u segment (less prominent changes of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document } parameter shown in fig. 4 in comparison with maps of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document } parameter presented in fig. displacement of the electrode v2 in direction to v3 causes slight shrinking of qrs complex in time (decrease in the values of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\alpha } $ $ \end{document } parameter shown in fig. 2). there were no observed scaling effect in time for st - t - u wave (reported by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\alpha } $ $ \end{document } parameter), as well as only slight changes in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document } parameter observed in the distance of 5 cm from v2 in direction to v3 electrode with more evident changes in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{nrmse}}$$\end{document } values. the changes of st - t - u segment are probably more connected to effect of signal amplitude scaling than to changes in the main waveform pattern. displacement of v3 and v4 electrodes in direction to v2 position is crucial for qrs complex morphology. moving electrodes in this direction causes slight stretching of qrs wave as shown in fig. 2. shifting v3 and v4 electrodes along a left diagonal more significantly affects st - t - u segment than displacement along right diagonal as observed in the sdm (figs. 3, 4). changes observed in maps of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{nrmse}}$$\end{document } parameter for v5 and v6 are not confirmed by the values of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\alpha } $ $ \end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{rmse}}$$\end{document }, and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{r}$$\end{document } parameters. these suggest that differences in ecg signal in v5 and v6 are more connected to ecg amplitude scaling than to morphology changes. high values of inter - individual relative variability were observed in the precordium from 0.639 (v6) to 0.886 (v3) for qrs complex and from 0.693 (v2) to 0.989 (v5) for st - t - u wave. in anterior leads v2 and v3, higher values of rv were found for qrs complex than for st - t - u segment. in the remaining precordial leads, higher rv hoekema computed relative variability index for 25 healthy subjects and found that the rv of qrs complex in close distance (up to 3.8 cm in the vertical direction) to v2 electrode was 0.503. the large variability of measured ecg signal depends on the anatomical differences between studied patients, e.g., different position and orientation of the heart in the chest or different torso geometry. the high inter - individual variability found in ecg signal affects the mean values of non - normalized shape difference parameters like root - mean - square error. therefore, more appropriate ecg parameters for subjects comparison are normalized descriptors like \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\text{nrmse}}$$\end{document }, and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{r}$$\end{document}. there is an accepted rule of ecg electrodes positioning according to the anatomical landmarks like intercostal spaces. however, in women, precordial electrodes are often positioned under the breast what could be the reason for unsatisfactory reproducibility. it comes from concerns that the ecg amplitudes are attenuated substantially by the breast tissue.. showed that breast size accounted only for < 1 % of ecg amplitude variations. they recommended placing electrodes on the breast in standardized positions and propose to use special device to deal with not trivial task of electrodes positioning, especially in women with large breast tissue. in the present study, we examined only male patients, but in light of mentioned study, our results could be also valid for assessment of electrodes displacement effect in women. experimental studies have shown that heart in the chest may be situated in the frontal plane at a distance up to 3 cm from the position of precordial electrode v2 on the chest. this difference in distance between surface sensor and the source inside the torso caused change in distribution and amplitudes of heart potentials measured on the body surface [17, 34 ]. thus, placing electrodes even in accordance with the existing standard does not ensure that for all subjects, they are located in the same relation to the heart position. the use of body surface potential mapping might be considered to avoid not precise positioning of ecg electrodes in relation to heart location. this was already pointed out in bspm studies showing a more complete view of the electrical activity of the heart and valuable diagnostic information not visible in standard 12-lead ecg [2, 11, 15 ]. our study demonstrated complexity of the problem of ecg morphology distortion as a consequence of precordial electrodes displacements. we focused on detailed description of the influence of precordial electrodes displacement in any direction on ecg morphology in qrs and st - t - u segments. whether observed changes have significant effect on clinical diagnosis still remains under question and need clinically oriented studies. it was already shown that shifts of precordial leads by 2 cm can result in altered r wave progression and shift in the precordial transition zone, respectively, in 20 and 75 % of patients as well as altered qrs complex and t wave leading to misinterpretation regarding anteroseptal infarction and ventricular hypertrophy, or false statements about appearance of myocardial ischemia or right bundle branch block. the analysis of ecg signals recorded from vertically displaced v1 and v2 leads could also give false impression of brugada syndrome. [26, 28 ] studied the relation of horizontal simultaneous displacement of v1v3 leads on the changes of computer - based diagnosis concerning mi and left ventricular hypertrophy (lvh). in their experiment, v1 was shifted rightward up to 3 cm, v2 was shifted leftward up to 3 cm, and v3 was moved leftward half of this distance. important classification changes caused by such lead displacements were observed in less than 1.5 % cases (for mi) and less than 1 % (for lvh). our results showed that in such constructed experiment, the most affected lead in the sense of morphology changes could be only lead v2, where the changes of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document } were visible in contrast to lead v1 and v2 (fig. the worst effect could be expected if the leads v1 and v2 will be shifted in direction to each other. the high inter - individual variability of ecg amplitude in studied patients group suggests important influence of specific human anatomy on measured ecg signals. therefore, non - normalized ecg parameters like rmse should be avoided for comparison of data from different subjects. the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\overline{\updelta } $ $ \end{document } parameter calculated using the dfm gives additional information about the shape changes of ecg signal being more sensitive to real morphology changes. multiparameter analysis performed gives complete view, showing time and amplitude s scaling effects of ecg signal due to electrode displacements. obtained results may help to choose alternative locations of precordial electrodes when there is need to expose the space on the body surface for other diagnostic procedures.	inaccurate electrode placement and differences in inter - individual human anatomies can lead to misinterpretation of ecg examination. the aim of the study was to investigate the effect of precordial electrodes displacement on morphology of the ecg signal in a group of 60 patients with diagnosed cardiac disease. shapes of ecg signals recorded from precordial leads were compared with signals interpolated at the points located at a distance up to 5 cm from lead location. shape differences of the qrs and st - t - u complexes were quantified using the distribution function method, correlation coefficient, root - mean - square error (rmse), and normalized rmse. the relative variability (rv) index was calculated to quantify inter - individual variability. ecg morphology changes were prominent in all shape parameters beyond 2 cm distance to precordial leads. lead v2 was the most sensitive to displacement errors, followed by leads v3, v1, and v4, for which the direction of electrodes displacement plays a key role. no visible changes in ecg morphology were observed in leads v5 and v6, only scaling effect of signal amplitude. the rv ranged from 0.639 to 0.989. distortions in ecg tracings increase with the distance from precordial lead, which are specific to chosen electrode, direction of displacement, and for ecg segment selected for calculations.
deliberate self - harm (dsh) is a relevant phenomenon that includes several expressions with different import of injury on body, repetitiveness, duration etc., producing anomalies about inclusion or exclusion of behaviors in the classification. in the literature, dsh consists of parasuicidal behaviors, which means a deliberate destruction of body tissue, with or without suicidal intent (1), where a definite intention to die discriminates a suicide attempt from the dsh. the most important classifications are self - mutilation (sm), dsh, non suicidal self - injury (nssi). indirect methods as self - starvation, drunken driving, alcohol or drug abuse, smoking (2), as well as more socially acceptable expressions, such as piercing or tattooing, are not considered (3). the set of behaviors consistently changes, depending on the severity of the injury, the repetitiveness and pattern of behaviors. two prevalent expressions, consisting of slight injuries on the body, are observed : the compulsive type, manifesting with trichotillomania and association with obsessive - compulsive disorder, and the episodic - repetitive type, manifesting as skin cutting and burning, interfering with wound healing, inserting objects under the skin, hitting the body with objects (4). pattison and kahan explain that self - injury occurs within a short time frame and people are fully aware of the effects on their body (5). the low lethality of dsh separates it from conscious suicide attempts, because it appears to be a maladaptive coping strategy, unlike a real purpose to die. several previous research trials stated that the suicidal intent is difficult to recognize, because people often conceal it ; therefore, a clear discrimination between the two phenomena is impossible (5). the high comorbidity between dsh and suicidal behaviors (6), as well as the increased suicide risk for people with dsh history, support this matter. furthermore, in italy, an official non - suicidal dsh index is not yet present. this is a limitation for the studies, since references and data are only international. the experiential avoidance model (7) provides a model merging the previous theoretical models. the model asserts that dsh (without suicidal intent) is a negatively reinforced strategy to reduce or end an unwanted emotional arousal ; this consists of any behavior suitable to avoid eliciting stressful internal and external experiences (e.g. thoughts, memories, somatic sensations). multiple strategies can be used : thought suppression, drug and alcohol use, avoidance of feared settings and objects. although it is a maladaptive coping strategy, it is suitable to escape unwanted feelings, and to act as a strong negative reinforcement. two functions are more frequent : the affect - regulation function is a strategy to control negative feelings and restore well - being ; the self - punishment function is a strategy where anger is addressed against oneself. the precipitating risk factors of dsh and suicide are school dropout, poor family and social support, early puberty, impulse control disorder, use, abuse or addiction to substances, breach of law, living in marginal risk districts, belonging to deviant groups, psychiatric parents (9). along with psycho - pathological factors, it is included a problematic integration, detachment experiences, unworked breakdown and frustration, unclear sexual orientation, social exclusion and withdrawal. besides intrapsychic factors, it is important to analyze adolescent sense of life and death, looking at suicides as a possible answer to conflicts. the body becomes the symbolic expression of internal and relational conflicts, as well as a means to reach both social and gender identity. the failure of the body mentalization process could be the reason of the aggressive behaviors against the self. specific interventions for dsh are still missing, and primary prevention of dsh is rare. the first step could be to realize actions for protection and risk factors survey (10), besides focusing on life skills improvement, by educational programs, and increasing coping strategies (11), emotional expression (3) and stress management (12, 13). school could be a preventive context, where planning specific programs can be easily done. because of this, our study involved three secondary schools in the area of florence. the purpose was to perform and analysis of risk factors for dsh, to plan preventive actions. deliberate self - harm (dsh) is a relevant phenomenon that includes several expressions with different import of injury on body, repetitiveness, duration etc., producing anomalies about inclusion or exclusion of behaviors in the classification. in the literature, dsh consists of parasuicidal behaviors, which means a deliberate destruction of body tissue, with or without suicidal intent (1), where a definite intention to die discriminates a suicide attempt from the dsh. the most important classifications are self - mutilation (sm), dsh, non suicidal self - injury (nssi). indirect methods as self - starvation, drunken driving, alcohol or drug abuse, smoking (2), as well as more socially acceptable expressions, such as piercing or tattooing, are not considered (3). the set of behaviors consistently changes, depending on the severity of the injury, the repetitiveness and pattern of behaviors. two prevalent expressions, consisting of slight injuries on the body, are observed : the compulsive type, manifesting with trichotillomania and association with obsessive - compulsive disorder, and the episodic - repetitive type, manifesting as skin cutting and burning, interfering with wound healing, inserting objects under the skin, hitting the body with objects (4). pattison and kahan explain that self - injury occurs within a short time frame and people are fully aware of the effects on their body (5). the low lethality of dsh separates it from conscious suicide attempts, because it appears to be a maladaptive coping strategy, unlike a real purpose to die. several previous research trials stated that the suicidal intent is difficult to recognize, because people often conceal it ; therefore, a clear discrimination between the two phenomena is impossible (5). the high comorbidity between dsh and suicidal behaviors (6), as well as the increased suicide risk for people with dsh history, support this matter. furthermore, in italy, an official non - suicidal dsh index is not yet present. this is a limitation for the studies, since references and data are only international. the experiential avoidance model (7) provides a model merging the previous theoretical models. the model asserts that dsh (without suicidal intent) is a negatively reinforced strategy to reduce or end an unwanted emotional arousal ; this consists of any behavior suitable to avoid eliciting stressful internal and external experiences (e.g. thoughts, memories, somatic sensations). multiple strategies can be used : thought suppression, drug and alcohol use, avoidance of feared settings and objects. although it is a maladaptive coping strategy, it is suitable to escape unwanted feelings, and to act as a strong negative reinforcement. klonsky, (8) found seven functions for dsh. two functions are more frequent : the affect - regulation function is a strategy to control negative feelings and restore well - being ; the self - punishment function is a strategy where anger is addressed against oneself. the precipitating risk factors of dsh and suicide are school dropout, poor family and social support, early puberty, impulse control disorder, use, abuse or addiction to substances, breach of law, living in marginal risk districts, belonging to deviant groups, psychiatric parents (9). along with psycho - pathological factors, it is included a problematic integration, detachment experiences, unworked breakdown and frustration, unclear sexual orientation, social exclusion and withdrawal. besides intrapsychic factors, it is important to analyze adolescent sense of life and death, looking at suicides as a possible answer to conflicts. the body becomes the symbolic expression of internal and relational conflicts, as well as a means to reach both social and gender identity. the failure of the body mentalization process could be the reason of the aggressive behaviors against the self. specific interventions for dsh are still missing, and primary prevention of dsh is rare. the first step could be to realize actions for protection and risk factors survey (10), besides focusing on life skills improvement, by educational programs, and increasing coping strategies (11), emotional expression (3) and stress management (12, 13). school could be a preventive context, where planning specific programs can be easily done. because of this, our study involved three secondary schools in the area of florence. the purpose was to perform and analysis of risk factors for dsh, to plan preventive actions. the research analyses young adults well - being, in particular the relationship with the body, social and family support and life approach. the aim is to analyze risk factors of dsh and to promote awareness about the behavior. a total of 313 students, 18 - 22 years old (mean age = 20.8 years), of which 63% were male and 37% were female. the sampling is snowball and voluntary, but questionnaires compilation was held exclusively in the fifth section classes, as participants should be adults. teachers of the three different secondary schools in florence (from where the students were selected) also participated in the study by filling special interviews. the close ended questionnaire was divided into two sections : a first section concerned everyday life topics (free time activities and fun, family communication and relationships, risk behaviors, externalizing / internalizing behaviors, stressful events) and a second section with specific scales : the bat : body attitudes test (no italian version is yet validated. ten out of 20 items were translated ; discarded items were not suitable to males) ; the italian validation of mspss : multidimensional scale of perceived social support ; the mast : multi - attitude suicide tendency scale (only 14 items were picked from the italian validation) ; the italian validation of pwb : psychological well - being scales by ryff (14). to avoid bias, the research was described as a study on young adults life styles. results were discussed with students and teachers. to investigate the topic from the teachers point of view, a semi - structured interview was submitted to health education teachers. the interview focused on the following topics : professional history, distress perception in school (particularly for dsh) cases histories, recommended actions, criticalities and resources in the school and area. a quantitative descriptive analysis was conducted and the associations between variables were examined by one - way anova and pearson s correlation. in particular, anova analyzed different answers on the cutting item among groups (i.e. school, sex, family communication and stressful events). correlations were analyzed among the cutting item and risk behaviors (i.e. have you ever had unprotected sex ?), internalizing behaviors (i.e. have you ever eaten as much as you should ?) and externalizing behaviors (i.e. have you ever threatened other people ?). the contents of the interview were analyzed with the atlas.ti software (atlas.ti scientific software development gmbh, berlin, germany). a total of 313 students, 18 - 22 years old (mean age = 20.8 years), of which 63% were male and 37% were female. the sampling is snowball and voluntary, but questionnaires compilation was held exclusively in the fifth section classes, as participants should be adults. teachers of the three different secondary schools in florence (from where the students were selected) also participated in the study by filling special interviews. the close ended questionnaire was divided into two sections : a first section concerned everyday life topics (free time activities and fun, family communication and relationships, risk behaviors, externalizing / internalizing behaviors, stressful events) and a second section with specific scales : the bat : body attitudes test (no italian version is yet validated. ten out of 20 items were translated ; discarded items were not suitable to males) ; the italian validation of mspss : multidimensional scale of perceived social support ; the mast : multi - attitude suicide tendency scale (only 14 items were picked from the italian validation) ; the italian validation of pwb : psychological well - being scales by ryff (14). the questionnaire was given to students during school time. to avoid bias, results were discussed with students and teachers. to investigate the topic from the teachers point of view, the interview focused on the following topics : professional history, distress perception in school (particularly for dsh) cases histories, recommended actions, criticalities and resources in the school and area. a quantitative descriptive analysis was conducted and the associations between variables were examined by one - way anova and pearson s correlation. in particular, anova analyzed different answers on the cutting item among groups (i.e. school, sex, family communication and stressful events). if possible, by the bonferroni index, a second analysis was conducted. correlations were analyzed among the cutting item and risk behaviors (i.e. have you ever had unprotected sex ?), internalizing behaviors (i.e. have you ever eaten as much as you should ?) and externalizing behaviors (i.e. have you ever threatened other people ?). the contents of the interview were analyzed with the atlas.ti software (atlas.ti scientific software development gmbh, berlin, germany). they spend their free - time surfing the internet, chatting, watching tv and listening to the music on their own, which is the only recurring activity they also enjoy a lot. everyday activities are more lonely and of average fun, while they show more pleasure in occasional and more cohesive pastimes, such as doing sport, going to the disco and concerts, being with boyfriend / girlfriend and going to the cinema. some of them belong to associations (44%) : half of them to a sport association, the others to cultural, health, religious, social associations. through anova, we observe an association between failing a school year and self - cutting (see table 1) ; this agrees with the literature, where school distress is an important risk factor (15). p < 0.05 represents the statistical significance. of them, 5.4% acknowledge engaging intentional self - cutting at least sometimes, which supports referential literature (16 - 18), and 64.7% of them are male. this contrasts literature, which only attributes self - harm behavior to females (3, 15, 17 - 26). nevertheless, threatening other people is correlated with cutting item, which appears to be explained by the highest incidence of boys revealing dsh. the research finds no association between social support and cutting ; enhancing the topic is suggested, considering the wide range the literature gives to social support as a protective factor (27 - 29). the research observes an association between family incommunicability and dsh, supporting the evidence in literature (30) (see table 2). the presence of tattoos and piercings was examined, although no significant association was found with dsh. concerning substances and alcohol use, a statistical association between getting drunk and cutting was found, partially validating the literature, which attests alcohol and the bonferroni index. substances use / abuse, along with smoking cigarettes, are strong risk factors for dsh (15, 31, 32). sexual harassments in the past 3 years associate with cutting (see table 3), supporting the hypothesis which considers it the main risk factor for dsh (32 - 36). other risk behaviors, such as dangerous driving and gambling, or internalizing behaviors, are not related with self - cutting. nevertheless, self - cutting - related variables, such as getting drunk and threatening other people, are associated with other risk behaviors. this situation could be of considerable proportions, according to the hypothesis that dsh could represent the presence of additional risk behaviors (15). among stressful and traumatic events, except sexual harassments, no association with cutting several data where self - harm is a nonfunctional strategy to cope with stress, were not confirmed (7, 8). the bat scale was significantly related to cutting, confirming the difficult relationship with the body to be a risk factor and the need for preventive actions on this topic. an association between the life repulsion subscale, mast scale, and self - cutting is found (see figure 1), which appears to support literature asserting self - harm and suicide attempts are related (4, 6, 10, 15, 26, 28, 37 - 41). teachers highlight that dsh is a dysfunctional relationship with the body. eating disorders and substance abuse they run into this type of distress : she showed me, under her gloves, she had signs indeed. their role is critical to express the problem and to manage it further by the school, by the family and, then, by the services. they highlight distress markers : discipline difficulties (clear provocative attitudes : the teacher comes in, she had unsuitable attitudes), rules respect difficulties, important social introversion, substance use and abuse and family troubles. the role of the teacher is critical, first in noticing these signs i perceived distress, i saw her standing on her own all the time, she never employed her body, she was not joyful, she showed me the cuts. then establishing a trust relationship in order to talk frankly with students. we try carefully to make them feel we are close to them, it can be a moment where we start talking. they state that it is a teacher s duty to lead students to psychological consultant and to family : at least, let s look at them : how can you act as if nothing has happened ? let the parents come, let s talk to them, or they say is there any problem, how are you ? then i immediately say there is a service, because i absolutely give no kind of advice, my work is noticing. they also observe peers support and their involvement is important the class is usually completely welcoming towards distressed people. greater criticalities are connected with bureaucratic features, such as the teachers turnover, and the needed fulfillments to start an intervention. a marked taboo on open distress expression is present and the determined prejudice in italian culture about psychic distress. they are still suspicious to go to the listening desk, because going to the psychologist is insane. i did not want him to respond, such as mrs, what ? do you think i need it ? besides, it is not clear what they can do. as we are still afraid of making a mistake and getting in trouble, sometimes, in doubt, we keep back rather than take a step forward. teachers gave valuable advice : besides already started interventions, such as the psychological consultant and the health education classes, they consider the clarification and formalization of the class coordinator survey and handling very important, and they highlight that a referential action protocol is recommended. according to these teachers, open attitude, attention to distress signs recognition and relational and communicative abilities are basic features to start dealing with these problems, to involve the family and to start a supporting extracurricular networking. to improve life quality in the school community the comments reproduce current teenagers behaviors, and the study usefulness was acknowledged. someone identified in the questionnaire a way to better understand one and the others. other participants observed an incongruity among the results and the personal perception of the phenomenon, which is higher. they spend their free - time surfing the internet, chatting, watching tv and listening to the music on their own, which is the only recurring activity they also enjoy a lot. everyday activities are more lonely and of average fun, while they show more pleasure in occasional and more cohesive pastimes, such as doing sport, going to the disco and concerts, being with boyfriend / girlfriend and going to the cinema. some of them belong to associations (44%) : half of them to a sport association, the others to cultural, health, religious, social associations. through anova, we observe an association between failing a school year and self - cutting (see table 1) ; this agrees with the literature, where school distress is an important risk factor (15). p < 0.05 represents the statistical significance. of them, 5.4% acknowledge engaging intentional self - cutting at least sometimes, which supports referential literature (16 - 18), and 64.7% of them are male. this contrasts literature, which only attributes self - harm behavior to females (3, 15, 17 - 26). nevertheless, threatening other people is correlated with cutting item, which appears to be explained by the highest incidence of boys revealing dsh. the research finds no association between social support and cutting ; enhancing the topic is suggested, considering the wide range the literature gives to social support as a protective factor (27 - 29). the research observes an association between family incommunicability and dsh, supporting the evidence in literature (30) (see table 2). the presence of tattoos and piercings was examined, although no significant association was found with dsh. concerning substances and alcohol use, a statistical association between getting drunk and cutting was found, partially validating the literature, which attests alcohol and the bonferroni index. substances use / abuse, along with smoking cigarettes, are strong risk factors for dsh (15, 31, 32). sexual harassments in the past 3 years associate with cutting (see table 3), supporting the hypothesis which considers it the main risk factor for dsh (32 - 36). other risk behaviors, such as dangerous driving and gambling, or internalizing behaviors, are not related with self - cutting. nevertheless, self - cutting - related variables, such as getting drunk and threatening other people, are associated with other risk behaviors. this situation could be of considerable proportions, according to the hypothesis that dsh could represent the presence of additional risk behaviors (15). among stressful and traumatic events, except sexual harassments, no association with cutting several data where self - harm is a nonfunctional strategy to cope with stress, were not confirmed (7, 8). the bat scale was significantly related to cutting, confirming the difficult relationship with the body to be a risk factor and the need for preventive actions on this topic. an association between the life repulsion subscale, mast scale, and self - cutting is found (see figure 1), which appears to support literature asserting self - harm and suicide attempts are related (4, 6, 10, 15, 26, 28, 37 - 41). teachers highlight that dsh is a dysfunctional relationship with the body. eating disorders and substance abuse they run into this type of distress : she showed me, under her gloves, she had signs indeed. their role is critical to express the problem and to manage it further by the school, by the family and, then, by the services. they highlight distress markers : discipline difficulties (clear provocative attitudes : the teacher comes in, she had unsuitable attitudes), rules respect difficulties, important social introversion, substance use and abuse and family troubles. the role of the teacher is critical, first in noticing these signs i perceived distress, i saw her standing on her own all the time, she never employed her body, she was not joyful, she showed me the cuts. then establishing a trust relationship in order to talk frankly with students. we try carefully to make them feel we are close to them, it can be a moment where we start talking. they state that it is a teacher s duty to lead students to psychological consultant and to family : at least, let s look at them : how can you act as if nothing has happened ? let the parents come, let s talk to them, or they say is there any problem, how are you ? then i immediately say there is a service, because i absolutely give no kind of advice, my work is noticing. they also observe peers support and their involvement is important the class is usually completely welcoming towards distressed people. greater criticalities are connected with bureaucratic features, such as the teachers turnover, and the needed fulfillments to start an intervention. a marked taboo on open distress expression is present and the determined prejudice in italian culture about psychic distress. they are still suspicious to go to the listening desk, because going to the psychologist is insane. i did not want him to respond, such as mrs, what ? do you think i need it ? besides, it is not clear what they can do. as we are still afraid of making a mistake and getting in trouble, sometimes, in doubt, we keep back rather than take a step forward. teachers gave valuable advice : besides already started interventions, such as the psychological consultant and the health education classes, they consider the clarification and formalization of the class coordinator survey and handling very important, and they highlight that a referential action protocol is recommended. according to these teachers, open attitude, attention to distress signs recognition and relational and communicative abilities are basic features to start dealing with these problems, to involve the family and to start a supporting extracurricular networking. to improve life quality in the school community, results were discussed with participating students and teachers. to rouse a feedback, students expressed their opinions on nameless post - its. the comments reproduce current teenagers behaviors, and the study usefulness was acknowledged. someone identified in the questionnaire a way to better understand one and the others. other participants observed an incongruity among the results and the personal perception of the phenomenon, which is higher. this study confirms existent data in the literature, as in the case of the association between self - cutting and alcohol use, and between self - cutting and sexual harassments. self - injury and the mast scale were related, highlighting a relation with suicide attempts and suggesting an analysis of the risk factors mediating these behaviors. the present research also emphasizes the role of the school, by finding associations between self - cutting and school dropout. this is consistent with teachers assertions about school, as an educational community, where they actively have to operate. however, a psychological counselor must be present, to handle situations with increased risks. moreover, suggesting the creation of opportunities to instruct teachers with appropriate tools may also prove fundamental. this could be useful to fight resistances due to negative stereotypes of psychological distress and stigmatization. finally, this research builds a link between pure survey and psychological and educational action. a cooperation among researchers, teachers and psychologists is still present, to start further comparison opportunities on observed topics, involving families.	background : self - harm behaviors consist of parasuicidal behaviors, which represent a deliberate destruction of body tissue, with or without suicidal intent. a theoretical model is the experiential avoidance model. the most frequent risk factors are school distress, poor social integration, poor social and family support, drugs use, sexual abuse, altered sense of life and death, bad relationship with the body and unsolved body mentalization process.objectives:the objective of the present study was to perform an analysis of risk factors for self - harm behaviors, to help plan preventive actions.patients and methods : one questionnaire with specific scales was employed for students, whereas three semi - structured interviews were employed for teachers, all on distress perception and self - harm in school.results:data analysis confirms an association between self- cutting and alcohol use, sexual harassments, school dropout, threatening people, incommunicability with family members and negative relationship with the body and suicide attempts, with a clear tendency for males. in the interviews, teachers highlight self - injury as a dysfunctional relationship with the body and observe several risk markers of psychological distress.conclusions:the results confirm the available literature data, while noting that self - harming is a preponderantly male behavior. the results also signal the need to create opportunities to instruct teachers to combat the resistances and stereotypes of psychological distress.
the nucleotide binding and oligomerization domain 2 protein (nod2) is a member of the nucleotide - binding and oligomerization domain (nod)-like receptor (nlr) family. it is also known as caspase recruitment domain - containing protein 15 (card15) and nucleotide - binding oligomerization domain (nod)-like receptor with a card (nlrc2). the nod2 gene is located on chromosome 16q12 - 21, and encoded by the card5 gene. the nod2 protein consists of two n - terminal caspase recruitment domains (card), a central nucleotide - binding domain (nbd ; also known as a nod domain), and a series of c - terminal leucine - rich repeats (lrps). the lrp is similar to the repeats found in toll - like receptors (tlrs), and senses muramyl dipeptide (mdp), a component of gram - positive bacterial peptidoglycan. the nbd domain contains a nacht domain, winged helix and superhelical subdomains, which mediate nod2 oligomerization. nod2 oligomerization promotes the recruitment of receptor - interacting serine / threonine - protein kinase 2 (rip2). nod2 is an intracellular bacterial - derived mdp sensing receptor, and is expressed in various cell subsets, particularly myeloid cells. alteration of these innate immune responses can result in several chronic inflammatory diseases, including crohn 's disease. the first report on nod2 addressed the association of nod2 variants with crohn 's disease, therefore, extensive studies have focused on the role of nod2 with inflammatory bowel disease. the three nod2 gene mutations, the arginine to tryptophan conversion at amino acid residue 702 (r702w), the glycine to arginine change at 908 (g908r), and the frame shift - stop codon mutation at 1007 (1007fs), are commonly reported. because g908r and 1007fs mutants are located in the lrp domain, these mutants can not effectively recognize of microbial components, thus preventing nod2 dimerization. because the r702w mutant is located in the nbd domain, this mutant results in defective oligomerization. the association of various nod2 gene mutations with several other rheumatic diseases has been studied in depth. however, the association of these three common variants and rheumatoid disease has not been demonstrated. this review focuses on nod2 immune regulation and the role of nod2 in the pathogenesis of atherosclerosis. atherosclerosis is the most common pathological process of coronary artery and cerebrovascular disease, which, collectively, are the primary cause of sudden death worldwide. atherosclerosis is strongly associated with hypercholesterolaemia, hypertension, diabetes, smoking and genetic factors. atherosclerosis begins with lipid deposition on the vessel wall and the alteration of endothelium hemostatic functions. such changes in endothelium hemostatic function promote inflammatory responses, including increased expression of adhesion molecules on endothelial cell, which leads to leukocytes penetration. penetration of monocytes / macrophages into the intima of arteries is an important process in atherogenesis. reportedly, others inflammatory cells, including lymphocytes and mast cells, also contribute to plaque progression. atherosclerotic plaque is comprised of the necrotic core, calcified regions, accumulated modified lipids, and fibrous cap. atheroma progression is mediated by the production of inflammatory cells, such as vasoactive mediators, cytokines, and proteinases. two major complications of atherosclerosis, plaque rupture and thrombosis, are regulated by inflammatory cells. plaque rupture is caused by the disruption of fibrous cap integrity, which is mediated by matrix degrading enzymes from inflammatory cells ; thrombosis is generally mediated by procoagulant molecules, such as tissue factor, but is also mediated by inflammatory cells. these points demonstrate that atherosclerosis is a chronic and multifactorial inflammatory disease and that disease progression is modulated by monocytes and macrophages, which are major players in innate immunity. prps (pattern recognition proteins) recognize common pathogen components known as pathogen - associated molecular patterns (pamps or microorganism associated molecular patterns, mamps). at least 50 prps have been reported in the mammalian system, and are calssified in to three groups : toll - like receptors (tlrs), retinoic acid inducible gene i (rig - i)-like receptors (rlrs), and nucleotide - binding oligomerization domain (nod)-like receptors (nlrs). various microbial agents have been reported in atheroma plaque, including c. pneumonia, h. pylori, herpes simplex virus, epstein - barr virus, porphyromonas gingivalis, etc. however, antibiotics do not attenuate disease severity, and atherosclerosis is not altered in the experimental germ - free environment. pamps recognition by tlr induces the myeloid differentiation primary response gene 88 (myd88) activation, which then promotes nf-b nuclear translocation via signaling cascades. in apolipoprotein e (apoe) and myd88 double deficient mice or apoe and tlr2/tlr4 double - deficient mice, atherosclerotic plaque formation and macrophage recruitment is reduced. the effect of tlr1, tlr3, and tlr6 in atherogenesis is not clear, and that of tlr5, tlr8, and tlr9 is unknown. taken together, these points suggest that atherosclerosis development is characterized by heterogeneous and complex immune balance. nod2 deficient mice are susceptible to bacterial infection, including s. aureus, c. pneumonia, h. pylori, and b. anthracis. mdp, the ligand of nod2 as described above, present in atherosclerotic plaque. taken together, we hypothesize that nod2 may play some role as an immune regulator in atherosclerotic plaque. it is reported that apoe and nod2 double - deficient mice are more susceptible to atherosclerosis. total serum cholesterol levels, serum inflammatory cytokine levels, leukocyte infiltration, and atherosclerotic plaque are elevated in apoe and nod2 double - deficient mice than apoe deficient mice. atherosclerosis development is enhanced by p. gingivalis infection, and this enhancement is more pronounced in apoe and nod2 double - deficient mice. when mdp is used to activate nod2, this is in contrast to tlrs, even though both nod2 and tlrs are bacterial sensing receptors that initiate innate immune responses. however, the molecular mechanism of nod2-mediated inflammatory regulation is not known. in this review, we will discuss a potential common signaling pathway that is both triggered by nod2 and involved in atherosclerosis. mdp - nod2 interaction triggers homotypic dimerization via card - card binding ; this dimerization follows rip2 recruitment and activation. in a step - wise fashion, rip2 activates nf-b, and ap-1 by tak1 (tgf- activated kinase 1) complex, and nemo, p38, jnk and erk mapk pathway. this results in the expression of several inflammatory cytokines, including tnf-, il-6, il-8, cc - chemokine ligand 2 (ccl2), and cxc - chemokine ligand 2 (cxcl 2). these cytokines induce the recruitment and priming of innate immune cells, including neutrophils and monocytes. it has also been reported that nod2 signaling inhibits activation of tlr2-induced c - rel, an nf-b subunit ; tlr2-induced c - rel activation is enhanced in nod2 deficient mice. these observations suggest that nod2 works as both a positive and negative regulator of nf-b activation. the molecular mechanism of this ambivalent property of nod2 is not clear, and may depend on microenvironmental conditions or activation context. in experiments with apoe and myd88 double - deficient mice, the inhibition of tlr2- or tlr4-induced p65 however, inhibition of nf-b activation by deletion of i-b kinase 2 (ikk2), which is essential for nf-b activation, in macrophages, increases atherosclerosis in ldl receptor deficient mice. it is contradictory that both tnf-, a pro - inflammatory cytokine, and il-10, an anti - inflammatory cytokine, production are decreased in ikk2-deleted macrophages. these results suggest that pro- and anti - inflammatory balance is important to control the development of atherosclerosis. taken together, we hypothesize that nod2 works as a key player to regulate immune balance in atherosclerosis according to microenvironmental conditions. understanding of nod2 regulatory signaling in atherosclerosis may lead us towards the development of atherosclerosis treatment. il-10 is produced by macrophages, t cells, and b cells, and is associated with the differentiation of th2 and treg cells, therefore, it works as a broad - spectrum inflammation inhibitor. administration of il-10 delays atherosclerosis development, and il-10 and apoe double deficient mice are more susceptible to atherosclerosis. however, co - stimulation with pgn (peptidoglycan, tlr2 ligand), but not lps (tlr4 ligand), induces il-10 secretion. in this pathway, il-10 transcription is regulated by phosphorylation of heterogeneous nucleoribonuclear protein a1 (hnrnp a1), and p38 mapk. it is also reported that the three major nod2 mutants, particularly 1007fs, inhibit il-10 transcription following tlr2 activation. crohn 's disease patients carrying the 1007fs mutant have impaired hnrnp a1 phosphoylation, and il-10 secretion. normal mdp - nod2 signal can not inhibit il-10 production followed by tlr2 stimulation, but 1007fs patients have il-10 production impairment followed the strong tlr2 stimulation. apoe and nod2 double - deficient mice produce more il-10, despite having large atherosclerotic lesions. based on the impairment of il-10 production in 1007fs mutant patients, it is possible that the 1007fs mutant may show a gain of function to inhibit il-10 suggesting that patients with the 1007fs nod2 mutant may be more susceptible to atherosclerosis. although this has not been reported, it has been shown that inflammatory bowel disease patients have a greater risk of developing atherosclerosis. prostaglandin e2 (pge2) is an inflammatory lipid molecule in the eicosanoid family, the synthesis of which is initiated by the release of arachidonic acid (aa) from membrane glycerophospholipids through cytosolic phospholipase a2a. pge2 synthesis depends on the activity of cyclooxygenase-1, cyclooxygenase-2, and pge2 synthases (pgess). it has been reported that tlr4 activation in macrophages drives pge2 production by the upregulation of cyclooxygenase-2 through the nf-b pathway, and that pge2 has a proinflammtory role. however, the effect of pge2 in atherosclerosis is still controversial. c - reactive protein (crp) which inhibits pge2 expression increase in atherosclerotic plaque ; ppar- induces pge2 expression, also increases in atherosclerotic plaque. pge2 can modulate platelets adhesion on atherosclerotic plaque, however, pge2 receptor antagonists does not modulate atherosclerosis. considering these results, it is possible that pge2 can play different roles, either anti- or pro - atherogenic, depending on the disease state. nod2 is highly expressed in atherosclerotic lesions and pge2 is upregulated by mdp treatment in ex vivo cultured human carotid atherosclerotic plaque. pge2 has both proinflammatory and anti - inflammatory effects depending on cell types and its receptor subtypes, suggesting that the function of nod2 in atherosclerosis is ambivalent. from these reports, the question remains, as to how nod2 regulates this immune balance, and what confers susceptibility against some diseases. this suggests that the effect of mdp - nod2 signal may be more important on vulnerable plaque formation not whole atherosclerosis pathogenesis. because the most critical and fatal event in atherosclerosis is vulnerable plaque rupture, understanding this mechanism will provide new insights to predict and treat atherosclerosis and its complications. atherosclerosis is a chronic inflammatory disease, with both innate and adaptive immune responses contributing to disease initiation and progression. it has been demonstrated that foxp3 expressing treg cells have a protective effect against atherosclerosis. similarly, il-10 or tgf-, which is important for treg differentiation, has an anti - atherogenic effect. treg cells are known to inhibit t cell activation and maintain immune balance, thereby modulating disease progression. th1 response is known as a pro - atherogenic process by producing proinflammatory cytokines, such as ifn-, and il-6, and regulating antibody production from b cells. ifn- injection induces plaque growth, and ifn- or and ifn- receptor deficient mice have smaller atherosclerotic plaques. severe hypercholesterolemia in apoe deficient mice induces a switch in the th2 immune response ; however, il-4 production does not prevent the development of atherosclerosis. il-17 production is increased in ldlr or apoe deficient mice fed a western diet to develop atherosclerosis. these results suggest that th17 immunity is pro - atherogenic. as an intracellular receptor against microbes, nod2 mediates innate immune response. in contrast, nod signaling does not strongly induce co - stimulatory molecule expression on dcs. whereas tlrs primarily induce a th1 immune response, nod2 promotes a th2 immune response. secretion of il-12, a major cytokine that induces th1 differentiation from macrophages, by pgn treatment is reduced by mdp co stimulation. these results suggest that nod2 may shift the immune balance to th2 from th1, thus causing the anti - atherogenic th2 immune response. nod2 signaling affects the production of il-23, an important cytokine for th17 differentiation, from dendritic cells by mir-29 upregulation. in regards to atherosclerosis, it is possible that nod2 reduction of the th17 immune response in atheroma plaque contributes to its anti - atherogenic effect. systemic immune network balance determines atherosclerosis progression, but the mechanism is not clear. the amar group reported that atherosclerosis development is increased when nod2 signaling is impaired. it is similar that the 1007fs mutant disrupts tlr2 signaling, even though downstream signaling through rip2 is impaired. further studies are required to address the association of the gain of function (inhibit tlr2 signal) of 1007fs, or other mutants, with atherosclerosis development. the yan group reported that mdp mediated innate immune signaling induces pge2 production in atherosclerotic plaque. can nod2 signaling mediate the vulnerable plaque formation ? if nod2 induces the vulnerable plaque formation, it will work as pro - atherogenic. however, it is not consistent with the study on nod2 deficient mice as it shows the anti - atherogenic effect. these findings 118 nod2 variants have been associated with various diseases, including crohn 's disease and other rheumatoid diseases. however, the genetic association of nod2 variants with atherosclerosis has not been reported until recently. the risk of atherosclerosis development is higher in inflammatory bowel disease patients. based on these data, it is possible that a relationship exists between nod2 mutation and atherosclerosis development. particularly, nod1 has a similar structure to nod2 ; nod1 has only one card domain, while nod2 has two. nod1 is widely expressed in whole tissue, nod2 expression is limited to myeloid cells, keratinocytes, and epithelial cells of intestine, lung, and oral cavities. only one paper reported on nod1 and atherosclerosis in 2015 ; administration of the nod1 ligand, fk565, to apoe deficient mice accelerated atherosclerosis development. nod2 has quite different effects, leading to the question of what causes such differences. in this review, we discussed the possible role of nod2 signaling in atherosclerosis pathogenesis ; we also summarized two recent studies revealing the relationship between nod2 and atherosclerosis. undoubtedly, elucidating these questions will provide new insights into the mechanisms of host defense and the pathogenesis of inflammatory diseases.	nod2 (nucleotide - binding and oligomerization domain 2) was initially reported as a susceptibility gene for crohn 's disease, with several studies focused on elucidating its molecular mechanism in the progression of crohn 's disease. we now know that nod2 is an intracellular bacterial sensing receptor, and that mdp - mediated nod2 activation drives inflammatory signaling. various mutations in nod2 have been reported, with nod2 loss of function being associated with the development of crohn 's disease and other autoimmune diseases. these results suggest that nod2 not only has an immune stimulatory function, but also an immune regulatory function. atherosclerosis is a chronic inflammatory disease of the arterial wall ; its pathologic progression is highly dependent on the immune balance. this immune balance is regulated by infiltrating monocytes and macrophages, both of which express nod2. these findings indicate a potential role of nod2 in atherosclerosis. the purpose of this review is to outline the known roles of nod2 signaling in the pathogenesis of atherosclerosis.
atherosclerosis is a common cause of morbidity and mortality in type 1 diabetes mellitus (t1 dm). the mechanism involved in acceleration of cardiovascular disease (cvd) and atherosclerosis in these patients is still poorly understood. in young patients with t1 dm and good glycemic control, however, even well - controlled diabetic youth may present subtle lipid abnormalities and serum cholesterol within the normal to borderline concentration coexistent with hyperglycemia may accelerate vascular lesions [1, 2 ]. disorders of carbohydrate metabolism were described in women and adolescents with polycystic ovary syndrome (pcos) [3, 4 ]. significant positive correlation between insulin and androgen levels suggests that insulin resistance plays an important role in the pathogenesis of pcos. in adult women with hyperandrogenism, adverse lipid profile such as increased concentration of triglycerides (tg) and non - hdl - cholesterol (non - hdl - ch) as well as decreased level of hdl - cholesterol (hdl - ch) were found. in patients with polycystic ovary syndrome (pcos), pirwany. reported increased hepatic lipase activity, enzyme responsible for the excessive production of atherogenic small dense ldl particles. the activity of this enzyme is regulated by insulin resistance, estrogens, and also the androgens. insulin resistance and hyperinsulinaemia, together with high androgens concentration, can therefore increase the risk of atherogenic lipid profile. increased incidence of hyperandrogenic disorders, hirsutism, and pcos were described in adult women with t1 dm [5, 6 ]. however, in adolescent girls clinical features of hyperandrogenism can be mild and hyperandrogenemia may be manifested only biochemically [79 ]. in these patients, high androgens concentration may contribute to dyslipidaemia, which, in coexistence with poor glycemic control, can increase the risk of cardiovascular disease. there are also data suggesting that androgen excess together with poor glycemic control may accompany development of microalbuminuria in pubertal girls. the aim of the study was to establish whether hyperandrogenemia in adolescent girls with t1 dm is associated with atherogenic lipid profile. all adolescent girls attending the pediatric diabetes clinic of the university hospital in katowice who menstruated for more than 1 year were consecutively invited to participate in the study. the exclusion criteria were honeymoon period, monogenic types of diabetes, type 2 diabetes mellitus, abnormal thyroid function, hyperprolactinaemia, congenital adrenal hyperplasia, consumption of medications known to influence sex steroids or shbg in the last 3 months, chronic systemic disease, and malnutrition. t1 dm diagnosis was confirmed (c - peptide level 0.05). in 16 (30%) diabetic girls, increased androgens levels were found (t1dm - h) ; in 38 (70%) all the androgens concentrations remained within the normal range (t1dm - n). fifteen healthy, regularly menstruating adolescent girls with no clinical signs of hyperandrogenism and with androgens level within the normal range, matched for chronological and gynecological age, served as the control group (cg). the study was conducted according to the declaration of helsinki and approved by the ethics committee of the medical university of silesia. informed consent was obtained from each subject or / and parent or guardian. in all participants, menstrual pattern was evaluated and oligomenorrhea was defined as menstrual cycles longer than 45 days in the last six months. weight was measured with seca scale with a precision of 100 g and height with harpenden stadiometer to 0.1 cm. waist circumference (wc) and hip circumference (hc) were measured with nonelastic flexible tape and waist - to - hip ratio (whr) was calculated. hirsutism was evaluated by one of the two authors (agnieszka zachurzok and aneta gawlik) by means of the modified ferriman - gallwey score. in each girl, the transabdominal pelvic ultrasound examination was performed by the same observer (agnieszka drosdzol - cop) with 5 mhz convex transducer (siemens acuson antares 5.0), and volume and structure of the ovaries were evaluated. ovaries were considered polycystic if 12 or more cysts 29 mm in diameter were present at least in one ovary and/or if increased ovarian volume (> 10 ml) occurred. in each subject with t1 dm, detailed medical history was obtained, including age at the onset of t1 dm, hba1c records from the beginning of the disease, and dir for the last 12 months expressed as insulin units per kilograms of body weight per day. serum concentration of lipids (total cholesterol (tc), tg, and hdl - ch), gonadotropins (lh, fsh), androstenedione (a), testosterone (t), 17-hydroxyprogesterone (17ohp), estradiol (e2), and sex hormone binding globulin (shbg) were measured. calculation of ldl - cholesterol (ldl - ch) concentration was performed according to friedewald 's equation. non - hdl - ch and ldl / hdl - cholesterol ratio (ldl / hdl) as well as free androgen index (fai = t 100/shbg) and lh / fsh ratio were also calculated. blood tests were performed during the follicular phase of menstrual cycle (37 days of cycle) or after 3 months of amenorrhea. biochemical hyperandrogenemia was defined if t level exceeded 58 ng / dl, a exceeded 4.1 ng / ml, or fai exceeded 4.5. plasma tc and tg levels were analyzed enzymatically, and hdl - ch concentration was measured by direct procedure using synthetic polymer and detergent (spd procedure, daichi). serum levels of lh, fsh, and dheas were measured using chemiluminescent immunoassay by immulite 2000 analyzer (dpc, usa). for dheas assay polyclonal rabbit anti - dheas antibody was used, with intra- and interassay variation coefficients : 7.1% and 8.2%, respectively. t and e2 were analyzed by electrochemiluminescence immunoassay by cobas e 411 analyzer (roche, switzerland). for t assay, biotinylated monoclonal anti - t antibody (sheep) was used, with intra- and interassay variation coefficients : 5.3% and 6.3%, respectively. hba1c was measured by high - performance liquid chromatography method, and shbg was determined by immunoradiometric assay (radim, roma, italy). 17ohp and a were analyzed by enzyme - linked immunosorbent assay (drg diagnostics gmbh, germany). for 17ohp assay, polyclonal goat anti-17ohp antibody was used, with intra- and interassay variation coefficients : 5.8% and 6.8%, respectively. for a assay, polyclonal rabbit anti - a antibody was used, with intra- and interassay variation coefficients : 7.1% and 8.4%, respectively. all values were expressed as mean standard deviation for normal or median (interquartile range) for skewed distribution. the normality of the empirical distributions of variables was verified by shapiro - wilk 's test and graphically by histograms. correlation analysis was performed using pearson correlation coefficient for normally distributed samples and spearman correlation coefficient for nonnormally distributed data. comparison between independent groups was performed using student 's t - test for normally distributed data and mann - whitney u test for skewed distributions. differences between the parameters in the three groups (t1 dm with normal androgen level, t1 dm with hyperandrogenemia, and control group) were assessed by one - way anova for normally distributed variables, followed by the post hoc test least - significant difference (lsd) for multiple comparisons. homogeneity of variances in this analysis was verified by levene 's test. for nonnormal distributions kruskal - wallis test was used. to evaluate the relationship between independent anthropometric, metabolic, and hormonal variables (bmi z - score, whr, hba1c from the diagnosis of t1 dm, dir, t, a, and fai) and dependent lipid variables (tc, tg, ldl - ch, ldl / hdl, and non - hdl - ch) in all t1 dm girls methods applied for statistical analysis were adequate for the sample size used in the study. p value 175 mg / dl) was present in 10 (55.5%) t1dm - h and in 20 (52%) non - hdl - ch above 130 mg / dl was found in 7 (39%) and 11 (29%) diabetic groups, respectively (p > 0.05), and ldl - ch exceeding 100 mg / dl in 11 (61%) and 18 (47%) groups, respectively (p > 0.05). in none of the girls from the study and control group ldl - ch lipid profiles in the three groups of girls are presented in figures 1(a) and 1(b). tc and ldl - ch concentration in t1dm - h were significantly higher than in cg (196.1 41.2 versus 162.7 31.7 mg / dl, p = 0.01 ; 117.3 33.1 versus 91.3 27.8 mg / dl, p = 0.01, resp.) moreover, ldl - ch, non - hdl - ch concentration, and ldl / hdl ratio were also significantly higher in t1dm - h than in t1dm - n (117.3 33.1 versus 97.7 26.7 mg / dl, p = 0.03 ; 137.3 42.9 versus 113.3 40.4 mg / dl, p = 0.04 ; 2.8 3.7 versus 1.6 0.5, p = 0.04, resp.). hdl - ch concentration was similar in both diabetic groups and higher than in cg ; however, significant difference was seen only between t1dm - n and cg (61.2 12.4 l versus 50.6 10.4 mg / dl, p = 0.01). in diabetic girls wc correlated positively with tc (r = 0.36, p = 0.03) and whr correlated significantly with tc (r = 0.43, p = 0.007), ldl (r = 0.45, p = 0.005), and non - hdl - cholesterol (r = 0.42, p = 0.01). we found no significant relationship between lipids and bmi or bmi z - score. only a tendency toward significant correlation of mean hba1c from the onset of t1 dm and tg was found (r = 0.28, p = 0.054). correlation between lipids concentration and dir per kg of body weight, age at t1 dm diagnosis, t1 dm duration, and mean hba1c were not statistically significant. there was a significant relationship between fai and ldl (r = 0.37, p = 0.01) and a tendency toward relationship between fai and tc (r = 0.28, p = 0.07). moreover, hyperandrogenemia correlated significantly with ldl (r = 0.35, p = 0.01) and with ldl / hdl ratio (r = 0.28, p = 0.05). to identify anthropometric (wc, hc, and bmi z - score), metabolic (dir for last 12 months, mean hba1c from the t1 dm onset), and hormonal parameters (t, a, and fai) and their influence on lipid concentration in girls with diabetes, stepwise multiple linear regression was performed (table 3). the regression models showed that fai and whr were significantly associated with tc (r = 0.4, p < 0.001) as well as with non - hdl - ch (r = 0.4, p < 0.001) and ldl - ch (r = 0.4, p < 0.001). tg concentration and ldl / hdl were significantly (r = 0.7, p < 0.001 ; r = 0.6, p < 0.001, resp.) related to the mean hba1c, whr, t, and fai. to validate the influence of glycemic control on lipid profile, we divided girls with t1 dm, according to ispad guidelines, into two groups : well - controlled (38 girls, mean hba1c < 7.5%, mean : 6.6 0.5% (48.8 5.9 mmol / mol)) and poorly controlled (16 girls, mean hba1c 7.5%, mean : 9.4 1.2% (79.4 13.3 mmol / mol), p < 0.001). in poorly controlled diabetic girls bmi z - score and wc were higher than in well - controlled ones (1.0 (0.31.3) versus 0.4 (0.00.7), p = 0.02 ; 0.81 (0.770.85) cm versus 0.77 (0.750.79) cm, p = 0.04). there were no significant differences with respect to hormonal profile between the groups ; however shbg concentration was significantly higher in well - controlled group (50.6 18.8 versus 37.9 15.2 nmol / l, p = 0.04). significantly higher tg level was found in girls with poorly controlled t1 dm than in well - controlled ones (113.0 (85.0141.0) versus 89.0 (63.0115.0) mg / dl, p = 0.03). also the tendency to higher non - hdl - ch was seen in this group (126.7 (107.9165.3) versus 115.0 (99.6133.1) mg / dl, p = 0.07). there were no significant differences with respect to other lipids between the two groups of diabetic girls. the relationship of hyperandrogenic disturbances with atherogenic lipid profile is documented in women with pcos with and without t1 dm [36, 15, 16 ]. however, to our knowledge, this is the first report presenting the relationship of hyperandrogenemia and lipid profile in diabetic adolescent girls. marked dyslipidaemia is more characteristic for type 2 diabetes, and in t1 dm lipid disturbances are rather mild. however in both types of diabetes, poor glycemic control increases serum triglyceride levels and decreases hdl. it can also result in a modest increase in ldl - cholesterol, which because of the elevation in triglycerides is often in the small dense subfraction. it is therefore important to optimize glycemic control in patients with diabetes because this will have secondary beneficial effects on lipid levels. in our diabetic patients, mean tc exceeded ispad norms in both subgroups but the mean level of non - hdl - ch and ldl - ch was increased only in girls with biochemical hyperandrogenemia. on the other hand, diabetic girls in both subgroups presented hdl - ch level higher than in control group ; however, only in girls without androgen excess the difference was significant. we did not assess apolipoprotein concentration, but the ldl / hdl ratio is believed to be a good apob / apoa surrogate. we found significantly higher ldl / hdl ratio in t1dm - h girls with respect to the other groups which can reflect more atherogenic lipid profile in these patients compared to the girls without hyperandrogenemia. described an adverse metabolic phenotype in women with pcos and high testosterone level but not in those having elevated androstenedione only. moreover, higher androstenedione / free testosterone ratio was associated with a beneficial metabolic profile. according to wild and bartholomew, women with pcos had tc and non - hdl - ch levels twice as high as and hdl level significantly lower than non - pcos controls. elevated ldl concentration was found even if women with pcos were neither overweight nor obese. demonstrated in women with pcos higher hepatic lipase activity that correlated not only positively with wc and whr but also negatively with shbg concentration. in accordance with these data, we have also found the correlation between wc and whr and atherogenic lipid profile, but we did not observe any relationship between lipid disturbances and shbg level. however, there was a positive correlation between fai and ldl concentration as well as between hyperandrogenemia and ldl and ldl / hdl ratio. similar relationship was reported by lerchbaum., who described in women with pcos a positive correlation between increased free testosterone concentration and high tc and tg as well as low hdl. we did not find any differences in hba1c from the beginning of the disease as well as dir for the last 12 months between diabetic groups with or without hyperandrogenemia. therefore, we believe that we were able to examine the real influence of androgen excess on lipid pattern. on the other hand, we found the relationship between the glucose homeostasis and lipid profile, as in girls with poor glycemic control tg level was significantly higher. moreover, wild and bartholomew revealed that in women with pcos altered glucose - insulin homeostasis is a stronger contributor to dyslipidaemia than hyperandrogenemia or chronic high estrogen exposure. these two mechanism - hyperandrogenemia, which can increase hepatic lipase activity coexisting with poor glycemic control implicated in hypertriglyceridaemia, could lead to more atherogenic lipid profile in this specific group of diabetic girls. another interesting finding in our research was lower dheas concentration in diabetic girls with no hyperandrogenemia while in hyperandrogenic girls it was comparable to the control group. found that 58% of diabetic children have abnormal response to acth test as well as lower dheas concentration without any signs of hypocortisolism. in type 2 diabetes, lower levels of dheas were also reported. serum levels of the entire sulphoconjugated steroids were found significantly lower in well - controlled patients in comparison with poorly controlled type 2 diabetics. in our diabetic subjects, there was no difference between well- and poorly controlled girls with respect to dheas concentration. moreover, in diabetic hyperandrogenic girls, testosterone and androstenedione concentration as well as fai were increased, whereas 17ohp level was similar to controls. this suggests that although the main source of androgens in girls with diabetes and hyperandrogenemia is the ovary, there may be also the upregulation of adrenal androgens production. however, this finding needs to be confirmed on a larger group of adolescents with t1 dm as in the study by meyer. dheas in diabetic adolescents was not different than that in the controls. in patients with insulin - dependent diabetes, to deliver appropriate amount of insulin to the liver and achieve good metabolic control, most often supraphysiological doses of insulin are injected subcutaneously. hyperinsulinaemia, due mainly to cogonadotropin action, can stimulate androgens synthesis in the ovaries. in our study, girls with hyperandrogenemia had higher dir with respect to girls with normal androgens, but the difference was statistically insignificant. however, lack of significance could have been a result of the insufficient number of patients in the groups examined. our study limitation is a small number of diabetic girls with elevated androgen level compared to a larger group of girls without hyperandrogenemia. considering that girls with t1 dm are less affected by clinical hyperandrogenism and the risk of pcos development is lower than in their healthy peers, it is evident that a single center may not have a large number of patients. we also acknowledge the fact of methodological problems regarding androgens determination in young girls due to inadequate assays sensitivity to measure low concentration, interference of other steroid molecules with similar structure, and the lack of well - defined normative values. however, all the assays were performed in the same laboratory using the same kits in girls from the study and from the control group. we have also included some girls with gynecological age below 24 months in whom irregular menses could be due to the incomplete maturation of hypothalamic - pituitary - ovarian axis. although we were not evaluating pcos symptoms but only biochemical hyperandrogenism in our patients, according to rosenfield pcos should be considered when menstrual irregularity persists for more than 1 year. we conclude that hyperandrogenemia may unfavorably influence lipid profile in adolescent girls with t1 dm, particularly in those with high whr and poor glycemic control. the issue of high androgens in diabetic adolescent girls is important, not only because of adverse effect on fertility in the future life of young women, but also with respect to increased risk of cardiovascular disease and microalbuminuria. identification of the next modifiable risk factor, in the perspective of longer life with t1 dm, which is now possible with intensive diabetes care, could give this particular group of patients some practical advantages. the study results need to be confirmed in multicenter studies with a larger number of patients.	study objectives. the study aim was to evaluate whether hyperandrogenemia in adolescent girls with type 1 diabetes mellitus (t1 dm) may adversely influence lipid profile. design and participants. lipid levels in 16 diabetic girls with biochemical hyperandrogenemia (t1dm - h) aged 16.3 1.2 years were compared to 38 diabetic girls with normal androgen levels (t1dm - n) aged 15.8 1.2 years. 15 healthy girls served as controls (cg). in all patients, anthropometric measurements were done, and androgens and shbg were assessed. results. in t1dm - h, total cholesterol (tc) and low density cholesterol (ldl - ch) were significantly higher than in cg (196.1 41.2 versus 162.7 31.7 mg / dl, p = 0.01 ; 117.3 33.1 versus 91.3 27.8 mg / dl, p = 0.01, resp.). their ldl - ch, non - high density cholesterol (non - hdl - ch) concentrations, and ldl / hdl ratio were also significantly higher than in t1dm - n (117.3 33.1 versus 97.7 26.7 mg / dl, p = 0.03 ; 137.3 42.9 versus 113.3 40.4 mg / dl, p = 0.04 ; 2.8 3.7 versus 1.6 0.5, p = 0.04, resp.). in stepwise multiple linear regression, free androgen index (fai) and waist - to - hip ratio (whr) were associated with tc (r2 = 0.4, p < 0.0006), non - hdl - ch (r2 = 0.4, p < 0.0003), and ldl - ch (r2 = 0.4, p < 0.0008). triglycerides and ldl / hdl ratio were (r2 = 0.7, p < 0.0001, r2 = 0.6, p < 0.0003 resp.) related to testosterone, fai, whr, and mean hba1c. conclusion. lipid profile in diabetic adolescent girls is adversely influenced by the androgens level, particularly in the group with higher whr and poorer glycemic control.
diffuse axonal injury (dai) is a brain injury characterized mainly as axonal injury of the white matter. it often follows brain trauma, which causes wide - ranging denaturation of white matter, focal hemorrhage, emergence of axonal retraction balls, and microglia clusters. dai is often accompanied by other brain injuries, and this has caused patients severe brain damage or even placed them in a persistent vegetative state. according to reports made in recent years, the mortality rate of dai is 42%62% [1, 2 ]. dai has been as an independent category of disease accepted by neurosurgery academic. however, there are currently no standard diagnostic criteria, and the relationship to other brain injuries needs to be investigated further in order to develop better clinical treatments for dai. below, the authors review the concept, pathological mechanism, and methods of clinical diagnosis of dai. it has gone through three conceptual stages in its history. the first period began in 1956, when strich studied autopsies from 5 patients with severe closed brain trauma and proposed that degeneration of the diffuse white matter might be attributed to the physical damage to nerve fibers. the second period began in 1961, when this strich studied 20 patients who had died of brain trauma. he found that the shearing force of the rotational acceleration of head movement (one of the main causes of brain injury) caused the nerve fibers to break and evoked diffuse degeneration of hemisphere and brainstem. the third period began in the 1980s, when adams and gennerelli studied the mechanism of development and clinical pathology of dai thoroughly and made prominent achievements, which were given great consideration when the international academic community selected a final name for this condition. it takes place after external injury involving shearing force, and it mainly manifests in the form of focal axonal changes and axonal breakage. and it can be divided into primary and secondary axonal injury. the pathological mechanism of dai is very complicated, but a clear understanding of the pathological mechanism is very important to diagnosis, clinical treatment, and prognosis ; pathological characterization has become a hot topic in neurosurgical research. the main cause of primary axonal injury was axonal breakage, retraction, and the formation of what is called axon retraction balls because of the shape of the swelling at the end of the axonal axis, which was caused by the external shear force and tension. the formation of these axon retraction balls was believed to lead to the final breakage of the axon. currently, it is thought that the axon retraction balls cause axon breakage, so interrupting protein transport, and the individual axon retraction ball has been observed under microscopy at the end of broken axons. however, multiple recent studies have shown that the site of instant, strong shearing force or tension within the brain does not always match the site of actual injury. animal studies have shown there to be no axon breakage immediately after brain trauma, and pathological examination suggested that the myelin of the axons had remained intact [36 ]. this has sparked debate over whether it is suitable to assess the number of injured axons by determining the total number of axon retraction balls after onset of dai. after external instant shear force and tension act on the brain, the permeability of the axon membrane changes, and large amounts of ca enter the cells. the anterograde transport of axon plasma is gradually converted to retrograde transport, so activating the cysteine protein signal pathway and caspase-3. the inherent cellular calpain inhibitor calpastatin is hydrolyzed. a relatively high level of activated calpain accumulates within the cell, and this degrades the axonal cytoskeleton network. recent studies have shown that influx of ca and degradation of the axonal cytoskeleton network are progressive events, during which axons usually maintain their morphology several hours after injury [710 ]. spectrin, also called cell ghost, is a structural protein found on the inner side of the membrane. it not only supports the lipid bilayer but also maintains the shape of red blood cells. it forms a transformable network beneath the plasma side of the membrane and so maintains the biconcave disk shape of red blood cells. during the early stage of injury, calpain - mediated hydrolysis of spectrin in focal axon was observed, as indicated by single and double markers ' under immunohistological examination via light microscopy and electromicroscopy. most axons show signs of calpain - mediated hydrolysis of spectrin 1 - 2 h after the injury. related pathological changes include loss of microtubules, swelling of the mitochondria, and neurofilamentous knots, which indicate that calpain - mediated hydrolysis of structural protein and degradation of the cytoskeleton play important roles in the development and progression of dai pathology [1113 ]. mitochondrial damage after onset of dai mainly includes swelling and breakage of the mitochondrial crest and membrane. ca influx leads to changes in the permeability of the mitochondrial membrane and affects the opening of the switching pore in said membrane. the intake of small molecules causes the mitochondria to swell and break, which further not only disrupts the energy metabolism and ion homeostasis but also releases caspases and the activators of apoptosis, so triggering caspase - mediated progressive cell death. impairment of the mitochondria, imbalance in ion homeostasis, the release of proapoptotic factors, and activation of caspases are key contributors to the high mortality and poor prognosis of dai. it has drawn a great deal of attention because it can be converted to toxic -amyloid (a) after protease hydrolysis. the use of immunohistology to assess changes in app in axons is the gold standard of neuropathology and trauma model diagnosis of dai [18, 19 ]. once pathological changes take place, the anterograde transport of app becomes disrupted, which causes focal aggregation of app. increasing amounts of evidence show that changes in glia cells play very important roles in the development and progression of dai. the morphological and functional changes in astrocytes, microglia, and oligodendrocytes that take place after onset of dai and are called glial reaction. glial cells become activated and involved in eliminating and engulfing particles expelled from the site of injury, extend projections to fill in cavities, form glial scars, and produce matrix metal proteins (mmps) to reconstruct damaged extracellular matrices after the progression of dai. glia cells also express insulin - like growth factor-1, epithelial growth factor, and other neurotrophic growth factors in order to decrease the rate of neuronal death and neural injury after the progression of dai [20, 21 ]. astroglia (as) is a major type of glial cells in the central nervous system (cns) originating from neural ectoderm. the distribution of as in the brain was regular (gfap positive cells in hippocampus and dentate gyrus in obvious rules). this kind of order contributes to the position of the fixed relationship and the function of stable relationship between as and neuron. and as may also be involved in the complex functions of the brain activity, including learning and memory. recently, it showed that as clears hemorrhage in the early damage and degeneration necrosis tissue with macrophages and thereby promotes wound repair [22, 23 ]. corresponding to different neurotransmitters and neuropeptide, there are many receptors in as, such as 5-ht and -gaba. in recent years we thought that it (at least under the condition of in vitro) has almost all possible neurotransmitters functional receptors. after being damaged, neurons produce more neurotransmitters than normal, so the receptors on the as can upregulate and produce more growth factors to promote repairing of injury. oligodendrocyte (olg) is myelin glial cells in the central nervous system and rich in grey and white matter of brain and spinal cord. mechanical damage, ischemia, or axonal degeneration can cause the damage and apoptosis of olg ; otherwise, there is great relevance between axonal degeneration after brain injury and the apoptosis of olg. and the fas and p75 receptor activation may be involved in apoptosis. however, glial cells become activated further, to the point of overactivation, as dai progresses. overactivated glial cells continuously release inflammatory factors, such as il-1 and tnf-, and they release oxygen free radicals and cytotoxic substances, which elicits inflammatory responses, causes oxidative stress in brain tissue, and directly or indirectly induces neuronal death. overactivation of glia cells causes the release of chondroitin sulfate proteoglycan, prevents the glia cells from reconstructing the extracellular matrix, inhibits axon growth, and weakens the ability of glial cells to eliminate products expelled from the site or injury. in this way, overactivated glial cells promote neuronal injury. activation of glia cells can also promote neuron - glia and glia - glia interactions. previous studies have demonstrated that the chemokine cxcl-12, which is released from astrocytes, promotes the release of glutamate, which further promotes the release of large amounts of tnf- from microglia. high concentrations of tnf- impair the ability of microglia to eliminate glutamate, and this causes excitatory toxicity and injures neurons. astrocytes also release the anti - inflammatory factor il-10, which inhibits the release of tgf- from microglia and promotes the maturation of oligodendrocytes [2730 ]. however, it remains unclear whether the activation of glial cells promotes injury or repair. ct allows rapid and reliable location of focal hemorrhages related to axonal injury, but it is difficult to find injuries other than hemorrhages, especially if they are small in size or involve needle - like bleeding. traditional mri examination not only allows rapid location of hemorrhages, but it is also a sensitive and reliable way of locating nonhemorrhages. it has better resolution than ct scans and it is especially suitable for injuries to the posterior cranial fossa and deep white matter. however, it still has a high rate of false negative results for small lesions and mild dai. moreover, patients are often unable to complete the examination due to the long time requirements. as medical science has progressed, more accurate methods of diagnosing dai have been developed. dwi involves using the anisotropy of protein to identify changes in white matter after onset of dai. studies have shown dwi to be an accurate method of examining nonhemorrhage injuries, especially at the sites within the cranial vault. however, this method is often not sufficiently accurate for the examination and diagnosis of injuries to the corpus callosum and grey matter. dti, which was developed as an improved form of dwi, can be used to evaluate nerve alignment, injury context, and the microstructure of white matter effectively. it can also allow direct observation of the nerve alignment and the collection of abnormal morphology information regarding major nerve fibers. in this way, dti can detect dai in a highly sensitive way and allow estimation of the time elapsed from injury to examination. gre - swi can detect more minor hemorrhages and so indicate the severity of dai more accurately than other methods can, which makes it especially suitable for early diagnosis of dai. this new imaging method is the use of magnetic susceptibility which is different between different organizations and imaging technology. and the key to imaging is magnetic sensitive material ; in some tissues, such as venous blood, bleeding, and calcification, the magnetic susceptibility is different from that of surrounding tissues. on the one hand it can shorten t2 ; on the other hand, it can lead to blood vessels and surrounding tissues of different phase contrast. diffuse axonal injury (dai) accounts for more than 30% of severe craniocerebral injury and is the main causes leading to a vegetative state or serious nerve dysfunction. further clinical study found hemorrhage of dai with worse prognosis than less bleeding. however, both ct and routine mri are not sensitive to the smaller hemorrhage stove. gre - swi is very sensitive to hemoglobin metabolites, such as dna, methemoglobin, hemoglobin, and hemosiderin. so, gre - swi can detect these metabolites more effectively than conventional mri [31, 32 ]. so the gre - swi play an important role in the evaluating, treating of traumatic brain injury, and prognosis judging. although gre - swi is valuable for finding the minor hemorrhage in brain clinically, it still can not make difference between other minor hemorrhages caused by patients related diseases, such as hypertension. and the acquisition and processing technology still needed further improvement, to improve the scanning speed, reduce artifacts, and improve the signal - to - noise ratio. animal studies have shown that rats with mild dai have abnormal neural electrophysiology regardless of whether they have sustained any axonal injury. other studies have shown pathological changes and decreases in action potential in the axonal axis of the corpus callosum of mice with brain trauma. the action potential of both myelinated nerve fibers and unmyelinated nerve fibers in the corpus callosum has been found to decrease. among those nerve fibers, myelinated fibers were found to recover their action potential gradually as their axons were repaired, while unmyelinated nerve fibers did not [3438 ]. these findings indicated that the abnormal action potential of unmyelinated nerve fibers may play an important role in the disability associated with dai. currently, commonly used biochemical markers for acute dai diagnosis and analysis of the conditions and prognosis associated with dai include -app, spectrin, and its decomposition products sbdp145 and sbdp150. other markers include neurofilaments and the phosphorylated products of their tau subunits and hydrolyzation of myelin basic protein. the detection of -app is currently considered the gold standard of dai examination in forensic and laboratory settings. it is often used for early diagnosis of dai. under normal conditions, the -app present in axons can not be detected using immunohistochemistry. however, after onset of dai, the disruption of transportation through the axoplasm causes -app to aggregate in the axons, bringing its concentration up to detectable level. this makes it suitable for use as a marker for early diagnosis of dai. however, detection of -app by immunohistochemistry after onset of dai can cause underestimation of the scope of axonal injury. through more in - depth studies, detection of -app695, an isoform of -app, could provide more reliable and sensitive diagnosis of dai. attention must be paid to diseases that can cause clinically abnormal axonal metabolism, in which -app has been shown present via immunohistochemistry. in this way, patients ' disease history must be taken into consideration, which would increase the accuracy of diagnosis via immunohistochemical examination of -app. the spectrin - ii subunit is present within the neuron body, dendrite, and axons. along with neurofilaments and microtubule - associated proteins the spectrin - ii subunit of calpain degradation products (sbdp) detected in cerebral cortex, cortex medullary junction, corpus callosum, and cerebrospinal fluid following dai mainly include sbdp-150 and sbdp-120. the trends in the changes of the concentrations of sbdp-150 and sbdp-120 in the cerebral cortex and corpus callosum have been shown to be similar, which indicate that, after onset of dai, calpain - induced necrosis is an important pathological mechanism of dai. however, the trends in the concentrations of sbdps in cerebrospinal fluid are not synchronous with those of the brain, and the trends in the concentrations of degradation products from different subunits of spectrin are also different. one possible reason for this is that the proteins released from the brain parenchyma must be transported into the cerebrospinal fluid via the intercellular fluid, while proteins released from injured neurons in the subarachnoid space can be released directly into the brain. in this way, the measurement of the expression of different subunits of spectrin expression could be used to assess the severity of dai, show whether it is associated with focal or diffuse functional impairment, and provide some basis for predicting the pathological mechanism of dai. neurofilaments are composed mainly of light chains (nf - l), medium chains (nf - m), and heavy chains (nf - h). after onset of dai, the spatial configurations of nf - l, nf - m, and nf - h peptides were different, according to the severity of dai. in mild and moderate dai, three types of nf subunits presented focal disorder. phosphorylated neurofilament was hydrolyzed and finally resulted in neurofilament collapse. because nf - h can be detected in serum after onset of dai and increased from 6 h, peaked at 12 h and 48 h, and decreased to normal level on day 7 [42, 43 ]. nf - m must be investigated further if it can be used as a specific marker of dai diagnosis. each molecule of tau contains 2 - 3 phosphoric acid groups. overphosphorylated tau groups lose their normal transport function in axons and in turn inhibit the assembly and promote dissemble of microtubule, finally causing axonal breakage. after onset of dai, tau was depolymerized to c - tau by calpain, which can be detected in large amounts in cerebrospinal fluid. the detection level of c - tau in the cerebrospinal fluid is negatively correlated to the severity of dai of patients in clinical settings [44, 45 ]. in this way, the detection of c - tau in cerebrospinal fluid was used to quantitatively evaluate the severity of axonal injury. investigation has shown that once the c - tau level in patients ' cerebrospinal fluid reaches 2.126 mg / ml, the accuracy of prognosis of the mortality rate reaches 100% and specificity rises above 80%. however, c - tau detected in serum was not found to facilitate effective evaluation of prognosis. for this reason, the detection of c - tau levels in the cerebrospinal fluid is considered one of the most suitable biochemical markers for clinical diagnosis of dai. myelin basic protein (mbp) is the main protein in myelin in the central nervous system (cns). it is present on the plasma side of myelin, where it keeps the protein 's structure and function stable. it is specific to nerve tissue. because of the blood - brain barrier (bbb), mbp is readily released into cerebrospinal fluid, and a very small amount of mbp is released into the blood. after onset of dai, the cns is damaged and the bbb can be completely destroyed. the changes in the permeability of bbb result in the increase of mbp levels in serum. determination of mbp level in serum can indicate its quantity in a timely manner, and the samples for determination are easy to collect. scholars both within and outside of china have reported that mbp could be a suitable index of the severity of cns injury. in the same way, the determination of mbp levels in serum and cerebrospinal fluid could facilitate preliminary judgement of the severity of dai and allow objective evaluation of the progression and prognosis of dai. however, the sensitivity of the detection of serum mbp is not currently ideal and the use of mbp detection in clinical settings is limited. other biomarkers for diagnosis of dai include cyclooxygenase-2, aquaporin-4, inflammatory reaction factors (such as il-1, il-6, and tnf), and basic fibroblast growth factor. these factors can facilitate diagnosis of continued injury, inflammatory responses, and the development and progression of dai. although neuropsychological assessment as a noninvasive form of diagnosis can not be used to quantify dai, it can be used to indirectly show the efficacy of clinical treatment according to the differences in consciousness and cognitive disorders of patients in acute and subacute states. studies have shown that cognitive disorder is related to the site of injury, correlated in some extent to the state of the white matter connected to specific functional areas. increasing numbers of investigators have attempted to discern clinical efficacy directly through digitalized neuronal evaluation. according to the different standards, a variety of partition can be made to the neuropsychological test. the most common ones are divided into a single test and battery of tests. and the test concludes infants, children, and adults, three versions. and the test is divided into part for verbal test and others for nonverbal test. the revised hrb test battery mainly surveys the following ten aspects : category test, touch operation test, music rhythm test, finger tapping test, halstead - wepman aphasia screening test, voice perception test, on one side of the edge test, grip strength test, the attachment test, and perceptual disorder test. this set of tests use demarcation points as the norm (the critical points) to distinguish pathology. then according to the abnormal test counting damage index damage index = abnormal test number / total number. the first version includes 269 projects, a total of 11 subtests. the second version added intermediate memory subtest. there are 11 subtests that constituted the first edition of lnnb and include sports test, rhythm test, touch test, visual test, feeling type words, expressive words, writing test, reading test, math quiz, memory test, and intellectual processes test. and lnnb has three additional scales, as the disease symptoms characteristic scale (qualitative scale), the left hemisphere lateralization of scale, and right side of the scale. each project of lnnb adopted 3-level scoring mode : 0 is normal, 1 represents borderline state, and 2 indicates exception.	the current work reviews the concept, pathological mechanism, and process of diagnosing of dai. the pathological mechanism underlying dai is complicated, including axonal breakage caused by axonal retraction balls, discontinued protein transport along the axonal axis, calcium influx, and calpain - mediated hydrolysis of structural protein, degradation of axonal cytoskeleton network, the changes of transport proteins such as amyloid precursor protein, and changes of glia cells. based on the above pathological mechanism, the diagnosis of dai is usually made using methods such as ct, traditional and new mri, biochemical markers, and neuropsychological assessment. this review provides a basis in literature for further investigation and discusses the pathological mechanism. it may also facilitate improvement of the accuracy of diagnosis for dai, which may come to play a critical role in breaking through the bottleneck of the clinical treatment of dai and improving the survival and quality of life of patients through clear understanding of pathological mechanisms and accurate diagnosis.
	we report class a carbapenemase (kpc)-3-producing klebsiella pneumoniae meningitis in a 6-month - old child in algeria. multilocus sequence typing showed that the sequence type obtained corresponded to st512, an allelic single - locus variant of the pandemic st258 widely distributed in kpc producers from europe. to our knowledge, this is the first report of kpc-3-producing k. pneumoniae st512 in a north african country.
myocardial depression in the setting of sepsis and septic shock is common and has been recognized for a long time [15 ]. it has been shown that cardiovascular involvement increased mortality from 70% to 90%, compared to 20% in septic patients without myocardial impairment [2, 6 ]. thus, cardiac dysfunction in sepsis is thought to have bad prognostic value as it coincides with its severity. septic cardiomyopathy is characterized by reversible biventricular dilatation, decreased ejection fraction, and impaired response to fluid resuscitation and catecholamine stimulation. however, septic myocardial impairment remains a clinical enigma [7, 8 ], and even its real incidence is uncertain due to the imprecision with which it is clinically described, the heterogeneity in patient selection in the published studies, and, finally, the lack of universally accepted definition of septic myocardial depression. a reduced left ventricular ejection fraction (lvef) is often used ; on the other hand, septic cardiomyopathy can be defined as a global (systolic and diastolic) but reversible dysfunction of both the left and right sides of the heart [7, 10 ]. in recent years, the hypothesis that the myocardial dysfunction during sepsis could be mediated by ischemia related to decreased coronary blood flow waned and a complex mechanism was invoked to explain cardiac dysfunction in sepsis [11, 12 ]. in fact, septic shock is characterized by circulatory compromise, microcirculatory alterations, and mitochondrial damage, which all reduce cellular energy production. in order to reduce the risk of major cell death and a diminished likelihood of recovery, adaptive changes appear to be activated in sepsis. as a result furthermore, the effects of the host 's immune - inflammatory response with particular focus on depressant molecules (i.e., tnf - alfa, il-1), complement molecules, cellular adhesion molecules, and altered intracellular energetic and dysregulated intracellular calcium fluxes have been called upon in the pathophysiology of myocardial depression in sepsis [1419 ]. a role of unbalance in oxidative status leading to high production of reactive oxygen species (ros) has been hypothesized as playing a pivotal role in myocardial depression in sepsis. nitric oxide (no), a mediator involved in sepsis, is known to have a strong multifaceted influence on cardiac function since it affects the systemic and cardiac vascular tone and has direct effects on cardiomyocytes. finally, in the recent years, mitochondrial dysfunction has been considered as a crucial mechanism of heart impairment in sepsis. conclusively, a very complex pathogenesis involving a combination of hemodynamic, molecular, genetic, and metabolic cardiac alterations underlies cardiac involvement in sepsis. in this paper, we review the current literature regarding the pathophysiology of cardiac dysfunction in sepsis focusing on the possible role of oxidative - nitrosative stress unbalance and mitochondria dysfunction. we discuss these mechanisms within the broad scenario of cardiac involvement in sepsis, presenting also our related data obtained on postmortem cardiac samples of septic patients. oxidative stress (os) arises as a result of an imbalance between free radical production and antioxidant defense. when antioxidant strategies are overwhelmed, os results and excessive ros and reactive nitrosative species (rns) are produced. ros can cause oxidation damage to all cellular components, including lipids, proteins, and dna. the latter is the most detrimental, since replication of damaged dna can lead to genetic mutations or apoptosis [2226 ]. some of these species interfere with signaling cascades, while others provoke deleterious effects on various biological molecules and structures. it is clear that the increased production of signaling species and strong oxidants act in synergy with collapse in energy metabolism to provoke cell dysfunction, which may result in organ failure and death. os in patients with sepsis has been widely described over the last years [2729 ], and it is now widely accepted that oxidative stress is central to the etiology of cell and organ dysfunction and tissue damage in sepsis [3033 ]. although several sources of ros may be involved, a family of the nadph oxidases appears to be especially important for redox signaling ; during sepsis, a major source of ros is the nadph oxidases that are present in a variety of cells, especially the professional phagocytes and endothelial cells, and that are central to the genesis of the inflammatory response (figure 1). nadph oxidase is a superoxide - generating enzyme comprising a membrane - bound catalytic subunit (nox) and several cytosolic regulatory subunits. nox2 is the catalytic subunit of phagocyte nadph oxidase [35, 36 ]. on activation, the cytosolic components translocate to the transmembrane catalytic protein gp91, which results in the formation of functional nadph oxidase complex., nadph oxidase is a pivotal source of ros that subsequently triggers the release of ros by other enzymes, thus playing a pivotal determinant role of the redox state of the myocardium [37, 38, 40, 41 ], and it has also been implicated in the tnf- production induced by lps. experimental data derived from animal models showed a strong increase in nadph oxidase activity and o2 in the heart in response to lps (lipopolysaccharides) [43, 44 ]. however, the role of this enzyme complex in myocardial septic depression is not completely clarified, partially due to the fact that the membrane subunit of the nadph oxidase gp91 (nox2) has at least 3 other homologs, nox1, nox3, and nox4, which are expressed in a cell- and tissue - specific fashion, are subject to independent activation and regulation, and may subserve distinct functions. nox2-derived ros were likely to mediate hyperinflammatory responses and sepsis - induced mortality in mice. in particular, peng. demonstrated that the subunit gp91 of nadph oxidase plays a critical role in myocardial depression induced by endotoxemia and that gp91-containing nadph oxidase signaling contributes to lps - stimulated tnf- expression in cardiomyocytes. also the role of nox1/nadph oxidase in septic myocardial dysfunction has been investigated, and recently matsuno. investigated the involvement of nox1-derived ros in endotoxemia - induced cardiac dysfunction. using an animal model, the authors demonstrated a correlation between the increase of nox1 mrna and the production of ros in cardiac tissue of septic mice and that the increase in cardiomyocytes apoptosis and activation of caspase-3 induced by lps were attenuated in mice deficient in nox1. in particular, ros derived from nadph oxidase are known to induce cardiomyocytes apoptosis, and it has been suggested that nox1-induced ros might cause apoptosis by reducing akt signaling in the heart. apoptosis may be directly involved in cardiac dysfunction in sepsis as demonstrated in studies on animal models, showing that endotoxin may induce a tnf - alpha - dependent apoptotic cascade in the myocardium. finally, it is noteworthy that complex interactions among different ros sources exist and that nadph oxidase - produced ros may induce ros production by other sources, thus increasing the total level of ros. mitochondrial ros production can be increased by ros produced by different sources and it was demonstrated that mitochondrial ros production may, in turn, stimulate nadph oxidase ros production in endothelial cells. mitochondria, which occupy 3050% of the cardiomyocyte cytoplasmic volume, are critical in cardiac energy balance since energy supply for cardiomyocytes is mostly derived from mitochondrial oxidative phosphorylation (oxphos). on the other hand mitochondrial dysfunction, reflected in the structure, function, and number of mitochondria within the cardiomyocyte, leads to diminished energy production, loss of myocyte contractility, altered electrical properties, and eventual cardiomyocyte cell death. furthermore, since 1966 when jensen was among the first investigators to demonstrate that mitochondria produce ros, a solid evidence exists regarding ros production in mitochondria [6064 ]. mitochondrial dysfunction and its consequence, oxidative stress, have long been considered contributory factors in cardiac tissue damage [6568 ]. in sepsis, mitochondrial dysfunction exists [6972 ] and mitochondrial damage is thought to play a pivotal role in cardiac dysfunction during sepsis [73, 74 ]. several animal models of sepsis have demonstrated cardiac mitochondrial dysfunction during sepsis [7582 ]. evidence exists that mitochondrial dysfunction is a key feature in endotoxemia and the associated multiorgan failure syndrome including heart failure. soriano. studied twenty - five patients presenting with severe sepsis or septic shock and histologically demonstrated on heart sections derangements of mitochondrial cristae in patients who died. found mitochondrial abnormalities in patients who died from sepsis : hydropic change (edema of the mitochondrial matrix), cystic alterations of the cristae, and collapse into small myelin - like clusters were described in septic patients who died in surgical and medical intensive care units. conclusively, a large body of evidence supports the hypothesis that mitochondrial dysfunction and mitochondria - induced ros are key factors in cardiac impairment in sepsis. no is a free gaseous radical with function of messenger and effector molecule, synthesized by a family of enzymes (nitric oxide synthase (nos)). no synthesis is activated by one of the three isoforms of nos that are obligated homodimers that catalyze nadph - dependent oxidation of l - arginine to no and l - citrulline : nos1 (neuronal or nnos), nos2 (inducible or inos), and nos3 (endothelial or enos). no is an important bioactive substance which plays an important role in the regulation of normal body function and disease occurrence, and it is recognized to be a ubiquitous signaling molecule with a multitude of biological actions and targets. signaling may involve direct reactions between no and a molecular target or can occur through indirect reactions of secondary ros. in fact, actions of no are multifaceted, and its interactions with oxygen or oxygen - related reactive intermediates (e.g., superoxide) yield numerous reactive nitrogen as well as oxygen species. these account for most of the so - called indirect effects attributed to no through oxidation, nitrosation, and nitrate reactions referred to as oxidative, nitrosative, and nitrative stress, respectively. however, much about no biological actions remains contradictory, especially with regard to pathophysiologic disturbances in no signaling. there is an ongoing debate about the levels of no involved, whether there is a clearly defined threshold at which no crosses from being beneficial to being destructive. some authors hypothesized that the biological function of no depends mostly on concentration and time course of exposure to no, supposing that cytotoxic events, such as arrest of the cell cycle, cell senescence, or apoptosis, can occur at high no concentrations. however, other authors suggested that the chemical and biological reactivity of no that has been studied using very high no concentrations is of doubtful physiological relevance. vascular bioavailability of no is a critical factor in regulation of many physiological processes including blood pressure, vascular tone [9295 ], vascular permeability, adhesion and aggregation of platelets, and smooth muscle cell proliferation. the physiological production of no in the heart maintains coronary vasodilator tone and inhibits platelet aggregation and neutrophil and platelet adhesion, so performing an active role in cardioprotection. it is now determined that no protects the heart against ischemia - reperfusion injury [99, 100 ] ; however, excessive no formation is thought to contribute to contractile dysfunction [101, 102 ]. the inducible nos (inos / nos2) is generally believed to be the high - capacity no - producing enzyme in sepsis since endotoxin and cytokines and various mediators are demonstrated to overstimulate the inos which is inactive under physiological conditions. unlike the other two nos isoforms, inos is not constitutively expressed in cells, and its production is elicited by several stimuli like bacterial lipopolysaccharide and cytokines. primarly identified in macrophages, this enzyme may be expressed in virtually any cell or tissue, such as in the myocardium, and once expressed, inos is constantly active. although some studies demonstrated that high levels of no in sepsis could be beneficial due to a bactericidal effect, excessive production of no is an important player during hypotension and catecholamine - resistant septic shock and contributes to myocardial dysfunction [106109 ] (figure 2). in the inflammatory condition that is central in endotoxemia, no and peroxynitrite (onoo) have a central role in the development of mitochondrial dysfunction. several experimental studies performed on animals subjected to endotoxemia demonstrated that no production, production of o2 and h2o2, global protein nitration, nitrotyrosine content, protein carbonylation, and lipid peroxidation are increased in cardiac mitochondria [76, 80, 81, 110, 111 ]. moreover, the antioxidant systems seem to be inhibited as shown by decreased activity of mn - superoxide dismutase and glutathione peroxidase and depletion of glutathione [112, 113 ]. in their elegant experiment, escames. demonstrated that increased oxidative stress, impairment in oxphos function, and a decrease in atp production were restored by genetic deletion of inos (inos mice). this argument is further supported by the fact that treatment with melatonin, an inhibitor of inos, prevented the impairment of mitochondrial homeostasis after sepsis, restored atp production, and improved survival. other experimental studies conducted in septic animal models demonstrated an improvement of cardiac function by pharmacological inhibition or genetic deletion of nos. finally, the possible role of enos in cardiac involvement during sepsis has to be discussed. nos 3 is expressed in endothelial cells and in cardiac myocytes ; nos3-derived no physiologically has a positive inotropic and lusitropic effect, thus contributing to optimal cardiac performance and filling [116, 117 ]. studies demonstrated that during sepsis an increase in inos is induced by endotoxins and cytokines while a decrease in enos activity would be likely to happen. functioning of nos3 in sepsis is not yet completely clarified and controversial results are reported with studies showing that nos3 has no proinflammatory or anti - inflammatory effects in sepsis. for example, yamashita. examined the effect of chronic enos overexpression and the role of enos - derived no in lps - induced septic shock using enos transgenic mice and demonstrated that chronic enos overexpression in the endothelium of mice resulted in resistance to lps - induced hypotension, lung injury, and death. these effects are associated with the reduced vascular reactivity to no and the reduced anti - inflammatory effects of no. other studies postulated a proinflammatory role for enos and that enos - derived no is critical for maximal inos expression in the vasculature [121, 122 ]. more recently, bougaki. examined the impact of nos3 deficiency on systemic inflammation and myocardial dysfunction in vivo and in cardiomyocytes isolated from mice subjected to peritonitis - induced polymicrobial sepsis and reported that nos3 protects against systemic inflammation and myocardial dysfunction during polymicrobial sepsis. the detrimental effects of nos3 deficiency on myocardial function appeared to be caused by impaired ca2 + handling of isolated cardiomyocytes obtained from mice subjected to colon ascendens stent peritonitis. depressed ca2 + handling of cardiomyocytes of nos3ko mice were associated with impairment of mitochondrial integrity and marked depression of the ability of mitochondria to produced atp, a determinant of the function of the sr ca2 + -atpase pump. moreover, ichinose. demonstrated that cardiomyocyte - specific overexpression of enos prevented cardiac impairment and death after sepsis induction in mice, thus underscoring an important protective role of myocardial nos3 against endotoxin - induced myocardial dysfunction and death. finally, a recent study by van de sandt. demonstrated that endothelial nos plays a key role in the development of sepsis. in their experiment on male nos3 and c57bl/6 wildtype mice rendered septic by cecum ligation and puncture, the authors found that nos3 promoted a drop in mean arterial blood pressure and systemic vascular resistance, a hyperdinamc state despite impaired left ventricular function, a rapid deterioration of cardiac output, and limited coronary flow reserve, thus leading to short survival times. these findings were not observed in septic nos3 mice which showed that survival times extended more than twofold. although a two - step oxidation of l - arginine to l - citrulline, with concomitant production of no, represents the reaction assumed to be catalyzed by nos, these enzymes are also capable of catalyzing the production of additional products, notably superoxide anion (o2), depending on the conditions. during the reaction of molecular oxygen with the amino acid substrate l - arginine to produce l - citrulline and no, electrons donated by nadph at the carboxy - terminal reductase domain of nos are passed to the heme catalytic center of the oxidase domain, where activation of molecular oxygen is coupled to no synthesis by two successive monooxygenations of l - arginine. the cofactor 6r-5,6,7,8-tetrahydrobiopterin (bh4) is required for these reactions ; in its absence, electron flow to molecular oxygen becomes uncoupled from l - arginine oxidation, resulting in production of o2 instead of no [126, 127 ]. furthermore, low concentrations or absence of l - arginine and accumulation of endogenous methylarginines are supposed to cause the uncoupled reduction of molecular oxygen. both enos and inos are thought to be involved in the uncoupled reduction of oxygen, leading to the production of o2 and h2o2. superoxide derived from uncoupled nos or other mechanisms is an important source of cellular oxidative stress, including bh4 oxidation which may occur both directly by superoxide and through the oxidization to bh2 by onoo [129, 130 ]. conclusively, nos uncoupling leads to reduced de novo no production ; sequestration of bioactive no by superoxide anions via peroxynitrite formation ; and peroxynitrite - mediated oxidation of bh4 to bh2, resulting in further propagation of nos uncoupling. the production of onoo is a crucial pathophysiological event which occurs during sepsis since it represents a critical cytotoxic factor in oxidative stress - mediated tissue damage, supposed to be the no toxicity mediator [131134 ], which, in turn, exhibits multiple inhibitory actions in the mitochondrial respiratory chain. onoo and its derivatives are able to enter the cell membrane and consequently oxidize multiple target molecules, either directly or through the generation of highly reactive radicals, resulting in structural modification and dysfunctions in lipids, proteins, and nucleic acids with significant cytotoxic consequences [136139 ]. onoo can also react with carbon dioxide (in equilibrium with physiological levels of bicarbonate anion) leading to the formation of carbonate (co3) and nitrogen dioxide (no2) radicals, which are one - electron oxidants. no2 can undergo diffusion - controlled radical - radical termination reactions, resulting in nitrated species, such as nitrotyrosine (figure 3). alternatively, it can undergo homolytic fission to generate one - electron oxidants hydroxyl (oh) and no2 radicals. the proton - catalyzed decomposition to form oh and no2 radicals may become relevant in hydrophobic phases resulting in the initiation of lipid peroxidation processes [140, 141 ]. another important interaction of onoo occurs with nucleic acids, with the production of 8-hydroxydeoxyguanosine or 8-nitroguanine (figure 4). onoo can disrupt dna integrity, impair the activity of ion channel, break down mitochondrial respiratory chain, and induce cell death. it mediates nitration of tyrosine and cysteine residues in proteins which is one of the crucial pathways contributing to its cytotoxicity. several studies suggested that onoo is responsible, at least in part, for the development of endotoxin - induced hypotension, endothelial injury, multiple organ dysfunction, and subsequent death [145, 146 ]. in their nice experiment in sepsis model mice induced by lps, okazaki. found that the lps - treated mice were under oxidative stress and that species, such as superoxide and peroxynitrite, were mainly involved in the oxidative stress. in support of these results, superoxide, nitric oxide, and peroxynitrite cardiac formation has been demonstrated in septic hearts, which has been implicated in the pathogenesis of the myocardial depression and cell death in sepsis [148, 149 ]. finally, data from the literature showed that onoo could mimic many of the cardiovascular alterations associated with shock (endothelial dysfunction, vascular hyporeactivity, myocardial impairment, and cellular energetic failure) and that peroxynitrite neutralizers reduced peroxynitrite accumulation and improved myocardial contractile dysfunction and inflammation in septic animal models [136, 151 ]. peroxynitrite contributes to the cardiovascular collapse of septic shock, promoting vascular contractile failure and endothelial and myocardial dysfunction, and is also implicated in the occurrence of multiple organ dysfunction in this setting [152154 ]. collectively, these findings broaden our understanding of the role of oxidative and nitrosative stress unbalance in the pathophysiology of cardiac dysfunction in sepsis, thus contributing to establishment of a relationship between these settings and the occurrence of oxidative stress. cardiac impairment is common in severe sepsis and septic shock and it is demonstrated that it strongly contributes to the high rate of mortality in these subjects. oxidative - nitrosative stress may contribute to cardiac dysfunction in sepsis, and mitochondria are one of the major sites for generation of ros / rns as a detrimental side product of oxidative energy metabolism. in turn, damaged mitochondria may increase the production of ros / rns. to improve mortality and morbidity in the septic patient, the most important focus are a prompt and specific management of the infectious finalized to eradicate the causative pathogen as well as supportive therapy to maintain and restore organ function. this strategy is also recognized as the standard therapy for sepsis - induced cardiomyopathy ; however, attempts to reduce high mortality rates of patients with sepsis, severe sepsis, and septic shock by manipulating these functional alterations have provided limited success. the complex pathogenesis of septic cardiac failure, involving a combination of interconnected hemodynamic, structural, metabolic, molecular, and genetic alterations, may explain why despite all these therapeutic strategies, sepsis remains a big clinical challenge for effectively managing the disease to minimize mortality. numerous basic research and clinical trials have been undertaken to evaluate the possible modulation of the uncontrolled response in sepsis. the knowledge that unbalanced oxidative stress could be critical in the pathophysiology of cardiac impairment in sepsis has naturally led to consideration of the potential therapeutic effects of antioxidant agents. over the years, treatment of the impaired myocardial energetics rather than cardiac inflammation has been postulated to curb the lethal tools of sepsis with drugs that target the oxidative stress unbalance and the deep mechanisms within mitochondria [21, 160167 ].	background. myocardial depression in sepsis is common, and it is associated with higher mortality. in recent years, the hypothesis that the myocardial dysfunction during sepsis could be mediated by ischemia related to decreased coronary blood flow waned and a complex mechanism was invoked to explain cardiac dysfunction in sepsis. oxidative stress unbalance is thought to play a critical role in the pathogenesis of cardiac impairment in septic patients. aim. in this paper, we review the current literature regarding the pathophysiology of cardiac dysfunction in sepsis, focusing on the possible role of oxidative - nitrosative stress unbalance and mitochondria dysfunction. we discuss these mechanisms within the broad scenario of cardiac involvement in sepsis. conclusions. findings from the current literature broaden our understanding of the role of oxidative and nitrosative stress unbalance in the pathophysiology of cardiac dysfunction in sepsis, thus contributing to the establishment of a relationship between these settings and the occurrence of oxidative stress. the complex pathogenesis of septic cardiac failure may explain why, despite the therapeutic strategies, sepsis remains a big clinical challenge for effectively managing the disease to minimize mortality, leading to consideration of the potential therapeutic effects of antioxidant agents.
graphene was mechanically exfoliated from highly - oriented pyrolytic graphite onto si / sio2 substrates. surface hybrids were obtained by immersion in a solution of [fe4(l)2(dpm)6 ] in 1,2-dichloroethane : dichloromethane (9/1, v / v) at room temperature, followed by washing with isopropanol. bulk samples of the hybrids were prepared by first sonicating 8.4 mg of graphite powder in 20 ml of the solvent mixture for 48 hrs, followed by adding 11.5 mg of [fe4(l)2(dpm)6 ] in 20 ml of the same mixture, followed by stirring for 45 min and final filtering through ptfe membranes (0.2 m pore size). magnetic measurements were corrected for the diamagnetism of the sample and sample holder, as independently determined. a home - built micro - squid susceptometer inside a dilution refrigerator was used for zero - field measurements, with extremely small ac field amplitudes (50 moe) and the data corrected by the bare graphene signal. afm images were acquired with a bruker icon system in tapping mode, using olympus ac200ts cantilevers. maldi - tof spectra were recorded using a laser pulsed at ~4 hz, while the xps spectra were obtained with a monochromatized al - k source (1486.6 ev), see si for details. electronic devices were fabricated using a pmma resist, e - beam lithography and thermally evaporated au / ti.	controlling the dynamics of spins on surfaces is pivotal to the design of spintronic1 and quantum computing2 devices. proposed schemes involve the interaction of spins with graphene to enable surface - state spintronics3,4, and electrical spin - manipulation4 - 11. however, the influence of the graphene environment on the spin systems has yet to be unraveled12. here we explore the spin - graphene interaction by studying the classical and quantum dynamics of molecular magnets13 on graphene. while the static spin response remains unaltered, the quantum spin dynamics and associated selection rules are profoundly modulated. the couplings to graphene phonons, to other spins, and to dirac fermions are quantified using a newly - developed model. coupling to dirac electrons introduces a dominant quantum - relaxation channel that, by driving the spins over villain s threshold, gives rise to fully - coherent, resonant spin tunneling. our findings provide fundamental insight into the interaction between spins and graphene, establishing the basis for electrical spin - manipulation in graphene nanodevices.
magnetic particle imaging (mpi) is a new method for imaging the spatial distribution of magnetic nanoparticles, as tracers, with high resolution. the method was proposed by gleich and weizenecker and exploits the nonlinear magnetization response of the nanoparticles to a time - variable magnetic field and allows for fast image acquisition. mpi has many applications, especially in medical diagnosis such as blood flow visualization for coronary artery diseases, cancer detection [2, 3 ], stem cell tracking, and molecular imaging. mpi uses an oscillating drive field (excitation field) of sufficient amplitude to change the magnetization of the nanoparticles, which induces a voltage signal in the receive coils. to enable spatial encoding of the information, a static magnetic gradient field, also known as the selection field, this field contains a spatial location named a field - free point (ffp) that has zero field magnitude, and only the particles located at the ffp induce the mpi signal in the receive coils [68 ]. this method, also known as frequency space reconstruction, employs a large system matrix and requires its inversion and some postprocessing to deal with poor conditioning. the approach has high computational load and requires the system matrix to be estimated before imaging [1012 ]. the second approach is the x - space method, which was introduced by goodwill. this approach does not require matrix inversion or precharacterization and hence provides a robust reconstruction algorithm with a potential for real - time imaging. linearity and shift invariance (lsi) are important characteristics of most medical diagnostic systems. for example, since x - ray computed tomography (ct) is lsi, the ct images of tissue attenuation coefficient maps provide quantitative lumen diameters for cardiovascular diagnosis. it has been theoretically proven that the mpi is an lsi system. however, in practice, to detect mpi signal, it is necessary to utilize a filter to remove the drive field signal at its fundamental excitation frequency, which is an unavoidable phenomenon in current mpi techniques. this filtering action also removes the induced particle signal at the excitation frequency and hence the complete mpi signal is not available in practice which destroys the lsi properties of the mpi image. to recover the complete mpi signal to facilitate the use of the fast x - space imaging method in qualitative medical diagnosis, an algorithm is presented in and is optimized in. the approaches use the partial field of views (pfovs), which are primarily generated for enlarging the fov. it is shown that the lost information can be recovered by matching the two successive overlapped pfovs, provided the particle concentration at one reference location is known. although the methods provide satisfactory results, the computational load is reported in to be relatively high, which increases the imaging time and so this can not be used for ultrafast imaging. in this paper, the effect of the filtered mpi image is analyzed and it is shown that its value is constant provided that the trajectory of the ffp is generated by a harmonic function, a property that has to be satisfied in both one - dimensional (1d) and multidimensional imaging. assuming a constant image loss, a fast analytical algorithm is proposed to restore the lost information. the approach can be real time since its complexity depends on solution of a small - sized system of linear equations and does not require overlapping of pfovs. the method utilizes the derivative of the magnetization curve of the particles and a mathematical model of the x - space image. it also requires a priori information regarding the system, which can be obtained experimentally or estimated theoretically prior to imaging. this is another advantage of the proposed approach in comparison to previous works, which require a boundary condition to be obtained at the start of imaging. several 1d and 2d imaging simulations are presented to demonstrate the effectiveness of the proposed algorithm. the developed recovery algorithm is based on the mathematical model of the mpi signal. in this section, the signal model of the x - space method is presented and a model of the term removed from the signal as a result of filtering the fundamental frequency of the excitation signal is derived. then, a new algorithm is proposed to recover the lost image. in this section, the mathematical model of the 1d mpi signal is derived which is used in section 2.2 to model the x - space reconstruction method and to analyze the lsi properties of the x - space. when the excitation field in mpi is periodic, the voltage signal v(t), induced by the particles ' magnetization, is periodic as well, and therefore it can be expanded into a fourier series as follows:(1)vt=k=vkeik0t, where vk are the fourier coefficients and 0 = 2f0 with f0 as the frequency of the excitation field. the coefficients can be written as(2)vk=1tt/2t/2vteik0tdt, where t = 1/f0. assuming a 1d distribution of the particles in the x - direction and a constant sensitivity x for the receive coil in this direction, the induced voltage by the time - varying magnetization of the particles can be written as follows : (3)vt=0xobjectmx, ttdx, where 0 denotes the permeability of free space and m is the particle magnetization, which depends on the magnetic field h(x, t). the particle magnetization can be modeled using the langevin function () as follows:(4)mh = mcxlh, where m [am ] is the particle magnetic moment, [ma ] is a property of the magnetic particle, and c(x) [particles / m ] is the particle concentration that has to be measured in an mpi image. according to (3), the time derivative of magnetization is of interest and can be written as(5)mt = mcxlhhht.on the other hand, the magnetic field that particles experience in mpi is as follows:(6)hx, t = hsxhdt, where hs is the selection field and hd is the excitation or drive field. therefore, using (6), (5) can be written as(7)mt = mcxlhhdt, where h=h/h. now, using (3) and (7), (2) can be written as follows:(8)vk=0xmtt/2t/2fovcxlhhdtdx eik0tdtvk=0xmtfovcxt/2t/2lhhdteik0tdt dx.by defining the function(9)skx0xmtt/2t/2lhhdteik0tdt,(8) can be written as(10)vk=fovskxcxdx.sk(x) are the so - called system function, which in fact are the fourier coefficients of an induced voltage by a point - like distribution of particles at position x. remark 1. if the particles are ideal (i.e., have a step - like magnetization cure). when the particle magnetization is modeled by the langevin function, the field of view is slightly different from the object to be imaged. therefore, in the remaining equations, the region of integration is replaced by fov. if the particles are ideal (i.e., have a step - like magnetization cure). when the particle magnetization is modeled by the langevin function, the field of view is slightly different from the object to be imaged. therefore, in the remaining equations, the region of integration is replaced by fov. for a homogeneous harmonic drive field with cosine waveform as(11)hdt = acos0tand a linear selection field hs(x) = gxx, where gx is the gradient of the selection field in the x - direction, it has been shown in that sk(x) can be written by a convolution as(12)skx=20xmtilgxxuk1gxxa1gxxa2,where(13)ukx = sink+1arccosxsinarccosxis the chebyshev polynomial of order k. now, substituting (12) in (10), one has(14)vk=20xmtifovlgxxuk1gxxa1gxxa2cxdxvk=12fovlgxxskidealxcxdx, where sk represent the system function for the ideal particles, which are defined as(15)skidealx=40xmtiuk1gxxa1gxxa2.now, one can rewrite (14) as follows:(16)vk=12fova / gxa / gxlgxxx~skidealx~dx~ cxdxvk=12a / gxa / gxskidealx~fovlgxx~xcxdx dx~vk=12a / gxa / gxskidealxc^xdx, where(17)c^xlgxxcx.from (15) and (16), one can conclude that vk correspond to coefficients of a chebyshev series (see, e.g.,), and therefore c^x can be written based on the chebyshev series as follows:(18)c^x = gxta0xmik=1vkuk1gxxa. this equation is used in the following subsection to describe the 1d x - space reconstruction method. in the x - space reconstruction method, the image is defined to be c^xt at position x(t) = xffp(t), where xffp(t) denotes the position of the ffp. c^xffpt can be measured from the induced voltage v(t) through the following relation : (19)c^xffpt = vt0xmgxxffptt=1/0arccosgxxffp / a, where xffpt is the time derivative of xffp(t), that is, the velocity of the ffp. the formulation of c^x by a convolution in (17) demonstrates that the x - space mpi image is a linear and shift - invariant image, properties which are characteristic of most clinical imaging systems, for example, ultrasound, ct, and magnetic resonance imaging (mri). however, to acquire an mpi image while retaining its lsi properties, one needs the measurement of the true value of v(t), which is not available in practice. in fact, a band - stop filter is used in practice to segregate v(t) from the signal induced by the drive field (11) in the receive coil. the filter suppresses the first harmonic of the signal at the frequency of the drive field and leaves the signal at higher harmonics. according to (13) and (18), the filtered first harmonic can be written as (20)c^xlost = gxta0xmiv1which is a constant value in the image. it should be noted that since v1 is the fourier coefficient of an odd real function (as a result of the sinusoidal drive field, v(t) is an odd function), its value is imaginary and therefore, according to (20), the lost image is a real constant value. using (15) and (16), one can write (20) as follows:(21)c^xlost=2gxtaa / gxa / gxc^x1gxxa2dxwhich shows that the lost value varies when c^x is shifted due to the velocity term 1-gxx / a2, and hence its envelope at different positions resembles the sinusoidal excitation pattern (note that sinarccosx=1-x2). this means that the lost value is maximal at the center of the fov and minimal at its edges. in the next subsections, the effect of this constant loss is analyzed and a compensation algorithm is proposed. according to the definition, the mpi image is linear when the image intensity is linearly proportional to the concentration of the particles and is shift - invariant when the image intensity is independent of the location of the particles. considering the expression of the lost image in (21) and the fact that the image loss depends on the location of the particles, it can be verified that the lost information destroys the lsi properties of the mpi image. to visualize the abovementioned phenomenon, consider figure 1 which shows the x - space mpi images of a set of magnetic nanoparticles located at different positions of the fov with concentration c1(x). as demonstrated, there is a difference in the maximum value of the acquired image intensity for the same particles at different locations. also, it is clear from this figure that the lost constant value is larger when the particles are located at the center of the fov, since the velocity of the ffp is maximum at the center. based on the results in the previous section, it can be verified that an x - space mpi without first harmonic information does not exhibit lsi and, to provide an image for quantitative medical diagnosis, the lost constant image should be recovered. an algorithm is presented in [16, 18 ], which can recover the lost image by dividing the fov mechanically or electronically, into several pfovs having sufficient overlap to reliably match the successive pfovs. the algorithm also requires an image of a location having no particle, for example, a location outside the patient. although the method fully recovers the lost image, its computational load may be prohibitive for fast and real - time applications. if the fov includes a zero - particle area (the reference location for the previous algorithms), finding the lost image is a straightforward task. for example, in mpi images of figure 1, fovs have at least one edge at which no particle is located. therefore, in these cases, finding the global minimum of the image is enough to completely recover the lost image (see figure 2 for an example). for example, for the sets of particles shown in figure 8, only a partial image loss can be compensated by using this method. the proposed method in this paper is a model - based compensation algorithm, which can be applied in general cases. the approach does not require overlapping of successive fovs to cover the object, and accordingly it can reduce the imaging time. the method utilizes the model of the point spread function (psf) of 1d x - space image (denoted by), which, as noted in, can be obtained theoretically with precise matching with experiment. the algorithm also assumes that the particle distribution can be described as the sum of discrete point - like particles with known locations in the fov. this is achievable through deconvolution of the original image and looking at the local maxima of the signal to determine the location of particles (a common approach to find the local maxima is to find the first - order difference information of the signal ; whenever a smooth signal is available, it is easy to use the available basic algorithms to find the particles, which are very robust ; in the simulation results, the findpeaks command of matlab is used to find the particles and their positions). the reported experimental results of mpi signal show an acceptable noise level to roughly deconvolve the signal for the proposed algorithm [13, 22 ]. in addition, the approach requires a priori information, which can be obtained theoretically or from a reference image, having the same property as used in for initialization. based on the above explanation, the following assumptions, besides the general assumptions of the x - space reconstruction, turn out to be crucial within the development of the recovery algorithm. the noise level of the signal is attenuated enough before applying the recovery algorithm. the noise level of the signal is attenuated enough before applying the recovery algorithm. a robust deconvolution method is applied to roughly represent the mpi signal as the sum of signals of discrete particles with definite positions. a robust deconvolution method is applied to roughly represent the mpi signal as the sum of signals of discrete particles with definite positions. in the following, first, an approach is developed for the case in which the particles in the fov are not in close proximity ; that is, the tails of the image for one set do not affect the image of the other sets. then, the approach is modified for the more general case. according to (17), the image for the set of point - like particles can be written as(22)c^ixi = lgxxici, where the subscript i denotes the ith particle. also, it is obvious from (21) that the image loss for this set of particles (voxel) is proportional to the particle concentration and an integral term depending on and the velocity of the ffp. for point - like particles, the derivative of the langevin function is nonzero only in a small region, and therefore the velocity term can be assumed to be constant in this region (however, for a case in which the psf is not very narrow, this assumption is valid up to a certain accuracy). as a consequence, the lost image for the set of particles with concentration ci located at xi can be approximated as(23)c^ixilostkcii, with i1gxxia2,where k includes the constant terms in (21) and also the integral of the derivative of the langevin function (which is the psf of the image). equation (23) indicates that the image loss for a set of particles is linearly proportional to the velocity of the ffp at the position of the particles, and hence its envelope, throughout the fov, resembles a sinusoidal pattern. using (22), one can write (23) as in the following:(24)c^ixilostkc^ixii, where kk/gxxi is a constant value for all positions in the fov (the value of psf,, is maximum and constant at xi). this value, which is required in the subsequent development, can be identified theoretically or experimentally through the imaging of a simple set of particles, whose image loss can be easily obtained by finding the global minimum of the signal. in other words, a boundary condition as satisfied in figure 2, similar to the previous pfov methods, is enough to experimentally measure this value. it should also be noted that, unlike the pfov methods, this measurement is only required to be done once. based on the above result, assuming that the x - space imaging conditions are satisfied, the image loss for the particles with concentration cj located at position xj can be related to the image loss of the particles with concentration ci at position xi, as follows:(25)c^jxjlostc^jxjjc^ixiic^ixilost. since image c^x is linear, it can be considered as the sum of the images of the point - like particles distributed throughout the imaging axis. the lost image c^xlost associated with c^x, therefore, can also be considered as the sum of the image losses associated with each set of particles. then, assuming that c^x is the image of the n sets of particles located in the fov, it can be written as(26)c^x=i=1nc^ixi. also, for each individual set, one has(27)c^ixiread = c^ixic^xlostc^ixilostkic^xlost, where c^ixiread is the measured value for the particles noted by index i at position xi. therefore, it can be verified that, for two particle sets of i and j, the subtraction of measured values, defined by ij, is equal to the subtraction of the real image values at xi and xj, respectively ; that is,(28)ijc^ixireadc^jxjread = c^ixic^jxj, which can be measured from the filtered image, based on the assumption that the particles can be identified in the fov. therefore, considering an initial particle set, noted here by index 1, the total image loss can be written based on c^1x1lost as follows:(30)c^xlostc^1x1lost1 + 11i=2niki=2ni1i.now, substituting (27) in (30), the image loss can be measured based on available information as follows:(31)c^xlostc^1x1read1k1i=2ni1iwith(32)ki=1ni. equation (31) can be applied for cases in which the discrete particles are sufficiently distant from each other such that the tails of the image for one set do not affect the images of other particles. however, this issue is discussed below for the case of two particles ; however, it can be easily extended to cases involving multiple particles. consider the particles i and j to be very close to each other ; therefore, for these particles, (27) will be as follows:(33)c^ixiread = c^ixi+c^jxic^xlost, c^jxjread = c^jxj+c^ixjc^xlost, where c^ixj denotes the effect of the image of the particles at position xi on the image of the particles at xj. therefore, to find the lost image, the values of c^ixj and c^jxi should be identified. knowing the psf, the relations for these values are given by(34)c^ixj = c^ixilgxxixj, c^jxi = c^jxjlgxxjxi.substituting for c^ixi and c^jxj from (33), (34) can be written as(35)c^ixj = c^ixireadc^jxi+c^xlostlgxxixj, c^jxi = c^jxjreadc^ixj+c^xlostlgxxjxi, which is a system of linear equations for the variables c^ixj and c^jxi. the solution of this system gives the values of interaction terms which are a function of c^(x)lost. finally, these terms should be subtracted from the read values in (28) and (31), and the total lost image can be obtained from (31). in the case of multiple particles with multiple interactions, a similar system of equations as in (35) can be developed, where the number of equations is equal to the number of interaction terms. the model of the 1d psf:(1)in an initialization scanning, find the image loss for one set of particles by measuring the global minimum of the image signal, and then compute k in (24). this step can be omitted when the value of k is measured theoretically.(2)deconvolve the original image to find the particles and their positions in the fov through a searching algorithm, and then compute i using (23) and c^ixiread for each set of particles.(3)compute the interaction values of each particle set on the neighbors by solving the system of linear equations, such as (35).(4)consider a particle set (indexed 1) as the reference set and measure 1i for all sets using (28), taking into account the effect of the neighbor particles.(5)compute the image loss through (31). in an initialization scanning, find the image loss for one set of particles by measuring the global minimum of the image signal, and then compute k in (24). deconvolve the original image to find the particles and their positions in the fov through a searching algorithm, and then compute i using (23) and c^ixiread for each set of particles. compute the interaction values of each particle set on the neighbors by solving the system of linear equations, such as (35). consider a particle set (indexed 1) as the reference set and measure 1i for all sets using (28), taking into account the effect of the neighbor particles. compute the image loss through (31). an important note which should be mentioned is that the algorithm requires knowing as narrow convolution kernel (psf) model (and its image loss to measure k) as possible, which can be used to deconvolve the mpi signal to roughly represent it as the sum of discrete point - like particles. apparently, when the noise level of the signal allows using a narrower psf, then the error of approximation in (23) would be less, and the overall performance of the algorithm would be improved. an important note which should be mentioned is that the algorithm requires knowing as narrow convolution kernel (psf) model (and its image loss to measure k) as possible, which can be used to deconvolve the mpi signal to roughly represent it as the sum of discrete point - like particles. apparently, when the noise level of the signal allows using a narrower psf, then the error of approximation in (23) would be less, and the overall performance of the algorithm would be improved. any particle set can be considered as the initial set, indexed by 1 in (31). however, in case that a nonideal filter is implemented in practice, the initial set may affect the accuracy of the algorithm. selecting this set from those in the center of fov with a medium concentration can be a proper choice. but, in general, the best choice, which depends on the performance of the filter, can be determined from the results of the first experiments (which can be considered as a calibration procedure). from the theoretical point of view, any particle set can be considered as the initial set, indexed by 1 in (31). however, in case that a nonideal filter is implemented in practice, the initial set may affect the accuracy of the algorithm. selecting this set from those in the center of fov with a medium concentration can be a proper choice. but, in general, the best choice, which depends on the performance of the filter, can be determined from the results of the first experiments (which can be considered as a calibration procedure). considering the case of a nonideal filter, the same procedure as in remark 6 can be applied to measure the value of k ; that is, an appropriate choice is to measure it for the particle at the center of trajectory. considering the case of a nonideal filter, the same procedure as in remark 6 can be applied to measure the value of k ; that is, an appropriate choice is to measure it for the particle at the center of trajectory. the approach of uses the average of several scans to find the image loss, and it is guaranteed to be robust with respect to the noise, provided that the image noise has zero mean. similarly, if this condition is satisfied, the assumption of having smooth signal (introduced in assumption 3), and accordingly the robustness property, for the proposed approach can be readily achieved by using multiple scans. the approach of uses the average of several scans to find the image loss, and it is guaranteed to be robust with respect to the noise, provided that the image noise has zero mean. similarly, if this condition is satisfied, the assumption of having smooth signal (introduced in assumption 3), and accordingly the robustness property, for the proposed approach can be readily achieved by using multiple scans. before studying multidimensional mpi, it is worth mentioning that the reason that the lost image is constant in 1d mpi is that the chebyshev polynomial of order zero u0(x) is constant and does not depend on the spatial position. on the other hand, the x - space image can be written as the chebyshev series (18) since the system function is expressed by the chebyshev polynomials. this notation for the system function is the direct effect of a cosine (harmonic) drive field. this means that, for other waveforms of the drive field, the system function can not be written as the chebyshev polynomials. based on the above discussion, it can be verified that, in general, the image loss in multidimensional mpi is not a dc value. however, one method of maintaining a constant image loss in multidimensional x - space is to combine the 1d images of the object to generate a 2d/3d image. this method is experimentally studied in, which requires only one pair of drive coils and one receive coil. using the above method, the image loss would be constant and recoverable for 1d mpi images, but the signal equation of multidimensional mpi differs from that of 1d mpi. then, a question arises of whether it is possible to use the proposed recovery algorithm for 2d/3d mpi. the generalized 3d mpi signal is given by (36)vt=rmcrhrrffpr = rffpt, where r = [x y z ] and h(r) is a matrix psf which is convolved in 3d (denoted by) with the particle distribution. when the multidimensional image is reconstructed from 1d images, which is by a collinear arrangement of detection coil and drive coil trajectory, then the collinear psf h\|\|(r) should be considered. assuming the drive field trajectory is in the x - direction, one has (37)h\|\|r = lhrgx3x2hr2+lhrhr1gx3x2hr2with hr = gxx2+gyy2+gzz2. supposing the particles are located at line y = z = 0, the second term in (37) will disappear when a drive field trajectory is on this line. the remaining psf is identical to the 1d psf, which depends on the time derivative of the langevin function, and is assumed to be known by the proposed algorithm. when the trajectory moves away from this line, the resultant psf depends not only on the derivative of the langevin function, but also on the function itself. therefore, in different spatial positions for the drive field, the psf would no longer be the same as the one used by the algorithm. however, the collinear psf is similar to gaussian curves, which has a narrow shape at line y = z = 0 and becomes broader with distance from this line. figure 3 illustrates a collinear psf for a point - like sample of particles at different positions of the drive field. it is well investigated in that it is always possible to reconstruct a broad gaussian - like curve from linear superposition of a narrow one, which is why multicolor imaging is not possible in 1d mpi of langevin particles with ffp motion being a single line. this property provides the possibility of describing an mpi signal, obtained from a linear ffp trajectory, based on the linear superposition of 1d mpi images resulting from the spatial distribution of the particles along the trajectory line. this distribution can be obtained by deconvolution of the 1d x - space psf from the obtained signal. to clarify this property, an example the dashed mpi signal is the output of the band - stop filter, which is induced by the particles at the center of imaging area and the available large set of particles at the border, due to a linear trajectory of the drive field at the center. one should note that although the drive field in figure 4 does not pass through the large particle set, this induces a signal in the receive coil because the ffp is not only a small area for medium - level selection field power, which is generally used in practice. using a deconvolution algorithm, the solid line in the figure shows a new representation of the signal based on the 1d psf, which can be interpreted as the superposition of 1d mpi images of several particles with the same psf. therefore, this representation property shows that the proposed recovery algorithm is applicable in multidimensional mpi. remark 9. image of particles with broad psf, similar to the case where they are close to each other or, in the extreme case, cover the entire fov, can be expressed as the sum of images of particles with narrow psf. in fact, the algorithm, regardless of the dimension of particle distribution or imaging, simply tries to find the level of the received signal by expressing the signal as the sum of images of point - like particles (like figure 4), and then, knowing the effect of a sample set of particles (k in (24)), the algorithm estimates the total image loss. image of particles with broad psf, similar to the case where they are close to each other or, in the extreme case, cover the entire fov, can be expressed as the sum of images of particles with narrow psf. in fact, the algorithm, regardless of the dimension of particle distribution or imaging, simply tries to find the level of the received signal by expressing the signal as the sum of images of point - like particles (like figure 4), and then, knowing the effect of a sample set of particles (k in (24)), the algorithm estimates the total image loss. nonlinearity of the gradient field or the relaxation effects may cause an asymmetry in the psf. considering the point in remark 9, as long as a model of the psf is available and the image loss is quasi - constant, the algorithm can still be applied regardless of the asymmetry of the signal. keep in mind that when the magnetic fields are far from being ideal (ideal magnetic fields : a linear selection field and a homogeneous drive field), the mpi image can not be formulated as a convolution like (17), and therefore the assumption of constant image loss is not also valid. nonlinearity of the gradient field or the relaxation effects may cause an asymmetry in the psf. considering the point in remark 9, as long as a model of the psf is available and the image loss is quasi - constant, the algorithm can still be applied regardless of the asymmetry of the signal. keep in mind that when the magnetic fields are far from being ideal (ideal magnetic fields : a linear selection field and a homogeneous drive field), the mpi image can not be formulated as a convolution like (17), and therefore the assumption of constant image loss is not also valid. in this section, simulation results are presented to evaluate the effectiveness of the recovery algorithm proposed in section 2.4. in the following, simulations are first presented for 1d imaging, and then the result for application of the algorithm to a 2d mpi image is illustrated. matlab codes are used to simulate both 1d and 2d images. in 1d simulations, point - like particles are considered in the fov, and since the algorithm is derived based on the shape / model of the psf, all images are normalized according to the maximum level of concentration ; this means the results are reported regardless of the concentration level of the particles. 1d simulations are conducted in three cases for the distribution of particles, and two different particle sizes are analyzed : particles with a core diameter of 30 nm which have a narrow psf (considered in the first two cases) and particles with a core diameter of 22 nm (considered in the third case). this is to evaluate the error of the approximation used to derive (23). three sets of particles with different concentrations are considered in the fov, having no interaction. since the image of particles does not affect the image of other particles, then the location of particles can be easily determined, and step (3) of image loss recovery for 1d x - space mpi is not required. however, since the particles do not have similar concentrations, ij must be computed. the result for this case as the figure demonstrates, in this case, the algorithm can detect the image loss with high accuracy. as the first study, three sets of particles with different concentrations are considered in the fov, having no interaction. since the image of particles does not affect the image of other particles, then the location of particles can be easily determined, and step (3) of image loss recovery for 1d x - space mpi is not required. however, since the particles do not have similar concentrations, ij must be computed. the result for this case as the figure demonstrates, in this case, the algorithm can detect the image loss with high accuracy. four sets of particles are considered in the fov, where particles indexed by 2 and 3 are very close to each other and interact. by deconvolving the image, it is possible to easily determine the position of particles. for this case, it is required to use (35) to cancel the interaction effect. the result for this case is shown in figure 7, which demonstrates the proper compensation of the lost information. it should be noted that if the particles were closer, then deconvolving could not segregate the particles and they would be considered as one set of particles with a higher concentration. in the second case, four sets of particles are considered in the fov, where particles indexed by 2 and 3 are very close to each other and interact. by deconvolving the image, the result for this case is shown in figure 7, which demonstrates the proper compensation of the lost information. it should be noted that if the particles were closer, then deconvolving could not segregate the particles and they would be considered as one set of particles with a higher concentration. four sets of particles with a wide psf are considered in the fov. in this simulation, the images of particles indexed by 1, 2, and 3 undergo interactions with each other. also, there are a total of eight interactions in the image ; therefore, the equivalent model for (35) includes a system of eight linear equations. to simulate the measurement errors, the level of interactions, obtained by (34), is considered to be 90% of its true value. the result of using the algorithm for this case is illustrated in figure 8, which shows satisfactory recovery of the image, with a small deviation, despite the wide psf and error in the measurements. in the final case, four sets of particles with a wide psf are considered in the fov. in this simulation, the images of particles indexed by 1, there are a total of eight interactions in the image ; therefore, the equivalent model for (35) includes a system of eight linear equations. to simulate the measurement errors, the level of interactions, obtained by (34), the result of using the algorithm for this case is illustrated in figure 8, which shows satisfactory recovery of the image, with a small deviation, despite the wide psf and error in the measurements. to simulate the algorithm for 2d imaging, the mpi simulation toolbox developed in this matlab toolbox simulates the magnetic fields and magnetization of the particles and uses the system function method to reconstruct the image. this program is appropriately modified for the x - space imaging and the ffp trajectory is changed to meet the requirements for constant image loss in multidimensional imaging. measurement error, similar to case 3, is also considered in this simulation. it is worth mentioning that, in the result, one may expect that figure 9(c) is better that figure 9(d), but in fact the concentration of particles shown by figure 9(d) is more close to the true value. in terms of the image quality, it can be easily improved by combining the images of two orthogonal line - scan drive fields. this paper analyzes the lsi characteristics of the x - space mpi image and proposes an algorithm to recover these characteristics, which are important for quantitative medical diagnosis. although the theoretical model of the x - space image is known to exhibit lsi, it is shown that filtering the fundamental excitation frequency, which is necessary in practice to manifest the particle signal, destroys the lsi properties. the lost image could be constant and hence recoverable when a 1d harmonic drive field is applied in both 1d and multidimensional imaging. to recover the lost information, an algorithm is proposed using x - space psf model of the particles and the properties of the mathematical model of the image. the algorithm tries to find the level of the received signal by expressing it as the sum of images of point - like particles, and then, knowing the image loss of a sample set of particles, the algorithm estimates the total image loss. the initial image loss information can be obtained offline and is enough to be measured for one time. in this regard, the proposed approach has an advantage over the previous methods, where a priori information is required to be measured online at the beginning of each test. the complexity of the proposed approach depends on a small - sized system of linear equations, and therefore it has low computational load, which preserves the real - time advantage of x - space imaging. this is another advantage of the proposed approach with respect to current available approaches, where overlapping of successive fovs is required which increases the imaging time to cover the entire object. the results of simulations for 1d and 2d imaging, for both narrow and broad psfs, demonstrate the effectiveness of the algorithm. although in simulation study these assumptions are released up to some level and the results are still promising, they seem to be the most challenging parts of implementing the algorithm in practice. in this regard, the future work is devoted to the study, through experiments, of the sensitivity of the algorithm to derive a robustness level with respect to the assumptions. the future work is also devoted to the comparison of the processing times of the proposed algorithm with the previous ones.	linearity and shift invariance (lsi) characteristics of magnetic particle imaging (mpi) are important properties for quantitative medical diagnosis applications. the mpi image equations have been theoretically shown to exhibit lsi ; however, in practice, the necessary filtering action removes the first harmonic information, which destroys the lsi characteristics. this lost information can be constant in the x - space reconstruction method. available recovery algorithms, which are based on signal matching of multiple partial field of views (pfovs), require much processing time and a priori information at the start of imaging. in this paper, a fast analytical recovery algorithm is proposed to restore the lsi properties of the x - space mpi images, representable as an image of discrete concentrations of magnetic material. the method utilizes the one - dimensional (1d) x - space imaging kernel and properties of the image and lost image equations. the approach does not require overlapping of pfovs, and its complexity depends only on a small - sized system of linear equations ; therefore, it can reduce the processing time. moreover, the algorithm only needs a priori information which can be obtained at one imaging process. considering different particle distributions, several simulations are conducted, and results of 1d and 2d imaging demonstrate the effectiveness of the proposed approach.
in spite of the low complication rates related to laparoscopic bariatrics, there is an increasing attention in endoluminal and/or transgastric instruments for weight loss procedures. interest in endoluminal treatments for the obese has been present for many years. the first breakthrough in endoluminal therapy has been the intragastric balloon (igb), currently the orbera balloon, but previously referred to as the bioenterics intragastric balloon (bib). they are intended to act as a bridging therapy to bariatric surgery by decreasing the patients body mass index (bmi) with the using of a balloon in the gastric space [1 - 6 ]. the first clinically reliable balloon received contrast enhancement (ce) mark in 1998 and has become the world leader in intraluminal weight loss devices. the orbera balloon also known as the bib has been placed in over 250,000 patients. this balloon was approved by the food and drug administration (fda) for us use in 2015. the balloon is inserted into the stomach through the oral cavity and esophagus using an endoscopic procedure. during procedure, the patient is under sedation. after balloon insertion, balloon is filled with methylene blue mixed saline water, therefore, it expands into a spherical shape balloon. the balloon can be filled with various amounts of saline (from 400 to 700 cc) to best match the patient s gastric structure. the intra - gastric balloon should be removed approximately after 6 months. the bib system is currently being tested (allergan, irvine, ca, usa). fig. 1 shows this system. this system is a saline - filled balloon that is placed into the gastric space with using gastroscopy. there was large scaled data about the bib system. total about 2,500 patients were enrolled with a mean bmi of 44.4. after 6 months, mean bmi and excess body weight loss (ewl) were 35.411.8 and 33.918.9. obesity - related parameters such as fasting low density lipoprotein cholesterol, blood glucose, triglyceride, and blood pressure was significantly improved. there were two mortality cases associated with balloon placement and these patients had a history of gastric surgery. bib system within a multidisciplinary weight management program is a relatively short term effective procedure for weight control, but it is not possible to certify its potential to maintain the weight for a long time. fulfillium (napa, ca, usa) is developing a balloon having multiple compartments. this system has failure detection systems to enable early detection, therefore, endoscopist can remove a leaking balloon prior to collapse, and avoid passage of it to the duodenum (fig. 2). based on previous experience with balloon technology, this method is appropriate ; however, it is the safety profile that contributes to its limited use. other available balloons are the reshape balloon, which can provide similar weight loss as the orbera. unlike the orbera, these balloons are in a fixed position within the stomach and as a result there is 26% incidence of significant ulceration along with a 2% incidence of spontaneous balloon deflation. reshape medical (san clemente, ca, usa) performs to a prospective, non - randomized, multicenter study designed to implant up to 20 ce - marked reshape balloons in twenty patients with bmi between 30 and 40. enrolled patients will be followed after videotaped endoscopic placement of the 900 cc dual balloon design at 180 days and again 210 days for an additional endoscopy documenting post - removal mucosa. from the clinical view, the igb can cause three dominant intolerance symptoms : nausea (vomiting), reflux and abdominal pain, all of which are prophylactically treated before and after balloon placement to avoid early removal. at present, intolerance symptoms which can lead to early removal occur in 26% of patients. using only the large orbera data, the incidence of ulceration, deflation with migration, and small bowel obstruction are negligible. baronova therapeutics (goleta, ca, usa) is making the transpyloric shuttle (tps) (fig. 3). this system restricts food ingestion with repeatedly blocking of the pylorus. and pilot studies have proven safety and initial efficacy of the product with significant ewl at three months. barosense (redwood city, ca, usa) has developed the trans - oral endoscopic restrictive implant system (teris) (fig. this system is located in stomach cardia area, therefore, this system can induce early and prolonged satiety. this system is currently enrolling patients in a phase 1 trial with a secondary objective to perform a preliminary evaluation of the efficacy to guide their pivotal trial. endosphere (redwood city, ca, usa) has developed satisphere, an endoscopically placed device increasing the food contact time (fig. its length is about 2025 cm, this system is self - anchored in the distal stomach or proximal duodenum, and, it makes increased hormonal signaling, thereby causing early satiety and downregulation of glucose production. ultimately, this system would be removed between 6 and 18 months and act as an adjunct to diet and exercise. a small study of 11 patients was performed and showed 12% ewl at 1 month without any adverse events. they are intended to act as a bridging therapy to bariatric surgery by decreasing the patients body mass index (bmi) with the using of a balloon in the gastric space [1 - 6 ]. the first clinically reliable balloon received contrast enhancement (ce) mark in 1998 and has become the world leader in intraluminal weight loss devices. the orbera balloon also known as the bib has been placed in over 250,000 patients. this balloon was approved by the food and drug administration (fda) for us use in 2015. the balloon is inserted into the stomach through the oral cavity and esophagus using an endoscopic procedure. during procedure, the patient is under sedation. after balloon insertion, balloon is filled with methylene blue mixed saline water, therefore, it expands into a spherical shape balloon. the balloon can be filled with various amounts of saline (from 400 to 700 cc) to best match the patient s gastric structure. the intra - gastric balloon should be removed approximately after 6 months. the bib system is currently being tested (allergan, irvine, ca, usa). fig. 1 shows this system. this system is a saline - filled balloon that is placed into the gastric space with using gastroscopy. there was large scaled data about the bib system. total about 2,500 patients were enrolled with a mean bmi of 44.4. after 6 months, mean bmi and excess body weight loss (ewl) were 35.411.8 and 33.918.9. obesity - related parameters such as fasting low density lipoprotein cholesterol, blood glucose, triglyceride, and blood pressure was significantly improved. there were two mortality cases associated with balloon placement and these patients had a history of gastric surgery. bib system within a multidisciplinary weight management program is a relatively short term effective procedure for weight control, but it is not possible to certify its potential to maintain the weight for a long time. fulfillium (napa, ca, usa) is developing a balloon having multiple compartments. this system has failure detection systems to enable early detection, therefore, endoscopist can remove a leaking balloon prior to collapse, and avoid passage of it to the duodenum (fig. 2). based on previous experience with balloon technology, this method is appropriate ; however, it is the safety profile that contributes to its limited use. other available balloons are the reshape balloon, which can provide similar weight loss as the orbera. unlike the orbera, these balloons are in a fixed position within the stomach and as a result there is 26% incidence of significant ulceration along with a 2% incidence of spontaneous balloon deflation. reshape medical (san clemente, ca, usa) performs to a prospective, non - randomized, multicenter study designed to implant up to 20 ce - marked reshape balloons in twenty patients with bmi between 30 and 40. enrolled patients will be followed after videotaped endoscopic placement of the 900 cc dual balloon design at 180 days and again 210 days for an additional endoscopy documenting post - removal mucosa. from the clinical view, the igb can cause three dominant intolerance symptoms : nausea (vomiting), reflux and abdominal pain, all of which are prophylactically treated before and after balloon placement to avoid early removal. at present, intolerance symptoms which can lead to early removal occur in 26% of patients. using only the large orbera data, the incidence of ulceration, deflation with migration, and small bowel obstruction are negligible. baronova therapeutics (goleta, ca, usa) is making the transpyloric shuttle (tps) (fig. 3). this system restricts food ingestion with repeatedly blocking of the pylorus. and therefore, it makes food to increase the contact time within stomach. pilot studies have proven safety and initial efficacy of the product with significant ewl at three months. barosense (redwood city, ca, usa) has developed the trans - oral endoscopic restrictive implant system (teris) (fig. this system is located in stomach cardia area, therefore, this system can induce early and prolonged satiety. this system is currently enrolling patients in a phase 1 trial with a secondary objective to perform a preliminary evaluation of the efficacy to guide their pivotal trial. endosphere (redwood city, ca, usa) has developed satisphere, an endoscopically placed device increasing the food contact time (fig. its length is about 2025 cm, this system is self - anchored in the distal stomach or proximal duodenum, and, it makes increased hormonal signaling, thereby causing early satiety and downregulation of glucose production. ultimately, this system would be removed between 6 and 18 months and act as an adjunct to diet and exercise. a small study of 11 patients was performed and showed 12% ewl at 1 month without any adverse events. endoscopic endoluminal treatment of obesity can be a noninvasive, cost - effective, and relatively safer option than bariatric surgery. however, there are many debates according to the long efficacy, economic feasibility and durability of these procedures. anyway, role of therapeutic endoscopist is very important in the obesity management in aspects of the primary modality for managing complications that occur after bariatric surgery as well as endoscopic endoluminal treatment of obesity. in the near future, role of gastroenterologists will be more important in the management of obesity and its related problems.	it is well recognized that obesity is a big problem and it can induce large economic burden. obesity affects about 40% people in the america alone and obesity also is the worldwide problem, with about 400 million obese adults. moreover, another problem of obesity is the increasing prevalence of overweight children. though bariatric surgery remains the gold treatment modality in the obesity treatment, endoluminal approaches may have the meaningful role for weight control. endoscopists should have a role in the management of obesity because endoluminal therapies demonstrate their safety and efficacy over the coming years. endoluminal therapies can be summarized by above methods : space occupying, malabsorption method, and reduction of gastric volume. in this review, we will introduce various restrictive endoscopic procedures in obesity treatment.
even if these conventional methods have been improved, there are still requirements for novel treatments that ccould destroy cancer cells without harming the healthy tissue [15 ]. nanooncology represent a rapidly developing research field with great potential in fighting against a complex disease like cancer. although nanoparticles are small in size, nanometric drug delivery carriers and bio - active nanomaterials are large enough to attach multiple functional moieties to the cell surface, allowing significant enhancement of their properties and distributions in living systems. generally, the safety / toxicity of the nanoparticles depends on shape, surface coating and their electrical charge. it has been stated that the attachment of a targeting moiety, may optimize these nano - biosystems for a specific biomedical application and help prevent unwanted side effects [69 ]. gold nanoparticles mediated thermal ablation of tumors following exposure to nir light is superior to conventional techniques, since is minimally invasive and easy to apply, with great capacity to destroy malignant lesions in vital regions where surgical resection is not feasible. radiofrequency techniques or other thermally destructive methods can not differentiate between tumor and surrounding normal tissue, often affecting the healthy tissue as well [1012 ]. photothermal therapy employing gold nanoparticles (irradiated with near - infrared laser that excites electrons of different atomic levels and sub - levels to ground state by emitting the energy in the form heat) can destroy cancerous cells and is able to produce selective necrosis at cellular levels [1318 ]. with reference to the recent research data, we address here the current role of gold nanoparticles as photothermal anticancer agents. zhang and co - workers used peg - coated gold nanoparticles to enhance the in vitro and in vivo radiation response of gold nps. they used multiple size nanoparticles such as 4.8, 12.1, 27.3, and 46.6 nm peg - coated gold nps in hela cells. the toxicities of the peg - coated gold nps with different sizes were investigated 24 and 48 hours after treatment. their conclusion was that peg - coated gold nanoparticles can cause a significant decrease in cancer cell survival after gamma radiation. 12.1 and 27.3 nm peg - coated gold nanoparticles proved to be highly dispersive in the cells and displayed stronger sensitization effects than 4.8 and 46.6 nm particles by both cell apoptosis and necrosis. the authors also performed in vivo by evaluating tumor size and weight after 5 gy gamma radiations radiosensitization effects on 4.8, 12.1, 27.3, and 46.6 nm peg - coated gold particles showing that 12.1 and 27.3 nm peg - coated gold nanoparticles show high radiosensitivity and may be used for cancer phothermal ablation. in one study performed by kuo. 35 nm gold nanorods (au nrs) and 50 nm gold nanoparticles (au nps) were used to test two different approaches of cancer thermal therapy : photodynamic therapy (pdt) and photothermal therapy (ptt). in this au nrs and au the authors showed that combination of ptt and pdt proved to be efficient in killing cancer cells as compared to ptt or pdt treatment alone because its stability. a novel, 4-component bio - nanosystem compose of a phthalocyanine photosensitiser drug and a primary antibody (anti - her2) specific to cell surface receptors attached on a 4 nm gold nanoparticle was used to enhance drug targeting of breast cancer cells that overexpress the her2 receptor on the cellular surface. the authors showed that the targeting capability of the 4-component nanoparticle conjugates enhances the efficacy of pdt cell death when tumor - associated antigens are present on the cytoplasmic membrane of the malignant cells. multifunctional doxorubicin (dox)-loaded hollow gold nanospheres (dox@hauns) against ephb4 receptors were administered in ephb4-positive tumors both in vitro and in vivo. in vivo release of dox from dox@hauns, triggered by nir laser, was assessed by dual - radiotracer technique suggesting significantly decreased tumor growth when compared with treatments with nontargeted dox@hauns plus laser or hauns plus laser. the authors obtained encouraging results may have promise as a new anticancer therapy : tumors in 6 of the 8 mice treated with t - dox@hauns plus laser regressed completely with only residual scar tissue by 22 days following injection, and none of the treatment groups experienced a loss in body weight. a nanoparticle - based probe (composed of 10 nm spherical gold nanoparticle (np) with ph - responsive ligands and raman probes on the surface) was tested as turn - on theragnostic agent for simultaneous raman imaging / diagnosis and photothermal therapy. the system was developed to provide the surface with both positive and negative charges upon mildly acidic condition, which subsequently results in rapid aggregations of the gold nps that provides hot spots for the sers probe. another advantage of this rapid aggregation was the accumulation mainly in b16 f10 mouse melanoma cells. as the result, both raman imaging and photothermal efficacy were enhanced in treated malign cells. in a study conducted by puvanakrishnan. the effect of multiple dosing of gnps and nanoparticle type to improve tumor - targeting efficiency was analyzed. arge pegylated gold nanoshells (gnss) and small pegylated gold nanorods (gnrs) were used to compare the effect of the size of these particles on tumor targeting efficiency. specifically, they compared the effect of single and multiple doses of gnrs and gnss on in vivo tumor targeting. (four- to six - week old nude mice (swiss nu / nu) were inoculated with the a431 cells). neutron activation analysis (naa) was used to determine the amount of gnps accumulated for the different doses and the percent accumulation of gnrs and gnss in a squamous cell carcinoma tumor model. the authors showed a straight relationship between the efficacy of multiple dosing with increased accumulation of gnps in tumors with low toxicity following administration. iron - oxide core / gold - shell nanoparticles 30 nm diameter were used to demonstrate that gold nanoparticles are phagocytosed by pancreatic cancer cells, thus permitting magnetic resonance imaging (mri) of sensitizer delivery and photothermal ablation. using panc-1 cells followed by photothermal ablation, the cell proliferation percentage changed from 100% to 71.3% and 47.0% for cells treated with 25 and 50 g / ml goldmag. according to the authors, photothermal ablation of panc-1 cells demonstrated an effective treatment response, specifically a reduction to only 61%, 21.9%, and 2.3% cell proliferation for cells treated with 0, 25, and 50 g / ml goldmag. the conclusion of the study is that goldmag nanoparticles could serve as photothermal sensitizers, and mri is feasible to quantify intracellular internalization inside pancreatic malign cells. in another study 15 nm aunps conjugated to covalently coupled to anti - egfr antibodies were taken up by a431 cells in culture that catalytically aggregate them (by enzyme degradation of antibodies and ph effects), shifting their absorption into the nir region, thus amplifying their photonic absorption. treatment of human squamous cell carcinoma a431 (positive for epidermal growth factor receptor egfr) murine xenografts with anti - egfr antibodies conjugated to aunps and nir resulted in complete tumor ablation in most cases with no adverse effects on the surrounding healthy tissue. several authors developed an aptamer switch probe (asp) linking chlorin e6 (ce6), to the surface of gold nanorods (aunrs) to target a leukemia cancer cell line for photodynamic therapy (pdt) and photothermal therapy (ptt) applications. in the presence of target cancer cells, the asp changes conformation to drive ce6 away from the gold surface, thereby producing singlet oxygen for pdt upon light irradiation. in addition, absorption of radiation by the gold nanorods enables further cell destruction by the photothermal effect. the authors claims that their approach offers a remarkably improved and synergistic therapeutic effect compared to ptt or pdt alone, providing high specificity and therapeutic efficiency. several authors developed a bionanosystem composed of polydopamine polymerized onto gold nanorods, and egf receptor antibodies (anti - egfr) immobilized onto the layer. antibody - functionalized nrs were significantly more toxic to cancer cells (oscc15, mda - mb-231 and mcf7) in vitro compared with untargeted nrs when irradiated with a broadband light source. pd - mediated surface modification is suggested to be a useful strategy for the conjugation of cancer - specific ligands to nanoparticle surfaces, enabling the formation of biofunctional diagnostic and therapeutic metal nanoparticles. polydopamine - enabled surface functionalization of gold nanorods provided a potent light - activated photothermal therapeutic response, producing significant photoinduced toxicity of breast and oral cancer cells following nir irradiation ((480850 nm), 60 w / cm2 for 5, 10 or15 min. in another study, gold nanoshells were encapsulated in silica epilayers doped with iron oxide and further labeled with antibodies targeting ngal in aspc-1-derived xenografts in nude mice. the authors showed that antingal - conjugated tgns specifically targeted pancreatic cancer cells - aspc-1 (human, pancreas, adenocarcinoma, derived from metastatic ascites) in vitro and in vivo providing contrast for both nir fluorescence (808 nm for 10 min at a power density of 5.81 w / cm2) and t2 weighted mr imaging with higher tumor contrast than can be obtained using long - circulating but non - targeted pegylated nanoparticles. the nanocomplexes also enabled highly specific cancer cell death via nir photothermal therapy in vitro. in a very recent paper several authors report the construct of a biodegradable plasmon resonant liposome gold nanoparticles system (liposau nps) with high thermal response in nir irradiation. the pharmacokinetic study of liposau nps were conducted on a small animal model showing that degradation of this nanoparticles occurs in hepatocytes and are excreted through the hepato - biliary and renal route. the authors next studied the therapeutic potential of liposau nps on a mouse tumor xenograft model using nir laser (750 nm) illumination. the authors claim that resulting destruction of tumor mass was obtained combined with the prolonging of disease - free interval. their ability to generate massive amount of h2a.x foci is a strong indicator of the mode of tumor ablation by dna double strand breaks. applicability of such novel biodegradable hybrid nanoparticle system holds great promise in cancer nanotherapeutics. in another study, the authors used a thermosensitizing cytokine - tumor necrosis factor - alpha conjugated to 30 nm gold nanospheres (au - tnf) to treat murine sck and 4t1-gfp breast carcinomas and 2h11 lymphatic endothelial cells that were next heated by laser pulses (at various wave lenghts). to enhance photothermal efficiency in near - infrared window of tissue transparency the authors analyzed slightly ellipsoidal nanoparticles, their clustering, and laser - induced nonlinear dynamic phenomena leading to amplification and spectral sharpening of photothermal and photoacoustic resonances red - shifted relatively to linear plasmonic resonances. using a murine carcinoma model, they obtained higher therapy efficacy of au - tnf conjugates compared to laser and au - tnf alone or laser with tnf - free gold nanospheres. we have recently showed that laser photoexcited gold nanoparticles interfere with the mitochondrial electron transfer chain through interaction of the activated electrons from the surface of pre - gnp with the electrons within the mitochondrial membrane in pancreatic cancer cells. following this interaction, panc-1 cells exhibit increased mitochondrial permeability transition (mpt) that generates immediate dissipation of the mitochondrial membrane potential and osmotic mitochondrial lysis. in addition we have used quantitative proteomic techniques to prove that treatment with activated gold nanoparticles leads to inactivation of bcl-2 anti - apoptotic proteins causing a decrease of mitochondrial transmembrane potential (mtp) followed by cellular necrosis. we also identified an upregulation of the cox 5b factor following the treatment with photoexcited gold nanoparticles strongly suggesting that mitochondria enter a hypoxic state when treated with gnps having electrons in a photoexcited state. the observation was correlated with the downregulation of a subunit of complex v (atp5h) which is known to be involved in oxidative phosphorylation (since it generates an electrochemical gradient of protons across the inner membrane) leading to defective mitochondrial oxidative phosphorylation (oxphos). since cancer because malign cells exhibit increased metabolic activity, their mitochondria are fundamentally different compared with normal cells and exhibit an extensive metabolic reprogramming. this represents the underlying mechanism that renders malign cells more susceptible to mitochondrial dysfunction than benign cells. zhang and co - workers used peg - coated gold nanoparticles to enhance the in vitro and in vivo radiation response of gold nps. they used multiple size nanoparticles such as 4.8, 12.1, 27.3, and 46.6 nm peg - coated gold nps in hela cells. the toxicities of the peg - coated gold nps with different sizes were investigated 24 and 48 hours after treatment. their conclusion was that peg - coated gold nanoparticles can cause a significant decrease in cancer cell survival after gamma radiation. 12.1 and 27.3 nm peg - coated gold nanoparticles proved to be highly dispersive in the cells and displayed stronger sensitization effects than 4.8 and 46.6 nm particles by both cell apoptosis and necrosis. the authors also performed in vivo by evaluating tumor size and weight after 5 gy gamma radiations radiosensitization effects on 4.8, 12.1, 27.3, and 46.6 nm peg - coated gold particles showing that 12.1 and 27.3 nm peg - coated gold nanoparticles show high radiosensitivity and may be used for cancer phothermal ablation. in one study performed by kuo. 35 nm gold nanorods (au nrs) and 50 nm gold nanoparticles (au nps) were used to test two different approaches of cancer thermal therapy : photodynamic therapy (pdt) and photothermal therapy (ptt). in this au nrs and au the authors showed that combination of ptt and pdt proved to be efficient in killing cancer cells as compared to ptt or pdt treatment alone because its stability. a novel, 4-component bio - nanosystem compose of a phthalocyanine photosensitiser drug and a primary antibody (anti - her2) specific to cell surface receptors attached on a 4 nm gold nanoparticle was used to enhance drug targeting of breast cancer cells that overexpress the her2 receptor on the cellular surface. the authors showed that the targeting capability of the 4-component nanoparticle conjugates enhances the efficacy of pdt cell death when tumor - associated antigens are present on the cytoplasmic membrane of the malignant cells. multifunctional doxorubicin (dox)-loaded hollow gold nanospheres (dox@hauns) against ephb4 receptors were administered in ephb4-positive tumors both in vitro and in vivo. in vivo release of dox from dox@hauns, triggered by nir laser, was assessed by dual - radiotracer technique suggesting significantly decreased tumor growth when compared with treatments with nontargeted dox@hauns plus laser or hauns plus laser. the authors obtained encouraging results may have promise as a new anticancer therapy : tumors in 6 of the 8 mice treated with t - dox@hauns plus laser regressed completely with only residual scar tissue by 22 days following injection, and none of the treatment groups experienced a loss in body weight. a nanoparticle - based probe (composed of 10 nm spherical gold nanoparticle (np) with ph - responsive ligands and raman probes on the surface) was tested as turn - on theragnostic agent for simultaneous raman imaging / diagnosis and photothermal therapy. the system was developed to provide the surface with both positive and negative charges upon mildly acidic condition, which subsequently results in rapid aggregations of the gold nps that provides hot spots for the sers probe. another advantage of this rapid aggregation was the accumulation mainly in b16 f10 mouse melanoma cells. as the result, both raman imaging and photothermal efficacy were enhanced in treated malign cells. in a study conducted by puvanakrishnan. the effect of multiple dosing of gnps and nanoparticle type to improve tumor - targeting efficiency was analyzed. arge pegylated gold nanoshells (gnss) and small pegylated gold nanorods (gnrs) were used to compare the effect of the size of these particles on tumor targeting efficiency. specifically, they compared the effect of single and multiple doses of gnrs and gnss on in vivo tumor targeting. (four- to six - week old nude mice (swiss nu / nu) were inoculated with the a431 cells). neutron activation analysis (naa) was used to determine the amount of gnps accumulated for the different doses and the percent accumulation of gnrs and gnss in a squamous cell carcinoma tumor model. the authors showed a straight relationship between the efficacy of multiple dosing with increased accumulation of gnps in tumors with low toxicity following administration. iron - oxide core / gold - shell nanoparticles 30 nm diameter were used to demonstrate that gold nanoparticles are phagocytosed by pancreatic cancer cells, thus permitting magnetic resonance imaging (mri) of sensitizer delivery and photothermal ablation. using panc-1 cells followed by photothermal ablation, the cell proliferation percentage changed from 100% to 71.3% and 47.0% for cells treated with 25 and 50 g / ml goldmag. according to the authors, photothermal ablation of panc-1 cells demonstrated an effective treatment response, specifically a reduction to only 61%, 21.9%, and 2.3% cell proliferation for cells treated with 0, 25, and 50 g / ml goldmag. the conclusion of the study is that goldmag nanoparticles could serve as photothermal sensitizers, and mri is feasible to quantify intracellular internalization inside pancreatic malign cells. in another study 15 nm aunps conjugated to covalently coupled to anti - egfr antibodies were taken up by a431 cells in culture that catalytically aggregate them (by enzyme degradation of antibodies and ph effects), shifting their absorption into the nir region, thus amplifying their photonic absorption. treatment of human squamous cell carcinoma a431 (positive for epidermal growth factor receptor egfr) murine xenografts with anti - egfr antibodies conjugated to aunps and nir resulted in complete tumor ablation in most cases with no adverse effects on the surrounding healthy tissue. several authors developed an aptamer switch probe (asp) linking chlorin e6 (ce6), to the surface of gold nanorods (aunrs) to target a leukemia cancer cell line for photodynamic therapy (pdt) and photothermal therapy (ptt) applications. in the presence of target cancer cells, the asp changes conformation to drive ce6 away from the gold surface, thereby producing singlet oxygen for pdt upon light irradiation. in addition, absorption of radiation by the gold nanorods enables further cell destruction by the photothermal effect. the authors claims that their approach offers a remarkably improved and synergistic therapeutic effect compared to ptt or pdt alone, providing high specificity and therapeutic efficiency. several authors developed a bionanosystem composed of polydopamine polymerized onto gold nanorods, and egf receptor antibodies (anti - egfr) immobilized onto the layer. antibody - functionalized nrs were significantly more toxic to cancer cells (oscc15, mda - mb-231 and mcf7) in vitro compared with untargeted nrs when irradiated with a broadband light source. pd - mediated surface modification is suggested to be a useful strategy for the conjugation of cancer - specific ligands to nanoparticle surfaces, enabling the formation of biofunctional diagnostic and therapeutic metal nanoparticles. polydopamine - enabled surface functionalization of gold nanorods provided a potent light - activated photothermal therapeutic response, producing significant photoinduced toxicity of breast and oral cancer cells following nir irradiation ((480850 nm), 60 w / cm2 for 5, 10 or15 min. in another study, gold nanoshells were encapsulated in silica epilayers doped with iron oxide and further labeled with antibodies targeting ngal in aspc-1-derived xenografts in nude mice. the authors showed that antingal - conjugated tgns specifically targeted pancreatic cancer cells - aspc-1 (human, pancreas, adenocarcinoma, derived from metastatic ascites) in vitro and in vivo providing contrast for both nir fluorescence (808 nm for 10 min at a power density of 5.81 w / cm2) and t2 weighted mr imaging with higher tumor contrast than can be obtained using long - circulating but non - targeted pegylated nanoparticles. the nanocomplexes also enabled highly specific cancer cell death via nir photothermal therapy in vitro. in a very recent paper several authors report the construct of a biodegradable plasmon resonant liposome gold nanoparticles system (liposau nps) with high thermal response in nir irradiation. the pharmacokinetic study of liposau nps were conducted on a small animal model showing that degradation of this nanoparticles occurs in hepatocytes and are excreted through the hepato - biliary and renal route. the authors next studied the therapeutic potential of liposau nps on a mouse tumor xenograft model using nir laser (750 nm) illumination. the authors claim that resulting destruction of tumor mass was obtained combined with the prolonging of disease - free interval. their ability to generate massive amount of h2a.x foci is a strong indicator of the mode of tumor ablation by dna double strand breaks. applicability of such novel biodegradable hybrid nanoparticle system holds great promise in cancer nanotherapeutics. in another study, the authors used a thermosensitizing cytokine - tumor necrosis factor - alpha conjugated to 30 nm gold nanospheres (au - tnf) to treat murine sck and 4t1-gfp breast carcinomas and 2h11 lymphatic endothelial cells that were next heated by laser pulses (at various wave lenghts). to enhance photothermal efficiency in near - infrared window of tissue transparency the authors analyzed slightly ellipsoidal nanoparticles, their clustering, and laser - induced nonlinear dynamic phenomena leading to amplification and spectral sharpening of photothermal and photoacoustic resonances red - shifted relatively to linear plasmonic resonances. using a murine carcinoma model, they obtained higher therapy efficacy of au - tnf conjugates compared to laser and au - tnf alone or laser with tnf - free gold nanospheres. we have recently showed that laser photoexcited gold nanoparticles interfere with the mitochondrial electron transfer chain through interaction of the activated electrons from the surface of pre - gnp with the electrons within the mitochondrial membrane in pancreatic cancer cells. following this interaction, panc-1 cells exhibit increased mitochondrial permeability transition (mpt) that generates immediate dissipation of the mitochondrial membrane potential and osmotic mitochondrial lysis. in addition we have used quantitative proteomic techniques to prove that treatment with activated gold nanoparticles leads to inactivation of bcl-2 anti - apoptotic proteins causing a decrease of mitochondrial transmembrane potential (mtp) followed by cellular necrosis. we also identified an upregulation of the cox 5b factor following the treatment with photoexcited gold nanoparticles strongly suggesting that mitochondria enter a hypoxic state when treated with gnps having electrons in a photoexcited state. the observation was correlated with the downregulation of a subunit of complex v (atp5h) which is known to be involved in oxidative phosphorylation (since it generates an electrochemical gradient of protons across the inner membrane) leading to defective mitochondrial oxidative phosphorylation (oxphos). since cancer because malign cells exhibit increased metabolic activity, their mitochondria are fundamentally different compared with normal cells and exhibit an extensive metabolic reprogramming. this represents the underlying mechanism that renders malign cells more susceptible to mitochondrial dysfunction than benign cells. we may assert that photothermal therapy using functionalized gold nanoparticles represents a feasible therapeutic method against human malignancies. however further studies are required in order to assess the interactions of nanoparticles with biological systems.	recent research suggests that nanotechnologies may lead to the development of novel cancer treatment. gold nanoparticles with their unique physical and chemical properties hold great hopes for the development of thermal - based therapies against human malignancies. this review will focus on various strategies that have been developed to use gold nanoparticles as photothermal agents against human cancers.
pulmonary alveolar proteinosis (pap) is a rare disorder characterized by abundant accumulation of surfactant within the alveoli. the disease presents clinically in one of three forms : congenital, primary acquired (idiopathic), or secondary. the acquired or idiopathic form accounts for more than 90% of all cases and is believed to be caused by autoantibodies targeting granulocyte - macrophage colony stimulating factor (gm - csf). secondary pap develops in association with hematological malignancies, chronic pulmonary infections, inhalation exposure, and hiv infection. recent studies have reported the detection of neutralizing autoantibodies against gm - csf in the serum as well as bal fluid of patients with idiopathic pap which suggests that gm - csf may be a serologic marker for idiopathic pap. spontaneous remission may occur in up to one third of the patients. in this paper, we report a case of secondary alveolar proteinosis associated with behet 's disease, which was diagnosed by bal findings and lack of circulating anti - gm - csf antibody, with spontanoeus resolution. a 51-year - old man with stable behet 's disease reported dry cough and dyspnea upon exertion which had developed three weeks earlier. past medical history was unremarkable except for behet 's disease diagnosed at the age of 45. the patient commenced on topical steroid and cyclosporine treatment for one month that was completed two weeks before admission for uveitis. physical examination revealed fever (37.4c), papular skin lesions and fine rales at the base of both lungs. laboratory data on admission were as follows : hemoglobin, 13.1 g / dl ; hematocrit, 40.2% ; leukocytes, 6400/mm ; platelets, 223,000/mm ; erythrocyte sedimentation rate, 42 mm / h ; and c - reactive protein, 12.4 mg / dl. chest x - ray showed ground glass opacification and interstitial pattern particularly in the right lung. arterial blood gases at room air were as follows : ph : 7.42, po2 : 74 mm hg, and pco2 : 36.8 mm hg. pulmonary function tests were normal : forced ventilatory capacity (fvc), 3700 ml, 96% predicted ; forced expiratory volume in 1 second (fev1), 2800 ml, 91% predicted ; diffusion lung capacity (dlco), 82% predicted. ct scan of the chest revealed ground glass opacification of alveolar spaces with thickening of inter - obular and intralobular septa typical of the crazy paving pattern (figure 1). serologies for cytomegalovirus, respiratory syncytial virus, adenovirus, influenza, legionella and mycoplasma pneumoniae were also negative. empirical treatment with moxifloxacin was instituted. bal differential cytology showed the composition to be 74% macrophages, 21% neutrophils, and 5% lymphocytes. light microscopic examination of the fluid showed large amounts of amorphous, lipoproteinaceous material that was periodic acid - schiff stain (pas) positive (figure 2). smear, special stains, culture of the bal fluid, and tissue were negative for tuberculosis, bacteria, pneumocystis carinii, fungi or malignant cells. histology of the transbronchial biopsy specimen demonstrated a pas - positive intra - alveolar eosinophilic material consistent with pap. previous studies have reported anti - gm - csf antibody as being present in the serum of patients with idiopathic pap. anti - gm - csf antibodies were not detected in the serum and bal fluid. based on these findings the patient was diagnosed as secondary pap associated with behet 's disease. while the patient was followed as an outpatient at our department his clinical status was stable with progressive recovery of dyspnea and cough. chest ct performed four weeks later revealed complete resolution of the ground glass opacification and septal thickening (figure 3). chest ct demonstrating ground glass opacities with thickened interlobular septa in both lungs, the typical crazy paving pattern seen in pulmonary alveolar proteinosis. eosinophilic material in the alveolar spaces with scattered alveolar macrophages and lymphocytes (hematoxylyn - eosin stain 100). ct of the chest showing complete resolution of the ground glass pattern and interstitial thickening of the interlobular septa. pap is a rare disorder characterized by abundant accumulation of phospholipids and proteinaceous material within the alveoli of the lungs. since this disease was first described in 1958, fewer than 500 cases have been reported in the medical literature. advances in the cellular and molecular basis of the disease have led to a better understanding of the pathogenesis of pap. the link between gm - csf and human idiopathic pap was revealed when anti - gm - csf antibodies were detected in the serum and bal fluid of patients with the disease. the idiopathic form accounts for more than 90% of cases and is believed to be caused by autoantibodies targeting granulocyte - macrophage - colonystimulating factor. secondary pap develops in the setting of a predisposing factor such as hematological malignancies, immunodeficiency syndromes, chronic infection or immunosuppressive medications. pap has only rarely been associated with autoimmune diseases. in a review of over 400 published cases of pap, a diagnosis of behet 's disease coexisted in only one patient reported by uchiyama.. we believe that the presence of alveolar proteinosis may be underestimated because of three factors. one, alveolar proteinosis associated with behet 's syndrome may easily be missed as there are no specific features of alveolar consolidation to suggest the diagnosis. second, the diagnosis can only be made with confidence on the basis of high resolution computed tomography (hrct) appearence. consequently, alveolar proteinosis may be overlooked or misdiagnosed as another inflammatory disease such as pneumonia, alveolar hemorrhage or drug reaction which are common in the setting of autoimmune disease. the absence of anti - gm - csf antibodies strongly suggested that our patient had developed secondary pap in the setting of behet 's disease. the development of pap may have been associated with infection but there was no evidence of current or earlier infection. although few studies have described an association between secondary pap and prolonged corticosteroid therapy, the effect of local steroid treatment may be considered negligible for immunosuppression as there are no data to suggest immunosuppression due to short course local steroid treatment. furthermore, patients who receive corticosteroid treatment for other conditions do not develop alveolar proteinosis because only a few studies have described an association between secondary pap and long - term corticosteroid therapy. inhibition of cd4-t cells by cyclosporine may have produced deficiency of macrophage recruitment and phagocytosis causing impairment of surfactant clearance in this case. autoimmune disease and vasculitis may also have contributed to the development of alveolar proteinosis to a variable extent by unknown mechanisms. although alveolar proteinosis may run a fatal course, spontaneous recovery may also occur. to our knowledge, this is only the second reported case of secondary pap associated with behet 's disease and the first case of a spontaneous remission. our patient draws attention to the fact that secondary alveolar proteinosis with spontaneous resolution may occur in the setting of behet 's disease. therefore, unnecessary diagnostic interventions or treatment modalities for secondary alveolar proteinosis associated with behet 's disease should be reconsidered in stable patients. further case database and multicenter trials are needed to establish the real incidence and prognosis of alveolar proteinosis in behet 's disease. none of the authors has any conflict of interest to declare in relation to the subject matter of this manuscript.	a 51-year - old man with behet 's disease complained of fever, dry cough and dyspnea during exertion. chest ct showed ground glass opacities with interstitial septal thickening in both lungs. bronchoalveolar lavage (bal) revealed amorphous and lipoproteinaceous material that was periodic acid - schiff (pas) stain positive. transbronchial biopsy specimen demonstrated pas positive alveolar eosinophilic material consistent with pulmonary alveolar proteinosis. serum anti - granulocyte - macrophage colony stimulating factor (gm - csf) antibody was negative. recent studies have reported anti - gmcsf not present in the the serum of patients with secondary pulmonary alveolar proteinosis (pap) but they have not reported so in patients with idiopathic pap. we report a case of alveolar proteinosis in the setting of behet 's disease with spontaneous remission.
total joint arthroplasties of the hip and knee represent a remarkable feat of modern medicine in terms of reducing pain and restoring function to millions of patients afflicted with severe arthritis. while the evolution of implants in many cases has resulted in marked improvements for patients, there are noticeable, high profile failures that have occurred during the same period. often times the performance and longevity of new implants and devices are based on limited data. these data are commonly in the form of reported case series in the literature where many of these studies are funded by the manufacturers of the implant and/or conducted by designers of the implant, both of whom have an inherent conflict of interest. this undoubtedly can introduce bias into the results of these studies.12345 mobile bearing (mb) knee design has got remarkable success when followed for more than 20 years,6 low contact stress (lcs) design introduced in 1977, has been proved very successful with maximum followup of 31 years in the hands of designer, buechel.7 based on its success, same manufacturer (depuy orthopaedics inc., warsaw, in, united states) introduced press - fit condylar (pfc) knee with rotating platform (rp) with two main variants pfc - rp - cruciate retaining (pfc - rp - cr) and pfc - rp - posterior stabilized (pfc - rp - ps) in year of 2000. while there has been enough literature on cr variant, we could only come across two papers, with minimum 5 years followup with at least 100 knees in the cohort for ps variant. these are meftah.8 (designer for depuy) with 97.7% success rate at the end of 10 years of followup maniar.9 lead author (designer for depuy pfc - ps - rp) with 100% success rate at the end of 8 years followup. incidentally, for the available studies of ps variant of pfc - rp one of the co - authors is the designer himself for depuy (ranawat cs, maniar, rn respectively). the design features of this implant are an improvement over the lcs - rp by depuy orthopaedics inc., the 4.8 mm thick chrome baseplate was designed to accept a unidirectional tibial insert with 16 mm high post, and full conformity in both coronal and sagittal planes. this is the first study by a non - designer on the pfc - rp - ps design with 100% success. this has a relevance, vis - - vis bias that one may have in terms of reproducibility of technique and funding from the manufacturer. we associate our excellent mid - term results to intraoperative technical aspects and stringent intraoperative exclusion criteria. we started performing pfc - rp - ps knees since 2003, with very selective intraoperative criteria, in choosing the patients for this implant. one hundred and twenty one selective knees in 83 patients were operated for pfc - rp - ps total knee arthroplasty (tka) between january 2003 and october 2010. a written informed consent was taken for every knee that if there were unsatisfactory intraoperative technical conditions, we would not go ahead with pfc - rp - ps design and may have to revert to fixed bearing (fb) design. all knees having an inadequate balancing of flexion and extension gap, knees with severe osteolysis and those with trapezoidal gap were excluded intraoperatively and an fb design was implanted. moreover, knees having extreme deformities where balancing could not be up to a satisfactory level were excluded intraoperatively in favor of fb design. we used cemented, ps, rp prosthesis from the same manufacturer (pfc - rp - ps sigma, depuy orthopaedics inc., the patella was resurfaced in 100% knees with a cemented polyethylene component. after losing 1 patient (1 knee) to followup as patient died of myocardial infarction, we retrospectively analyzed 120 knees in 82 patients in our study [figures 1 and 2 ]. the average age of the patient at the time of surgery was 62.5 years (range 3883 years). there were fifty five female patients (66.3%) and 28 male patients (33.7%). the diagnosis was osteoarthritis in 68 knees (80.8%) and rheumatoid arthritis in 15 knees (19.2%). varus deformity was seen in 67 knees (82.0%) and valgus deformity was seen in 11 knees (18.0%). about 44 patients (53%) were either overweight (body mass index [bmi ] 25.0029.99) or obese (bmi 30.00). the mean bmi of the patients in the study was 31.7 (range 23.939.6). minimum followup was of 5 years and 1 month (range 5 years and 1 month to 11 years and 10 months). a bar diagram showing year wise distribution of different types tkas a bar diagram showing year wise distribution of all tkas all the surgeries were performed by the lead author through a standard central skin incision with a midvastus approach. an intramedullary guide was used in case of a femur while an extramedullary jig was used for tibia. balancing was done first in extension using spacer blocks. for creation of a rectangular flexion space, posterior condyle cut parallel to tibial condyle or the gap balancing technique was used, the efficacy of which has been published by the lead author.10 this is a modification of the original gap - balancing technique. in this technique, the proximal tibia is the first cut at 90. the distal femur is cut at 5 of valgus for a varus knee or 3 of valgus for a valgus knee. the soft tissues are first balanced in extension (up to 2 mm of equal mediolateral opening was accepted). then, the knee is flexed to 90. all the osteophytes were thoroughly removed, especially the posterior ones, prior to soft tissue releases for coronal plane balancing. an anteroposterior (ap) femoral cutting block of appropriate size is placed on the cut surface of distal femur and preliminarily fixed with pins. a lamina spreader is then applied between posterior margins of the block and cut tibial surface with the knee at 90 flexion to create even tension in the collaterals. the block is then rotated and/or shifted anteroposteriorly with stylus on the upper edge (to make sure that the notching is prevented) until a rectangular gap is created equal to the extension gap. in an ideal case, both extension and flexion gaps form a perfect rectangle. equal laxity of 12 mm mediolateral, in flexion of 90 with spacer block in, with varus and valgus stress testing, respectively, should be desirable. any flexion gap of more than 2 mm in the formation of a rectangular flexion space on stretching with lamina spreader was not accepted. the balanced extension and flexion gaps so obtained were confirmed by corroborating with transepicondylar axis and whiteside 's line (ap axis), which are other accepted methods of confirming rotation of the femoral component. an fb implant was used in the following situations : the balancing in flexion was not satisfactory despite efforts to fit appropriate size femoral implant and moving the ap cutting block as needed or the flexion balancing remaining more than 2 mm loose. obviously, the ap cutter block can not be shifted further posteriorly (which would tighten the flexion space) for fear of notchinga trapezoidal flexion gap developed (on the medial side height more than lateral, in typical varus deformity, after it is released, and balanced in extension) despite rotating the femoral ap cutting jig. this can particularly happen in extreme deformities or combination of flexion + varus deformitiessubstantial incompetence of the collateral ligaments, especially in cases of severe bone loss or extensive posteromedial release, including semimembranosus. the balancing in flexion was not satisfactory despite efforts to fit appropriate size femoral implant and moving the ap cutting block as needed or the flexion balancing remaining more than 2 mm loose. obviously, the ap cutter block can not be shifted further posteriorly (which would tighten the flexion space) for fear of notching a trapezoidal flexion gap developed (on the medial side height more than lateral, in typical varus deformity, after it is released, and balanced in extension) despite rotating the femoral ap cutting jig. this can particularly happen in extreme deformities or combination of flexion + varus deformities substantial incompetence of the collateral ligaments, especially in cases of severe bone loss or extensive posteromedial release, including semimembranosus. no undue attempt was made to fit in the pfc - rp - ps prosthesis in any of these unacceptable scenarios. authors abandoned the pfc - rp - ps prosthesis and went ahead to perform fb implant if the above criteria were not met. the advantage of the instrumentation allowed the switch over to fb implant, efficiently, as the femoral component for both, the rp and the fb implant, are the same, requiring only tibial fin cut changes intraoperatively. thus, all the knees were either pfc sigma rp ps or pfc sigma fb ps for this project. we resurfaced patellar component in all the cases and it was done with a free - hand technique. in all the cases, the patellar thickness was measured with a vernier caliper and suprapatellar synovial fold was resected. patella was held with the help of two towel clips, in an everted position so that it lies with its equator parallel to the floor. saw blade was passed through substance at a particular level so that after patellar component implantation the original thickness of patella is created ; taking care that at least 14 mm of native patellar bone is left behind after the cut. final patellar button implantation was done a little medially so that the button lies adequately medial to the anterior lateral flange of the femoral component ; in an effort to recreate the normal patellofemoral biomechanics. under no circumstances, was the patella undercut or overcut or the patellar button put eccentrically. we do this step with a logical presumption that every time we overcut to leave a thinner or smaller patella than before, the quadriceps tendon will have natural tendency to rub its surface against the metal at the femoral component notch. this, we feel, is the most common cause related to the surgical technique, which can lead to crepitus or clunk, postoperatively. all components were cemented with palacos - r (a nonantibiotic impregnated low - viscosity cement). drain was kept for all knees for 48 h. postoperative protocols were the same for all the patients. all patients received routine antibiotics and thrombosis prophylaxis therapy and were usually discharged on the 3 postoperative day and asked to followup as per routine schedule. the patients were discharged on 3 postoperative day and were called for followup on 10 day and 3 week for inspection of surgical site and stich removal. thus, patients were evaluated biannually with calculation of their knee society scores (ksss) and a radiological assessment for loosening / osteolysis (by dividing the periarticular region into various zones as described in the knee society x - ray scoring system), spin - offs or any other abnormalities by getting true - sized ap, lateral and oblique weight - bearing radiographs, and an axial view for patella. statistical test was done for statistical analysis of our results [figures 3 and 4 ]. suggested guidelines for assignment of zones are : 1 and 2 for medial plateau, 3 and 4 for lateral plateau, and 5 to 7 for the stem fixation or the central fixation if there is no stem lateral view of the femoral component. suggested guidelines for the assignment of zones are : 1 and 2 for the anterior flange, 3 and 4 for the posterior part, and 5 to 7 for the stem fixation or the central fixation if there is no stem all the surgeries were performed by the lead author through a standard central skin incision with a midvastus approach. an intramedullary guide was used in case of a femur while an extramedullary jig was used for tibia. balancing was done first in extension using spacer blocks. for creation of a rectangular flexion space, posterior condyle cut parallel to tibial condyle or the gap balancing technique was used, the efficacy of which has been published by the lead author.10 this is a modification of the original gap - balancing technique. in this technique, the proximal tibia is the first cut at 90. the distal femur is cut at 5 of valgus for a varus knee or 3 of valgus for a valgus knee. the soft tissues are first balanced in extension (up to 2 mm of equal mediolateral opening was accepted). then, the knee is flexed to 90. all the osteophytes were thoroughly removed, especially the posterior ones, prior to soft tissue releases for coronal plane balancing. an anteroposterior (ap) femoral cutting block of appropriate size is placed on the cut surface of distal femur and preliminarily fixed with pins. a lamina spreader is then applied between posterior margins of the block and cut tibial surface with the knee at 90 flexion to create even tension in the collaterals. the block is then rotated and/or shifted anteroposteriorly with stylus on the upper edge (to make sure that the notching is prevented) until a rectangular gap is created equal to the extension gap. in an ideal case, equal laxity of 12 mm mediolateral, in flexion of 90 with spacer block in, with varus and valgus stress testing, respectively, should be desirable. any flexion gap of more than 2 mm in the formation of a rectangular flexion space on stretching with lamina spreader was not accepted. the balanced extension and flexion gaps so obtained were confirmed by corroborating with transepicondylar axis and whiteside 's line (ap axis), which are other accepted methods of confirming rotation of the femoral component. an fb implant was used in the following situations : the balancing in flexion was not satisfactory despite efforts to fit appropriate size femoral implant and moving the ap cutting block as needed or the flexion balancing remaining more than 2 mm loose. obviously, the ap cutter block can not be shifted further posteriorly (which would tighten the flexion space) for fear of notchinga trapezoidal flexion gap developed (on the medial side height more than lateral, in typical varus deformity, after it is released, and balanced in extension) despite rotating the femoral ap cutting jig. this can particularly happen in extreme deformities or combination of flexion + varus deformitiessubstantial incompetence of the collateral ligaments, especially in cases of severe bone loss or extensive posteromedial release, including semimembranosus. the balancing in flexion was not satisfactory despite efforts to fit appropriate size femoral implant and moving the ap cutting block as needed or the flexion balancing remaining more than 2 mm loose. obviously, the ap cutter block can not be shifted further posteriorly (which would tighten the flexion space) for fear of notching a trapezoidal flexion gap developed (on the medial side height more than lateral, in typical varus deformity, after it is released, and balanced in extension) despite rotating the femoral ap cutting jig. this can particularly happen in extreme deformities or combination of flexion + varus deformities substantial incompetence of the collateral ligaments, especially in cases of severe bone loss or extensive posteromedial release, including semimembranosus. no undue attempt was made to fit in the pfc - rp - ps prosthesis in any of these unacceptable scenarios. authors abandoned the pfc - rp - ps prosthesis and went ahead to perform fb implant if the above criteria were not met. the advantage of the instrumentation allowed the switch over to fb implant, efficiently, as the femoral component for both, the rp and the fb implant, are the same, requiring only tibial fin cut changes intraoperatively. thus, all the knees were either pfc sigma rp ps or pfc sigma fb ps for this project. we resurfaced patellar component in all the cases and it was done with a free - hand technique. in all the cases, the patellar thickness was measured with a vernier caliper and suprapatellar synovial fold was resected. patella was held with the help of two towel clips, in an everted position so that it lies with its equator parallel to the floor. saw blade was passed through substance at a particular level so that after patellar component implantation the original thickness of patella is created ; taking care that at least 14 mm of native patellar bone is left behind after the cut. final patellar button implantation was done a little medially so that the button lies adequately medial to the anterior lateral flange of the femoral component ; in an effort to recreate the normal patellofemoral biomechanics. under no circumstances, was the patella undercut or overcut or the patellar button put eccentrically. we do this step with a logical presumption that every time we overcut to leave a thinner or smaller patella than before, the quadriceps tendon will have natural tendency to rub its surface against the metal at the femoral component notch. this, we feel, is the most common cause related to the surgical technique, which can lead to crepitus or clunk, postoperatively. all components were cemented with palacos - r (a nonantibiotic impregnated low - viscosity cement). drain was kept for all knees for 48 h. postoperative protocols were the same for all the patients. all patients received routine antibiotics and thrombosis prophylaxis therapy and were usually discharged on the 3 postoperative day and asked to followup as per routine schedule. the patients were discharged on 3 postoperative day and were called for followup on 10 day and 3 week for inspection of surgical site and stich removal. thus, patients were evaluated biannually with calculation of their knee society scores (ksss) and a radiological assessment for loosening / osteolysis (by dividing the periarticular region into various zones as described in the knee society x - ray scoring system), spin - offs or any other abnormalities by getting true - sized ap, lateral and oblique weight - bearing radiographs, and an axial view for patella. statistical test was done for statistical analysis of our results [figures 3 and 4 ]. anteroposterior view of the tibial component. suggested guidelines for assignment of zones are : 1 and 2 for medial plateau, 3 and 4 for lateral plateau, and 5 to 7 for the stem fixation or the central fixation if there is no stem lateral view of the femoral component. suggested guidelines for the assignment of zones are : 1 and 2 for the anterior flange, 3 and 4 for the posterior part, and 5 to 7 for the stem fixation or the central fixation if there is no stem of the 121 knees (n = 83) included in the study, we could followup 120 knees (n = 82), one died due to myocardial infarction at 7 years followup. the average knee society clinical and functional scores, respectively, were 27 points (range : 750 points) and 40 points (range 560 points) preoperatively and 93 points (range 64100 points) and 95 points (range 70100 points) postoperatively. this indicates a mean increase of about 71% (mean absolute increase of 66 points) in the clinical score and about 58% (mean absolute increase of 55 points) in the functional score, which is statistically significant. the mean postoperative flexion was 124 (range 99134), a mean increase of 23 from the preoperative flexion of 101 (range 75125). three knees (3.61%) had anterior knee pain of which 2 resolved over a period of 69 months while 1 still has mild but persistent pain (6 years followup). two knees (2.40%) had crepitus which resolved over a period of 912 months after supervised physiotherapy and vastus medialis obliqus strengthening exercises. two knees (2.40%) had superficial skin infection (stitch abscess) which were treated with oral antibiotics and dressing and did not necessitate a wash. two knees (2.40%) showed a nonprogressive osteolytic line (1 in number) on the femoral side. one patient with bilateral knees done sequentially fared lower on her second (right) knee score as she developed a psychiatric illness during her rehabilitation period unrelated to the surgery, which prevented her from pursuing the physiotherapy. she developed a postoperative extension lag of 5 in her right knee, which lead to a deduction of 5 points from her good postoperative clinical score of 81. there were no revisions (kaplan meier survivorship of 100%), periprosthetic fractures, neurovascular complications, thromboembolic phenomena or deep infections, and the followup radiographs revealed no gross osteoysis / loosening. the comparison of the mean clinical score and functional score before and after surgery and the analysis of the results are given in tables 15 and figure 5. descriptive statistics wilcoxon signed ranks test statistical analysis - z value a bar diagram showing preoperative and postoperative functional and clinical scores thus, in the above statistical analysis it is clear that in all the 120 cases of pfc - rp - ps knees the postoperative clinical and functional scores are higher than the preoperative scores (negative ranks 0/120, positive ranks 120/120). using wilcoxon signed ranks test, the pfc - rp - ps knees, have a p = 0.000 (p < 0.05), giving a success rate of 100% in terms of significant improvement in clinical and functional postoperative scores. the kaplan meier analysis, done for the minimum followup of 5 years, gave a 100% survivorship (0/78 revision). the meta - analysis by hopley.11 and carothers.12 and the landmark study by buechel.7 on lcs - rp knees have demonstrated excellent results as good, if not better, as those with fb knees., one of the earliest designers of mb knees, have the longest followup of up to 31 years. these knees were introduced with the goal of providing an implant that would allow increased congruity without compromising motion. the implant was made more conforming, in both the sagittal and coronal planes, at the tibiofemoral articulation, thereby decreasing the contact stresses. motion at the polyethylene tibial component interface was designed to avoid the compromise in knee range of motion (rom) expected with increased articular conformity with fb designs. in theory, this design would provide an ideal biomechanical situation - decreased polyethylene back wear from increased articular conformity while improving knee rom with the addition articulation at the tibial polyethylene interface.1314 the pfc - rp - ps knee was introduced by the same manufacturer (depuy orthopaedics inc., warsaw, in, united states) in order to decrease the high spin out rates in some of the earlier studies. the pfc - rp - ps prosthesis is said to be an improvement over the lcs - rp variety. the tibial component is a highly polished 4.8 mm thick cobalt - chromium baseplate with the poly having nearly full conformity in the sagittal and coronal plane. there is a 16 mm post in the ps variety, apparently reducing the risk of spin - outs. the pfc - rp - ps knee was introduced after the success of its fb variant. clinically, the results of the limited number of pfc - rp - ps followup studies, including the kss and mean rom, have been better than that seen in many of the lcs studies.67 one of the main reasons for the failure of tka in the long run is the premature polyethylene wear (backwear)1516 with subsequent surrounding bone osteolysis as a result of condylar lift - off and subsurface movement at the back of tibial poly insert.171819 this is the place where the pfc - rp - ps knees can be highly advantageous. coronal conformity with regards to fb) and increased contact area without dramatically increasing the fixation stresses.20212223 they decrease the paradoxical sliding / shear and are more tolerable of femoral condylar lift off. it is a known fact that multidirectional motion accelerates the poly wears, while unidirectional motion reduces the same. there is multidirectional motion (rotational, translational, and flexion extension) of the femoral component relative to the tibial bearing surface in fb - tka, all occurring at the single superior surface of the poly. however, in the pfc - rp - ps knees, the superior surface experiences purely flexion extension (unidirectional) and the inferior surface has purely rotational (unidirectional) movements. thus, these movements are decoupled and the wear is largely diminished.2425 the phenomenon of rotational medial and lateral postimpingement in ps systems are reduced in pfc - rp - ps knees due to postrotating with the femoral box instead of rotating against it leading due reduction of cam - postwear. though pfc - rp - ps knees have an additional articulating surface between the poly and tibia, the concerns of generation of extra polyethylene debris and consequential rapid poly wear have not been validated till date,1516 which can be explained by the fact of decoupling of joint motion as mentioned earlier [figures 1 and 2 ]. the self aligning feature of pfc - rp - ps helps the facilitation of central patellar tracking.20 by means of bearing motion, the rotating poly provides for greater self correction of component malalignment in cases of substantial malrotation of tibial component (internal rotation). we believe that an important reason for such good results in our study was known exactly when not to do the pfc - rp - ps knee [table 6 ]. this is a very important factor in the success of pfc - rp - ps knees. most of the complications of this implant such as the spin - offs or patellofemoral pain can be attributed to imperfect balancing. total knee arthroplasties done by year we as a policy, as mentioned earlier, plan for pfc - rp - ps knees to begin with. however, no attempt is made to fit in the prosthesis if balancing is not satisfactory and the exclusion criteria, as mentioned earlier, are followed strictly. we compared our results with publications by two authors on pfc - rp - ps design, both members of the designer team [table 7 ]. this study differs from them by having stricter exclusion criteria for the selection of rp over fb knees. this has helped us achieve a significant improvement of the postoperative rom over the preoperative rom up to a mean of 23. this is higher by 8 than the previous similar study, having 100% survivorship, by maniar., the author attributes the improvement in postoperative rom to stricter knee selection and surgical steps like the removal of posterior femoral osteophytes, restoration of patellar thickness, and accepting up to 2 mm of extra space in flexion as compared to an extension. comparison with studies by designers we also compared our results with other studies done for rp knee designs, which include two meta - analyses with 10 years - plus duration of the index surgery. the outcome compares favorably for the pfc - rp - ps knees as illustrated in the following table [table 8 ]. comparison with other studies by non designers bedair. compared the survivorship of hip and knee implants recorded by the designers and the national registries separately, 32% of comparisons performed demonstrated greater survivorship in the designer series compared to the registry, while 0% reported lower, and 68% demonstrated no difference (p < 0.01).27 the performance and longevity of new implants and devices are based on limited data. these data are commonly in the form of reported case series in the literature where many of these studies are funded by the manufacturers of the implant and/or conducted by designers of the implant, both of whom have an inherent conflict of interest. a common and prevailing sentiment is that the results reported in these types of studies may portray an overly optimistic picture of the implant 's performance. the earlier studies on the followup and survivorship of pfc - rp - ps knees were designer series in essence. maniar and ranawat studies have excellent midterm results (100% and 97%, respectively), but both are by designers of the implant manufactured by depuy8 [table 7 ]. a recent study by lee. in 2016 reported excellent midterm results of a success rate of 96.7% over 10 years.28 as per bedair., there is no concordance in terms of bias and authority when the excellent results are published by designers. our study and its excellent results should stand out as it is not done by designers or manufacturers nor is it funded by them. one of the controversial points, whether to internally rotate the femoral component for the achievement of a flexion rectangular gap is highly debatable. as per boldt, most of the complications after internal rotation of femur resulted when it exceeded 5. they stated that optimal amount was up to 3. we in all the cases followed the same religiously, i.e. never internal rotate beyond the epicondylar line. the extra femoral cut during extension gap balancing which is the cause behind the patellar clunk can be avoided by proper technique and adhering to the exclusion criteria. the pfc - rp - ps knee was introduced by the same manufacturer (depuy orthopaedics inc., warsaw, in, united states) in order to decrease the high spin out rates in some of the earlier studies. the pfc - rp - ps prosthesis is said to be an improvement over the lcs - rp variety. the tibial component is a highly polished 4.8 mm thick cobalt - chromium baseplate with the poly having nearly full conformity in the sagittal and coronal plane. there is a 16 mm post in the ps variety, apparently reducing the risk of spin - outs. the pfc - rp - ps knee was introduced after the success of its fb variant. clinically, the results of the limited number of pfc - rp - ps followup studies, including the kss and mean rom, have been better than that seen in many of the lcs studies.67 one of the main reasons for the failure of tka in the long run is the premature polyethylene wear (backwear)1516 with subsequent surrounding bone osteolysis as a result of condylar lift - off and subsurface movement at the back of tibial poly insert.171819 this is the place where the pfc - rp - ps knees can be highly advantageous. coronal conformity with regards to fb) and increased contact area without dramatically increasing the fixation stresses.20212223 they decrease the paradoxical sliding / shear and are more tolerable of femoral condylar lift off. it is a known fact that multidirectional motion accelerates the poly wears, while unidirectional motion reduces the same. there is multidirectional motion (rotational, translational, and flexion extension) of the femoral component relative to the tibial bearing surface in fb - tka, all occurring at the single superior surface of the poly. however, in the pfc - rp - ps knees, the superior surface experiences purely flexion extension (unidirectional) and the inferior surface has purely rotational (unidirectional) movements. thus, these movements are decoupled and the wear is largely diminished.2425 the phenomenon of rotational medial and lateral postimpingement in ps systems are reduced in pfc - rp - ps knees due to postrotating with the femoral box instead of rotating against it leading due reduction of cam - postwear. however, the long term followups are awaited. though pfc - rp - ps knees have an additional articulating surface between the poly and tibia, the concerns of generation of extra polyethylene debris and consequential rapid poly wear have not been validated till date,1516 which can be explained by the fact of decoupling of joint motion as mentioned earlier [figures 1 and 2 ]. the self aligning feature of pfc - rp - ps helps the facilitation of central patellar tracking.20 by means of bearing motion, the rotating poly provides for greater self correction of component malalignment in cases of substantial malrotation of tibial component (internal rotation). we believe that an important reason for such good results in our study was known exactly when not to do the pfc - rp - ps knee [table 6 ]. this is a very important factor in the success of pfc - rp - ps knees. most of the complications of this implant such as the spin - offs or patellofemoral pain can be attributed to imperfect balancing. total knee arthroplasties done by year we as a policy, as mentioned earlier, plan for pfc - rp - ps knees to begin with. however, no attempt is made to fit in the prosthesis if balancing is not satisfactory and the exclusion criteria, as mentioned earlier, are followed strictly. we compared our results with publications by two authors on pfc - rp - ps design, both members of the designer team [table 7 ]. this study differs from them by having stricter exclusion criteria for the selection of rp over fb knees. this has helped us achieve a significant improvement of the postoperative rom over the preoperative rom up to a mean of 23. this is higher by 8 than the previous similar study, having 100% survivorship, by maniar., the author attributes the improvement in postoperative rom to stricter knee selection and surgical steps like the removal of posterior femoral osteophytes, restoration of patellar thickness, and accepting up to 2 mm of extra space in flexion as compared to an extension. comparison with studies by designers we also compared our results with other studies done for rp knee designs, which include two meta - analyses with 10 years - plus duration of the index surgery. the outcome compares favorably for the pfc - rp - ps knees as illustrated in the following table [table 8 ]. comparison with other studies by non designers bedair. compared the survivorship of hip and knee implants recorded by the designers and the national registries separately, 32% of comparisons performed demonstrated greater survivorship in the designer series compared to the registry, while 0% reported lower, and 68% demonstrated no difference (p < 0.01).27 the performance and longevity of new implants and devices are based on limited data. these data are commonly in the form of reported case series in the literature where many of these studies are funded by the manufacturers of the implant and/or conducted by designers of the implant, both of whom have an inherent conflict of interest. a common and prevailing sentiment is that the results reported in these types of studies may portray an overly optimistic picture of the implant 's performance. the earlier studies on the followup and survivorship of pfc - rp - ps knees were designer series in essence. maniar and ranawat studies have excellent midterm results (100% and 97%, respectively), but both are by designers of the implant manufactured by depuy8 [table 7 ]. a recent study by lee. in 2016 reported excellent midterm results of a success rate of 96.7% over 10 years.28 as per bedair., there is no concordance in terms of bias and authority when the excellent results are published by designers. our study and its excellent results should stand out as it is not done by designers or manufacturers nor is it funded by them. one of the controversial points, whether to internally rotate the femoral component for the achievement of a flexion rectangular gap is highly debatable. as per boldt, most of the complications after internal rotation of femur resulted when it exceeded 5. they stated that optimal amount was up to 3. we in all the cases followed the same religiously, i.e. never internal rotate beyond the epicondylar line. the extra femoral cut during extension gap balancing which is the cause behind the patellar clunk can be avoided by proper technique and adhering to the exclusion criteria. authors feel durable and reproducible results of pfc - rp - ps design knees are very technique sensitive. a surgeon should know before starting to operate, the possibility of changing over to fb implant, if stringent criteria of flexion extension spaces and patella - femoral joint balancing are to be met with. the way ahead with the pfc - rp - ps knees looks promising when the exclusion criteria for this design are given their fair share of importance. coming from a nondesigner, this study acquires a higher degree of relevance without any designer 's or manufacturer 's bias.	background : total joint arthroplasties of the hip and knee represent a remarkable feat of modern medicine in terms of reducing pain and restoring function to millions of patients afflicted with severe arthritis. oftentimes, the performance and longevity of new implants and devices are based on limited data. this is the first study by a non - designer on the press fit condylar rotating platform posterior stabilized (pfc - rp - ps) design with 100 success. this has a relevance, vis - -vis bias that one may have in terms of reproducibility of technique and funding from the manufacturer. we associate our excellent mid - term results to intra operative technical aspects and stringent intra operative exclusion criteria.materials and methods : our study includes a cohort of 121 selected knees operated between january 2003 and october 2010. we used cemented, posterior stabilized (ps), mobile bearing (mb), and rp prosthesis from the same manufacturer in all these 121 knees. the patients were evaluated bi - annually with the calculation of their knee society scores (kss) and a radiological assessment for loosening / osteolysis.results:120 knees were available for followup. the average knee society clinical and functional scores, respectively, were 27 points and 40 points preoperatively and 93 points and 95 points postoperatively. this indicates a mean increase of about 71 in the clinical score and about 58 in the functional score, which is statistically significant. the mean postoperative flexion was 124, a mean increase of 23 from the preoperative flexion of 101. there were no revisions (kaplan-meier survivorship of 100).conclusions : we feel durable and reproducible results of pfc - rp - ps design knees are very technique sensitive. the way ahead with the pfc - rp - ps knees looks promising when the exclusion criteria for this design are strictly met. coming from a non - designer, this study acquires a higher degree of relevance without any designer 's or manufacturer 's bias.
obesity is a major public health problem in developing and developed countries across the world. maternal nutritional status (quality and/or quantity) during the critical periods of life, such as gestation and/or lactation, is the major cause of obesity and predisposes offspring to non - communicable and metabolism related chronic diseases. maternal dietary changes during gestation and lactation may cause chronic diseases in adulthood due to maladaptive phenotypic plasticity. nutritional factors such as energy, fatty acids, protein, and micronutrients affect several aspects of fetal growth. in addition to energy intake, the macronutrient composition of a diet may play an important role in insulin homeostasis. inadequate distribution of macronutrients, including overconsumption of carbohydrates, fats, or both may lead to lipid disturbances associated with obesity and insulin resistance. obesity and high - fat diet during gestation play pathogenic roles in the development of obese phenotype in offspring. the children who are born to obese mothers are likely to be obese than those from lean mothers. increasing evidence suggests that the type and amount of maternal dietary fatty acids have an effect on metabolism. only a few studies have assessed the long - term effects of maternal dietary fatty acid changes on offspring, but no studies have been conducted to assess the long - term effects of different types and amounts of maternal dietary oil with similar energetic values on the offspring at adolescence. several studies have shown that maternal omega-6 consumption leads to obesity, but the latest review in this context has reported that there are limited studies evaluating the effects of maternal monounsaturated fatty acid (mufa) intake in offspring until the adolescence. the purpose of this study was to compare different amounts of extra virgin olive oil versus soy oil in the maternal isocaloric diet during gestation and lactation on obesity, serum glucose, and lipid profile in female mice offspring at adolescence. the experimental protocol was approved by the animal research committee of iran university of medical sciences, tehran, iran. all institutional and national guidelines for the care and use of laboratory animals were followed. eight - week - old inbred adult female c57bl/6 mice (211.5 g) were obtained from the razi vaccine and serum research institute, tehran, iran. each mouse was individually housed at 21 - 23 c and controlled humidity (505%) under a 12-h artificial light cycle (7 am to 7 pm). to adapt, all animals received ain93 m diet two weeks before the beginning of the study. the ain93 m diet composition (per 1 kg of diet) was 140 g of protein as casein lactate (iranian caseinate industry, iran) and 1.8 g as l - cystein (w326305, sigma aldrich, germany), 40 g of lipid as soy oil or evoo (kamzit company, iran), 630 g of carbohydrate as corn starch (corn dextrin from corn refinery, iran) and 100 g as sugar (local confectionery, iran), 35 g of ain93 g mineral mix (296040002, mp biomedicals, usa), 10 g of ain93 vitamin mix (296040201, mp biomedicals, usa), 2.5 g of choline bitartrate (c1629, sigma aldrich, germany), 0.008 g as tert - butyl hydroquinone (112941, sigma aldrich, germany) and 50 g of fiber (wheat bran, iran). after vaginal plug confirmation, mothers were randomly assigned to four different dietary groups (n=10 in each group) as shown in table 1. for the control group, 16% soy oil diet (lso) was considered. to create an isocaloric diet (same kcal / g) ; the amount of dietary fiber all mothers were isocalorically fed with the same kcal/(g body weight) of the group that ate less (pair - fed model). in this model, the least amount of diet eaten by the groups is determined and given to the other group of animals housed under identical conditions the following day. composition of the experimental diets per 1 kg during the study (ain93 g diet) lso : low soy oil ; loo : low olive oil ; hso : high soy oil ; hoo : high olive oil ; evoo : extra virgin olive oil ; l - cys : l - cystein mothers were fed by the above - mentioned diets throughout the gestation and lactation periods. after labor, offspring were reduced to 2 pups in each cage through simple randomization and nursed by birth mothers and weaned on day 21. animals were weighed weekly on a calibrated balance scale (marte scale (ek-3000i, usa)) to record the weight change to the nearest 0.1 gram. mothers were sacrificed one day after offspring weaning, as well as pups at 6 weeks of life by 40 mg / kg ketamine and 8 mg / kg xylasine. one day after weaning, maternal fasting blood samples were collected in a tube without anticoagulant, but offspring followed the study until adolescence (6 weeks). at 6 weeks, fasting blood samples of the offspring were collected. the samples were centrifuged at 4000 g for 15 minutes and then the serum was separated and stored at -80 c until the biochemical analyses. for the mothers and offspring, blood levels of total cholesterol (tc), high - density lipoprotein (hdl), low - density lipoprotein (ldl), and triglycerides (tg) were measured photometrically in an automatic analyzer (cobas integra 400, roche, mannheim, germany). serum glucose was measured by an enzymatic method (pars azmoon kit, pars azmoon co., tehran, for the comparison of continuous variables between groups, one - way anova was performed with post - hoc test and data were expressed by meansd (standard deviation). to adjust for potentially confounding variables and to assess the interactions between different variables, this test was performed to assess the effect of type and amount of dietary oils on maternal weight gain, biochemical parameters, birth weight of offspring, as well as weight and biochemical parameters at adolescence. whenever the interaction was significant repeated measures anova test was performed to compare the weight gain trend in the offspring, followed by scheffe post - hoc test for pair - group comparison. statistical analyses were performed with the ibm spss package (version 20, ibm corporation). the experimental protocol was approved by the animal research committee of iran university of medical sciences, tehran, iran. all institutional and national guidelines for the care and use of laboratory animals were followed. eight - week - old inbred adult female c57bl/6 mice (211.5 g) were obtained from the razi vaccine and serum research institute, tehran, iran. each mouse was individually housed at 21 - 23 c and controlled humidity (505%) under a 12-h artificial light cycle (7 am to 7 pm). to adapt, all animals received ain93 m diet two weeks before the beginning of the study. the ain93 m diet composition (per 1 kg of diet) was 140 g of protein as casein lactate (iranian caseinate industry, iran) and 1.8 g as l - cystein (w326305, sigma aldrich, germany), 40 g of lipid as soy oil or evoo (kamzit company, iran), 630 g of carbohydrate as corn starch (corn dextrin from corn refinery, iran) and 100 g as sugar (local confectionery, iran), 35 g of ain93 g mineral mix (296040002, mp biomedicals, usa), 10 g of ain93 vitamin mix (296040201, mp biomedicals, usa), 2.5 g of choline bitartrate (c1629, sigma aldrich, germany), 0.008 g as tert - butyl hydroquinone (112941, sigma aldrich, germany) and 50 g of fiber (wheat bran, iran). after vaginal plug confirmation, mothers were randomly assigned to four different dietary groups (n=10 in each group) as shown in table 1. for the control group, 16% soy oil diet (lso) was considered. to create an isocaloric diet (same kcal / g) ; the amount of dietary fiber all mothers were isocalorically fed with the same kcal/(g body weight) of the group that ate less (pair - fed model). in this model, the least amount of diet eaten by the groups is determined and given to the other group of animals housed under identical conditions the following day. composition of the experimental diets per 1 kg during the study (ain93 g diet) lso : low soy oil ; loo : low olive oil ; hso : high soy oil ; hoo : high olive oil ; evoo : extra virgin olive oil ; l - cys : l - cystein mothers were fed by the above - mentioned diets throughout the gestation and lactation periods. after labor, offspring were reduced to 2 pups in each cage through simple randomization and nursed by birth mothers and weaned on day 21. animals were weighed weekly on a calibrated balance scale (marte scale (ek-3000i, usa)) to record the weight change to the nearest 0.1 gram. mothers were sacrificed one day after offspring weaning, as well as pups at 6 weeks of life by 40 mg / kg ketamine and 8 mg / kg xylasine. one day after weaning, maternal fasting blood samples were collected in a tube without anticoagulant, but offspring followed the study until adolescence (6 weeks). at 6 weeks, fasting blood samples of the offspring were collected. the samples were centrifuged at 4000 g for 15 minutes and then the serum was separated and stored at -80 c until the biochemical analyses. for the mothers and offspring, blood levels of total cholesterol (tc), high - density lipoprotein (hdl), low - density lipoprotein (ldl), and triglycerides (tg) were measured photometrically in an automatic analyzer (cobas integra 400, roche, mannheim, germany). serum glucose was measured by an enzymatic method (pars azmoon kit, pars azmoon co., tehran, for the comparison of continuous variables between groups, one - way anova was performed with post - hoc test and data were expressed by meansd (standard deviation). to adjust for potentially confounding variables and to assess the interactions between different variables, this test was performed to assess the effect of type and amount of dietary oils on maternal weight gain, biochemical parameters, birth weight of offspring, as well as weight and biochemical parameters at adolescence. whenever the interaction was significant, repeated measures anova test was performed to compare the weight gain trend in the offspring, followed by scheffe post - hoc test for pair - group comparison. statistical analyses were performed with the ibm spss package (version 20, ibm corporation). the effects of diets containing different amounts of evoo and/or soy oil on maternal body weight, serum lipid profile, and glucose were investigated. the results showed that the interaction between type and amount of dietary oil on maternal weight was not significant during the three weeks of gestation and lactation. the main effect of soy oil on maternal weight, adjusted for the amount of dietary oils, was significantly higher than evoo throughout the three weeks of gestation and lactation (p=0.002, p<0.001, p<0.001, p=0.001, p<0.001, and p<0.001, respectively). except for the first week of gestation, the main effect of 16% oil fed diets on maternal weight was significantly higher than the 45% oil fed diets (p<0.001, p<0.001, p=0.02, p=0.02, and p=0.01, respectively). as shown in figure 1, at the first week of gestation, maternal weight was significantly higher in the 45% than the 16% oil fed groups (p<0.001). 16% and 45% oil fed mothers mean of maternal weight during three weeks of gestation and three weeks of lactation (fed with soy oil and/or olive oil, 16% and/or 45% amounts, n=10 in each group). significant difference between soy oil and olive oil fed diets ; significant difference between the 16% and 45% oil fed mothers. the interaction between the type and amount of dietary oil on maternal biochemical parameters was not significant after lactation. the main effect of soy oil was significantly higher on serum tc, ldl and glucose levels (p<0.001, p=0.001, and p=0.002, respectively), but it was lower on hdl.c (p=0.023). serum tg was significantly higher in the 16% oil fed diets (p=0.001), but ldl.c was significantly higher in the 45% oil fed diets (p=0.009) (figure 2). mean of maternal serum glucose and lipid profile (fed with soy oil and/or olive oil, 16% and/or 45% amounts, n=10 in each group). significant difference between the soy oil and olive oil fed diets ; significant difference between the 16% and 45% oil fed mothers. the effects of maternal diet on the trend of weight gain and weight of female offspring at birth and adolescence were investigated. the results showed that the interaction between type and amount of dietary oil on birth weight of female offspring was not significant. the main effect of soy oil on birth weight, adjusted for the amount of dietary oils, was significantly higher than evoo (1.290.2 vs. 10.09) (p<0.001), but it was not significantly different between the offspring born to mothers fed with 16% and 45% oil (1.130.3 vs. 1.160.2) (p=0.63). the interaction between type and amount of dietary oils on weight was not significant at adolescence. the main effect of soy oil on weight was significantly higher than evoo at adolescence, adjusted for the amount of dietary oil (18.51.7 vs. 13.71.05) (p<0.001). adjusting for the type of dietary oils, the main effect of the amount of maternal dietary oil on the weight at adolescence was significantly higher in the low - fat than high - fat diet group (16.83.08 vs. 15.42.4) (p=0.001). scheffe post - hoc test showed that all pair - group comparisons for weight gain were significant (p<0.001) except for the offspring born to mothers fed with 16% and 45% olive oil (p=0.79). the results showed that the interaction between the type and amount of dietary oil on serum glucose, tc, and tg of offspring were significant (p<0.001, p<0.001, and p<0.001, respectively). serum glucose was significantly higher in the lso than loo (p<0.001), lso than hso (p<0.001), and loo than hoo (p<0.001) groups. serum tc was significantly higher in the lso than loo (p<0.001), hso than hoo (p<0.001), lso than hso (p<0.001), and loo than hoo (p<0.001) groups. serum tg was significantly higher in the lso than loo (p<0.001), hso than hoo (p<0.001), lso than hso (p=0.03), and loo than hoo (p<0.001) groups. the interaction between the type and amount of dietary oil on serum ldl / hdl ratio was not significant. the mean of serum ldl / hdl ratio was higher in the soy oil than evoo, adjusted for the amounts of dietary oils (2.83.9 vs. 2.21.3) (p=0.6). adjusting for the type of dietary oil, serum ldl / hdl ratio was higher in the 16% than 45% oil diet groups (3.33.9 vs. 1.70.69) (p=0.11). however, these differences were not significant. serum biochemical parameters in female offspring comparison of four dietary groups evaluated by one - way anova followed by scheffe post - hoc test ; significant difference between the lso and loo groups ; significant difference between the hso and hoo groups ; significant difference between the lso and hso groups ; significant difference between the loo and hoo groups ; lso : low soy oil diet ; loo : low olive oil diet ; hso : high soy oil diet ; hoo : high olive oil diet ; tc : total cholesterol ; tg : triglyceride ; values are reported as meansd (n=10 in each group) the effects of diets containing different amounts of evoo and/or soy oil on maternal body weight, serum lipid profile, and glucose were investigated. the results showed that the interaction between type and amount of dietary oil on maternal weight was not significant during the three weeks of gestation and lactation. the main effect of soy oil on maternal weight, adjusted for the amount of dietary oils, was significantly higher than evoo throughout the three weeks of gestation and lactation (p=0.002, p<0.001, p<0.001, p=0.001, p<0.001, and p<0.001, respectively). except for the first week of gestation, the main effect of 16% oil fed diets on maternal weight was significantly higher than the 45% oil fed diets (p<0.001, p<0.001, p=0.02, p=0.02, and p=0.01, respectively). as shown in figure 1, at the first week of gestation, maternal weight was significantly higher in the 45% than the 16% oil fed groups (p<0.001). 16% and 45% oil fed mothers mean of maternal weight during three weeks of gestation and three weeks of lactation (fed with soy oil and/or olive oil, 16% and/or 45% amounts, n=10 in each group). significant difference between soy oil and olive oil fed diets ; significant difference between the 16% and 45% oil fed mothers. the interaction between the type and amount of dietary oil on maternal biochemical parameters was not significant after lactation. the main effect of soy oil was significantly higher on serum tc, ldl and glucose levels (p<0.001, p=0.001, and p=0.002, respectively), but it was lower on hdl.c (p=0.023). serum tg was significantly higher in the 16% oil fed diets (p=0.001), but ldl.c was significantly higher in the 45% oil fed diets (p=0.009) (figure 2). mean of maternal serum glucose and lipid profile (fed with soy oil and/or olive oil, 16% and/or 45% amounts, n=10 in each group). significant difference between the soy oil and olive oil fed diets ; significant difference between the 16% and 45% oil fed mothers. the effects of maternal diet on the trend of weight gain and weight of female offspring at birth and adolescence were investigated. the results showed that the interaction between type and amount of dietary oil on birth weight of female offspring was not significant. the main effect of soy oil on birth weight, adjusted for the amount of dietary oils, was significantly higher than evoo (1.290.2 vs. 10.09) (p<0.001), but it was not significantly different between the offspring born to mothers fed with 16% and 45% oil (1.130.3 vs. 1.160.2) (p=0.63). the interaction between type and amount of dietary oils on weight was not significant at adolescence. the main effect of soy oil on weight was significantly higher than evoo at adolescence, adjusted for the amount of dietary oil (18.51.7 vs. 13.71.05) (p<0.001). adjusting for the type of dietary oils, the main effect of the amount of maternal dietary oil on the weight at adolescence was significantly higher in the low - fat than high - fat diet group (16.83.08 vs. 15.42.4) (p=0.001). scheffe post - hoc test showed that all pair - group comparisons for weight gain were significant (p<0.001) except for the offspring born to mothers fed with 16% and 45% olive oil (p=0.79). the results showed that the interaction between the type and amount of dietary oil on serum glucose, tc, and tg of offspring were significant (p<0.001, p<0.001, and p<0.001, respectively). serum glucose was significantly higher in the lso than loo (p<0.001), lso than hso (p<0.001), and loo than hoo (p<0.001) groups. serum tc was significantly higher in the lso than loo (p<0.001), hso than hoo (p<0.001), lso than hso (p<0.001), and loo than hoo (p<0.001) groups. serum tg was significantly higher in the lso than loo (p<0.001), hso than hoo (p<0.001), lso than hso (p=0.03), and loo than hoo (p<0.001) groups. the interaction between the type and amount of dietary oil on serum ldl / hdl ratio was not significant. the mean of serum ldl / hdl ratio was higher in the soy oil than evoo, adjusted for the amounts of dietary oils (2.83.9 vs. 2.21.3) (p=0.6). adjusting for the type of dietary oil, serum ldl / hdl ratio was higher in the 16% than 45% oil diet groups (3.33.9 vs. 1.70.69) (p=0.11). however, these differences were not significant. serum biochemical parameters in female offspring comparison of four dietary groups evaluated by one - way anova followed by scheffe post - hoc test ; significant difference between the lso and loo groups ; significant difference between the hso and hoo groups ; significant difference between the lso and hso groups ; significant difference between the loo and hoo groups ; lso : low soy oil diet ; loo : low olive oil diet ; hso : high soy oil diet ; hoo : high olive oil diet ; tc : total cholesterol ; tg : triglyceride ; values are reported as meansd (n=10 in each group) offspring chronic disease susceptibility is determined by maternal dietary status during gestation and/or lactation. to the best of our knowledge, this is the first animal study that has investigated the effects of different types and amounts of maternal dietary oil during gestation and lactation in an isocaloric diet ; with a focus on evoo and coupled with offspring post - weaning control diet on weight, fasting glucose, and lipid profile of female offspring at adolescence. several studies have reported that maternal high - fat diet during gestation and lactation leads to obesity and insulin resistance due to pancreatic remodeling, but no study to date has examined the effects of type and amount of maternal dietary oil during gestation and lactation in an isocaloric diet on offspring at adolescence. the present study showed that in isocaloric diet, maternal evoo consumption during gestation and lactation reduces weight, serum glucose, and lipid profile in the female offspring at adolescence. in addition, the weight, serum glucose and lipid profile were lower in the offspring born to mothers fed with a high - fat than low - fat diet. in this study, we had to increase the fiber content of high - fat diets in order to create an isocaloric diet. therefore, the best effects of these diets on the variables could be due to the high - fiber content. although the prevalence of obesity is increasing, but no recent changes in the amount of fat consumption have been observed. while high - fat intake has been shown to play a role in promoting adipose tissue, the findings from carefully controlled weight - gain and weight - loss trials, comparing low-, moderate- and high - fat diets, have suggested that the amount of fat in the diet is less important than the type of fat consumed. a rapidly growing literature indicates that, in western - style diet, an increase in dietary omega-6 is due to increased consumption of vegetable oil (e.g., soy, sunflower, and corn). based on the currently available evidence, high levels of n-6 fatty acids in a diet lead to the elevation of blood lipid profiles, insulin resistance, prothrombosis, blood viscosity, vasospasm, and vasoconstriction. recent studies revealed that maternal high - fat diet could modulate the expression of hepatic fatty acid -oxidation - related genes. besides similar to these findings, we also showed that maternal diet containing low or high amounts of soy oil lead to obesity and lipid profile disturbances in offspring at adolescence in comparison to a diet containing evoo. hydroxytyrosol and oleuropein as phenolic components of olive oil affect fasting glucose and lipid serum levels. human studies have shown that the administration of a diet supplemented with olive leaf polyphenols (51.1 mg ol, 9.7 mg ht per day) ameliorated insulin action and secretion that regulate glucose homeostasis.. phenolic components of evoo lead to such benefits. in a study with high - sucrose and high - fat - sucrose diet, a similar effect on weight gain and adipose deposition has also been found after isocalorically feeding in wistar rats. however, a pair - fed model with high - fat feeding in c57bl/6j mice did not produce statistically significant changes in body weight gain. these results indicate that not only the amount of calorie but also the macronutrient composition of a diet may lead to obesity. inconclusive results are due to different compositions of the diets and the type of oils in all studies. in addition, none of the mentioned pair - fed studies were done during critical periods of life. our study differs from previous pair - fed studies in terms of the type and amount of dietary oil and the time of intervention. in the present study, we have evaluated the effect of isocaloric diets with different amounts of carbohydrate - to - fat ratio (1:1, 2:1, or 3:1) on body size and body composition in growing wistar rats. it is stated that the 3:1 and 2:1 diets increase the weight and progressively decreased body fat composition. this is in agreement with our results whereby dietary substitution of fat with carbohydrates led to a higher weight at adolescence in low - fat and high - carbohydrate (lso and loo) diets. the weight of offspring in both groups of evoo fed diet was lower than soy oil diet. studies have shown that omega-6 fatty acids lead to an increase in fat mass, resulting in insulin resistance and elevation in serum lipid profile related to obesity. however, omega-9 fatty acids activate the peroxisome proliferator - activated receptor (ppar) and reduce the activation of the sterol regulatory element - binding proteins (srebps), then inhibit lipogenesis and reduce insulin resistance. in another study, a diet containing 7% sunflower oil (n-6 diet), linseed oil (n-3 diet), or soybean oil (n-6/n-3 diet) during pregnancy and lactation were compared. maternal consumption of omega-6 increased inguinal weight relative to body weight and leptin levels in the offspring. the weight of offspring was higher in soy oil than the evoo fed group. in a study by xiaoling li, the effects of high - fat diet (hf) and maternal obesity on the metabolic programming of liver in offspring were assessed. it was shown that maternal high - fat diet leads to suppression of glucose and lipid metabolism - related genes in offspring. maternal obesity and/or high - calorie and high - fat diets were assessed, whereas we compared isocaloric diets with high - fat and/or high - carbohydrate components with different oils. in our study, high - carbohydrate - low - fat diet impaired glucose homeostasis and lipid profile in offspring at adolescence. used maternal diet supplemented with 10% evoo (rich in mufa) in the first or second half of gestation in piglets. in their study, mufa reduced the incidence of low birth weight. showed that maternal olive oil supplementation decreased body weight gain through an increase in uncoupling protein-1 protein level in brown adipose tissue of offspring at weaning. they proposed that oleic acid prevents the development of obesity and steatosis by stimulating thermogenic capacity. similar to priego and colleagues, the results of our study showed that the weight of offspring in evoo groups was significantly lower than soy oil groups ; both at birth and at adolescence. one of the limitations of our study was apparently the amount of fiber in different groups. the fiber content of the low - fat diet was lower than high - fat diet increating isoenergitic values. therefore, some benefits of high - fat diet versus low - fat diet may be due to fiber content. based on our results, a maternal diet containing evoo has better effects on birth weight as well as weight and serum biochemical parameters of offspring at adolescence. maternal high - fat diet reduced weight, serum glucose and lipid profile in offspring at adolescence. one of the main limitations of our study was the fact that weight and lipid profile were measured only at adolescence. additionally, while periodic measures from birth until aging are suggested, we did not directly measure whether maternal dietary with different types and amounts of oils induced epigenetic changes in metabolism. in future studies, direct assessment of epigenetic changes such as histone modification or dna methylation will allow us to examine the hypothesis that perinatal programming of the weight and lipid profile occur via epigenetic mechanisms. a free - fed group along with pair - fed groups is recommended in future studies. since the present study was an animal model investigation, the effects of the diets can not be directly transposed to humans. rather, it provides indications of possible effects of such dietary manipulations on the metabolism and may give better insights for future research regarding the underlying mechanisms. in general, maternal dietary macronutrient components are the most determinant of obesity in the next generation. offspring of high - fat and low - carbohydrate fed mothers had a lower weight than those with low - fat and high - carbohydrate. a maternal diet containing evoo decreased weight and improved serum glucose and lipid profile in female offspring at adolescence.	background : health status of offspring is programmed by maternal diet throughout gestation and lactation. the present study investigates the lasting effects of maternal supplementation with different amounts of soy oil or extra virgin olive oil (evoo) on weight and biochemical parameters during gestation and lactation of female mice offspring.methods:eight weeks old female c57bl/6 mice (n=40) were assigned through simple randomization into four isocaloric dietary groups (16% of calories as soy oil (lso) or evoo (loo) and 45% of calories as soy oil (hso) or evoo (hoo)) during three weeks of gestation and lactation. after weaning (at 3 weeks), all offspring received a diet containing 16% of calories as soy oil and were sacrificed at 6 weeks. two - way anova was used to adjust for confounding variables and repeated measures test for weight gain trend. statistical analyses were performed with the ibm spss package.results:at birth and adolescence, the weight of offspring was significantly higher in the soy oil than the olive oil groups (p<0.001 and p<0.001, respectively). adolescence weight was significantly higher in the offspring born to mothers fed with 16% oil than those with 45% oil (p=0.001). serum glucose, triglyceride and total cholesterol were significantly higher in the lso than loo (p<0.001, p<0.001 and p<0.001), lso than hso (p<0.001, p=0.03 and p<0.001), and loo than hoo (p<0.001, p<0.001 and p<0.001) dietary groups, respectively. serum triglyceride and total cholesterol were significantly higher in the offspring of hso than hoo fed mothers (p<0.001 and p<0.001, respectively).conclusion : a maternal diet containing evoo has better effects on birth weight, as well as weight and serum biochemical parameters in offspring at adolescence.
hereditary hypotrichosis simplex of the scalp (hss) is a rare autosomal dominant genotrichosis characterized by a hair defect confined to the scalp in the absence of other ectodermal or systemic abnormalities. hereditary hypotrichosis simplex of the scalp (hss) is a rare autosomal dominant genotrichosis characterized by a hair defect confined to the scalp in the absence of other ectodermal or systemic abnormalities. a 26-year - old girl referred to our dermatology clinic because of sparse and short hair. she had normal hair at birth, but hair loss began when she was four months old. she had short hairs of varying length, its length, never exceeds 7 cm. on physical examination, she had normal physical and mental development with no neurological impairment. in addition, her scalp hair was sparse and fragile with no signs of cicatricial alopecia [figure 1 ]. sparse and fragile hair with no sign of cicatricial alopecia eyebrows and eyelashes, nails, dental, eyes and sweat glands were normal. the following investigation : blood count, erythrocyte sedimentation rate, liver, thyroid and renal function tests, serum electrolytes, urinalysis, blood glucose, the glucose tolerance test, hb a1c, copper, zinc, and ferritin. trichogram of the patient 's hair show multiple abnormalities : distorted hair bulbs, ruffling and fraying of hair cortex with fracturing of hair shaft, resembling trichorrhexis nodosa [figure 2 ]. distorted hair bulbs, ruffling and fraying of hair cortex with fracturing of hair shaft a scalp biopsy show marked decrease number of hair follicles ; most of them are in anagen phase, accompanied by mild chronic predominantly lymphocytic perifollicular infiltration, consistent with hypotrichosis simplex.. marked decrease number of hair follicles, most of them are in anagen phase, accompanied by mild chronic predominantly lymphocytic perifollicular infiltration, consistent with hypotrichosis simplex hereditary hypotrichosis simplex of the scalp (hss) is a rare autosomal dominant genotrichosis characterized by a hair defect confined to the scalp in the absence of other ectodermal or systemic abnormalities. usually, patients with the (hss) present with normal hair at birth and in the first years of life. they experience a progressive, gradual loss of hair that is limited to the scalp, beginning at the middle of the first decade and leading to almost complete loss of scalp hair by the third decade. a few sparse, fine the body hair, beard, eyebrows, axillary hair, teeth, and nails are normally developed. scalp skin biopsy revealed a decreased number of follicles, especially in the telogen phase, with no specific pattern and no structural changes. before, structural hair defects may be presented with a genetic disorder affecting hair growth or part of a congenital syndrome or may indicate underlying metabolic disorders, or may be associated with other diseases. abnormalities of the hair shaft are varied and often confusing and can be congenital or acquired. they do require accurate recognition, which can be helpful in the diagnosis and management of a hair disorder, or in the detection of underlying disease. trichorrhexis nodosa is a common shaft defect affecting scalp hair, in which there is a distinctive response to injury. the hair is fragile and on examination regularly, spaced pale node - like swellings may be observed. it may affect normal hair following excessive or repeated trauma, or may occur after minimal trauma if there is an inherent defect in keratin synthesis causing abnormally brittle hair. blume - peytavi u,. suggested the triad of hypotrichosis, structural hair - shaft defects (dysplastic and broken hair shafts as seen in pili torti, trichorrhexis nodosa and pseudomonilethrix), and abnormal amino - acid composition (hypercysteine hair), associated with glucosuria without diabetes, may represent a new genetic syndrome due to an enzyme defect or deficiency. in 2004, a novel hidrotic ectodermal dysplasia (hypotrichosis, trichorrhexis nodosa and nail dystrophy) in a 3-year - old girl was reported. we reported a 26-year - old iranian girl affected by hss associated with trichorrhexis nodosa with history of the same disease in six members of her family. investigation suggested mutations in lipase h (liph) were responsible for autosomal recessive hypotrichosis simplex with woolly hair in three families. human hair as a key phenotypic marker can lead us to the underlying metabolic or genetic syndromes. the finding of hypotrichosis simplex of the scalp, associated with structural hair - shaft defects in seven adults of one family who were otherwise healthy, in the latest generation may represent a new genetic syndrome. the finding of hypotrichosis simplex of the scalp, associated with structural hair - shaft defects in seven adults of one family who were otherwise healthy, in the latest generation may represent a new genetic syndrome.	hereditary hypotrichosis simplex of the scalp is a genetic disorder, characterized by sparse or absent scalp hair without structural defects, in the absence of other ectodermal or systemic abnormalities. structural hair defects may be presented with a genetic disorder affecting hair growth or part of a congenital syndrome or may indicate underlying metabolic disorders, or may be associated with other diseases. we describe a 26-years - old persian girl suffering from hypotrichosis simplex of the scalp with trichorrhexis nodosa who had no ectodermal defects and systemic disease.
pediatric tissue engineering is subjected to special requirements : the scaffolds need to be suitable for and adapted to mechanical and biological changes during childhood. as recently described, different types of scaffold materials such as hydrogels, synthetic, or natural scaffolds have already been studied in tissue engineering, for example, long - gap esophageal atresia [25 ]. in general, decellularized extracellular matrix (ecm) scaffolds can easily be obtained from donors (humans or animals). all cellular components can be removed from the scaffolds by the use of chemical, biological, or mechanical methods and allowing an off - the - shelf production. ecm scaffolds are rich in structural proteins and have an intact three - dimensional structure. by removing cells and cellular antigens, the ecm scaffolds are obviously biocompatible and are thought not to provoke a chronic rejection reaction after implantation into another species. however, ecm proteins can also provide costimulatory signals to immune cells [712 ]. the degree of inflammation or remodeling is strongly dependent on the scaffold material and chemical treatment, so - called cross - linking [6, 7, 1316 ]. it is supposed that native scaffolds are subjected to fast enzymatic degradation in the recipient 's body, accompanied by the loss of mechanical properties. on the one hand, degradation is necessary for the development of a constructive remodeling process. however, on the other hand, after scaffold implantation in vivo, it needs to maintain its mechanical properties and the scaffold should be used as a matrix for reseeding by different tissue - typical cells. after successful in - growth by, for example, muscle cells or fibroblasts, remodeling processes in the scaffold will occur according to the new biological environment (e.g., the subcutaneous region). a compromise has to be effected to lower the rate of degradation (without completely blocking), downregulate inflammation, and to increase the rate of cellular infiltration. both depend strongly on the biological properties of the scaffold. a common approach for increasing the mechanical strength, inhibiting inflammatory processes, and decreasing the rate of degradation of biologic scaffolds is the use of chemical cross - linking agents. the common principle of cross - linking is based on the presumption that free amino (nh2), carboxyl (cooh), and hydroxyl groups (oh) on collagen may have antigenic effects. these free groups were cross - linked and masked with appropriate chemical reagents such as glutaraldehyde, genipin, or carbodiimides. some cross - linking agents are toxic (e.g., formalin) or tend to calcificate the implant (e.g., glutaraldehyde (ga)) ; others have been described to be well tolerated (e.g., genipin (gp)) or to be metabolized residue - free in the cross - linking reaction (e.g., carbodiimide (cdi)) [1719 ]. in the present study, we investigated in an animal model whether an acellular esophagus provokes an inflammatory response, rejection or is well tolerated. in detail, we implanted small circular pieces of esophagus scaffolds into rats subcutaneously. chemically pre - treated scaffolds were cross - linked either with ga, gp, or cdi. as control, commercially available bovine pericardium (bp ; st. furthermore, the polarization of macrophages is important to the remodeling outcome and was therefore investigated. m1-activated macrophages express il-12, il-23, il-10 and produce inflammatory cytokines such as il-1, il-6, and tnf-, which promote active inflammation. they are inducer and effector cells in th1-type inflammatory responses and express ccr7 as a surface cell marker [6, 21 ]. in contrast, m2-activated macrophages have an il-12, il-23 and il10 phenotype and are able to facilitate tissue repair and constructive remodeling [20, 22, 23 ]. m2 macrophages predominantly induce the th2 response, which is particularly beneficial for the constructive tissue remodeling. in addition, macrophages of anti - inflammatory phenotype inhibit proinflammatory cytokines and express the surface marker cd163 [15, 20, 24, 25 ]. macrophages are able to change their polarization in response to local stimuli during the process of wound healing. the recognition of the predominant phenotype of macrophages provides an indication of scaffold rejection, inflammation or acceptance after implantation. however, the anti - inflammatory phenotype may be simultaneously detected with the general macrophage marker cd68 by immunohistochemical methods. furthermore, the general macrophage marker cd68 also stains pro - inflammatory and not activated or polarized macrophages. therefore, the cd163/cd68 ratio can be used to calculate the amount of m2 macrophages over time. thus, the objective of this work was to characterize the host response to different cross - linked ecm esophageal scaffolds, in particular aspects of inflammation or rejection and the macrophage polarization to clarify the most suitable cross - linking agent for the integration of ecm esophageal scaffolds into its surrounding tissue after implantation. all experiments were performed with esophagi of pigs (deutsche landrasse, 2565 kg) in cooperation with the heart center leipzig, department of cardiac surgery. the organs were obtained under sterile conditions and stored at 4c in a 0.9% nacl solution. for decellularization, esophagi were cut into pieces of 8 cm length, and the tunica adventitia was removed mechanically. esophagi were then placed in a 5% sodium dodecyl sulfate - solution (sds ; roth, karlsruhe, germany) for 7 days. after complete removal of all cells, the scaffolds were washed in phosphate buffered saline (pbs) for 2 days. after finishing the decellularization, the tissue was digested enzymatically by adding dnase (200 g / ml ; sigma, deisenhofen, germany) in pbs + mgcl2 (50 mm) and incubated in 37c for 12 h. after washing in pbs, the scaffolds were sterilized by gamma radiation (25 kgy from a co source) and stored in pbs at 4c for maximum 4 weeks. following decellularization and after explantation at day 1, 9, and 30 postimplantation, representative areas were embedded in paraffin wax, cut into slices (5 m thickness), and routinely stained by azan and he staining. in azan stained slides, the matrix morphology of decellularized porcine scaffolds was compared to that of native esophagi using light microscopy. the assessment of he - stained neutrophils, fibroblasts, giant cells, and microvessels were performed by light microscopy on explanted scaffolds. cells were scored as follows : 0 = no cells ; 1 = 1100 cells ; 2 100200 cells ; 3 200 cells/5 mm. immunohistochemical analysis of decellularized esophagus was performed with a commercially available envision dab staining kit (dako, carpinteria, usa). briefly, formalin - fixed, paraffin - embedded esophagus tissue sections of 5 m thickness were deparaffinized. subsequently, slices were heated in 50 mm tris buffered saline solution at 95c for 15 min. after cooling, the slides were incubated with proteinase k (250 g / ml) for 10 min and washed in distilled water. endogenous enzyme activity was blocked (10 min, dako staining kit), and the tissue slides were incubated with primary antibodies. the staining steps with anti - collagen iii (acris antibodies, herford, germany), anti - collagen iv (acris antibodies), anti - fibronectin (dianova, berlin, germany), and anti - elastin (acris antibodies) were performed following the manufactures instruction (envision dab staining kit, dako). in stained slices, extracellular matrix composition of decellularized and native scaffolds was investigated by light microscopy. to investigate cellular infiltration in explanted scaffolds, the following antibodies were used : anti - cd3, anti - cd68, anti - cd163 (all serotec, oxford, uk), and anti - hydroxyl - prolyl - hydrogenase (hph, bma biomedical, augst, switzerland). control experiments were carried out without primary antibodies. to visualize the nuclei, all slices were counterstained with mayer 's hemalun solution. in stained slices, from each section, the cd3, cd68, and cd163 positive cells as well as nuclei were counted in 3 5 microscopic fields by two blinded observers (magnification 1000). the data are represented as a ratio of cd - positive cells / nuclei (mean standard error of mean (sem)). the isolation and quantification of dna in the decellularized tissue scaffolds was performed using the protocol of qiagen (dneasy, hilden, germany). in brief, decellularized esophagus matrix scaffolds were cut into small cross - sectional pieces of 25 mg. lysis buffer and proteinase k (qiagen, hilden, germany) were added, and samples were incubated overnight in a shaking water bath (56c). after successful tissue lysis, the dna was purified and measured spectrophotometrically using a nanodrop spectrophotometer (peqlab, erlangen, germany). the dna - content of matrix scaffolds undergoing enzymatic digestion was compared to matrix scaffolds without enzymatic digestion (both n = 6). native esophagus tissue served as positive control (n = 6). for carbodiimide cross - linking, circular pieces of esophagus scaffolds (3 mm thickness) were immersed in 2-(n - morpholino)ethanesulfonic acid buffer (mes buffer ; 0.2 m, ph 5.0 ; sigma, munich, germany). after 1 h, the mes buffer was discarded and the scaffolds were incubated in a solution consisting of mes buffer (0.2 m, ph 5.0), n - hydroxysuccinimide (nhs ; 0.12 m), and n-(3-dimethylaminopropyl)-n - ethylcarbodiimide (edc ; 0.3 m). after 16 h, the scaffolds were removed and rinsed in mes buffer for 24 h and in pbs for at least 24 h [17, 29 ]. for gp cross - linking, the scaffolds were incubated in a 0.33% genipin / ethanol solution (alexis, lausen, switzerland) for 3 days at 37c. then the scaffolds were removed and rinsed in 75% ethanol for 2 h and in pbs for 3 days [21, 30, 31 ]. for glutaraldehyde cross - linking, the scaffolds were immersed in 0.625% glutaraldehyde / distilled water (sigma) for 3 days at 37c. 60 sprague - dawley rats were grouped according to scaffold cross - linking : untreated, ga, gp, cdi, bp, and sham group (each treatment group : n = 9 ; sham group : n = 15 ; for details see table 1). animals of the sham group underwent the same surgical procedure but received no implants. before subcutaneous implantation of scaffolds, the animals were anesthetized with 0.15 mg / kg medetomidin (pfizer, berlin, germany), 2 mg / kg midazolam (ratiopharm, ulm, germany), and 0.005 mg / kg fentanyl (janssen - cilag, neuss, germany). after 15 min, circular pieces of cell - free esophagus scaffolds (3 mm thickness) were implanted into a subcutaneous back pocket of the rat (1 cm length). finally, the anesthesia was antagonized with 0.75 mg / kg atipamezol (pfizer), 0.2 mg / kg flumazenil (roche, penzberg, germany), and 0.12 mg / kg naloxon (ratiopharm). the rats were given carprofen (5 mg / kg s.c, pfizer) postoperative for 3 days. after postoperative care for 1, 9, and 30 days, the animals were narcotized, euthanized, and the scaffolds were explanted. tissue was immersed in 4% paraformaldehyde solution and embedded in paraffin for further histological investigations (see above). all procedures were approved by the committee of animal care and use of the relevant local governmental body (tvv03/09) in accordance to the law of experimental animal protection. the statistical evaluation of the immunohistology was performed by kruskal - wallis with post hoc mann - u - whitney. effect of decellularization on the dna - content was evaluated using a one - way anova, followed by post hoc tukey test for pairwise comparisons. furthermore, the lower and upper 95% confidence intervals (ci) were measured for the calculation of an area, where the true value is of a probability of at least 95%. differences were considered significant if the lower and the upper ci among two groups did not overlap. to investigate whether the ecm composition of the decellularized esophagus scaffold (figure 1(a) ; top) is similar to native esophagus tissue (figure 1(a) ; bottom), both tissues were histologically and immunohistologically characterized. azan staining of natural (figure 1(b)) and acellular esophagus scaffolds (figure 1(c)) revealed anatomical intact structures, optimal matrix geometry, and no remaining cellular structures. as demonstrated in figure 2, collagen iii (figure 2(a)) and fibronectin (figure 2(b)) could be observed in large amounts in all tissue areas (submucosa, muscular layer) in acellular similar to natural esophagi (small pictures, figures 2(a)2(d)). the vessels expressed collagen iv (figure 2(c)) and elastin, which is additionally located in tela submucosa (figure 2(d)). a one - way anova revealed a significant effect of decellularization on the dna - content compared to controls [f(2,15) = 15.918 ; p < 0.001 ]. the remaining dna in the decellularized matrix scaffolds undergoing dna digestion was 8.05 2.01 ng / mg versus 756.96 49.07 the dna content of decellularized matrix scaffolds without nucleic acid digestion was 10.04 3.01 ng / mg (n = 6), see figure 3. the post hoc comparison did not reveal significant differences in the remaining dna content of decellularized scaffolds with and without dna digestion (p = 0.757). the percentage of remaining dna content after both procedures was decreased by about 99% compared with native esophagus tissue (p < 0.001 ; without digestion : 98.7 0.4%, respectively, with digestion : 98.9 0.3%). a set of representative pictures of cell infiltration into untreated (figures 4(a)4(c)) and cross - linked scaffolds (gp, figures 4(d)4(f)) is shown in figure 4. at day 1 postimplantation, untreated scaffolds (figure 4(a)) displayed a cellular infiltration from the periphery to the center of the tissue. in gp cross - linked scaffolds (figure 4(d)), only a cellular layer was detectable at the periphery. at day 9 postimplantation, an increase of infiltrating cells into untreated scaffolds (figure 4(b)) was observed compared to gp cross - linked (figure 4(e)) scaffolds. at day 30 postimplantation, whole implants of the untreated scaffold group (figure 4(c)) were infiltrated with cells, whereas gp scaffolds (figure 4(f)) showed only an immaterial cellular infiltration. furthermore, in all cross - linked groups (gp, ga, cdi, bp) the infiltrating rate was decreased compared to the untreated scaffold group. in addition, untreated scaffolds were degraded largely compared to cross - linked scaffolds at day 30 postimplantation. furthermore, all scaffolds were infiltrated with granulocytes migrating from the periphery to the central region at day 1 postimplantation, without detecting any differences among the groups. they also migrated from the periphery to the center of the tissue. in terms of chronic - granulating inflammation, no significant differences were detected. at day 30, scaffolds were infiltrated by fibroblasts and sporadically by lymphocytes or granulocytes. in terms of chronic - granulating inflammation, collagen fibers and macrophages were increased accompanied by decreased amount of capillaries. all data are summarized in figure 5. for detailed analysis of infiltration of macrophages, lymphocytes, and fibroblasts, scaffold slides were stained immunohistologically with anti - cd3, anti - cd68, anti - cd163, and anti - hph antibodies. for estimating the effects, the data of bp, gp, ga, and cdi were compared to those of the untreated group. at day 1, no cd3-, cd163-, or hph - positive cells were detected in any scaffold. scaffold analysis at day 9 revealed a significantly lower number of cd163 macrophages in bp, whereas at day 30 in bp, gp, ga, and cdi cd163 macrophages were significantly decreased as compared to the untreated group (figure 6(a)). also, at day 9 only in the bp group cd68-positive cells were observed to a significantly lesser extent. however, at day 30, cd68-positive cells were also significantly decreased in the gp group (figure 6(b)). additionally, in most groups the amount of anti - inflammatory, proremodeling macrophage m2 phenotype increased from day 9 to day 30, indicated by a higher cd163/cd68 ratio (untreated : + 26.88% ; bp : + 82.14% ; gp : + 23.51%), except the ga (28.65%) and cdi (74.79%) group (figure 6(c)). analysis of cd3-positive lymphocytes reveals in all groups only a marginal infiltration into the scaffold. the percentage was less than 0.5%, and no significant differences between the groups were detected (not shown). immunohistological hph staining demonstrated a tendency to increase in number of fibroblasts in the ga and gp groups at day 9. at day 30, in the gp and cdi group, the numbers of fibroblasts were significantly increased compared to the untreated group (figure 6(d)). these scaffolds have the advantage of possessing intact structural proteins and growth factors that reduce inflammatory responses [3336 ]. the response of the host to several xenogenic ecm scaffolds has been well characterized and understood ; however, it is only partially studied in animal models for esophageal cross - linked ecm scaffolds [713, 22, 24, 25, 3541 ]. additionally, previously published data suggest that the host response to the ecm scaffolds is strongly dependent on the species and chemical pretreatment [15, 42 ] thus, it is essential to find out the host response after subcutaneous implantation of porcine esophagus scaffolds. furthermore, the outcome of the host response after implantation of different cross - linked ecm scaffolds was studied. since the scaffolds were decellularized and morphologically intact, we assume that a chronic rejection will not occur. this is not common, because in studies with small intestinal submucosa (sis)-ecm t - lymphocyte infiltrations, encapsulations and necrosis were observed as signs of rejection [13, 14 ]. in the present study, only in one animal, encapsulation was observed at day 9 postimplantation. none of the used scaffolds showed signs of necrosis or t - cell infiltration (cd3 0.5%). signs of inflammation were granulocyte infiltration at day 1, encapsulation by macrophages and fibroblasts at day 9, and scaffold infiltration of macrophages and fibroblasts at day 30 postimplantation. this is characteristic for structural remodeling processes such as scarring after foreign body implantation and was consistent with our expectations. we do not assume that dna remnants of the scaffolds were the cause of inflammatory reactions in our experiment, though in the recent literature such a reaction was described. in most biological material, remaining dna consisted of fragments less than 300 bp. the remnant dna is subject to fast enzymatic degradation in vivo [15, 43 ]. instead of remnant dna as cause of inflammation, it is more plausible that free amino (nh2), carboxyl (cooh), and hydroxyl (oh) groups of the remaining collagen scaffold may be responsible for the immunological reactions [18, 44 ]. to avoid or inhibit such reactions, these free groups can be bound by functional groups of chemical cross - linking, which precludes the antigenic properties of collagen [17, 18, 45 ]. in the present study, the scaffolds were pretreated by three different cross - linking agents : cdi, ga, and gp. the cross - linkers were selected in accordance with descriptions of the biological compatibility in the recent literature [18, 34 ]. an alternative to the use of ga and gp may be the application of cdi. first, cdi reacts with the free carboxyl- followed by the amine groups of collagen, generating the cross - link and representing a treatment method without residual chemicals [17, 18 ]. by fixation with cdi, tissue quality was improved, the grade of calcification was lowered (compared to ga), and no toxicity was observed. cdi was also used as a cross - linking agent in commercially available tissue products. nevertheless, in the present study a fixation of the esophagus scaffolds with cdi did not indicate the expected results. unfortunately, in the cdi group, the cd163/cd68 ratio was decreased at day 30 postimplantation, indicating a switch to a proinflammatory and destructive m1 macrophage phenotype. in contrast to ga and gp fixed tissue as well as to untreated scaffolds, we observed a chronic inflammation, which was maintained and increased after the cdi cross - link. interestingly, other studies described similar effects in cdi - fixed sis - ecm tissue [15, 46 ]. after implantation of cdi - fixed sis - ecm, badylak and colleagues could also observe a predominant m1 macrophage phenotype, characterized by chronic inflammation at week 16 postimplantation. furthermore, it is reported that cdi cross - linking caused a decrease in elasticity and mechanical toughness, which is essential for esophageal function. on the basis of these data, cdi can not be considered as an optimal cross - linker in the present study. ga is the most common cross - linking agent and often used in commercially available tissues. tissue that is cross - linked with ga exhibits a decreased immunological reaction and stabilized collagen scaffold [41, 48, 49 ]. we also observed that the immunological response in ga cross - linked esophagus scaffolds was lower in comparison to chemically untreated scaffolds. at day 30 postimplantation, the rate of cd163 macrophages was decreased, but the total number of macrophages (cd68) remained constant [15, 20, 24, 40 ]. interestingly, in contrast to gp, bp, and the untreated scaffold group, cd163/cd68 ratio was decreased at day 30 postimplantation. however, the suppression of immunological actions of xenografts by ga is not complete ; cellular toxicity and host immune response (cytotoxic t - cell activation) have been described [21, 35, 37, 45 ]. furthermore, cytotoxicity of ga cross - linked bioprostheses for host fibrocytes, fibroblasts, and macrophages has been described [50, 51 ]. in the present study, we did not observe any cytotoxic effect of ga cross - linked esophagus scaffolds on host fibroblasts or macrophages compared to the untreated group. however, the use of ga as a cross - linker to reduce the inflammatory response comprises further disadvantages. depolymerization of ga cross - links as well as calcification of implants has been reported. however, in our study, we did not find signs of calcification and the process of depolymerization was not investigated. the sum of our data suggests that ga was not an optimal cross - linker for constructive remodeling in the present study (e.g., cd68+macrophage infiltration), which corresponds with other disadvantages described in the literature [35, 37, 50, 51 ]. it reacts with collagen amino groups and is approximately 10.000 times less cytotoxic than ga [21, 34, 45 ]. it forms stabile cross - linking products, reacts antiphlogistically, protects against inflammatory degradation, and causes faster tissue regeneration in comparison to ga [30, 39, 52 ]. an in vitro degradation assay demonstrated that gp - fixed acellular scaffold tissue kept burst pressures equivalent to ga cross - linked scaffold tissue. furthermore, recent literature showed that proliferative capacity of infiltrated cells was greater than that after ga cross - linking [31, 34 ]. in in vivo experiments in dogs, decreased inflammation around implanted gp - fixed acellular arteries was observed compared to ga - fixed implants ; however, the degree of inflammation has not been clearly described. signs of calcification were not detected in gp - fixed tissue [19, 45 ]. these results correspond to findings of our studies. in an in vivo experiment in rats, a minimal macrophage infiltration or localization outside untreated and treated scaffolds however, in a study that compared the host inflammatory response to subcutaneously implanted gp and ga cross - linked acellular bovine pericardia, the cross - linking with ga caused a significantly increased inflammatory response compared to untreated and gp cross - linked tissue scaffolds. we could show that macrophages were present in untreated and cross - linked scaffolds at day 1 postimplantation. however, at day 30 postimplantation, pretreatment with gp caused a significant decrease of infiltrating macrophages (compared to untreated scaffolds). moreover, immunohistochemical staining revealed that pan - macrophage marker cd68 were significantly decreased in gp, similar to the reference scaffold bp (compared to untreated scaffolds). in scaffolds treated with ga or cdi, a decrease of cd68 macrophages one might assume that gp prevented scaffolds from inflammation processes in contrast to cdi or ga or untreated scaffolds. in addition, we calculated the cd163/cd68 ratio to define the change in the amount of the proremodeling m2 macrophages over time. at day 30 postimplantation, we detected an m2 macrophage phenotype switch, which is known to be associated with constructive remodeling and tissue repair [20, 22, 23 ], whereas in ga and cdi cross - linked scaffolds a destructive m1 macrophage phenotype was detected. furthermore, gp - fixed scaffolds exhibited a moderate degradation, extremely low lymphocyte infiltration and a significantly increased fibroblast infiltration compared to untreated scaffolds. the presence of proremodeling macrophages and fibroblasts (which are some of the typical cells in the subcutaneous region) cells might be suggestive of host repair and constructive tissue remodeling [23, 53 ]. furthermore, the infiltration, survival, and living of the tissue - fibroblasts suggested a biocompatibility of gp scaffolds. the present subcutaneous rat model proved to be an appropriate experimental tool to investigate the influence of different cross - linking agents on host response (after implantation of porcine ecm scaffolds) and to identify the best tolerated cross - linking agent. signs of graft rejection such as encapsulation or lymphocyte infiltration did not occur in any group. however, differences in inflammatory processes and infiltration of cells such as macrophages or fibroblasts could be observed depending on scaffold pretreatment with different crosslinking agents. high rates of proinflammatory macrophages were detected in untreated, cdi, and ga scaffolds. this indicated inflammatory processes resulting in fast scaffold degradation and a low rate of integration of the scaffold in the surrounding tissue. in contrast, scaffolds treated with gp were only mildly infiltrated by macrophages (cd68), similar to the established reference standard bp. moreover, in gp, the proinflammatory cd163 macrophage phenotype was significantly decreased, lymphocytes were at the limit of detection, and the rate of subcutaneous tissue - typical fibroblasts was increased. this might suggest that gp was an agent that scaffolds prevented for graft rejection and is efficient to attenuate inflammatory processes. low rates of macrophages and infiltration of fibroblasts (in the subcutaneous region) in gp scaffolds imply a better scaffold tolerance and an improved integration of esophageal scaffolds into their surrounding tissue after implantation. this suggested the practicability of gp as a cross - linking agent for implants in clinical application. we consider gp an adequate compromise between the rate of degradation, inflammation, and the infiltration by tissue typical cells. the results of the present study help to provide a new piece of the puzzle in the development of esophageal xenografts. the use of acellular esophagus scaffolds could be an important therapeutic tool in the near future. the development of a large animal model seems to be the next step to prove the functionality and host response (constructive remodeling processes) after the implantation of segmental gp cross - linked ecm esophagus scaffolds in the esophageal location. if this approach is successful, there is a wide range of applications possible (e.g., esophagus atresia, esophageal trauma, long - segment barrett 's esophagus, etc.) to increase the quality of life of patients and minimize the complications in severe esophageal diseases.	most infants with long - gap esophageal atresia receive an esophageal replacement with tissue from stomach or colon, because the native esophagus is too short for true primary repair. tissue - engineered esophageal conducts could present an attractive alternative. in this paper, circular decellularized porcine esophageal scaffold tissues were implanted subcutaneously into sprague - dawley rats. depending on scaffold cross - linking with genipin, glutaraldehyde, and carbodiimide (untreated scaffolds : positive control ; bovine pericardium : gold standard), the number of infiltrating fibroblasts, lymphocytes, macrophages, giant cells, and capillaries was determined to quantify the host response after 1, 9, and 30 days. decellularized esophagus scaffolds were shown to maintain native matrix morphology and extracellular matrix composition. typical inflammatory reactions were observed in all implants ; however, the cellular infiltration was reduced in the genipin group. we conclude that genipin is the most efficient and best tolerated cross - linking agent to attenuate inflammation and to improve the integration of esophageal scaffolds into its surrounding tissue after implantation.
low flow anesthesia (fresh gas flow [fgf ] 1 l / min) is gaining in popularity because of its advantages (vis - - vis economy, less pollution, and conservation of heat and humidity)1 in addition to the availability of modern anesthetic machines and anesthetic gas monitors. desflurane, with its properties of low blood and fat solubility and no limitation of fgf even with older co2 absorbers, is most suitable for low flow anesthesia.2 low flow anesthesia needs an initial wash - in phase to build up the required concentration of desflurane in the circle circuit (fid) and alveoli (fad).3 most reported wash - in schemes need a very high fgf or vaporizer concentration of desflurane, yet achieve only some of the targeted fad.2,4,5 there is one scheme that can achieve every fad, but it is a complex logistic regression equation needing a computer to perform the calculation,6 so is inconvenient for everyday use. recently, the authors reported a simple, single step 1 - 1 - 12 wash - in scheme using an fgf of n2o : o2 1:1 l / min and desflurane 12%, that can rapidly yield every fad from 1% to 6% within 4 minutes without tachycardia.7 although n2o is widely used because of its desirable effects (eg, analgesia and additive effect to the minimum alveolar concentration [mac ] of desflurane), there are still some situations where n2o should not be used,811 which effectively limits the generalizability of this scheme. the objective of the present study was to evaluate the 1 - 1 - 12 wash - in scheme in situations where n2o is currently excluded. this study was approved by the institutional review board of khon kaen university (he561247) and was registered at www.clinicaltrials.gov (nct01348984). the sample size comprising 106 patients was determined in the same way as in our previous related study.7 we included patients with an american society of anesthesiologists physical status of i or ii, aged 1664 years, and scheduled for elective surgery under general anesthesia with endotracheal intubation and controlled ventilation. patients with pulmonary or cardiac disease, a body mass index > 30 kg / m, or a contraindication to use of succinylcholine were excluded. all patients were managed by standard intraoperative monitoring and care in the same way as in our previous study.7 they were monitored with electrocardiography, pulse oximetry, noninvasive blood pressure measurement, capnography, and anesthetic gas analyzing equipment. the anesthetic machine, integrated with an anesthetic gas analyzer, used in this study was a primus (drger ag, lbeck, germany). each patient s heart rate and blood pressure were recorded before induction as a baseline measurement. the patients were then premedicated with intravenous fentanyl 1 g / kg and induced with propofol 2 mg / kg. ventilation was controlled using an fgf of air : o2 1:1 l / min and desflurane 12%. the initial ventilator setting was at a tidal volume 7 ml / kg and a respiratory rate of 12 per minute adjusted to keep the paco2 around 3035 mmhg. the time to achieve an fad at 1%, 2%, 3%, 4%, 5%, 6%, and 7% was recorded as the primary outcome. after an fad of 7% was achieved, fgf was reduced to 0.51 l / min and desflurane adjusted according to the judgment of the anesthesiologist. the statistical analysis was performed using statistical package for the social sciences for windows version 16.0 software (spss inc, chicago, il, usa). continuous demographic data are presented as the mean standard deviation and the categorical data as the number of patients (percentage). the primary outcome is presented as the mean standard deviation and 95% confidence interval. the unpaired student s t - test was used to compare the time and fid to achieve each fad in this study, consistent with our previous study.7 heart rate and blood pressure values at different time points were compared using repeated measures analysis of variance. the statistical analysis was performed using statistical package for the social sciences for windows version 16.0 software (spss inc, chicago, il, usa). continuous demographic data are presented as the mean standard deviation and the categorical data as the number of patients (percentage). the primary outcome is presented as the mean standard deviation and 95% confidence interval. the unpaired student s t - test was used to compare the time and fid to achieve each fad in this study, consistent with our previous study.7 heart rate and blood pressure values at different time points were compared using repeated measures analysis of variance. in total, 106 patients participated in and completed the study. the demographic data for these patients are presented in table 1. the trajectories of time taken to reach each fad by each patient during wash - in are presented in figure 1. the times taken to achieve fad from 1% to 7% and the 95% confidence intervals are presented in table 2. an fad of 1%, 2%, 3%, 4%, 5%, 6%, and 7% can thus be expected at 0.6, 1, 1.5, 2, 3, 4, and 6 minutes, respectively. the rising pattern of fad and fid is similar to that observed in our previous study,7 except for some statistically but not clinically significant differences at 5%, 6% and 4%, 5%, 6%, respectively (figures 2 and 3). the rise in heart rate during wash - in was statistically but not clinically significant (figure 4). blood pressure decreased slightly ; this was statistically significant but not clinically so (figure 5). the pattern of the trajectories of time taken to achieve each fad in figure 1 reveals that this scheme has acceptable intrasubject and intersubject variability. although there are statistically significant differences in the increasing pattern of time and fid at some points of fad, these are in the order of a few seconds and not clinically significant. the times taken to achieve fad from 1% to 6% in this study (without n2o as the carrying gas) is the same as that reported in our previous study, which used n2o as a part of fgf.7 the results of the present study show that the performance of the 1 - 1 - 12 wash - in scheme is not affected by the use of n2o. the second gas effect of n2o12 does not influence the uptake of desflurane because both n2o and desflurane have nearly the same very low blood - gas solubility (0.47 versus 0.42). we extended the end point of fad in this study to 7%, which took 6 minutes to achieve, because without n2o, which has an additive effect on the mac of desflurane,13 a higher fad may be required to control the depth of anesthesia. the time required to increase fad from 6% to 7% was 2 minutes, which is double the time required to increase each 1% from 4% to 6%. this may be due to the narrower gradient between fid and fad at the high end of fad. our scheme requires less fgf, but can achieve each fad from 1% to 7% within 6 minutes, which is more rapid and simpler than most reported wash - in schemes. baum used an fgf of 4.4 l / min and found that fad reached 90%95% of the fresh gas concentration within 1015 minutes.2 mapleson reported a spreadsheet model comprising two components : a circle circuit and a 70 kg anesthetized standard man, using an fgf equal to the total ventilation with 3 mac of desflurane that could achieve an fad of one mac in 1 minute.4 hendrickx used an fgf of o2:n2o 2:4 l / min with desflurane 6.5% and found that an fad of 4.5% could be achieved in 15 minutes.5 the aforementioned schemes used higher fgfs yet achieved only some specific fads. hendrickx thus proposed an empirical model that could be used to predict fgf - desflurane combinations that achieved a target fad within the first 5 minutes.6 their model, however, is a complex logistic regression equation requiring a computer to calculate, making it impractical for daily use. in comparison, our scheme is simpler and more practical, and yields a rapid wash - in with an expected fad from 1% to 7% at 0.6, 1, 1.5, 2, 3, 4, and 6 minutes, respectively. this scheme can be applied with or without n2o. while nyktari reported that a rapid increase in fid to 1.5 mac without the support of premedication caused a significant increase in airway resistance, we did not find such a problem. the explanation is that, with a lower fgf, an fid of 1.5 mac was gradually achieved in 7 minutes and fentanyl was used as a premedication. in the current study, there was a statistically significant but not clinically increase in heart rate and decrease in blood pressure. this result is similar to our previous study using n2o as a carrying gas7 and also consistent with the report by warltier and pagel,15 but different from the study by ebert and muzi,16 who reported hypertension and tachycardia in healthy volunteers receiving titration of desflurane from 1 to 1.5 mac following thiopental induction. the explanation is that fentanyl, used as a premedication, attenuates sympathetic stimulation.17 moreover, the rapid increase in fid in our scheme (to the level exceeding the mac of desflurane before the therapeutic level of the induction agent is on the wane) maintains the patient in the surgical stage throughout the study without sympathetic overactivity. the 1 - 1 - 12 wash - in scheme has many advantages : simplicity (just one step for setting) ; coverage (every targeted fad from 1% to 6% [7% where n2o is omitted ]) ; rapidity (achieving the targets within 0.6 to 4 minutes [6 minutes for 7% ]) ; flexibility (applicable to situations both with or without n2o) ; safety (wash - in without sympathetic overactivity) ; and economy (just 2 l / min of fgf and 12% desflurane). after achieving the required fad, the fgf can be reduced to a low flow anesthesia range of 0.51 l / min and the target fad can be simply maintained by setting the desflurane above the fad by 1%2%.3,18 given that we excluded patients with a body mass index > 30 kg / m, this scheme may not be generalized to such conditions, so further study is required. the 1 - 1 - 12 wash - in scheme using a simple, single step fgf of n2o or air : o2 1:1 l / min and desflurane 12% for low flow anesthesia in patients requiring general anesthesia with endotracheal intubation and controlled ventilation has the same performance with or without n2o. each concentration of fad from 1% to 7% can be expected at 0.6, 1, 1.5, 2, 3, 4, and 6 minutes, respectively. the 1 - 1 - 12 wash - in scheme covers the fad required for both balanced and pure inhalation anesthesia. there were nonclinically significant increases in heart rate and decreases in blood pressure during this wash - in period.	backgroundwe reported a 1 - 1 - 12 wash - in scheme for desflurane - nitrous oxide (n2o) low flow anesthesia that is simple, rapid, and predictable. there remain some situations where n2o should be avoided, which limits the generalizability of this wash - in scheme. the objective of our study was to determine the performance of this scheme in contexts where n2o is not used.methodswe recruited 106 patients scheduled for elective surgery under general anesthesia. after induction and intubation, wash - in was started with a fresh gas flow of air : o2 1:1 l / min and a vaporizer concentration of desflurane of 12%. controlled ventilation was then adjusted to maintain paco2 at 3035 mmhg.resultsthe alveolar concentration of desflurane (fad) rose rapidly from 0% to 6% in 4 minutes in the same pattern as observed in our previous study in which n2o was used. an fad of 7% was achieved in 6 minutes. an fad of 1% to 7% occurred at 0.6, 1, 1.5, 2, 3, 4, and 6 minutes. the rise in heart rate during wash - in was statistically significant, although not clinically so. there was a slight but statistically significant decrease in blood pressure, but this had no clinical significance.conclusionperformance of the 1 - 1 - 12 wash - in scheme is independent of the use of n2o. respective fads of 1%, 2%, 3%, 4%, 5%, 6%, and 7% can be expected at 0.6, 1, 1.5, 2, 3, 4, and 6 minutes.
hyponatremia can be asymptomatic or can cause a variety of symptoms such as nausea, vomiting, malaise, lethargy, confusion, headache, decreased level of consciousness, seizures, and coma, depending on its severity. in their study on hyponatremia in hospitalized cancer patients and its impact on clinical outcomes, doshi. concluded that hyponatremia in a patient with cancer is associated with longer hospital stay and higher mortality. patients referred for palliative care have multiple symptoms and tiredness or fatigue is one of the most frequent symptoms in palliative care. hyponatremia can be counted as one of the many contributing factors for fatigue in these patients. hyponatremia can be classified, according to volume status, as - hypovolemic hyponatremia : decrease in total body water with greater decrease in total body sodium, euvolemic hyponatremia : normal body sodium with increase in total body water, hypervolemic hyponatremia : increase in total body sodium with greater increase in total body water. hyponatremia can be further sub - classified according to effective osmolality as hypotonic hyponatremia, isotonic hyponatremia, and hypertonic hyponatremia. hypovolemic causes of hyponatremia include acute corticosteroid withdrawal, cerebral salt wasting, diuretic use, gastrointestinal loss, iatrogenic (insufficient volume, use of hypotonic solutions), and skin loss. normovolemic causes include adrenal insufficiency, drug - induced : selective serotonin reuptake inhibitors, opioids, and oxytocin. hypothyroidism and syndrome of inappropriate secretion of antidiuretic hormone (siadh) are other important causes of normovolemic hyponatremia. hypervolemic causes of hyponatremia include acute renal failure, cirrhosis, and congestive heart failure. in cancer patients, which form a majority of palliative care patients cancers of the lung, breast, head and neck are the ones commonly associated with hyponatremia caused mostly by siadh. among these, lung cancer has the worst prognosis which can be compounded by the increase in mortality caused by hyponatremia. establishing the cause of hyponatremia in palliative care patients is often not undertaken as they have multiple co - morbidities and there could be several factors involved. in a developing country like india, financial implication is a major factor that contributes to physician 's reluctance in ordering multitude of tests required to establish the exact cause of hyponatremia such as serum osmolality, urine osmolality, and imaging studies. a literature search reveals that hyponatremia is prevalent in patients and often is an incidental finding which is undertreated. a retrospective manual analysis of charts of consecutive patients, who were referred to the department of pain and palliative medicine, st. john 's medical college hospital, bengaluru for a total of 5 years, was carried out. all patients whose sodium levels were below 135 meq / l were included in the analysis. statistical package spss 16 (ibm spss statistics) was used for analysis of data. hyponatremia was recorded in 222 which is 28.8 % of the patients whose sodium levels were documented. patients age varied from 3 to 94 years, and 125 men and 97 women demonstrated hyponatremia [figure 1b ]. (a) documentation of sodium levels, (b) demographics of the 28.8% of patients, who had documented hyponatremia, 61.1 % had malignant disease compared to 39.9 % with nonmalignant disease [figure 2 ]. prevalence of hyponatremia in malignant and nonmalignant diseases in the malignant group, gastrointestinal cancers showed the highest percentage (36.7%), followed by hematological cancers (16.1%), genitourinary cancers (14.7%), lung cancers (12.5%), head and neck cancers (8.8%), breast cancers (5.1%), and endocrine, renal and soft tissue cancers (5.7%) forming the remaining group [figure 3 ]. types of cancers with hyponatremia the nonmalignant group of patients had different diseases such as fractures, amputations, stroke, peripheral vascular disease, renal failure, cardiac failure, and chronic obstructive pulmonary disease. of the 222 patients, who had hyponatremia, 117 (52.7%) had mild hyponatremia with a sodium of 130134 meq / l, 65 (29.2%) had moderate hyponatremia with a sodium of 125129 meq / l, and 40 (19.1%) had severe hyponatremia with a sodium value of 124 meq / l and less [figure 4a ]. (a) grades of hyponatremia, (b) percentage of malignant and nonmalignant diseases in different grades of hyponatremia malignant patients formed 88.8% in the mild hyponatremia group, 75.4% in the moderate group, and 82.5 % in the severe hyponatremia group. the nonmalignant patients formed 11.2%, 24.6%, and 17.5 5% in the mild, moderate, and severe hyponatremia group, respectively [figure 4b ]. hyponatremia was recorded in 222 which is 28.8 % of the patients whose sodium levels were documented. patients age varied from 3 to 94 years, and 125 men and 97 women demonstrated hyponatremia [figure 1b ]. of the 28.8% of patients, who had documented hyponatremia, 61.1 % had malignant disease compared to 39.9 % with nonmalignant disease [figure 2 ]. in the malignant group, gastrointestinal cancers showed the highest percentage (36.7%), followed by hematological cancers (16.1%), genitourinary cancers (14.7%), lung cancers (12.5%), head and neck cancers (8.8%), breast cancers (5.1%), and endocrine, renal and soft tissue cancers (5.7%) forming the remaining group [figure 3 ]. types of cancers with hyponatremia the nonmalignant group of patients had different diseases such as fractures, amputations, stroke, peripheral vascular disease, renal failure, cardiac failure, and chronic obstructive pulmonary disease. of the 222 patients, who had hyponatremia, 117 (52.7%) had mild hyponatremia with a sodium of 130134 meq / l, 65 (29.2%) had moderate hyponatremia with a sodium of 125129 meq / l, and 40 (19.1%) had severe hyponatremia with a sodium value of 124 meq / l and less [figure 4a ]. (a) grades of hyponatremia, (b) percentage of malignant and nonmalignant diseases in different grades of hyponatremia malignant patients formed 88.8% in the mild hyponatremia group, 75.4% in the moderate group, and 82.5 % in the severe hyponatremia group. the nonmalignant patients formed 11.2%, 24.6%, and 17.5 5% in the mild, moderate, and severe hyponatremia group, respectively [figure 4b ]. true occurrence of hyponatremia in various clinical settings is difficult to determine. a literature search (cochrane database and embase) for the prevalence of hyponatremia in palliative care patients did not yield any result. this is true, especially with elderly patients. in their study looking at predictors of inpatient mortality in an acute palliative care unit, elsayem. correlation of hyponatremia to patient mortality was not done in the present study. in their study on prevalence, incidence, and etiology of hyponatremia in elderly patients with fragility fractures, they found that dehydration and prescription of thiazide diuretics and proton pump inhibitors were the potential causative factors and not siadh. in the present study too, the prevalence of hyponatremia was significant at 28.8% with a p value of 0.000. in another study, shapiro. found that the prevalence of severe hyponatremia in elderly hospitalized person was significant with a p < 0.001. they found that it was more frequent in women and multifactorial in etiology in 50% of the cases. in their group of patients, they found that it is most commonly caused by siadh. in the present study, more men (125) were found to have hyponatremia compared to women (97). states that hyponatremia is a predictor of mortality in the general population independently of age, gender, and co - morbid conditions. a study on ambulatory patients with mild hyponatremia followed up over a period of 8 years, found that the prevalence was 6.9% and was significantly associated with age, black ethnicity, presence of cirrhosis, cardiac failure or cancer and use of antiepileptic drugs and serotonin reuptake inhibitors. in the present study, 61.10% of patients with malignant diseases and 39.9% of patients with nonmalignant diseases were found to have hyponatremia. in a study that looked at hyponatremia in acute medical admissions this could indicate that hyponatremia might be a cause of acute admissions. in another study that looked at diagnostic codification of hyponatremia on patients admitted under internal medicine, the authors found that there was a low prevalence of hyponatremia codification and stated that hyponatremia was underreported and undertreated in spite of numerous studies showing devastating effects of hyponatremia. cancer patients form the majority of palliative care patients and cancer in itself and its different manifestations cause hyponatremia. an aging population also has more prevalence of cancer, thereby increasingly seeking palliative care. hyponatremia can be found associated with siadh in 1015% of patients with small cell lung cancer. in their article on tumor related hyponatremia, onitilo. hyponatremia may be detected by routine laboratory testing or by the manifestation of neurological symptoms in cancer patients. hospitalized cancer patients are prone to have a higher rate of hyponatremia when compared to hospitalized noncancer patients with the outcome in the cancer patients being poor. in a study result from the international metastatic renal cell cancer database consortium, hyponatremia was found to be independently associated with a worse outcome in metastatic renal cell cancer patients. most of the patients referred for palliative care in the present study population were cancer patients. worldwide, cancer patients still form the majority of palliative care patients. with an already bad prognosis, it is tough for patients to bear the added burden of hyponatremia and its complications. it is time that we recognized and treated hyponatremia diligently so that the patients and their relatives suffer less from the added burden. even today substantial amount of work needs to be done to assess the causes and management outcomes of hyponatremia in palliative care patients. prevalence of hyponatremia is significant in palliative care patients. although this study showed that hyponatremia is more in cancer patients, it can not be generalized, as the number of cancer patients included in the study was proportionately more than noncancer patients. the causes and clinical outcomes associated with hyponatremia in palliative care patients warrant analysis of a prospective cohort.	introduction : hyponatremia is an undertreated finding in clinical practice. it is the most common electrolyte abnormality. hyponatremia can be asymptomatic or can cause symptoms ranging from nausea and lethargy to convulsions and coma. palliative care patients have a multitude of symptoms and there are several contributing factors towards this. hyponatremia could be one of the contributing factors. looking at the prevalence of hyponatremia would highlight the magnitude of the problem and would prompt healthcare professionals to investigate and treat hyponatremia in palliative care patients, which in turn might reduce symptoms such as fatigue and nausea. this could improve the quality of life in palliative care patients.aim:to assess the prevalence of hyponatremia among patients referred for palliative care in a tertiary care hospital.methodology:this is a descriptive study, with retrospective analysis of consecutive patient charts for 5 years. the sodium levels at the time of referral for palliative care, was reviewed. inferential statistics for the result was calculated using the z-test.results:of the 2666 consecutive patient charts that were reviewed, sodium values were recorded in 796 charts. among the recorded charts, 28.8 % of patients showed hyponatremia at the time of referral which was significant with a p value of 0.000 (< 0.05). of these, 61.1 % had malignancy as their diagnosis and the rest had nonmalignant diseases, ranging from trauma to chronic obstructive pulmonary disease.conclusions:prevalence of hyponatremia is significant in palliative care patients. a prospective study looking at the causes and clinical outcomes associated with hyponatremia in palliative care patients is needed.
these include, firstly, the molecular - genetic hypotheses which hypothesize strong effects of schizophrenia susceptibility genes. a corollary of the molecular - genetic hypothesis is the proposal that targeting drugs at these genes might yield novel and more effective treatments for schizophrenia. secondly, the neuronal network hypotheses propose strong effects of altered neuronal integration in schizophrenia. the corollary of this hypothesis predicts that drugs which fundamentally reset the tone of networks of neuronal interactions will prove efficacious in treating schizophrenia. thirdly, the signal transduction hypothesis proposes that basic alterations in receptor - mediated signal transduction (cither at the receptor or post - receptor levels) induce schizophrenia - like pathology. it follows that ameliorating altered signaling via specific medications which target receptor / post - receptor molecules will prove efficacious in treating schizophrenia. thus, one could suggest, for instance, that schizophrenia arises because of mutation in a specific susceptibility gene - oc7 nicotinic receptors for instance. this mutation results in diminished oc7 expression which, in turn, leads to altered neuronal connectivity and signal transduction. these alterations in neuronal signaling and connectivity lead to some of the symptoms of schizophrenia. the corollary is the proposal that a7 agonists will improve schizophrenia, symptoms - a hypothesis that is now being tested. the underlying assumption of these lines of reasoning is that if one can identify the critical node (figure 1) in the pathogenesis of schizophrenia and alter its functioning, one will more effectively treat schizophrenia. the implicit assumption is that only one (or a small number) of molecular targets function as critical nodes in the pathogenesis of schizophrenia. the role of molecular biology in such an undertaking is relatively straightforward : (i) identify the disease - inducing molecules (genetic linkage studies, candidate gene approaches) ; (ii) express the molecule in a way suitable for high - throughput - screening of large chemical libraries to identify candidate ligands with appropriate pharmacology (agonist, antagonist, partial agonist, inverse agonist, allosteric modulator) ; (iii) provide molecular - target based assays for profiling candidate ligands at a large variety of other druggable targets to verify that the final lead compounds arc suitably selective (or suitably nonselective) ; and (iv) provide molecular - target based assays for profiling candidate ligands against various molecular targets which can lead to serious side effects. these can include prolongation of the qt interval via blockade of herg k+-channels, agonism of 5-ht 2b serotonin receptors which can lead to cardiovascular side effects, carcinogenicity, genotoxicity, and alteration of cytochrome p450 isoforms leading to altered pharmacokinetics (see ref 24 for instance). in the case of antipsychotic medications, weight gain and adverse metabolic side effects (likely mediated in part via h1 -histamine and 5-ht2c - serotonin receptor blockade34) and extrapyramidal side effects (due to d2-dopamine receptor blockade) indeed, much of preclinical drug discovery in both industry and academia is driven primarily via molecular target - based screening and profiling technologies. despite our ability to screen millions of drug - like compounds at hundreds of druggable targets which comprise the druggable genome, no novel molecularly targeted treatments for schizophrenia have been approved. indeed, as already mentioned, clozapine continues to be the gold - standard treatment for schizophrenia. assumption, a large number of potential nodes have been identified for therapeutic drug discovery. these have been identified via the three general strategies outlined above (eg, molecular genetic, neuronal network, or signal transduction) and a large number of these candidate nodes have been a theme of research over the past decade. as we have recently summarized as part of a larger study of psychiatric drug discovery, nearly 150 investigational compounds directed against many individual molecular targets (nodes) have been subjected to at least early - phase clinical trials (roth and conn, unpublished report). representative compounds for each node are listed in table i. in this table, antipsychotic drugs have been classified based on molecular target (eg, node)/targets (nodes) and whether the compounds were validated with preclinical and clinical studies. lastly, it is indicated whether the compounds were found, based on clinical trials, to be superior to a standard comparator medication (typically haloperidol). based on the currently available data, we were unable to find any evidence to support the hypothesis that targeting any single molecular target (node) other than d2 dopamine receptors will yield a drug which effectively treats the core symptoms of schizophrenia. additionally, we were unable to find any support for the hypothesis that drugs targeting a single node are more effective at treating schizophrenia than drugs targeting a large number of nodes. indeed, clozapine, which targets at least 50 nodes, remains superior to all other medications. it is unknown whether any single molecular target of greater promise will ever be found. there are many ways in which these findings can be interpreted, although each interpretation relies mainly on untested assertions. a typical criticism one can make of these findings is that we have not yet found the critical node and that once this key node is discovered, the pathway towards drugs with greater efficacy and fewer side effects will be clarified. the untested assumptions are (i) that such a special node associated with efficacy exists ; (ii) that it can be discovered ; and (iii) that, once discovered, using techniques of molecular biology, a drug can be designed to target it. an implicit assumption underlying this argument relates to the need for an enhanced understanding of the molecular pathogenesis of schizophrenia in order to discover and validate suitable molecular targets. based upon our current understanding of the molecular pathogenesis of schizophrenia, no critical, node other than the d2 dopamine receptor has yet been convincingly and reproducibly elucidated, although a large number of candidate genes and susceptibility factors have been described. these include neuregulin-1, dysbindin, disrupted in schizophrenia-1 (disc-1) and many others (eg, rcelin, regulator of g protein signaling-4, catccholo - methyltransferase, mglur3 glutamate receptor, and so on ; see ref 8 for recent review). as we and others have pointed out (figure 1.) these susceptibility gene products are found in a variety of cell types (both neuronal and glial) and show differential subcellular localizations. as figure i shows, the molecular targets identified are frequently found in circuits which are targeted by drugs with a promiscuous pharmacology (eg, clozapine). no single node is an obvious target for therapeutic drug discovery efforts, although nearly all of the identified nodes have been reported to be targets of therapeutic drug discovery (roth and conn, unpublished report). another possibility is that schizophrenia, can be most effectively treated by influencing several nodes simultaneously. indeed, based on the demonstrated superiority of clozapine for treatment - resistant schizophrenia and the relative inferiority of all other medications, there is strong support for this hypothesis. a great deal of effort has been expended to discover an optimal clozapinemimetic devoid of the side effects of clozapine which include agranulocytosis, seizures, sialorrhea, weight gain, sedation, and hypotension. we, and others, have suggested that the massively parallel screening of large numbers of molecular targets allows one to efficiently discover toxic vs therapeutic targets. antipsychotic drug - induced weight gain might be due to h1 -histamine and 5-ht7c - reccptor blockade, agranulocytosis to h4 histamine agonism, sedation to h1 histamine antagonism, and so on. thus far, these molecular targets implicated in clozapine 's side effects (h1 -histamine, -histamine, 5-ht2c serotonin) are not identical with those targets thought to be involved in its superiority as an antipsychotic drug (5-ht2a serotonin, d4-dopamine, 5ht6 and 5-ht7 serotonin). a problem with the approach of designing selectively nonselective drugs is that it is very difficult to rationally design in new pharmacological properties during the drug discovery process. this is an emerging paradigm, however, and some successful strategics have recently been elucidated. the neuronal systems approach similarly proposes that there might be crucial nodes in the network that are amenable to target - based discovery efforts. spedding and colleagues have cogently argued that a systems - level approach using animal models will lead to more effective treatment for psychiatric diseases. based on a model which involves specific alterations in hippocampal - cortical circuitry, they propose testing compounds in animals in which these circuits are disrupted by phenycyclidinc (pcp). in support of this systems - level approach, nearly every approved antipsychotic drug will ameliorate pcpinduced alterations in neuronal functioning. however, it is also true that drug classes with demonstrated ability to ameliorate pcp - induced deficits (eg, 5-ht2a antagonists) are only marginally effective in treating schizophrenia.- thus, in vivo systems - level screens can be highly effective tools to verify in vivo actions of putative atypical antipsychotic drugs. it does not appear that any of the available in vivo screening models are able to predict relative efficacy at treating schizophrenia, however. in addition, none of the available models appears to adequately recapitulate the entirety of the human phenotype. suitable animal model will eventually be found which recapitulates the schizophrenia phenotype, although it is also plausible that no suitable preclinical model will ever be found which adequately recapitulates schizophrenia, pathology. clearly, despite decades of research we have not yet discovered an adequate preclinical model, and it is within the realm of possibility that schizophrenia is a uniquely human disease which can not be adequately modeled in rodents. in large measure, this is likely to be due to the fact that a number of genetic hits as well as nongenomic factors converge to produce the final phenotype in humans. at present, we have no way to predict either way, and continued research in this arena will be based more on untested assumptions than on data. another possibility is that schizophrenia represents a complex disease with genetic and epigenetic factors and which is both chronic and progressive, resulting in irreversible end - organ damage - similar to hypertension. indeed, there is accumulating evidence for epigenetic factors involved in the etiology of schizophrenia - particularly relating to reelin. there has also been abundant evidence accumulated over the past several decades that schizophrenia is associated with subtle but reproducibly documented neurodegeneration (reviewed in refs 46,47). accordingly, optimal treatment of schizophrenia would be similar to that for other progressive and complex diseases such as hypertension, where individuals at risk would be identified and then treated to avoid end - organ damage. such an approach has already been attempted, with a mixed degree of success. in this study, individuals at risk were identified and then prophylactlcally treated with placebo or olanzapine. although the results were not statistically significant, there was a trend toward protection of conversion to overt psychosis among individuals treated with olanzapine. as is clear from the foregoing, the tools of molecular biology can, at least theoretically, accelerate drug discovery in schizophrenia. in the main, molecular biological approaches have been more useful in providing reagents for high - throughput screening campaigns than for providing better animal models - at least to date. with the continued discovery of schizophrenia susceptibility genes, it is at least conceivable that better preclinical models will be produced. to a great degree, lack of progress in developing more effective antipsychotic drugs has stemmed mainly from the failure both to fully appreciate the pharmacological robustness of clozapine and to discover medications which reproduce the essential features without producing serious side effects. it is not clear whether any of the paradigms outlined will lead to more effective medications, although it is likely that continued molecular target - based screening will eventually yield medications with fewer side effects.	this review summarizes the various conceptual paradigms for treating schizophrenia, and indicates how molecular biology and drug discovery technologies can accelerate the development of new medications. as yet, there is no convincing data that a crucial druggable molecular target exists which, if targeted, would yield medications with efficacies greater than any currently available. it is suggested, instead, that drugs which interact with a multiplicity of molecular targets are likely to show greater efficacy in treating the core symptoms of schizophrenia.
cadmium (cd), a heavy metal, is present in soils, sediments, air, and water. unlike most metals, cd use began fairly recently with its large - scale application dating from 1940s. today its main uses are for nickel cadmium battery manufacture, pigments, and plastic stabilizers (who, 1995). cadmium in soil and water is taken up by plants and is concentrated and transferred to upper links of the food chain, including humans (who, 1995 ; satarug., 2003). due to the long biological half - life of cd (i.e., 1030 years) its accumulation in the body can increase the risk of toxicity (sugita and tsuchiya, 1995). the principal determinants of human cd exposure are smoking habits, diet, and to a certain extent, occupational exposure, like that occurring in non - ferrous metal smelters, in the production and processing of cd alloys and compounds and, increasingly, in the recycling of electronic waste. non - occupational exposure is mainly from cigaret smoke which contains relatively high concentrations of this element. according to who (wakabayashi., 1987) one cigaret (containing 0.53 g cd / g of tobacco) can result in up to 3 g daily cd absorption via the lungs. chronic exposure to these low doses of cd causes neuroendocrine and neurobehavioral disturbances in animals and humans (viaene., 2000 ; 2003). at a high concentration cd increases oxidative damage in the rodent brain (lopez., 2006 ; yang., 2007) less information is available on the mediation by redox mechanisms of brain effects of a low concentration of cd. in a recent study we examined the effect of a low dose of cd (7.5 g / day) on 24-h changes in expression of redox pathway enzyme and circadian genes in rat medial basal hypothalamus (mbh ; jimnez - ortega., a disruption of 24-h pattern of mbh gene expression of nitric oxide synthase (nos)-1 and -2, heme oxygenase (ho)-1 and -2, mn - superoxide dismutase (mn - sod), catalase, glutathione peroxidase (gpx), and glutathione reductase (gsr) was detectable. mean 24-h levels of mbh mrna for ho-2, mn - sod, and catalase augmented after cd intake (presumably as a compensatory increase caused by the augmented oxidative load), whereas those of nos-2 decreased (presumably because cd causes toxicity independently of no formation ; wright and baccarelli, 2007). after cdcl2 intake, the 24-h pattern of clock gene expression in mbh seen in control rats was significantly disrupted, suggesting that a primary mechanism of action could be on circadian clock mechanisms (jimnez - ortega. melatonin, the major secretory product of the pineal gland, is released every day at night. in addition, melatonin is also locally synthesized in various cells, tissues, and organs including lymphocytes, human and murine bone marrow, the thymus, the gastrointestinal tract, skin, and the eyes where it plays either an autocrine or paracrine role (see for reference, pandi - perumal., 2006 ; reiter., 2009 ; hardeland., both in animals and in humans, melatonin participates in diverse physiological functions signaling not only the length of the night (the chronobiotic effect ; dawson and armstrong, 1996 ; arendt and skene, 2005) but also enhancing free radical scavenging, the immune response and cytoprotection (hardeland., 2011). cytoprotective role of melatonin on cd - induced changes in clock gene and redox enzyme gene expression in rat mbh. we also wished to assess whether the concomitant administration of melatonin could modify 24-h expression of mbh redox enzyme genes in a way compatible with the recently reported activity of melatonin on the same set of enzymes (jimnez - ortega., 2009). male wistar rats (45 days of age, n = 169) were maintained under standard conditions with controlled light (12:12 h light / dark schedule ; lights on at 0800 h) and temperature (22 2c). the rats received cdcl2 (5 ppm) and melatonin (3 g / ml) or vehicle in drinking water for 1 month. the stock solution of melatonin was prepared in 50% ethanol ; final ethanol concentration in drinking water was 0.015%. cd - administered animals and vehicle - treated controls received 0.015% ethanol in drinking water. since rats drank about 20 ml / day with 9095% of this total daily water taken up during the dark period, the melatonin dosage used provided approximately 60 g melatonin / day. this dose was 10 timed higher than that needed to obtain physiological circulating melatonin levels in pinealectomized rats (cardinali., 2004). after 1 month groups of six to eight rats were sacrificed by decapitation under conditions of minimal stress at six different time intervals, every 4 h throughout a 24-h cycle, starting at 0900 h. at night intervals animals were killed under red dim light. the brains were rapidly removed and the mbh including the median eminence was quickly dissected out following the landmarks of szentagothai. the mbh consisted of a 3-mm - thick block of tissue weighing 46 mg and extending from the rostral border of the optic chiasm to the rostral margin of the mammillary bodies, and laterally to the hypothalamic sulci (moreno., 1992). the care and use as well as all procedures involving animals were approved by the institutional animal care committee, faculty of medicine, complutense university, madrid. the study was in accordance with the guidelines of the institutional care and use committee of the national institute on drug abuse, national institutes of health and the guide for the care and use of laboratory animals (institute of laboratory animal resources, 1996). total rna extraction was performed using the rneasy protect mini kit and was analyzed using quantitec sybr green kit (qiagen, hielden, germany). the iscript cdna synthesis kit (bio - rad laboratories sa, madrid) was used to synthesize cdna from 1 g of total rna, according to the manufacturer s protocol. reactions were carried out in the presence of 200 nm of specific primers for clock, bmal1, per1, per2, cry1, and cry2, nos-1, nos-2, ho-1, ho-2, cu / zn - sod, mn - sod, catalase, gpx, and gsr. primers were designed using primer3 software (the whitehead institute, http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and are shown in table 1. polymerase chain reactions (pcr) were carried out in an eppendorf realplex mastercycler (eppendorf ag, hamburg, germany). the real - time quantitative pcr (qpcr) program included a 94c enzyme activation step for 2 min followed by 40 cycles of 95c denaturation for 15 s, 60c annealing for 30 s and 72c extension for 30 s. detection of fluorescent product was carried out at the end of the 72c extension period. serial dilutions of cdna from control mbh were used to perform calibration curves in order to determine amplification efficiencies. for the primers used there were no differences between transcription efficiencies, the amount of initial cdna in each sample being calculated by the 2 method (livak and schmittgen, 2001). fractional cycle at which the amount of amplified target becomes significant (ct) was automatically calculated by the pcr device. to estimate whether treatment or time of day modified the expression of -actin, in mbh pcr with serial dilutions of this housekeeping gene was performed. in this study ct did not vary significantly as a function of treatment or of time of day, indicating the validity to employ -actin as a housekeeping gene. it must be noted that in a previous study on -actin expression in a larger hypothalamic block than that used herein, which weighed 4263 mg and included the preoptic, suprachiasmatic, paraventricular, supraoptic, arcuate, dorsomedial, ventromedial, and mammillary areas and the median eminence revealed time of day changes with maxima at 0800 h (iovanna., 1990). after verifying normality of distribution of data in a normal distribution probability plot, the statistical analysis of the results was performed by a one - way or a two - way factorial analysis of variance (anova) followed by bonferroni s multiple comparison tests, as stated. male wistar rats (45 days of age, n = 169) were maintained under standard conditions with controlled light (12:12 h light / dark schedule ; lights on at 0800 h) and temperature (22 2c). the rats received cdcl2 (5 ppm) and melatonin (3 g / ml) or vehicle in drinking water for 1 month. the stock solution of melatonin was prepared in 50% ethanol ; final ethanol concentration in drinking water was 0.015%. cd - administered animals and vehicle - treated controls received 0.015% ethanol in drinking water. since rats drank about 20 ml / day with 9095% of this total daily water taken up during the dark period, the melatonin dosage used provided approximately 60 g melatonin / day. this dose was 10 timed higher than that needed to obtain physiological circulating melatonin levels in pinealectomized rats (cardinali., 2004). after 1 month groups of six to eight rats were sacrificed by decapitation under conditions of minimal stress at six different time intervals, every 4 h throughout a 24-h cycle, starting at 0900 h. at night intervals animals were killed under red dim light. the brains were rapidly removed and the mbh including the median eminence was quickly dissected out following the landmarks of szentagothai. the mbh consisted of a 3-mm - thick block of tissue weighing 46 mg and extending from the rostral border of the optic chiasm to the rostral margin of the mammillary bodies, and laterally to the hypothalamic sulci (moreno., 1992). the care and use as well as all procedures involving animals were approved by the institutional animal care committee, faculty of medicine, complutense university, madrid. the study was in accordance with the guidelines of the institutional care and use committee of the national institute on drug abuse, national institutes of health and the guide for the care and use of laboratory animals (institute of laboratory animal resources, 1996). total rna extraction was performed using the rneasy protect mini kit and was analyzed using quantitec sybr green kit (qiagen, hielden, germany). the iscript cdna synthesis kit (bio - rad laboratories sa, madrid) was used to synthesize cdna from 1 g of total rna, according to the manufacturer s protocol. reactions were carried out in the presence of 200 nm of specific primers for clock, bmal1, per1, per2, cry1, and cry2, nos-1, nos-2, ho-1, ho-2, cu / zn - sod, mn - sod, catalase, gpx, and gsr. primers were designed using primer3 software (the whitehead institute, http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and are shown in table 1. polymerase chain reactions (pcr) were carried out in an eppendorf realplex mastercycler (eppendorf ag, hamburg, germany). the real - time quantitative pcr (qpcr) program included a 94c enzyme activation step for 2 min followed by 40 cycles of 95c denaturation for 15 s, 60c annealing for 30 s and 72c extension for 30 s. detection of fluorescent product was carried out at the end of the 72c extension period. serial dilutions of cdna from control mbh were used to perform calibration curves in order to determine amplification efficiencies. for the primers used there were no differences between transcription efficiencies, the amount of initial cdna in each sample being calculated by the 2 method (livak and schmittgen, 2001). fractional cycle at which the amount of amplified target becomes significant (ct) was automatically calculated by the pcr device. to estimate whether treatment or time of day modified the expression of -actin, in mbh pcr with serial dilutions of this housekeeping gene was performed. in this study ct did not vary significantly as a function of treatment or of time of day, indicating the validity to employ -actin as a housekeeping gene. it must be noted that in a previous study on -actin expression in a larger hypothalamic block than that used herein, which weighed 4263 mg and included the preoptic, suprachiasmatic, paraventricular, supraoptic, arcuate, dorsomedial, ventromedial, and mammillary areas and the median eminence revealed time of day changes with maxima at 0800 h (iovanna., 1990). after verifying normality of distribution of data in a normal distribution probability plot, the statistical analysis of the results was performed by a one - way or a two - way factorial analysis of variance (anova) followed by bonferroni s multiple comparison tests, as stated. the effect of cd and/or melatonin on 24-h pattern of mbh mrna levels of the circadian clock genes examined is depicted in figure 1. in control rats, mbh mrna levels of bmal1 attained maximal values at the beginning of scotophase followed by a nadir at the middle of the phase and a second increase at the end (p < 0.01). mbh per1 expression peaked at later afternoon during photophase (p < 0.01) while cry2 expression showed maxima during the scotophase (p < 0.03). two maxima (at the middle of photophase and at the beginning and middle of scotophase) were seen in the case of mrna per2 levels (p < 0.01 ; figure 1). effect of melatonin on cd - induced changes in 24-h pattern in expression of clock, bmal1, per1, per2, cry1, and cry2 in rat mbh. the rats received cdcl2 (5 ppm) and melatonin (3 g / ml) or vehicle in drinking water for 1 month. groups of six to eight rats were killed by decapitation at six different time intervals throughout a 24-h cycle. are the mean sem of mrna determination as measured by triplicate real - time pcr analyses of rna samples. letters denote significant differences in a one - way anova followed by a bonferroni s multiple comparison test performed at every time interval, as follows : p < 0.05 vs. cd and melatonin groups ; p < 0.02 vs. cd and cd + melatonin groups ; p < 0.05 vs. cd ; p < 0.01 vs. the remaining groups ; p < 0.01 vs. cd and control groups ; p < 0.05 vs. control. one - way anovas within each experimental group indicated significant time - related changes in clock gene expression as follows : controls : bmal1, per1, per2, and cry2 (f = 11.3, p < 0.0001, f = 2.54, p < 0.04, f = 7.94, p < 0.001, and f = 3.41, p < 0.03, respectively). cd : clock, per1, per2, and cry2 (f = 4.71, p < 0.008, f = 4.12, p < 0.01, f = 5.84, p < 0.03, and f = 19.1, p < 0.0001, respectively). cd + melatonin : bmal1, per1, per2, cry1, and cry2 (f = 10.1, p < 0.0001, f = 3.11, p < 0.04, f = 4.48, p < 0.009, f = 7.82, p < 0.001, and f = 19.6, p < 0.0001, respectively. melatonin : per2 and cry2 (f = 14.2 and 50.8, p < 0.0001, respectively). for further statistical analysis, cdcl2 treatment significantly suppressed circadian rhythmicity in bmal1 expression, changed the phase of per1, per2, and cry2 expression and induced a late photophase peak in clock expression (p < 0.01). the concomitant administration of melatonin counteracted the changes in 24-h pattern of per1 expression caused by cd, whereas it did not affect significantly other changes found with cd (as for clock, per2, and cry2 expression) or induced new maxima (as for bmal1 or cry1 expression ; figure 1). in animals receiving melatonin alone the 24-h pattern in expression of mbh per2 and cry2 was significantly disrupted (p < 0.01), while that of clock, bmal1, per1, and cry1 remained unchanged (figure 1). figures 2 and 4 summarize the effect of treatment on 24-h pattern and mean levels of mbh mrna of the redox enzymes examined. control rats exhibited significant daily variations in mbh expression of cu / zn - sod, mn - sod, catalase, gpx, nos-2, ho-1, and ho-2 genes (figures 2 and 3). cdcl2 administration phase - delayed mn - sod gene expression and suppressed the maximum in gene expression of gpx seen at midday (p < 0.03 ; figure 2). cdcl2 also affected the 24-h pattern of expression of mbh nos and ho isoenzymes by inducing maxima at late scotophase (nos-1), at midday and late scotophase (nos-2), at midday, early and late scotophase (ho-1), and at early morning (ho-2 ; figure 3). effect of melatonin on cd - induced changes in 24-h pattern in expression of mrna for cu / zn - sod, mn - sod, catalase, gpx, and gsr in rat mbh. for experimental details are the mean sem of mrna determination as measured by triplicate real - time pcr analyses of rna samples. letters denote significant differences in a one - way anova followed by a bonferroni s multiple comparison test performed at every time interval, as follows : p < 0.02 vs. cd and control groups ; p < 0.05 vs. melatonin and control groups ; p < 0.01 vs. the remaining groups ; p < 0.05 vs. melatonin and cd groups ; p < 0.05 vs. melatonin and control groups ; p < 0.05 vs. the remaining groups ; p < 0.01 vs. cd ; p < 0.02 vs. control ; one - way anovas within each experimental group indicated significant time - related changes in enzyme gene expression as follows : controls : cu / zn - sod, mn - sod, catalase, and gpx (f = 6.21, p < 0.002, f = 3.33, p < 0.03, f = 3.17, p < 0.04, and f = 2.98, p < 0.04, respectively). cd : cu / zn - sod and mn - sod (f = 6.25, p < 0.002, and f = 7.01, p < 0.001, respectively). cd + melatonin : cu / zn - sod, mn - sod, catalase, and gsr (f = 14.1, p < 0.0001, f = 15.5, p < 0.0001, f = 10.8, p < 0.0001, and f = 8.24, p < 0.0001, respectively). melatonin : mn - sod, catalase, gpx, and gsr (f = 4.49, p < 0.002, f = 2.46, p < 0.05, f = 15.9, p < 0.0001, and f = 8.35, p < 0.0001, respectively). for further statistical analysis, see text. effect of melatonin on cd - induced changes in 24-h pattern of expression of mrna for nos-1, ho-1, nos-2, and nos-2 in rat mbh. for experimental details are the mean sem of mrna determination as measured by triplicate real - time pcr analyses of rna samples. letters denote significant differences in a one - way anova followed by a bonferroni s multiple comparison test performed at every time interval, as follows : p < 0.05 vs. cd + melatonin group ; p < 0.05 vs. the remaining groups ; p < 0.02 vs. cd ; p < 0.02 vs. control and cd groups ; p < 0.01 vs. the remaining groups ; p < 0.02 vs. control and cd + melatonin groups ; p < 0.01 vs. control ; p < 0.01 vs. melatonin. one - way anovas within each experimental group indicated significant time - related changes in enzyme gene expression as follows : controls : ho-1, nos-2, and ho-2 (f = 7.54, p < 0.001, f = 10.1, p < 0.0001, and f = 5.64, p < 0.002, respectively). cd : nos-1, ho-1, nos-2, and ho-2 (f = 5.88, p < 0.002, f = 8.41, p < 0.0001, f = 18.3, p < 0.0001, and f = 7.42, p < 0.001, respectively). cd + melatonin : ho-1, nos-2, and ho-2 (f = 8.45, p < 0.0001, f = 6.12, p < 0.002, and f = 4.04, p < 0.03, respectively). melatonin : nos-1, nos-2, and ho-1 (f = 10.7, p < 0.0001, 12.9, p < 0.0001, and 5.92, p < 0.004, respectively). for further statistical analysis, see text. effect of melatonin on cd - induced changes in mrna of redox enzymes in rat mbh. shown letters denote significant differences in a one - way anova followed by a bonferroni s multiple comparison as follows : p < 0.01 vs. control and cd groups ; p < 0.05 vs. control ; p < 0.02 vs. control ; p < 0.02 vs. the remaining groups ; p < 0.02 vs. control and cd groups ; p < 0.03 vs. the remaining groups. the concomitant administration of melatonin reversed the phase delay in mn - sod gene expression given by cdcl2 (p < 0.001 ; figure 2) and counteracted the effect of cd on 24 h pattern in expression of nos and ho isoenzymes (p < 0.02 ; figure 3). melatonin + cdcl2 administration disrupted the 24-h pattern of cu / zn - sod gene expression by inducing a late scotophase peak (p < 0.02 ; figure 2). in animals receiving melatonin alone, suppression of 24-h rhythmicity in gene expression (cu / zn - sod, ho-2) and induction of a mid - scotophase (gsr) or early morning peak of expression (nos-1, nos-2, ho-1) figure 4 depicts the mean 24-h values for gene expression of mbh redox enzymes in the four groups of animals studied. a factorial anova on the effects of treatment on mean gene expression indicated that cdcl2 administration increased significantly mn - sod, catalase, and ho-2 expression and decreased that of nos-2 gene. the concomitant administration of melatonin reversed the effect of cd on mean catalase, nos-2, and ho-2 gene expression, augmented 24-h mean values of cu / zn- and mn - sod mrna levels and decreased those of gpx, gsr, and ho-1. in animals receiving melatonin alone, significant increases in mean cu / zn and mn - sod gene expression, and decreases in that of gpx, gsr, nos-1, nos-2, ho-1, and ho-2, were found (figure 4). the mammalian circadian timing system comprises oscillators found in almost every cell of the body together with a central rhythm generator located in the hypothalamic suprachiasmatic nuclei (scn ; lincoln., 2006). at the cell level, circadian rhythms are driven by the self - regulatory interaction of a set of clock genes and their protein products (levi., 2010). the positive drive to the daily clock is constituted by helix - loop - helix, pas - domain containing transcription factor genes, called clock and bmal1 (or its paralog npas2). the protein products of these genes form heterodimeric complexes that control the transcription of other clock genes, notably period (per1/per2/per3) genes, and cryptochrome (cry1/cry2) genes, which in turn provide the negative feedback signal that shuts down the clock / bmal1 drive to complete the circadian cycle. other clock genes like rev - erb, ror, nr1d1, or timeless provide additional transcriptional / translational feedback loops to form the rest of the core clockwork (levi., 2010), which has been characterized in rodents by a transgenic gene deletion methodology. a major question concerns as about how the circadian apparatus is adjusted to maintain coordination between physiology and the changing environment. models including the relation between the redox state and the circadian clockwork have been proposed (rutter., 2002). in zebrafish, light, as a key entraining stimulus for the circadian clock, induces the production of hydrogen peroxide that acts as the second messenger coupling photoreception to the circadian clock, as shown by the induction of zcry1a and zper2 genes and the subsequent circadian oscillation of zper1. in z3 cells (hirayama., 2007). these findings support a link between the regulation circadian clock genes and the control of cellular redox state (rutter., 2002). the present study aimed to examine the relations between circadian clock gene expression and gene expression of redox enzymes in a complex, heterogeneous brain area like the mbh of rats receiving a low dose of cdcl2 and/or melatonin. in the mbh of control rats expression of bmal1 peaked at early scotophase, per1 at late afternoon, and per2 and cry2 at mid - scotophase, whereas neither clock nor cry1 expression showed significant 24-h variations. presumably, the nuclear heterogeneity of the mbh fragments employed precluded identification of the reciprocal relationship between clock / bmal1 and per1/per2 expression seen in isolated nuclei, like the scn (poirel., 2003 ; agez., 2007 ; challet, 2007). as shown previously (jimnez - ortega., 2010), a low amount of cdcl2 (i.e., 5 ppm in tap water, about 7.5 g / day) significantly suppressed (bmal1) or disrupted 24-h pattern of expression (per1, per2, cry2) while in the case of clock significant 24-h variations were induced. the results suggest that the inherent transcription modifications that give the clock its own natural rhythmicity are disrupted in rats drinking a low amount of cdcl2 in tap water. previous studies from this laboratory indicated that chronic exposure of rats to similar low doses of cd affected the circadian variation of pituitary hormone release (lafuente. it seems feasible that the changes in clock gene expression in mbh, a key region in hormone regulation, play a role in the circadian hormone disruption. in the present study, the concomitant administration of melatonin (3 g / ml ; a chronobiotic cytoprotective agent ; hardeland., 2011) in drinking water failed to counteract most effects of cd on clock genes, except for per1 expression this suggests that the transcription modifications through which cdcl2 administration disrupts the natural rhythmicity of the circadian clock are only slightly affected by melatonin administration. a survey of the scientific literature supports an effect of melatonin on circadian clock components in the mammalian scn (poirel., 2003 ; agez., 2007 ; challet, 2007), retina (dinet and korf, 2007 ; dinet., 2007), and striatal neurons in culture (imbesi., 2009) animals receiving melatonin alone showed significant phase - advances in mbh per2 and cry2 expression to peak at late afternoon and early scotophase, respectively. again, the nuclear heterogeneity of the mbh fragments makes it impossible any valid comparison with the published effects on melatonin activity in isolated brain nuclei. as an indication of the link between the redox status and the circadian system, 24-h variations in brain redox pathway enzymes have been described, including nos (ayers., 1996 ; tunctan., 2002 ; clemens., 2005), ho (artinian., 2001 ; rubio., 2003), sod (diaz - munoz., 1985 ; schaper., 1986 ; martin. our present results on the circadian variation in gene expression of nos and ho in mbh of control rats are consistent with previous publications (jimnez - ortega., 2009, 2010). in the case of the circadian rhythms in mbh mrna levels for cu / zn- and mn - sod and catalase, our previous data partially disagreed with them, e.g., the late afternoon peak in cu / zn - sod gene expression reported herein was only seen in one of our previous studies (jimnez - ortega., 2010) while the late afternoon peak in mn - sod was seen only as a trend in the two previous studies (jimnez - ortega., 2009, 2010). the administration of low amount of cdcl2 brought about significant changes in 24-h variation in gene expression of mbh cu / zn - sod, gpx, gsr, nos-2, ho-1, and ho-2. the concomitant administration of melatonin prevented most of the effects of cd on 24-h rhythmicity and overall expression of redox enzyme genes. in particular, the co - administration of melatonin and cdcl2, or the administration of melatonin alone, brought about a remarkable increase of cu / zn - sod gene expression. providing that this did reflect an increased enzyme protein activity, the elevated levels of reduced glutathione expected to occur may explain, by negative feedback regulation (griffith, 1999 ; lu, 2009), the very significant decrease in mbh mrna levels for gpx, gsr, and ho-2 found under those conditions. while some metals, like iron, copper, chromium, vanadium, or cobalt undergo redox - cycling reactions, a second group of metals including cd, mercury, and nickel cause toxicity mainly by depleting glutathione (wright and baccarelli, 2007). thus melatonin could be an effective antidote for the toxic effect of this second group of metals. the doses of cd employed in the present study may resemble real exposure level in active tobacco smokers, in moderately to heavily polluted areas or under occupational exposure conditions (brzska and moniuszko - jakoniuk, 2005). under this condition, cd intake was close to that proposed by the world health organization as a tolerable limit for humans [1 g / day for a life span of 60 years (1995) ]. it should be noted that because cd absorption in the gastrointestinal tract of rats is lower than in humans, rat models simulating human exposure need to increase exposure doses to be higher than the real daily human intake of cd (rogalska., 2009). in the present and a previous study (jimnez - ortega., 2010), a low dose of cd differentially affects nos-1 and nos-2 expression in mbh by disrupting their 24-h pattern and by decreasing the 24-h mean mrna levels for nos-2. the neuronal isoform of nos (nos-1) is constitutively expressed in neurons whereas expression of the inducible (macrophage) isoform nos-2 occurs mainly in glial cells (galea., 1992) and also in neurons nos has been detected in several hypothalamic areas including the scn (plano., 2007) and the supraoptic, paraventricular, ventromedial, and dorsomedial nuclei (ceccatelli, 1997). nos is also present in fibers at the median eminence, mainly in the internal layer and around blood vessels of the portal system (knauf. the present results reinforce the view that the toxic effect of a low dose of cd is independent on excessive no formation. it is interesting that melatonin counteracted the generally inhibitory and disrupting effects of cd on 24-h pattern of activity of nos whereas, when administered alone, melatonin decreased gene expression of mbh nos, as reported previously (jimnez - ortega., 2009) and seen again in the present series of experiments. heme oxygenase has an important role in controlling the redox state of the cell by functioning as a rate - limiting enzyme in the heme degradation process (mancuso., 2007). ho-1 is an inducible isoform that is responsive to various stimuli, including oxidative stress. ho-2 is a constitutive isoform that is highly concentrated in the brain is less inducible by oxidative stress. various hypothalamic nuclei displayed both ho-1 and ho-2 mrna proteins (ewing and maines, 1997) and enzymatic activities (rubio., 2003) explaining the high co production rate that the hypothalamus exhibits (laitinen and juvonen, 1995). in the present study cdcl2 augmented mbh mrna levels of ho-2 and affected the 24-h pattern of expression of both ho isoenzymes by inducing maxima at midday, early and late scotophase (ho-1), and at early morning (ho-2). the stimulatory effect of cdcl2 on ho-2 mrna reported in the present study can be interpreted in terms of an increased of oxidative load (i.e., more need of ho-1 expression). the administration of melatonin counteracted significantly the effect of cd on 24 h pattern in expression of both isoenzymes of ho. as shown earlier (jimnez - ortega., 2009) the inhibitory effect of melatonin on ho-1 and ho-2 mrna is remarkable. the mechanisms involved in regulation of redox enzyme gene expression by melatonin could involve receptor - mediated and receptor - independent phenomena (hardeland., 2011). among the latter inhibition of radical oxygen species since ros play a role in cellular signaling processes, including transcription factors like nuclear factor-b or activator protein-1, a decrease of free radical production by melatonin would allow the repression of redox - sensitive transcription factors, which could regulate gene transcription (lezoualch., 1998 ; beni., 2004 ; rodriguez., 2004). one important is that studies employing western blotting analysis of clock protein levels are further needed to understand cd. it should be also important to assess whether the changes in amplitude as well in timing of 24-h rhythm of gene expression discussed herein can be attributed to an effect on the circadian master clock or to a masking effect on some output(s) of the clock. the nuclear heterogeneity of the mbh fragments employed is a feasible explanation for the inability to uncover some the relationships seen between clock genes in isolated brain nuclei. summarizing, the results support the conclusion that the interfering effect of melatonin on the activity of a low dose of cd on 24-h rhythms of mbh clock and redox enzyme genes is mainly exerted at the level of redox enzyme gene expression. since most of published studies on neuroprotective activity of melatonin were performed by measuring specific targets at single time points, generally at morning hours, and in view of the 24-h changes in redox state that occurs in a number of tissues (hardeland., 2003 ; subramanian., 2008), it should be important to include a chronopharmacological approach for the full analysis of the above mentioned effects of melatonin. 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.	in a previous study we reported that a low daily p.o. dose of cadmium (cd) disrupted the circadian expression of clock and redox enzyme genes in rat medial basal hypothalamus (mbh). to assess whether melatonin could counteract cd activity, male wistar rats (45 days of age) received cdcl2 (5 ppm) and melatonin (3 g / ml) or vehicle (0.015% ethanol) in drinking water. groups of animals receiving melatonin or vehicle alone were also included. after 1 month, mbh mrna levels were measured by real - time pcr analysis at six time intervals in a 24-h cycle. in control mbh bmal1 expression peaked at early scotophase, per1 expression at late afternoon, and per2 and cry2 expression at mid - scotophase, whereas neither clock nor cry1 expression showed significant 24-h variations. this pattern was significantly disrupted (clock, bmal1) or changed in phase (per1, per2, cry2) by cdcl2 while melatonin counteracted the changes brought about by cd on per1 expression only. in animals receiving melatonin alone the 24-h pattern of mbh per2 and cry2 expression was disrupted. cdcl2 disrupted the 24-h rhythmicity of cu / zn- and mn - superoxide dismutase (sod), nitric oxide synthase (nos)-1, nos-2, heme oxygenase (ho)-1, and ho-2 gene expression, most of the effects being counteracted by melatonin. in particular, the co - administration of melatonin and cdcl2 increased cu / zn - sod gene expression and decreased that of glutathione peroxidase (gpx), glutathione reductase (gsr), and ho-2. in animals receiving melatonin alone, significant increases in mean cu / zn and mn - sod gene expression, and decreases in that of gpx, gsr, nos-1, nos-2, ho-1, and ho-2, were found. the results indicate that the interfering effect of melatonin on the activity of a low dose of cdcl2 on mbh clock and redox enzyme genes is mainly exerted at the level of redox enzyme gene expression.
preswallow pharyngeal bolus presence is viewed on videofluoroscopic swallow study (vfss) or fiberoptic endoscopic examination of swallowing (fees) amongst many patients presenting with oropharyngeal dysphagia. there are two main causes for this presentation : poor oral bolus containment with premature bolus spillage and/or a delayed pharyngeal swallow trigger. poor oral bolus containment results in passive or ineffective movement of a liquid or viscous bolus from the oral cavity into the pharynx prior to pharyngeal swallow onset. by definition this occurs whilst the oral preparatory or oral stage of swallow is still underway, that is, before or during lingual propulsion. separately, a delay in the pharyngeal swallow trigger is defined by a failure of a coordinated and timely pharyngeal response following a purposeful transfer of the bolus into the pharynx. poor oral containment leading to premature bolus spillage can occur in isolation or in combination with a delayed pharyngeal swallow trigger [13 ]. high resolution manometry with impedance (hrmi) with automated pressure flow analysis (pfa) is a new method to diagnostically interpret pharyngeal and ues function. pressure sensors detect activity of swallow musculature whilst impedance electrodes provide measures which indicate bolus flow. pfa derives a range of swallow metrics that indicate bolus flow timing, intrabolus pressure, contractile vigour, bolus presence, and ues luminal diameter, making it possible to measure and describe the function of different mechanical components of pharyngeal swallowing. a global swallow risk index (sri) generated from pfa metrics as a means to amplify dysfunction has shown to correlate with the presence of aspiration and/or postswallow residue as seen on videofluoroscopy [4, 5 ]. following on from this previous work, the purpose of this study was to use hrmi in combination with automated pfa to objectively describe pressure - flow patterns in the pharynx and ues in the event of preswallow pharyngeal bolus presence as seen on videofluoroscopy. we hypothesised that specific pfa metrics would be altered in patients with preswallow pharyngeal bolus presence compared to patients without preswallow pharyngeal bolus presence and controls. the aim of this study was to identify the altered pfa metrics which may provide a means to describe functional changes in the pharynx in the event of preswallow pharyngeal bolus presence. we analysed vfss investigations performed in 40 adult patients with broad dysphagia (24 males, mean age 46 yrs, and age range 2395 yrs) and 8 adult controls (3 males, mean age 38 yrs, and age range 2447 yrs). at the time of initial investigation, all subjects were enrolled in study protocols that were approved by the research ethics committee, university hospital leuven, belgium. in patients with dysphagia, underlying diseases / conditions were identified through a review of medical records. eighteen patients had a neurological history (10 patients were post stroke, 2 had parkinson 's disease, 1 huntington 's disease, 1 multiple sclerosis, 2 dementia, 1 spina bifida and 1 post - neurosurgery). of the remaining four patients 1 was post - cervical surgery, 1 wegener disease, 1 postseptic shock, and 1 diabetes. this database of patients with broad dysphagia has previously been reported on [49 ], and the diverse clinical presentations and dysphagia severity have been purposeful in order to concept - test pfa metrics in relation to radiological measures from vfss. previously this has included aspiration status, postswallow residue, ues opening, and in this case preswallow pharyngeal bolus presence. studies were performed in the radiology department, university hospital leuven, with a 3.2 mm diameter solid state high resolution manometry and impedance catheter incorporating 25 1 cm - spaced pressure sensors and 12 adjoining impedance segments, each of 2 cm (unisensor ag catheter, attikon switzerland). subjects were intubated after topical anaesthesia (lignocaine spray) and the catheter was positioned with sensors straddling the entire pharyngoesophageal segment (velopharynx to proximal esophagus). pressure and impedance data were acquired at 20 hz (solar gi acquisition system, mms, netherlands) with the subject sitting upright. most subjects were tested with at least 5 boluses in the lateral view : liquid (x3), semisolid (x1), and solid boluses (x1). a standard liquid contrast material (micropaqueh) was given as liquid bolus and used with thickener (thick & easy) for semisolid boluses. a low osmotic hydrosoluble iodium compound (ultravisth) the bingham viscosity of the liquid barium (micropaqueh) was 0.22 pascal seconds (pas), 4.50 pas for the semisolid bolus. all controls were given boluses of 10 ml volume while patients were given either 5 ml or 10 ml volumes as determined on clinical grounds by the attending specialist. solid boluses consisted of a 4 cm piece of bread soaked in the appropriate radiological marker which was chewed and swallowed. all swallows were prompted and the first swallow following bolus administration was marked for analysis. continuous videofluoroscopy sequences (25 frames / sec) of swallows were analysed by a speech pathologist (author lara ferris) who was not present during acquisition and was blinded to study functional measures, patient history, and clinical reports. each primary bolus swallow was reviewed to determine movement of the bolus and the level reached from the oral cavity into the distal pharynx prior to the onset of the pharyngeal swallow, defined by onset of rapid laryngeal excursion [1, 10, 11 ]. the levels reached were defined as follows : still in the oral cavity, base of tongue, valleculae, or pyriform sinuses. based on the results for all the analysed swallows patients were classified as follows : group 1 : patients who never demonstrated bolus movement to the pyriform sinuses prior to swallow onset.group 2 : patients who demonstrated bolus movement to the pyriform sinuses at least once prior to swallow onset. group 1 : patients who never demonstrated bolus movement to the pyriform sinuses prior to swallow onset. group 2 : patients who demonstrated bolus movement to the pyriform sinuses at least once prior to swallow onset. note : amongst controls, at swallow onset bolus movement was observed in the mouth or at the base of tongue for all swallows. pharyngeal pfa was performed using automated impedance manometry software (aimplot) to calculate pfa metrics and two global indices which have been previously validated against vfss, namely, the sri, indicative of global dysfunction, and the integrated ratio of nadir impedance to impedance (izn / z), indicative of postswallow residue. the calculations used to derive pfa metrics have been previously described [49, 12 ]. in brief, pressure impedance recordings are displayed as pressure topography plots with embedded impedance recordings which show bolus flow movements, the pharyngeal stripping wave, and relaxation and movement of the ues pressure zone (figure 1(b)). on selection of specific landmarks on the pressure topography space - time plot (with embedded impedance recordings), specific regions of interest (roi) are mapped. the landmarks identified are (1) time of onset of pharyngeal swallow, (2) position of the ues proximal margin postswallow, and (3) position of the velopharynx during the swallow (numbered 13 in figure 1(b)). there are three roi encompassing (1) the pharynx, (2) distal pharynx, and (3) ues. within each of the roi specifically, identification of peak pressure defines the maximum contraction in space and time, the nadir impedance (nadimp) defines the centre of the swallowed bolus in space and time (figures 1(b) and 1(c)), and the pressure at nadir impedance (pnadimp, figure 1(c)) defines pharyngeal intrabolus pressure. the time interval from nadir impedance to peak pressure (tnadimp to peakp, figure 1(c)) measures the time from bolus passage to pharyngeal contraction ; the flow interval (fi) correlates with pharyngeal bolus transit time. the ues nadir impedance (ues nadimp) is measured as a correlate of ues opening diameter and the ues intrabolus pressure (ues ibp) is measured using the established method of ghosh. the swallow risk index (sri) was empirically derived and designed to amplify difference in swallow metrics previously shown to be altered in relation to swallow dysfunction and aspiration risk [4, 5 ]. these validation studies using concurrent vfss showed that the sri for liquid and viscous swallows is significantly higher in patients demonstrating penetration - aspiration compared to patients with no penetration or aspiration [4, 5 ]. therefore the sri quantifies the overall level of swallowing dysfunction potentially predisposing to aspiration risk [4, 5 ]. a postswallow residue score was designed using the integrated ratio of nadir impedance to impedance (izn / z ratio) which relates postswallow impedance to the impedance during bolus passage. data gathered from multiple swallows were averaged for each subject. only liquid boluses (5 or 10 mls) were assessed. data distribution across the study cohort was nonparametric and therefore medians (interquartile range) are presented. mann - whitney u tests were used to compare controls and patients grouped on vfss assessment with bonferroni correction for multiple comparisons, p < 0.017. amongst controls a total of 24 liquid swallows were analysed and 102 swallows were analysed from 40 broad dysphagia patients. controls showed bolus in the mouth or at the base of tongue at the time of swallow onset in all cases. 20 of 40 patients showed bolus to the pyriform sinuses at least once and this was the most frequent bolus position at swallow onset in 75% of this patient group. as shown in figure 2 using mann - whitney u tests, in this cohort of broad dysphagia patients the sri, a global measure of swallowing dysfunction, was higher in relation to evidence of bolus to the pyriform sinuses prior to the pharyngeal swallow onset compared to controls (p < 0.001), and a trend was observed compared to patients who did not spill to the pyriform sinuses (p = 0.02). the tnadimp - peakp (poor ability for the bolus to be propelled ahead of the pharyngeal stripping wave) was shorter in both patient groups (group 1 p < 0.002 and group 2 p < 0.001) compared to controls ; the flow interval (suggesting extended pharyngeal bolus dwell time) was longer in both patient groups (group 1 p < 0.001 and group 2 p < 0.002) compared to controls ; and the izn / z (postswallow residue metric) was significantly higher in both patient groups (group 1 p < 0.000 and group 2 p two individual metrics in this data differentiate the patient groups : group 1 : patients without preswallow bolus presence to the pyriform sinuses prior to swallow onset and group 2 : patients with preswallow bolus presence to the pyriform sinuses prior to swallow onset. pharyngeal mean impedance (correlating with reduced pharyngeal distension) was significantly higher (p < 0.002) in group 2 compared to group 1 and controls (p < 0.001) and the ues mean impedance (correlating with reduced ues opening) was higher (p < 0.000) in group 2 compared to group 1 and controls (p < 0.001). collectively this pattern of pressure - flow differences is consistent with impairment of the mechanisms that drive bolus propulsion, pharyngeal distension, and relaxation and opening of the ues in accordance with laryngeal excursion. as has been shown previously, a higher ues impedance recording correlates with reduced ues opening diameter and therefore, in the case of preswallow pharyngeal bolus presence to the pyriform sinuses, an explanation for the increase in pharyngeal and ues mean impedance : bolus movement to the pyriform sinuses before swallow onset leads to a greater loss of bolus volume of the remaining propelled bolus. a smaller bolus volume for propulsion results in reduced distension of the pharynx and ues during the swallow. vfss and fees are currently the most widely used instrumental swallow assessments ; however differential diagnosis of oropharyngeal dysphagia can be difficult based on visualisation alone. the use of pharyngeal manometry for the assessment of dysphagia has its limitations and has been described by nativ - zeltzer. in 2012 ; however the integration of concurrent high resolution manometry with impedance allows for dynamic swallow function assessment, and its potential to assist in the evaluation of swallow function is beginning to emerge, as has been demonstrated in this and a number of other recent studies [49, 12 ]. within the adult population, there is discussion in the literature regarding normal variability for the presence of the bolus lower in the pharynx prior to swallow onset [1519 ]. however, the spillage or propulsion of all or part of the bolus to the pyriform sinuses prior to pharyngeal swallow trigger is a pathological event [13, 20 ] suggesting markedly altered mechanics of bolus transport through the pharynx. the clinical relevance in detecting preswallow pharyngeal bolus presence lies in the fact that recognising the causes for bolus presence in the pharynx prior to pharyngeal swallow onset is important for treatment of oropharyngeal dysphagia. our findings demonstrate how specific pfa metrics are altered in relation to a general pathological observation of preswallow pharyngeal bolus presence in dysphagia patients. this pilot study presents specific pressure flow analysis metrics (using integrated high resolution manometry and impedance) which are significantly associated with a known pathological presentation of oropharyngeal dysphagia, that is, preswallow pharyngeal bolus presence to the pyriform sinuses. these results provide reason to further explore the potential differences in pressure flow analysis metrics that may distinguish the causes for preswallow pharyngeal bolus presence, that is, poor oral containment and/or delayed pharyngeal trigger pressure flow analysis with aimplot software deriving metrics and a global measure of dysfunction, the swallow risk index, have the potential to guide diagnosis and treatment of patients with oropharyngeal dysphagia and may be used to track patient changes in swallow function over time.	objectives. preswallow pharyngeal bolus presence is evident in patients with oropharyngeal dysphagia. pressure flow analysis (pfa) using high resolution manometry with impedance (hrmi) with aimplot software is a method for objective interpretation of pharyngeal and upper esophageal sphincter (ues) pressures and bolus flow patterns during swallowing. this study aimed to observe alterations in pfa metrics in the event of preswallow pharyngeal bolus presence as seen on videofluoroscopy (vfss). methods. swallows from 40 broad dysphagia patients and 8 controls were recorded with a hrmi catheter during simultaneous vfss. evidence of bolus presence and level reached prior to pharyngeal swallow onset was recorded. aimplot software derived automated pfa functional metrics. results. patients with bolus movement to the pyriform sinuses had a higher sri, indicating greater swallow dysfunction. amongst individual metrics, tnadimp to peakp was shorter and flow interval longer in patient groups compared to controls. a higher pharyngeal mean impedance and ues mean impedance differentiated the two patient groups. conclusions. this pilot study identifies specific altered pfa metrics in patients demonstrating preswallow pharyngeal bolus presence to the pyriform sinuses. pfa metrics may be used to guide diagnosis and treatment of patients with oropharyngeal dysphagia and track changes in swallow function over time.
in middle - age, hypercholesterolemia (hc) is one of the major risk factors for heart disease patients and 10% serum cholesterol reduction is associated with 30% mortality rate among them. the prevalence rate of hc was not equal in two genders as far as varied in populations from 3% to 53% in men and from 4% to 40% in women. some plasma cholesterol correlated factors are included lifestyle, diet, smoking, central obesity, gender, physical activity and age. the association between waist circumference (wc) and cardiovascular disease (cvd) are established well in some studies besides a negative correlation between serum cholesterol level and height was observed in men. the alterations in lipid and lipoprotein concentration resulted in a changing of cvd risk factors. the mortality and morbidity rates due to cvd have been estimated by plasma cholesterol levels in young and adult people in framingham study. direct correlation between serum cholesterol level and age has been seen in men and women. waist to hip ratio (whr) was directly related to serum cholesterol. in pubertal period, the prevalence of hc increased with age and was significantly common in men more than in women at age range 24 - 29 years. in tibet adults, golestan province is in the north of iran (south east of caspian sea). of 1.7 million people in this area, 66.39% were 15 - 64 years old, also 71% and 29% are living in urban and rural areas, respectively. agriculture is the main job in a rural area and different ethnic groups such as fars (native), turkman and sisstani live in this region. the aim of this study is to evaluate the association between serum cholesterol level and abdominal obesity in men and women in 25 - 65-year - old people in golestan province in the north of iran. this is a cross - sectional study conducted on the 1956 subjects aged 25 - 65 years (990 men and 966 women). the required sample size based on the previous study, with estimation of 25% hc rate ; a confidence level of 95% and a maximum marginal error 0.02 was calculated 1800. the sample size increased to 2000 to compensate for an expected dropout rate of approximately 20%. subjects were chosen randomly from 100 clusters and each cluster comprised 20 cases with equal proportions of genders and age groups in 11 districts. in the first stage, the clusters were chosen randomly using systematic sampling technique based on postal code in urban areas and family health number in primary health centers in rural areas. in the second stage, we selected 20 subjects randomly in each cluster. the subjects who were pregnant, on lowering drugs and those who refused to participate in this study were excluded from the study. waist circumference was measured to the nearest 0.5 cm at the superior border of the iliac crest. abdominal obesity was defined based on who the classification with a wc 102 cm and 88 cm in men and women, respectively. the blood was drowned from each subject after 12 h fast in the morning in order to measure the serum cholesterol level. it was measured on commercial kits (pars azmoon, karaj, iran) by auto - analyzer. spss 16.0 software (chicago ii, usa) was used for the statistical analysis using chi - square test and t - test for comparing frequencies and the means, respectively. logistic regression analysis was applied in order to estimate the odds ratio (or) of hc considering the wc ranges at 95% significant level. this study was approved by ethical research committee and consent was received from all participants. mean and standard deviation of age and serum cholesterol level were 44.2 11.3 years and 203 11.3 mg / dl, respectively. the prevalence of abdominal obesity was seen in 42.6% (35.9% in men and 49.6% in women). hc was common in 50.8% with more prevalence in women (57.0%) than men (44.7%) [table 1 ]. characteristics of subjects, mean (sd), (total=1956, men=990, women=966) distribution of serum cholesterol levels and wc based on age in men were presented in table 2. as whole the mean of cholesterol was 20 mg / dl in abdominal obese more than in normal people and statistical differences was significant among all age groups (p < 0.001). results of logistic regression analysis showed that the or of hc is (or = 4.208 [95% confidence interval [ci ] : 1.9399.130 ]) in abdominal obese compared to normal subjects and (or = 3.956 [1.8218.592 ]) in men aged 2535 and 3545 years, respectively. or was not significant after age 45. the association between serum cholesterol level and wc based on age in men distribution of serum cholesterol levels and abdominal obesity based on age in women are presented in table 3. the mean of serum cholesterol levels differences was significant among normal and abdominal obese in all age groups (p < 0.001). the risk of hc is (or = 3.444 [95% ci : 1.9596.056 ]) in abdominal obese compared to normal subjects in women aged 2535 years. or was not significant in other age groups. the association between serum cholesterol level and wc based on age in women contrary to normal subjects, abdominal obese people 's mean serum cholesterol level remained significantly high in all of the age groups except for men aged 4555 years. the association between hc and abdominal obesity in men and women was not similar. in men aged under 45 years and in women aged under 35 years, the or for hc was seen in abdominal obese subjects more than in normal ones. the same as our study, the association between serum cholesterol level and abdominal based on age and sex has been reported in other studies. in a systematic review study, the direct association between serum cholesterol level and age was reported in two genders. among 25 - 64 years old, hc increased with age and it was more in men more than in women in 24 - 49 years age group. the association between age and serum cholesterol was not similar in all studies. besides, inverse relationship between dyslipidemia and both wc and age among turkish adult men, the decline trend has been shown between hc and body mass index when adjusted with age in who monica project. like above studies, we found the association between abdominal obesity and serum cholesterol level while it was weaker in upper age and less in women. with regard to different ethnic groups in the north of iran, genetic factors probably influence on changing of serum cholesterol level and it should be considered in future studies. lifestyle had a meaningful role in changing of serum cholesterol level in adults. look upon changing of lifestyle in iran as a developing country, we recommended to establish an educational planning for controlling the abdominal obesity and hc especially in early middle age. diet, physical activity, ethnicity, job and background disease were not assessed in our study. in addition, we did not provide a proper statistical test to evaluate the design effect resulted by cluster sampling. if we used a proper statistical test, it would change the narrow ci and low p value. hypercholesterolemia is a major health problem in the north of iran and abdominal obesity is associated with it. in the early middle age, the association between wc and serum cholesterol level in abdominal obese subjects was more than in normal subjects, while this association has not been revealed in men and women upper age 45 and 35 years, respectively.	background : the main aim of this study is to evaluate the association between serum cholesterol level and abdominal obesity in adult men and women in the north of iran.materials and methods : this cross - sectional and analytical study was conducted on the 1956 subjects (990 men and 966 women) between 25 and 65 years old chosen by cluster sampling. plasma cholesterol was measured in the morning after a 12 h fast and determined by auto - analyzer. hypercholesterolemia (hc) was defined by a total plasma cholesterol level over 200 mg / dl. waist circumference 102 cm and 88 cm in men and women were defined as abdominal obesity. spss 16.0 software was used for statistical analysis and p < 0.05 considered as statistical significance.results:averagely, the mean of age was 44.2 years and mean standard deviation of plasma total cholesterol level was 203 11.3 mg / dl. the hc was seen in 50.8% of subjects with a more common in women than in men. compared with normal subjects, in abdominal obese people, the odds ratio (or) of hc was (or = 4.208 [95% confidence interval [ci ] : 1.9399.130 ]) and (or = 3.956 [95% ci : 1.8218.592 ]) in men aged 2535 and 3545 years, respectively. in women aged 2535 years, it was (or = 3.444 [95% ci : 1.9596.056 ]) in abdominal obese compared with normal subjects.conclusion:hypercholesterolemia was revealed as a major health problem among adults, and it was associated with abdominal obesity especially in the early middle - age in the north of iran. this association was not significant in men and women after the age of 45 and 35, respectively.
rheumatoid arthritis (ra) is an autoimmune arthritis affecting joints mainly, chronic inflammatory, along with many other tissues and organs. the process produces an inflammatory response of the synovitis secondary to hyperplasia of synovial cells, excess synovial fluid, and the development of pannus in the synovium. the pathology of the disease process often leads to the destruction of articular cartilage and ankylosis of the joints. owing to the lung tissue has redundant connective tissue and the close relation with blood vessels, and also has a link - intensive cycle system, the lung is one of the primary target organs. interstitial lung disease (ild) is the most common manifestation of rheumatoid lung disease [13 ]. about 0.8% of the world 's population rheumatoid lung disease occurs in about 28% of patients, either in the beginning or during the course of their disease. the presence of rheumatoid lung disease is associated with severe active disease and increased mortality compared to the general population [4, 5 ]. its pathogenesis may be related to cd4 cd25 regulatory t cells (treg) (figure 2) and foxp3 expression in a previous study [6, 7 ]. tripterygium glycosides tablet comprises triptolide which is a traditional medicinal plant that has been used in china for many years to treat inflammatory conditions including ra. in this study we will be testing tripterygium glycosides tablet that is superior to methotrexate in improving the paw swelling degree, arthritis index (ai), pulmonary function, and so on. rats were purchased from the experimental animal center of nanjing medical university (nanjing, china). all animals were housed under specific pathogen free (spf) conditions and given free access to water and standard rat chow. tripterygium glycosides tablet (tpt), 10 mg per piece, was produced by the shanghai medical hongqi pharmaceutical factory, batch number no. 20110819.methotrexate (mtx), 2.5 mg per piece, was produced by the shanghai traditional chinese medicine co., ltd. tripterygium glycosides tablet (tpt), 10 mg per piece, was produced by the shanghai medical hongqi pharmaceutical factory, batch number no. 20110819. methotrexate (mtx), 2.5 mg per piece, was produced by the shanghai traditional chinese medicine co., ltd. 20111004. elisa kit was purchased from r & d company, usa. interleukin-10 (il-10, lot number 341225), tumor necrosis factor alpha (tnf-, lot number no. endothelin-1 (et-1, lot number 340918).freund 's complete adjuvant (fca, sigma, usa, lot number no. 098k8729).regulatory t cells kit : anti - mouse cd4-fitc (ebioscience, usa, lot : 11 - 0040, clone no. ox35), anti - mouse cd25-pe (biolegend, usa, lot number : 202105, clone no. ox-39).anti - mouse foxp3 monoclonal antibody (santa cruz, usa, lot : sc-130666).pcr master mix kit and m - mulv reverse kit (fermentas, canada, lot : k0171, k1622). elisa kit was purchased from r & d company, usa. interleukin-10 (il-10, lot number 341225), tumor necrosis factor alpha (tnf-, lot number no. 341012). freund 's complete adjuvant (fca, sigma, usa, lot number no. 098k8729). regulatory t cells kit : anti - mouse cd4-fitc (ebioscience, usa, lot : 11 - 0040, clone no. ox35), anti - mouse cd25-pe (biolegend, usa, lot number : 202105, clone no. ox-39). anti - mouse foxp3 monoclonal antibody (santa cruz, usa, lot : sc-130666). pcr master mix kit and m - mulv reverse kit (fermentas, canada, lot : k0171, k1622). microplate reader was produced by bio - tek corporation, usa (model : elx800).pcr amplification was produced by biometra inc., germany (model : t1-thermoblock, t - gradient thermoblock).anires 2003 animal lung function analysis system was produced from beijing bei lanbo technology co., ltd., china.electrophoresis was produced by amersham corporation, usa (model : eps-301). microplate reader was produced by bio - tek corporation, usa (model : elx800). anires 2003 animal lung function analysis system was produced from beijing bei lanbo technology co., ltd., china. electrophoresis was produced by amersham corporation, usa (model : eps-301). the rats were randomly divided into four groups, the norma control (nc) group, model control (mc), methotrexate (mtx), and tripterygium glycosides tablet (tpt) group, 12 rats in each group. except for the rats of nc group, the others were intracutaneously injected with 0.1 ml of freund 's complete adjuvant in the right hindlimb. administration from 19th after inflammation, nc group and mc group were treated with physiological saline (1 ml/100 g per day). mtx group and tpt group were treated with mtx, 1 ml/100 g per week, and tpt, 1 ml/100 g per day. 12th after inflammation, the joints were observed and recorded, once every three days. calculated ai by five scoring method. no swelling (0 point) ; swelling from little toe joint (1 point) ; swelling from toe joints and foot (2 points) ; swelling from ankle and below (3 points) ; swelling from all of ankle (4 points). we have (1)e(%)=(vtvn)vn100% (vn, vt represent the volume before and after modeling). we have (2)lung index (li)=lung wet weight (mg)body weight (g)100%. 30th after administration, remove the lungs tissue, and tissue was fixed in 4% paraformaldehyde 8 according to szapiel method to determine the extent of alveolitis : 0 points : without alveolitis () ; 1 points : mild alveolitis (+), lesions were confined to below 20% of total lung tissue ; 2 points : moderate alveolitis (+ +), lesions reach about 40%50% of total lung tissue ; 3 points : severe alveolitis (+ + +), lesions were more than 50 percent of total lung tissue. average expiratory flow (fev1/fvc %), 25% vital capacity of the peak expiratory flow (fef25), 50% vital capacity of the peak expiratory flow (fef50), 75% vital capacity of the peak expiratory flow (fef75), maximum mid - expiratory flow (mmf), peak expiratory flow (pef). process of the operation : the rats were anesthetized with chloral hydrate (10%, 0.35 ml/100 g) through intraperitoneal injection and underwent tracheotomy endotracheal intubation. the rats were put inside the body of description to keep head low, and the ventilator tube was connected to mechanical ventilation in order to test pulmonary function. in the determination process, use the way that external pressure to make animal deep inspiration / deep breath. whole blood samples were taken into k3-edta containing tubes and added to each tube with blood (10 cells per tube), anti - mouse cd4-fitc (0.25 ug), anti - mouse cd25-pe (1.0 ug) successively. add 1 ml rbc lysate to tube and incubate for 1525 minutes in dark again. rna was extracted by trizol (takara co., japan) from 100 mg lung tissue and quantified using a spectrophotometer (eppendorf co., german). three micrograms of total rna were reverse transcribed into cdna using murine moloney leukemia virus (m - mlv) reverse transcriptase (promega, usa). pcr was carried out according to the manufacturer 's instructions. the house keeping gene gapdh (genbank accession : nm-017008) : sense : 5-tcc acc acc ctg ttg ctg tag-3, and antisense : 5-cca cag tcc atg cca tca ct-3. amplified fragment 258 bp. the foxp3 gene (genbank accession : nm-001108250) : sense : 5-gca aac gga gtc tgc aag tg-3, and antisense : 5-gca gga gct ctt gtc cac tga-3, amplified fragment 450 bp. pcr products were analyzed using gel works software after scanning the ethidium bromide - stained 1.5% agarose gel. protein was lysed in gel - loading buffer containing 50 mm tris - hcl (ph 6.8), 100 mm dithiothreitol, 2% sodium dodecyl sulfate, 0.1% bromophenol blue, and 10% glycerol. fifty micrograms of total protein was resolved by sds polyacrylamide gel electrophoresis and electrically blotted onto a nitrocellulose membrane. the filters were blocked with phosphate buffered saline (pbs) containing 15% nonfat milks. detection of foxp3 or beta actin was carried out by western blot analysis with the mouse anti - foxp3 monoclonal antibody (1 : 500) or the rabbit anti - beta actin polyclonal antibody (1 : 5000) as the primary antibody, and goat anti - mouse or goat anti - rabbit igg - conjugated horseradish peroxidase as secondary antibody. levels of tnf-, il-10, and et-1 in supernatants of different groups were measured using commercially available elisa kits according to the test protocols. values were expressedas pg / ml. the elisa standard curve was prepared using a serial dilution of tnf-, il-10 and et-1 standard protein concentrations. the levels of recombinant tnf-, il-10, and et-1 in the supernatant of the cells culture were calculated from the od450 values according to the elisa curve of the commercial tnf-, il-10, and et-1 standards. lung tissue was dewaxed and rehydrated followed by antigen retrieval through microwaving in 2 mm edta (ph 9.0) for foxp3 antigen. sections were blocked with 5% bovine serum albumin (diluted in pbs) for 30 min and then incubated with each primary antibody in a moist chamber at 4c overnight. parallel sections from the same tissue block were used for the staining of all molecular variables. after washing in pbs, hrp polymer - linked secondary antibody was added for 60 min at room temperature. sections for the negative control were prepared using rabbit igg1 or mouse igg1 instead of primary antibody under the same experimental conditions. continuous variables are the mean standard deviation. all samples were tested to ascertain if they followed a normal distribution. before inflammation, there was no obvious difference between paw swelling degree and arthritis index in each group. before administration, the level of paw swelling degree and arthritis index of mc group, mtx group, tpt group were significantly higher than that in the nc group (p < 0.05 or p < 0.01). after administration, compared to the nc group, the level of paw swelling degree and arthritis index of mc group were increased significantly (p < 0.01). compared to the mc group, the level of swelling degree and arthritis index of mtx and tpt group were reduced significantly (p < 0.01). compared to the mtx group, the level of swelling degree and arthritis index of tpt group were reduced significantly (p < 0.01). 30th after administration, pulmonary function parameters such as fef50, fef25, fvc, fef75, mmf, and pef were significantly decreased, then fev1/fvc, li and score of alveolitis were increased in mc group. fvc, fef25, fef50, fef75, mmf, and pef were decreased and the score of alveolitis was increased with treatment of tpt. 30th after administration, compared to the nc group, the concentrations of tnf-, et-1 in serum and et-1 in lung tissue were significantly increased, and il-10 in serum was decreased obviously in the mc group (p < 0.01). the concentrations of tnf- and et-1 in tpt group were significantly lower than those in mc group, and the concentrations of il-10 were significantly higher than those in mc group (p < 0.01). the differences of tnf-, et-1 between tpt group and mtx group were statistically significant (p < 0.01). we detected treg expression by flow cytometry in peripheral blood (figures 1(a)1(d)), and immunohistochemistry, rt - pcr, and western blot were used to detect the expression of foxp3 in lung tissue. flow cytometry results showed that the expression of cd4 cd25 treg was decreased significantly in mc group and increased in tpt group. immunohistochemistry results showed that the expression of foxp3 in lung tissue was concentrated in nucleus and cytoplasm. the expression of foxp3 was decreased in the mc group and increased in the tpt group. rt - pcr and western blot results showed that the expressions of foxp3 mrna and protein were decreased significantly in mc group (figures 3 and 4). we detected that paw swelling, ai, alveolitis points, tnf, and et-1 expression were significantly increased in rats experimentally developed with adjuvant arthritis, while fvc, fef25, fef50, fef75, mmf, pef, il-10, cd4 treg, cd4 cd25 treg, and foxp3 expression in lung tissue were significantly decreased. these studies have shown inflammatory reaction was emerged in the joints of rats developed with aa, while their pulmonary function has been changed, the level of pulmonary function declines as a result. pulmonary function change was further indicated by the observation of pulmonary ventilation function disorder, particularly restrictive ventilatory disorder, accompanied by small airway obstruction. it is worthwhile to note that an imbalance between proinflammatory and anti - inflammatory cytokine existed, suggesting that the inflammation was occurred in both joints and lung. the abnormal expression of regulate t cells and foxp3 shows treg was probably involved in ra pathogenesis of lung injury [1317 ]. modern pharmacological studies show that triptolide has anti - inflammatory and immunomodulatory active ingredients effects in experimental animals. pharmacological and clinical experiments also show that the major effective component of tripterygium is alkaloids, may directly lead to the results that reduce capillary permeability, inhibit infiltration of inflammatory exudation, inhibit or counter modulate various types of inflammatory mediators as well as the anticoagulant, antiembolism, and reduce the damage on lung tissue [18, 19 ]. the immune modulatory effect of tripterygium ranged widely from the humoral immunity, cellular immunity, to macrophage phagocytosis. triptolide, its monomer, has inhibition on inflammatory joint edema was induced by carrageenan, croton oil, and freund 's complete adjuvant. a study also showed that triptolide has improved the role that could improve the recovery of alveolitis and fibrosis. triptolide can reduce the size of alveolitis and pulmonary fibrosis and increase alveolar space. in our results, compared with the aa rats, fvc, fef25, fef50, fef75, mmf, pef, il-10, and the expression of regulate t cells, foxp3 was significantly increased in the triptolide - treated group, while paw swelling, ai, tnf-, and et-1 decreased. there also found that the treatment effect of tripterygium was more obvious than that of triptolide. the levels of cd4 cd25 treg decreased in peripheral blood of the aa rats obviously. it may be because of that the diminished cd4 cd25 treg level is correlated to the breakdown of the autoimmune balance and the development of rheumatoid arthritis - induced lung injury, for low level of cd4 cd25 treg can not sufficiently convert cd4 cd25 t cells into regulatory cells through immune induction. we proposed that as a consequence, the cd4 cd25 t cells can be converted into treg that secretes il-10 in peripheral blood of the aa rats, thus promoting the expression of foxp3 in lung tissue, then making the high level expression of anti - inflammation cytokine and the low level expression of proinflammatory cytokines. finally the immunosuppression activity of cd4 cd25 treg is exemplified exclusively, as our data implied. our results showed that tripterygium can obviously improve pulmonary function in aa rats, and the mechanism may function through inhibiting the expression of tnf-, et-1, upregulating cd4 cd25 treg level, and promoting the expression of il-10 and foxp3 [2026 ]. it is suggested that tripterygium can upregulate cd4 cd25 treg and foxp3 expression, playing the role of the immune adjustment [2730 ]. tnf- is a upstream regulatory factor of this process, which can induce accumulation of macrophages, secrete large amount of tgf- and et-1, expand the inflammatory response, and promote the formation of synovitis inflammation and lung disease. tnf- not only induces the amplification of th2 cells, but also limits the immunosuppression activity of cd4 cd25 regulatory t cells, which suppress immune tolerance [3134 ]. in this study, we concluded that tripterygium plays a vital role of immune adjustment in lung injury.	objective. to observe the effects of tripterygium glycosides tablet (tpt) on swelling degree, arthritis index (ai), pulmonary function, cytokines, the expression of regulatory t cells (treg), and foxp3 in rats of adjuvant arthritis. methods. rats were averagely divided into normal control (nc) group, model control (mc) group, methotrexate (mtx) group, and tripterygium glycosides tablet (tpt) group. except for the rats of normal group, the others were intracutaneously injected with 0.1 ml of freund 's complete adjuvant in the right hindlimb. nc group and mc group were treated with physiological saline. mtx group and tpt group were treated with mtx, tpt, respectively. results. the levels of swelling degree, ai, the alveolar inflammation integral, tnf alpha (tnf-), and endothelium-1 (et-1) in mc group were significantly increased (p < 0.01), and the levels of forced vital capacity (fvc), 25% vital capacity of the peak expiratory flow (fef25), 50% vital capacity of the peak expiratory flow (fef50), 75% vital capacity of the peak expiratory flow (fef75), maximum midexpiratory flow (mmf), peak expiratory flow (pef), interleukin-10 (il-10), cd4 + cd25 + treg, and foxp3 were decreased (p < 0.01). the scores of alveolitis and et-1 were decreased with treatment of tpt. the levels of fvc, fef25, fef50, fef75, mmf, pef, il-10, and cd4 + cd25 + treg in peripheral blood were increased. the expressions of foxp3 protein and mrna in lung tissue were also increased in tpt group. conclusions. the paw swelling can be inhibited by tpt, and the inflammatory response in lung tissue was also decreased, which is a significant improvement in pulmonary function. the mechanism is probably associated with upregulating the expression of il-10, foxp3, and downregulating the level of tnf-.
changes in land systems, human - induced transformations of ecosystems and landscapes and the resulting changes in land cover, reach far beyond local alterations and are pervasive factors of global environmental change. today, more than 75% of the earth 's ice - free land shows significant evidence of land use induced alterations of many environmental processes, such as primary production, the water cycle, biogeochemical cycles, the climate system, and biodiversity. on the other hand, land provides vital socioeconomic resources to society, such as of food, fuel, fibres and many other ecosystem services that support production functions, regulate risks of natural hazards, or provide cultural and spiritual services. land system changes are the direct result of human decision making at multiple scales, with far reaching consequences for the earth system, that feedback on human well - being and decision making. thus, land system change is both a cause and consequence of socio - ecological processes that encompasses a huge range of spatio - temporal scales. land systems represent the terrestrial component of the earth system and encompass all processes and activities related to the human use of land, including socioeconomic, technological and organizational investments and arrangements, as well as the benefits gained from land and the unintended social and ecological outcomes of societal activities. thus, land system science has emerged to serve as a platform for integration of these different dimensions of global environmental change research, and aims at offering potential options for mitigation and adaption to environmental change, for example through modified land system architecture. by studying the mutual interplay between social and ecological systems that shape land use and land cover, land system science operates at the interface of the social and natural sciences and requires a high level of interdisciplinary collaboration across academic disciplines land system science has developed over the past twenty years from the study of land use and land cover change, which initially was dominated by monitoring and modelling of the ecological impacts of land cover changes such as deforestation and desertification on the natural system [24 ]. gradually, the research field has become more integrative, focusing on both the drivers and impacts of land change as part of global environmental change. the growing group of researchers engaged in this field led to the emergence of land change science as a separate, interdisciplinary, research field engaging scientists across the social, economic, geographical and natural sciences. the increasing attention for feedbacks between drivers and impacts including adaptive behaviour, the interactions between social and ecological systems and teleconnections between world regions and between cities and their rural hinterlands have motivated an integrated socio - ecological systems perspective. in this perception, land systems this perspective has also moved land system science from a focus on the most dramatic land cover changes to giving more attention to subtle changes of human interactions with the natural surroundings, including land management and the provisioning of a wide range of ecosystem services. the articles in this issue strongly reflect this shift in perspective by explicitly addressing changes in land management and the modes in which land is governed. the land system science community is organized within the global land project, one of the core projects of both the international geosphere biosphere programme (igbp) and the international human dimensions programme on global environmental change (ihdp) commissioned by the international council for science (icsu) and the international social science council (issc). in 2013, a new programme gathering all previous global environmental change programmes was established and named future earth in response to a visioning process on earth system science and global sustainability initiated by icsu. the aims of the new programme, that will also host the global land project, include a stronger interdisciplinary approach and a stronger focus on science that supports sustainability transitions through co - design and co - production of research together with important stakeholders. while interdisciplinarity is in the genes of land system science, a stronger engagement in the development of sustainability solutions provides an important opportunity for the researchers engaged in this field. traditionally land system science however, scientific insights are not always easily integrated in these processes and much land is owned and managed by private land owners that are not always responsive to planning and policy. therefore, new ways of linking science and practice need to be developed to effectively translate scientific findings into sustainability solutions and implementation in practice. important ways forward in this perspective include the evaluation and design of alternative ways to govern land resources [1214 ] and the use of land systems architecture in the design of novel land systems that more optimally use spatial and temporal interactions within the land system configuration to provide ecosystem services and adaptive capacity under conditions of global environmental change. many of the articles in this issue address these challenges and illustrate the role of land system science as a central and critical component of global sustainability science. the articles in this issue provide a synthesis of many, interrelated, topics in land system science, either from a thematic or methodological perspective. overall, the articles can be divided into four main approaches to studying land systems : land systems dynamics, land use intensity, impacts of land change and governance of land systems. the first four articles deal with linkages between the local and global, teleconnections and modelling approaches to understanding and influencing land system dynamics. use the concept of telecoupling to review how local processes of land system change are affecting other, often distant, regions and how demand and policy often have indirect consequences in different world regions. the authors argue for combining place - based research and global modelling to better understand the causal links between flows of goods and services and land system change. state that land systems research should address the interactions between urban and rural areas as well as the transformations within urban and peri - urban landscapes. they propose a conceptual framework that allows identifying and examining linkages between urbanization dynamics and the associated land system changes, which can occur in distant places, and discuss the sustainability implications of this increasingly important land system change. brown. discuss the ways in which different model types can help in understanding future land system dynamics and inform the formulation, implementation and monitoring of land use policy and planning processes. from a review of current models they conclude that a majority of models are focusing on exploring socio - ecological system function, scenario analysis and ex - ante assessment.. argue for a complementary use of exploratory modelling and optimization approaches to explore the options of land system configurations that best meet the goods and services demands of society. while such combinations of modelling concepts have been proposed before, little operational examples are yet available and the combined use of such approaches in co - designing land use planning and policy with stakeholders has not yet been fully explored. provide a conceptual framework for studying land use intensity based on a review of theoretical concepts and indicators available in the literature. they indicate that the integration of three dimensions of land - use intensity is needed, combining metrics for inputs and outputs of land based production systems as well as system parameters that can reflect the unintended outcomes of land use and represent powerful drivers of land system dynamics. such structured analysis of land use intensity is rarely conducted in the current land system science literature. address the extensification processes taking place in several regions around the world and argue that agricultural abandonment does not always result in a straightforward trajectory of forest recovery. agricultural abandonment is the starting point of many alternative pathways of changes in land management, vegetation and the use and governance of the natural resources. the different trajectories are strongly dependent on the local socio - economic and biophysical context, and impacts may have strong tradeoffs between ecosystem services provided by the changing land system. such tradeoffs are also key to the article of grau. who discuss alternative land development trajectories of land sparing and land sharing, that is, a separation of natural area conservation from intensive production systems versus a more multi - functional integration of service provisioning on the same area. while some of these discussions are dominated by strong generalizations, the article in this issue pleas for a more nuanced analysis, the integration of different development trajectories and a broadening of the scale of analysis while accounting for global teleconnections. key to such analysis is an improved understanding of land use intensity, and the consideration of local socioeconomic and ecological constraints. discuss strengths and weaknesses of remote sensing as provider of data on land cover change. many of the more subtle changes in land use that occur within a certain land cover type, such as changes in land - use intensity, management and spatial organization, can not be straightforwardly derived from remote sensing. hence, different methods and approaches need to be followed to acquire relevant information on these changes. in their review of the state of the art in land - use intensity monitoring, they outline major challenges, but also opportunities emerging from the availability of new technologies, for mapping across different land - use types. the impacts and feedbacks of changes in land use intensity and land cover extent are addressed by the next group of four articles. discuss how the land system plays a crucial role within the food system, dependent on the connections between local food production systems and regional to global markets. in their article they sketch a land systems approach for contributing to sustainable local food production systems in the context of a globalized food system. change in land systems also affects natural systems. outline how changes and modifications in land cover constitute the most dominant drivers of biodiversity loss globally. these changes affect the structure and function of ecosystems and alter their capacity to provide sustained ecosystem services for human well - being. moreover, there are challenges and critical gaps in our knowledge on the links between biodiversity, ecosystem function and ecosystem services. the authors suggest that research on ecosystem services needs to expand beyond its largely descriptive approach to a deeper understanding of socio - ecological functions. crossmann and colleagues discuss how land system science can contribute to mainstreaming and operationalizing the ecosystem service concept in land management and policy. it is argued that a further integration between socio - economic and biophysical approaches in ecosystem service assessments and land governance analysis is needed to support the development and implementation of more sustainable land management practices. while the impact of land system change on food systems, biodiversity, ecosystem services has been discussed frequently, the relationships between land system changes and human health issues have been given much less attention, especially due to the very different disciplinary communities addressing these issues. review the literature in this field and discuss the ways in which knowledge from both fields can complement each other and how problems due to the different ontologies of the different disciplines can be overcome. present an overview of alternative ways of governance of land resources and argue that land is now governed more on the basis of flows of resources or goods rather than on territorial arrangements. they conclude that this has generated new forms of social exclusion and inequity, and that future land policy needs to combine territorial and flow - centered arrangements. focus on the processes of large scale land acquisitions that have received a lot of media attention and an increasing interest from the scientific community. the impacts of such large scale land acquisition on social systems, the environmental system and the food system are still largely unknown and very variable depending on local context and the mode of acquisition and governance. however, the scale of these acquisitions is very large and spread across a large part of (mainly) the developing world. land acquisitions are one of the typically examples of how local realities of changes in land systems are intricately linked to the global challenge of sustainable and equitable development. finally, we acknowledge that a number of important aspects of land system science are not discussed in this issue either because reviews of those topics have been published recently or because contributions were unavailable or not yet ready for publication. examples of recent reviews on other important aspects of land system science include those on long term historic changes in land use, land use and the climate system, forest transitions, land use data and livestock systems. also, a series of meta - analysis of case studies on the drivers [3641 ] and consequences of land system change provide a useful synthesis of commonalities and context dependencies across place - based land systems research. the synthesis of land change, through review, meta - analysis and the development of conceptual frameworks and theory, helps to bring together findings on land system changes across different scales and from different disciplinary perspectives. all papers in this issue identify new challenges based on the knowledge gaps identified within the review. this way, synthesis of current knowledge translates into an updated research agenda for the land system science community.	this issue of current opinion in environmental sustainability provides an overview of recent advances in land system science while at the same time setting the research agenda for the land system science community. land system science is not just representing land system changes as either a driver or a consequence of global environmental change. land systems also offer solutions to global change through adaptation and mitigation and can play a key role in achieving a sustainable future earth. the special issue assembles 14 articles that entail different perspectives on land systems and their dynamics, synthesizing current knowledge, highlighting currently under - researched topics, exploring scientific frontiers and suggesting ways ahead, integrating a plethora of scientific disciplines.
we focused our analysis on mammalian hosts and viruses because they, more than any other host pathogen type, are likely to be associated with emerging infectious diseases of humans (3,6). we constructed a database of all emerging viruses of humans that were previously identified as originating in wildlife ; the database was supplemented with all zoonotic viruses with nonhuman mammalian hosts found in the international committee on the taxonomy of viruses database (www.ictvdb.org) (2). for each zoonotic virus, we conducted a literature search for reports of infection in any mammalian host, using the virus name and relevant synonyms (www.ictvdb.org) as keywords in web of knowledge (http://wokinfo.com/), wildlife disease association meeting abstracts (http://wildlifedisease.org/wda/conferences.aspx), google scholar (http://scholar.google.com/), and the global mammal parasites database (www.mammalparasites.org). the resulting 605 host pathogen relationships included 56 unique viruses classified in 17 taxonomic families and 325 unique mammals classified in 15 taxonomic orders. we excluded rabies from our analysis because the intense research effort on this virus and its high pathogenicity in almost all of its wide range of hosts (7) would skew the data disproportionately. we then conducted a secondary literature search to determine whether viruses in our database cause signs of disease in their wildlife hosts. for the search, we used an aggregate of all publications available in pubmed (www.ncbi.nlm.nih.gov/pubmed/), web of science (http://thomsonreuters.com/products_services/science/science_products/a-z/web_of_science/), biosis previews (http://thomsonreuters.com/products_services/science/science_products/a-z/biosis_previews/), and biologic & agricultural index plus (www.ebscohost.com/academic/biological-agricultural-index-plus) ; search terms consisted of virus names and international committee on the taxonomy of viruses synonyms, host genus and species names, and common names [reconciled to the 2005 version of mammal species of the world (8) ]. all resulting abstracts and available full text reports were examined until the first robust report of visible disease was encountered. viruses were identified as causing visible disease in a host if individual or epizootic death or grossly visible or otherwise observable signs of illness (e.g., high fever, loss of mobility, or severe decline in body condition) were reported. a report was considered robust only if infections were confirmed by pcr analysis or virus isolation and clinical signs were explicitly recorded to have occurred during active infection. we excluded studies reporting only serologic findings because of potential cross - reactivity among related viruses and poor correlation between serologic status and concurrent infection. our criteria of stopping a search once any evidence for visible disease was found meant that for mammal we considered diseases to be nonpathogenic in their hosts only if actively infected animals were explicitly reported to be free of visible disease. animals with less clear signs of disease, such as nasal discharge or death of neonates, were not considered asymptomatic because of the low detection probability associated with these traits in wild mammal surveillance. we rejected reports of experimentally induced disease because of the risk that dosage and inoculation technique would not be consistent with naturally occurring infections. however, we included experimental studies if actively infected animals remained asymptomatic, with the assumption that 1) clinical signs of infection were most likely to be seen in animals monitored in laboratory settings than in the wild and 2) stressful conditions in captivity would heighten the likelihood of a normally benign pathogen leading to clinical signs (9). furthermore, compared with naturally occurring infections, experimental infections often involve more direct routes of inoculation and are therefore more likely to induce disease. we conducted a logistic regression analysis, using firth s bias reduction procedure (10) as used by the brglm (bias reduction in generalized linear models) package of r v2.15 - 2 (http://cran.r-project.org/bin/windows/base/), of apparent host disease as a function of host taxonomy and virus taxonomy for the subset of mammal virus pairs for which the host order or virus family had at least 3 records in the database. we then calculated odds ratios for each host taxonomic order and virus family relative to the reference categories (artiodactyla and flaviviridae) and the predicted probability of being symptomatic for all species order virus family combinations. virus associations investigated yielded explicit information on host health in 52% of the 312 mammal virus pairs. of these, 28% (n = 88) of infected wildlife hosts were reported to have had visible disease and 72% (n = 224) were reported without evidence of visible disease (figure 1, panel a). the proportion of hosts that were symptomatic differed across host order (figure 1, panel b) and virus family (figure 1, panel c). virus pair reports describing symptomatic (observable) disease, asymptomatic disease (no observable disease), or no data (no description of disease included). c) percentage of viruses, by taxonomic family, for which hosts are reported symptomatic. the total number of each host order or virus family included in the database is given above each bar. all host orders and virus families in the database are included here, but analyses are limited to those host orders or virus families with at least 3 entries in the database. see the online technical appendix (wwwnc.cdc.gov/eid/article/19/5/12-1042-techapp1.xlsx) for the full database of host virus pairs and disease states. we found that virus family and host order were significant predictors of disease status (= 88.70, p<0.001 and = 59.45, p<0.001, respectively). species infected with paramyxoviruses, poxviruses, and reoviruses were more likely to have visible disease (p = 0.02, p = 0.001, and p = 0.04, respectively), and species infected with bunyaviruses were less likely to have visible disease relative to the reference category (p = 0.01) (table). hosts infected with filoviruses were marginally more likely to have visible disease (p = 0.08) (table). the subset of data used was selected by using a cutoff of at least 3 records in the database to avoid making inference about host orders or virus families, for which we had very little information. virus and host reference groups were selected as those for which sample size was sufficiently large and symptomatic infection was moderate (see figure 1). relative to the reference category, species classified in the order chiroptera were less likely to have visible disease (p<0.001), and species in the order rodentia were marginally less likely to have visible disease (p = 0.10) (table). compared with species in other orders, species in the order chiroptera had a lower probability of visible disease (figure 2), although all chiroptera species infected with nonrabies rhabdoviruses had a high probability of visible disease. in the dataset, all host pairs infected with rhabdoviruses were in the order chiroptera and were reported with visible disease in that host (figure 1). probability of being symptomatic as determined by logistic regression analysis, with bias reduction of whether a host is diseased, for 234 mammal probabilities are based on the predicted values of the logistic regression and are given on a 5-point gray scale (key on right). nonhuman primates (1113) and species classified within the taxonomic orders chiroptera and rodentia are the primary mammals targeted for zoonotic disease surveillance. our data suggest that species in the orders chiroptera and rodentia are less likely than species in other orders to have visible disease (figure 1)., we found that the probability of having visible disease depends on the taxonomic classification of the host and virus, and chiroptera is the only host order for which a single strategy (in this case, healthy animal surveillance) can be applied across nearly all virus families, excluding rhabdoviridae. therefore, particularly for the case of novel virus detection, our results point to a mixed strategy of targeted syndromic and healthy animal surveillance across host and virus taxonomies. a mixed strategy could combine apparently healthy animal surveillance (particularly in chiroptera) with syndromic surveillance in other wildlife and domestic animal hosts. syndromic surveillance has proven useful where secondary animal hosts are involved [e.g., surveillance for west nile virus (14), henipaviruses (15,16), and ebola virus (17) ]. there are limitations to our study, particularly ascertainment and reporting biases, as acknowledged in previous studies of emerging infectious diseases (2,3). in addition, differences in the number of species belonging to each order, the difficulty of testing inaccessible species, and limits to reliable diagnoses of emerging viruses have an effect, especially in resource - poor settings. furthermore, many disease states are not recognizable in free - ranging mammalian species under field conditions. last, there is a risk that an animal may be co - infected with several agents, only one of which causes disease ; that co - infection may have an additive or synergistic effect on clinical signs ; and that anthropozoonotic viruses artificially inflate the disease count of mammals in some taxonomic orders over others. however, our findings were determined on the basis of an aggregation of the best data available on host health as it relates to zoonotic viruses, and they have useful implications for public health. our analysis supports a holistic, probability - based approach to zoonotic virus discovery, specifically, continued analysis of passively and actively reported deaths and increased investment in broad surveillance of healthy wildlife. the latter could be targeted geographically to those regions most likely to generate novel emerging infectious diseases (2) or taxonomically to groups that are reservoirs for the highest proportion of zoonoses (3,18). these efforts could be envisaged as part of a strategy for smart surveillance, heightening the opportunity for discovery of novel zoonoses, particularly if wildlife are sampled at key interfaces where contact with human or domestic animals (and thus the opportunity for spillover) is highest.	we analyzed a database of mammal virus associations to ask whether surveillance targeting diseased animals is the best strategy to identify potentially zoonotic pathogens. although a mixed healthy and diseased animal surveillance strategy is generally best, surveillance of apparently healthy animals would likely maximize zoonotic virus discovery potential for bats and rodents.
the n3 (also called omega-3) long - chain fatty acid docosahexaenoic acid (dha, 22:6n3), with a 22-carbon chain and six double bonds, comprises about 10% of the dry weight of the human brain (svennerholm, 1968 ; rapoport, 2003). animal studies have shown that dha readily crosses the blood / brain barrier (ouellet., 2009) and plays a critical positive role in all aspects of neuronal growth, synaptic connections, and functioning (cockburn, 1994 ; jamieson., 1999 ; salem., 2001 this includes roles in regulating the activity of na + k + atpase in the neural membrane (bourre. 2011), neuron size (ahmad., 2002), neurogenesis (auestad and innis, 2000 ; coti bertrand. 2009 ; dagai., 2009 ; he., 2009), neurite growth (calderon and kim, 2004 ; sakamoto., 2007 ; liu., 2008 cao., 2009), synapse formation and function (yoshida., 1997 ; cansev and wurtman, 2007 ; wu., 2008 ; cao., 2009 ; wurtman., 2009), neuronal integrity and vitality (issa., 2006 ; 2009), gene expression in the brain (kitajka., 2002), brain glucose transport (pifferi., 2007) 2009), and learning ability (bourre., 1989 ; yoshida., 1997 ; greiner, 1999 ; salem., 2001 ; takeuchi., 2002 ; shirai and suzuki, 2004 ; garcia - calatayud., 2005 2008 ; holguin., 2008 ; fedorova., 2009 ; he., 2009 ; hooijmans. because animals lack the enzyme required to make an n3 double bond, at least the basal n3 fatty acid, alpha - linolenic acid (ala, 18:3n3), must be obtained from their diets. mammals can convert ala to eicosapentaenoic epa, 20:5n3), and can subsequently convert epa to docosapentaenoic (dpa, 22:5n3) and then to dha, though conversion efficiency is quite low and capacity limited, especially for epa to dha (pawlosky., 2001). as cited above, animal studies show that a deficiency of dietary n3 fatty acids leads to a decrease in neuronal size and synapse number and impaired learning ability. many studies in human infants have shown that levels of dha in the maternal diet or blood during pregnancy and in maternal milk or formula are positively related to cognitive and visual development in infants, as reviewed by mccann and ames (2005) ; eilander. studies involving the n3 content of the maternal diet may underestimate the effect of dha, because most dha delivered by a mother to her fetus or nursing infant derives from her fat stores rather than current intake (sauerwald. most studies relating the level of n3 fatty acids in the diet or blood to cognitive measures in older children have also found a positive relationship, including studies in italy (agostoni., 1997), scotland (whalley., 2004), maryland (ryan and nelson, 2008), finland (aberg., 2009), alabama (neggers., 2009), and wales (kirby., 2010), though a dutch study did not (de groot., 2007). two studies in which children were provided with short - term supplemental dietary n3 found a positive effect on cognitive measures (richardson and montgomery, 2005 ; dalton., 2009) and two did not (osendarp. none of these studies have considered sex differences which, for reasons we will outline below, are to be expected. the reliance of human and other mammalian brains on the neuronal functions of dha appears to be the result of a very ancient evolutionary contingency. though dha is now a relatively scarce and limiting resource for the development of large brains in terrestrial environments, neurons first evolved in an aquatic environment where high levels of dha were readily available. the first links in this chain of contingency apparently reach back more than 3 billion years. ancient cyanobacteria evolved the ability to synthesize n3 ala for incorporation into the thylakoid membrane where it plays an essential role in photosynthesis as it does today in the chloroplasts of all green plants. dinoflagellates and certain cyanobacteria and algae subsequently evolved a metabolic pathway to efficiently convert ala to dha using the enzyme delta-4-desaturase, and these phytoplankton are still the source of dha for all aquatic animal life. fossilized acritarchs suggest that dinoflagellates may have evolved more than 3 billion years ago (javaux., 2010). the first neurons evolved in precambrian cnidarians feeding on dinoflagellates and other phytoplankton rich in dha (nichols., 2003 ; putnam., 2007) ; hence neurons could evolve a design that was dependent on substantial supplies of dha. when larger and more elaborate brains evolved in marine vertebrates, their neurons could continue to rely on large amounts of dha because the phytoplankton producing this long - chain polyunsaturated fatty acid also lay at the base of their food chain. (dha s function is not limited to the vertebrate nervous system ; it also plays important roles in muscles, blood, and mitochondria.) marine arthropods also have a diet rich in dha, but when exclusively terrestrial arthropods first colonized the land, they were cut off from the dha supplied by phytoplankton and had minimal ability to synthesize longer - chain n3. although their bodies have significant amounts of ala obtained from plants, their nervous systems contain little or no dha (jerde. 2011) ; instead they use mainly ala (stark., 1993 ; their inability to convert alpha - linolenic to dha may limit the complexity of their nervous systems. reptiles subsequently evolved this conversion ability, although their synthetic pathway differs from that of phytoplankton (lacking delta-4 desaturase), and is very much less efficient. although allometrically small reptilian brains have some dha, the proportion of dha is quite low compared with mammalian brains (mitchell., 2007), and their limited n3 supply may have similarly constrained the growth and hence the evolution of a more complex nervous system. the evolution of endothermy in mammals greatly increased caloric requirements, and the concomitant 10-fold increase in consumption of plants and/or insects provided much larger amounts of ala, permitting the synthesis of larger amounts of dha despite the inefficiency of this process in terrestrial animals. these higher levels of dietary n3 allowed for a considerable expansion of the mammalian brain, which is not only allometrically much larger than a reptile brain but also contains a much higher proportion of dha (mitchell., 2007). higher dha levels in mammalian mitochondrial membranes also facilitated endothermic metabolism (brand. meanwhile, the evolution and diversification of flowering plants led to increases in the n3 content of terrestrial plants, especially in their fruits, nuts, and seeds, and permitted the co - evolution of many new species of herbivorous insects with high levels of ala. the first primates were insectivores occupying a nocturnal, arboreal niche, and their enhanced feeding skills and diet permitted the further expansion of the primate brain. (chimpanzee females continue to invest considerable amounts of time feeding on insects as shown by mcgrew, 1979.) frugivorous primates also obtain substantial amounts of insects in the fruit they eat (redford., 1984) as well as higher concentrations of n3 in nuts and seeds. folivorous primates, like gorillas, have relatively smaller brains compared to frugivores (clutton - brock and harvey, 1980 ; harvey., 1980), and must still ingest a large volume of plants to provide the necessary n3. as the hominid brain expanded to a size seven times larger than expected from the brain : body size relationship in mammals, the need for n3 and dha increased proportionately. because human synthesis of dha from ala remains very limited, like that of other terrestrial animals, sources of preformed dha in the diet are important. potential sources include the meat, organs, and eggs of herbivores and birds, and especially flesh from aquatic animals, which provides larger amounts of dha. some have argued that exploitation of aquatic nutritional resources was essential for the evolution of the large hominid brain (broadhurst., 2002), and there is evidence for significant amounts of aquatic foods in hominid diets from two million years ago (braun., 2010 ; stewart, 2010). in order to grow their very large brains, human fetuses, and nursing infants require much larger amounts of dha than can be reliably obtained from maternal daily intake, and most of the dha they receive comes from maternal fat stores. studies using radioisotope - labeled fatty acids show that approximately 80% of the dha and other essential long - chain fatty acids provided in human milk come from maternal fat rather than from the current diet (sauerwald., 2000). these fatty acids are stored mainly in women s gluteofemoral fat, and these depots are protected except during the third trimester and lactation when their fatty acids are mobilized (lassek and gaulin, 2006). because men do not make these physiological investments in offspring, women s need for these fatty acids greatly exceeds that of men, a fact that probably explains the unique human sex difference in body fat. there is no facilitated transport of dha into adipose, so the proportion of dha in fat stores depends on the concentration of dha in the blood. because this concentration is relatively low compared with other fatty acids, the percentage in adipose is also relatively low (0.20.3%). developing human females must therefore have substantial amounts of adipose tissue in order to store sufficient amounts of dha to support the growth of large brains in their children. in a study of dutch children, female fat increased from 14.8 to 25.5% of body weight during puberty, while male fat decreased from 10.5 to 9.3% (boot., 1997). in a sample of young american women, there was a mean of 16.2 kg of adipose tissue at the end of puberty (lassek and gaulin, 2006). assuming a dha percentage of 0.2%, this amount of adipose would contain 32 g of dha which would become available when adipose is mobilized during late pregnancy and lactation, when the fetal and infant dha requirement is 100200 mg / day (clandinin. a female child must store this dha at the same time that she requires substantial amounts of dha to support her own growth and development. because she must allocate some of her limited dietary n3 to storing dha for her future children, there is a competition between her need for dha for her own body and brain and her need to store dha for future reproduction. girls with proportionately larger amounts of gluteofemoral fat and lower waist - hip ratios have earlier menarche (lassek and gaulin, 2007). while human males usually have much less adipose than females, they have substantially more than typical primates (pond and mattacks, 1987). the optimal amount of dha per day in children has not been established, but participants in a 1999 workshop sponsored by national institutes of health recommended that at least 0.1% of calories should be dha (simopoulos and leaf, 1999), which would be 220 mg for a 2000 calorie (8.4 mj) diet (assuming 9 calories / g of fat). similar amounts are recommended for pregnant and nursing women (koletzko., 2007.) the per capita amount of dha and ala in the american food supply in 1990 was 70 mg and 2.4 g respectively (gerrior., 2004). while n3 fatty acids are known to have positive effects on cognition, less is known about the effects of n6 fatty acids, such as linoleic (la, 18:2n6) and arachidonic acid (aa, 20:4n6). as with n3, some form of n6 must come from the diet, and there is no interconversion of n3 and n6 fatty acids. in the terrestrial synthetic pathway that evolved to elongate ala, the la - to - arachidonic conversion competes for the same enzymes used to synthesize dha from ala (rubin and laposata, 1992 ; emken., 1994 ; innis., 2004 ; hibbeln., 2006 ; harnack. because of this metabolic competition, higher n6 fatty acid intake might be expected to have negative effects on cognition ; and four studies have shown this (agostoni., 1997 ; whalley., 2004 amounts of n3 fatty acids have declined in the american diet during the twentieth century while n6 have increased (blasbalg., 2011). reconstructions of the paleolithic diet suggest that over most of human evolution, there was more n3 than n6 in the diet (kuipers., linoleic acid in the american food supply was 29.3 g in 1990 and the ratio of total n6 to n3 was 12.3 (gerrior., 2004). because of their metabolic competition for necessary enzymes, the very high amount of n6 linoleic acid compared with n3 in the diet of american children is likely to decrease the conversion rate of ala to dha. larger, population - based samples may help to clarify the relationships between fatty acid consumption and cognitive performance. cognitive and dietary data collected in the third national health and nutrition examination survey (nhanes iii) conducted in 19881994 provides an opportunity to examine the relationship between dietary n3 and n6 fatty acid intake and cognitive outcomes in a large sample of american children, as well as the possible effects of other nutrients. hip ratios and cognitive performance which may be mediated by n3 fatty acids (lassek and gaulin, 2008). this leads us to predict that dietary n3 will also be positively related to cognitive outcomes in this sample, whereas dietary n6 will be inversely related to the same outcome measures. because of the much greater requirement for n3 fatty acids in human females, we predict that their cognitive performance will be more sensitive to the amount of n3 in the diet and to the competing effects of dietary n6. the reliance of human and other mammalian brains on the neuronal functions of dha appears to be the result of a very ancient evolutionary contingency. though dha is now a relatively scarce and limiting resource for the development of large brains in terrestrial environments, neurons first evolved in an aquatic environment where high levels of dha were readily available. the first links in this chain of contingency apparently reach back more than 3 billion years. ancient cyanobacteria evolved the ability to synthesize n3 ala for incorporation into the thylakoid membrane where it plays an essential role in photosynthesis as it does today in the chloroplasts of all green plants. dinoflagellates and certain cyanobacteria and algae subsequently evolved a metabolic pathway to efficiently convert ala to dha using the enzyme delta-4-desaturase, and these phytoplankton are still the source of dha for all aquatic animal life. fossilized acritarchs suggest that dinoflagellates may have evolved more than 3 billion years ago (javaux., the first neurons evolved in precambrian cnidarians feeding on dinoflagellates and other phytoplankton rich in dha (nichols., 2003 ; putnam., 2007) ; hence neurons could evolve a design that was dependent on substantial supplies of dha. when larger and more elaborate brains evolved in marine vertebrates, their neurons could continue to rely on large amounts of dha because the phytoplankton producing this long - chain polyunsaturated fatty acid also lay at the base of their food chain. (dha s function is not limited to the vertebrate nervous system ; it also plays important roles in muscles, blood, and mitochondria.) marine arthropods also have a diet rich in dha, but when exclusively terrestrial arthropods first colonized the land, they were cut off from the dha supplied by phytoplankton and had minimal ability to synthesize longer - chain n3. although their bodies have significant amounts of ala obtained from plants, their nervous systems contain little or no dha (jerde. 2011) ; instead they use mainly ala (stark., 1993 ; their inability to convert alpha - linolenic to dha may limit the complexity of their nervous systems. reptiles subsequently evolved this conversion ability, although their synthetic pathway differs from that of phytoplankton (lacking delta-4 desaturase), and is very much less efficient. although allometrically small reptilian brains have some dha, the proportion of dha is quite low compared with mammalian brains (mitchell., 2007), and their limited n3 supply may have similarly constrained the growth and hence the evolution of a more complex nervous system. the evolution of endothermy in mammals greatly increased caloric requirements, and the concomitant 10-fold increase in consumption of plants and/or insects provided much larger amounts of ala, permitting the synthesis of larger amounts of dha despite the inefficiency of this process in terrestrial animals. these higher levels of dietary n3 allowed for a considerable expansion of the mammalian brain, which is not only allometrically much larger than a reptile brain but also contains a much higher proportion of dha (mitchell., 2007). however, mammals still have a lower proportion than fish (stoknes., 2004 ; usda, 2011). higher dha levels in mammalian mitochondrial membranes also facilitated endothermic metabolism (brand. meanwhile, the evolution and diversification of flowering plants led to increases in the n3 content of terrestrial plants, especially in their fruits, nuts, and seeds, and permitted the co - evolution of many new species of herbivorous insects with high levels of ala. the first primates were insectivores occupying a nocturnal, arboreal niche, and their enhanced feeding skills and diet permitted the further expansion of the primate brain. (chimpanzee females continue to invest considerable amounts of time feeding on insects as shown by mcgrew, 1979.) frugivorous primates also obtain substantial amounts of insects in the fruit they eat (redford., 1984) as well as higher concentrations of n3 in nuts and seeds. folivorous primates, like gorillas, have relatively smaller brains compared to frugivores (clutton - brock and harvey, 1980 ; harvey., 1980), and must still ingest a large volume of plants to provide the necessary n3. as the hominid brain expanded to a size seven times larger than expected from the brain : body size relationship in mammals, the need for n3 and dha increased proportionately. because human synthesis of dha from ala remains very limited, like that of other terrestrial animals, sources of preformed dha in the diet are important. potential sources include the meat, organs, and eggs of herbivores and birds, and especially flesh from aquatic animals, which provides larger amounts of dha. some have argued that exploitation of aquatic nutritional resources was essential for the evolution of the large hominid brain (broadhurst., 2002), and there is evidence for significant amounts of aquatic foods in hominid diets from two million years ago (braun., 2010 ; stewart, 2010). in order to grow their very large brains, human fetuses, and nursing infants require much larger amounts of dha than can be reliably obtained from maternal daily intake, and most of the dha they receive comes from maternal fat stores. studies using radioisotope - labeled fatty acids show that approximately 80% of the dha and other essential long - chain fatty acids provided in human milk come from maternal fat rather than from the current diet (sauerwald., 2000). these fatty acids are stored mainly in women s gluteofemoral fat, and these depots are protected except during the third trimester and lactation when their fatty acids are mobilized (lassek and gaulin, 2006). because men do not make these physiological investments in offspring, women s need for these fatty acids greatly exceeds that of men, a fact that probably explains the unique human sex difference in body fat. there is no facilitated transport of dha into adipose, so the proportion of dha in fat stores depends on the concentration of dha in the blood. because this concentration is relatively low compared with other fatty acids, the percentage in adipose is also relatively low (0.20.3%). developing human females must therefore have substantial amounts of adipose tissue in order to store sufficient amounts of dha to support the growth of large brains in their children. in a study of dutch children, female fat increased from 14.8 to 25.5% of body weight during puberty, while male fat decreased from 10.5 to 9.3% (boot.,, there was a mean of 16.2 kg of adipose tissue at the end of puberty (lassek and gaulin, 2006). assuming a dha percentage of 0.2%, this amount of adipose would contain 32 g of dha which would become available when adipose is mobilized during late pregnancy and lactation, when the fetal and infant dha requirement is 100200 mg / day (clandinin., 1980a, b ; haggarty, 2004). a female child must store this dha at the same time that she requires substantial amounts of dha to support her own growth and development. because she must allocate some of her limited dietary n3 to storing dha for her future children, there is a competition between her need for dha for her own body and brain and her need to store dha for future reproduction. girls with proportionately larger amounts of gluteofemoral fat and lower waist - hip ratios have earlier menarche (lassek and gaulin, 2007). while human males usually have much less adipose than females, they have substantially more than typical primates (pond and mattacks, 1987). the optimal amount of dha per day in children has not been established, but participants in a 1999 workshop sponsored by national institutes of health recommended that at least 0.1% of calories should be dha (simopoulos and leaf, 1999), which would be 220 mg for a 2000 calorie (8.4 mj) diet (assuming 9 calories / g of fat). the per capita amount of dha and ala in the american food supply in 1990 was 70 mg and 2.4 g respectively (gerrior., 2004). while n3 fatty acids are known to have positive effects on cognition, less is known about the effects of n6 fatty acids, such as linoleic (la, 18:2n6) and arachidonic acid (aa, 20:4n6) as with n3, some form of n6 must come from the diet, and there is no interconversion of n3 and n6 fatty acids. in the terrestrial synthetic pathway that evolved to elongate ala, the la - to - arachidonic conversion competes for the same enzymes used to synthesize dha from ala (rubin and laposata, 1992 ; emken., 1994 ; innis., 2004 ; hibbeln., 2006 ; because of this metabolic competition, higher n6 fatty acid intake might be expected to have negative effects on cognition ; and four studies have shown this (agostoni., 1997 ; whalley., 2004 ; novak. amounts of n3 fatty acids have declined in the american diet during the twentieth century while n6 have increased (blasbalg., 2011). reconstructions of the paleolithic diet suggest that over most of human evolution, there was more n3 than n6 in the diet (kuipers., linoleic acid in the american food supply was 29.3 g in 1990 and the ratio of total n6 to n3 was 12.3 (gerrior., 2004). because of their metabolic competition for necessary enzymes, the very high amount of n6 linoleic acid compared with n3 in the diet of american children is likely to decrease the conversion rate of ala to dha. larger, population - based samples may help to clarify the relationships between fatty acid consumption and cognitive performance. cognitive and dietary data collected in the third national health and nutrition examination survey (nhanes iii) conducted in 19881994 provides an opportunity to examine the relationship between dietary n3 and n6 fatty acid intake and cognitive outcomes in a large sample of american children, as well as the possible effects of other nutrients. hip ratios and cognitive performance which may be mediated by n3 fatty acids (lassek and gaulin, 2008). this leads us to predict that dietary n3 will also be positively related to cognitive outcomes in this sample, whereas dietary n6 will be inversely related to the same outcome measures. because of the much greater requirement for n3 fatty acids in human females, we predict that their cognitive performance will be more sensitive to the amount of n3 in the diet and to the competing effects of dietary n6. detailed dietary histories based on 24-h recall were obtained by skilled interviewers for 13,923 males and 15,182 females aged 090 in the nhanes iii sample, 19881994. from this larger sample the child sample included 26% non - hispanic whites, 35% non - hispanic blacks, 35% mexican - americans, and 5% other. because of the oversampling of blacks and hispanics, specific fatty acid content of the foods consumed was estimated using the food database of the university of minnesota s nutrition coordinating center, and for other nutrients, the usda national nutrient database for standard reference (usda). nutrients used in the analysis which were estimated by conjoining the dietary histories and food composition databases in this way included vitamins a, b6, b12, c, and e, iron, folate, riboflavin, niacin, and thiamine, serum electrolytes, specific sugars, saturated fats with 10, 12, 14, 17, 18, 20, and 22 carbons, monounsaturated fats with 14, 16, 18, 20, and 22 carbons, the n6 fatty acids la and aa (summed for total n6), the n3 fatty acids ala, epa, dpa, and dha (added together for total n3), and total saturated, monounsaturated, and polyunsaturated fats. four cognitive tests were administered to 2253 males and 2309 females aged 616 in the nhanes iii sample, including the math and reading tests from the wide range achievement test - revised and the digit span and block design tests from the wechsler intelligence scale for children - revised. the mean scaled score across these four tests was used as a measure of cognitive performance for each of the 616 year - olds in this study ; both cognitive scores and dietary data were available for 2103 females and 2051 males, and these subgroups thus comprise our primary sample. other (non - dietary) measures included in the analyses as possible confounding variables include race / ethnicity, family income, family size, and years of education of the householder parent. serum lead was also included because it is known to have a significant negative relationship with cognitive performance in children (wasserman. sample weights and complex adjustments for the sampling methodology were not used ; this is in accordance with the recommendations of korn and graubard (1991). mean energy intake was 20% higher in males and dietary fatty acid intakes showed similar differences ; the mean test score and ratio of n6 to n3 was higher in females. p < 0.05 p < 0.001 p < 0.00001 for differences in means. based on stepwise multiple regression, table 2 shows the effect of significant nutrients on the cognitive performance of the children in the sample, controlling for race / ethnicity, family income and size, years of education of the householder parent, and serum lead. for both sexes, total dietary n3 fatty acids are significantly positively related to test scores, while for females, total n6 fatty acids are negatively related. none of the other 33 nutrients are significantly related, including iron and folate and total dietary saturated, monounsaturated, and polyunsaturated fatty acids. based on its unstandardized regression coefficient, an increase in daily n3 intake of 1 g increases the average test score by 0.38 points (0.09 sd) in females and by 0.19 points (0.10 sd) in males ; this represents 0.14 and 0.07 of the sds for the test scores. in females, but not males, when the ratio of n6 to n3 was used in place of the separate n3 and n6 intakes in the regression, it was significantly and negatively related to the cognitive score (beta = 0.071, p = 0.001). when the regression was run with both sexes, sex was not a significant variable. standardized regression coefficients for the effect of dietary fatty acids on performance on four cognitive tests in youth 616, nhanes iii. p < 0.05 p < 0.001 p < 0.00001. national health and nutrition examination survey may sample more than one member in participating households ; this creates a lack of independence among cases that is potentially problematic. to examine whether this lack of independence may have favored our hypothesis, we repeated the analysis using both sexes of children and limiting the sample to one child per household (1581 boys ; 1682 girls), and found that there was no difference in the results of the regression. using a large sample drawn from nhanes iii, dietary n3 fatty acids are positively related to cognitive performance in children 616 years of age, while n6 fatty acids are negatively related to cognitive performance in females in the same sample. as predicted, the contribution of dietary n3 to cognitive performance is much greater (two - fold) in females, and females also show a significant negative effect for n6 fatty acids which compete with n3 for enzymes needed in the biosynthesis of dha. the special effects of n3 and n6 are apparent because dietary consumption of 33 other fatty acids and nutrients are not related to cognitive outcome measures based on the same dietary data set and the same children. the positive cognitive effect of dietary n3 fatty acids, as measured by the imperfect method of 24-h recall, is of greater magnitude in girls than the negative effect of serum lead, a well known influence on cognition in children. dietary iron and folate, which have also been found to relate to cognitive performance in some studies of children (arija., because of the complex sampling method used in the nhanes, these results should be viewed with caution and should not be considered representative of the american population. also, the diet estimates used in this study were based on a single 24-h recall ; and while this type of assessment is related to the long - term diet, it is not a highly accurate measure (knutsen., 2003 ; sekula., 2005 ; slater., 2010 in addition, the children in the sample are past the ages of maximal brain growth, a period when the effects of dietary fatty acids would be expected to be greater. however, despite these limitations, their cognitive ability is still related to the amount of dietary n3 fatty acids, and of the 40 nutritional variables used in the analysis, only n3 and n6 fatty acids were significantly related to cognitive ability. the stronger effect of n3 and significantly negative effect of n6 in girls may reflect their greater need for n3 fatty acids to sustain future pregnancy and lactation, as explained above. because stored maternal fat is selectively used to support the development of the fetal and infant brain via the placenta and breast milk females must prepare for these demands by storing dha in fat at a much higher rate than males during their childhood and adolescence, while their own brains and bodies are still growing. this competition between growth and reproductive goals, absent in boys, may make girls more subject to the antagonism between the n6 and n3 fatty acid families in commandeering necessary synthetic enzymes. the effect of dietary fatty acids on cognition demonstrated here is relatively small but of similar magnitude to the negative effect of lead. both are environmental variables that can be altered, and clear steps have been taken in the case of lead. since more than half of the variance in cognition is heritable (plomin., 2000), and other environmental variables are often intractable (e.g., family income), dietary fatty acid intake may be of significant pragmatic importance. these findings on the relationship between dietary fatty acids and cognitive performance are of particular interest in relation to current american food consumption patterns. the cognitive effects of dietary fatty acids in american children may be greater than in other populations because of the limited amount of n3 fatty acids in the american diet combined with unusually high levels of n6 (blasbalg., 2011). based on the energy intake of the sample children, the recommended amount of 0.1% of energy for dha would be 250 in males and 210 mg in females per day. assuming a very generous conversion rate of 2% for ala to dha, the mean total daily amount of dha for the sample children would be just 70 mg for males and 54 mg for females, considerably short of the recommended amounts. a coordinated increase in n6 and decrease in n3 supplies are the hallmarks of modern american industrial food production, and reflected in the high n6/n3 ratios in the nhanes iii sample. corn, which increased by 78% in the us diet from 1970 to 2000 (putnam., soybean oil, especially when partially hydrogenated or made from prevalent low - ala varieties of soybeans, has a similarly high ratio. moreover, n3 in processed food is often removed because it is prone to spoil and thus reduces product shelf life (holman, 1998). an experimental study in rats using feeds corresponding to the japanese diet (high n3, low n6) and american diet (high n6, low n3) found both better learning and many more synapses in the hippocampus in the rats fed the japanese diet (yoshida., 1997). it thus seems possible that the high n6/n3 ratio in the american diet might contribute to the relatively low ranking of american children in international testing (nces, 2005) compared to children in countries with lower n6/n3 ratios, like japan. thus, evolutionary considerations may help lead to findings with considerable potential public health significance. 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.	because the first neurons evolved in an environment high in the n3 (omega-3) fatty acid docosahexaenoic acid (dha), this fatty acid became a major component of neural structure and function and makes up 10% of the dry weight of the human brain. since n3 fatty acids must come from the diet, this suggests a possible positive role for dietary n3 fatty acids in cognition and a possible negative role for n6 fatty acids, which compete with n3 for access to critical enzymes. because human females must provide dha for the growth of the unusually large brains of their offspring from maternal fat stored during childhood, their need for dha is especially great. we used stepwise regression to determine whether particular dietary fatty acids and other nutrients were related to cognitive performance in over 4000 american children aged 616 from the third national health and nutrition examination survey ; a variety of possible biological, social, and environmental risk factors were statistically controlled. in this context the only dietary factors related to cognitive performance were n3 and n6 fatty acids. dietary n3 fatty acids were positively related to cognitive test scores in male and female children, while n6 showed the reverse relationship, significantly so in females. in female children the positive effects of n3 intake were twice as strong as in males and exceeded the negative effects of lead exposure. this suggests that increasing dietary intake of n3 and decreasing n6 fatty acids may have cognitive benefits in children, especially in females.
lymphoblastoid cell lines (lcls), immortalized by epstein - barr virus transformation of lymphocytes isolated from whole blood, were derived from european - american participants in the cap trial, a six - week 40mg / day simvastatin trial (supplementary table 8). (whitehouse station, nj), converted to active form (beta - hydroxy simvastatin acid, sva) and quantified by liquid chromatography - tandem mass spectrometry as described. lcls were normalized to a uniform cell density and exposed to 2m sva (simvastatin - exposed) or control buffer (control - exposed) for twenty - four hours as described. this concentration was selected by assessing dose - response effects on expression profiles (n=8 lcls at 4 doses), wherein a more robust change in expression profiles was observed with 2m simvastatin exposure (7.8% of genes, q=0.001) than lower doses (3.24) and trans - eqtls (log10 bf > 7.20). for cis - eqtls, we considered the largest log10bf above the cis - cutoff for any snp within 1 mb of the transcription start site or the transcription end site of the gene under consideration. for trans - eqtls, we considered the largest log10bf above the trans - cutoff for any snp, and if that snp was in the cis - neighborhood of the gene being tested, we ignored any potential trans - associations ; there were 6130 for which the snp with the largest log10bf was not in cis with the associated gene. correspondingly, we only considered those 6130 genes when computing the permutation - based fdr for the trans - associations. we define cis - snps as being within 1 mb of the transcription start site or end site of that gene. to identify differential eqtls, we first computed associations between all snps and the log fold change using bimbam as above. 3 indicate that there are a few possible patterns of differential association. while these patterns may have different mechanistic or phenotypic interpretations, they are not distinguished by a test of log fold change. we used the interaction models introduced in maranville. to compute the statistical support (assessed with bayes factors, or bfs) for the four alternative eqtl models described in results versus the null model (no association with genotype). these methods are based on a bivariate normal model for the treated data (t) and control - treated data (u). note that simply quantile transforming t and u to a standard normal distribution is not sufficient to ensure that they are jointly bivariate normal, and so we employed the following more extensive normalization procedure. let d = qt - qu and s = qt+qu, where q indicates that the vector following it has been quantile normalized. we then quantile normalize and scale d and s to produce s = (sqs) and d = (dqd), where s, d are robust estimates of the standard deviations of s and d respectively (specifically, they are the median absolute deviation multiplied by 1.4826). note that this transformation ensures that s and d are univariate normal.. finally let u = (s d) and t = (s + d). the bf when the eqtl effect is identical in the two conditions (model 1) uses the linear model l(s ~ d + g), where g is the vector of genotypes at a single snp. the bf when the eqtl is only present in the control - treated samples (model 2) uses the model l(u ~ t + g). the bf when the eqtl is only present in the simvastatin - treated samples (model 3) uses the model l(t ~ u + g). the bf when the eqtl effect is in the same direction but unequal in strength (model 4) uses the model l(d ~ s + g). we averaged each bf for each gene and each cis - snp over four plausible effect size priors (0.05, 0.1, 0.2, 0.4). to find eqtls that interact with treatment (i.e., conform best to one of the differential models 2 - 4, rather than the null model or the stable model) we defined an interaction bayes factor (ibf) as ibf = 2(bf2 + bf3 + bf4) / 3(bf1 + 1), where bfi denotes the bf for model i compared with the null model (the 1 in the denominator represents the null model bf0). large values of the ibf represent strong support for at least one interaction model (2 - 4) compared with the two non - interacting models (0 - 1), and hence strong support for a differential association. marshfield cohort : cases of myopathy were identified from electronic medical records of patients treated at the marshfield clinic (wisconsin, usa) using a combination of automated natural language processing and manual review as described. 72 cases of incipient myopathy (creatine kinase concentrations > 3-fold normal with evidence in the charts of muscle complaints) were identified for which patients were not also undergoing treatment with concomitant drugs known to increase incidence of statin - induced myopathy (fibrates or niacin). the study population included residents living in central and northern wisconsin, served by the marshfield clinic, a large multispecialty group practice. search and heart protection study collaborative groups : a total of 100 myopathy cases were identified from participants with genotyping data in the search trial, including 39 definite myopathy cases (creatine kinase > 10 uln with muscle symptoms) and 61 incipient myopathy cases (defined as creatine kinase 5.0 times baseline value and alanine transaminase 1.7 times baseline value and creatine kinase > 3.0 uln). genotypes were available from the illumina human610-quad beadchip for 25 myopathy cases (12% of which had definite myopathy) and from the illumina humanhap300-duo beadchip for 75 myopathy cases (48% of which had definite myopathy). genotypes for rs9806699 were only available in individuals genotyped on the illumina human610-quad beadchip so proxy snps were used. all myopathy cases were compliant with statin therapy (95 myopathy cases occurred whilst the patient was taking simvastatin 80 mg daily, and 5 cases whilst taking simvastatin 20 mg daily). controls were identified from the search study and the heart protection study as well as from the heart protection study (where considerably more participants had been genotyped). controls from the heart protection study had similar baseline characteristics to those in the search study and inclusion of this large number of additional controls improved statistical power. multicentre ethics approval was obtained from the south east research ethics committee for the search study, and from the local ethics committees covering each of the 69 uk hospitals involved in the heart protection study. meta - analysis was performed using a random effects model and, for bayesian analysis, considering an expected effect size to 0.2. associations of rs9806699 with plasma creatine kinase in the cap and jupiter trials were also assessed using linear regression. the cap trial (clinicaltrials.gov number, nct00451828) was approved by the institutional review boards located at children s hospital oakland research institute (oakland, ca) and all enrollment sites. the jupiter trial (clinicaltrials.gov number, nct00239681) was approved by the institutional review board of brigham and women s hospital. gatm knockdown was achieved by 48-hour transfection of ambion silence select sirna or non - targeting control into 80,000 hepg2 or huh7 cells / well in 12-well plates. to assess the influence of sterol depletion, cell culture media was replaced with media containing 10% lipoprotein deficient serum (hyclone) or fetal bovine serum (omega scientific) at 24-hr transfection. cell culture media was collected from all samples at time of harvest, and apob (mp biomedicals), apoai (meridian life sciences), and apoe (biodesign) were quantified in triplicate by sandwich - style elisa. lymphoblastoid cell lines (lcls), immortalized by epstein - barr virus transformation of lymphocytes isolated from whole blood, were derived from european - american participants in the cap trial, a six - week 40mg / day simvastatin trial (supplementary table 8). (whitehouse station, nj), converted to active form (beta - hydroxy simvastatin acid, sva) and quantified by liquid chromatography - tandem mass spectrometry as described. lcls were normalized to a uniform cell density and exposed to 2m sva (simvastatin - exposed) or control buffer (control - exposed) for twenty - four hours as described. this concentration was selected by assessing dose - response effects on expression profiles (n=8 lcls at 4 doses), wherein a more robust change in expression profiles was observed with 2m simvastatin exposure (7.8% of genes, q=0.001) than lower doses (3.24) and trans - eqtls (log10 bf > 7.20). for cis - eqtls, we considered the largest log10bf above the cis - cutoff for any snp within 1 mb of the transcription start site or the transcription end site of the gene under consideration. for trans - eqtls, we considered the largest log10bf above the trans - cutoff for any snp, and if that snp was in the cis - neighborhood of the gene being tested, we ignored any potential trans - associations ; there were 6130 for which the snp with the largest log10bf was not in cis with the associated gene. correspondingly, we only considered those 6130 genes when computing the permutation - based fdr for the trans - associations. we define cis - snps as being within 1 mb of the transcription start site or end site of that gene. to identify differential eqtls, we first computed associations between all snps and the log fold change using bimbam as above. while these patterns may have different mechanistic or phenotypic interpretations, they are not distinguished by a test of log fold change. we used the interaction models introduced in maranville. to compute the statistical support (assessed with bayes factors, or bfs) for the four alternative eqtl models described in results versus the null model (no association with genotype). these methods are based on a bivariate normal model for the treated data (t) and control - treated data (u). note that simply quantile transforming t and u to a standard normal distribution is not sufficient to ensure that they are jointly bivariate normal, and so we employed the following more extensive normalization procedure. let d = qt - qu and s = qt+qu, where q indicates that the vector following it has been quantile normalized. we then quantile normalize and scale d and s to produce s = (sqs) and d = (dqd), where s, d are robust estimates of the standard deviations of s and d respectively (specifically, they are the median absolute deviation multiplied by 1.4826). note that this transformation ensures that s and d are univariate normal.. finally let u = (s d) and t = (s + d). the bf when the eqtl effect is identical in the two conditions (model 1) uses the linear model l(s ~ d + g), where g is the vector of genotypes at a single snp. the bf when the eqtl is only present in the control - treated samples (model 2) uses the model l(u ~ t + g). the bf when the eqtl is only present in the simvastatin - treated samples (model 3) uses the model l(t ~ u + g). the bf when the eqtl effect is in the same direction but unequal in strength (model 4) uses the model l(d ~ s + g). we averaged each bf for each gene and each cis - snp over four plausible effect size priors (0.05, 0.1, 0.2, 0.4). to find eqtls that interact with treatment (i.e., conform best to one of the differential models 2 - 4, rather than the null model or the stable model) we defined an interaction bayes factor (ibf) as ibf = 2(bf2 + bf3 + bf4) / 3(bf1 + 1), where bfi denotes the bf for model i compared with the null model (the 1 in the denominator represents the null model bf0). large values of the ibf represent strong support for at least one interaction model (2 - 4) compared with the two non - interacting models (0 - 1), and hence strong support for a differential association. marshfield cohort : cases of myopathy were identified from electronic medical records of patients treated at the marshfield clinic (wisconsin, usa) using a combination of automated natural language processing and manual review as described. 72 cases of incipient myopathy (creatine kinase concentrations > 3-fold normal with evidence in the charts of muscle complaints) were identified for which patients were not also undergoing treatment with concomitant drugs known to increase incidence of statin - induced myopathy (fibrates or niacin). the study population included residents living in central and northern wisconsin, served by the marshfield clinic, a large multispecialty group practice. search and heart protection study collaborative groups : a total of 100 myopathy cases were identified from participants with genotyping data in the search trial, including 39 definite myopathy cases (creatine kinase > 10 uln with muscle symptoms) and 61 incipient myopathy cases (defined as creatine kinase 5.0 times baseline value and alanine transaminase 1.7 times baseline value and creatine kinase > 3.0 uln). genotypes were available from the illumina human610-quad beadchip for 25 myopathy cases (12% of which had definite myopathy) and from the illumina humanhap300-duo beadchip for 75 myopathy cases (48% of which had definite myopathy). genotypes for rs9806699 were only available in individuals genotyped on the illumina human610-quad beadchip so proxy snps were used. all myopathy cases were compliant with statin therapy (95 myopathy cases occurred whilst the patient was taking simvastatin 80 mg daily, and 5 cases whilst taking simvastatin 20 mg daily). controls were identified from the search study and the heart protection study as well as from the heart protection study (where considerably more participants had been genotyped). controls from the heart protection study had similar baseline characteristics to those in the search study and inclusion of this large number of additional controls improved statistical power. multicentre ethics approval was obtained from the south east research ethics committee for the search study, and from the local ethics committees covering each of the 69 uk hospitals involved in the heart protection study. meta - analysis was performed using a random effects model and, for bayesian analysis, considering an expected effect size to 0.2. associations of rs9806699 with plasma creatine kinase in the cap and jupiter trials were also assessed using linear regression. the cap trial (clinicaltrials.gov number, nct00451828) was approved by the institutional review boards located at children s hospital oakland research institute (oakland, ca) and all enrollment sites. the jupiter trial (clinicaltrials.gov number, nct00239681) was approved by the institutional review board of brigham and women s hospital. gatm knockdown was achieved by 48-hour transfection of ambion silence select sirna or non - targeting control into 80,000 hepg2 or huh7 cells / well in 12-well plates. to assess the influence of sterol depletion, cell culture media was replaced with media containing 10% lipoprotein deficient serum (hyclone) or fetal bovine serum (omega scientific) at 24-hr transfection. cell culture media was collected from all samples at time of harvest, and apob (mp biomedicals), apoai (meridian life sciences), and apoe (biodesign) were quantified in triplicate by sandwich - style elisa.	statins are widely prescribed for lowering plasma low - density lipoprotein (ldl) concentrations and cardiovascular disease risk1, but there is considerable interindividual variation in treatment response2,3 and increasing concern regarding the potential for adverse effects, including myopathy4 and type 2 diabetes5. despite evidence for substantial genetic influence on ldl concentrations6, pharmacogenomic trials have failed to identify genetic variations with large effects on either statin efficacy7 - 9 or toxicity10, and have yielded little information regarding mechanisms that modulate statin response. here we identify a downstream target of statin treatment by screening for the effects of in vitro statin exposure on genetic associations with gene expression levels in lymphoblastoid cell lines derived from 480 participants of a clinical trial of simvastatin treatment7. this analysis identified six expression quantitative trait loci (eqtls) that interacted with simvastatin exposure including rs9806699, a cis - eqtl for the gene gatm that encodes glycine amidinotransferase, a rate - limiting enzyme in creatine synthesis. we found this locus to be associated with incidence of statin - induced myotoxicity in two separate populations (meta - analysis odds ratio = 0.60, 95% confidence interval = 0.45 - 0.81, p=6.010 - 4). furthermore, we found that gatm knockdown in hepatocyte - derived cell lines attenuated transcriptional response to sterol depletion, demonstrating that gatm may act as a functional link between statin - mediated cholesterol lowering and susceptibility to statin - induced myopathy.
a rare case of amyloidosis cutis dyschromica (acd) is being described in a middle - aged muslim man who had a strong family history of vitiligo. his patchy dyschromic lesions started from the age of 6 years and gradually increased to involve practically the whole trunk and all extremities but spared the face, palms and soles. individually they were hyperpigmented macules and few keratotic papules interspersed with very sharply marginated hypo and depigmented macules. such a clinical picture prompts a dermatologist to think of dowling degos disease, dyschromia universalis hereditaria, xeroderma pigmentosum (xp) and the rare acd or poikilodermatous cutaneous amyloidosis. a skin biopsy revealed amyloid pigment in the papillary dermis confirming the diagnosis of acd. acd is a rare form of primary cutaneous amyloidosis which is refractory to all forms of treatment with only anecdotal reports of treatment modalities like acitretin, topical steroids, keratolytics and co2 lasers. a 41-year - old muslim man living in south africa presented to this clinic with diffuse patchy dyschromic lesions on the body. he said he started developing gradually increasing diffuse hyperpigmentation starting from his trunk followed by patchy hypopigmentation and depigmentation from the age of 6 years all over the body. cutaneous examination revealed diffuse reticulate pigmentation of the entire body sparing palms, soles, dorsa of hands, feet and face and an island in the presternal area which he said was his original color. the hypopigmented macules were variably sized, well defined and ranged from tiny 4 mm to about 2 cm in diameter [figure 1a and b ]. some scattered hyperpigmented keratotic papules were found on the extremities and proximal inner arms abutting the axillae, which had been growing for the past 4 years [figure 2a and b ]. all lesions were asymptomatic with no blistering, ulceration, atrophy, poikilodermatous changes, scaling or oozing. the patient gave no history of photosensitivity, any systemic abnormality or any inflammatory dermatosis and there was no growth retardation. a 3-mm punch biopsy revealed amorphous pink masses of amyloid in the papillary dermis [figure 3 ]. (a) reticular pigmentation of the anterior trunk with patchy hypopigmented and depigmented macules. (b) reticular pigmentation of the posterior trunk with patchy hypopigmented and depigmented macules (a) keratotic papules in the most proximal part of right arm with multiple hyperpigmented keratotic papules. (b) keratotic papules in the most proximal part of left arm with multiple hyperpigmented keratotic papules congo red staining showing pinkish amorphous amyloid deposited in the papillary dermis acd is a rare variant of primary cutaneous amyloidosis. on evaluating data as recently as november 2012 acd is characterized by a typical reticulate hyperpigmentation interspersed with hypo and depigmented macules diffusely spread all over the body, occasional reports of associated mild itch, onset before puberty and deposition of amyloid immediately below the epidermis. scattered hyperpigmented keratotic papules that interspersed within the dyschromic lesions which when biopsied also showed deposition of amyloid have been reported. concomitant connective tissue disorders like systemic sclerosis, systemic lupus erythematosus and dermatomyositis and generalized morphoea as well as atypical parkinsonism with motor weakness and spasticity with no systemic amyloidosis and colon carcinoma have been reported in the literature too. genetic factors have been implicated owing to the fact that it occurs before puberty and shows a familial predilection. uvb- and uvc - induced damage to keratinocytes is said to take a longer time for repair in acd. the cytokeratin following keratinocyte apoptosis is phagocytosed by histiocytes and fibroblasts and that is responsible for the amyloid formation. the amyloid is said to be derived from keratinocytes with positive cytokeratin staining. however, the point against photoexposure is that sun - exposed areas in acd often show milder presentation compared to the trunk. hypopigmentation and hyperpigmentation is a novel finding in acd unlike what is seen in the other common variants of primary cutaneous amyloidosis like macular amyloidosis or lichen amyloidosis where only hyperpigmentation is a feature. a hypothesis for consideration would be that the dyschromia occurs in acd due to two factors, i.e., the balance between epidermal pigment retention and dermal pigment deposition. when epidermal melanin remains normal or increase the underlying melanophages, it will contribute to clinical hyperpigmentation. on the other hand, when epidermal melanin is drastically reduced due to decreased pigment donation to keratinocytes from the melanocyte, or due to damage to the melanocytes, clinical hypopigmentation can occur. since the deposits are in the papillary dermis they may stretch the basement membrane, thereby the basal cell layer reducing the density of melanocytes per mm resulting in hypo / depigmentation in that area. the amyloid deposition in papillary dermis and the absence of other histological features are easy to make the histological diagnosis of acd. additionally, acd lacks the marked photosensitivity, very premature actinic damage and the associated cutaneous malignancies seen in xp. though poikilodermatous amyloidosis can cause histological confusion with acd, the former presents with lichenoid papules, early blistering of extremities, photosensitivity, short stature and palmoplantar keratoderma in addition to poikiloderma. acd is known to be refractory to most forms of therapy despite scattered anectodal reports of efficacy of topical steroids, keratolytics, capsaicin, dimethyl sulfoxide and co2 laser. literature has shown variable efficacy of oral acitretin owing to its ability to induce apoptosis, thereby reducing the available keratinocytes, the most probable source of amyloid. our patient avoids sun exposure and uses broad spectrum sunscreens but has refused any active topical or systemic medication. it is not possible to explain the association of acd with such a strong family history of vitiligo in this patient except for the fact that genetic factors, consanguinity and involvement of melanocytes are evident in both these entities. we have tried to hypothesize as to the mechanism of concurrent hypopigmentation and hyperpigmentation in acd, which no other case report on this entity has done to the best of our knowledgekeratotic hyperpigmented papules though not common can be seen in this entity where the predominant and pathognomonic lesions are diffusely scattered hypopigmented and hyperpigmented macules. presence of such papules in areas like proximity of the axillary vault can confuse the clinician because it is reminiscent of dowling degos disease where hypo and hyperpigmented macules both are being increasingly documented in indian patients with fitzpatrick phototype iv and v skinour patient of acd had a strong family history of vitiligo. this observation may be noteworthy for future cases to further probe any remote association between the two. we have tried to hypothesize as to the mechanism of concurrent hypopigmentation and hyperpigmentation in acd, which no other case report on this entity has done to the best of our knowledge keratotic hyperpigmented papules though not common can be seen in this entity where the predominant and pathognomonic lesions are diffusely scattered hypopigmented and hyperpigmented macules. presence of such papules in areas like proximity of the axillary vault can confuse the clinician because it is reminiscent of dowling degos disease where hypo and hyperpigmented macules both are being increasingly documented in indian patients with fitzpatrick phototype iv and v skin our patient of acd had a strong family history of vitiligo. this observation may be noteworthy for future cases to further probe any remote association between the two. we have tried to hypothesize as to the mechanism of concurrent hypopigmentation and hyperpigmentation in acd, which no other case report on this entity has done to the best of our knowledgekeratotic hyperpigmented papules though not common can be seen in this entity where the predominant and pathognomonic lesions are diffusely scattered hypopigmented and hyperpigmented macules. presence of such papules in areas like proximity of the axillary vault can confuse the clinician because it is reminiscent of dowling degos disease where hypo and hyperpigmented macules both are being increasingly documented in indian patients with fitzpatrick phototype iv and v skinour patient of acd had a strong family history of vitiligo. this observation may be noteworthy for future cases to further probe any remote association between the two. we have tried to hypothesize as to the mechanism of concurrent hypopigmentation and hyperpigmentation in acd, which no other case report on this entity has done to the best of our knowledge keratotic hyperpigmented papules though not common can be seen in this entity where the predominant and pathognomonic lesions are diffusely scattered hypopigmented and hyperpigmented macules. presence of such papules in areas like proximity of the axillary vault can confuse the clinician because it is reminiscent of dowling degos disease where hypo and hyperpigmented macules both are being increasingly documented in indian patients with fitzpatrick phototype iv and v skin our patient of acd had a strong family history of vitiligo. this observation may be noteworthy for future cases to further probe any remote association between the two. we have tried to hypothesize as to the mechanism of concurrent hypopigmentation and hyperpigmentation in acd, which no other case report on this entity has done to the best of our knowledgekeratotic hyperpigmented papules though not common can be seen in this entity where the predominant and pathognomonic lesions are diffusely scattered hypopigmented and hyperpigmented macules. presence of such papules in areas like proximity of the axillary vault can confuse the clinician because it is reminiscent of dowling degos disease where hypo and hyperpigmented macules both are being increasingly documented in indian patients with fitzpatrick phototype iv and v skinour patient of acd had a strong family history of vitiligo. this observation may be noteworthy for future cases to further probe any remote association between the two. we have tried to hypothesize as to the mechanism of concurrent hypopigmentation and hyperpigmentation in acd, which no other case report on this entity has done to the best of our knowledge keratotic hyperpigmented papules though not common can be seen in this entity where the predominant and pathognomonic lesions are diffusely scattered hypopigmented and hyperpigmented macules. presence of such papules in areas like proximity of the axillary vault can confuse the clinician because it is reminiscent of dowling degos disease where hypo and hyperpigmented macules both are being increasingly documented in indian patients with fitzpatrick phototype iv and v skin our patient of acd had a strong family history of vitiligo. this observation may be noteworthy for future cases to further probe any remote association between the two. we have tried to hypothesize as to the mechanism of concurrent hypopigmentation and hyperpigmentation in acd, which no other case report on this entity has done to the best of our knowledgekeratotic hyperpigmented papules though not common can be seen in this entity where the predominant and pathognomonic lesions are diffusely scattered hypopigmented and hyperpigmented macules. presence of such papules in areas like proximity of the axillary vault can confuse the clinician because it is reminiscent of dowling degos disease where hypo and hyperpigmented macules both are being increasingly documented in indian patients with fitzpatrick phototype iv and v skinour patient of acd had a strong family history of vitiligo. this observation may be noteworthy for future cases to further probe any remote association between the two. we have tried to hypothesize as to the mechanism of concurrent hypopigmentation and hyperpigmentation in acd, which no other case report on this entity has done to the best of our knowledge keratotic hyperpigmented papules though not common can be seen in this entity where the predominant and pathognomonic lesions are diffusely scattered hypopigmented and hyperpigmented macules. presence of such papules in areas like proximity of the axillary vault can confuse the clinician because it is reminiscent of dowling degos disease where hypo and hyperpigmented macules both are being increasingly documented in indian patients with fitzpatrick phototype iv and v skin our patient of acd had a strong family history of vitiligo. this observation may be noteworthy for future cases to further probe any remote association between the two. we have tried to hypothesize as to the mechanism of concurrent hypopigmentation and hyperpigmentation in acd, which no other case report on this entity has done to the best of our knowledgekeratotic hyperpigmented papules though not common can be seen in this entity where the predominant and pathognomonic lesions are diffusely scattered hypopigmented and hyperpigmented macules. presence of such papules in areas like proximity of the axillary vault can confuse the clinician because it is reminiscent of dowling degos disease where hypo and hyperpigmented macules both are being increasingly documented in indian patients with fitzpatrick phototype iv and v skinour patient of acd had a strong family history of vitiligo. this observation may be noteworthy for future cases to further probe any remote association between the two. we have tried to hypothesize as to the mechanism of concurrent hypopigmentation and hyperpigmentation in acd, which no other case report on this entity has done to the best of our knowledge keratotic hyperpigmented papules though not common can be seen in this entity where the predominant and pathognomonic lesions are diffusely scattered hypopigmented and hyperpigmented macules. presence of such papules in areas like proximity of the axillary vault can confuse the clinician because it is reminiscent of dowling degos disease where hypo and hyperpigmented macules both are being increasingly documented in indian patients with fitzpatrick phototype iv and v skin our patient of acd had a strong family history of vitiligo. this observation may be noteworthy for future cases to further probe any remote association between the two. we have tried to hypothesize as to the mechanism of concurrent hypopigmentation and hyperpigmentation in acd, which no other case report on this entity has done to the best of our knowledgekeratotic hyperpigmented papules though not common can be seen in this entity where the predominant and pathognomonic lesions are diffusely scattered hypopigmented and hyperpigmented macules. presence of such papules in areas like proximity of the axillary vault can confuse the clinician because it is reminiscent of dowling degos disease where hypo and hyperpigmented macules both are being increasingly documented in indian patients with fitzpatrick phototype iv and v skinour patient of acd had a strong family history of vitiligo. this observation may be noteworthy for future cases to further probe any remote association between the two. we have tried to hypothesize as to the mechanism of concurrent hypopigmentation and hyperpigmentation in acd, which no other case report on this entity has done to the best of our knowledge keratotic hyperpigmented papules though not common can be seen in this entity where the predominant and pathognomonic lesions are diffusely scattered hypopigmented and hyperpigmented macules. presence of such papules in areas like proximity of the axillary vault can confuse the clinician because it is reminiscent of dowling degos disease where hypo and hyperpigmented macules both are being increasingly documented in indian patients with fitzpatrick phototype iv and v skin our patient of acd had a strong family history of vitiligo. this observation may be noteworthy for future cases to further probe any remote association between the two.	we are reporting a rare case of amyloidosis cutis dyschromica in a 41-year - old man. this is a rare form of primary cutaneous amyloidosis characterized by reticulate pigmentation with hypopigmented and hyperpigmented macules, onset in childhood, familial tendency in some, occasional mild itching and deposition of amyloid in the papillary dermis. our case also had multiple bilaterally symmetrical hyperpigmented keratotic papules abutting the axillary vault resembling those seen in dowling deogs disease. the other unusual feature in this patient was the strong family history of vitiligo, which we are unable to explain. we have also tried to explain the mechanism leading to the hyperpigmentation and hypopigmentation in amyloidosis cutis dyschromica.
this phenomenon is one of the postoperative outcomes that can be caused by heat loss due to exposure to cold weather, thermoregulation mechanism disorders and, consequently, vasodilatation and loss of muscle tone as a result of anesthetics or local anesthesia. body temperature reduction and, consequently, chills lead to an increase in heart rate, and catecholamine release, vasoconstriction, lower circulation, and metabolic acidosis. there are numerous risk factors in relation with development of intraoperative hypothermia, including aging, female gender, depth of anesthesia, type of surgery, length of anesthesia, operating room (or) temperature, patient 's low weight, and history of chronic diseases and cool fluids infusion. postoperative chills can be unpleasant and distressful, cause complications for the patients, and may lead to worsening of their postoperative pain, such that some patients indicate it as their worst hospitalization experience. this rate has been reported as 6.3 - 66% for the patients receiving general anesthesia and as an average of 56.7%, ranging from 40% to 60%, for the patients receiving spinal anesthesia. prevention of hypothermia is an absolute way of preventing the postoperative chills in response to hyperthermia. intraoperative hypothermia can be reduced by the methods which reduce skin heat emission to the environment because of a cold or, surgery incision evaporation, and by prevention of intravenous infusion of cold fluids. other non - medical methods employed in various studies include warming and humidifying the air way, warming the skin through warm covers, application of patient warming system through water circulation and forced air warming system, and infusion of warm fluids. among the above - mentioned strategies to prevent hypothermia, warming intravenous fluids to preserve patient 's body temperature in the or can be conveniently applied, as intravenous warm fluids are available in all ors in iran. one of the surgeries performed at a high rate is cesarean section (cs). exposure of these patients to anesthesia for cs leads to more temperature reduction because of the effect of anesthetics on vascular and body thermoregulation mechanisms, abdominal vast incisions, and wetting surgical covers with blood and amniotic fluid. various studies reported controversial results in relation to application of non - medical methods in the prevention of hypothermia among the mothers undergoing cs. (2012) in their study on the effect of warming of patients and infusion of pre - warmed fluids in cs on the prevention of hypothermia and postoperative chills showed that core temperature was higher in the study groups (infusion of warm fluids and forced air warming system) compared to the control group (p = 0.004). incidence of chills was also lower in the study groups compared to the control group (p = 0.035). they concluded that forced air warming system and infusion of pre - warmed intravenous fluids prevented patients postoperative chills. on the contrary, butwick. (2007) in a study on 30 healthy mothers who had undergone elective cs with spinal anesthesia divided into two groups of compressed air and conventional or air conditioning showed that core temperature changes were similar in the two groups (p = 0.8), and concluded that application of forced air warming system could not prevent hypothermia among the women who had undergone cs. as patients support is one of the main duties of nurses, anesthesia nurses are responsible for this important issue. therefore, this study aimed to apply an appropriate, cost - effective, complication - free and available method of warming intravenous fluids to prevent hypothermia during cs. this quasi - experimental study was conducted on 62 mothers selected as candidates for elective cs under general anesthesia in shahid beheshti hospital in kashan from february to september 2010. the subjects were selected through purposive sampling, and the selection was based on the inclusion criteria. from 84 mothers who assessed for eligibility, 22 mothers excluded because of not meeting inclusion criteria (n = 16) or declined to participate (n = 8). the subjects (n = 62) were randomly assigned to study (receiving pre - warmed fluid, n = 31) and control (receiving room temperature fluid, n = 31) groups. firstly, the goal and method of study were explained to the eligible subjects and written consents were obtained from them for ethical considerations. the inclusion criteria were : termination of pregnancy in 37 - 42 weeks of gestational age ; undergoing surgery with general anesthesia ; having a tracheal tube ; surgery length less than 1 h ; not receiving corticosteroids, non - steroidal sedatives, mg sulfate, or anti - hypertension drugs ; and lack of endocrine disorders, vascular diseases, pregnancy hypertension, fever, amniotic bag rupture, polyhydramnios, and oligohydramnios. the exclusion criteria were : having received intraoperative blood transfusion, surgery length more than 1 h, receiving medications other than conventional medication, intraoperative hypotension due to any reason leading to infusion of more fluid than calculated, or therapeutic program changes. the candidate mothers for cs received infusion of warmed ringer 's lactate at 37c (kept in bon marry serological water bath for up to 24 h before surgery) in the study group and ringer 's lactate at room temperature (25.5c) in the control group through angiocatheter no. 16, immediately after arrival at the or and lying on the surgical bed. or temperature and humidity were measured and recorded using tympanic infrared thermometer (beurer medical ft55, ulm, germany) and humidity meter (tfa dostman / wertheim, germany. or temperature and humidity were identical in both study and control groups. the required amount of fluids was calculated based on length of fasting time, maintenance fluid, amount of bleeding and urine output, and length of surgery. anesthesia device used for both the groups was dragger fabious (moislinger allee 53 - 55, germany). after they were monitored and administered primary fluids, the subjects underwent pre - operative care. after pre - oxygenation, anesthesia was induced by administering na thiopental (5 ml / kg), and succinylcoline (1 mg / kg) was administered to facilitate intubation. anesthesia was continued by administering isoflurane at alveolar concentration of 0.6%, a mixture of oxygen 50% and nitrous oxide 50%, and with atracurium (0.2 mg / kg) continuing the muscle relaxation. bp, pulse, and arterial o2 saturation of the subjects were measured and recorded using saadat monitoring device (alborz - b model made in iran) before and after the induction of anesthesia. patients core temperature was measured and recorded using tympanic infrared thermometer at the tympana before and at 15-min intervals after the induction of anesthesia (a total of five times). after surgery, the effect of relaxants was neutralized by administering atropine (0.02 mg / kg) and neostigmine (0.04 mg / kg). data collection tool contained a questionnaire to record patients demographic information, a checklist for recording medications during anesthesia, and a checklist of the recorded intraoperative parameters. validity of the information recording form was confirmed by content validity through references and guides of university professors. reliability of the measurement tools was obtained by careful measurement and confirmation of their calibration and sensitivity. the devices were calibrated, while their specificity and sensitivity had been determined by the manufacturing company and the medical technologist engineer in the related hospital. the research coworkers filling the forms of data record and the subjects were blinded to the study to increase the validity of the data. after the data were collected, they were analyzed by t - test, mann whitney u test, chi - square test, and analysis of variance (anova) with repeated measurements through spss version 16. lower - volume pre - warmed serum bags (500 ml), which could be infused more quickly, were used to prevent temperature loss in the orprecise determination of bleeding volume is difficult in surgeries, and this issue is even more difficult in cs and cases of amniotic bag rapture. to decrease this effect, the amount of bleeding was estimated by collection of amniotic fluid and by using counting and weighing surgical gauzes and long gauzes. lower - volume pre - warmed serum bags (500 ml), which could be infused more quickly, were used to prevent temperature loss in the or precise determination of bleeding volume is difficult in surgeries, and this issue is even more difficult in cs and cases of amniotic bag rapture. to decrease this effect, the amount of bleeding was estimated by collection of amniotic fluid and by using counting and weighing surgical gauzes and long gauzes. lower - volume pre - warmed serum bags (500 ml), which could be infused more quickly, were used to prevent temperature loss in the orprecise determination of bleeding volume is difficult in surgeries, and this issue is even more difficult in cs and cases of amniotic bag rapture. to decrease this effect, the amount of bleeding was estimated by collection of amniotic fluid and by using counting and weighing surgical gauzes and long gauzes. lower - volume pre - warmed serum bags (500 ml), which could be infused more quickly, were used to prevent temperature loss in the or precise determination of bleeding volume is difficult in surgeries, and this issue is even more difficult in cs and cases of amniotic bag rapture. to decrease this effect, the amount of bleeding was estimated by collection of amniotic fluid and by using counting and weighing surgical gauzes and long gauzes. variables of age, weight, time interval between arrival to or and beginning of anesthesia, lengths of surgery, temperature and humidity of or, mothers core temperature in the first minutes of their arrival to or, and the amount of mothers fluid intake in the two groups of study and control were analyzed by statistical tests and showed no significant difference [table 1 ]. chi - square test showed no significant difference between the cause of cs in the mothers of study and control groups (p = 0.98) [table 2 ]. comparison of some characteristics of mothers in the study and control groups comparison of causes of cs for mothers in the study and control groups core temperature of the mothers was measured in the first minutes of their arrival to or until the beginning of anesthesia in the two groups at their tympana and showed no significant reduction (p = 0.10), but the measurements taken every 15 min to the end of surgery and anesthesia showed a reduction in both the groups. this reduction was more in the control group, such that at 15 min of surgery, the temperatures were 36.04 0.43c and 35.63 0.6c in the study and control groups, respectively, which showed a significant difference (p = 0.003). the core temperatures at the 30 min were 35.98 0.5c and 35.41 0.6c in the study and control groups, respectively, which showed a significant difference (p = 0.001). in addition, the core temperatures of the mothers at the end of anesthesia were 36 0.5c and 35.34 0.6c in the study and control groups, respectively, which showed a significant difference (p = 0.000) [table 3 ]. comparison of core temperatures of the mothers in the study and control groups during surgery comparison of the core temperatures of mothers in the two groups of study and control before and during anesthesia showed a significant difference by repeated - measures anova (p = 0.008) [table 4 ]. comparison of core temperatures of the mothers in the study and control groups during surgery mothers core temperatures showed no significant difference at the beginning of anesthesia in the two groups (p = 0.10), but the measurements taken at the 15 (p = 0.003) and 30 min of surgery (p = 0.001) and at the end of surgery (p = 0.000) showed a significant difference [table 3 ]. it reveals that pre - warmed serum led to higher core temperature in the mothers of the study group compared to those of the control group [table 4 and figure 1 ]. mothers core temperatures during surgery in our study, the core temperature of mothers in the study group (receiving pre - warmed fluid) was 0.5c higher compared to those in the control group (receiving room temperature fluids). (2009) who studied the effect of pre - warmed intravenous fluid (38c) among mothers as candidates for cs by spinal anesthesia on prevention of their hypothermia. in their study, woolnough (2009) compared the two methods of active fluid warming (warming during surgery) and passive fluid warming (warming before surgery) in women undergoing cs and concluded that warm fluids slowed down the reduction of body temperature and increased mothers comfort. finally, this study revealed the efficiency of passive fluid warming and its cost efficacy. (2012) studied the effect of keeping the patients warm before cs on hypothermia and postoperative chills. a total of 45 patients were randomly assigned to three groups : receiving warm intravenous fluid (40c), active air conditioning by compressed air, and the control group. core temperatures of patients in the groups that received warm fluid and compressed air conditioning showed a lesser reduction than in the control group (p = 0.004). also, the incidence of chills in these groups was less that in the control group (p = 0.035). they concluded that keeping the patient warm (with either compressed air or administering warm fluids) could prevent hypothermia and postoperative chills among the patients undergoing cs with spinal anesthesia. (2011) studied the effect of warming intravenous fluids in the maintenance of core body temperature during cs under spinal anesthesia. one group received room temperature fluid (22c) and the other warm intravenous fluid (39c). the core temperature reduction was less among the mothers who received warm fluids compared to the other group (p < 0.01), but there was no significant difference in the incidence of chills in the two groups. (2004) randomly assigned 60 orthopedic patients to two groups of study and control (37c fluid and room temperature fluid, respectively). mean differences of patients esophageal temperatures on the 15, 30, 45, and 60 min during anesthesia were significant in both the groups. the core temperature had a descending trend from the beginning of the surgery, but this reduction and descending trend was observed more in the control group. there was also a significant difference in the mean skin temperature (p < 0.001). their findings were consistent with our findings indicating the positive effect of warm fluids infusion on the prevention and reduction of hypothermia during cs. (2007) conducted a study on 30 patients undergoing cs in two groups that received compressed air and conventional or temperature, respectively. the results showed that the changes in core temperature were similar in the two groups (p = 0.8) and core hypothermia occurred in 8 out of 15 mothers in the compressed air group and in 10 out of 15 mothers in the conventional op temperature group (p = 0.5). they concluded that application of compressed air could not prevent hypothermia in patients undergoing cs. (2009) showed that the incidence of chills in elective cs patients receiving warm intravenous fluids and in those receiving room temperature fluids showed no difference. other studies also showed that warming intravenous fluids could not prevent hypothermia among women undergoing elective cs. these results are not consistent with ours, possibly due to low sample size of the above - mentioned study. previous studies showed that even minor hypothermia might lead to serious unexpected outcomes in many of the patients, including surgery incision infection, prolonged hospitalization, increased bleeding during surgery, more demand for blood transfusion, postoperative ventricular tachycardia, postoperative chills, and prolonged recovery period, as the main complications that result from hypothermia in human beings. the most important methods are infusion of warmed intravenous fluids and application of forced air warming system. each liter of low - temperature infused fluid can diminish the body temperature of a person by 0.25c. infused warmed intravenous fluids can enhance the core temperature by 0.5c-0.7c and reduce the risk of hypothermia. to keep the patient warm before and during surgery, the two main methods of forced air warming system and infusion of warmed intravenous fluids are used to maintain the patient 's core temperature during surgery. the main advantages of body temperature preservation in normal range include low risks of incision infection, coagulation disorders, and myocardial ischemia. based on the obtained results, it can be concluded that application of the convenient, easy, and low - cost method of warming intravenous fluids (ringer 's serum) can be helpful in prevention of hypothermia resulting from general anesthesia among mothers undergoing cs and its potential hazardous complications (prolonged hospitalization, increased bleeding during surgery, more demand for blood transfusion, postoperative ventricular tachycardia, postoperative chills, prolonged recovery period, infection, and myocardial ischemia during surgery). therefore, nurses as patients advocate should prevent the incidence of patients complications in op through precisely checking the vital signs, especially temperature, and by undertaking other preventive interventions such as infusion of warmed intravenous fluids.	background : hypothermia is one of the problems occurring during surgery, which can happen due to thermoregulation mechanism disorders and intake of low temperature iv fluids, and may cause increase in blood pressure, heart rate, intracranial pressure, oxygen consumption, pain, and discomfort to the patient. the rate of cesarean section in our country is three times more than the global standard. as one of the responsibilities of the nurse is patient 's advocacy, s / he should support them. this study aimed to investigate the effect of pre - warmed intravenous fluids on prevention of hypothermia during general anesthesia in cesarean section.materials and methods : sixty - two women undergoing elective cesarean section by general anesthesia were randomly allocated in two groups of intervention and control. women in the intervention group received pre - warmed serum (37c) while those in the control group received serum at room temperature (25.5c). the core body temperature and some hemodynamic parameters of the participants were assessed during the operation.results:the mean of pulse rate, systolic blood pressure, diastolic blood pressure, and arterial o2 saturation in the two groups were not statistically significant (p > 0.05). but the mean of mothers core body temperature at the end of anesthesia in the intervention and control groups were 36 0.5c and 35.34 0.6c, respectively (p < 0.05).conclusion : infusion of pre - warmed serum (37c) would prevent intraoperative hypothermia and improve the nursing care for women who undergo cesarean section by general anesthesia.
significant among the advances of the last 3 decades in breast cancer diagnosis and management are the broad access to mammographic service screening by asymptomatic individuals [15 ], diagnosis by minimally invasive needle biopsy techniques, and the widespread acceptance of breast conservation surgery. surgical management, specifically the complete surgical removal of all detectable breast carcinoma, remains the preferred first therapeutic step for the majority of men and women with stage 0iii breast cancer [6, 7 ]. breast - conserving procedures, as an evidence - based alternative to mastectomy, have gained widespread acceptance since the nsabp b-04 and b-06 trials over 30 years ago [8, 9 ]. pathologic examination of resected tissue continues to serve as the definitive means of establishing adequacy of breast conservation surgery (bcs) with the reporting of surgical margin status. as such, margin evaluation remains a key data point in the clinical decision of whether to administer radiation therapy postoperatively or to perform an additional excision to obtain negative margins. neither the nsabp b-04 or b-06 trials defined margin negativity beyond the simple absence of neoplasm on the margin itself, so although margin negativity was required for accrual into the bcs - radiation therapy arm, proximity of tumor or tumor subtype to surgical margins was neither reported nor controlled. the last 30 years have yielded confusing and contradictory information regarding the risk of local or systemic recurrence and survival following bcs. numerous authors have sought to identify variables useful in the prediction of breast cancer recurrence or at least to serve as criteria for surgical reexcision. many of these variables have been drawn from the characteristics of the neoplasm itself or the proximity of the neoplasm to surgical margins. investigators have reported the relationship between recurrence (or the presence of residual cancer in reexcision specimens) and the proximity of invasive cancer to negative bcs margins [1113 ], the number of involved margins, multifocality, and the presence of an extensive intraductal component (eic) associated with an invasive cancer [15, 16 ]. unfortunately, many reports attempting to correlate recurrence risk with proximity to surgical margins failed to define margin status relative to duct carcinoma in situ (dcis) [1719 ], and so it was not known to what extent margin positivity for dcis may have contributed to observed recurrence rates. gradually, dcis was identified as an independent risk factor in breast cancer recurrence after bcs. more recent studies have demonstrated varying degrees of correlation between the overall extent of dcis [2022 ], nuclear grade, and proximity to surgical margins [2427 ] and risk of breast cancer recurrence. in some series, over 50% of women undergoing breast conservation surgery required additional surgery to achieve satisfactory margin status. most of these reports have been inspired by one or more persistent clinical questions, relevant to the clinical management and prognosis of a patient diagnosed with breast cancer (table 1). it has been difficult to draw a clear consensus from much of the breast cancer recurrence literature published over the last 3 decades ; however some general conclusions are reasonable. positive margins for both dcis and invasive cancer place a patient at high risk for local recurrence and are to be avoided. secondly, negative margins are not a guarantee against recurrence, yet increasing margin width crudely correlates with decreasing risk of recurrence by reducing the likelihood of residual carcinoma in the adjacent breast. greater extent of dcis and higher nuclear grade are positively correlated with recurrence, even with negative margins (21, 23). despite these broad generalizations, which in retrospect appear intuitive, a high degree of concordance across these studies is not observed with covariables such as margin width of dcis and risk of local failure or dcis extent and risk of recurrence. the lack of consensus in this literature is frustrating to surgeons and oncologists alike who seek reproducible data to support evidence - based management protocols. it seems obvious that margin evaluation is not an exact science, but how much of the conflicting data is related to the biology of breast cancer itself ? some authors have pointed to difficulties in standardizing, optimizing, and reporting breast specimen examinations by pathologists [31, 32 ]. this general lack of concordance suggests that the pathology data collection itself is not optimized for reproducibility ; a reasonable conclusion considering the conventional methods of gross specimen examination used to report breast cancers is essentially unchanged from the preimaging era. the literature addressing tumor attributes and risk of recurrence mostly relies upon data abstracted from pathology reports based on traditional methods of gross and microscopic examination. in the vast majority of these laboratories, surgical specimens are sectioned to conform to industry - standard 25 mm 175 mm glass microscope slides. measurement methodologies for dcis extent vary from laboratory to laboratory and are often not specified in the publication. describe a protocol sanctioned by the college of american pathologists for the examination of specimens from patients with dcis. the protocol is a welcome effort to assist pathologists in the identification and reporting of dcis in resected tissue. the authors appropriately enumerate the varied methods used by pathologists to measure dcis extent but no recommendation was made to standardize the process or correlate imaging data into specimen examination. commonly, pathologists measure the largest single focus on an individual slide or the span of dcis across a single slide and report the result as dcis extent. other pathologists calculate extent by counting the number of slides involved with dcis. some pathologists add the gross section width (e.g., 3 mm, 5 mm) of involved slides to estimate cumulative dcis extent, a practice found by dadmanesh. to underestimate extent in 72% of cases. rarely, a pathologist will attempt to correlate histopathology with imaging studies and measure dcis extent across images using the locations of slides positive for dcis. reconciliation of presurgical imaging characteristics with pathologic measurements of size, extent, or margin status prior to finalizing the pathology report seems to be the exception, rather than the rule. in clinical practice, reporting of 6-axis bcs margins is standard practice in most centers ; however numerous technical limitations plague the process. breast tissue is inherently pliable, and the dimensions of the specimen and associated neoplasia can change dramatically with changes in patient position from mammogram to mri, operating suite and pathology work station. pathologists or their assistants typically apply multicolored inks to the specimen circumference to approximate the orienting clips or sutures placed surgically ; however this process typically occurs without surgeon validation of the designated margins. the resulting margin boundaries (e.g., anterior - medial or posterior - lateral) may or may not represent the surgeon 's view of the same boundaries. the risk for the patient is that a reexcision of a close or involved margin may not align with the margin as identified by the pathologist essentially leaving a critical margin unresected. despite remarkable advances in breast imaging technology since the early screening trials including digital mammography, 3d automated ultrasound, digital tomosynthesis, magnetic resonance imaging (mri), and breast - specific gamma imaging (bsgi), the techniques employed by pathologists to grossly examine resected breast tissue have changed little, save for the measurement of margins on oriented specimens. specimen radiographs obtained for wire - localization bcs procedures are usually available for pathologist review ; however the contribution of these images to the gross and microscopic examination is only occasionally documented in pathology reports. the degree to which wire - localization radiographs guide the pathologist 's determination of cancer size, extent, and proximity to margins is unknown. in a study of 135 bcs procedures for dcis, mammography was found to significantly underestimate the extent of dcis as measured pathologically even when performing complete specimen radiography and extensive tissue sampling. compared to mammography, presurgical mri has significantly higher sensitivity in the detection of dcis ; however presurgical mri studies are typically not available to pathologists at the time of specimen gross examination. lacking knowledge of the imaging extent and distribution of a neoplasm in three dimensions (as is possible with mri), gross surgical specimens are sectioned without reference to the axis of greatest cancer extent or mammographically occult encroachment on surgical margins. pathologists intentionally select 6-axis sections for microscopic study when a grossly palpable or visible area of concern is identified, when wire - localization images suggest margin compromise or when a surgeon identifies margins of concern. otherwise, sections are randomly selected based on gross inspection to represent 6-axis margins. the specimen is further morcellated until the individual tissue blocks conform to the industry - standard tissue cassette, an area no larger than 25 mm 35 mm. improved characterization of dcis extent and margin status has resulted from protocols requiring submission of 100% of lumpectomy tissue, notably the serial subgross techniques utilized by holland., macdonald. [21, 23 ], cheng. three - dimensional reconstruction techniques to improve margin evaluation were reported by mai. and ichihara.. these techniques typically maintain the spatial orientation of the tissue sections for subsequent three - dimensional reconstruction. unfortunately, these methods require that additional breast tissue is trimmed from the submitted tissue blocks so they conform to standard tissue processing methods. the result is a diminution or loss of local breast and tumor anatomic relationships. in some centers, mammographic examinations are routinely performed on intact tissue slices [22, 23, 35 ] ; in others, radiographic examination of gross specimen sections is either performed on a subset of sections or not at all. a significant limitation to the accurate reporting of dcis extent and margin status results from poor information flow among the breast care team members at the time of surgery. modern imaging techniques provide the breast imager and surgeon with an increasingly lucid presurgical depiction of the extent and distribution of breast neoplasia. functional imaging, particularly breast mri, gives insight into the anatomic distribution of neoplasm in three dimensions [44, 45 ] and can identify disease, particularly dcis extent beyond the sensitivity and specificity of mammography or ultrasound [46, 47 ]. many studies have attempted to evaluate the sensitivity or specificity of mammography, ultrasound, and mri using reported pathologic tumor characteristics as the reference standard. most of the published imaging - pathology correlation studies import tumor measurements directly from cancer registry or pathology reports. the data thus obtained is subject to all of the limitations associated with conventional pathologic techniques. as an example, ichihara. found mri was not only more sensitive than mammography in detection of dcis (88% versus 27%) but maximum extent of dcis measured by mammogram (90 mm) or mri - detection (110 mm) far exceeded maximum pathology extent measurements (25 mm). schouten van der velden. in comparing reported pathology cancer size measurements of 49 cancers with estimates by mammogram and mri found agreement within 5 mm in only 27% of mammogram and 38% of mri studies. some authors regard the discrepant size and extent measurements as evidence that mri overestimates the extent of breast carcinoma and raise concerns of unnecessary or additional surgery [47, 48 ]. notably absent from the current debate on the sensitivity and specificity of breast imaging technology and the clinical utility of breast mri as a presurgical planning procedure is a discussion of the limitations of the pathologic techniques used to validate modern imaging studies. the practical limitations of conventional breast pathology reporting techniques [31, 49, 50 ] are summarized in table 2. prior to the widespread clinical use of breast mri, breast pathologists using large - format contiguous histologic sections measuring up to 8 10 cm visualized breast cancer in three dimensions [33, 51 ] and improved correlation between imaging and pathologic measurements of cancer size and extent [49, 52 ]. subsequent reports have confirmed the clinical advantages of large - format breast specimen processing with improved imaging - pathology correlation of cancer distribution and optimized evaluation of surgical margins [50, 53, 54 ]. the suitability of these techniques to the community hospital setting and the benefit to both imager and pathologist were described by biesemier and alexander and mchine - neuville.. the image - guided large - format breast pathology (lbp) initiative at carilion clinic necessitated preimplementation development of several key program elements (table 3). the specimen processing methods at carilion clinic were developed over a six - month period. they were adapted from procedures in place at falun central hospital, falun, sweden and central virginia pathology consultants, lynchburg, virginia following site visits to those locations. capital and operating budgets were prepared, and all laboratory capital and operating expenses were funded exclusively from laboratory operating revenue. a separate cost center was not created within the histology laboratory for the large - format program ; however expenses attributed to histology labor and consumables were recorded for analysis of incremental costs in comparison with conventional pathology processing. specimen radiography and radiograph interpretation were performed in the breast imaging department in a cost center created for the purpose of supporting the large - format pathology program. the technical and professional component billing and revenue capture for specimen radiography and interpretation using cpt codes 76098-tc and 76098 - 26 were booked to the specimen radiography cost center. this weekly conference, attended by breast imagers, surgeons, and pathologists, was created to optimize surgical outcomes through the continuous correlation of large - format pathology with clinical findings and all pre- and postimaging data, including mri. as an adjunct to the existing weekly interdisciplinary breast pretreatment planning conference, this conference a detailed postsurgical analysis of tumor size, extent, multifocality, and proximity to surgical margins was performed by correlating all presurgical breast imaging studies with subgross and histopathologic findings. this was accomplished with real - time projection of large - format pathology slides alongside mri and other projected presurgical imaging studies. presurgical imaging estimates of cancer extent, geographic distribution, and proximity to anatomic landmarks were reviewed and revised with large - format pathologic validation. final surgical margin measurements were scrutinized in view of presurgical procedure planning goals and intraoperative surgical findings. secondly, presurgical planning was conducted with a goal of optimizing the utilization and outcomes of breast - conserving procedures whenever possible. the conference differed from the usual presurgical planning conference by leveraging the team 's experience gained from the post - surgical large - format - imaging correlations. breast mri was routinely performed on most cases diagnosed and treated in the lbp era and was generally not available in this breast program prior to implementation of lbp. all mastectomy, lumpectomy, and reexcision specimens are included in the large - format program with specialized processing procedures designed for each specimen type. mastectomy specimens were phased - in beginning august 2004 in order to gain experience with the technique. nearly 400 surgical specimens representing between 250 and 275 new breast cancer diagnoses are accessioned and processed annually. upon receipt in the surgical pathology suite, all specimens are checked for proper surgical orientation, and any deficiencies identified are promptly corrected by the surgeon. all surgical margins are marked before sectioning with india ink with a discrete color coded to each of six axial margins. mastectomy specimens are sectioned in the sagittal plane at uniform 5 mm increments with preservation of medial - to - lateral sequence and cephalocaudad orientation. initially experience permitted introduction of time and cost conserving measures so that approximately one - half to two - thirds of each specimen was submitted for radiography, focusing on the location of previous biopsy or lumpectomy sites and any additional guidance provided by breast surgeons and imagers. breast - conserving specimens and reexcisions are inked for margins and sectioned at 5 mm increments. the plane of sectioning varies with the requirements of the case. occasionally, demonstration of maximum extent of disease or multifocality in one plane is of paramount importance. in these cases, the plane of sectioning is guided by image interpretation of size, extent, and multifocality, and sections are typically made in the coronal (frontal) plane. in cases where the status of chest wall or anterior cutaneous margins (or both) are of particular concern or the sagittal plane shows maximum extent, the sagittal plane is selected for sectioning and large - format section submission. this approach allows convenient correlation of sagittal large histology sections with mri sagittal - reconstructed images. orientation of all specimens is maintained with the use of radioopaque alphabet characters to denote axis margins in each plane as well as the sequence of sections, for example, superficial to deep in the coronal plane or tissue slices are placed on previously exposed radiographic film with orienting characters and transported expeditiously to the breast imaging facility for radiography. following specimen radiography and radiographic interpretation (vide infra), the specimen with accompanying radiographs is returned for pathologic examination with a large - format specimen checklist detailing the clinical and specimen radiographic findings in written narrative and using template diagrams of both breasts. this worksheet communicates the clinical and radiographic findings of concern with specific questions to be addressed pathologically. imaging abnormalities visible mammographically are marked directly on the films with a wax pencil as a guide for histopathologic section submission (figure 1). the location and extent of ultrasound or mri - detected findings, not visible mammographically or with specimen radiography, section(s) best demonstrating critical findings such as proximity to margins or maximum extent of neoplasm are encircled for possible large - format submission. following traditional pathologic specimen examination with gross inspection and palpation, sections are selected for histologic study to incorporate gross pathologic findings as well as findings of concern communicated by imagers and surgeons (figure 2). microscopic evaluation of surgical margins is typically accomplished with a combination of both large - format and conventionally sized tissue blocks. large sections usually permit margin evaluation along 100% of the circumference of a bcs specimen and are selected to encompass critical margins whenever close proximity to a surgical line of excision is suspected. margins perpendicular to the large - format plane of section are usually submitted as conventional tissue blocks. orientation of all conventionally sized margin sections is perpendicular to the specimen surface to allow measurement of margin width to the nearest whole millimeter. the precise location of these perpendicular margins is selected with guidance from both imaging and clinical information and conventional pathologic examination. reexcision specimens, including surgical shave excisions of biopsy cavities, are sectioned and oriented perpendicular to the surgical margin so that margin distance from the cavity to the new surgical margins remains measureable. the anatomic location of all tissue blocks removed from the specimen is recorded in the gross dictation narrative of the pathology report as well as on the specimen radiographs which are preserved as a permanent record of pathology slide origin. this record is provided to the pathologist at microscopic sign - out as an aid in the reconstruction of the specimen in three dimensions. upon receipt in the imaging center, specimens are placed in an analog specimen radiography unit (faxitron bioptics, llc). specimen film images are examined by a breast imager, and all relevant mammograms, core biopsy specimen radiographs, wire - localization images, ultrasound, and mri studies are reviewed. mammographically detected lesions of concern are marked on the radiograph films, and the location of mammogram occult abnormalities identified with ultrasonography and mri is encircled. to facilitate communication of clinical, presurgical imaging and specimen radiography findings to the pathologist, the large - format specimen checklist a graphical depiction of the location and extent of clinical and imaging - suspected neoplasia is marked on a template bilateral breast diagram. this form is returned with the specimen and annotated specimen radiographs to the surgical pathology suite. upon receipt in the histopathology laboratory, conventional tissue blocks are separated from the large - format tissue blocks and separately processed in the routine manner. large - format blocks are fixed for an additional 2432 hours in 10% neutral buffered formalin. after fixation, all breast tissue is processed on automated processors (tissue - tek vip, sakura finetek usa, inc.). paraffin embedding of large tissue sections requires reusable cassette forms fabricated on - site from 14 ga 1 1/4 wide aluminum bar stock (figure 3). paraffin block dimensions are thereby conformable to the size of the tissue block and measure up to 8 10 cm. sections are prepared at 3 to 4 microns on a leica sliding microtome model sm2500 (leica microsystems). slide staining is performed on an automated histology slide stainer with slide baskets customized to hold large - format glass slides (shandon varistain 24 - 4, thermo electron corporation). pathologists are provided with transcribed gross dictation, all slides including large - format slides, annotated specimen radiographs, and the large - format specimen checklist completed by the breast imager. with attention to the specific questions communicated by the breast imager, histopathology slides and radiographs are analyzed to complete all relevant data fields following the college of american pathologists ' recommended synoptic reporting protocol. when maximum size of invasive carcinoma or extent of in situ carcinoma is represented by the large - format slide, that dimension is directly measured from the slide and reported. when clinical or imaging information indicates a greater size / extent than represented on the large - format slide, a three - dimensional reconstruction of tumor size / extent is performed. following guidance provided by the breast imager on the large - format specimen worksheet, size and extent are either directly measured or calculated using the uniform 5 mm section thickness as a guide and reported to the nearest whole millimeter. margin evaluation of bcs specimens is accomplished with similar reference to imager guidance. in sagittal - plane sections, circumferential microscopic evaluation of margins is correlated between large - format slides and specimen radiographs depicting anterior, inferior, posterior, and superior margins. medial and lateral margin sections, whether conventional or of the large - format type, are similarly correlated with images. proximity to each of six axial margins is measured to the nearest whole millimeter and recorded as 0 mm (positive), 110 mm in 1 mm increments, or > 10 mm. margin involvement of a boundary between two margins is reported as involvement of both margins at their junction, for example, anterior - lateral or superior - medial. a series of 135 consecutive lumpectomy specimens with a diagnosis of dcis without invasive carcinoma were analyzed for margin status using the above techniques. ninety - two specimens processed following the lbp protocol including presurgical mri were compared to forty - three specimens processed using conventional pathologic techniques without presurgical mri. the surgical reexcision rate and breast conservation rate in each category were retrieved from cancer registry data, and the volume of each lumpectomy specimen was computed with dimensions obtained from the gross pathology specimen description. the average number of conventional pathology blocks produced per mastectomy and lumpectomy specimen was tabulated for a total of 100 resections. the block totals were compared between cases submitted in calendar year 2004 with conventional pathology processing and in 2005 with large - format processing. an average of one large - format block per case was submitted in 2005. technical expense for labor and materials associated with the processing of breast pathology specimens were calculated for 50 consecutive cases in calendar year 2005. histotechnologist time devoted to the processing of conventional and large - format breast tissue from the time of receipt to delivery of stained and coverslipped slides was separately recorded for conventional and large - format processed tissue. total technical time, expressed in fractional hours in each category was multiplied by the average hourly wage, including all benefits, of the histotechnologists directly involved with the project to arrive at total labor expense (tle) for conventional and large - format processing. unit labor expense per slide was calculated by dividing tle by the total number of finished slides produced in each category. all materials used in processing and slide production were allocated to each category as appropriate. for liquid reagents and stains shared in processing, a pro - rata allocation between categories was estimated using the surface area of conventional versus large - format slides multiplied by the number of slides in each category. total cost of consumables in each category was divided by the number of slides produced to calculate consumable cost per completed slide for each group. finally, tle and material cost per finished slide in each category were combined to calculate total technical expense for both conventional and large - format slides. amortization of capital equipment and indirect costs, such as overhead and staff training, were not included in the calculation of technical expense. professional time devoted to the examination and reporting of conventional and large - format sections a searchable breast database was developed at the inception of the program using microsoft access (microsoft corporation). all data collections and analyses were performed in compliance with institutional review board approved research protocols. separate pages in the database were created for clinical, mammography and ultrasound, mri, and pathology data fields. training of cancer registry and breast center staff in the population of data fields and search methodology was conducted by information technology staff. conventional histopathologic evaluation of surgical margins is typically accomplished in bcs specimens with six tissue blocks, one selected in each margin axis. in any one plane of section, four conventionally sized tissue blocks are usually removed for microscopic analysis (figures 4 and 5). the relationship between histologic section orientation and percent of the surface margin available for microscopic study in a hypothetical 8.0 cm diameter bcs specimen is presented in table 4. if each of the conventional margin sections spans 1.0 cm of arc, approximately 16% of the circumference in one plane of section is available for microscopic study. alternatively, if the margin sections each span 2.0 cm of margin circumference, the proportion represented rises to 32%. in contrast, the large - format method permits microscopic examination of 100% of the circumference of the selected plane of section of a bcs specimen. the margin evaluation of a three - dimensional resection specimen is more complex than the evaluation of margins in a single plane, however. en - face margin sampling theoretically permits the examination of a greater percentage of the specimen surface ; however this method does not allow for the measurement of margin width. perpendicular orientation of margin blocks facilitates measurement of margin distance at the expense of proportionate sampling of the specimen surface. as seen in table 4, routine histologic sections of 4-micron thickness represent a very small percentage of the specimen surface area, even when compared with the amount of tissue theoretically available in a 3 mm thick paraffin block. the representative nature of margin evaluation, as evidenced by the small proportion of surface margin actually examined, illustrates the necessity of obtaining the greatest yield and relevance possible from each margin section removed from a specimen. in the lbp program, large - format section selection was guided by review of imaging and clinical data conveyed at the time of specimen gross examination. in approximately 10% of cases, two or more large - format sections were submitted to gain experience with the technique and to correlate the extent of neoplasia with mri. with experience however, one optimally selected large - format section supplemented with conventionally sized blocks proved sufficient to evaluate margins, demonstrate maximum extent of neoplasia, and correlate with imaging studies. the influence of the lbp program on reported surgical margins and reexcision rate for dcis was compared with preimplementation conventional pathologic reporting. the effect of the lbp initiative was evaluated in the context of a fully integrated interdisciplinary breast program characterized by meticulous presurgical planning, routine use of large - format sections, and correlative postsurgical interdisciplinary analysis of tumor size, extent, multifocality, and proximity to surgical margins. adequacy of breast conserving surgery was expressed as margin width in millimeters exceeding a selected distance in each of six axes. the integrated lbp program with presurgical mri is compared with conventional breast pathology without presurgical mri in table 5. india ink applied to the surface of a breast specimen to mark the surgical margin may migrate into cracks and clefts in the specimen surface as noted by campbell. the pathologist encountering an inked tissue edge on a conventional pathology slide may regard it as a true inked margin and measure margin width from a focus of cancer to the visible ink, unaware that the true margin is at greater distance (figure 7). the migration of india ink into the interior of breast surgical specimens is relatively common and appears to occur along fissures in the specimen exposed or created by the surgical procedure or specimen handling. with conventional histologic sections, tissue blocks removed from a surgical specimen exhibiting an inked tissue edge a potential for misinterpretation of the margin width exists if the tissue block was taken from an area of ink migration. in such a circumstance, the margin measurement would be lower than the true margin width. preservation of the anatomy of the specimen with large - format sections permits clear identification of the peripheral margin contour so that ink migration into the interior of the tissue block is recognized and not confused with india ink on a true peripheral surgical margin (figure 8). imaging studies, particularly specimen radiographs, are correlated with histopathology in the determination of greatest extent of dcis and maximum size of invasive carcinoma at the time of pathologist signout in all cases. in many but not all instances, in such cases, extent can be measured directly from the large section slides themselves. frequently, however, it is necessary to reconstruct the specimen in three dimensions. three - dimensional reconstructions are made by correlating the annotated specimen radiographs with individual tissue blocks keyed to the radiographs. reference is made to imaging estimates of tumor size and extent. in these reconstructions, calculations of dcis extent are based on a combination of direct slide measurement and calculation of maximum distance of histologically confirmed dcis across uniform 5 mm tissue slices. a comparison of dcis extent measurements between conventional pathologic techniques and imaging - guided large - format breast pathology (lbp) is given in table 6. a typical case depicting tumor size and extent measurement and margin analysis using the lbp technique is shown in figures 9 and 10. in this example, the 71-year - old patient presented with a palpable 1 - 2 cm nodule of the upper - inner quadrant. mammogram showed a birads 4 stellate density 14 mm in diameter corresponding to the palpable lesion. ultrasonography disclosed an 15 mm hypoechoic lesion amenable to ultrasound - guided core needle biopsy, which was positive for grade 3 invasive ductal carcinoma. presurgical mri obtained for surgical planning purposes confirmed the presence of the biopsied lesion and additionally demonstrated a 60 mm area of enhancement extending anteriorly and superiorly from the index lesion. the specimen was sectioned in the sagittal plane to correlate with mri sagittal - reconstructed images and to best demonstrate extent of mri enhancement and proximity to anterior, posterior, superior, and inferior margins. gross examination of the specimen disclosed a firm 15 mm nodule ; however, most of the enhancing 60 mm mri - detected lesion consisted of clinically, surgically, and pathologically impalpable grade 3 dcis. final margin analysis reported dcis present 2.0 mm from the anterior - superior margin. the primary capital expense is for a sliding microtome which can be purchased new for $ 55,000$65,000 (leica microsystems). existing laboratories may configure their tissue processors and staining equipment to accommodate large - format specimens if excess capacity exists. otherwise, conventional automated processors (e.g., sakura tissue - tek vip, $ 50,000) and stainers (sakura tissue - tek drs, $ 30,000) may be acquired for this purpose and are easily adapted to large sections. the direct labor and materials expense for lbp is moderated by cost savings resulting from a reduction in the number of conventional pathology sections submitted per case (table 7). the focused, image - guided nature of the lbp technique results in the elimination of low - yield sections typically submitted in mastectomy and lumpectomy cases. the direct technical component labor and materials expense for conventional and large - format slides is given in table 8. this expense calculation does not include indirect expenses such as allocated overhead and staff training or amortization of capital equipment. also not included in the expense calculation is an allowance for additional professional time for lbp gross specimen examination by pathologists or pathology assistants. the net incremental technical expense per case is derived by subtracting the savings realized in reducing the number of conventional pathology sections per case from the increased expense associated with large section submission (table 9). for nearly three decades, numerous reports have analyzed pathologist - derived data such as dcis extent or distance to surgical margins to predict risk for cancer recurrence or provide an evidence - based rationale for surgical reexcision following breast conservation surgery. the pathologic methods used to generate this data have been poorly standardized and largely bereft of imaging correlation. the reproducibility of the methods typically used by pathologists to examine surgical specimens and report breast cancer attributes has been largely unchallenged until recently. the analysis and reporting of surgical margins, as it is practiced today, remains a crude science. for example, dcis may escape gross detection in the dense as well as fat - replaced breast. without gross findings to provide guidance, margin sections are often randomly submitted without reference to imaging studies. on all but the very smallest of excisions, the typical margin analysis may not accurately and completely document proximity of neoplasm to the surface margin. to its credit, the 2005 international consensus committee panel on image - detected breast cancer made general recommendations for the examination of surgical specimens including the routine use of correlative specimen radiography or ultrasonography. the panel also recommended rigorous and documented specimen sampling to allow for a targeted return for additional sampling or, better still, processing of the specimen in its entirety. these recommendations, however welcome, have not resolved the barriers to information flow between imaging and pathology departments nor were they intended to address the additional costs associated with complete specimen processing. the panel did not reference large - format techniques as described by tot and others [50, 56 ]. in clinical practice, the college of american pathologists, as part of its voluntary q - probes quality assessment program, recently reported a retrospective pathology - imaging correlation study from 48 institutions. this study is an important step in the right direction but was restricted to self - reported pathology departmental processes from core needle biopsies and did not purport to evaluate routines involving surgical excision specimens. even so, it provides valuable insight into the prevailing attitudes and practices among the subset of pathologists participating in the program. in the cap q - probes study, most pathology departments (65%) did not have a formal mechanism in place to correlate imaging with core biopsy results. the frequency with which pathologists conduct formal imaging correlation with lumpectomy or mastectomy results prior to finalizing the pathology report is unknown and may be even lower. many breast pathologists currently use specimen radiography to identify calcifications in surgical specimens, needle biopsy cores, or paraffin blocks ; breast imager involvement with these investigations is variable and largely unknown. the imaging - guided large - format breast pathology techniques described here require imager involvement and eliminate barriers to the communication of relevant clinical and imaging data to the pathologist. these techniques involve the imager prospectively in the identification and reporting of multiple tumor foci, axis of greatest cancer extent, and the proximity of impalpable neoplasm to surgical margins. the addition of imaging guidance to the pathologist 's examination is not only relevant but timely. the best opportunity for pathologist identification and anatomic localization of these findings comes before the specimen is morcellated and the 3d relationships are lost. preservation of the breast anatomy in three dimensions is an essential aspect of this technique. not only is the pathologist able to reconstruct the extent of the neoplasm in three dimensions, but the breast imager is better able to reconcile true cancer extent with imaging findings, especially mri enhancing lesions. the long - recognized discordance between reported pathologic tumor characteristics and imaging estimates of tumor size and extent has persisted despite a better presurgical impression of the complexity of breast cancers in three dimensions. this discordance has been attributed by some to a lack of specificity on the part of the imaging studies themselves without reference to the limitations inherent in the pathology techniques long held as the gold standard for measuring breast cancer characteristics. a formal validation study of breast mri using benchmark large - format examination techniques is beyond the scope of this paper but could contribute significantly to our understanding of the sensitivity and specificity of presurgical mri in predicting breast cancer extent and multifocality. although it is generally agreed that most breast cancer recurrences after bcs are related to residual cancer in the ipsilateral breast, large - format specimen analysis illustrates how complex the 3d architecture of breast cancer, particularly dcis, can be. clearly, any margin sampling method short of including 100% of a specimen surface will be representative by nature. to improve the sensitivity of margin analysis, a strategy to improve the yield and specificity of margin section selection is required. at the present time, imaging guidance combined with large - format histopathology offers the most accessible technique for the routine clinical laboratory. the prevalence of ink migration into the interior of resection specimens and its influence on margin reporting is unknown ; it may contribute to the underreporting of margin width in some cases. a fair question is whether a more thorough approach to margin evaluation resulting from lbp results in a higher incidence of close or inadequate surgical margins. in this paper, the opposite was found to be the case (table 5). as an isolated undertaking, an lbp program would hypothetically result in the reporting of close or positive margins with greater frequency. the reported outcomes occurred, not from a large - format program in isolation, but from an lbp program fully integrated into an interdisciplinary breast center. meticulous interdisciplinary presurgical planning with post - surgical outcome analysis and correlation of pre- and postsurgical imaging studies routinely occurred in a context of large - format pathology mapping of tumor extent and margin proximity. optimized in this way, large - format - imaging correlations can influence not only the selection of surgical procedure, but the extent and volume of breast tissue removed to follow the anatomic distribution of cancer in the breast. imagers become more conversant with the biologic behavior of breast cancer subtypes and their varied appearance in imaging studies, particularly mri. it seems likely that including a breast surgeon in the enhanced imaging large - format correlation process can improve the cross - disciplinary understanding of case - specific nuances in cancer distribution and optimize utilization of presurgical imaging studies to plan breast - conserving surgical procedures. the present - day clinical reliance on nonstandardized measurements of dcis extent and proximity of dcis to surgical margins to guide management decision making should raise theoretical and practical concerns. most breast pathologists are aware of the challenges faced in the gross specimen identification of dcis, the difficulty in measuring overall dcis extent in resections and the practical necessity of limiting the number of sections taken from surgical specimens. as a result, the resulting data presented in the literature is a confusing blend of methodologies and is neither comparable across studies nor is it applicable to individual practice environments. the implication for future investigative work is that nonstandardized pathologist - generated breast cancer data will continue to obfuscate clinical research. all of these limitations existed well before the advent of breast conservation surgery, but their relevance is greater today. many clinical decisions, such as whether to offer breast conservation, surgical reexcision, or radiation therapy, are based on pathologic parameters not deemed relevant to report a generation ago. in the present era of screen - detected breast cancer in asymptomatic women, the unnecessary limitations posed by traditional examination methods are a relevant concern not only for the occult component of a symptomatic or palpable lesion (as they undoubtedly were in the past), but also for the entire neoplastic process involving some patients ' breasts. pathologists are understandably reluctant to intentionally increase costs and effort associated with specimen processing and reporting, especially in an environment of fixed reimbursement. objectively, the incremental direct costs associated with the lbp program (table 9) are moderated by a reduction in low - yield, random histopathology sections. expressed as dollar expense per unit surface area examined, the technical costs of conventional and large section pathology are quite similar (table 8). with a targeted, imaging - assisted specimen examination protocol, the additional expense associated with an lbp program must be viewed in the broader context of interdisciplinary improvements in clinical efficiency and compassionate care and not as the sole burden of the pathology laboratory or department. the clinical consequences of improved margin status and lower reexcision rates for individual patients are readily apparent ; viewed from the perspective of enhanced cost - efficiency on a national or global scale the gains could be substantial. this paper describes the successful incorporation of a large - format breast pathology program into an existing comprehensive breast care center based on a non - profit health system in the united states. from its inception, the program was designed to enhance the bidirectional information flow among breast imagers, surgeons, and pathologists. a goal of the initiative was to provide the breast team members with a more precise characterization of breast cancer attributes relevant to prognosis and clinical management through improved imaging, pathology, and clinical correlation. these measures were deemed appropriate in view of the increasing clinical reliance upon pathology - reported breast cancer attributes, wherein relatively small increments in reported tumor variables can translate into significant changes in clinical management and perceived prognosis. the experience derived from the development of this program indicates it is feasible and desirable for community hospital - based pathology and breast imaging departments to adopt these processes for the benefit of all patients in a comprehensive, interdisciplinary breast center.	modern breast imaging, including magnetic resonance imaging, provides an increasingly clear depiction of breast cancer extent, often with suboptimal pathologic confirmation. pathologic findings guide management decisions, and small increments in reported tumor characteristics may rationalize significant changes in therapy and staging. pathologic techniques to grossly examine resected breast tissue have changed little during this era of improved breast imaging and still rely primarily on the techniques of gross inspection and specimen palpation. only limited imaging information is typically conveyed to pathologists, typically in the form of wire - localization images from breast - conserving procedures. conventional techniques of specimen dissection and section submission destroy the three - dimensional integrity of the breast anatomy and tumor distribution. these traditional methods of breast specimen examination impose unnecessary limitations on correlation with imaging studies, measurement of cancer extent, multifocality, and margin distance. improvements in pathologic diagnosis, reporting, and correlation of breast cancer characteristics can be achieved by integrating breast imagers into the specimen examination process and the use of large - format sections which preserve local anatomy. this paper describes the successful creation of a large - format pathology program to routinely serve all patients in a busy interdisciplinary breast center associated with a community - based nonprofit health system in the united states.
type 2 diabetes is a strong risk factor for cardiovascular outcomes, and the incidence of these outcomes rises with the degree of hyperglycemia (1). however, large randomized controlled trials have yielded mixed findings with respect to the cardiovascular effects of glucose lowering. four of these trials explicitly tested the effect of intensive versus standard glucose lowering on cardiovascular outcomes in people with type 2 diabetes (25). a meta - analysis of these four trials suggested a beneficial or neutral effect on one or more cardiovascular outcomes (6). however, one of these, the action to control cardiovascular risk in diabetes (accord) trial reported both increased total and cardiovascular mortality and reduced nonfatal myocardial infarction in the intensive glucose - lowering group during a mean follow - up period of 3.7 years (2). the reasons for these discrepant findings remain unknown despite many analyses. also not known is whether the effect on mortality or myocardial infarctions was sustained or limited by time. this uncertainty was addressed by the accord follow - on (accordion) study, which consented and monitored accord participants for up to 7 years after the intensive glycemic intervention was stopped. the design of the accord trial and the effect of intensive versus standard glucose - lowering approaches on cardiovascular outcomes have been previously reported (2,7,8). briefly, accord enrolled 10,251 people whose mean age was 62 years, who had type 2 diabetes for a median duration of 10 years with a mean glycated hemoglobin (hba1c) level of 8.3%, and who had previous cardiovascular disease or cardiovascular disease risk factors. canada were randomly assigned to intensive glucose - lowering approaches targeting a glycated hemoglobin level 8%, white vs. nonwhite, and blood pressure trial vs. lipid trial) was assessed with the use of statistical tests of interactions between the treatment effect and the subgroup within the cox models. the glycemic portion of the accordion study was designed to assess the effect of 3.7 years of exposure to the accord intensive versus the standard glycemic glucose - lowering approach on the long - term incidence of cardiovascular outcomes. canada on up to seven occasions between may 2011 (1.5 years after the conclusion of the accord trial) and october 2014. during four telephone and three clinic visits, the occurrence of cardiovascular outcomes, deaths, dialysis, all hospitalizations, severe hypoglycemia, medication usage, and related information was ascertained. documentation supporting the diagnosis of myocardial infarction, unstable angina, strokes, and deaths was sought and verified by the site. weight, height, waist circumference, blood pressure, pulse, and a neuropathy examination were conducted at each clinic visit. glycated hemoglobin, a lipid profile, serum creatinine, alanine aminotransferase level, and a spot urine albumin - to - creatinine ratio were centrally measured at the first and third visits, during which an electrocardiogram and data concerning health - related quality of life were also collected. were also ascertained using the national death index. as in accord, the primary outcome was the composite of cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke. prespecified secondary outcomes included an expanded composite outcome comprising the primary end point, any revascularization, or hospitalization for heart failure ; a composite outcome comprising the primary end point or death from any cause ; a coronary heart disease composite comprising fatal or nonfatal myocardial infarction, death occurring unexpectedly or after a cardiovascular procedure or noncardiovascular surgery, or unstable angina ; nonfatal myocardial infarction ; stroke ; nonfatal stroke ; all - cause mortality ; cardiovascular mortality ; and heart failure death or hospitalization. all of the reported cardiovascular outcomes during the accord trial were adjudicated by a masked, independent committee. conversely, in light of evidence showing that adjudication does not materially affect the estimate of the effect size in randomized trials (11,12), only a randomly selected 10% of the reported outcomes were similarly adjudicated during the accordion follow - up phase (to ensure quality control). analyses in accordion were conducted on all of the cardiovascular outcomes that occurred after randomization and that were reported by the sites during the active or follow - up period, regardless of the final adjudication status. outcome definitions were identical to those used in accord and are available online (13). statistical analyses were undertaken by the accord coordinating centre using sas 9.4 software (sas institute inc., cary, nc), according to a prespecified plan that was finalized before any analyses began. a nominal level of significance of p 8%, white vs. nonwhite, and blood pressure trial vs. lipid trial) was assessed with the use of statistical tests of interactions between the treatment effect and the subgroup within the cox models. ninety percent (n = 8,601) of these living individuals, representing 98% of the 8,777 individuals without a primary outcome event during the active phase of the accord trial, consented to further, posttrial follow - up. of 10,251 randomized accord participants, 9,533 (93%) were known to be alive at the end of the full accord trial (when both the lipid and blood pressure interventions were discontinued), and 8,777 remained free of a myocardial infarction of stroke (i.e., the first occurrence of a nonfatal component of the primary outcome) at that time. participants who agreed to ongoing follow - up were younger and at the time of randomization had lower serum creatinine and ldl levels, had a lower prevalence of smoking and prior cardiovascular events, were less likely to be on insulin, and were more likely to be taking metformin, statins, and aspirin than the 1,650 accord participants who had died or did not agree to further follow - up (table 1). baseline characteristics according to follow - up data are n (%), mean (sd), or median (interquartile range). 2) achieved during a mean active treatment period of 3.7 years was clearly smaller but persisted well after transition of all participants to the standard glycemic treatment strategy. thus, a mean of 1.2 years after transition (i.e., when the accord trial ended), the mean (sd) glycated hemoglobin level was 7.4% (1.2%) in the intensive group and 7.8% (1.3%) in the standard group (p < 0.001), and at the end of the accordion follow - up, these levels were 7.8% (1.4%) and 8.0% (1.4%), respectively (p = 0.005). this hba1c difference persisted during the accordion follow - up period despite similar reported glucose - lowering regimens (supplementary table 1) and similar weights of 91.8 (20.4) kg and 91.2 (20.4) kg (p = 0.4) in people who had been allocated to the intensive and standard groups, respectively. during the accordion phase of the study, there were similar rates of severe hypoglycemia requiring medical assistance or any severe hypoglycemia in each allocated group (supplementary table 2). the incidence of reported clinical outcomes and the hazard of allocation to the intensive versus the standard glucose - lowering regimen during the accord trial and the full accordion median follow - up period of 8.8 years are shown in fig. 1 and listed in table 2. allocation to an intensive glucose - lowering regimen for a mean of 3.7 years had a neutral long - term effect on the first occurrence of the primary composite outcome comprising nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death ; death from any cause ; an expanded composite of nonfatal myocardial infarction, nonfatal stroke, or any death ; a composite of fatal coronary heart disease, nonfatal myocardial infarction, or unstable angina ; and heart failure hospitalizations. conversely, the increase in death from cardiovascular causes that was noted at the end of the active treatment period persisted. however, as a result of similar rates after the intervention, the hazard ratio was attenuated to 1.20 (95% ci 1.03, 1.39 ; p = 0.02). product - limit estimates of time to event for the primary outcome, death (fig. 3) suggested that any differences in incidence were confined to the active treatment period. no differences in the long - term effect of the intervention on the primary outcome or mortality were noted for predefined subgroups (supplementary figs. 3 and 4). the event rates and hazard ratios (hrs,) with 95% cis (horizontal lines) are shown for prespecified outcomes that occurred from randomization until the end of the accord trial and until the end of prolonged follow - up comprising the accord and accordion phase. first occurrence of outcomes during different study phases chd, coronary heart disease ; chf, congestive heart failure ; cvd, cardiovascular death ; mi, myocardial infarction ; revasc, revascularization ; ua, unstable angina. identified using the national death index. the kaplan - meier curves display the time to event for the primary outcome (a) and total mortality (b) during follow - up from randomization until the end of accordion. the inset for each graph displays the same curve with a magnified y - axis. the kaplan - meier curves display the time to event for the three components of the primary outcome, including nonfatal myocardial infarction (mi) (a), nonfatal stroke (b), and cardiovascular (cv) death (c) during follow - up from randomization until the end of accordion. intensive glycemic control may have long - term effects on serious health outcomes in people with type 2 diabetes. such legacy effects may be beneficial or harmful and may emerge, be attenuated, or become magnified with time. these data from the long - term follow - up of the accord trial show that intensive glycemic control for a median of 3.7 years had a prolonged effect on hba1c levels, possibly due to some effect on -cell function (14). at the same time, it had a neutral effect on the primary cardiovascular composite outcome of nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death during a median follow - up period of 8.8 years. it also had a neutral effect on an expanded composite outcome that included death from any cause and the individual outcomes of nonfatal myocardial infarction, nonfatal stroke, and/or congestive heart failure. intensive glycemic control had a neutral long - term effect on all - cause mortality during this 8.8-year period, despite the higher mortality during the 3.7-year active phase of the accord trial. 2) and accrued data (table 2) show that this was apparently due to clustering of the excess of cardiovascular deaths between 2 and 3 years after randomization and subsequent attenuation or reversal of this pattern. the reduction in nonfatal myocardial infarction noted during the active phase of the trial was also attenuated over the entire follow - up period. the excess of cardiovascular - specific mortality accompanying intensive therapy that was noted during the active phase of the trial remains unexplained despite many analyses. possible explanations that have not been supported include severe hypoglycemia (15) ; weight gain (r.p. byington, unpublished analysis) ; the specific therapies used to lower glucose, including insulin (16) or other glucose - lowering drugs (unpublished analysis) ; or the differential effects of intensive control in people with underlying cardiac autonomic neuropathy (17). the degree of glucose lowering that was achieved in the intensive group was also not responsible for the mortality signal (18). indeed, two accord analyses found that the excess mortality in the intensive group was most apparent in participants whose hba1c levels remained high despite allocation to the intensive group and use of therapeutic strategies seeking nearly normal glucose levels (18,19). other analyses have suggested that the excess of mortality may have been due to a harmful effect of the intervention in subgroups of individuals with peripheral neuropathy, aspirin use, or hba1c 8.5% at baseline (20), or in the subgroup with the highest discrepancy between baseline hba1c and the hba1c levels that would be predicted from fasting plasma glucose levels (due to higher protein glycation, higher prandial glucose levels, or other reasons) (21). finally, that the excess mortality during accord may have been a rare chance occurrence has been suggested (22). the absence of excess mortality in the active phase of the three other trials of intensive glucose control (6), the concomitant reduction in nonfatal myocardial infarction and various indices of ischemic heart disease during this phase (6,23), and attenuation of the mortality signal during the full 9 years of follow - up provide some support for this possibility. the 10- to 20-year effect of up to 6 years of intensive glycemic control on cardiovascular outcomes has also been reported in the three other large outcomes trials of intensive glucose control. in the uk prospective diabetes study (ukpds), 10 years of active therapy achieving a median hba1c difference of 0.9% reduced the 18-year incidence of myocardial infarction by 15% and death by 13% in people with newly diagnosed type 2 diabetes (24). similarly, 6 years of active therapy achieving an hba1c difference of 1.5% reduced the 10-year incidence of cardiovascular outcomes by 17% in male veterans who participated in the veterans affairs diabetes trial (vadt) (25). the action in diabetes and vascular disease : preterax and diamicron mr controlled evaluation (advance) trial, with a lesser glycemic difference, reported that 5 years of intensive glycemic control had a neutral effect on cardiovascular outcomes during 10 years of follow - up (26). two of these posttrial intention - to - treat follow - up analyses reported a neutral effect on mortality, and one reported a reduction. a meta - analysis of the odds ratios for death from accordion and these other three studies using a fixed - effects variance estimate (27) with heterogeneity assessed using the i statistic (28) (supplementary table 3) yields an overall mortality odds ratio of 0.98 (95% ci 0.92, 1.04). taken together, the findings of accordion demonstrate neither a beneficial legacy effect of 3.7 years of intensive glucose - lowering therapy on cardiovascular end points nor a continued excess of all - cause mortality. the unexplained 20% higher relative risk of cardiovascular death in accordion corresponds to an absolute risk difference of 0.13%/year or 1.3% over 10 years. when deciding on therapy for an individual patient, these findings need to be balanced against a neutral effect on overall mortality and a large reduction in eye disease in response to intensive glucose lowering (29). these findings suggest that for people with type 2 diabetes and additional cardiovascular risk factors, the main benefits of intensive glycemic control are noncardiovascular. they also support current recommendations (30,31) to tailor the degree of glucose control to the overall health status of individual patients, taking their overall frailty and burden of other illnesses into account.	objectivein the action to control cardiovascular risk in diabetes (accord) trial, 4 years of intensive versus standard glycemic control in participants with type 2 diabetes and other cardiovascular risk factors had a neutral effect on the composite cardiovascular outcome, increased cardiovascular and total mortality, and reduced nonfatal myocardial infarction. effects of the intervention during prolonged follow - up were analyzed.research design and methodsall surviving accord participants were invited to participate in the accord follow - on (accordion) study, during which participants were treated according to their health care provider s judgment. cardiovascular and other health - related outcomes were prospectively collected and analyzed using an intention - to - treat approach according to the group to which participants were originally allocated.resultsa total of 8,601 people, representing 98% of those who did not suffer a primary outcome or death during the accord trial, were monitored for a median of 8.8 years and a mean of 7.7 years from randomization. intensive glucose lowering for a mean of 3.7 years had a neutral long - term effect on the primary composite outcome (nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death), death from any cause, and an expanded composite outcome that included all - cause death. moreover, the risk of cardiovascular mortality noted during the active phase (hazard ratio 1.49 ; 95% ci 1.19, 1.87 ; p < 0.0001) decreased (hr 1.20 ; 95% ci 1.03, 1.39 ; p = 0.02).conclusionsin high - risk people with type 2 diabetes monitored for 9 years, a mean of 3.7 years of intensive glycemic control had a neutral effect on death and nonfatal cardiovascular events but increased cardiovascular - related death.
the role of a diet with a higher content of unsaturated fatty acids, in place or concomitant to a diet with high content of lipids, has been appointed as an effective strategy to control metabolic disorders. monounsaturated fatty acids (mufa) rich diet has been reported to decrease plasma total cholesterol and ldl - cholesterol and increase hdl - cholesterol levels [25 ]. moreover, when saturated fatty acids are replaced by mufa in the diet of obese women, levels of inflammatory markers decrease, including il-6 and visfatin in serum. macadamia nut oil is rich in monounsaturated fatty acids, containing approximately 65% of oleic acid (c18:1) and 18% palmitoleic acid (c16:1) of the total content of fatty acids. some studies have shown that diet rich in macadamia can improve the lipid profile [2, 810 ], but to date there is no studies on the effect of supplementation of macadamia oil on adipocyte hypertrophy and inflammation. in 2008, cao and colleagues showed that mice deficient in lipid chaperones ap2 and mal1 present increased levels of palmitoleic acid in serum. elevated levels of circulating palmitoleic acid restored sensitivity of insulin in liver and skeletal muscle, hepatosteatosis, and hyperglycemia, generated by high - fat diet. with this, the authors named this fatty acid as a lipokine, since palmitoleic acid has a hormonal - like effect. the administration of high - fat diet in c57bl6 mice induces metabolic perturbations similar to those observed in humans. in fact, consumption of the high levels of saturated fatty acids is associated with overweight, visceral obesity, inflammation, dyslipidemia, and insulin resistance, in skeletal muscle, liver, and adipose tissue [1217 ]. saturated ffa promotes inflammation by interaction with toll - like receptor 4 (tlr4), activating nfb, jnk, and ap-1 pathways [18, 19 ]. a low grade inflammation is established with increase in plasma levels of il-6, il-1, prostaglandins, tnf-, and leptin and decrease in the production and secretion of adiponectin, il-10, and il-4 [20, 21 ]. the increase in local inflammation is potentiated by the recruitment of macrophages to adipose tissue and polarization of m2 macrophages (macrophages type 2) to m1 macrophages (macrophages type 1) [16, 22, 23 ]. the aim of our study was to evaluate the effect of macadamia oil supplementation, rich in mufa (palmitoleic and oleic acids), on adipose tissue and peritoneal macrophages inflammation in mice fed a balanced diet or high - fat diet rich in saturated fatty acids. we measured glucose uptake (2 - 6 deoxyglucose uptake) and mrna content of proteins (glut-4 ; irs-1) involved in insulin signaling in soleus muscle. the contents of il-10, il-6, tnf-, and il-1 in peritoneal macrophages and adipose tissue were also determined. all experiments were performed according to protocols approved by the animal care and use committee of the institute of biomedical sciences, university of so paulo. c57bl/6 male mice (8 weeks old) were used in this study. animals were housed with light - dark cycle of 12 - 12 h and temperature of 23 2c. animals were divided into four groups : (a) control diet (cd), (b) high - fat diet (hfd), (c) control diet supplemented with macadamia nut oil (vital tman, uchoa, sp, brazil) (cd + mo), and (d) high - fat diet supplemented with macadamia oil (hf + mo). the oil composition is shown in table 1. during the first 4 weeks preceding the induction of obesity by hfd, all groups were ad libitum fed a control diet (76% carbohydrates, 9% fat, and 15% proteins). cd + mo and hf + mo were supplemented by oral gavage at 2 g per kg of body weight, three times per week, during 12 weeks. this dosage of oil was chosen based on previous studies from our group using different oils with no signs of hepatic toxicity. serum triacylglycerol, total cholesterol, ldl - cholesterol, and hdl - cholesterol were determined by colorimetric assays (labtest diagnostics, lagoa santa, mg, brazil). serum glucose and insulin were measured using labtest colorimetric assay and radioimmunoassay (millipore, billerica, ma, usa), respectively, as described by masi. the homa index was determined by calculating fasting serum insulin (u / ml) fasting plasma glucose (mmol l)/22.5. glucose tolerance test (gtt) and insulin tolerance test (itt) were carried out in all groups after 6 h fasting at the end of the 10th and 11th weeks of treatment, respectively. the methodologies used for gtt and itt were similar to that described by masi. animals were euthanized on co2 chamber and soleus muscles rapidly and carefully isolated and weighed (810 mg). haematoxylin and eosin (h&e) staining was conducted using leica autostainer xl and leica cv5030. sections were mounted using dpx media (fisher scientific, ireland) and analyzed using nikon 80i transmission light microscope. gastrocnemius muscle fragments (100 mg) were subjected to lipid extraction. for this, 0.5 ml chloroform / methanol (2 : 1 ; v / v) was added to 100 mg of gastrocnemius sample, well - vortexed and incubated at room temperature for 5 min. additional volumes of 1.25 ml chloroform and 1.25 ml deionized h2o were then added, and finally, following vigorous homogenization for 3 min, samples were centrifuged at 1200 g for 5 min, at room temperature to obtain two phases : aqueous phase in the top and organic phase in the bottom containing. after that, for fatty acid composition determination, gastrocnemius lipid extracts were dried using atmospheric n2 for evaporation of the solvent without fatty acid oxidation. the fractions of neutral and polar lipids were separated from these extracts by using a column chromatography. the polar (phospholipids) and neutral (triglycerides) fractions were methylated (for formation of methyl esters), using acetyl chloride and methanol. the methyl esters were analyzed in a gas chromatographer coupled to a flame ionizer detector (fid) (varian gc 3900). fatty acid composition was then determined by using standard mixtures of fatty acids with known retention times (supelco, 37 components). for the analysis of fatty acids, the reading was initiated at 170c temperature for 1 minute and then a ramp of 2.5c / min was employed to reach a final temperature of 220c that was maintained for 5 min. we used the cp wax 52 cb column, with a 0.25 mm thickness, internal diameter of 0.25 mm, and 30 mm long, with hydrogen as the carrier gas. about 100 mg of retroperitoneal adipose tissue was used for the determination of tnf-, il-6, and il-10 content. adipose tissue was homogenized in ripa buffer (0.625% nonidet p-40, 0.625% sodium deoxycholate, 6.25 mm sodium phosphate, and 1 mm ethylenediaminetetraacetic acid at ph 7.4), containing 10 g / ml of a protease inhibitor cocktail (sigma - aldrich, st. homogenates were centrifuged at 12.000 g for 10 min at 4c, supernatant was collected, and protein concentration was determined using bradford assay (bio - rad, hercules, ca, usa). retroperitoneal adipose tissue explants (about 100 mg) were cultured in dmem sterile medium (gibco), containing 10% fbs, 2 mm glutamine, streptomycin, and penicillin for 24 h, at 37c and 5% co2, humidified air environment. thereafter, medium culture was collected and used for the determination of il-1 and il-10, using elisa assays (duoset kits, r&d system). cytokine and nitric oxide (no) production were evaluated in macrophages obtained by washing the peritoneal cavity with 6 ml rpmi culture medium (gibco), containing 10% fbs and 4 mm glutamine. macrophage - rich cultures (more than 90% of the cells were f4/80) were obtained by incubating peritoneal cells in 24-well polystyrene culture plates for 2 h at 37c in a 5% co2, humidified air environment. g / ml of lps (e. coli, serotype 0111:b4, sigma chemical company, usa) for 24 h. medium was collected for determination of il-6, il-10, il-1b and tnf- by elisa and nitrite content by griess method. total rna from the gastrocnemius muscle was extracted with trizol reagent (invitrogen life technologies, grand island, ny, usa), following the method described by chomczynski and sacchi. reverse transcription to cdna was performed using the high - capacity cdna kit (applied biosystems, foster, ca, usa). gene expression was evaluated by real - time pcr, using rotor gene (qiagen) and sybr green (invitrogen life technologies) as fluorescent dye. quantification of gene expression was carried out using the rpl-19 gene as internal control, as previously described. mice fed high - fat diet showed increased body weight gain, hypercholesterolemia, and insulin resistance. these modifications were similar to those observed in our previous studies [24, 25 ]. animals fed the high - fat diet (hf and hf + mo) for eight weeks showed increased (by 2-fold) body weight gain and visceral adiposity index as compared with cd and cd + mo (table 3). the weights of the liver and the brown adipose tissue depot were not altered with diet or supplementation (table 3). although the visceral adiposity index of mice fed high - fat diet (hf and hf + mo) was greater than in animals that received control diet (cd and cd + mo), the hf group had an increase (by 1,62-fold) of adipocytes size compared to the control diet (figures 1(a) and 1(b)), with statistical difference not evidenced in hf + mo group. no difference was evidenced by diet or supplementation in ldl - c, nefa, and glycerol (data not shown). moreover, the basal glycemia and kitt were increased in both groups treated with high - fat diet (data not shown). homa - ir index was increased in the hf group (by 3-fold) as compared to the other groups including the hf + mo (figure 2(a)). this result suggests a beneficial effect of macadamia oil supplementation on insulin responsiveness in the hf group. the peripheral insulin resistance was confirmed by glucose uptake in incubated soleus muscle (data not shown), as also shown previously [24, 25 ]. in addition, both groups treated with high - fat diet showed decrease in glut-4 mrna expression (figure 2(b)). the pgc-1, irs-1, cpt-1 and perilipin 5 mrna expression were not modulated in our treatment. the hf group showed an increase in triacylglycerol content in gastrocnemius muscle, but this effect was blunted in hf + mo (figure 2(c)). the fatty acid composition in neutral or polar lipid fractions remains unchanged regardless of the diet given and mo supplementation (see table 1 in supplementary material available online at http://dx.doi.org/10.1155/2014/870634). the contents of the anti - inflammatory cytokine il-10 were increased in the hf + mo group (approximately 4,09-fold) (figure 3(a)), while il-1b concentration in the medium of adipose tissue explants was increased in the hf group (figure 3(b)). when stimulated with lps, macrophages from all groups showed increased il-6 production (by 2.97-fold) (figure 4(d)), whereas il-10 and no production were elevated in cells from the hf and ct + mo groups (2.39- and 4.08-fold compared to base line, resp.) (figures 4(a) and 4(e)). no effect of lps stimulation was observed on tnf- production by macrophages from all groups (figure 4(c)). moreover, macrophages from the hf group showed an increase (by 2,41-fold) of il-1 production compared to unstimulated cells whereas the supplementation with mo abolished this elevation (figure 4(d)). mo attenuated nitrate production by lps stimulation on macrophages from the hf group (figure 4(a)). the production of il-10 was decreased in the ct group compared to ct + mo and hf groups (by 1,88-fold) (figure 4(e)). tnf- and il-6 production remained unchanged by diet or supplementation (figures 4(c) and 4(d)). no significant difference was found in serum levels of adiponectin after 12 weeks of treatment (data not shown). as expected, leptin concentration was increased (by 5.69-fold) in the groups fed the high - fat diet (data not shown). the significant difference between the cd + mo and the cd + ct groups suggests that mo can enhance circulant leptin. we showed herein that twelve weeks of macadamia oil supplementation attenuate the increase in inflammation and adipocyte hypertrophy in mice fed a high - fat diet that exhibit signs of the metabolic syndrome. high consumption of fat, sucrose, and industrialized foods in association with sedentary lifestyle is the main contributor to obesity and its related comorbidities, including dyslipidemias, insulin resistance, and cardiovascular diseases ; evidence has been accumulated that low grade inflammation plays a key role in the obesity induced comorbidities [11, 3137 ]. the increase in adipocyte diameter has been associated with disturbances in cellular homeostasis, such as insulin resistance, inflammation, and hypoxia. the prevalence of large adipocytes increases leptin production and secretion, as observed in our study. leptin is known to stimulate proinflammatory cytokines production in lymphocytes [4042 ], monocytes, and macrophages. mice fed the hfd for 8 weeks exhibited increased il-10 content in retroperitoneal adipose tissue. this result may be associated with the increase in peroxisome proliferator activated receptor- (ppar-) gamma activity. this nuclear receptor increased the number of small adipocytes and raised the il-10 [45, 46 ]. the increase of il-10 content in adipose tissue leads to macrophage polarization (type 2) that is important for remodeling and tissue repair [47, 48 ]. moreover, the increase in il-10 content in adipocytes is associated with increased insulin sensitivity in adipose tissue [49, 50 ]. il-1 strongly induces the inflammatory response in innate immune cells, via jnk and nfb pathway. patients with high level of the circulating il-1 are associated with greater risk on development of type 2 diabetes. adipose tissue and peritoneal macrophages are two sources of il-1, and macadamia oil supplementation was effective in decreasing the production of this cytokine in both. however, unexpectedly, the cdm showed an increased il-1 production after lps stimulation in peritoneal macrophages. no production is increased in lps - stimulated macrophages being more pronounced in mice fed high - fat diet. we demonstrated herein that the same pattern and the supplementation with macadamia oil prevented the production of no by peritoneal macrophages from hf mice. other bioactive compounds, such as epigallocatechin gallate and resveratrol, decrease no production by macrophage inhibition through of map kinase, jnk, and nfb signaling. in conclusion, macadamia oil supplementation attenuated inflammation and adipocyte hypertrophy in obese mice.	excess of saturated fatty acids in the diet has been associated with obesity, leading to systemic disruption of insulin signaling, glucose intolerance, and inflammation. macadamia oil administration has been shown to improve lipid profile in humans. we evaluated the effect of macadamia oil supplementation on insulin sensitivity, inflammation, lipid profile, and adipocyte size in high - fat diet (hf) induced obesity in mice. c57bl/6 male mice (8 weeks) were divided into four groups : (a) control diet (cd), (b) hf, (c) cd supplemented with macadamia oil by gavage at 2 g / kg of body weight, three times per week, for 12 weeks (cd + mo), and (d) hf diet supplemented with macadamia oil (hf + mo). cd and hf mice were supplemented with water. hf mice showed hypercholesterolemia and decreased insulin sensitivity as also previously shown. hf induced inflammation in adipose tissue and peritoneal macrophages, as well as adipocyte hypertrophy. macadamia oil supplementation attenuated hypertrophy of adipocytes and inflammation in the adipose tissue and macrophages.

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